Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof

专利摘要:

公开号:ES2774320T9
申请号:ES17797429T
申请日:2017-08-17
公开日:2020-07-21
发明作者:Mark White；Sandeep Kumar；Christopher Chan；Spencer Liang；Lance Stapleton；Andrew W Drake；Yosi Gozlan；Ilan Vaknin；Shirley Sameah-Greenwald；Liat Dassa；Zohar Tiran；Gad S Cojocaru；Maya Kotturi；Hsin-Yuan Cheng；Kyle Hansen；David Nisim Giladi；Einav Safyon；Eran Ophir；Leonard Presta；Richard Theolis；Radhika Desai；Patrick Wall
申请人:Compugen Ltd；
IPC主号:

专利说明:

[0002] Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof
[0004] II. Background of the invention
[0006] Unstimulated T cells must receive two independent signals from antigen presenting cells (APCs) in order to be productively activated. The first, Signal 1, is antigen-specific and occurs when antigen receptors on T cells encounter the appropriate antigen-MHC complex on the APC. The fate of the immune response is determined by a second, antigen-independent signal (Signal 2) that is delivered through a T-cell costimulatory molecule that binds to its APC-expressed ligand. This second signal could be either stimulatory (positive costimulation) or inhibitory (negative costimulation or co-inhibition). In the absence of a costimulatory signal, or in the presence of a co-inhibitory signal, T cell activation is impaired or aborted, which can lead to a state of antigen-specific nonresponsiveness (known as T-cell anergy) , or it can lead to apoptotic death of T cells.
[0008] Pairs of costimulatory molecules usually consist of ligands expressed on APCs and their cognate receptors expressed on T cells. Prototype ligand / receptor pairs of costimulatory molecules are B7 / CD28 and CD40 / CD40L. The B7 family consists of structurally related cell surface protein ligands that can provide a stimulatory or inhibitory signal to an immune response. Members of the B7 family are structurally related, with the extracellular domain containing at least one variable or constant immunoglobulin domain.
[0010] Both positive and negative costimulatory signals play critical roles in the regulation of cell-mediated immune responses, and molecules that mediate these signals have been shown to be effective targets for immunomodulation. Based on this knowledge, several therapeutic strategies have been developed that involve the targeting of costimulatory molecules, and have been shown to be useful for the prevention and treatment of cancer by activating, or preventing inactivation, of immune responses in cancer patients and for the prevention and treatment of autoimmune diseases and inflammatory diseases, as well as allogeneic transplant rejection, each by inactivating uncontrolled immune responses, or inducing "signal inactivation" by negative costimulation (or co-inhibition) in subjects with these pathological conditions.
[0011] The manipulation of the signals supplied by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases, and transplant rejection. Therapeutic strategies include blocking costimulation using monoclonal antibodies to the ligand or receptor of a costimulatory partner, or using soluble fusion proteins composed of the costimulatory receptor that can bind to and block its appropriate ligand. Another strategy is the induction of co-inhibition using soluble fusion protein of an inhibitory ligand. These strategies are based, at least partially, on the eventual deletion of auto or allo-reactive T cells (which are responsible for the pathogenic processes in autoimmune or transplantation diseases, respectively), presumably because, in the absence of costimulation (which induces genes survival), T cells become highly susceptible to the induction of apoptosis. Thus, new agents that are capable of modulating costimulatory signals, without compromising the capacity of the immune system to defend against pathogens, are highly advantageous for the treatment and prevention of said pathological conditions.
[0012] Costimulatory pathways play an important role in tumor development. Interestingly, tumors have been shown to evade immune destruction by preventing T cell activation through inhibition of costimulatory factors in the B7-CD28 and TNF families, as well as by attracting regulatory T cells, which inhibit antitumor responses. of T cells (see Wang (2006), "Immune Suppression by Tumor Specific CD4 + Regulatory T cells in Cancer", Semin. Cancer. Biol. 16: 73-79; Greenwald, et al. (2005), "The B7 Family Revisited ", Ann. Rev. Immunol. 23: 515-48; Watts (2005)," TNF / TNFR Family Members in Co-stimulation of T Cell Responses ", Ann. Rev. Immunol. 23: 23-68; Sadum, et al., (2007) "Immune Signatures of Murine and Human Cancers Reveal Unique Mechanisms of Tumor Escape and New Targets for Cancer Immunotherapy", Clin. Canc. Res. 13 (13): 4016-4025). Such costimulatory molecules expressed in tumors have become attractive biomarkers for cancer and can serve as tumor associated antigens (TAAs). In addition, costimulatory pathways have been identified as immunological checkpoints that attenuate T-cell-dependent immune responses, both at the initiation and effector function level in tumor metastases. As engineered cancer vaccines continue to improve, it is becoming increasingly clear that such immune checkpoints are a significant barrier to the ability of vaccines to induce therapeutic anti-tumor responses. In this regard, costimulatory molecules can serve as adjuvants for active (vaccination) and passive (antibody-mediated) cancer immunotherapy, providing strategies to thwart immune tolerance and stimulate the immune system.
[0014] Over the past decade, agonists and / or antagonists of various costimulatory proteins have been developed to treat autoimmune diseases, graft rejection, allergy, and cancer. For example, CTLA4-Ig (Abatacept, Orencia®) is approved for the treatment of RA, mutated CTLA4-Ig (Belatacept, Nulojix®) for the prevention of acute kidney transplant rejection, and the anti-CTLA4 antibody (Ipilimumab, Yervoy ®), has recently been approved for the treatment of melanoma. Other costimulation regulators have been approved, such as antibodies Merck's antiPD-1 (Keytruda®) and BMS (Opdivo®) have been approved for cancer treatments and are also being tested for viral infections.
[0016] However, although monotherapy with inhibitory anti-checkpoint antibodies has shown promise, several studies (Ahmadzadeh et al., Blood 114: 1537 (2009), Matsuzaki et al., PNAS 107 (17): 7875-7880 ( 2010), Fourcade et al., Cancer Res. 72 (4): 887-896 (2012) and Gros et al., J. Clinical Invest. 124 (5): 2246 (2014)) examining tumor-infiltrating lymphocytes ( TIL) have shown that TILs commonly express multiple checkpoint receptors. Furthermore, it is likely that TILs expressing multiple checkpoints are in fact the most reactive with tumors. In contrast, T cells not reactive with tumors in the periphery more likely express a single checkpoint. Blocking of checkpoints with full-length monospecific antibodies is probably non-discriminatory with respect to derepression of tumor-reactive TILs against autoantigen-expressing T-cells that are assumed to contribute to autoimmune toxicities.
[0018] One target of interest is PVRIG. PVRIG, also called Poliovirus Receptor-Related Immunoglobulin Domain-Containing Protein, Q6DKI7 or C7orf15, is a 326 amino acid long transmembrane domain protein with a signal peptide (ranging from amino acids 1 to 40), a domain extracellular (spanning amino acid 41 to 171), a transmembrane domain (spanning amino acid 172 to 190), and a cytoplasmic domain (spanning amino acid 191 to 326). PVRIG binds to the Poliovirus 2 receptor-related protein (PVLR2, also known as nectin-2, CD112, or mediator of herpesvirus B entry, (HVEB) a human plasma membrane glycoprotein), the binding partner of PVRIG .
[0020] Another target of interest is TIGIT. TIGIT is a co-inhibitory receptor that is highly expressed on CD4 + effector and regulatory (Treg) T cells, CD8 + effector T cells, and Nk cells. TIGIT has been shown to attenuate the immune response by (1) direct signaling, (2) ligand-induced signaling, and (3) competition with and disruption of signaling by the costimulatory receptor CD226 (also known as DNAM-1). TIGIT signaling has been the best studied in NK cells, where it has been shown that binding with its cognate ligand, poliovirus receptor (PVR, also known as CD155), directly suppresses the cytotoxicity of NK cells through its cytoplasmic domain ITIM. Inactivation of the TIGIT gene or blocking of the TIGIT / PVR interaction with antibodies has been shown to increase NK cell death in vitro, as well as exacerbate autoimmune diseases in vivo. In addition to its direct effects on T and NK cells, TIGIT can induce PVR-mediated signaling in dendritic or tumor cells, leading to increased production of anti-inflammatory cytokines, such as IL10. In T cells, TIGIT can also inhibit lymphocyte responses by disrupting the homodimerization of the costimulatory receptor CD226, and competing with it to bind to PVR.
[0022] TIGIT is highly expressed in lymphocytes, including Tumor Infiltrating Lymphocytes (TIL) and Tregs, that infiltrate different types of tumors. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a dominant immune escape mechanism for cancer. Notably, the expression of TIGIT is strongly correlated with the expression of another important co-inhibitory receptor, PD1. TIGIT and PD1 are co-expressed in the TILs of numerous human and murine tumors. Unlike TIGIT and CTLA4, PD1 inhibition of T cell responses does not involve competition for ligand binding with a costimulatory receptor.
[0024] Consequently, TIGIT is an attractive target for monoclonal antibody therapy, and further in combination with additional antibodies including anti-PVRIG antibodies.
[0026] III. Brief summary of the invention
[0028] The invention is defined by the claims and any other aspects or embodiments shown herein that are not within the scope of the claims are for information only.
[0030] Accordingly, in one aspect, the invention provides compositions comprising an antigen-binding domain that binds to human TIGIT (SEQ ID NO: 97) comprising a variable heavy domain comprising SEQ ID NO: 160 and a light domain variable comprising SEQ ID NO: 165. An antigen-binding domain comprising a heavy variable domain comprising SEQ ID NO: 150 and a light variable domain comprising SEQ ID NO: 155 or the binding domain The antigen comprises a variable heavy domain comprising SEQ ID NO: 560 and a variable light domain comprising SEQ ID NO: 565.
[0032] In a further aspect, the invention provides a composition comprising antibodies comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said VH comprises SEQ ID NO: 160 and a light chain comprising VL-VC, wherein said VL comprising SEQ ID NO: 165 and VC is either kappa or lambda. The disclosed antibody may comprise a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein the VH comprises SEQ ID NO: 150; and a light chain comprising VL-VC, wherein said VL comprising SEQ ID NO: 159 and VC is either kappa or lambda. Additionally, the disclosed antibody may comprise a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein VH comprises SEQ ID NO: 560; and a light chain comprising VL-VC, wherein said VL comprising SEQ ID NO: 565 and VC is either kappa or lambda.
[0033] In some aspects, the sequence of CH1-hinge-CH2-CH3 is selected from human IgG1, IgG2 and IgG4, and variants thereof. In some aspects, the heavy chain has SEQ ID NO: 164 and the light chain has SEQ ID NO: 169.
[0035] In a further aspect, the compositions may further comprise a second antibody that binds to a human checkpoint receptor protein, which may be human PD-1 or human PVRIG. The second antibody may comprise an antigen-binding domain comprising a variable heavy domain comprising SEQ ID NO: 5 and a variable light domain comprising SEQ ID NO: 10, or a heavy chain having SEQ ID NO: 9 and a light chain having SEQ ID NO: 14.
[0037] In a further aspect, the invention provides nucleic acid compositions comprising a first nucleic acid encoding a variable heavy domain comprising SEQ ID NO: 160 and a second nucleic acid encoding a variable light domain comprising SEQ ID NO: 165. Alternatively, the disclosed nucleic acid compositions comprise a first nucleic acid encoding a variable heavy domain comprising SEQ ID NO: 150 and a second nucleic acid encoding a variable light domain comprising SEQ ID NO: 155. Alternatively , the disclosed nucleic acid compositions comprise a first nucleic acid encoding a variable heavy domain comprising SEQ ID NO: 560 and a second nucleic acid encoding a variable light domain comprising SEQ ID NO: 565.
[0039] In a further aspect, the invention provides expression vector compositions comprising these nucleic acid compositions are also provided, such as a first expression vector comprising a first nucleic acid and a second expression vector comprising a second nucleic acid, or alternatively an expression vector comprising both a first and a second nucleic acid.
[0041] In a further aspect, the invention provides host cells comprising the expression vector compositions, and methods for preparing the antibodies which comprise culturing the host cells under conditions where antibodies are produced and recovering the antibody.
[0043] In a further aspect, the invention provides anti-PVRIG antibodies comprising a heavy chain having SEQ ID NO: 9 and a light chain having SEQ ID NO: 14. The disclosure further provides antibodies having a heavy chain having SEQ ID NO: 19; and a light chain having SEQ ID NO: 24.
[0045] In a further aspect, an anti-PVRIG antibody (either CHA.7.518.1.H4 (S241P) or CHA.7.538.1.2.H4 (S241P) is co-administered with a second antibody that binds to a checkpoint receptor protein human, such as an antibody that binds to PD-1.
[0047] In a further aspect, an anti-PVRIG antibody (either CHA.7.518.1.H4 (S241P) or CHA.7.538.1.2.H4 (S241P)) is co-administered with a second antibody that binds to a receptor protein point of human control, such as an antibody that binds to human TIGIT, such as CPA.9.086 or CPA.9.083 or c Ha .9.547.13.
[0049] In a further aspect, the disclosure provides nucleic acid compositions comprising a first nucleic acid encoding the heavy chain of either CHA.7.518.1.H4 (S241 P) or CHA.7.538.1.2.H4 (S241 P)) and a second nucleic acid encoding the light chain of either CHA.7.518.1.H4 (S241P) or CHA.7.538.1.2.H4 (S241P), respectively.
[0051] In a further aspect, the invention provides expression vector compositions comprising these nucleic acid compositions are also provided, such as a first expression vector comprising a first nucleic acid and a second expression vector comprising a second nucleic acid, or alternatively an expression vector comprising both the first and second nucleic acids.
[0053] In a further aspect, the invention provides host cells comprising the expression vector compositions, and methods for preparing the antibodies which comprise culturing the host cells under conditions where the antibodies are produced and recovering the antibody.
[0055] In a further aspect, the description provides methods comprising: a) providing a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind to: i) the TIGIT protein; ii) the PVR protein; iii) the PD-1 protein; iv) the PD-L1 protein; and v) an isotype control; c) operating fluorescence activated cell sorting (FACS); d) for each of TIGIT, PVR, PD-1 and PD-L1, determining the percentage of cells in said population that express the protein relative to said isotype control antibody; where if the percentage of positive cells is> 1% for the 4 receptors, e) administering antibodies against TIGIT and PD-1 to said patient.
[0057] In a further aspect, the description provides methods comprising: a) providing a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind to: i) the PVRIG protein; ii) the PVRL2 protein; iii) the PD-1 protein; iv) the PD-L1 protein; and v) an isotype control; c) operating fluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2, PD-1 and PD-L1, determine the percentage of cells in said population that express the protein in relation to said antibody of isotype control; where if the percentage of positive cells is> 1% for the 4 receptors, e) administering antibodies against PVRIG and PD-1 to said patient.
[0059] In a further aspect, the disclosure provides methods comprising a) providing a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind to: i) the PVRIG protein; ii) the PVRL2 protein; iii) the TIGIT protein; iv) the PVR protein; and v) an isotype control; c) operating fluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2, TIGIT and PVR, determining the percentage of cells in said population that express the protein relative to said isotype control antibody; where if the percentage of positive cells is> 1% for the 4 receptors, e) administering antibodies against PVRIG and TIGIT to said patient.
[0061] IV. Brief description of the drawings
[0063] Figure 1 depicts the full length sequence of human PVRIG (showing two different methionine starting points). Signal peptide is underlined, ECD is double underlined.
[0065] Figure 2 depicts the sequence of the human poliovirus receptor 2-related protein (PVLR2, also known as nectin-2, CD112 or herpesvirus B entry mediator, (HVEB)), the binding partner of PVRIG. PVLR2 is a glycoprotein of the human plasma membrane.
[0067] Figure 3A and B represent the heavy and light variable chains, as well as the sequences of vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 of each of the CHA antibodies listed of the invention, CHA.7.518.1.H4 (S241 P), and CHA.7.538.1.2.H4 (S241 P).
[0069] Figure 4A and B antibodies to PVRIG increase proliferation of T cells in MLR. Low CFSE cell percentages are shown from MLR assays treated with the indicated PVRIG antibodies. Each graph represents an individual CD3 T cell donor. The experiments are described in Example 23 of USSN 15 / 048,967, incorporated by reference herein.
[0071] Figure 5 A and B binding characteristics of hybridoma antibody to PVRIG to He K hPVRIG engineered cell lines, HEK parent cells, and Jurkat cells. HEK OE denotes HEK hPVRIG cells, HEK pair denotes parental HEK cells. For the data in Jurkat, gMFIr indicates the fold difference in geometric MFI of the PVRIG antibody staining from its controls. The concentration indicates which at which the gMFIr was calculated. Non-binding indicates that the antibody does not bind to the cell line tested. The highlighted antibodies are the 'best four' antibodies of interest.
[0073] Figure 6A and B binding characteristics of the hybridoma antibody to PVRIG to primary human PBMC, cyno overexpressing cells, and primary cyno PBMC. Expi cyno OE denotes expi cells transiently transfected with cPVRIG, expi pair denotes expi parental cells. gMFIr indicates the fold difference in geometric MFI of the PVRIG antibody staining compared to its controls. The concentrations indicate which at which the gMFIr was calculated. Not assayed indicates that the antibodies were not assayed due to a lack of binding to HEK hPVRIG cells, expi human cPVRIG, or failure to meet the requirements for binding to PBMC subsets. The highlighted antibodies are the 'best four' antibodies of interest. The experiments are described in Example 21 of USSN 15 / 048,967, incorporated by reference herein.
[0075] Figure 7A and B Summary of the blocking ability of the PVRIG antibodies in the FACS-based competition assay. The IC50 of inhibition is indicated. Absence of IC50 indicates that these antibodies are not blocking. The highlighted antibodies are the 'best four' antibodies of interest. The experiments are described in Example 21 of USSN 15 / 048,967, incorporated by reference herein.
[0077] Figure 8A and B The TILs were co-cultured with melanoma 624 cells at 1: 1 E: T for 18hr in the presence of Ab anti-PVRIG (c Pa .7.021; 10ug / ml), anti-TIGIT (clone 10A7; 10ug / ml ) or in combination. The supernatant was collected and assayed in a Th1 Th2 Th17 cytometric bead microarray assay for secreted cytokines. The levels of IFNy ( A ) and TNF ( B ) were detected. Treatments were compared by Student's t test (* P <0.05, ** P <0.01) of triplicate samples.
[0079] Figure 9A to F TIL MART-1 or 209 were co-cultured with 624 melanoma cells at 1: 1 E: T for 18hr in the presence of Ab anti-PVRIG (CPA.7.021; 10ug / ml), anti-DNAM1 (clone DX11, BD Biosciences Cat. No. 559787; 10ug / ml) or in combination. The supernatant was collected and assayed in a Th1 Th2 Th17 bead microarray cytometric assay for secreted cytokines. The levels of IFNy ( A, D ) and TNF ( B, E ) were detected. TILs were stained for surface expression of CD137 ( C, F ).
[0081] Figure 10A and B The TIL (F4) were co-cultured with 624 melanoma cells at 1: 3 E: T for 18hr in the presence of Ab anti-PVRIG (CPA.7.021; 10ug / ml), anti-TIGIT (clone 10A7; 10ug / ml), anti-PD1 (mAb 1B8, Merck; 10ug / ml) or in combination. The supernatant was collected and assayed in a Th1 Th2 Th17 bead microarray cytometric assay for secreted cytokines. I FNy ( A ) and TNF ( B ) levels were detected
[0082] Figure 11 A to E represent four humanized sequences for each of CHA.7.518, CHA.7.524, CHA.7.530, CHA.7.538_1 and CHA.7.538_2. All humanized antibodies comprise the H4 substitution (S241 P). Note that the light chain for CHA.7.538_2 is the same as for CHA.7.538_1. The "HI" of each is a "CDR exchange" with no changes in the human frame. Subsequent sequences alter frame changes displayed in larger bold type size. CDR sequences are indicated in bold. The definitions of the CDRs are AbM from the website www.bioinf.org.uk/abs. Human germline and junction sequences from IMGT® the international ImMunoGeneTics® information system www.imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France). Residue numbering shown as sequential (seq) or according to Chothia from the website www.bioinf.org.uk/abs/ (AbM). "b" indicates buried side chain; "p" indicates partially buried; "i" indicates side chain at the interface between the VH and VL domains. Differences in sequences between human and murine germ lines indicated by an asterisk (*). Additional potential mutations in the frames are indicated below the sequence. Potential changes in the CDR sequences are indicated below each CDR sequence as indicated in the figure (#deamidation substitutions: Q / S / A; these can prevent deamidation of asparagine (N). @ Substitutions of tryptophan oxidation: Y / F / H; these can prevent tryptophan oxidation; @ methionine oxidation substitutions: L / F / A).
[0084] Figure 12A to E represents a compilation of the humanized sequences of three CHA antibodies: CHA.7.518, CHA.7.538.1, and CHA.7.538.2.
[0086] Figure 13 depicts schemes for combining humanized VH and VL CHA antibodies. The "VH quim" and "VL quim" are the mouse heavy and light variable sequences linked to a constant domain of human IgG.
[0088] Figure 14. Binding characteristics of hybridoma antibody to PVRIG to primary human PBMC, cyno overexpressing cells, and primary cyno PBMC. Expi cyno OE denotes expi cells transiently transfected with cPVRIG, expi pair denotes expi parental cells. gMFIr indicates the fold difference in geometric MFI of the PVRIG antibody staining compared to its controls. The concentrations indicate which at which the gMFIr was calculated. Not assayed indicates that the antibodies were not assayed due to a lack of binding to HEK hPVRIG cells, expi human cPVRIG, or failure to meet the requirements for binding to PBMC subsets. The highlighted antibodies are four antibodies for which humanization was done (See Figure 24). The experiments are described in Example 21 of USSN 15 / 048,967, incorporated by reference herein.
[0089] Figure 15. Summary of the blocking ability of antibodies against PVRIG in the FACS-based competition assay. The IC50 of inhibition is indicated. Absence of IC50 indicates that these antibodies are not blocking. The highlighted antibodies are four antibodies for which humanization was done (See Figure 24).
[0091] Figure 16. Summary of the activity of antibodies against PVRIG selected in cytotoxicity assays of NK cells against Reh cells and MOLM-13. The times of change in cytotoxicity from the control were calculated by dividing the absolute level of death (%) in the condition with antibody against PVRIG, by the absolute level of death (%) with the control antibody. The times of change are calculated from the 5: 1 effector to target ratio.
[0092] Figure 17. PVRIG ortholog sequence alignment. The aligned sequences of the extracellular domain of human PVRIG, cynomolgus, marmot, and rhesus. The differences between human and cynomolgus are highlighted in yellow.
[0094] Figure 18. Binding of human anti-PVRIG antibodies to cyno, human, hybrid cyno / human PVRIG variants. Binding of antibodies to wild-type PVRIG of cyno (•), PVRIG H61R cyno (■), PVRIG P67S cyno (▲), PVRIG L95R / T97I cyno (▼), and human PVRIG wild-type (♦) is shown. ELISA signals are plotted as a function of antibody concentration.
[0096] Figure 19. Correlation of epitope pool and cyno cross-reactivity of anti-human PVRIG antibodies.
[0097] Figure 20A to B (A) Specificity of CHA.7.518.1.H4 (S241P) against HEK cells engineered to overexpress PVRIG and parental HEK cells. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration. (B) Specificity of CHA.7.538.1.2.H4 (S241 P) against HEK cells engineered to overexpress PVRIG and parental HEK cells. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration.
[0099] Figure 21 A to B illustrates the ability of CHA.7.518.1.H4 (S241 P) (A) and CHA.7.538.1.2.H4 (S241 P) (B) to bind to Jurkat cells that endogenously express PVRIG confirmed by RNA expression. (A) Binding of CHA.7.518.1.H4 (S241P) to Jurkat cells. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration. Isotype staining is shown as a negative control. (B) Binding of CHA.7.538.1.2.H4 (S241P) to Jurkat cells. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration. Isotype staining is shown as a negative control. Both antibodies are capable of binding Jurkat cells with comparable affinity to HEK hPVRIG cells.
[0100] Figure 22 illustrates the ability of CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) to bind to CD8 T cells that were expanded by exposure to the CMV peptide (494-503, NLVPMVATV) and that endogenously express PVRIG confirmed by the expression of a Rn. Binding of CHA.7.518.1.H4 (S241 P) and c Ha .7.538.1.2.H4 (S241 P) to CD8 T cells expanded with the CMV peptide. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration. Isotype staining is shown as a negative control.
[0101] Figure 23A to B. (A) Specificity of CHA.7.518.1.H4 (S241P) against expi cells engineered to overexpress PVRIG from cynomolgus and parental expi cells. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration. Specificity of CHA.7.538.1.2.H4 (S241 P) against expi cells engineered to overexpress PVRIG from cynomolgus and parental expi cells. The data shows measurements of absolute geometric MFI (gMFI) as a function of increasing antibody concentration.
[0103] Figure 24A to B. (A) Blocking of PVRIG Fc in HEK cells by CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P). The data show the percentage of PVRIG Fc binding to HEK cells as a function of increasing antibody concentration relative to the maximum signal induced by PVRIG Fc and secondary only background. (B) Effect of CHA.7.544 on PVRIG Fc binding to HEK cells. The data shows the absolute gMFI derived from the binding of PVRIG Fc to HEK cells in the presence of increasing concentrations of CHA.7.544. The amount of PVRIG Fc binding was detected by an Alexa 647-conjugated anti-mouse Fc secondary antibody.
[0105] Figure 25 A to B. (A) Blocking of PVRL2 Fc to HEK hPVRIG cells by CHA.7.518.1.H4 (S241P), CHA.7.538.1.2.H4 (S241P), and CHA.7.530.3. The data show the percentage of PVRL2 Fc binding to HEK hPVRIG cells as a function of increasing antibody concentration relative to the maximum signal induced by PVRL2 Fc and secondary only background. (B) Effect of CHA.7.544 on PVRL2 Fc binding to HEK hPVRIG cells. The data show the percentage of PVRL2 Fc binding to HEK hPVRIG cells as a function of increasing antibody concentration relative to the maximum signal induced by PVRL2 Fc and secondary only background.
[0107] Figure 26. Shows the percentage of binding of CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) conjugated to Alexa 647 with respect to their maximum signal after preincubation of Jurkat cells with CHA.7.518.1.H4 (S241P), CHA.7.538.1.2.H4 (S241 P) unconjugated and an isotype control.
[0109] Figure 27A to B A) Humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), increase proliferation of CD4 + T cells. Representative data (n> 2) show the percentage of low CFSE proliferating CD4 + T cells (mean plus standard deviation) from a single human CD4 + T cell donor when co-cultured with CHO-S OKT3 hPVRL2 cells in the presence of an antibody anti-DNAM-1 or different anti-PVRIG antibodies or IgG isotype controls. The dashed line indicates the percentage at baseline of CD4 + T cells with low CFSE, proliferating after treatment with the human IgG4 isotype control antibody. The numbers refer to the percentage increase or decrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the relevant isotype control antibodies (B) Humanized PVRIG antibodies , CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), increase the proliferation of CD4 + T cells in an hPVRL2-dependent manner. Representative data (n> 2) show the percentage of low CFSE proliferating CD4 + T cells (mean plus standard deviation) from a single human CD4 + T cell donor in response to coculture with parental CHO-S OKT3 cells, or CHO- S OKT3 hPVRL2 in the presence of an anti-DNAM-1 antibody or different anti-PVRIG antibodies or IgG isotype controls. The dashed line indicates the percentage at baseline of CD4 + T cells with low CFSE, proliferating after treatment with either the human IgG4 isotype or mouse IgG1 isotype antibodies. The numbers refer to the percentage increase or decrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the relevant isotype control antibodies.
[0111] Figure 28A to C. (A) Humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), increase the proliferation of CD8 + T cells. Representative data (n> 2) show the percentage of low CFSE proliferating CD8 + T cells (mean plus standard deviation) from a single human CD8 + T cell donor (Donor 232) when co-cultured with CHO-S OKT3 hPVRL2 cells in presence of an anti-DNAM-1 antibody or different anti-PVRIG antibodies or IgG isotype controls. The dashed line indicates the percentage at baseline of CD8 + T cells with low CFSE, proliferating after treatment with mouse IgG1 or human IgG4 isotype control antibodies. The numbers refer to the percentage increase or decrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the relevant isotype control antibodies. (B) Humanized PVRIG antibodies, CHA.7.518.1.H4 (S241 P) and c Ha .7.538.1.2.H4 (S241 P), increase the proliferation of CD8 + T cells. Representative data (n> 2) show the percentage of low CFSE proliferating CD8 + T cells (mean plus standard deviation) from a single human CD8 + T cell donor (Donor 234) when co-cultured with CHO-S OKT3 hPVRL2 cells in presence of an anti-DNAM-1 antibody or different anti-PVRIG antibodies or IgG isotype controls. The dashed line indicates the percentage at baseline of CD8 + T cells with low CFSE, proliferating after treatment with mouse IgG1 or human IgG4 isotype antibodies. The numbers refer to the percentage of increase or decrease in the proliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the isotype control antibodies. relevant. (C) Humanized PVRIG antibodies, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P), increase IFN and CD8 + T cell secretion. Representative data (n> 2) show pg / ml IFNy produced (mean plus standard deviation) by three different human donors of CD8 + T cells (Donors 231, 232, and 234) when co-cultured with CHO-S OKT3 cells. hPVRL2 in the presence of an anti-DNAM-1 antibody or different anti-PVRIG antibodies or IgG isotype controls. The dashed line indicates baseline I FNy production after treatment with human IgG4 isotype antibody. The numbers refer to the percentage increase in the secretion of I FNy of the treatments with the anti-PVRIG antibody compared to the IgG4 isotype control.
[0113] Figure 29. Humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), consistently increase proliferation of CD4 + T cells in multiple donors, while CHA. 7.530.3 and CHA.7.544 do not. The percentage of proliferation relative to the isotype control was calculated by dividing the percentage of CD4 + T cells with low CFSE after the PVRIG antibody treatment over the isotype antibody treatment for each donor. Percent proliferation for isotype antibody treatment was set to zero. Each symbol on the graph represents a different donor.
[0115] Figure 30A to D. (A) Dose-dependent effect of humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), on T cell proliferation CD4 +. Representative data (n> 2) with 2 different human donors show the mean percentage of proliferating CD4 + T cells after dose titration from 66nM to 0.726nM with either human IgG4 isotype antibodies, CHA.7.518.1.H4 (S241 P), or CHA.7.538.1.2.H4 (S241 P). The estimated EC50 is in the single digit nM range. (B) Dose-dependent effect of humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241 P), on CD8 + T cell proliferation. Representative data (n> 2) with 2 different human donors show the mean percentage of proliferating CD8 + T cells after dose titration from 66nM to 0.264nM with either human IgG4 isotype antibodies, CHA.7.518.1.H4 (S241P ), CHA.7.38.1.2, or CHA.7.544. The estimated EC50 is in the single digit nM range.
[0117] Figure 31 A to C. (A) Flow cytometric analysis of the expression of TIGIT and PVRIG in TIL and expression of PVR, PVRL2 in the melanoma cell line 624. The values represent the ratio of the mean fluorescence intensity (MFI ) versus the isotype control. (BC) Representative experiment showing the secretion of I FNy (B) and TNF (C) by TILs after co-culturing with melanoma 624 cells at 1: 3 E: T for 18hr in the presence of isotype control Ab, anti -TIGIT (30pg / ml) or anti-PVRIG (10 pg / ml) as a single treatment (blue histograms) or in combination with anti-TIGIT (green histograms). The percentage of the effect of Ab mono treatment was compared to the mIgG1 isotype control treatment and the percentage of the combined Ab treatment effect was compared to the anti-TIGIT mono treatment.
[0119] Figure 32A to H. TIL (209-gp100 / 463-F4-gp100) were co-cultured with melanoma 624 cells at 1: 3 E: T for 18 h in the presence of the Ab anti-PVRIG c Ha .7.518.1.H4 ( S241P) or CHA.7.538 with or without anti-TIGIT (aTIGIT) in combination and assayed for cytokine secretion. The percentage effect of Ab treatment was compared with isotype control treatment and the mean of 5 experiments (F4) or 6 experiments (209) was plotted. Two-tailed paired T-test was calculated for each treatment compared to isotype or in combination compared to anti-TIGIT alone, p-values are indicated.
[0121] Figure 33A to B. (A) Humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), and an anti-TIGIT antibody increase proliferation of CD4 + T cells compared to single antibody treatments. Representative data (n> 2) show the percentage of low CFSE proliferating CD4 + T cells (mean plus standard deviation) from a single human CD3 + T cell donor (Donor 143) when co-cultured with CHO-S OKT3 hPVRL2 cells. The dashed line indicates the percentage at baseline of proliferating CD4 + T cells with low CFSE, after treatment with the human IgG4 isotype control antibody. (B) Humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), and anti-TIGIT antibody increase proliferation of CD4 + T cells compared to single antibody treatments. Representative data (n> 2) show the percentage of low CFSE proliferating CD4 + T cells (mean plus standard deviation) from a single human CD4 + T cell donor (Donor 201) when co-cultured with CHO-S OKT3 hPVRL2 cells. The dashed line indicates the percentage at baseline of proliferating CD4 + T cells with low CFSE, after treatment with the human IgG4 isotype control antibody. The numbers refer to the percentage increase or decrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the relevant isotype control antibodies.
[0123] Figure 34A to B. (A): The combination of the humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), and the anti-TIGIT antibody increases the proliferation of CD8 + T cells. Representative data (n> 2) show the percentage of low CFSE proliferating CD8 + T cells (mean plus standard deviation) from a representative human CD8 + T cell donor (Donor 232) when co-cultured with CHO-S OKT3 hPVRL2 cells. The dashed line indicates the percentage at baseline of proliferating CD8 + T cells with low CFSE, after treatment with the human IgG4 isotype control antibody. The numbers refer to the percentage increase or decrease in proliferation of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the relevant isotype control antibodies. (B) Combination of humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), and anti-TIGIT antibody increases IFNy secretion from T cells CD8 +. Representative data (n> 2) show pg / ml IFNy produced (mean plus standard deviation) by a representative human CD8 + T cell donor (Donor 232) when co-cultured with CHO-S OKT3 hPVRL2 cells. The dashed line indicates baseline I FNy production after treatment with human IgG4 isotype antibody. The numbers refer to the percentage increase or decrease in I FNy secretion of the anti-PVRIG or anti-DNAM-1 antibody treatments, respectively, compared to the relevant isotype control antibodies.
[0125] Figure 35 represents the design of the experimental system of Example 2 (3).
[0127] Figure 36A to C. shows a histogram representing the expression levels of PVRIG (using Anti-human PVRIG CHA.7.538.AF647), TIGIT (using Anti-human TIGIT Cat. 17-9500-41 eBioscience) and d Na M- 1 (using Biolegend Cat.338312 Human Anti-CD226-APC) in TIL. The expression times are compared with the isotype control (Iso).
[0129] Figure 37. Summary representation of the effect of anti-PVRIG antibodies on the secretion of I FN and TIL. TILs were cocultured with CHO-S HLA-A2 / B2M cells that overexpress PVRL2 in an E: T ratio of 1: 3 for 18 h in the presence of anti-PVRIG antibodies (c518, c538 and 544) or with an anti-TIGIT antibody. . Each point represents an average of the IFNy secretion data from the same TILs from different experiments. The percentage indicated is the difference between each antibody treatment compared to the isotype control. Paired two-tailed T-test was calculated for each treatment compared to 544 or in combinations, compared to anti-TIGIT alone, p-values are indicated. The number of experiments performed for each TIL; 209 (N = 3), F4 (N = 2), F5 (N = 3) and MART1 (N = 2).
[0131] Figure 38. Summary representation of the effect of c518 and c538 dose response on TNF-α secretion from TILs. TILs were co-cultured with CHO-S HLA-A2 / B2M cells that overexpress PVRL2 in an effector-to-target ratio of 1: 3 for 18 h in the presence of anti-PVRIG antibodies (c518, c538 or isotype control) as described in the Example 2 (3).
[0133] Figure 39A to C. TILs were co-cultured with CHO-S HLA-A2 / B2M target cells that overexpress PVRL2 in an E: T ratio of 1: 3 for 18hr in the presence of anti-PVRIG antibodies (c518, c538 and 544) or with anti-TIGIT antibody. The percentage indicated in the tables above is the difference in the effect of TIL cytokine secretion from each antibody treatment compared to its isotype control. The first experiment is represented in Figure A and B, and the second experiment in Figure C.
[0135] Figure 40. CHO-S OKT3 co-culture assay design. CFSE-labeled CD3 + T cells were co-cultured with CHO-S-OKT3-PVRL2 cells or mock transfection for 5d. The effect of anti-PVRIG Ab on T cell proliferation and cytokine secretion was analyzed.
[0137] Figure 41. Effect of anti-PVRIG antibodies on the secretion of I FNy in CHO-OKT3 PVRL2 cells in responding versus non-responding donor. CD3 + cells from 2 different donors were co-cultured with CHO-S-PVRL2 cells in a 5: 1 E: T ratio for 5d in the presence of anti-PVRIG Ab and assayed for cytokine secretion and T cell proliferation. (A) 'responder donor' in which we observed an effect to anti-PVRIG Ab. (B) 'non-responding donor' in which we observed no effects to Ab treatment.
[0139] Figure 42. Effect of anti-PVRIG antibodies on the proliferation of CD4 and CD8 from a responding donor. CFSE-labeled CD3 + T cells were co-cultured with CHO-S-PVRL2 cells in a 5: 1 E: T ratio for 5d in the presence of anti-PVRIG Ab or anti-TIGIT Ab. The effect on the proliferation of T cells selecting for CD4 or CD8 was evaluated by flow cytometry. The percentage of proliferating cells (low CFSE) (A) or total cell number (B) of CD4 + low CFSE or CD8 + low CFSE are presented.
[0141] Figure 43. Shows the effect of anti-PVRIG antibodies on IFNy secretion or CD8 proliferation from a responding donor. CD3 + cells were co-cultured with CHO-S-PVRL2 cells at 5: 1 E: T for 5d in the presence of anti-PVRIG Ab and assayed for (A) cytokine secretion and (B) T cell proliferation. The effect of Ab treatment was compared with isotype control treatment and the mean of 5 donors 'responders' (responders) is presented. (C) Levels of IFNy secretion from the same 5 donors after coculture with CHOS-OKT3 PVRL2 as described in section A and B after treatment with isotype Ab against anti-PVRIG. The p-value represents the ratio of the paired T test.
[0143] Figure 44 is a summary table of the effect of Ab treatment on donors tested (n = 10). The percentages indicated represent the effect of Ab treatment on a specific outcome (indicated in the column headings) compared to the relevant isotype control. The 'responder' donors (donor no. 3, 72,226,345 and ES_001) considered as 'responder' in which some anti-PVRIG Ab (mainly CHA.7.518) increased I FNy or proliferation compared to isotype controls.
[0145] Figures A to B represent the results of experiments with various antibodies. The affinities (nM) are shown in A, with the HEK hPVRIG cells being the HEK cells transformed with hPVRIG as discussed herein and the Jurkat cells expressing endogenous hPVRIG. (B) Represent the gMFI using 4 different antibodies to the primary CD8 T cells of Donor 1 and (C) being the primary CD8 T cells of Donor 2.
[0147] Figures 46A to B depict interactions of TIGIT with CHO cells. (A) Human TIGIT Fc protein binds to CHO cells. Graded concentrations of human TIGIT Fc and IgG1 synagis control were evaluated for their ability to bind to CHO cells in a FACS-based binding assay. (B) Human PVR is expressed on activated CD4 T cells. CD4 T cells were co-cultured with CHO cells expressing the OKT3 antibody scFv and activated for 5 days. On day 5, CD4 T cells were analyzed for PVR expression and CFSE dilution.
[0149] Figures 47A to C depict anti-mPVRlg and anti-PDL-1 antibody antitumor responses in a CT26 tumor model. A-B. Groups of 10 BALB / c mice were injected subcutaneously with 5 x 105 CT26 cells. After measuring tumors on day 4, mice were randomized (mean tumor volume 40 mm3 per group) and then treated with the designated mAb (100 or 200 pg / IP dose) followed by additional doses on days 7, 11, 14, 18 and 21. A. The groups were treated with 6 doses of single agents. Anti-PDL-1 vs control *** p <0.0001. Tumor volumes are represented as the Mean Volume SEM. B. Tumor volumes were measured twice weekly. The number of tumor-free (TF) mice per group is indicated. C. Survival ratios of assigned groups; Anti-PDL-1 vs. control ** p = 0.005.
[0151] Figures 48A to C depict antitumor responses of a combination of anti-PVRIG and anti-PDL-1 antibodies in a CT26 tumor model. A-B. Groups of 10 BALB / c mice were injected subcutaneously with 5 x 105 CT26 cells. After measuring tumors on day 7, mice were randomized (mean tumor volume 75 mm3 per group) and then treated with the designated mAb (300 pg / IP dose) followed by additional doses on days 11, 14, 18, 21 and 25. A. The groups were treated with 6 doses of combined agents. Anti-PDL-1 mAb 407 vs. control p = 0.0005; anti-PDL-1 and mAb 406 vs. control p = 0.056. B. Tumor volumes x3 were measured weekly. The number of tumor-free (TF) mice per group is indicated. C. Survival ratios of assigned groups; Anti-PDL-1 mAb 407 vs. control * p = 0.0088.
[0153] Figure 49A to D depict the amino acid sequences and nucleic acid sequence for the variable heavy chain (A and B, respectively) and the amino acid sequences and nucleic acid sequence for the variable light chain (C and D, respectively ) for AB-407 (BOJ-5G4-F4).
[0155] Figure 50 depicts the amino acid sequences of the constant domains of IgG1 (with some useful amino acid substitutions), human IgG2, IgG3, IgG4, IgG4 with a hinge variant that finds particular use in the present invention, and the constant domains of the kappa and lambda light chains.
[0157] Figure 51 depicts the ECD sequences of human and cynomolgus macaque TIGIT (referred to as cyno) and of the human PVR ECD proteins.
[0159] Figure 52. Shows the flow cytometric binding summary for anti-TIGIT fabs. All single ELISA positive fabs were analyzed by flow cytometry. Mean fluorescence intensity (MFI) was measured for Expi293 cells overexpressing human or cyno TIGIT, as well as parental Expi293 cells. The MFI ratio for specific target versus non-target binding was calculated. Data for selected clones are shown.
[0160] Figure 53A and B depict the anti-TIGIT antibody sequences. Unless otherwise indicated, CDRs use IMGT numbering (including sequence listing antibodies.
[0162] Figure 54. Shows the FACS KD results of the binding of anti-TIGIT mAbs to Expi293 cells that overexpress human TIGIT as described in Example 12.
[0164] Figure 55. Shows the FACS KD results of mAb binding to Expi293 cells overexpressing cyno TIGIT.
[0166] Figure 56. Shows the results of Example 14, showing the resulting kinetic rate constants and the resulting equilibrium dissociation constants, where the data was reliable enough to estimate the binding constants.
[0168] Figure 57A to B show the results of the binding of the human PVR-Fc variant to Expi293 cells that overexpress human TIGIT in Example 4. Figure A (left): Binding curve generated for the cell-titrated human PVR-m2aFc construct Expi293 that overexpress human TIGIT. KD and 95% confidence interval are shown. Figure B (right): Binding curve generated for the human PVR-h1 Fc construct titrated with Expi293 cells that overexpress human TIGIT. KD and 95% confidence interval are shown.
[0169] Figure 58. Shows a table of phage antibodies that inhibit the binding of human PVR-m2aFc to overexpressed human TIGIT in Expi293 cells. The mAbs were tested against a known blocking reference antibody (BM26), and human IgG4 isotype control antibody (Synagis). A "Yes" indicates that the mAb inhibited hPVR in a manner analogous to BM26.
[0170] Figure 59. Shows a table of IC50 values of anti-TIGIT hybridoma antibodies that inhibit the binding of human PVR-h1Fc to overexpressed human TIGIT in Expi293 cells. Values are representative of one or two independent experiments. The IC50 results for the two independently performed experiments showed a range of only 1.2-2 times difference.
[0172] Figure 60. Shows the results of Example 6, that anti-human TIGIT BM and phage-derived antibodies, CPA.9.027, CPA.9.049, CPA.9.059, BM26, and BM29 increase IL-2 signaling. Bm 26 and BM29 are both of the human IgG4 isotype (hIgG4 with a S241P variant). Representative data (n> 2) show the RLU (mean / - standard deviation) of the luciferase signal from a 6-hour coculture of Jurkat IL-2-RE luciferase cells with human TIGIT and aAPC CHO-K1 cells with human PVR. . The concentration of each antibody was 10 gg / ml.
[0174] Figure 61. Shows additional results from Example 6, that anti-human TIGIT phage and BM hIgG4-derived antibodies, CPA.9.027, CPA.9.049, CPA.9.059, BM26, and BM29 increase IL-2 signaling of a dose-dependent manner. BM26 and BM29 are both of the hIgG4 isotype. Representative data (n> 2) show the RLU (mean / - standard deviation) of the luciferase signal from a 6-hour coculture of Jurkat IL-2-RE luciferase cells with human TIGIT and aAPC CHO-K1 cells with human PVR. . A 10 point 2-fold dilution series starting at 20 gg / ml was used for each antibody.
[0176] Figure 62. Shows the results of Example 6, that the anti-human TIGIT BM and hybridoma-derived antibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, CHA.9.560, Bm 26, and BM29 increase IL-2 signaling. BM26 and BM29 are both of the mIgG1 isotype. The non-blocking human anti-TIGIT antibody, CHA.9.543 does not increase IL-2 signaling. Representative data (n> 2) show the RLU (mean / - standard deviation) of the luciferase signal from a 6-hour coculture of Jurkat IL-2-RE luciferase cells with human TIGIT and aAPC CHO-K1 cells with human PVR. . The concentration of each antibody was 10 gg / ml.
[0178] Figure 63. Shows the results of Example 6, that the reference and hybridoma-derived anti-human TIGIT antibodies mIgG1, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, CHA.9.560, and BM26 increase the IL-2 signaling in a dose-dependent manner. BM26 is of the mIgG1 isotype. Representative data (n> 2) show the RLU (mean / - standard deviation) of the luciferase signal from a 6-hour coculture of Jurkat IL-2-RE luciferase cells with human TIGIT and aAPC CHO-K1 cells with human PVR. . A 10 point 2-fold dilution series starting at 20 gg / ml was used for each antibody.
[0180] Figure 64. Shows that the anti-human TIGIT phage, hybridoma, and BM-derived antibodies, CPA.9.027, CPA.9.049, CPA.9.059, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, CHA.9.560 , BM26, and BM29 increase antigen-specific IFNy signaling. BM26 is tested as both the hIgG4 isotype and mIgG1, while BM29 is only tested as the hIgG4 isotype. Representative data (n = 2) show the amount of IFNy (mean / - standard deviation) in the culture supernatant after 24 hours of co-culturing CMV-specific CD8 + T cells with human Mel624 PVR cells. The concentration of each antibody was 10 gg / ml. The human Mel624 PVR used in the assay was pulsed with 0.0033 gg / ml or 0.001 gg / ml of peptide.
[0182] Figure 65. Shows that the anti-human TIGIT phage, hybridoma and BM antibodies, CPA.9.027, CPA.9.049, CPA.9.059, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, and CHA.9.560 , as well as BM26, increase antigen-specific IFNy signaling either alone (open bars) or in combination with an anti-PVRIG antibody, CHA.7.518.1.H4 (S241P) (hatched bars). BM26 is of the mIgG1 isotype. For isotype control antibody treatments, the open bar refers to the isotype antibody alone, and the hatched bar refers to the isotype antibody in combination with CHA.7.518.1.H4 (S241 P). Representative data (n = 2) show the amount of I FNy (mean / - standard deviation) in the culture supernatant after a 24-hour coculture of CMV-specific CD8 + T cells with Mel624 cells that overexpress human PVR and PVRL2. human. The concentration of each antibody was 10 gg / ml. The Me1624 human PVR / human PVRL2 cells used in the assay were pulsed with 0.0033 gg / ml or 0.001 gg / ml of peptide.
[0184] Figure 66. Shows the percentage increase in I FNy secretion with anti-human TIGIT antibodies, CHA.7.518.1.H4 (S241P), and the combination of anti-human TIGIT and CHA.7.518.1.H4 antibodies ( S241P), on the respective isotype control antibodies.
[0186] Figure 67 is the dendrogram for the epitope pre-classification experiments of Example 7.
[0188] Figure 68 is the grouping of antibodies from the epitope pre-classification experiments of Example 7.
[0190] Figure 69. Shows high affinity binding to cells overexpressing human TIGIT in a dose titration of affinity matured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7, CHA.9.547.13), reference antibodies (BM26, BM29), and the hIgG4 isotype control (anti-Synagis) in Expi293 cells overexpressing human TIGIT, as described in the experiments of Example 3. All antibodies were titrated using a 2-fold serial dilution at 11 points starting at 10 gg / ml (133.33 nM [binding site]). AF647-labeled goat anti-human F (ab ') (Jackson Immunoresearch) was added to the cells to detect the binding of anti-TIGIT antibodies. The gMFIs of the anti-TIGIT antibodies bound to the Expi293 cells overexpressing human TIGIT (black line), and to the parental Expi293 cells (gray line). K ^d values +/- 95% CI, and curve fits are indicated below each graph.
[0192] Figure 70. Shows that anti-TIGIT antibodies cross-react with cyno TIGIT in a dose titration of affinity matured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547. 7, CHA.9.547.13), reference antibodies (BM26, BM29), and the hIgG4 isotype control (anti-Synagis) in Expi293 cells that overexpress cyno TIGIT, as described in the experiments of Example 3. All Antibodies were titrated using a 2-fold 11-point serial dilution starting at 10 pg / ml (133.33 nM [binding site]). AF647-labeled goat anti-human F (ab ') (Jackson Immunoresearch) was added to the cells to detect the binding of anti-TIGIT antibodies. GMFIs of anti-TIGIT antibodies bound to Expi293 cells that overexpress cyno TIGIT (black line), and to parental Expi293 cells (gray line) are shown. K ^d values +/- 95% CI, and curve fits are indicated below each graph.
[0194] Figure 71. Shows that affinity matured phage antibodies cross-react with mouse TIGIT in a dose titration of affinity matured phage antibodies reformatted as mouse IgG1 (mIgG1) (CPA.9.083, CPA.9.086 ), mouse anti-TIGIT reference antibodies (BM27 mIgG1, BM30 mIgG1), and the mIgG1 isotype control (anti-Synagis), are shown, as described in the experiments of Example 3. A) The gMFI of the antibodies anti-TIGIT bound to HEK cells that overexpress mouse TIGIT (black line), and parental HEK cells (gray line). B) The gMFI of anti-TIGIT (black line) or Synagis mIgG1 (gray line) antibodies bound to regulatory CD4 + CD25 + Foxp3 + T cells isolated from Renca tumors implanted sc in Balb / c mice. Anti-TIGIT antibodies were well titrated using a series of 2 or 3-fold serial dilutions starting at 15 pg / ml (200 nM [binding site]), or 10 pg / ml (132 nM [binding site]), respectively. AF647-labeled goat anti-mouse IgG-Fc (Southern Biotech) was added to the cells to detect the binding of anti-TIGIT antibodies in cells that overexpress mouse TIGIT. Anti-TIGIT antibodies were directly conjugated to AF647 for binding of mouse Treg. The K ^d values for each anti-TIGIT antibody are indicated.
[0196] Figure 72. Shows a dose titer of affinity matured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7, CHA.9.547.13), and reference antibodies ( BM26, BM29) in human memory effector CD95 + CD28-CD8 + CD3 + T cells from 3 healthy PBMC donors (Donors 321, 322, and 334), as described in the experiments of Example 3. PBMCs were surface stained with antibodies against the following lineage markers CD3, CD4, CD8, CD14, CD16, CD28, CD56, and CD95 (BD Biosciences, BioLegend), as well as live / dead fixable aqua staining (Life Technologies). AF647-labeled anti-TIGIT antibodies and hIgG4 isotype control antibody (anti-Synagis) were then titrated using a 12-point serial 3-fold dilution starting at 30 pg / ml (396 nM [binding site]). The gMFIs of anti-TIGIT antibodies bound to memory effector T cells are shown. The Kd values for each antibody in the 3 different donors are reported in the table. The affinity matured phage antibodies (CPA.9.083 and CPA.9.086) had the highest binding affinity to human memory effector T cells.
[0198] Figure 73. Shows a dose titration of the affinity matured phage antibodies (CPA.9.083, CPA.9.086, CPA.9.103), humanized hybridoma antibody (Ch A.9.547.1), and reference antibody (BM26 ) in PBMC cyno memory effector CD95 + CD28-CD8 + CD3 + T cells isolated from 2 unstimulated cyno monkeys (BioreclamationlVT), as described in the experiments of Example 3. PBMC were surface stained with antibodies against following lineage markers CD3, CD4, CD8, CD14, CD16, CD28, CD56, and CD95 (BD Biosciences, BioLegend), as well as live / dead fixable aqua staining (Life Technologies). AF647-labeled anti-TIGIT antibodies and hIgG4 isotype control antibody (anti-Synagis) were then titrated using a 12-point serial 3-fold dilution starting at 30 pg / ml (396 nM [binding site]). The gMFIs of the anti-TIGIT antibodies bound to the memory effector T cells are shown with the gMFI of the anti-Synagis isotype control antibody hIgG4 subtracted. The K ^d values for each antibody in the 2 donors are reported in the table. Affinity-matured phage antibodies (CPA.9.083 and CPA.9.086) had the highest binding affinity to cyno memory effector T cells.
[0200] Figure 74. Shows the SPR kinetics of anti-TIGIT antibody binding to human, cyno, and mouse TIGIT, as described in the experiments of Example 5. The equilibrium kinetic rate and dissociation constants for antibodies to Affinity matured phage (CPA.9.083, CPA.9.086, CPA.9.103), humanized hybridoma antibodies (CHA.9.547.1 and CHA.9.547.7), and reference antibodies (BM26, BM29) were determined by SPR on the ProteOn instrument.
[0202] Figure 75. Shows that anti-TIGIT antibodies block PVR / TIGIT interactions, as described in the experiments of Example 4. Expi293 cells overexpressing human TIGIT were preincubated well with the affinity matured phage antibodies (CPA.9.083, CPA .9.086), humanized hybridoma antibodies (CHA.9.547.7, CHA.9.547.13), reference antibodies (BM26, BM29), or the hIgG4 isotype control (anti-Synagis). All antibodies were titrated using a serial 11-point 2.5-fold dilution starting at 10 pg / ml (133.33 nM [binding site]). After preincubation of the antibody, human PVR-m2aFc was added to the cells at 158 nM [binding site] or EC90. AF647-labeled goat anti-mouse IgG-Fc (Southern Biotech) was then added to the cells to detect the binding of anti-TIGIT antibodies. The percentage of inhibition of the binding of PVR-m2aFc to Expi293 cells that overexpress human TIGIT for each antibody. IC50 values for each human anti-TIGIT antibody are reported in the table (n = 2 experiments).
[0204] Figure 76. Shows the results of Example 6, that affinity matured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7, Ch A.9.547.13), and antibody Reference (BM26) increase IL-2 signaling in a dose-dependent manner. Synagis hIgG4 is the isotype control antibody. Representative data (n> 2) show the RLU (mean / - standard deviation) of the luciferase signal from a 6 hour coculture of Jurkat IL-2-RE luciferase cells with human TIGIT and CHO-K1 cells with human PVR. A 19 point 1.5 fold dilution series was used for each antibody starting at 20 pg / ml.
[0206] Figure 77. Shows that anti-TIGIT antibodies induce IFNy in CMV-specific CD8 + T cells. An in vitro coculture assay with CMV-specific human CD8 + T cells was used to evaluate the effect of affinity matured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7, CHA .9.547.13), and reference antibodies (BM26, BM29) on antigen-specific cytokine secretion, as described in the experiments of Example 6. The target cell line used in the assay was HLA-pancreatic adenocarcinoma cells. A2 +, Panc.05.04 which endogenously expresses human PVR and PVRL2. Panc.05.04 cells were pulsed with the CMV pp65 peptide at 0.03 pg / ml or 0.01 pg / ml at 37 ° C for 1 hour. Cells were then washed and plated at 50,000 cells / well in 96-well round bottom tissue culture treated plates. Anti-human TIGIT antibodies or hIgG4 isotype control antibody (anti-Synagis) were added at a concentration of 0.1 pg / ml. CMV-specific human CD8 + T cells from a single donor were expanded according to the above protocol. 50,000 human CD8 + T cells were added to each well. The cocultures were incubated at 37 ° C with 5% CO2 for 24 hours. The amount of human interferon gamma (IFNy) in the coculture supernatant was measured by flow cytometry using a bead cytometric assay (BD Biosciences). The percentage increase in IFNy secretion for each antibody on the hIgG4 isotype is summarized in the table (n = 2 experiments).
[0208] Figure 78. Shows that anti-TIGIT antibodies increase IFNy when combined with an antibody to PVRIG, CHA.7.518.1.H4 (S241P). An in vitro coculture assay with CMV-specific human CD8 + T cells was used to evaluate the effect of affinity matured phage antibodies (CPA.9.083, CPA.9.086), humanized hybridoma antibodies (CHA.9.547.7, CHA .9.547.13), and reference antibodies (BM26, BM29) on antigen-specific cytokine secretion in combination with an anti-PVRIG antibody, CHA.7.518.1. The target cell line used in this assay was HLA-A2 + pancreatic adenocarcinoma cells, Panc.05.04 that endogenously express human PVR and PVRL2. Panc.05.04 cells were pulsed with the CMV pp65 peptide at 0.03 pg / ml or 0.01 pg / ml at 37 ° C for 1 hour. Cells were then washed and plated at 50,000 cells / well in 96-well round bottom tissue culture treated plates. Anti-human TIGIT antibodies or hIgG4 isotype control antibody (anti-Synagis) were added at a concentration of 0.1 pg / ml in combination with CHA.7.518.1 (hatched bars) or an isotype control antibody hIgG4 at 10 pg / ml (solid bars). CMV-specific human CD8 + T cells from a single donor were expanded according to the above protocol. 50,000 human CD8 + T cells were added to each well. The cocultures were incubated at 37 ° C with 5% CO2 for 24 hours. The amount of human IFNy in the coculture supernatant was measured by flow cytometry using a bead cytometric assay (BD Biosciences). The percentage increase in IFNy secretion for each antibody on the hIgG4 isotype is summarized in the table (n = 2 experiments).
[0210] Figure 79. Shows the correlation analysis of the expression of PVRIG and TIGIT in CD4 + and CD8 + T cells of dissociated tumors. For each tumor sample, a mean fluorescence intensity ratio (MFIr) was calculated, and a Spearman correlation analysis was performed, and an r2 and p value were reported.
[0212] Figure 80. Shows the results of tumor growth and survival inhibition in TIGIT KO mice treated with a mouse anti-PVRIG antibody. They were injected s.c. to groups of 7-10 TIGIT KO and C57BL / 6 WT mice 1 x 105 B16 / Db-hmgp100 cells. Mice were treated twice weekly for 3 weeks, starting on the day of inoculation (day 0) with the designated antibody. A) Mean tumor volumes / -standard error of the mean (SEM) are shown in the upper graph, with *** indicating a p-value <0.001 for TIGIT KO treated with mouse anti-PVRIG antibody (Clone 407) in comparison with C57BL / 6 WT treated with the mIgG1 isotype control antibody. The tumor volumes for individual mice in each antibody treatment group are shown as spider graphs in the lower graphs. B) Table summarizing GIT as measured on indicated days compared to control C57BL / 6 WT mice treated with mIgG1 isotype control. C) Survival of mice after injection s.c. of B16 / Db-hmgp100 cells.
[0214] Figure 81A to C represents combination treatments with the indicated antibodies compared to the control in Mel-624, Colo205, and Panc.05.04 cells. T cells specific for gp100 or CMVpp65 were co-cultured with Mel-624, Colo205, and Panc.05.04 cells, gp100 or CMVpp65 peptide, and the indicated antibodies at 10 mg / ml. The IFN-y concentration in the conditioned medium was determined at 24 hrs. The Average Std Dev of triplicates is shown. The% change in I FN-y for each condition from hIgG4 is shown.
[0216] Figure 82 A to C represent the expression of PD-1 / TIGIT / PVRIG in CD8 T cells and the expression of PD-L1, PVR, PVRL2 in Colo205, Panc.05.04 cells. A) Expression of PVRIG, TIGIT, and PD-1 in CMVpp65-reactive T cells expanded with the peptide pp65 with IL-2 and IL-7 for 10 days. Expression of PVRIG, TIGIT, and PD-1 is shown in T cells reactive for CMVpp65. B) Expression of PD-L1, PVR, and PVRL2 in Colo205 and Panc.05.04 cells is shown. C) T cells specific for CMVpp65 were co-cultured with Colo205 and Panc.05.04 cells, CMVpp65 peptide, and the indicated antibodies at 10 mg / ml. The IFN-y concentration in the conditioned medium was determined at 24 hrs. The Average Std Dev of triplicates is shown. The% change in I FN-y for each condition from hIgG4 is shown.
[0218] Figure 83. PVRIG is expressed at its highest level in human cancer cytotoxic lymphocyte subsets. A) Expression of PVRIG in leukocyte cell subsets from 5-8 healthy PBMC donors is shown. Expression of PVRIG is defined as the ratio of the MFI of PVRIG to the MFI of the isotype control. B) Expression of PVRIG, TIGIT, CD96, and PD-1 in peripheral blood Tregs is shown in comparison to subsets of CD8 T cells from 5 healthy PBMC donors. C) CMV pp65-specific T cells from 3 healthy donors were expanded in vitro with pp65 peptide (495-503), IL-2, and IL-7 for up to 7 days. Expression of TIGIT (blue) and PVRIG (black) is shown in cells positive for the HLA-A2 / pp65 tetramer (495-503). D) Human T cells were cultured with allogeneic DC and the expression of TIGIT and PVRIG on CD4 + T cells is shown on day 0, 1, 2, and 7 after activation. E) Representative FACS plots showing expression of PVRIG (blue) compared to isotype control (red) in TIL (CD4 T cells, CD8 T cells, and NK cells) from a representative lung and kidney cancer. F) The co-expression of PVRIG, TIGIT, and PD-1 in TIL CD4 and CD8 from a lung cancer sample is shown. G) The expression of PVRIG in TIL CD8 + and CD4 + of human tumors dissociated from various types of cancer is shown. Each point represents a different tumor from an individual patient. H) The relative expression in TIL CD8 versus TIL Treg was evaluated for PVRIG, TIGIT, and PD-1 from endometrial, kidney, and lung tumors. For each tumor, the fold of expression in TIL CD8 was normalized to the fold of expression in TIL Treg and plotted. For A, B, C, G, and H, the mean ± SEM is shown by the error bars.
[0220] Figure 84. Expression of PVRL2 increases in the tumor microenvironment. A) The expression of PVRL2 was evaluated by IHC in tumors of the lung, ovary / endometrium, breast, colon, kidney, and melanoma. Bars represent mean ± SEM. For each tumor, 2 nuclei were evaluated by a pathologist and scored based on the prevalence and intensity of membranous staining in tumor cells as described in the supplemental methods. For each tumor, the average score of 2 nuclei is shown. B) A representative melanoma tumor is shown showing PVRL2 expression in tumor cells (arrow) and immune cells (*) in the stroma. C) Expression of PVRL2 on a log2 scale of dissociated tumors determined by FACS in TAM CD45-, CD14 +, and subsets of high mDC Lin-CD14-CD33 cells is shown. The mean ± SEM for each type of cancer is shown. The dotted line represents that no staining was observed. For cell type, at least 100 events were required in order to be analyzed. D) Representative FACS plots are shown for the expression of PVRL2 (blue) compared to IgG (red) for a lung cancer. E) For tumor samples in which both PVRIG and PVRL2 expression could be assessed, PVRIG expression in CD8 + T cells is plotted against PVRL2 expression in CD14 + TAM and CD45- cells for each tumor. Each point represents an individual tumor sample. The red line represents a 2-fold expression of PVRIG or PVRL2 compared to IgG. The Table in Figure 84F shows the prevalence of PVRL2 in various tumor samples.
[0222] Figure 85. Different regulation of PVRL2 and PD-L1 in tumor cells. A) The expression of PD-L1 and PVRL2 was evaluated by IHC in serial sections. Tumor samples in Figure 84A were grouped on the basis of tissue type and PVRL2 expression is shown as negative for PD-L1 and positive for PD-L1. PD-L1 negative tumors were defined as no membranous staining in tumor or immune cells of duplicate nuclei for a given tumor. Positive staining for PD-L1 was defined as membranous staining in at least 1 nucleus of a tumor. Bars represent the mean ± SEM for each group. B, C) Representative expression of a PVRL2 + PD-L1- (B) endometrial tumor and a PVRL2 + PD-L1- (C) lung tumor. d) Immature BM-DCs were grown with the indicated stimuli and the expression of PVR, PVRL2, and PD-L1 was evaluated by FACS on day 2 of culture. For each condition, the expression was normalized to the medium-only control condition. E) Expression of PVR, PVRL2, and PD-L1 in HT-29 cells treated with I FN-y or medium alone is shown. PD-L1 or PVRL2 is shown in blue and the IgG isotype control staining is shown in red.
[0224] Figure 86. CHA.7.518.1.H4 (S241P) is a high-affinity antibody that increases T-cell activation. A) Binding of c Ha .7.518.1.H4 (S241P) or isotype control is shown IgG to HEK293 PVRIG or parental HEK293 cells by FACS. KD values by FACS for the binding of CHA.7.518.1.H4 (S241P) to HEK293 hPVRIG, HEK293 cPVRIG, and Jurkat cells are shown. B) CHA.7.518.1.H4 (S241 P) disrupts PVRL2 Fc binding to HEK293 cells that ectopically express PVRIG. The Mean ± Std Dev of values in triplicate is shown. C) CHA.7.518.1.H4 (S241P) blocks the binding of PVRIG Fc to HEK293 cells that endogenously express PVRL2. D) Human CD4 T cells were co-cultured with aAPC CHO cells expressing an anti-CD3 antibody bound to the cell surface and hPVRL2 in the presence of 10 pg / ml anti-PVRIG antibody and human IgG isotype control antibodies. The effect of anti-PVRIG Ab on the proliferation of CD4 T cells isolated from 11 different donors is shown. Bars represent mean ± SEM. E) T-cell lines specific for gp100 (TIL-209, TIL-463) were co-cultured with CHO cells engineered to express HLA-A2 and PVRL2 along with 10 pg / ml anti-PVRIG antibody or IgG isotype control . The production of I FN-y and TNF-α was assayed at 24 hours after coculture. The mean ± Std Dev of values in triplicate is shown. The percentage of change in I FN-y and TNF-a for each condition with respect to the isotype control is represented by the number above each bar F) The expression of PVR, PVRL2, and PD-L1 (red) is shown with respect to IgG (blue) in MEL624, Colo205, and Panc.05.04 cells. For T cells, the expression of PVRIG, TIGIT, and PD-1 (red) is shown relative to IgG (blue) in TIL-209 and TIL-463 T cells specific for gp100, and on T cells specific for CMVpp65. To expand CMVpp65 reactive T cells, PBMC were cultured with the peptide pp65 (495-503), IL-2, and IL-7 for 10 days. The expression of PVRIG, TIGIT, PD-1 in cells positive for the HLA-A2 / pp65 tetramer is shown. G) Gp100-specific T cells (TIL-209, TIL-463) expanded from TIL derived from melanoma tumors were co-cultured with MEL624 cells in the presence of 10 pg / ml of the indicated antibodies. The concentration of IFN-y was determined in the conditioned medium at 24 hours. H, I) Expanded CMVpp65-specific T cells were cocultured with Colo205 and Panc.05.04 cells, CMVpp65 peptide, and the indicated antibodies at 10 pg / ml. The concentration of I FN-y in the conditioned medium was determined at 24 hrs. For E, G, H, I, the average ± StDev of triplicates is shown. The percent change in I FN-y for each condition relative to the isotype control is represented by the number above each bar.
[0226] Figure 87. Mice deficient in PVRIG have increased T cell function. A) Expression of PVRIG RNA was evaluated as measured by qRT-PCR of purified mouse immune cell subsets. The relative expression with respect to the constitutive one was determined by the Ct method. B) Pmel CD8 + TCR transgenic T cells were activated with gp100 (25-33) and the levels of PVRIG and TIGIT RNA transcripts were evaluated by qRT-PCR at the indicated time points. The graph shows the mean ± SEM of results from 5 different experiments. C) Spleens from PVRIG - / - and WT littermates were collected and analyzed by flow cytometry for expression of PVRIG in NK, CD4 + T and CD8 + cells ("Quiet" cells). Furthermore, CD3 + T cells were isolated from splenocytes and activated for 11 days with anti-CD3 / anti-CD28 beads. After activation, the expression of PVRIG on CD4 + and CD8 + T cells ("activated" cells) was analyzed by flow cytometry. Each point represents cells derived from an individual mouse. D) Splenocytes derived from WT and PVRIG - / - were labeled with eFluor450 Cell Proliferation Stain and cultured in the presence of Control-Fc (mouse IgG2a) or with mouse PVRL2 Fc. After 4 d of culture, cell division was analyzed by flow cytometry. Representative FACS plots from one experiment (left) and summary of percent inhibition by PVRL2 Fc (defined as% Control-Fc proliferation subtracted from% PVRL2 Fc proliferation) from 3 independent experiments (right) are presented. . * indicates p-value <0.05, paired student's t-test for change in proliferation in the presence of PVRL2-FC versus proliferation in the presence of control protein in WT T cells versus PVRIG - / -. E) Pmel CD8 + T cells derived from pmel PVRIG - / - or pmel PVRIG WT mice were activated for 11 days with their cognate peptide and IL2. The activated CD8 + pmel cells were then co-cultured with B16-Db / gp100 cells for 18 hours and after co-cultivation were evaluated for CD107 expression and for cytokine production. Four independent experiments are presented as indicated by each paired point. * indicates p-value <0.05, Student's t test comparing PVRIG - / - versus WT.
[0228] Figure 88. PVRIG deficiency results in reduced tumor growth and increased mechanism of CD8 + effector T cells. A) Mice were injected subcutaneously with C57BL / 6 WT or PVRIG - / - 5 x 10 5 MC38 cells. Tumor volumes x2 were measured weekly. n = 10 mice per group, Average ± SEM is shown, * Indicates p value <0.05 by unpaired Student's t-test for WT mice vs. PVRIG - / - mice (ANOVA). B) Individual tumor growth curves are shown. n = 10 mice per group, a representative experiment (n = 2) is shown. C) Mice were injected subcutaneously with C57BL / 6 WT or PVRIG - / - 5 x 10 5 MC38 cells. On day 14 after inoculation, mice were treated with anti-PD-L1, x2 weekly for 2 weeks. Tumor volumes x2 were measured weekly. n = 10 mice per group, Average ± SEM is shown, p-value = 0.052 by unpaired Student's t-test for WT mice versus PVRIG - / - mice, both treated with anti-PD-L1. D) Individual tumor growth curves are shown. A representative experiment (n = 2) is shown. E-H) In separate duplicate experiments, tumors were harvested on day 18 after the mice had received 2 doses of anti-PD-L1 or the relevant isotype control. Dissociated tumors were enriched for CD45 + cells prior to stimulation for 4 hours with PMA and Ionomycin in the presence of Brefeldin A. The graphs illustrate the total numbers per mg of tumor tissue of CD45 + immune cells, CD8 + T cells and CD8 + T cells producing Interferon-Y from mice treated with wild-type isotype and PVRIG - / - (E) and from mice treated with anti-PD-L1 wild-type and PVRIG - / - (F). G-H) The frequency of CD8 + IFN-Y + TNF-a + effector cells in tumor draining lymph nodes of PVRIG - / - treated with isotype and anti-PD-L1 mice, relative to their corresponding wild-type cohort, is shown. For E-H, Mean ± SEM is shown and p-values from an unpaired Student's t-test are shown.
[0230] Figure 89. Antagonist anti-PVRIG antibodies synergistically inhibit tumor growth in combination with PD-1 inhibitors or TIGIT genetic deficiency. A) Binding of mPVRL2 Fc fusion protein to mPVRIG HEK293 engineered cells that were preincubated with serial dilutions of anti-imPVRIG mAb or IgG isotype control Ab is shown. B) BALB / c mice were injected subcutaneously with 5 x 105 CT26 cells. On day 14 after inoculation, the mice were sacrificed and the spleen, draining lymph nodes and tumors were harvested. Cells were analyzed by flow cytometry to determine the expression of PVRIG in CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD3-CD49b + NK cells, Myeloid Derived Suppressor Cells (MDSC) CD11b + Gr-1 + and CD11b + macrophages. F4 / 80 +. C, D) BALB / c mice were injected subcutaneously with 5 x 105 CT26 cells. On day 7 after inoculation, mice were treated with anti-PD-L1 and / or anti-PVRIG Ab, 2x weekly for 3 weeks (arrows indicate Ab treatment). C) Tumor volumes are shown. *** indicates p-value <0.001 (ANOVA) for rat aPD-L1 + IgG2b compared to PD-L1 + aPVRIG treated groups. Arrows indicate when antibodies were dosed. D. Survival analysis of complete responder mice. * indicates p-value <0.05 (Logarithmic rank test) for rat PD-L1 IgG2b compared to groups treated with PD-L1 PVRIG. A representative study of 3 studies is shown. E. C57BL / 6 or TIGIT - / - mice were injected subcutaneously with 1 x 105 B16 / Db-hmgp100 cells. Mice were treated 2x weekly for 3 weeks with the designated mAb beginning on the day of inoculation (day 0). E. Tumor volumes were measured 2x weekly and the mean ± SEM is shown. Inhibition of tumor growth as measured on indicated days compared to WT control + mIgG1 isotype control. *** indicates p-value <0.001 for TIGIT - / - aPVRIG compared to WT control of isotype mIgG1. Arrows indicate when antibodies were dosed. F. Individual tumor growth curves are shown for each mouse. A representative experiment of 2 performed is shown.
[0232] Figure 90. PVRIG is expressed on TILS T and NK cells in human cancer. A) Expression of PVRIG, TIGIT, CD96, and PD-1 in PBMC CD4 T cell subsets from healthy donors is shown. The mean ± SEM is shown. B) Human T cells were co-cultured with allogeneic PBMC and the expression of the PVRIG protein on CD4 and CD8 T cells (top) is shown. C) Tumors dissociated and single cells were activated with anti-CD3 and anti-CD28. The expression of PVRIG (blue) relative to the IgG isotype control (red) was evaluated on day 0 (directly ex vivo) and on day 5 after activation. D) Expression of PVRIG in dissociated human tumor NK cells is shown. Each point represents a different tumor from an individual patient. The mean ± 95% confidence interval is shown. D) Dissociated tumor cells were activated with anti-CD3 and anti-CD28 beads for 5 days. Expression of PVRIG (blue) versus IgG control (red) on CD4 and CD8 T cells is shown on day 0 directly ex vivo and on day 5 after activation for 2 dissociated tumor samples. E) Expression of PVRIG was assessed on CD4 and CD8 T cells from dissociated tumors and from dissociated normal adjacent tissue concordant with the donor. Each line represents concordant tissues from an individual patient. A paired student's t test was performed. F) A correlation analysis of the magnitude of the times of the expression of PVRIG, TIGIT, and PD-1 is shown with respect to the IgG isotype control in CD4 and CD8 T cells of the tumors. Each point represents an individual tumor sample. A Spearman correlation coefficient and p-value are shown.
[0234] Figure 91. Expression of PVRL2 is increased in colon, skin, and breast cancers. A) Photomicrographs showing the binding of Sigma anti-human PVRL2 antibody to FFPE sections of positive cells, CHO-S human PVRL2 (right) compared to negative cells, CHO-S (left), after antigen recovery a pH9. B) Anti-PVRL2 antibody was tested on a panel of PVRL2 + (HT29, MCF7, PC3, PANC1, rT4, NCI-H1573) and PVRL2- (Jurkat, OPM2, Daudi, CA46) cell lines. C-F) Example of PVRL2 expression in normal and cancerous lung tissues. C) Normal tissue showing no staining. D) Lung adenocarcinoma showing partial positive staining. E) Lung adenocarcinoma showing positive staining. F) Lung adenocarcinoma showing strong positive staining.
[0236] Figure 92. PVRL2 is upregulated in TAM and CD45- cells in tumor compared to normal adjacent tissue. Expression of PVRL2 in CD45- and TAM cells from concordant donor tumor and adjacent normal tissue is shown. A p-value from the paired student's t-test is shown.
[0238] Figure 93. PVRIG and PVRL2 are coexpressed in the same tumor sample. The expression of PVRIG in CD4 T cells (A) and NK cells (B) is plotted against the expression of PVRL2 in TAM for an individual tumor.
[0240] Figure 94. Activity of CHA.7.518.1.H4 (S241P) in human T cells. A) Expression of PVRIG in CD4 T cells activated with CHO cells expressing anti-CD3 and PVRL2 bound to the cell surface. B) Expression of HLA-A2, B-2m, and PVRL2 in engineered CHO-S and CHO-S cell lines is shown. The times of expression with respect to the isotype are represented by the number. C) CHO cells ectopically expressing anti-CD3 and PVRL2 bound to the cell surface were co-cultured with purified CD8 T cells in the presence of various concentrations of anti-PVRIG Ab or relevant IgG control. The% proliferation is shown. Each point represents an average of triplicate values. D) CHO cells ectopically expressing HLA-A2 / B2m and PVRL2 were co-cultured with 2 gp100-specific T-cell lines (TIL F4, TIL 209) in the presence of 1 ug / ml of gp100 and various concentrations of anti- PVRIG or relevant IgG control. TNF-α concentrations on day 3 of coculture are shown. Each value represents an average of triplicates.
[0242] Figure 95. Characterization of the binding interactions of mPVRIG and a substituted anti-mPVRIG antibody. A, B) The binding of mPVRIG to mPVRL2 was assessed by surface plasmon resonance. C) Soluble Fc receptor or control proteins were incubated in a dose response with immobilized mPVRL2 HIS in an ELISA format. Bound Fc receptor is shown. D) Soluble PVRL2 HIS protein was incubated in a dose response with PVRIG Fc or DNAM Fc coated plates. E) Binding of mPVRIG Fc or control Fc fusion protein to cell line B16-F10 transfected with mPVRL2 siRNA, mPVRsRNA, or random sequence siRNA transfection is shown. F) The affinity characterization of rat anti-mouse PVRIG mAb was performed by examining the binding of anti-mPVRIG to HEK293 cells that overexpress mPVRIG. G) The affinity characterization of rat anti-mouse PVRIG mAb is shown by examining the binding of anti-mPVRIG to the D10.G4.1 cell line that endogenously expresses mPVRIG versus the rat IgG isotype control . H) Binding of anti-mPVRIG to D 10.G4.1 cells transfected with mouse PVRIG siRNA (green histogram) vs. scr siRNA (orange histogram). I) Binding of mPVRIG Fc preincubated with anti-mPVRIG Ab to B16-F10 cells, which endogenously express PVRL2.
[0244] Figure 96. Generation of transgenic mice with inactivation of PVRIG and TIGIT. Mice lines with conditional PVRIG inactivation and Tigit inactivation were generated by Ozgene Pty Ltd (Bentley WA, Australia). A) The targeting construct in which PVRIG exons 1 to 4 were flanked by loxP sites was electroporated in a C57BL / 6 ES cell line, Bruce4 (Koentgen et al., Int Immunol 5: 957-964, 1993). B). The targeting construct in which the Tigit exon 1 coding region (including ATG) and exons 2 and 3 were replaced with a FRT-flanked neo cassette was electroporated into a C57BL / 6 ES cell line, Bruce4. Homologous recombinant ES cell clones were identified by Southern hybridization and injected into goGermline blasts (Koentgen et al., Genesis 54: 326-333, 2016). Male chimeric mice were obtained and crossed with C57BL / 6J females to establish heterozygous germline offspring on a C57BL / 6 background. Germline mice were crossed with a ubiquitous FLP C57BL / 6 mouse line to remove the FRT-flanked selectable marker cassette and generate the conditional or inactivated alleles (for PVRIG and Tigit, respectively). For inactivation of PVRIG, mice were further crossed with a ubiquitous mouse line Cre C57BL / 6 to remove loxP-flanked exons and generate the inactivated allele.
[0246] Figure 97. Mice with inactivated PVRIG are immunophenotypically similar to wild-type mice. Mice (n = 5 per wild-type cohort and with inactivated PVRIG) were euthanized prior to collection of venous blood in anticoagulant-coated tubes and harvesting of organs. Single cells were recovered from freshly collected bone marrow, thymus, spleen, cutaneous and mesenteric lymph nodes. Cells were stained with fluorochrome-conjugated surface marker antibodies and acquired on a BD LSR Fortessa flow cytometer. Panels illustrate comparable frequencies of myeloid cells (A), dendritic cells (B), B cells (C), T cells (D), CD4 T cells (E), D8 T cells (F), and NK cells (G). ) in lymphoid tissue types. (H-I) Whole venous blood is run on a Hemavet 950 Veterinary Hematology System to compare differential counts and blood cell subsets frequencies from wild-type and PVRIG-deficient mice.
[0247] Figure 98. Increased effector function of T cells in PVRIG - / - mice treated with anti-PDL1 compared to WT with anti-PD-L1. MC38 tumors were inoculated into WT or PVRIG - / - mice and subsequently treated with anti-PD-L1 or rat IgG2b isotype control. On day 18, tumor-infiltrating CD45 + lymphocytes were purified from tumors, RNA was extracted, and transcript profiling was performed. Several genes related to T cells are shown, with each dot representing an individual mouse. Student's t-test p-values are shown.
[0248] Figure 99. Anti-TIGIT and anti-PVRIG antibodies induce tumor cell death. An in vitro co-culture assay with expanded CMV-specific human CD8 + T cells was used to assess the effect of the anti-TIGIT reference antibody and CHA.7.518.1.H4 (S241 P) on antigen-specific tumor cell death. . The target HLA-A2 + cell lines used in the assay were Mel624 (A) and Panc05.04 (B). Synagis hIgG4 is the isotype control antibody. Luciferase activity in target cells was measured with Bio-Glo luciferase substrate. Representative data (n> 2) show the percentage of specific death (mean / - standard deviation) of Mel624 or Panc05.04 cells after a 16 hour coculture with CMV-specific human CD8 + T cells from three different donors.
[0250] Figure 100. Dose-dependent tumor cell death of anti-TIGIT antibodies with CHA.7.518.1.H4 (S241 P). An in vitro coculture assay with CMV-specific human CD8 + T cells was used to evaluate the effect of two different anti-TIGIT antibodies, BM26 and CPA.9.086 when combined with CHA.7.518.1.H4 (S241 P) on the antigen-specific Mel624 cell death. Luciferase activity in target cells was measured with Bio-Glo luciferase substrate. Representative data (n> 2) show the percentage of specific death (mean / - standard deviation) of Mel624 cells after a 16 hour coculture with CMV-specific human CD8 + T cells from a donor.
[0252] Figure 101. CPA.9.086 CDR sequences, IMGT and Kabat numbering.
[0254] Figure 102. The combination of hIgG4 anti-TIGIT CHA.7.518.1.H4 (S241 P) induces tumor cell death. Coculture of CMV-reactive CD8 + T cells with Mel624 PVR, PVRL2 and luciferase OE Single dose of 10 pg / ml of aTIGIT Ab and 10 pg / ml of CHA.7.518.1.H4 (S241P) with CMV-reactive donor 4, while the titration of the dose started at 0.5 pg / ml Ab aTIGIT and 10 pg / ml CHA.7.518.1.H4 (S241P) with donor 156 reactive for CMV.
[0256] V. Detailed description of the invention
[0258] A. Global vision
[0260] The present disclosure provides several useful antibodies, for use alone or in combination, for the treatment of cancer. Cancer can be viewed as an inability of the patient to recognize and kill cancer cells. In many cases, these transformed (eg, cancer) cells counteract immune surveillance. There are natural control mechanisms that limit the activation of T cells in the body to prevent unrestricted activity of T cells, which can be exploited by cancer cells to escape or suppress the immune response. Restoring the ability of immune effector cells - especially T cells - to recognize and kill cancer is the goal of immunotherapy. The field of immuno-oncology, sometimes referred to as "immunotherapy," is developing rapidly, with several recent approvals of T-cell checkpoint inhibitor antibodies such as Yervoy, Keytruda, and Opdivo. These antibodies are generally referred to as "checkpoint inhibitors" because they normally block the negative regulators of T cell immunity It is generally understood that a variety of immunomodulatory signals, both costimulatory and co-inhibitory, can be used to orchestrate an optimal antigen-specific immune response. These antibodies generally bind to checkpoint inhibitory proteins such as CTLA-4 or PD-1, which, under normal circumstances, prevent or suppress the activation of cytotoxic T cells (CTL). By inhibiting the checkpoint protein, for example by using antibodies that bind to these proteins, an increased T-cell response to tumors can be achieved. That is, these cancer checkpoint proteins suppress the immune response; when proteins are blocked, for example, using antibodies to the checkpoint protein, the immune system is activated, leading to immune stimulation, leading to the treatment of conditions such as cancer and infectious diseases.
[0262] The present invention is directed to the use of antibodies to additional checkpoint proteins, PVRIG and TIGIT. PVRIG is expressed on the cell surface of NK and T-cells and shares several similarities with other known immune checkpoints. Identification and methods used to show that PVRIG is a checkpoint receptor are discussed in WO2016 / 134333. Provided herein are antibodies to human PVRIG that block the interaction and / or binding of PVLR2. When PVRIG binds to its ligand (PVRL2), it elicits an inhibitory signal that acts to attenuate the immune response of NK and T cells against a target cell (ie, analogous to PD-1 / PDL1). Blocking the binding of PVRL2 to PVRIG turns off this inhibitory signal of PVRIG and as a result modulates the immune response of NK and T cells. The use of an antibody to PVRIG that blocks the binding to PVRL2 is a therapeutic strategy that increases the death of cancer cells by NK and T cells. Blocking antibodies have been generated that bind to PVRIG and block the binding of its ligand, PVRL2. Anti-PVRIG antibodies are provided in combination with other checkpoint inhibitory antibodies, such as PD-1.
[0264] Similarly, TIGIT has been shown to also have attributes of a checkpoint receptor, and the present invention provides anti-TIGIT antibodies that block the interaction and / or binding of TIGIT to PVRs are provided. When TIGIT binds to its ligand (PVR), it elicits an inhibitory signal that acts to attenuate the immune response of NK and T cells against a target cell (ie, analogous to PD-1 / PDL1). Blocking the binding of PVR to TIGIT turns off this inhibitory signal of TIGIT and as a result modulates the immune response of NK and T cells. The use of an antibody to TIGIT that blocks binding to PVR is a therapeutic strategy that increases the cancer cell death by Nk and T cells. Blocking antibodies have been generated that bind to TIGIT and block the binding of its ligand, PVR. Anti-TIGIT antibodies are provided in combination with other checkpoint inhibitory antibodies, such as PD-1.
[0266] Additionally, the invention provides combinations of anti-PVRIG and anti-TIGIT antibodies for use in treating cancer.
[0268] B. Definitions
[0270] In order for the application to be more fully understood, various definitions are shown below. Such definitions are intended to encompass grammatical equivalents.
[0272] By "ablation" is meant herein a decrease or elimination of activity. In some embodiments, it is useful to eliminate the activity of the constant domains of the antibodies. Thus, for example, "ablation of the Fc and R binding" means that the amino acid variant of the Fc region has less than 50% of the starting binding compared to an Fc region that does not contain the specific variant, with less than 70-80 90-95-98% loss of activity, and, in general, the activity being below the level of detectable binding in a Biacore assay. As shown in Figure 50, an ablation variant in the constant region of IgG1 is the N297A variant, which removes the native glycosylation site and significantly reduces the binding of FcYRIIIa and thus reduces antibody-dependent cell-mediated cytotoxicity ( ADCC).
[0274] By "antigen-binding domain" or "ABD" is meant herein a set of six Complementarity Determining Regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds to an antigen. target as discussed herein. Thus, a "TIGIT antigen-binding domain" binds to the TIGIT antigen (the sequence of which is shown in Figure 51) as indicated herein. Similarly, a "PVRIG antigen-binding domain" binds to the PVRIG antigen (the sequence of which is shown in Figure 1) as indicated herein. As is known in the art, these CDRs are generally present as a first set of variable heavy CDRs (vhCDR or V ^h CDR) and a second set of variable light CDRs (vlCDR or V ^l CDR), each comprising three CDRs: vhCDR1 , vhCDR2, vhCDR3 for the heavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light. The CDRs are present in the variable heavy and variable light domains, respectively, and together form an Fv region. Thus, in some cases, the six CDRs of the antigen-binding domain are contributed by a variable heavy and variable light chain. In a "Fab" format, the set of 6 CDRs are provided by two sequences of different polypeptide, variable heavy domain (V H or V ^h; containing vhCDR1, VHCDR2 and VHCDR3) and the variable light domain (VL or V ^l ; containing vlCDR1, vlCDR2 and vlCDR3), the C-terminus of the vh domain being attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain being attached to the N-terminus of the constant light domain (and thus forming the chain light).
[0275] By "modification" is meant herein a substitution, insertion, and / or deletion of amino acids in a polypeptide sequence or an alteration in a residue chemically linked to a protein. For example, a modification can be an altered carbohydrate or a protein-bound PEG backbone. By "amino acid modification" is meant herein a substitution, insertion, and / or deletion of amino acids in a polypeptide sequence. For the sake of clarity, unless otherwise indicated, the amino acid modification is always at an amino acid encoded by DNA, e.g. eg, the 20 amino acids that have codons in DNA and RNA.
[0276] By "amino acid substitution" or "substitution" is meant herein the replacement of one amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is with an amino acid that is not natural in the particular position, either not natural in the body or not at all. For example, the substitution N297A refers to a variant polypeptide, in this case an Fc variant, in which the asparagine at position 297 is replaced with alanine. For clarity, a protein that has been engineered to change the nucleic acid coding sequence, but not change the starting amino acid (for example, swapping CGG (encoding arginine) for CGA (still encoding arginine) to increase expression levels in the host organism) is not an "amino acid substitution"; that is, despite the creation of a new gene that codes for the same protein, if the protein has the same amino acid in the particular position it started with, it is not an amino acid substitution.
[0278] By "amino acid insertion" or "insertion", as used herein, it is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E designates a glutamic acid insert after position 233 and before position 234. Additionally, -233ADE or A233ADE designates an AlaAspGlu insert after position 233 and before position 234.
[0280] By "amino acid deletion" or "deletion", as used herein, it is meant the deletion of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233 #, E233 () or E233del designates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233 # designates a deletion of the GluAspAla sequence starting at position 233.
[0282] By "variant protein" or "protein variant", or "variant", as used herein, it is meant a protein that differs from a parent protein by at least one amino acid modification. The protein variant can refer to the protein itself, to a composition comprising the protein, or to the amino acid sequence that encodes it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, e.g. eg, from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent. As described below, in some embodiments, the parental polypeptide, eg, a parental Fc polypeptide, is a human wild-type sequence, such as the Fc region of IgG1, IgG2, IgG3, or IgG4, although human sequences may also serve. with variants such as "parental polypeptides". The sequence of the protein variant herein will preferably possess at least about 80% identity to a parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98. -99% identity. The variant protein also refers to the variant protein itself, to compositions comprising the variant protein, or to the DNA sequence that encodes it. Accordingly, by "antibody variant" or "variant antibody", as used herein, is meant an antibody that differs from a parent antibody by at least one amino acid modification, "IgG variant "or" variant IgG ", as used herein, means an antibody that differs from a parental IgG (again, in many cases, from a human IgG sequence) thanks to at least one modification of amino acids, and "immunoglobulin variant" or "variant immunoglobulin", as used herein, means an immunoglobulin sequence that differs from that of a parental immunoglobulin sequence by at least one amino acid modification. "Fc variant" or "variant Fc", as used herein, means a protein comprising an amino acid modification in an Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that compose them. Thus, for example, S241P or S228P is a hinge variant with the proline substitution at position 228 relative to the parental IgG4 hinge polypeptide, where the S228P numbering is according to the EU index and S241P is the Kabat numbering. The EU index or EU index as in Kabat or the EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85, hereby incorporated in full by reference ). The modification can be an addition, deletion, or substitution. The substitutions can include natural amino acids and, in some cases, synthetic amino acids. Examples include US Pat. US No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO 05 / 35727A2; WO 05 / 74524A2; J. W. Chin et al., (2002), Journal of the American Chemical Society 124: 9026-9027; J. W. Chin, and P. G. Schultz, (2002), ChemBioChem 11: 1135-1137; J. W. Chin, et al., (2002), PICAS United States of America 99: 11020-11024; and, L. Wang, and P. G. Schultz, (2002), Chem. 1-10.
[0284] As used herein, "protein" means herein at least two amino acids covalently linked, including proteins, polypeptides, oligopeptides, and peptides. The peptidyl group can comprise natural amino acids and peptide bonds, or synthetic peptidomimetic structures, ie "analogs", such as peptoids (see, Simon et al., PNAS USA 89 (20): 9367 (1992)). Amino acids can be either natural or synthetic (eg, an amino acid that is not encoded by DNA); as will be appreciated by those skilled in the art. For example, homophenylalanine, citrulline, ornithine, and norleucine are considered synthetic amino acids for the purposes of the invention, and amino acids can be used in both the D and L (R or S) configurations. Variants of the present invention may comprise modifications that include the use of synthetic amino acids incorporated using, for example, technologies developed by Schultz et al., Including, but not limited to, the methods described by Cropp and Shultz, 2004, Trends Genet. 20 (12): 625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101 (2): 7566-71, Zhang et al., 2003, 303 (5656): 371-3, and Chin et al. ., 2003, Science 301 (5635): 964-7. In addition, polypeptides can include synthetic derivatization of one or more side or end chains, glycosylation, PEGylation, loop permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or markers.
[0286] By "residue", as used herein, it is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in human IgG1 antibody.
[0288] By "Fab" or "Fab region", as used herein, is meant the polypeptide that comprises the immunoglobulin domains VH, CH1, VL, and CL. Fab can refer to this isolated region, or to this region in the context of a full-length antibody or antibody fragment.
[0290] By "Fv" or "Fv fragment" or "Fv region", as used herein, is meant a polypeptide comprising the VL and VH domains of a single antibody. As those skilled in the art will appreciate, these are generally composed of two chains.
[0292] By "single chain Fv" or "scFv" is meant herein a variable heavy domain covalently linked to a variable light domain, generally using an scFv linker as discussed herein, to form a scFv or domain of scFv. A scFv domain can be in any orientation from the N to the C terminus (vh-connectpr-vl or vl-connector-vh). In general, the linker is a scFv linker as is generally known in the art, with the linker peptide predominantly including the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide must be of a length that is adequate to connect two molecules in such a way that they assume the correct conformation with respect to each other so as to retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers with a length of 1 to 20 amino acids can be used, with those of about 5 to about 10 amino acids finding use in some embodiments. Useful linkers include glycine-serine polymers, including, for example, (GS) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least one (and generally of 3-4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-proteinaceous polymers may find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and prolpropylene glycol, that is, they may find use as linkers.
[0294] By "IgG subclass modification" or "isotype modification", as used herein, is meant an amino acid modification that converts an amino acid from an IgG isotype to the corresponding amino acid in an IgG isotype. different aligned. For example, since IgG1 comprises a tyrosine and IgG2 a phenylalanine at position EU 296, a F296Y substitution in IgG2 is considered a subclass modification of IgG. Similarly, since IgG1 has a proline at position 241 and IgG4 has a serine there, an IgG4 molecule with an S241P is considered a subclass modification of IgG. Note that such subclass modifications are considered amino acid substitutions herein.
[0296] By "unnatural modification", as used herein, it is meant an amino acid modification that is not isotypic. For example, since none of the IgGs comprise An asparagine at position 297, the N297A substitution in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered an unnatural modification.
[0297] By "amino acid" and "amino acid identity", as used herein, it is meant one of the 20 naturally occurring amino acids that are encoded by DNA and RNA.
[0299] By "effector function", as used herein, it is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.
[0301] By "IgG Fc ligand", as used herein, is meant a molecule, preferably a polypeptide, from any organism, that binds to the Fc region of an IgG antibody to form a Fc complex. / Fc ligand. Fc ligands include, but are not limited to, FcyRI, FcyRII, FcyRMI, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to Fc and R (Davis et al., 2002, Immunological Reviews 190: 123-136). Fc ligands can include undiscovered molecules that bind to Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. By "Fc ligand", as used herein, is meant a molecule, preferably a polypeptide, from any organism, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.
[0303] By "parent polypeptide", as used herein, it is meant a starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide can be a natural polypeptide, or a variant or engineered version of a natural polypeptide. The parent polypeptide can refer to the polypeptide itself, to compositions comprising the parent polypeptide, or to the amino acid sequence that encodes it. Accordingly, by "parental immunoglobulin", as used herein, is meant an unmodified immunoglobulin polypeptide that is modified to generate a variant, and by "parental antibody", as used herein. Herein, it is meant an unmodified antibody that is modified to generate a variant antibody. It should be noted that "parental antibody" includes known commercial antibodies, produced recombinantly, as noted below.
[0305] By "Fc" or "Fc region" or "Fc domain", as used herein, is meant the polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain and, in some cases, part of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the N-end of the flexible hinge of these domains. For IgA and IgM, Fc can include the J chain. For IgG, the Fc domain comprises the immunoglobulin domains C and 2 and C and 3 (C and 2 and C and 3) and the lower hinge region between C and 1 ( C y 1) and C y 2 (C y 2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is commonly defined to include residues C226 or P230 at its carboxyl terminus, where the numbering is according to the EU index as in Kabat. In some embodiments, as more fully described below, amino acid modifications are made in the Fc region, for example, to alter binding to one or more Fc and R receptors or to the FcRn receptor.
[0307] By "heavy constant region" is meant herein the CH1-hinge-CH2-CH3 portion of an antibody.
[0309] By "position", as used herein, is meant a location in the sequence of a protein. Positions can be numbered sequentially, or according to an established format, for example the EU index for antibody numbering.
[0311] By "target antigen", as used herein. is meant the molecule that is specifically bound by the variable region of a given antibody. In the present case, a target antigen of interest herein is TIGIT, typically human TIGIT, and optionally cyno TIGIT, as defined below. Another target antigen of interest is PVRIG, typically human PVRIG, and optionally cyno PVRIG, as defined below.
[0313] By "target cell", as used herein, it is meant a cell that expresses a target antigen.
[0315] By "variable region", as used herein, is meant the region of an immunoglobulin that comprises one or more Ig domains encoded substantially by any of the V k (V. kappa), VA ( V.lamda), and / or VH that make up the kappa, lambda, and immunoglobulin heavy layer genetic loci, respectively.
[0317] By "wild-type or WT" is meant herein an amino acid sequence or a nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.
[0319] The antibodies of the present invention are generally isolated or recombinant. "Isolated", when used to describe the various polypeptides described herein, means a polypeptide that has been identified and separated and / or recovered from a cell or cell culture in which it is expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies that have different antigenic specificities. "Recombinant" means that the antibodies are generated using recombinant nucleic acid techniques in exogenous host cells.
[0321] "Specific binding" or "specifically binds to" or is "specific for" a particular antigen or epitope, means a binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule with a similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
[0323] Specific binding for a particular antigen or epitope can be displayed, for example, by an antibody having a KD for an antigen or epitope of at least about 10-9 M, at least about 10-10 M, at least about 10-11 M, at least about 10-12 M, at least about 10-13 M, at least about 10-14 M, at least about 10-15 M, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD that is 20, 50, 100, 500, 1,000, 5,000, 10,000 times or more for a control molecule relative to the antigen or epitope.
[0325] Also, specific binding for a particular antigen or epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20, 50, 100, 500, 1,000, 5,000, 10,000 times or more. higher for the epitope over a control, where KA or Ka refers to a rate of association of a particular antibody-antigen interaction. Binding affinity is generally measured using surface plasmon resonance (eg, Biacore assay) and flow cytometry with cells expressing the antigen.
[0327] C. Sequences
[0329] The sequence listing provides various sequences based on the Format of Figure 53; Reference is made to Figure 4 of USSN 62 / 513,916 as a guide for labeling the sequences. The variable heavy domain is marked with the identifier (eg, "CPA.0.86"), following the following sequence the format of Figure 53 of the present specification (identical to the format of Figure 4, referenced above), where the identifier of the next sequence is vhCDR1, the next one is vhCDR2, with vhCDR3, the full-length heavy chain, the variable light domain, vlCDR1, vlCDR2, vlCDR3, and the full-length light chain. Thus, a single antibody has 10 associated sequence identifiers. The sequence listing includes the sequences of mouse IgG1 BM26 (BM26-M1) (WO2016 / 028656A1, Clone 31C6) and mouse IgG1 BM29 (BM29-M1) (US2016 / 0176963 A1, Clone 22G2). Unless noted, full-length HC sequences for TIGIT antibodies are in the H4 (S241 P) format.
[0331] D. PVRIG proteins
[0333] The present disclosure provides antibodies that specifically bind to PVRIG proteins and prevent activation by their ligand protein, PVRL2, a glycoprotein of the human plasma membrane. PVRIG, also called Protein Containing the Poliovirus Receptor-Related Immunoglobulin Domain, Q6DKI7 or C7orf15, refers to the amino acid and nucleic acid sequences shown in RefSeq with the access identifier NP_076975, shown in Figure 1. The sequence of the human poliovirus receptor-related protein 2 (PVLR2, also known as nectin-2, CD112 or mediator of herpesvirus B entry, (HVEB)), the binding partner of PVRIG (as shown in Example 5 from the US Publication.
[0334] 2016/0244521), is shown in Figure 2. The antibodies of the invention are specific for the extracellular domain of PVRIG so that the binding of PVRIG and PVLR2 is blocked.
[0336] PVRIG is a 326 amino acid long transmembrane domain protein, with a signal peptide (spanning amino acid 1 to 40), an extracellular domain (spanning amino acid 41 to 171), a transmembrane domain (spanning amino acid amino acid 172 to 190) and a cytoplasmic domain (spanning from amino acid 191 to 326). There are two methionines that can be start codons, but the mature proteins are identical.
[0338] Accordingly, as used herein, the term "PVRIG" or "PVRIG protein" or "PVRIG polypeptide" can optionally include any such protein, or variant, conjugated, or fragment thereof, including, but not limited to not limited to known or wild-type PVRIG, as described herein, as well as any natural splice variants, amino acid variants or isoforms, and, in particular, the ECD fragment of PVRIG.
[0340] As noted herein and more fully described below, anti-PVRIG antibodies (including antigen-binding fragments) that bind to PVRIG and prevent activation by PVRL2 (eg, most commonly by blocking PVRIG interaction of PVRIG and PVLR2), are used to increase the activation of T cells and / or NK cells and are used to treat diseases such as cancer and infection by pathogens.
[0342] E. TIGIT proteins
[0344] The present invention provides antibodies that specifically bind to TIGIT proteins and prevent activation by its ligand protein, PVR, poliovirus receptor (also known as CD155), a human plasma membrane glycoprotein. TIGIT, or T-cell immunoreceptor with Ig and ITIM domains, is a co-inhibitory receptor protein also known as WUCAM, Vstm3, or Vsig9. TIGIT has an immunoglobulin variable domain, a transmembrane domain, and an immunoreceptor based on an inhibitory tyrosine-rich moiety (ITIM) and contains signature sequence elements of the PVR protein family. The extracellular domain (ECD) sequences of TIGIT and PVR are shown in Figure 51. Antibodies of the invention are specific for the TIGIT ECD so that the binding of TIGIT and PVR is blocked.
[0346] Accordingly, as used herein, the term "TIGIT" or "TIGIT protein" or "TIGIT polypeptide" may optionally include any such proteins, or variants, conjugates, or fragments thereof, including, but not limited to not limited to known or wild-type TIGIT, as described herein, as well as any natural splice variants, amino acid variants or isoforms, and in particular the ECD fragment of TIGIT.
[0347] As indicated herein and more fully described below, anti-TIGIT antibodies (including antigen-binding fragments) that bind to TIGIT and prevent activation by PVR (eg, most commonly by blocking the interaction of TIGIT and PVR), are used to increase the activation of T cells and / or NK cells and are used to treat diseases such as cancer and infection by pathogens.
[0349] SAW. Antibodies
[0351] As discussed below, the term "antibody" is generally used. The building blocks of traditional antibodies typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50- 70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention is directed to monoclonal antibodies that are generally based on the IgG class, which has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. In general, IgG1, IgG2 and IgG4 are used more frequently than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). The sequences depicted herein use the 356D / 358M allotype, however the other allotype is included herein. That is, any sequence that includes an IgG1 Fc domain included herein may have 356E / 358L replacing the 356D / 358M allotype.
[0353] The amino terminal part of each chain includes a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition, generally referred to in the art and herein as the "Fv domain" or "Fv region". In the variable region, three loops meet for each of the heavy chain and light chain V domains to form an antigen-binding site. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as a "CDR"), wherein the variation in amino acid sequence is highly significant. "Variable" refers to the fact that certain segments of the variable region differ widely in sequence between antibodies. The variability in the variable region is not uniformly distributed. Instead, the V regions consist of relatively invariant chains called 15-30 amino acid framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-15 amino acids long or longer.
[0355] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDR") and four FRs, arranged from the amino end to the carboxy end in the following order: FR 1-CDR1-FR2-CDR2-FR3 -CDR3 -FR4.
[0357] The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in and around the light chain variable region. from about 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEReSt, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues that form a hypervariable loop (eg, residues 26 -32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the region heavy chain variable: Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 The specific CDRs of the invention are described below.
[0359] As those skilled in the art will appreciate, the numbering and exact positioning of CDRs may be different between different numbering systems. However, the description of a variable heavy and / or variable light sequence should be understood to include the description of the associated (inherent) CDRs. Accordingly, the description of each variable heavy region is a description of the vhCDRs (eg, vhCDR1, vhCDR2, and vhCDR3) and the description of each variable light region is a description of the vlCDRs (eg, vlCDR1, vlCDR2 and vlCDR3). A useful comparison of CDR numbering is as follows, see Lafranc et al., Dev. Comp. Immunol.
[0360] 27 (1): 55-77 (2003):
[0363] Throughout the present specification, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the hinge and EU numbering system for Fc regions (eg, Kabat et al., supra (1991)).
[0365] The present description provides a large number of different sets of CDRs. In this case, a "complete set of CDRs" comprises the three variable light CDRs and three variable heavy CDRs, e.g. eg to vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a light variable or heavy variable major domain, respectively. Furthermore, as more fully indicated herein, the variable heavy and variable light domains may be on separate polypeptide chains, when using a heavy and light chain, or on a single polypeptide chain in the case of scFv sequences.
[0367] CDRs contribute to the formation of the antigen-binding site or, more specifically, the epitope-binding site of antibodies. "Epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics as well as specific charge characteristics. A single antigen can have more than one epitope.
[0369] The epitope can comprise amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in binding, such as amino acid residues that are effectively blocked by the peptide of binding to specific antigen; in other words, the amino acid residue is in the coat of the specific antigen-binding peptide.
[0371] Epitopes can be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished because binding to the former, but not the latter, is lost in the presence of denaturing solvents.
[0373] An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a single spatial conformation. Antibodies that recognize the same epitope can be checked in a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to a target antigen, eg, "pre-sorting". As indicated below, the invention includes not only the antigen and antibody binding domains listed herein, but those that compete for binding with the epitopes bound by the listed antigen-binding domains.
[0375] The carboxy terminal part of each chain defines a constant region, mainly responsible for the effector function. Kabat et al. collected numerous primary sequences from the variable regions of heavy chains and light chains. On the basis of the degree of conservation of the sequences, they classified the individual primary sequences in the CDRs and the framework and made a list of them (see, SEQUENCES OF IMMUNo Lo GICAL INTEREST, 5th edition, NIH publication, No. 91-3242 , EA Kabat et al., Incorporated in its entirety by reference).
[0376] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin domain (Ig)" is meant herein a region of an immunoglobulin that has a distinct tertiary structure. Of interest in the present invention are the heavy chain domains, including, the constant heavy domains (CH) and the hinge domains. In the context of IgG antibodies, IgG isotypes each have three CH regions. Accordingly, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat.
[0377] Another type of heavy chain Ig domain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" is meant herein the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the CH1 domain of IgG ends at position EU 220, and the CH2 domain of IgG begins at the residue at position EU 237. Thus, for IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering is according to the EU index as in Kabat.
[0379] The light chain generally comprises two domains, the variable light domain (which contains the light chain CDRs and together with the variable heavy domains forms the Fv region), and a constant light chain region (frequently referred to as CL or C k ). In general, either the constant lambda or constant kappa domain can be used, with lambda generally finding use in the invention.
[0381] Another region of interest for additional substitutions, noted below, is the Fc region.
[0383] A. Chimeric and humanized antibodies
[0385] In some embodiments, the antibodies herein can be derived from a mixture of different species, e.g. eg, a chimeric antibody and / or a humanized antibody. In general, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from more than one species. For example, "chimeric antibodies" traditionally comprise one or more variable regions of a mouse (or rat, in some cases) and the constant region (s) of a human. "Humanized antibodies" generally refer to non-human antibodies to which the framework regions of the variable domain have been changed by sequences found in human antibodies. Generally, in a humanized antibody, the whole antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to said antibody except for its CDRs. CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted onto the beta-sheet framework of a variable region of a human antibody to create an antibody, the specificity of which is determined by the grafted CDRs. The creation of such antibodies is described, e.g. eg, in WO 92/11018, Jones, 1986, Nature 321: 522-525, Verhoeyen et al., 1988, Science 239: 1534-1536. "Back-mutation" of selected acceptor framework residues to corresponding donor residues is frequently required to regain affinity that is lost in the initial grafted construct (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205 ; US 5821337; US 6054297; US 6407213). The humanized antibody will optimally also comprise at least a part, and usually all, of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region. Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20: 639-654. A variety of techniques and methods for humanizing and rearranging non-human antibodies are well known in the art (See, Tsurushita and Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein). Humanization methods include, but are not limited to, the methods described in Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988; Nature 332: 323-329; Verhoeyen et al., 1988, Science, 239: 1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86: 10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89: 4285-9, Presta et al., 1997, Cancer Res. 57 (20): 4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88: 4181-4185; O'Connor et al., 1998, Protein Eng 11: 321-8. Humanization or other methods of reducing the immunogenicity of non-human antibody variable regions may include surface modification methods, as described, for example, in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969-973.
[0387] Thus, the vhCDRs and vlCDRs of any of the antibodies listed herein can be humanized (or "rehumanized", for those that have already been humanized).
[0389] In certain embodiments, the antibodies of the disclosure comprise a heavy chain variable region from a particular germline immunoglobulin heavy chain gene and / or a light chain variable region from a particular germline immunoglobulin light chain gene. For example, such antibodies may comprise or consist of a human antibody comprising heavy or light chain variable regions that are "the product of" or "derived from" a particular germline sequence. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting the a human germline immunoglobulin sequence that is closest in sequence (ie, greater% identity) to the human antibody sequence. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain differences in amino acids compared to the germline sequence, due to, for example, natural somatic mutations or intentional introduction of site-directed mutation. However, a humanized antibody is typically at least 90% identical in amino acid sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains residues of amino acids that identify the antibody as being derived from human sequences when compared to the germline immunoglobulin amino acid sequences of other species (eg, murine germline sequences). In certain cases, a humanized antibody can be at least 95, 96, 97, 98, or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the sequence of amino acids encoded by the germline immunoglobulin gene excluding CDRs. That is, the CDRs can be murine, but the framework regions of the variable region (either heavy or light) can be at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the framework amino acids. encoded by a human germline immunoglobulin gene.
[0391] Typically, a humanized antibody derived from a particular human germline sequence will exhibit no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the humanized antibody may have no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene (again , prior to the introduction of any variant herein; that is, the number of variants is generally low).
[0393] In one embodiment, the parent antibody has been affinity matured, as is known in the art. Structure-based methods for humanization and affinity maturation can be employed, for example, as described in USSN 11 / 004,590. Selection-based methods can be employed to humanize and / or affinity mature the variable regions of the antibody, including, but not limited to, the methods described in Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272 (16): 10678-10684; Rosok et al., 1996, J. Biol. Chem. 271 (37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16 (10): 753-759. Other methods of humanization may involve grafting only parts of the CDRs, including, but not limited to, the methods described in USSN 09 / 810,510; Tan et al., 2002, J. Immunol. 169: 1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084.
[0395] B. Optional Antibody Engineering Preparation
[0397] The antibodies of the disclosure can be modified, or engineered, to alter amino acid sequences by amino acid substitutions. As discussed herein, amino acid substitutions can be made to alter the affinity of the CDRs for the protein (eg, TIGIT or PVRIG, including both increasing and decreasing binding), as well as to alter additional functional properties of antibodies. For example, antibodies can be engineered to include modifications to the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or cellular cytotoxicity. antigen-dependent. Furthermore, an antibody according to at least some embodiments of the disclosure can be chemically modified (eg, one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of the residues in the Fc region is that of the Kabat EU index.
[0399] In one example, the hinge region of Ch1 is modified such that the number of cysteine residues in the hinge region is altered, e.g. eg, it is increased or decreased. This strategy is further described in US Pat. US No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of Ch1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
[0400] In yet another example, the antibody can be modified to suppress Fab arm exchange in vivo, in particular when using the constant domains of IgG4. Specifically, this process involves the exchange of half IgG4 molecules (one heavy chain plus one light chain) between other IgG4 antibodies, effectively giving rise to bispecific antibodies that are functionally monovalent. Mutations in the hinge region and heavy chain constant domains can suppress this exchange (see, Aalberse, RC, Schuurman J., 2002, Immunology 105: 9-19). As indicated herein, a mutation that finds particular use in the present invention is S241P in the context of a constant domain of IgG4. IgG4 finds use in the present invention as it does not have a significant effector function, and is thus used to block the binding of the receptor to its ligand without cell depletion (eg, PVRIG to PVRL2 or TIGIT to PVR).
[0402] In some examples, amino acid substitutions can be made in the Fc region, in general, to alter binding to the Fc and R receptors. By "Fc gamma receptor", "FcyR" or "FcgammaR", as used in herein, it is meant any member of the family of proteins that binds to the Fc region of the IgG antibody and is encoded by an Fc and R gene. In humans, this family includes, but is not limited to, Fc and RI (CD64), including the FcYRIa, FcYRIb, and Fc and RIc isoforms; Fc and RII (CD32), including the isoforms FcYRIIa (including allotypes H131 and R131), FcYRIIb (including FcYRIIb-1 and FcyRiib-2), and Fc and RIIc; and Fc and RIII (c D16), including the FcyRIIIa isoforms (including the V158 and F158 allotypes) and FcyRIIIb (including the FcyRNIb-NA1 and FcyRIIIb-NA2 allotypes) (Jefferis et al., 2002, Immunol Lett 82: 57-65 ), as well as any undiscovered human FcyRs or isoforms or allotypes of FcyRs. An FcyR can be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII-1 (CD 16), and FcyRIII-2 (CD 16-2), as well as any undiscovered mouse FcyRs or isoforms or allotypes of FcyR.
[0403] There are several useful Fc substitutions that can be made to alter binding to one or more of the Fc and R receptors. Substitutions that result in increased binding as well as decreased binding may be useful. For example, it is known that increased binding to FcyRIIIa generally results in increased ADCC (antibody-dependent cell-mediated cytotoxicity; the cell-mediated reaction where non-specific cytotoxic cells expressing FcyR recognize the bound antibody in a target cell and they subsequently cause lysis of the target cell. Similarly, decreased binding to FcYRIIb (an inhibitory receptor) may also be beneficial in some circumstances. Amino acid substitutions that find use in the present invention include those listed in Nos. of Ser US 11 / 124,620 (particularly FIG. 41) and US Patent No. 6,737,056.
[0404] In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcy receptor, and / or to increase FcRn binding, by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283 , 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331 , 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This strategy is further described in PCT Publication WO 00/42072 to Presta. Furthermore, the binding sites in human IgG1 for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with enhanced binding have been described (see, Shields, RL et al. (2001) J. Biol. Chem. 276: 6591 -6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to enhance binding to FcyRNI. Additionally, the following combination mutants are shown to enhance FcyRNI binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A. Furthermore, mutations such as M252Y / S254T / T256E or M428L / N434S enhance FcRn binding and increase the circulating half-life of the antibody (see, Chan CA and Carter PJ (2010) Nature Rev Immunol 10: 301-316).
[0406] Furthermore, the antibodies of the disclosure are modified to increase their biological half-life. Several strategies are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US Pat. US No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered in the C ^h1 or C ^{l region} to contain a wild-type receptor binding epitope taken from two loops of a CH2 domain of an IgG Fc region, as described in the Pat. US Nos. 5,869,046 and 6,121,022 to Presta et al. Additional mutations to increase serum half-life are described in US Patent Nos. 8,883,973, 6,737,056, and 7,371,826 and include 428L, 434A, 434S, and 428L / 434S.
[0408] In yet another example, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be prepared (ie the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen or to reduce effector function such as ADCC. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence, for example, N297. For example, one or more amino acid substitutions that result in the removal of one or more glycosylation sites within the variable region can be made to thereby eliminate glycosylation at that site, with an alanine replacement finding use in some realizations.
[0409] Additionally or alternatively, an antibody that has an altered type of glycosylation can be prepared, such as a hypofucosylated antibody that has reduced amounts of fucosyl residues or an antibody that has increased bisected GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies according to at least some embodiments of the invention to thereby produce an antibody with altered glycosylation. See, for example, US Patent Publication No. 20040110704 and WO 2003/035835.
[0411] Another modification of the antibodies herein that is contemplated by the description is PEGylation or the addition of other water soluble moieties, typically polymers, e.g. eg, in order to increase the half-life. An antibody can be PEGylated, for example, to increase the biological half-life (eg, in serum) of the antibody as is known in the art.
[0413] In addition to substitutions made to alter the binding affinity to FcyR and / or FcRn and / or to increase serum half-life in vivo, additional modifications can be made to antibodies, as described in more detail below.
[0415] In some cases, affinity maturation is done. Amino acid modifications in CDRs are sometimes referred to as "affinity maturation". An "affinity matured" antibody is one that has one or more alterations in one or more CDRs resulting in an improvement in the affinity of the antibody for the antigen, compared to a parent antibody that does not possess this or these alterations. In some cases, it may be desirable to lower the affinity of an antibody for its antigen.
[0417] In some examples, one or more amino acid modifications are made to one or more of the CDRs of the antibodies of the invention (antibodies to PVRIG or TIGIT). In general, only 1 or 2 or 3-amino acids are substituted in any Single CDR, and generally no more than 1,2,3,4, 5, 6, 7, 8, 9, or 10 changes are made in a set of 6 CDRs (eg, vhCDRI -3 and vlCDR1 -3) . However, it should be appreciated that any combination of no substitutions, 1,2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitution.
[0419] Affinity maturation can be done to increase the binding affinity of the antibody for the antigen by at least about 10% to 50-100-150% or more, or 1 to 5-fold compared to the "parent" antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the antigen. Affinity matured antibodies are produced by known procedures. The affinity and efficacy correlation is discussed later.
[0421] Alternatively, amino acid modifications may be made to one or more of the antibody CDRs of the disclosure that are "silent", e.g. eg, they do not significantly alter the affinity of the antibody for the antigen. These can be done for a number of reasons, including optimizing expression (as can be done for nucleic acids encoding the antibodies of the disclosure).
[0423] Thus, the definition of CDRs and antibodies of the disclosure includes variant CDRs and antibodies; that is, the antibodies of the disclosure may include amino acid modifications in one or more of the CDRs of the listed antibodies of the invention. In addition, as noted below, amino acid modifications can also be made independently and optionally in any region outside the CDRs, including the framework and constant regions.
[0425] to. Generation of additional antibodies
[0427] Additional antibodies to human PVRIG can be prepared as is well known in the art, using well known methods such as those set forth in the examples. Thus, additional anti-PVRIG antibodies can be generated by traditional methods such as immunization of mice (sometimes using DNA immunization, for example, as used by Aldevron), followed by screening against the human PVRIG protein and generation of hybridomas, with the purification and recovery of the antibodies.
[0429] VII. TIGIT Antibodies of the Invention
[0431] The present invention provides anti-TIGIT antibodies, as defined in the claims. (For convenience, "anti-TIGIT antibodies" and "TIGIT antibodies" are used interchangeably). The anti-TIGIT antibodies of the invention specifically bind human TIGIT, and preferably human TIGIT ECD. The invention further provides antigen-binding domains, including full-length antibodies, containing several specific enumerated sets of 6 CDRs, that bind to TIGIT.
[0433] Specific binding for TIGIT or a TIGIT epitope may be presented, for example, by an antibody having a K ^d of at least about 10-4 M, at least about 10-5 M, at least about 10-6 M, at least about 10-7M, at least about 10-8M, at least about 10-9M, alternatively at least about 10-10M, at least about 10-11M, at least about 10-12M, at least about 10-13 M, at least about 10-14 M, at least about 10-15 M, or greater, where K ^d refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a K ^d that is 20, 50, 100, 500, 1,000, 5,000, 10,000 or more times greater for a control molecule relative to the antigen or TIGIT epitope.
[0434] However, for optimal binding to TIGIT expressed on the surface of NK and T cells, the antibodies preferably have a KD of less than 50 nM and most preferably less than 1 nM, finding use in the methods of the invention of less than 0. , 1 nM and less than 1 pM.
[0436] Also, specific binding for a particular antigen or epitope can be presented, for example, by an antibody having a ka (which refers to the rate of association constant) for an antigen or TIGIT epitope of at least 20, 50, 100, 500, 1,000, 5,000, 10,000 times or more for the epitope relative to a control, where ka refers to the constant of the rate of association of a particular antibody-antigen interaction.
[0438] In some embodiments, the anti-TIGIT antibodies of the invention bind to human TIGIT with a K ^d of 100 nM or less, 50 nM or less, 10 nM or less, or 1 nM or less (that is, higher binding affinity ), or 1pM or less, where K ^d is determined by known methods, e.g. eg, surface plasmon resonance (SPR, eg, Biacore assays), ELISA, KINEXA, and most typically SPR at 25 ° or 37 ° C.
[0440] Antibodies to TIGIT described herein are labeled as follows. Antibodies have reference numbers, for example "CPA.9.086". This represents the combination of variable heavy and variable light chains, as depicted in Figure 53, for example, knowing that these antibodies include two heavy chains and two light chains. "CPA.9.086.VH" refers to the variable heavy part of CPA. 9. 086, while "CPA. 9. 086.VL" is the variable light chain. "CPA. 9.086.vhCDR1", "CPA. 9. 086.vhCDR2", "CPA. 9.
[0441] 086.vhCDR3 "," CPA. 9. 086.vlCDR1 "," CPA. 9. 086.vlCDR2 ", and" CPA. 9. 086.vlCDR3 ", refers to the Cd R as indicated." CPA. 9.086.HC "refers to the complete heavy chain (eg, variable and constant domain) of this molecule, and "CPA. 9. 086.LC" refers to the entire light chain (eg, variable and constant domain) of the same molecule. In general, the human kappa light chain is used for the constant domain of each phage antibody (or humanized hybridoma) herein, although the lambda light constant domain is used in some embodiments. "CPA. 9. 086.H1" refers to a full-length antibody that comprises the variable heavy and light domains, including the constant domain of human IgG1 (therefore, the H1; IgG1, IgG2, IgG3, and IgG4 sequences are shown in Figure 50). Therefore, "CPA. 9. 086. H2" would be the variable domains of CPA. 9. 086 bound to a human IgG2. "CPA. 9. 086.H3" would be the variable domains of CPA. 9. 086 bound to a human IgG3, and "CPA. 9. 086.H4" would be the variable domains of CPA. 9. 086 bound to a human IgG4. Note that, in some cases, human IgGs may have additional mutations, such as those described below, and this can be noted. For example, in many embodiments, there may be a S241P mutation in human IgG4, and this may be noted as "CPA.9.086.H4 (S241 P)", for example. The sequence of human IgG4 with this S241P hinge variant is shown in Figure 50. Other potential variants are IgG1 (N297A), (or other variants that eliminate glycosylation at this site and thus many of the effector functions associated with binding a FcyRIIIa), and IgG1 (D265A), which reduces binding to FcyR receptors.
[0443] The invention further provides variable heavy and light domains, as well as full length heavy and light chains, as defined in the claims.
[0445] In some examples, the description provides scFvs that bind to TIGIT that comprise a variable heavy domain and a variable light domain joined by an scFv linker as noted above. The VL and VH domains can be in any orientation, e.g. eg, from the N to the C end "VH-connector-VL" or "VL-connector" VH. They are named for their component parts, for example, "scFv-CPA. 9,086. VH-connector-VL "or" scFv-CPA.9.086.VL-connector-VH ". Thus," scFv-CPA.9.086 "can be in any orientation.
[0447] In many embodiments, the antibodies of the invention are human (derived from phage) and block the binding of TIGIT and PVR. As shown in Figures 58 and 75, CPA antibodies that bind and block receptor-ligand interaction are as indicated below, with their components also indicated (as discussed in the "Sequence" section, the sequences of all except the scFv constructs are in the sequence listing):
[0449] CPA.9.018, CPA.9.018.VH, CPA.9.018.VL, CPA.9.018.HC, CPA.9.018.LC, CPA.9.018.H1, CPA.9.018.H2, CPA.9.018.H3, CPA.9.018. H4; CPA.9.018.H4 (S241P); CPA.9.018.vhCDR1, CPA.9.018.vhCDR2, CPA.9.018.vhCDR3, CPA.9.018.vlCDR1, CPA.9.018.vlCDR2, CPA.9.018.vlCDR3 and scFv-CPA.9.018;
[0451] CPA.9.027, CPA.9.027.VH, CPA.9.027.VL, CPA.9.027.HC, CPA.9.027.LC, CPA.9.027.H1, CPA.9.027.H2, CPA.9.027.H3, CPA.9.027. H4; CPA.9.018.H4 (S241P); CPA.9.027.vhCDR1, CPA.9.027.vhCDR2, CPA.9.027. vhCDR3, CPA.9.027.vlCDR1, CPA.9.027.vlCDR2, CPA.9.027.vlCDR3 and scFv-CPA.9.027;
[0453] CPA.9.049, CPA.9.049.VH, CPA.9.049.VL, CPA.9.049.HC, CPA.9.049.LC, CPA.9.049.H1, CPA.9.049.H2, CPA.9.049.H3; CPA.9.049.H4; CPA.9.049.H4 (S241P); CPA.9.049.vhCDR1, CPA.9.049.vhCDR2, CPA.9.049.vhCDR3, CPA.9.049.vlCDR1, CPA.9.049.vlCDR2, CPA.9.049.vlCDR3 and scFv-CPA.9.049;
[0455] CPA.9.057, CPA.9.057.VH, CPA.9.057.VL, CPA.9.057.HC, CPA.9.057.LC, CPA.9.057.H1, CPA.9.057.H2, CPA.9.057.H3; CPA.9.057.H4; CPA.9.057.H4 (S241P); CPA.9.057.vhCDR1, CPA.9.057.vhCDR2, CPA.9.057. vhCDR3, CPA.9.057.vlCDR1, CPA.9.057.vlCDR2, CPA.9.057.vlCDR3 and scFv-CPA.9.057;
[0457] CPA.9.059, CPA.9.059.VH, CPA.9.059.VL, CPA.9.059.HC, CPA.9.059.LC, CPA.9.059.H1, CPA.9.059.H2, CPA.9.059.H3; CPA.9.059.H4; CPA.9.059.H4 (S241P); CPA.9.059.vhCDR1, CPA.9.059.vhCDR2, CPA.9.059.vhCDR3, CPA.9.059.vlCDR1, CPA.9.059.vlCDR2, CPA.9.059.vlCDR3 and scFv-CPA.9.059;
[0459] CPA.9.083, CPA.9.083.VH, CPA.9.083.VL, CPA.9.083.HC, CPA.9.083.LC, CPA.9.083.H1, CPA.9.083.H2, CPA.9.083.H3; CPA.9.083.H4; CPA.9.083.H4 (S241P); CPA.9.083.vhCDR1, CPA.9.083.vhCDR2, CPA.9.083.vhCDR3, CPA.9.083.vlCDR1, CPA.9.083.vlCDR2, CPA.9.083.vlCDR3 and scFv-CPA.9.083;
[0461] CPA.9.086, CPA.9.086.VH, CPA.9.086.VL, CPA.9.086.HC, CPA.9.086.LC, CPA.9.086.H1, CPA.9.086.H2, CPA.9.086.H3; CPA.9.086.H4; CPA.9.086.H4 (S241P); CPA.9.086.vhCDR1, CPA.9.086.vhCDR2, CPA.9.086.vhCDR3, CPA.9.086.vlCDR1, CPA.9.086.vlCDR2, CPA.9.086.vlCDR3 and scFv-CPA.9.086;
[0463] CPA.9.089, CPA.9.089.VH, CPA.9.089.VL, CPA.9.089.HC, CPA.9.089.LC, CPA.9.089.H1, CPA.9.089.H2, CPA.9.089.H3; CPA.9.089.H4; CPA.9.089.H4 (S241P); CPA.9.089.vhCDR1, CPA.9.089.vhCDR2, CPA.9.089.vhCDR3, CPA.9.089.vlCDR1, CPA.9.089.vlCDR2, CPA.9.089.vlCDR3 and scFv-CPA.9.089;
[0465] CPA.9.093, CPA.9.093.VH, CPA.9.093.VL, CPA.9.093.HC, CPA.9.093.LC, CPA.9.093.H1, CPA.9.093.H2, CPA.9.093.H3; CPA.9.093.H4; CPA.9.093.H4 (S241P); CPA.9.093.vhCDR1, CPA.9.093.vhCDR2, CPA.9.093.vhCDR3, CPA.9.093.vlCDR1, CPA.9.093.vlCDR2, CPA.9.093.vlCDR3 and scFv-CPA.9.093; CPA.9.101, CPA.9.101.VH, CPA.9.101.VL, CPA.9.101.HC, CPA.9.101.LC, CPA.9.101.H1, CPA.9.101.H2, CPA.9.101.H3; CPA.9.101.H4; CPA.9.101.H4 (S241P); CPA.9.101.vhCDR1, CPA.9.101.vhCDR2, CPA.9.101.vhCDR3, CPA.9.101.vlCDR1, CPA.9.101.vlCDR2, CPA.9.101.vlCDR3 and scFv-CPA.9.101; and
[0466] CPA.9.103, CPA.9.103.VH, CPA.9.103.VL, CPA.9.103.HC, CPA.9.103.LC, CPA.9.103.H1, CPA.9.103.H2, CPA.9.103.H3; CPA.9.103.H4; CPA.9.103.H4 (S241P); CPA.9.103.vhCDR1, CPA.9.103.vhCDR2, CPA.9.103.vhCDR3, CPA.9.103.vlCDR1, CPA.9.103.vlCDR2, CPA.9.103.vlCDR3 and scFv-CPA.9.103.
[0468] Furthermore, the present disclosure provides a number of CHA antibodies, which are murine antibodies generated from hybridomas. As is well known in the art, the six CDRs are useful when placed in variable heavy and variable light regions of the human framework or when the variable heavy and light domains are humanized.
[0469] Accordingly, the present disclosure provides antibodies, usually full-length or scFv domains, comprising the following sets of CDRs, the sequences of which are shown in Figure 53 and / or in the sequence listing:
[0471] CHA.9.536.1, CHA.9.536.1.VH, CHA.9.536.1.VL, CHA.9.536.1.HC, CHA.9.536.1.LC, CHA.9.536.1.H1, CHA.9.536. 1.H2, CHA.9.536.1.H3; CHA.9.536.1.H4, CHA.9.536.1. H4 (S241P), CHA.9.536.1.vhCDR1, CHA.9.536.1. vhCDR2, CHA.9.536.1.vhCDR3, CHA.9.536.1.vlCDR1, CHA.9.536.1.vlCDR2 and CHA.9.536.1. vhCDR3;
[0473] CHA.9.536.3, CHA.9.536.3.VH, CHA.9.536.3.VL, CHA.9.536.3.HC, CHA.9.536.3.LC, CHA.9.536.3.H1, CHA.9.536. 3.H2, CHA.9.536.3.H3; CHA.9.536.3.H4, CHA.9.536.3.H4 (S241P); CHA.9.536.3.vhCDR1, CHA.9.536.3.vhCDR2, CHA.9.536.3.vhCDR3, CHA.9.536.3.vlCDR1, CHA.9.536.3.vlCDR2 and CHA.9.536.3.vhCDR3;
[0474] CHA.9.536.4, CHA.9.536.4.VH, CHA.9.536.4.VL, CHA.9.536.4.HC, CHA.9.536.4.LC, CHA.9.536.4.H1, CHA.9.536. 4.H2, CHA.9.536.4.H3; CHA.9.536.4.H4, CHA.9.536.4.H4 (S241P), CHA.9.536.4.vhCDR1, CHA.9.536.4.vhCDR2, CHA.9.536.4.vhCDR3, CHA.9.536.4.vlCDR1 , CHA.9.536.4.vlCDR2 and CHA.9.536.4.vhCDR3;
[0475] CHA.9.536.5, CHA.9.536.5.VH, CHA.9.536.5.VL, CHA.9.536.5.HC, CHA.9.536.5.LC, CHA.9.536.5.H1, CHA.9.536. 5.H2, CHA.9.536.5.H3; CHA.9.536.5.H4, CHA.9.536.5.H4 (S241P), CHA.9.536.5.vhCDR1, CHA.9.536.5.vhCDR2, CHA.9.536.5.vhCDR3, CHA.9.536.5.vlCDR1 , CHA.9.536.5.vlCDR2 and CHA.9.536.5.vhCDR3;
[0476] CHA.9.536.6, CHA.9.536.6.VH, CHA.9.536.6.VL, CHA.9.536.6.HC, CHA.9.536.6.LC, CHA.9.536.6.H1, CHA.9.536. 6.H2, CHA.9.536.6.H3; CHA.9.536.6.H4, CHA.9.536.6.vhCDR1, CHA.9.536.6.vhCDR2, CHA.9.536.6.vhCDR3, CHA.9.536.6.vlCDR1, CHA.9.536.6.vlCDR2 and CHA. 9.536.6.vhCDR3;
[0478] CHA.9.536.7, CHA.9.536.7.VH, CHA.9.536.7.VL, CHA.9.536.7.HC, CHA.9.536.7.LC, CHA.9.536.7.H1, CHA.9.536. 7.H2, CHA.9.536.7.H3; CHA.9.536.7.H4, CHA.9.536.5.H4 (S241P); CHA.9.536.7.vhCDR1, CHA.9.536.7.vhCDR2, CHA.9.536.7.vhCDR3, CHA.9.536.7.vlCDR1, CHA.9.536.7.vlCDR2 and CHA.9.536.7.vhCDR3;
[0479] CHA.9.536.8, CHA.9.536.8.VH, CHA.9.536.8.VL, CHA.9.536.8.HC, CHA.9.536.8.LC, CHA.9.536.8.H1, CHA.9.536. 8.H2, CHA.9.536.8.H3; CHA.9.536.8.H4, CHA.9.536.8.H4 (S241P), CHA.9.536.8.vhCDR1, CHA.9.536.8.vhCDR2, CHA.9.536.8.vhCDR3, CHA.9.536.8.vlCDR1 , CHA.9.536.8.vlCDR2 and CHA.9.536.8.vhCDR3;
[0480] CHA.9.560.1, CHA. 9.560.1.VH, CHA. 9.560.1.VL, CHA. 9.560.1.HC, CHA. 9.560.1.LC, CHA. 9.560.1.H1, CHA.
[0481] 9.560.1.H2, CHA. 9.560.1.H3; CHA. 9.560.1.H4, CHA.9.560.1.H4 (S241P), CHA. 9.560.1.vhCDR1, CHA.
[0482] 9.560.1.vhCDR2, CHA. 9.560.1.vhCDR3, CHA. 9.560.1.vlCDR1, CHA. 9.560.1.vlCDR2 and CHA. 9.560.1.vhCDR3;
[0483] CHA.9.560.3, CHA. 9,560. 3VH, CHA. 9,560. 3.VL, CHA. 9,560. 3.HC, CHA.9.560. 3.LC, CHA. 9,560. 3.H1, CHA.
[0484] 9,560. 3.H2, CHA. 9,560. 3.H3; CHA.9.560.3.H4, CHA.9.560.3.H4 (S241P); CHA. 9,560. 3.vhCDR1, CHA. 9,560.
[0485] 3. vhCDR2, CHA. 9,560. 3.vhCDR3, CHA. 9,560. 3.vlCDR1, CHA. 9,560. 3.vlCDR2 and CHA. 9,560. 3.vhCDR3;
[0486] CHA.9.560.4, CHA. 9,560. 4VH, CHA. 9,560. 4.VL, CHA. 9,560. 4.HC, CHA.9.560. 4.LC, CHA. 9,560. 4.H1, CHA.
[0487] 9,560. 4.H2, CHA. 9,560. 4.H3; CHA.9.560.4.H4, CHA.9.560.4.H4 (S241P), CHA. 9,560. 4.vhCDR1, CHA. 9,560.
[0488] 4. vhCDR2, CHA. 9,560. 4.vhCDR3, CHA. 9,560. 4.vlCDR1, CHA. 9,560. 4.vlCDR2 and CHA. 9,560. 4.vhCDR3;
[0489] CHA.9.560.5, CHA. 9,560. 5VH, CHA. 9,560. 5.VL, CHA. 9,560. 5.HC, CHA.9.560. 5.LC, CHA. 9,560. 5.H1, CHA.
[0490] 9,560. 5.H2, CHA. 9,560. 5.H3; CHA. 9,560. 5.H4, CHA. 9,560. 5.vhCDR1, CHA. 9,560. 5.vhCDR2, CHA. 9,560.
[0491] 5. vhCDR3, CHA. 9,560. 5.vlCDR1, CHA. 9,560. 5.vlCDR2 and CHA. 9,560. 5.vhCDR3;
[0493] CHA.9.560.6, CHA. 9,560. 6VH, CHA. 9,560. 6.VL, CHA. 9,560. 6.HC, CHA.9.560. 6.LC, CHA. 9,560. 6.H1, CHA.
[0494] 9,560. 6.H2, CHA. 9,560. 6.H3; CHA.9.560.6.H4, CHA.9.560.6.H4 (S241P), CHA. 9,560. 6.vhCDR1, CHA. 9,560.
[0495] 6. vhCDR2, CHA. 9,560. 6.vhCDR3, CHA. 9,560. 6.vlCDR1, CHA. 9,560. 6.vlCDR2 and CHA. 9,560. 6.vhCDR3;
[0496] CHA.9.560.7, CHA. 9,560. 7VH, CHA. 9,560. 7.VL, CHA. 9,560. 7.HC, CHA.9.560. 7.LC, CHA. 9,560. 7.H1, CHA.
[0497] 9,560. 7.H2, CHA. 9,560. 7.H3; CHA.9.560.7.H4; CHA.9.560.7.H4 (S241P); CHA. 9,560. 7.vhCDR1, CHA. 9,560.
[0498] 7. vhCDR2, CHA. 9,560. 7.vhCDR3, CHA. 9,560. 7.vlCDR1, CHA. 9,560. 7.vlCDR2 and CHA. 9,560. 7.vhCDR3; CHA.9.560.8, CHA. 9,560. 8VH, CHA. 9,560. 8.VL, CHA. 9,560. 8.HC, CHA.9.560. 8.LC, CHA. 9,560. 8.H1, CHA.
[0499] 9,560. 8.H2, CHA. 9,560. 8.H3; CHA.9.560.8.H4, CHA.9.560.8.H4 (S241P); CHA. 9,560. 8.vhCDR1, CHA. 9,560.
[0500] 8. vhCDR2, CHA. 9,560. 8.vhCDR3, CHA. 9,560. 8.vlCDR1, CHA. 9,560. 8.vlCDR2 and CHA. 9,560. 8.vhCDR3;
[0502] CHA.9.546.1, CHA. 9. 546.1VH, CHA. 9. 546.1.VL, CHA. 9. 546.1.HC, CHA. 9.546.1.LC, CHA. 9. 546.1.H1, CHA. 9.
[0503] 546.1. H2, CHA. 9. 546.1.H3; CHA.9.546.1. H4, CHA.9.546.1.H4 (S241P), CHA. 9. 546.1.vhCDR1, CHA. 9.
[0504] 546.1.vhCDR2, CHA. 9. 546.1.vhCDR3, CHA. 9. 546.1.vlCDR1, CHA. 9. 546.1.vlCDR2 and CHA. 9. 546.1.vhCDR3;
[0505] CHA.9.547.1, CHA. 9. 547.1VH, CHA. 9. 547.1.VL, CHA. 9. 547.1.HC, CHA. 9.547.1.LC, CHA. 9. 547.1.H1, CHA. 9.
[0506] 547.1. H2, CHA. 9. 547.1.H3; CHA.9.547.1. H4, CHA.9.547.1.H4 (S241P), CHA. 9. 547.1.vhCDR1, CHA. 9.
[0507] 547.1.vhCDR2, CHA. 9. 547.1.vhCDR3, CHA. 9. 547.1.vlCDR1, CHA. 9. 547.1.vlCDR2 and CHA. 9. 547.1.vhCDR3;
[0508] CHA.9.547.2, CHA. 9. 547. 2VH, CHA. 9. 547. 2.VL, CHA. 9. 547. 2.HC, CHA. 9,547. 2.LC, CHA. 9. 547. 2.H1, CHA.
[0509] 9. 547. 2.H2, CHA. 9. 547. 2.H3; CHA.9.547.2.H4, CHA.9.547.2.H4 (S241P), CHA. 9. 547. 2.vhCDR1, CHA. 9. 547.
[0510] 2. vhCDR2, CHA. 9. 547.2. vhCDR3, CHA. 9. 547. 2.vlCDR1, CHA. 9. 547. 2.vlCDR2 and CHA. 9. 547. 2.vhCDR3;
[0511] CHA.9.547.3, CHA. 9. 547. 3VH, CHA. 9. 547. 3.VL, CHA. 9. 547. 3.HC, CHA. 9,547. 3.LC, CHA. 9. 547. 3.H1, CHA.
[0512] 9. 547. 3.H2, CHA. 9. 547. 3.H3; CHA.9.547.3.H4, CHA.9.547.3.H4 (S241P), CHA. 9. 547. 3.vhCDR1, CHA. 9,547.
[0513] 3. vhCDR2, CHA. 9. 547.3. vhCDR3, CHA. 9. 547. 3.vlCDR1, CHA. 9. 547. 3.vlCDR2 and CHA. 9. 547. 3.vhCDR3;
[0514] CHA.9.547.4, CHA. 9. 547. 4VH, CHA. 9. 547. 4.VL, CHA. 9. 547. 4.HC, CHA.9.547. 4.LC, CHA. 9. 547. 4.H1, CHA.
[0515] 9. 547. 4.H2, CHA. 9. 547. 4.H3; CHA.9.547.4.H4, CHA.9.547.4.H4 (S241P), CHA. 9. 547. 4.vhCDR1, CHA. 9. 547.
[0516] 4. vhCDR2, CHA. 9. 547.4. vhCDR3, CHA. 9. 547. 4.vlCDR1, CHA. 9. 547. 4.vlCDR2 and CHA. 9. 547. 4.vhCDR3;
[0517] CHA.9.547.6, CHA. 9. 547. 6 VH, CHA. 9. 547. 6.VL, CHA. 9. 547. 6.HC, CHA. 9,547. 6.LC, CHA. 9. 547. 6.H1, CHA.
[0518] 9. 547. 6.H2, CHA. 9. 547. 6.H3; CHA.9.547.6.H4, CHA.9.547.6.H4 (S241P), CHA. 9. 547. 6.vhCDR1, CHA. 9. 547.
[0519] 6. vhCDR2, CHA. 9. 547.6. vhCDR3, CHA. 9. 547. 6.vlCDR1, CHA. 9. 547. 6.vlCDR2 and CHA. 9. 547. 6.vhCDR3;
[0520] CHA.9.547.7, CHA. 9. 547. 7VH, CHA. 9. 547. 7.VL, CHA. 9. 547. 7.HC, CHA. 9,547. 7.LC, CHA. 9. 547. 7.H1, CHA.
[0521] 9. 547. 7.H2, CHA. 9. 547. 7.H3; CHA.9.547.7.H4, CHA.9.547.7.H4 (S241P), CHA. 9. 547. 7.vhCDR1, CHA. 9. 547.
[0522] 7. vhCDR2, CHA. 9. 547.7. vhCDR3, CHA. 9. 547. 7.vlCDR1, CHA. 9. 547. 7.vlCDR2 and CHA. 9. 547. 7.vhCDR3;
[0523] CHA.9.547.8, CHA. 9. 547. 8VH, CHA. 9. 547. 8.VL, CHA. 9. 547. 8.HC, CHA.9.547.8.LC, CHA. 9. 547. 8.H1, CHA. 9.
[0524] 547. 8.H2, CHA. 9. 547. 8.H3; CHA.9.547.8.H4, CHA.9.547.8.H4 (S241P), CHA. 9. 547. 8.vhCDR1, CHA. 9. 547.
[0525] 8. vhCDR2, CHA. 9. 547.8. vhCDR3, CHA. 9. 547. 8.vlCDR1, CHA. 9. 547. 8.vlCDR2 and CHA. 9. 547. 8.vhCDR3;
[0526] CHA.9.547.9, CHA.9.547.9, CHA.9.547.9VH, CHA.9.547.9.VL, CHA.9. 547.9.HC, CHA.9.547.9.LC, CHA.9.547.9.H1, CHA.9.547.9.H2, CHA.9.547.9.H3; CHA.9.547.9.H4, CHA.9.547.9.H4, CHA.9.547.9.H4 (S241 P), CHA.9.547.9.H4 (S241P), CHA.9.547.9.vhCDR1, CHA.9.547 .9.vhCDR2, CHA.9.547.9.vhCDR3, CHA.9.547.9.vlCDR1, CHA.9.547.9.vlCDR2 and CHA.9.547.9.vhCDR3;
[0528] CHA.9.547.13, CHA.9.547.13, CHA.9.547. 13VH, CHA.9. 547.13.VL, CHA.9.547.13.HC, CHA. 9.547.13.LC, CHA.
[0529] 9.547.13. H1, CHA.9.547.13.H2, CHA.9. 547.13.H3; CHA.9.547.13.H4, CHA.9.547.13.H4, CHA.9.547.13.H4 (S241P), CHA.9.547.13.H4 (S241P), CHA. 9. 547.13.vhCDR1, CHA.9.547.13.vhCDR2, CHA.9.547. 13.vhCDR3, CHA. 9.
[0530] 547.13. vlCDR1, CHA. 9. 547.13.vlCDR2 and CHA. 9. 547. 13.vhCDR3;
[0532] CHA.9.541.1, CHA. 9. 541.1.VH, CHA. 9. 541.1.VL, CHA. 9. 541.1.HC, CHA. 9,541. LLC, CHA. 9. 541.1.H1, CHA. 9.
[0533] 541.1. H2, CHA. 9. 541.1.H3; CHA.9.541.1.H4, CHA.9.541.1.H4 (S241P), CHA. 9. 541.1.vhCDR1, CHA. 9.
[0534] 541.1. vhCDR2, CHA. 9. 541.1.vhCDR3, CHA. 9. 541.1.VlCDR1, CHA. 9. 541.1.vlCDR2 and CHA. 9,541. 1.vhCDR3;
[0535] CHA.9.541.3, CHA. 9. 541.3.VH, CHA. 9. 541.3.VL, CHA. 9. 541.3.HC, CHA. 9.541.3.LC, CHA. 9. 541.3.H1, CHA.
[0536] 9. 541. 3.H2, CHA. 9. 541. 3.H3; CHA.9.541.3.H4, CHA.9.541.3.H4 (S241P), CHA. 9. 541. 3.vhCDR1, CHA. 9. 541.
[0537] 3. vhCDR2, CHA. 9. 541.3. vhCDR3, CHA. 9. 541.3.vlCDR1, CHA. 9. 541.3.vlCDR2 and CHA. 9.541.3.vhCDR3;
[0538] CHA.9.541.4, CHA. 9. 541.4.VH, CHA. 9. 541.4.VL, CHA. 9. 541.4.HC, CHA. 9.541.4.LC, CHA. 9. 541.4.H1, CHA.
[0539] 9. 541.4.H2, CHA. 9. 541. 4.H3; CHA.9.541.4.H4, CHA.9.541.4.H4 (S241P), CHA. 9. 541. 4.vhCDR1, CHA. 9. 541.
[0540] 4. vhCDR2, CHA. 9. 541.4. vhCDR3, CHA. 9. 541.4.vlCDR1, CHA. 9. 541.4.vlCDR2 and CHA. 9.541.4.vhCDR3;
[0541] CHA.9.541.5, CHA. 9. 541.5.VH, CHA. 9. 541.5.VL, CHA. 9. 541.5.HC, CHA. 9.541.5.LC, CHA. 9. 541.5.H1, CHA.
[0542] 9. 541. 5.H2, CHA. 9. 541. 5.H3; CHA.9.541.5.H4, CHA.9.541.5.H4 (S241P), CHA. 9. 541. 5.vhCDR1, CHA. 9. 541.
[0543] 5. vhCDR2, CHA. 9. 541.5. vhCDR3, CHA. 9. 541.5.vlCDR1, CHA. 9. 541.5.vlCDR2 and CHA. 9.541.5.vhCDR3;
[0544] CHA.9.541.6, CHA. 9. 541.6.VH, CHA. 9. 541.6.VL, CHA. 9. 541.6.HC, CHA. 9.541.6.LC, CHA. 9. 541.6.H1, CHA.
[0545] 9. 541. 6.H2, CHA. 9. 541.6.H3; CHA.9.541.6.H4, CHA.9.541.6.H4 (S241P), CHA. 9. 541. 6.vhCDR1, CHA. 9. 541.
[0546] 6. vhCDR2, CHA. 9. 541.6. vhCDR3, CHA. 9. 541.6.vlCDR1, CHA. 9. 541.6.vlCDR2 and CHA. 9.541.6.vhCDR3;
[0547] CHA.9.541.7, CHA. 9. 541.7.VH, CHA. 9. 541.7.VL, CHA. 9. 541.7.HC, CHA. 9.541.7.LC, CHA. 9. 541.7.H1, CHA.
[0548] 9. 541. 7.H2, CHA. 9. 541. 7.H3; CHA.9.541.7.H4, CHA.9.541.7.H4 (S241P), CHA. 9. 541. 7.vhCDR1, CHA. 9. 541.
[0549] 7. vhCDR2, CHA. 9. 541.7. vhCDR3, CHA. 9. 541.7.vlCDR1, CHA. 9. 541.7.vlCDR2 and CHA. 9.541.7.vhCDR3; and CHA.9.541.8, CHA. 9. 541.8.VH, CHA. 9. 541.8.VL, CHA. 9. 541.8.HC, CHA. 9.541.8.LC, CHA. 9. 541.8.H1, CHA.
[0550] 9. 541. 8.H2, CHA. 9. 541. 8.H3; CHA.9.541.8.H4, CHA.9.541.8.H4 (S241P); CHA. 9. 541. 8vhCDR1, CHA. 9. 541.
[0551] 8.vhCDR2, CHA. 9. 541.8.vhCDR3, CHA. 9. 541.8.vlCDR1, CHA. 9. 541.8.vlCDR2 and CHA. 9.541.8.vhCDR3.
[0553] In the case of scFvs comprising the CDRs of the above antibodies, these are labeled scFvs that include a scFv comprising a variable heavy domain with the vhCDRs, a linker and a variable light domain with the vlCDRs, again as above in any orientation. Thus the description includes scFv-CHA.9.536.3.1, scFv-CHA.9.536.3, scFv-CHA.9.536.4, scFv-CHA.9.536.5, scFv-CHA.9.536.7, scFv-CHA.9.536. 8, scFv-CHA.9.560.1, scFv-CHA.9.560.3, scFv-CHA.9.560.4, scFv-CHA.9.560.5, scFv-CHA.9.560.6, scFv-CHA.9.560.7, scFv-CHA.9.560.8, scFv-CHA.9.546.1, scFv-CHA.9.547.1, scFv-CHA.9.547.2, scFv-CHA.9.547.3, scFv-CHA.9.547.4, scFv- CHA.9.547.6, scFv-CHA.9.547.7, scFv-CHA.9.547.8, scFv-CHA.9.547.9, scFv-CHA.9.547.13, scFv-CHA.9.541.1, scFv-CHA. 9.541.3, scFv-CHA.9.541.4, scFv-CHA.9.541.5, scFv-CHA.9.541.6, scFv-CHA.9.541.7, and scFv-CHA.9.541.8.
[0554] Furthermore, CHA.9.543 binds to TIGIT, but does not block the TIGIT-PVR interaction.
[0556] As discussed herein, the description further provides variants of the above components (CPA and CHA), including variants in the CDRs, as indicated above. Thus, the disclosure provides antibodies that comprise a set of 6 CDRs, as indicated herein that may contain one, two, or three amino acid differences in the set of CDRs, as long as the antibody still binds TIGIT. Suitable assays for testing whether an anti-TIGIT antibody contains mutations compared to the CDR sequences listed herein are known in the art, such as the Biacore assays.
[0557] Furthermore, the description further provides variants of the above variable heavy and light chains. In this case, the variable heavy chains can be 80%, 90%, 95%, 98%, or 99% identical to the "VH" sequences herein, and / or contain 1,2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variants are used. Variable light chains are provided that can be 80%, 90%, 95%, 98%, or 99% identical to the "VL" sequences herein (and, in particular, CPA.9.086), and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variants are used. The description includes these variants as long as the antibody still binds TIGIT. Suitable assays for testing whether an anti-TIGIT antibody contains mutations compared to the CDR sequences listed herein are known in the art, such as the Biacore assays.
[0559] Similarly, heavy and light chains are provided that are 80%, 90%, 95%, 98%, or 99% identical to the full-length "HC" and "LC" sequences herein (and, in particular CPA.9.086), and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variants are used. In these cases, the description includes these variants as long as the antibody still binds to TIGIT. Suitable assays for testing whether an anti-TIGIT antibody contains mutations compared to the CDR sequences listed herein are known in the art, such as the Biacore assays.
[0561] Furthermore, the framework regions of the variable heavy and variable light chains of the CPA or CHA antibodies herein can be humanized (or, in the case of CHA antibodies, "rehumanized", to the extent that the methods can humanization variants) as is known in the art (with occasional variants generated in the CDRs, as needed), and thus humanized variants of the VH and VL chains of Figure 53 (and, in particular, CPA.9.086) can be generated . In addition, the humanized variable heavy and light domains can then be fused with human constant regions, such as the constant regions of IgG1, IgG2, IgG3 and IgG4 (including IgG4 (S241P)).
[0563] In particular, as is known in the art, murine VH and VL chains can be humanized as is known in the art, for example, using the IgBLAST program from the NCBI website, as indicated in Ye et al. Nucleic Acids Res. 41: W34-W40 (2013). IgBLAST takes a murine VH and / or VL sequence and compares it to a library of known human germline sequences. As shown herein, for the humanized sequences generated herein, the databases used were IMGT human VH genes (F + ORF, 273 germline sequences) and IMGT human VL kappa genes (F + ORF, 74 germline sequences). Five exemplary CHA sequences were chosen: CHA.9.536, CHA9.560, CHA.9.546, CHA.9.547, and CHA.9.541 (see Figure 53). For this embodiment of humanization, human germline IGHV1-46 (allele1) was chosen for all 5 as the acceptor sequence and human heavy chain binding region IGHJ4 (allele 1) (J gene). For three of the four (CHA.7.518, CHA.7.530, CHA.7.538_1 and CHA.7.538_2), human germline IGKV1 -39 (allele 1) was chosen as the acceptor sequence and human light chain was chosen IGKJ2 (allele 1) (J gene). The J gene was chosen from human binding region sequences compiled on IMGT® the international information system ImMunoGeneTics as www.imgt.org. The CDRs were defined according to the definition of AbM (see, www.bioinfo.org.uk/abs/).
[0565] In some embodiments, the anti-TIGIT antibodies of the present disclosure include anti-TIGIT antibodies where the V ^h and V ^l sequences of different anti-TIGIT antibodies can be "mixed and matched" to create other anti-TIGIT antibodies. TIGIT binding of such "mixed and matched" antibodies can be assayed using the binding assays described above, e.g. g., ELISA or Biacore assays). In some embodiments, when the V ^h and V ^l chains are mixed and matched, a V ^h sequence of a particular V ^h / V ^l pair is replaced with a structurally similar V ^h sequence. Also, in some embodiments, a V ¹ sequence from a particular V ^h / V ¹ pair is replaced with a structurally similar V ¹ sequence. For example, the V ^h and V ^l sequences of homologous antibodies are particularly well suited for mixing and matching.
[0567] Accordingly, the TIGIT antibodies of the invention comprise CDR amino acid sequences selected from the group consisting of (a) sequences as listed herein; (b) sequences that differ from the CDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; (c) amino acid sequences that have 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to the sequences specified in (a) or (b); (d) a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding the amino acids as listed herein. In particular, the CPA.9.086 antibody can have sequences selected from (a), (b), (c) or (d).
[0569] The definition of antibodies to TIGIT further includes antibodies that share identity with the antibodies to TIGIT listed herein. That is, in certain embodiments, an anti-TIGIT antibody according to the disclosure comprises heavy and light chain variable regions comprising amino acid sequences that are identical to all or part of the anti-TIGIT amino acid sequences of preferred anti-TIGIT antibodies. , respectively, wherein the antibodies retain the desired functional properties of the parental anti-TIGIT antibodies. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (that is,% homology = no. Of identical positions / total no. Of positions X 100), taking into account the number of gaps , and the length of each gap, which needs to be entered for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
[0571] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller ( Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 ), using a table of PAM120 residue weights, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch ( J. Mol. Biol. 48: 444-453 (1970)) which has been incorporated into the GAP program in the GCG software package (commercially available), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16 , 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.
[0573] Additionally or alternatively, the protein sequences of the present disclosure may further be used as a "search sequence" to search against public databases, eg, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) from Altschul, et al. (1990) J Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to antibody molecules according to at least some embodiments of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.
[0575] In general, the percent identity for the comparison between antibodies to TIGIT is at least 75%, at least 80%, at least 90%, with at least about 95, 96, 97, 98 or 99% being preferred. percent identity. The percent identity can be along the entire amino acid sequence, eg, the entire heavy or light chain, or along a portion of the chains. For example, the definition of anti-TIGIT antibodies in the disclosure includes those that share identity throughout the entire variable region (for example, when the identity is 95 or 98% identical throughout the variable regions ), or along the entire constant region, or along just the Fc domain. In particular, the disclosure provides antibodies to TIGIT that have at least 75%, at least 80%, at least 90%, with at least about 95, 96, 97, 98 or 99% percent identity being preferred, with the CPA.9.086 antibody.
[0577] Furthermore, sequences that may have identical CDRs, but changes in the framework parts of the variable domain (or complete heavy or light chain) are also included. For example, antibodies to TIGIT include those with CDRs identical to those shown in Figure 53, but whose identity throughout the variable region may be less, eg, 95 or 98% percent identical. In particular, the disclosure provides antibodies to TIGIT that have CDRs identical to CPA.9.086, but with framework regions that are 95 or 98% identical to CPA.9.086.
[0578] A. Antibodies to TIGIT that compete for binding
[0580] The present disclosure provides not only the listed antibodies, but additional antibodies that compete with the listed antibodies (the CPA numbers listed herein that specifically bind to TIGIT) that specifically bind to the TIGIT molecule. As shown in Example 16, the TIGIT antibodies of the description are "pre-classified" into different classes of epitopes. Among the 44 antibodies to TIGIT in the epitope pre-classification study, there are four communities, each having related pairwise blocking patterns, separating them into 12 total discrete classes indicated herein and shown. in Figures 67 and 68. There are twelve discrete classes indicated herein; 1) BM9-H4, CHA.9.525, CPA.9.081 -H4, CHA.9.538, CHA.9.553, CPA.9.069-H4, CHA.9.543, CHA.9.556, CPA.9.077-H4 and CHA.9.561; 2) CHA.9.560 and CHA.9.528; 3) CHA.9.552, CHA.9.521, CHA.9.541, CHA.9.529, CHA.9.519, CHA.9.527 and CHA.9.549; 4) CPA.9.057-H4 and CHA.9.554; 5) CHA.9.546, CPA.9.012-H4, CHA.9.547, CPA.9.013-H4, CPA.9.018-H4, MBSA43-M1, Sino PVR-Fc (ligand), CHA.9.555, PVR-Fc M2A (ligand ), BM29-H4, CPA.9.027-H4, CPA.9.049-H4 and CPA.9.053-H4; 6) CPA.9.064-H4; 7) BM26-H4; 8) CPA.9.059-H4; 9) CHA.9.535 and CPA.9.009-H4; 10) CHA.9.536, CHA.9.522 and CPA.9.015-H4; 11) CPA.9.011-H4 and BM8-H4 and 12) CPA.9.071 -H4.
[0582] Thus, the disclosure provides anti-TIGIT antibodies that compete for binding with antibodies that are in discrete epitope classes 1 to 12. In a particular embodiment, the disclosure provides anti-TIGIT antibodies that compete for binding with CPA.9.086 and are at least 95, 96, 97, 98 or 99% identical to CPA.9.086.
[0584] Additional antibodies are generated that compete with the listed antibodies, as is known in the art and generally noted below. Competitive binding assays can be done as is known in the art, generally using SPR / Biacore® binding assays, as well as ELISA and cell-based assays.
[0586] VIII. Antibodies to PVRIG
[0588] The present invention provides anti-PVRIG antibodies. (For convenience, "anti-PVRIG antibodies" and "anti-PVRIG antibodies" are used interchangeably). The anti-PVRIG antibodies of the invention specifically bind to human PVRIG, and preferably to the human PVRIG ECD.
[0590] Specific binding for PVRIG or a PVRIG epitope can be presented, for example, by an antibody having a KD of at least about 10-4 M, at least about 10-5 M, at least about 10-6 M, at least about 10-7 M, at least about 10-8 M, at least about 10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, at least about 10-12 M, or greater, where KD refers to a rate of dissociation of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD that is 20, 50, 100, 500, 1,000, 5,000, 10,000 times greater or more for a control molecule relative to the antigen or PVRIG epitope.
[0592] However, as shown in the Examples of WO2016 / 134333, for optimal binding to PVRIG expressed on the surface of NK and T cells, the antibodies preferably have a KD less than 50 nM and most preferably less than 1 nM, finding use in the methods of the invention less than 0.1 nM and less than 1 pM and 0.1 pM.
[0594] Also, specific binding for a particular antigen or epitope can be displayed, for example, by an antibody having a KA or Ka for a PVRIG antigen or epitope of at least 20, 50, 100, 500, 1,000, 5,000, 10,000 times. greater or greater for the epitope relative to a control, where KA or Ka refers to a rate of association of a particular antibody-antigen interaction.
[0596] In some embodiments, the anti-PVRIG antibodies of the invention bind to human PVRIG with a K ^d of 100 nM or less, 50 nM or less, 10 nM or less, or 1 nM or less (that is, higher binding affinity ), or 1 pM or less, where K ^d is determined by known methods, e.g. eg, surface plasmon resonance (SPR, eg, Biacore assays), ELISA, KINEXA, and most typically SPR at 25 ° or 37 ° C.
[0598] It is important to note that the binding affinity for anti-PVRIG antibodies surprisingly correlates with activity. A cumulative analysis of screening data shows that the affinity of the anti-PVRIG antibodies of the invention highly correlates with their ability to bind to primary human T cells. More specifically, the antibodies that provided the highest maximum signal in T cells were those with affinities in the picomolar range. Antibodies that had affinities in the low nanomolar range and above provided relatively weak peak signals in T cells. Thus, the data indicate that the utility of anti-PVRIG antibodies for T cell-based immunotherapy can probably be defined, in part, based on their affinity. Reference is made to the antibody sequences of WO2016 / 134333 and, in particular, to the antigen-binding domains of anti-PVRIG indicated in Figures 38 (representing sequences that bind to PVRIG and block the interaction of PVRIG and PVRL2 ), Figure 39 (representing sequences that bind to PVRIG and do not block the interaction of PVRIG and PVRL2), Figure 40 (representing CDRs and data from these antibodies), and Figure 41 (representing CDRs from hybridomas that bind and block). That is, the Figures and Legends, as well as the particular sequences and SEQ ID NO: s of all CPA.7 and CHA.7 antibodies (including CDR, VH and VL and full length sequences) of WO2016 / 134333 They are expressly incorporated herein.
[0599] Figure 45 illustrates the ability of two anti-PVRIG antibodies with different affinities to bind to primary CD8 T cells. As shown in Figure 45, CHA.7.518 has approximately 8-fold higher affinity than CP A.7.021 (sequence in WO2016 / 13433), as measured by binding to HEK cells engineered to overexpress PVRIG (HEK hPVRIG) . Consistent with this, CHA.7.518 has approximately 13-fold higher affinity than CPA.7.021, as measured by binding to Jurkat cells. The higher affinity of CHA.7.518 corresponded to a higher maximum binding signal from HEK hPVRIG cells, but not from Jurkat cells.
[0600] In contrast, CHA.7.518 consistently provided a higher maximal binding signal from primary CD8 T cells, compared to CPA.7.021. This is illustrated in a binding titer experiment where different concentrations of isotype or anti-PVRIG antibodies were added to primary CD8 T cells, and the resulting maximum binding signal was measured. In the two illustrated donors (Figure 45), CHA.7.518 consistently provided a higher peak signal (geometric mean fluorescence intensity, gMFI) than CPA.7.021 in a titer dependent manner. gMFIr = intensity of the geometric fluorescence of the antibody of interest / intensity of the geometric fluorescence of the control antibody. The gMFIr measures the signal that the antibody of interest provides relative to an isotype antibody at a fixed concentration of both.
[0602] Accordingly, the anti-PVRIG antibodies of the invention have binding affinities (as measured using techniques outlined herein) in the picomolar range, e.g. g., 0.1 to 9 pM, with about 0.2 to about 2 being preferred, and about 0.2 to about 0.5 being of particular use.
[0604] As for antibodies to TIGIT, antibodies to PVRIG are similarly labeled as follows. Antibodies have reference numbers, for example "CHA.7.518.1". This represents the combination of variable heavy and variable light chains, as depicted in Figure 3, for example, knowing that these antibodies include two heavy chains and two light chains. "CPA.7.518.1.VH" refers to the variable heavy part of CpA.7.518.1, while "CPA.7.518.1.VL" is the variable light chain. "CPA.7.518.1.vhCDR1", "CPA.7.518.1.vhCDR2", "CPA.7.518.1.vhCDR3", "CPA.7.518.1.vlCDR1", "CPA.7.518.1.vlCDR2", and "CPA.7.518.1.vlCDR3", refers to the indicated CDRs. "CPA.7.518.1.HC" refers to the complete heavy chain (eg, variable and constant domain) of this molecule, and "CPA.7.518.1.LC" refers to the complete light chain (p g., variable and constant domain) of the same molecule. In general, the human kappa light chain is used for the constant domain of each phage antibody (or humanized hybridoma) herein, although, in some embodiments, the lambda light constant domain is used. "CPA.7.518.1.H1" refers to a full-length antibody that comprises the variable heavy and light domains, including the constant domain of human IgG1 (hence the sequences of H1; IgG1, IgG2, IgG3, and IgG4 are shown in Figure 50). Therefore, "CPA.7.518.1.H2" would be the variable domains of CPA.7.518.1 bound to human IgG2. "CPA.7.518.1.H3" would be the variable domains of CPA.7.518.1 bound to human IgG3, and "CPA.7.518.1.H4" would be the variable domains of CPA.7.518.1 bound to human IgG4. Note that, in some cases, human IgGs may have additional mutations, such as those described below, and this can be noted. For example, in many embodiments, there may be a S241P mutation in human IgG4, and this may be noted as "CPA.7.518.1.H4 (S241P)", for example. The sequence of human IgG4 in this S241P hinge variant is shown in Figure 50. Other potential variants are IgG1 (N297A), (or other variants that eliminate glycosylation at this site and thus many of the effector functions associated with binding to FcYRIIIa), and IgG1 (D265A), which reduces binding to FcyR receptors.
[0605] The invention further provides variable heavy and light domains, as well as full length heavy and light chains.
[0607] In some embodiments, the invention provides PVRIG-binding scFvs comprising a variable heavy domain and a variable light domain linked by an scFv linker as noted above. The VL and VH domains can be in any orientation, e.g. eg, from end N to C "VH-connector-VL" or "VL-connector" VH ' These are named for their component parts; for example, "scFv-CH A.7.518.1 VH-connector-VL" or "scFv-CPA.
[0608] 7.518.1 .VL-connector-VH ". Thus," scFv-CPA.7.518.1 "can be in any orientation.
[0610] IX. Nucleic acids encoding antibodies
[0612] Also provided are nucleic acid compositions encoding the antibodies of the invention, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and / or expression vector compositions. As will be appreciated by those skilled in the art, the protein sequences depicted herein may be encoded by any possible number of nucleic acid sequences, due to the degeneracy of the genetic code.
[0614] The nucleic acid compositions encoding the antibodies will depend on the format of the antibody. For traditional tetrameric antibodies that contain two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a single expression vector or into two expression vectors, as is known in the art, transformed into host cells, where they are expressed to form the antibodies of the invention. In some embodiments, for example, when using scFv constructs, a single nucleic acid encoding the variable heavy chain-linker-variable light chain is generally used, which can be inserted into an expression vector for transformation into host cells. Nucleic acids can be put into expression vectors containing the appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretory sequences, regulatory sequences, promoters, origins of replication, selection genes, etc. .
[0615] Preferred mammalian host cells for expressing the recombinant antibodies according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293, and others, as is known in the art.
[0617] Nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "becomes substantially pure" when it is purified from other cellular components or other contaminants, eg. g., other nucleic acids or cellular proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others known in the art.
[0619] To create a scFv gene, DNA fragments encoding V ^h and V ¹ are operably linked to another fragment encoding a flexible linker, e.g. g., which encodes the amino acid sequence (Gly4-Ser) 3 and others discussed herein, so that the V ^h and V ^l sequences can be expressed as a contiguous single chain protein, with the V ^l and V ^h joined by the flexible connector.
[0621] X. Formulations
[0623] The therapeutic compositions used in the practice of the foregoing methods (and, in particular, CHA.7.518.1.H4 (S241P) and CPA.9.086) can be formulated into pharmaceutical compositions comprising a suitable carrier for the desired method of administration. Suitable carriers include any material that, when combined with the therapeutic composition, retains the antitumor function of the therapeutic composition and is generally not reactive with the immune system of the patient. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers, such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptable carriers, excipients, or stabilizers are not toxic to recipients at the dosages and concentrations employed and may include buffers.
[0625] In a preferred embodiment, the pharmaceutical composition comprising the antibodies of the invention may be in a water soluble form, such as being present as pharmaceutically acceptable salts, which are intended to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases and the like.
[0627] Administration of the pharmaceutical composition comprising antibodies of the present invention, preferably in the form of a sterile aqueous solution, can be done in a number of ways, including, but not limited to, subcutaneously and intravenously.
[0629] Dosage amounts and frequency of administration, in a preferred embodiment, are selected to be therapeutically or prophylactically effective. As is known in the art, adjustments may be necessary for protein degradation, systemic versus localized administration, and rate of new protease synthesis, as well as age, body weight, general health, sex, diet, time of life. administration, drug interaction, and severity of condition, and will be ascertained by those of skill in the art with routine experimentation.
[0630] In order to treat a patient, a therapeutically effective dose of the Fc variant of the present invention can be administered. By "therapeutically effective dose" is meant herein a dose that produces the effects for which it is administered. The exact dosage will depend on the purpose of the treatment, and can be ascertained by those skilled in the art using known techniques.
[0632] XI. Methods for using the antibodies
[0634] Antibodies of the invention, including both PVRIG and TIGIT antibodies, can be used in various diagnostic and therapeutic applications. In some cases, the decision of which antibody to administer to a patient is made using an assessment of expression levels (either gene expression levels or protein expression levels, the latter being preferred) of tumor sample biopsies to determine if the sample overexpress either TIGIT or PVRIG, or both, to determine which therapeutic antibodies to administer.
[0636] A. Diagnostic uses
[0638] Accordingly, the antibodies of the invention also find use in in vitro or in vivo diagnosis, including imaging of tumors that overexpress either PVRIG or TIGIT, respectively. It should be noted, however, that, as discussed herein, both TIGIT and PVRIG, as the target protein of immuno-oncology, are not necessarily overexpressed in cancer cells, but rather in immune infiltrates in cancer. This is the mechanism of action, p. eg, activation of immune cells, such as T cells and NK cells, leading to the diagnosis of cancer. Consequently, these antibodies can be used to diagnose cancer. Diagnosis using antibodies to PVRIG is also indicated in WO 2016/134333, [0434-0459].
[0639] Generally, the diagnosis can be made in several ways. In one embodiment, a tissue from a patient, such as a biopsy sample, is contacted with an antibody to TIGIT, generally labeled, such that the antibody binds to endogenous TIGIT. The level of the signal is compared to that of normal non-cancerous tissue from either the same patient or a reference sample to determine the presence or absence of cancer. The biopsy sample can be from a solid tumor, a blood sample (for lymphomas and leukemias such as ALL, T-cell lymphoma, etc).
[0641] In general, in this embodiment, the anti-TIGIT is labeled, for example, with a fluorophore or other optical marker, which is detected using a fluorimeter or other optical detection system, as is well known in the art. In an alternative embodiment, a labeled secondary antibody is contacted with the sample, for example, using an anti-human IgG antibody from a different mammal (mouse, rat, rabbit, goat, etc.) to form a sandwich assay like is known in the art. Alternatively, the anti-TIGIT mAb could be directly labeled (ie, biotin) and detection can be done by a secondary Ab targeted to the labeling agent in the art.
[0643] Once the overexpression of TIGIT has been observed, the treatment can continue with the administration of an anti-TIGIT antibody according to the invention as indicated herein.
[0645] In other cases, an in vivo diagnosis is made. Generally, the anti-TIGIT antibody (including antibody fragments) is injected into the patient and imaging is performed. For example, the antibody is generally labeled with an optical label or an MRI label, such as a gadolinium chelate, radiolabel of mAbs (including fragments).
[0647] In some embodiments, the antibodies described herein are used for both diagnosis and treatment, or for diagnosis only. When anti-TIGIT antibodies are used for both diagnosis and treatment, some embodiments rely on two different anti-TIGIT antibodies against two different epitopes, such that the diagnostic antibody does not compete for binding with the therapeutic antibody, although , in some cases the same antibody can be used for both. For example, this can be done using antibodies that are in different classes, e.g. g., that bind to different epitopes in TIGIT, as indicated herein. Thus, the description includes compositions comprising a diagnostic antibody and a therapeutic antibody, and, in some embodiments, the diagnostic antibody is labeled as described herein. In addition, the therapeutic and diagnostic antibody composition can also be co-administered with other drugs as indicated herein.
[0649] Particularly useful antibodies for diagnostic use include, but are not limited to, those listed antibodies, or antibodies that use CDRs with variant sequences, or those that compete for binding with any of the antibodies in Figure 53.
[0651] In many embodiments, a diagnostic antibody is labeled. By "labeling" it is meant herein that the antibodies described herein have one or more elements, isotopes, or chemical compounds attached to allow detection in a screening or diagnostic procedure. In general, labels fall into several classes: a) immune labels, which can be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which can be radioactive or heavy isotopes, c) labels which are small molecules, which may include fluorescent and colorimetric staining agents, or molecules such as biotin that allow other labeling methods, and d) labels such as particles (including bubbles for ultrasound marking) or paramagnetic labels that allow body imaging . Labels can be incorporated into antibodies at any position and can be incorporated in vitro or in vivo during expression of the protein, as is known in the art.
[0653] The diagnosis can be made either in vivo, by the administration of a diagnostic antibody that enables whole-body imaging as described below, or in vitro, in samples taken from a patient. "Sample", in this context, includes any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen) , as well as tissue samples, such as the result of relevant tissue biopsies.
[0655] In addition, as indicated below and in the Examples and Figures, information regarding protein expression levels may be used from either PVRIG or TIGIT, or both, or PVRIG and PD-1, or TIGIT and PD-1, to determine which antibodies should be administered to a patient.
[0657] B. Cancer treatment
[0659] The antibodies of the invention find particular use in the treatment of cancer. In general, the antibodies of the invention are immunomodulatory, since, instead of directly attacking cancer cells, the antibodies of the invention stimulate the immune system, generally by inhibiting the action of the checkpoint receptor (e.g. e.g. PVRIG or TIGIT). Thus, unlike tumor-directed therapies, which aim to inhibit molecular pathways that are crucial for tumor growth and development, and / or deplete tumor cells, cancer immunotherapy aims to stimulate the patient's own immune system. patient to kill cancer cells, providing lasting tumor destruction. Several strategies can be used in cancer immunotherapy, including therapeutic cancer vaccines to induce cancer responses. tumor-specific T cells, and immunostimulatory antibodies (ie, inhibitory receptor antagonists = immune checkpoints) to eliminate immunosuppressive pathways.
[0661] Clinical responses with targeted therapy or conventional anticancer therapies tend to be transient as cancer cells develop resistance, and tumor recurrence occurs. However, the clinical use of cancer immunotherapy in recent years has shown that this type of therapy can have long-lasting clinical responses, showing a dramatic impact on long-term survival. However, although the responses are long-lasting, only a small number of patients respond (unlike conventional or targeted therapy, where a large number of patients respond, but the responses are transient).
[0663] By the time a tumor is clinically detected, it has already bypassed the immune defense system by acquiring immune resistance and immunosuppressive properties and creating an immunosuppressive tumor microenvironment through various mechanisms and a variety of immune cells.
[0665] Accordingly, the antibodies of the invention are useful for treating cancer. Due to the nature of an immuno-oncology mechanism of action, the checkpoint receptor (TIGIT or PVRIG) need not be overexpressed in or correlated with a particular cancer type; that is, the objective is to have the antibodies that eliminate the suppression of the activation of T cells and NK cells, so that the immune system will fight against cancers.
[0667] "Cancer", as used herein, broadly refers to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing growth or malignant tumor (eg, growth unregulated cell). The term "cancer" or "cancerous", as used herein, should be understood to encompass any neoplastic disease (whether invasive, non-invasive, or metastatic) that is characterized by abnormal and uncontrolled cell division causing the malignant growth or tumor, the non-limiting examples of which are described herein. This includes any physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer examples are exemplified in the working examples and are also described in the specification.
[0669] Non-limiting examples of cancer that can be treated using the antibodies of the invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer. , gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), liver cancer, bladder cancer , hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular grade; Intermediate grade diffuse NHL; High grade immunoblastic NHL; High-grade lymphoblastic NHL; High grade small uncleaved cell NHL; NHL with bulky disease; Mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; Multiple myeloma and post-transplant lymphoproliferative disorder (PTLD).
[0671] As shown in the Examples of WO2016 / 134333, PVRIG is overexpressed in and / or correlates with lymphocyte infiltration into tumors (as demonstrated by correlation with the expression of CD3, CD4, CD8 and PD-1) in various different tumors of various origins, and thus useful in the treatment of any cancer, including, but not limited to, prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HD)), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), large B-cell lymphoma diffuse, testicular germ cell tumors, meso telioma, and esophageal cancer.
[0673] In particular, CHA.7.518.1H4 (S241P) finds use in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cancer. cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, skin cancer other than melanoma (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), acute T-cell lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer.
[0675] In particular, CHA.7.538.1.2.H4 (S241P) finds use in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, cancer of the stomach, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer ( RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer .
[0677] In particular, CPA.9.086H4 (S241 P) finds use in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cancer. cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, skin cancer other than melanoma (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer.
[0679] In particular, CPA.9.083H4 (S241 P) finds use in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cancer. cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, skin cancer other than melanoma (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer.
[0681] In particular, CHA.9.547.7.H4 (S241 P) finds use in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, cancer of the stomach, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer ( RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer .
[0683] In particular CHA.9.547.13.H4 (S241P) finds use in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC) , lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer.
[0685] C. TIGIT antibody monotherapy
[0687] The TIGIT antibodies of the invention find particular use in the treatment of cancer as a monotherapy. Due to the nature of the immuno-oncology mechanism of action, it is not necessary for TIGIT to be overexpressed in or correlated with a particular cancer type; that is, the objective is to have anti-TIGIT antibodies that eliminate the suppression of the activation of T cells and NK cells, so that the immune system will fight against cancers.
[0689] Although any anti-TIGIT antibody in Figure 53 finds use in the treatment of cancer (including activating T cells as noted below), CPA.9.086.H4 (S241P), CPA.9.083.H4 (S241P), CHA.9.547.7.H4 (S241 P), and CHA.9.547.13.H4 (S241 P), find a particular use in some embodiments.
[0691] D. PVRIG Antibody Monotherapy
[0693] The PVRIG antibodies of the invention find particular use in the treatment of cancer as a monotherapy. Due to the nature of the immuno-oncology mechanism of action, it is not necessary for TIGIT to be overexpressed in or correlated with a particular cancer type; that is, the objective is to have anti-TIGIT antibodies that eliminate the suppression of the activation of T cells and NK cells, so that the immune system will fight against cancers.
[0695] In particular, CHA.7.518.1 H4 (S241 P) finds use as a monotherapy.
[0697] Similarly, in particular, CHA.7.538.1.2.H4 (S241 P) finds use as a monotherapy, in the treatment of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, cancer breast cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, skin cancer other than melanoma (squamous cell carcinoma and basal), glioma, renal cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, bladder cancer, and esophageal cancer.
[0698] E. Combination therapies
[0700] As is known in the art, combination therapies comprising a therapeutic antibody directed to an immunotherapy target and an additional therapeutic agent, specific for the pathological condition, are showing great promise. For example, in the area of immunotherapy, there are several promising combination therapies that use a chemotherapeutic agent (either a drug that is a small molecule or an anti-tumor antibody) or with an immuno-oncology antibody.
[0702] The terms "in combination with" and "co-administration" are not limited to the administration of said prophylactic or therapeutic agents at exactly the same time. Rather, it is meant that the antibody and the other agent (s) are administered in a sequence and at a time interval, such that they can act together to provide increased benefit over treatment alone with either antibody of the present invention or the other agent or agents. It is preferred that the antibody and the other agent or agents act additively, and it is especially preferred that they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the intended purpose. The skilled practitioner can determine empirically, or by considering the pharmacokinetics and modes of action of agents, the appropriate dose (s) of each therapeutic agent, as well as appropriate time schedules and methods of administration.
[0704] Accordingly, the antibodies of the present invention can be administered concomitantly with one or more other therapeutic regimens or agents. Additional therapeutic regimens or agents can be used to improve the efficacy or safety of the antibody. Also, additional therapeutic regimens or agents can be used to treat the same disease or a comorbidity rather than alter the action of the antibody. For example, an antibody of the present invention can be administered to the patient in conjunction with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.
[0706] 1. Antibodies against TIGIT with chemotherapeutic small molecules
[0708] The TIGIT antibodies of the present invention can be administered in combination with one or more other prophylactic or therapeutic agents, including, but not limited to, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, inhibitors. of kinase, anti-angiogenic agents, cardioprotectors, immunostimulatory agents, immunosuppressive agents, agents that promote hematological cell proliferation, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, or other therapeutic agents.
[0710] In this context, a "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide, alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, methuredopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL ');beta-lapacone;lapacol;colchicines; betulinic acid; a camptothecin (including the synthetic analog topotecan (HYCAMTN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobine; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, collophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediin antibiotics (eg, calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, eg, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin, including dynemycin A; a speramycin; as well as neocarzinostatin chromophore and related chromoprotein enediin antibiotic chromophores), aclacinomysins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carminomycin, daunophyllin, cromolynomycin, 6-Diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomofolino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mycofomycin, mitomycin, mitomycin, mitomycin, such as mitomycin nogalamycin, olivomycin, peplomycin, porphyromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorrub icin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; antiadrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid replenisher, such as frolinic acid; aceglatone; Aldophosphamide Glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucilo; bisantreno; edatraxate; defofamin; demecolcin; diaziquone; elfornithine; elliptinium acetate; an epothilone; ethoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pyrarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizophran; spirogermanium; tenuazonic acid; triaziquone; 2,2 ', 2 "-trichlorotriethylamine; trichothecenes (especially toxin T-2, verracurin A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine ; arabinoside ("Ara-C");thiotepa; taxoids, eg, paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE®, no cremophor, nanoparticle formulation of albumin engineered from paclitaxel (American Pharmaceutical Partners, Schaumberg, I11.), and docetaxel (TAXOTERE®; Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZARM®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; aminoidopterin inhibitor; daunomycin; RFS 2000 topoisomerase; difluoromethylornithine (DMFO); retin oids, such as retinoic acid; Capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; as well as combinations of two or more of the foregoing, such as CHOP, an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; CVP, an abbreviation for a combination therapy of cyclophosphamide, vincristine, and prednisolone; and FOLFOX, an abbreviation for an oxaliplatin (ELOXATIN®) treatment regimen combined with 5-FU and leucovorin.
[0712] According to at least some embodiments, the anti-TIGIT immune molecules could be used in combination with any of the standards of cancer care known in the art (as can be found, for example, at http://www.cancer.gov/cancertopics ).
[0714] Thus, in some instances, the anti-PVRIG antibodies listed herein (particularly, including CHA.7.538.1.2.H4 (S241 P) or CHA.7.518.1.H4 (S241 P)) can be combined with chemotherapeutic agents. Similarly, the anti-TIGIT antibodies listed herein (particularly, including CPA.9.086H4 (S241P), CPA.9.083H4 (S241P), and CHA.9.547.13.H4 (S241P)) can be combined with chemotherapeutic agents. .
[0716] Furthermore, the anti-PVRIG and anti-TIGIT antibodies of the invention can also be administered with other checkpoint inhibitors or activators.
[0718] 2. Combination therapy of antibody to TIGIT and checkpoint
[0720] As shown herein, the TIGIT antibodies of the invention can be combined with one of several checkpoint receptor antibodies. In some embodiments, a patient's tumor can be evaluated for receptor expression and the results are then used to inform a physician as to which antibodies to administer: PVRIG and PD-1, TIGIT and PD-1, or TIGIT and PVRIG. These tests are described later.
[0722] to. Anti-TIGIT antibodies in combination with anti-PD-1 antibodies
[0724] In one embodiment, the invention provides combinations of the anti-TIGIT antibodies of the invention and anti-PD-1 antibodies.
[0726] In one embodiment, a tumor biopsy is taken from a cancer patient, and dissociated as is known in the art for FACS analysis. Cells are stained with labeled antibodies against (1) TIGIT (eg, using any described herein or others in the art, such as MBSA43); (2) PD-1 (eg, using those known in the art, including EH12.2H7, Keytruda®, Opdivo®, etc.); (3) PD-L1 (for example, using those known in the art, such as BM-1 indicated herein) and (4) PVR (for example, using those known in the art, such as SKII.4) ; and (5) an isotype control antibody. FACS is performed and, for each receptor, the percentage of cells expressing the receptor relative to the control antibody is calculated. If the percentage of cells positive for TIGIT, PD-1, PD-1, and PVR is> 1% for all 4 receptors, then the patient is treated with antibodies to TIGIT and PD-1 as indicated herein.
[0728] Accordingly, the invention provides combinations of the anti-TIGIT antibodies of the invention and anti-PD-1 antibodies. There are two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®) and nivolumab (Opdivo®) and many more in development that can be used in combination with the anti-TIGIT antibodies of the invention.
[0730] Accordingly, the description provides the specific combinations of: CPA.9.083.H4 (S241P) (as shown in Figure 53B) with pembrolizumab; CPA.9.083.H4 (S241P) as shown in Figure 53B with nivolumab; CPA.9.086.H4 (S241P) as shown in Figure 53A with pembrolizumab; CPA.9.086.H4 (S241P) as shown in Figure 53A with nivolumab; CHA.9.547.7H4 (S241P) with pembrolizumab; CHA.9.547.7H4 (S241P) with nivolumab; CHA.9.547.13.H4 (S241P) with pembrolizumab and CHA.9.547.13.H4 (S241P) with nivolumab. (Reference is made to the sequence listing).
[0732] b. Anti-TIGIT antibodies in combination with anti-CTLA-4 antibodies
[0733] In another embodiment, the invention provides combinations of the anti-TIGIT antibodies of the invention and anti-CTLA-4 antibodies. There are two approved anti-CTLA-4 antibodies, ipilimumab (Yervoy®), and tremelimumab, as well as others in development, that can be used in combination with the anti-TIGIT antibodies of the invention.
[0735] Accordingly, the description provides the specific combinations of: CPA.9.083.H4 (S241 P) with ipilimumab; CPA.9.083.H4 (S241P) with tremelimumab; CPA.9.086.H4 (S241P) with ipilimumab; CPA.9.086.H4 (s 241 P) with tremelimumab; Ch A.9.547.7H4 (S241P) with ipilimumab; Ch A.9.547.7H4 (S241P) with tremelimumab; CHA.9.547.13.H4 (S241 P) with ipilimumab and CHA.9.547.13.H4 (S241 P) with tremelimumab.
[0737] c. Anti-TIGIT antibodies in combination with anti-PD-L1 antibodies
[0739] In another embodiment, the disclosure provides combinations of the disclosure's anti-TIGIT antibodies and anti-PD-L1 antibodies. There are three approved anti-PD-L1 antibodies, atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZITM), as well as other anti-PD-L1 antibodies in development, that can be used in combination with anti-PD-L1 antibodies. TIGIT from the description.
[0741] Accordingly, the invention provides the specific combinations of: CPA.9.083.H4 (S241P) with atezolizumab; CpA.9.083.H4 (S241P) with avelumab; CPA.9.083.H4 (S241P) with durvalumab; CPA.9.086.H4 (S241P) with atezolizumab; CPA.9.086.H4 (S241P) with avelumab; CPA.9.086.H4 (S241P) with durvalumab; CHA.9.547.7H4 (S241P) with atezolizumab; Ch A.9.547.7H4 (S241P) with avelumab; CHA.9.547.7H4 (S241 P) with durvalumab; CHA.9.547.13.H4 (S241P) with atezolizumab; CHA.9.547.13.H4 (S241P) with avelumab; and CHA.9.547.13.H4 (S241 P) with durvalumab.
[0743] d. Anti-TIGIT antibodies in combination with anti-LAG-3 antibodies
[0745] In another embodiment, the disclosure provides combinations of the anti-TIGIT antibodies of the invention and anti-LAG-3 antibodies. There are several anti-LAG-3 antibodies in development, including BMS-986016 (see International Patent Application No. WO2010 / 019570A2), GSK2831781 (see US Patent Application No.
[0746] 2016 / 0017037A), and the Merck clones 22D2, 11C9, 4A10, and / or 19E8 (see, WO2016 / 028672A1) and GSK2831781, as well as others in development, that can be used in combination with the anti-TIGIT antibodies of the invention .
[0748] Accordingly, the description provides the specific combinations of: CPA.9.083.H4 (S241P) with BMS-986016; CPA.9.083.H4 (S241 P) with GSK2831781; CPA.9.086.H4 (S241P) with BMS-986016; CPA.9.086.H4 (S241P) with GSK2831781; CHA.9.547.7H4 (S241P) with BMS-986016; CHA.9.547.7H4 (S241P) with GSK2831781; CHA.9.547.13.H4 (S241 P) with BMS-986016 and CHA.9.547.13.H4 (S241 P) with GSK2831781.
[0750] Accordingly, the description also provides the specific combinations of: CPA.9.083.H4 (S241P) with Merck clones 22D2, 11C9, and / or 4A10; CPA.9.086.H4 (S241P) with Merck clones 22D2, 11C9, and / or 4A10; CHA.9.547.7H4 (S241 P) with Merck clones 22D2, 11C9, and / or 4A10; CHA.9.547.13.H4 (S241 P) with Merck clones 22D2, 11C9, and / or 4A10.
[0752] and. Anti-TIGIT antibodies in combination with anti-TIM-3 antibodies
[0754] In another embodiment, the disclosure provides combinations of the anti-TIGIT antibodies of the invention and anti-TIM-3 antibodies. There is at least one anti-TIM-3 antibody in development, TSR-022, as well as others in development, that can be used in combination with the anti-TIGIT antibodies of the invention.
[0756] Accordingly, the description provides the specific combinations of: CPA.9.083.H4 (S241 P) with TSR-022; CPA.9.086.H4 (S241 P) with TSR-0226; CHA.9.547.7H4 (S241P) with TSR-022; and CHA.9.547.13.H4 (S241P) with TSR-022.
[0758] F. Anti-TIGIT antibodies in combination with anti-BTLA antibodies
[0760] In another embodiment, the description provides combinations of the anti-TIGIT antibodies of the invention and anti-BTLA antibodies, see, WO2011 / 014438, and particularly for the CDRs and full-length sequences of the anti-BTLA antibodies described therein. Accordingly, the description provides the specific combinations of: CPA.9.083.H4 (S241P) with an anti-BTLA antibody; CPA.9.086.H4 (S241P) with an anti-BTLA antibody; CHA.9.547.7H4 (S241P) with an anti-BTLA antibody; and CHA.9.547.13.H4 (S241P) with an anti-BTLA antibody.
[0762] g. Antibodies to TIGIT with antitumor antibodies
[0764] In some cases, the anti-TIGIT antibodies of the invention are co-administered with antibodies that, unlike immuno-oncology / checkpoint inhibitors that generally act on the immune system to increase the patient's native immune response, are instead directed against a tumor specific target antigen (TTA). There are a large number of well-approved or under development anti-TTA antibodies that can be combined with the present TIGIT antibodies. Currently approved antibodies include, but are not limited to, cetuximab, panitumumab, nimotuzumab (all versus EGFR), rituximab (CD20), trastuzumab and pertuzumab (HER2), alemtuzumab (CD52), bevacizumab (VEGF), ofatumumab (CD20) , denosumab (ligand of RANK), brentuximab (CD30), daratumumab (CD38), ibritumomab (CD20), and ipilimumab (CTLA-4). The target-specific oncology antibodies in clinical trials that can be combined with the anti-TIGIT antibodies herein include, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-1, such as nivolumab BMS-936558 / MDX-1106 / ONO-4538, AMP224, CT-011, MK-3475, anti-PDL-1 antagonists, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-H3, anti-VISTA; agonist antibodies directed at immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs, such as BMS-663513 urelumab (anti-4-1 BB; see, for example, US Patent Nos. 7,288,638 and 8,962,804); PF-05082566 utomilumab (see, for example, US Patent Nos. 8,821,867; 8,337,850; and 9,468,678, as well as International Patent Application Publication No. WO 2012/032433); anti-OX40 mAbs, such as anti-OX40 (see, for example, WO2006 / 029879 or WO2010096418); anti-GITR mAbs, such as TRX518 (see, for example, US Patent No. 7,812,135); Anti-CD27 mAbs, such as varlilumab CDX-1127 (see, for example, WO 2016/145085 and US Patent Publication Nos. US 2011/0274685 and US 2012/0213771), anti-ICOS mAb (for For example, MEDI-570, JTX-2011, and anti-TIM3 antibodies (see, for example, WO 2013/006490 or US Patent Application No. US 2016/0257758), as well as monoclonal antibodies against cancer of prostate, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphomas, lung cancers, including small cell lung cancer, kidney cancer, colorectal cancer, pancreatic cancer, gastric cancer, brain cancer, ( see, generally www.clinicaltrials.gov).
[0766] 3. Combination therapy of PVRIG and PD-1
[0768] As shown herein, the PVRIG antibodies of the invention can be combined with one of a number of checkpoint receptor antibodies.
[0770] to. Anti-PVRIG antibodies in combination with anti-PD-1 antibodies
[0772] In another embodiment, the invention provides combinations of the anti-PVRIG antibodies of the invention and anti-PD-1 antibodies.
[0774] In one embodiment, a tumor biopsy is taken from a cancer patient, and dissociated as is known in the art for FACS analysis. Cells are stained with labeled antibodies against (1) PVRIG (generally using CHA.7.518.1H4 (S241P), for example, although any indicated in WO2016 / 134333 can be used (specifically including any that bind, even if they do not block) or WO2017 / 041004)); (2) PD-1 (eg, using those known in the art, including EH12.2H7, Keytruda®, Opdivo®, etc.); (3) PD-L1 (for example, using those known in the art, such as BM-1 indicated herein) and (4) PVRL2 (for example, using those known in the art, such as TX11); and (5) an isotype control antibody. FACS is performed and, for each receptor, the percentage of cells expressing the receptor relative to the control antibody is calculated. If the percentage of cells positive for PVRIG, PD-1, PD-1, and PVRL2 is> 1% for all 4 receptors, then the patient is treated with antibodies to PVRIG and PD-1 as indicated herein.
[0776] There are two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®) and nivolumab (Opdivo®) and many more in development that can be used in combination with the anti-PVRIG antibodies of the invention.
[0778] Accordingly, the description provides the specific combinations of: CHA.7.518.1.H4 (S241P) (as shown in Figure 3) with pembrolizumab; CHA.7.518.1.H4 (S241 P) as shown in Figure 3 with nivolumab; CHA.7.538.1.2. H4 (S241 P) as shown in Figure 3 with pembrolizumab and CHA.7.538.1.2.H4 (S241 P) as shown in with nivolumab.
[0780] b. Anti-PVRIG antibodies in combination with anti-CTLA-4 antibodies
[0782] In another embodiment, the disclosure provides combinations of the anti-PVRIG antibodies of the invention and anti-CTLA-4 antibodies. There are two approved anti-CTLA-4 antibodies, ipilimumab (Yervoy®), and tremelimumab, as well as others in development, that can be used in combination with the anti-TIGIT antibodies of the invention.
[0784] Accordingly, the description provides the specific combinations of: CHA.7.518.1.H4 (S241P) with ipilimumab; CHA.7.518.1.H4 (S241p) with tremelimumab; CHA.7.538.1.2.H4 (S241P) with ipilimumab and Ch A.7.538.1.2.H4 (S241 P) with tremelimumab.
[0786] c. Anti-PVRIG antibodies in combination with anti-PD-L1 antibodies
[0788] In another embodiment, the disclosure provides combinations of the anti-PVRIG antibodies of the invention and anti-PD-L1 antibodies. There are three approved anti-PD-L1 antibodies, atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab, as well as other anti-PD-L1 antibodies in development, that can be used in combination with anti-TIGIT antibodies from the invention.
[0790] Accordingly, the description provides the specific combinations of: CHA.7.518.1.H4 (S241P) with atezolizumab; CPA.7518.1.H4 (S241P) with avelumab; CHA.7.518.1.H4 (S241P) with durvalumab; CHA.7.538.1.2.H4 (S241 P) with atezolizumab; CHA.7.538.1.2.H4 (S241 P) with avelumab and CHA.7.538.1.2.H4 (S241 P) with durvalumab.
[0792] d. Anti-PVRIG antibodies in combination with anti-LAG-3 antibodies
[0794] In another embodiment, the disclosure provides combinations of the anti-PVRIG antibodies of the invention and anti-LAG-3 antibodies. There are several anti-LAG-3 antibodies in development, including BMS-986016 (see WO2010 / 019570A2), GSK2831781 (see US Patent Application No. 2016 / 0017037A), and Merck clones 22D2, 11C9 , 4A10, and / or 19E8 {see, WO2016 / 028672A1) and GSK2831781, as well as others in development, which can be used in combination with the anti-PVRIG antibodies of the invention.
[0796] Accordingly, the description provides the specific combinations of: CHA.7.518.1.H4 (S241P) with BMS-986016; CHA.7.518.1.H4 (S241P) with GSK2831781; CHA.7.538.1.2.H4 (S241P) with BMS-986016 and CHA.7.538.1.2.H4 (S241 P) with GSK2831781.
[0798] Accordingly, the description also provides the specific combinations of: CHA.7.518.1.H4 (S241 P) with the Merck clones 22D2, 11C9, and / or 4A10 and CHA.7.538.1.2.H4 (S241P) with the clones from Merck 22D2, 11C9, and / or 4A10.
[0800] and. Anti-PVRIG antibodies in combination with anti-TIM-3 antibodies
[0802] In another embodiment, the disclosure provides combinations of the anti-PVRIG antibodies of the invention and anti-TIM-3 antibodies. There is at least one anti-TIM-3 antibody in development, TSR-022, as well as others in development, that can be used in combination with the anti-PVRIG antibodies of the invention.
[0804] Accordingly, the description provides the specific combinations of: CHA.7.518.1.H4 (S241P) with TSR-022 and CHA.7.538.1.2.H4 (S241 P) with TSR-0226.
[0806] F. Anti-PVRIG antibodies in combination with anti-BTLA antibodies
[0808] In another embodiment, the disclosure provides combinations of the anti-PVRIG antibodies of the invention and anti-BTLA antibodies, see WO2011 / 014438, and particularly for the CDRs and full-length sequences of the anti-BTLA antibodies disclosed therein. Accordingly, the description provides the specific combinations of: CHA.7.518.1.H4 (S241 P) with an anti-BTLA antibody and CHA.7.538.1.2.H4 (S241 P) with an anti-BTLA antibody.
[0810] g. Antibodies to PVRIG with antitumor antibodies
[0812] In some cases, the anti-PVRIG antibodies of the invention are co-administered with antibodies that, unlike immuno-oncology / checkpoint inhibitors that generally act on the immune system to increase the patient's native immune response, are instead directed against a tumor specific target antigen (TTA). There are a wide number of well-approved or developing anti-TTA antibodies that can be combined with the present antibodies to PVRIG, including CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P). Currently approved antibodies include, but are not limited to, cetuximab, panitumumab, nimotuzumab (all vs EGFR), rituximab (CD20), trastuzumab and pertuzumab (HER2), alemtuzumab (CD52), bevacizumab (VEGF), ofatumumab (CD20) , denosumab (RANK ligand), brentuximab (CD30), daratumumab (CD38), ibritumomab (CD20), and ipilimumab (CTLA-4). Target-specific oncology antibodies in clinical trials that can be combined with the anti-PVRIG antibodies herein include, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-1, such as nivolumab BMS-936558 / MDX-1106 / ONO-4538, AMP224, CT-011, MK-3475, anti-PDL-1 antagonists, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-H3, anti-VISTA; agonist antibodies directed at immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs, such as BMS-663513 urelumab (anti-4-1 BB; see, for example, US Patent Nos. 7,288,638 and 8,962,804); PF-05082566 utomilumab (see, for example, US Patent Nos. 8,821,867; 8,337,850; and 9,468,678, as well as International Patent Application Publication No. WO 2012/032433); anti-OX40 mAbs, such as anti-OX40 (see, for example, WO2006 / 029879 or WO2010096418); anti-GITR mAbs, such as TRX518 (see, for example, US Patent No. 7,812,135); anti-CD27 mAbs, such as varlilumab CDX-1127 (see, for example, Wo 2016/145085 and US Patent Publication Nos. US 2011/0274685 and US 2012/0213771), anti-ICOS mAb (for For example, MEDI-570, JTX-2011, and anti-TIM3 antibodies (see, for example, WO 2013/006490 or US Patent Publication No. US 2016/0257758), as well as monoclonal antibodies against cancer of prostate, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphomas, lung cancers including small cell lung cancer, kidney cancer, colorectal cancer, pancreatic cancer, gastric cancer, brain cancer, (see , generally www.clinicaltrials.gov).
[0814] 4. Combination therapy of PVRIG and TIGIT
[0815] There are specific combinations of anti-TIGIT and anti-PVRIG antibodies that find use in particular embodiments.
[0817] In one case, a tumor biopsy is taken from a cancer patient, and dissociated as is known in the art for FACS analysis. Cells are stained with labeled antibodies against (1) PVRIG (generally using CHA.7.518.1H4 (S241P), for example, although any indicated in WO2016 / 134333 can be used (specifically including any that bind, even if they do not block) or WO2017 / 041004)); (2) TIGIT (eg, using any described herein or others in the art, such as MBSA43); (3) PVR (eg, using those known in the art, such as SKII.4) and (4) PVRL2 (eg, using those known in the art, such as TX11); and (5) an isotype control antibody. FACS is performed and, for each receptor, the percentage of cells expressing the receptor relative to the control antibody is calculated. If the percentage of cells positive for PVRIG, TIGIT, PVR, and PVRL2 is> 1% for all 4 recipients, then the patient is treated with antibodies to PVRIG and TIGIT. Preferred combinations in this regard are CHA.7.518.1.H4 (S241 P) and CPA.9.086.
[0819] In one instance, antibodies containing the CDR sets of the anti-TIGIT CPA.9.086 antibody are combined with antibodies containing the CDR sets of the anti-PVRIG CHA.7.518.1 antibody. In a particular case, antibodies containing the VH and VL sequences of the anti-TIGIT CPA.9.086 antibody are combined with antibodies containing the VL and VL of the anti-PVRIG CHA.7.518.1 antibody. In one case, CPA.9.086.H4 (S241P) is combined as shown in Figure 53 with CHA.7.518.1 H4 (S241 P) as shown in Figure 3.
[0821] In one case, antibodies containing the CDR sets of the anti-TIGIT CPA.9.083 antibody are combined with antibodies containing the CDR sets of the anti-PVRIG CHA.7.518.1 antibody. In a particular case, antibodies containing the VH and VL sequences of the anti-TIGIT CPA.9.083 antibody are combined with antibodies containing the VL and VL of the anti-PVRIG CHA.7.518.1 antibody. In one case, CPA.9.086.H4 (S241P) is combined with CHA.7.518.1 H4 (S241 P).
[0823] In one instance, antibodies containing the anti-TIGIT CPA.9.086 antibody CDR sets are combined with antibodies containing the anti-PVRIG CHA.7.538.1.2.H4 (S241P) antibody CDR sets. In a particular case, antibodies containing the VH and VL sequences of the anti-TIGIT CPA.9.086 antibody are combined with antibodies containing the VL and VL of the anti-PVRIG antibody CHA.7.538.1.2.H4 (S241P). In one case, CPA.9.086.H4 (S241 P) is combined with CHA.7.538.1.2.H4 (S241 P).
[0825] In one case, CHA.518.1.H4 (S241 P) is combined with an anti-TIGIT antibody as listed in the sequence listing (with reference to all antibodies listed in Figure 4 of USSN 62 / 513.916), specifically CPA .9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.086, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA .9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560 .4, CHA.9.560.5, CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.546.1, CHA.9.547.2, CHA.9.547.3 , CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3. CHA.9.541.4. CHA.9.541.5, CHA.9.541.6. CHA.9.541.7 and CHA.9.541.8.
[0826] In one case, CPA.9.086 is combined with an anti-PVRIG antibody as outlined in WO2017 / 041004, including, but not limited to, those having a) a HC sequence of SEQ ID NO: 5 and LC sequence of SEQ ID NO: 3 (or the sets of CDRs contained therein) b) a HC sequence of SEQ ID NO: 32 and LC sequence of SeQ ID NO: 33 (or the sets of CDRs contained therein ); and c) a HC sequence of SEQ ID NO: 32 and an LC sequence of SEQ ID NO: 40 (or the sets of CDRs contained therein).
[0828] In some cases, the combination comprises an anti-TIGIT antibody selected from the group consisting of CPA.9.086, CPA.9.083, CHA.9.547.7, and CHA.9.547.13 and the antibody to PVRIG is selected from the group consisting in CHA7.518.1 and CHA.7.538.1.2. In some cases, the combination comprises an anti-TIGIT antibody selected from the group consisting of CPA.9.086, CPA.9.083, CHA.9.547.7, and CHA.9.547.13 and the antibody to PVRIG is CHA7.518.1. In some cases, the combination comprises an anti-TIGIT antibody selected from the group consisting of CPA.9.086, CPA.9.083, CHA.9.547.7, and CHA.9.547.13 and the antibody to PVRIG is CHA7.538.1.2 . In some cases, the combination comprises the anti-TIGIT CPA.9.086 antibody and the PVRIG CHA7.518.1 antibody. In some cases, the combination comprises the anti-TIGIT CPA.9.083 antibody and the PVRIG CHA7.518.1 antibody. In some cases, the combination comprises the anti-TIGIT CHA.9.547.7 antibody and the PVRIG CHA7.518 antibody. In some cases, the combination comprises the anti-TIGIT CHA.9.547.13 antibody and the PVRIG CHA7.518.1 antibody. In some cases, the combination comprises the anti-TIGIT CPA.9.086 antibody and the PVRIG CHA7.538.1.2 antibody. In some cases, the combination comprises the anti-TIGIT CPA.9.083 antibody and the PVRIG CHA7.538.1.2 antibody. In some cases, the combination comprises the anti-TIGIT CHA.9.547.7 antibody and the PVRIG CHA7.538.1.2 antibody. In some cases, the combination comprises the anti-TIGIT CHA.9.547.13 antibody and the PVRIG CHA7.538.1.2 antibody.
[0830] Figures 20-24 provide antibodies to PVRIG, as described in U.S. Patent Application No. 15 / 277,978, filed September 27, 2016. Antibodies to TIGIT herein The invention can be used in combination with antibodies to PVRIG as described in these figures, as well as those described throughout this application.
[0832] 5. Treatment evaluation
[0834] Generally, the antibodies of the invention, alone or in combination (PVRIG with PD-1, TIGIT with PD-1, or TIGIT with PVRIG) are administered to cancer patients, and efficacy is evaluated in several ways, as described in present memory. Thus, although standard efficacy tests can be performed, such as cancer load, tumor size, evaluation of the presence or degree of metastasis, etc., immuno-oncological treatments can also be evaluated on the basis of evaluations of immune status. This can be done in a number of ways, including both in vitro and in vivo testing. For example, assessment of changes in immune status (eg, presence of ICOS + CD4 + T cells after ipi treatment) can be done in conjunction with "traditional" measurements, such as tumor burden, size, invasiveness, involvement of LN, metastasis, etc. Thus, any or all of the following can be evaluated: the inhibitory effects of PVRIG or TIGIT on the activation or proliferation of CD4 + T cells, activation or proliferation of CD8 + T cells (CTL), cytotoxic activity mediated by CD8 + T cells, and / or CTL-mediated cell depletion, NK cell activity and NK-mediated cell depletion, the potentiating effects of PVRIG or TIGIT on Treg cell differentiation and proliferation, and Treg or myeloid-derived suppressor cell-mediated immunosuppression or immune tolerance ( MDSC), and / or the effects of PVRIG or TIGIT on the production of pro-inflammatory cytokines by immune cells, e.g. eg, IL-2, IFN-y, or TNF-a production by T cells or other immune cells.
[0836] In some embodiments, evaluation of treatment is done by evaluating proliferation of immune cells, using, for example, the CFSE dilution method, Ki67 intracellular staining of immune effector cells, and the 3H-Thymidine incorporation method.
[0838] In some embodiments, evaluation of treatment is done by evaluating increased gene expression or increased protein levels of markers associated with activation, including one or more of: CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surface expression of CD107A.
[0840] In some embodiments, evaluation of treatment is made by evaluating the amount of T cell proliferation in the absence of treatment, for example, prior to administration of the antibodies of the invention. If, after administration, the patient has an increase in T-cell proliferation, e.g. For example, a subset of the patient's T cells are proliferating, this is an indication that the T cells have been activated.
[0842] Similarly, evaluation of treatment with the antibodies of the invention can be done by measuring the patient's IFNy levels before administration and after administration to evaluate the efficacy of the treatment. This can be done in hours or days.
[0844] In general, gene expression assays are done as is known in the art. See, for example, Goodkind et al., Computers and Chem. Eng. 29 (3): 589 (2005), Han et al., Bioinform. Biol. Insights 11/15/15 9 (Suppl 1): 29-46, Campo et al., Nod. Pathol. 2013 Jan; 26 Suppl. 1: S97-S110.
[0846] In general, measurements of protein expression are also made in a similar manner as is known in the art, see, for example, Wang et al., Recent Advances in Capillary Electrophoresis-Based Proteomic Techniques for Biomarker Discovery, Methods. Mol. Biol. 2013: 984: 1-12; Taylor et al, BioMed Res. Volume 2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011 June 17: 722 (2): 171-182.
[0848] In some embodiments, the evaluation of the treatment is done by evaluating the cytotoxic activity measured by detecting the viability of the target cell through the estimation of numerous cellular parameters, such as enzymatic activity (including protease activity), permeability of the cell membrane. , cell adhesion, ATP production, coenzyme production, and nucleotide uptake activity. Specific examples of these assays include, but are not limited to, trypan blue or PI staining, 51Cr or 35S release method, LDH activity, MTT and / or WST assays, Calcein-AM assay, luminescence-based assay. , and others.
[0850] In some embodiments, evaluation of treatment is done by evaluating T cell activity as measured by cytokine production, measured intracellularly in the culture supernatant using cytokines including, but not limited to, IFNy, TNFa, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well known techniques.
[0852] Accordingly, evaluation of treatment can be done using assays evaluating one or more of the following: (i) increases in immune response, (ii) increases in ap and / or yS T cell activation, (iii) increases in the activity of cytotoxic T cells, (iv) increases in the activity of NK and / or NKT cells, (v) alleviation of suppression of ap and / or and S T cells, (vi) increases in secretion of pro-inflammatory cytokines, (vii) increases in IL-2 secretion; (viii) increases in the production of interferon-Y, (ix) increases in the Th1 response, (x) decreases in the Th2 response, (xi) decrease or elimination of the number and / or activity of cells of at least one of the regulatory T cells (Tregs).
[0854] Trials to measure efficacy
[0855] In some embodiments, T cell activation is assessed using a Mixed Lymphocyte Reaction (MLR) assay as described in the Examples. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0857] In one embodiment, the signaling pathway assay measures increases or decreases in immune response as measured for one example by phosphorylation or dephosphorylation of different factors, or by measuring other post-translational modifications. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0859] In one embodiment, the signaling pathway assay measures increases or decreases in activation of ap and / or y5 T cells as measured for one example by cytokine secretion or by proliferation or by changes in marker expression. activation, such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0860] In one embodiment, the signaling pathway assay measures increases or decreases in the activity of cytotoxic T cells as measured for an example by the direct death of target cells such as cancer cells or by the secretion of cytokines or by proliferation or by changes in the expression of activation markers such as, for example, CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0862] In one embodiment, the signaling pathway assay measures increases or decreases in the activity of NK and / or NKT cells as measured for an example by direct death of target cells such as cancer cells or by secretion of cytokines or by changes in the expression of activation markers such as, for example, CD107a, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0864] In one embodiment, the signaling pathway assay measures increases or decreases in suppression of ap and / or y5 T cells, as measured for one example by cytokine secretion or by proliferation or by changes in expression of activation markers, such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0865] In one embodiment, the signaling pathway assay measures increases or decreases in the secretion of pro-inflammatory cytokines as measured, for example, by ELISA or Luminex or by Multiplex bead-based methods or by intracellular staining and analysis by FACS or by Alispot etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0867] In one embodiment, the signaling pathway assay measures increases or decreases in IL-2 secretion as measured, for example, by ELISA or Luminex or by Multiplex bead-based methods or by intracellular staining and analysis by FACS or by Alispot etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0869] In one embodiment, the signaling pathway assay measures increases or decreases in interferon-Y production as measured, for example, by ELISA or Luminex or by Multiplex bead-based methods or by intracellular staining and analysis by FACS or by Alispot etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0871] In one embodiment, the signaling pathway assay measures increases or decreases in the Th1 response as measured for one example by cytokine secretion or by changes in the expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0873] In one embodiment, the signaling pathway assay measures increases or decreases in the Th2 response as measured for one example by cytokine secretion or by changes in the expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0875] In one embodiment, the signaling pathway assay measures increases or decreases in the cell number and / or activity of at least one regulatory T cell (T reg), as measured, for example, by flow cytometry or IHC. . A decrease in response indicates immunostimulatory activity. The appropriate decreases are the same as for the increases, listed below.
[0877] In one embodiment, the signaling pathway assay measures increases or decreases in M2 macrophage cell numbers, as measured, for example, by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity. The appropriate decreases are the same as for the increases, listed below.
[0879] In one embodiment, the signaling pathway assay measures increases or decreases in the protumorogenic activity of M2 macrophages, as measured for one example by cytokine secretion or by changes in the expression of activation markers. A decrease in response indicates immunostimulatory activity. The appropriate decreases are the same as for the increases, listed below.
[0881] In one embodiment, the signaling pathway assay measures increases or decreases in the increase of N2 neutrophils, as measured, for example, by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity. The appropriate decreases are the same as for the increases, listed below.
[0883] In one embodiment, the signaling pathway assay measures increases or decreases in the protumorogenic activity of N2 neutrophils, as measured for one example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity. The appropriate decreases are the same as for the increases, listed below.
[0885] In one embodiment, the signaling pathway assay measures increases or decreases in inhibition of T-cell activation, as measured for one example by cytokine secretion or by proliferation or by changes in the expression of markers of activation, such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0887] In one embodiment, the signaling pathway assay measures increases or decreases in inhibition of CTL activation as measured for an example by the direct death of target cells such as cancer cells or by the secretion of cytokines or by proliferation or by changes in the expression of activation markers, such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0889] In one embodiment, the signaling pathway assay measures increases or decreases in ap and / or yS T cell depletion as measured for one example by changes in the expression of activation markers. A decrease in response indicates immunostimulatory activity. The appropriate decreases are the same as for the increases, listed below.
[0891] In one embodiment, the signaling pathway assay measures increases or decreases in ap and / or yS T cell response as measured for one example by cytokine secretion or by proliferation or by changes in marker expression. activation, such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0892] In one embodiment, the signaling pathway assay measures increases or decreases in the stimulation of antigen-specific memory responses as measured for an example by cytokine secretion or by proliferation or by changes in the expression of activation markers. , such as CD45RA, CCR7 etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0894] In one embodiment, the signaling pathway assay measures increases or decreases in apoptosis or lysis of cancer cells as measured for an example by cytotoxic assays, such as, for example, MTT, Cr release, Calcin AM, or assays. based on flow cytometry such as CFSE dilution or propidium iodide staining, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0896] In one embodiment, the signaling pathway assay measures increases or decreases in the stimulation of the cytotoxic or cytostatic effect in cancer cells, as measured for an example by cytotoxic assays, such as, for example, MTT, Cr release, Calcin AM, or assays based on flow cytometry such as, for example, CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0898] In one embodiment, the signaling pathway assay measures increases or decreases in direct cancer cell death as measured for one example by cytotoxic assays, such as, for example, MTT, Cr release, Calcin AM, or based assays. in flow cytometry such as, for example, CFSE dilution or propidium iodide staining etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0900] In one embodiment, the signaling pathway assay measures increases or decreases in Th17 activity as measured for one example by cytokine secretion or by proliferation or by changes in the expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0902] In one embodiment, the signaling pathway assay measures increases or decreases in the induction of complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity, as measured for one example by cytotoxic assays, such as, for example , MTT, Cr release, Calcin AM, or flow cytometry based assays such as CFSE dilution or propidium iodide staining etc. A increased activity indicates immunostimulatory activity. Appropriate increases in activity are listed below.
[0904] In one embodiment, T cell activation is measured for example by the direct death of target cells such as cancer cells or by cytokine secretion or by proliferation or by changes in the expression of activation markers, such as CD137, CD107a, PD1, etc. For T cells, increases in proliferation, cell surface activation markers (e.g., CD25, CD69, CD137, PD1), cytotoxicity (ability to kill target cells), and cytokine production (p eg, IL-2, IL-4, IL-6, IFNy, TNF-a, IL-10, IL-17A) would be indicative of immune modulation that would be consistent with increased cancer cell death.
[0905] In one embodiment, NK cell activation is measured, for example, by direct death of target cells such as cancer cells or by cytokine secretion or by changes in the expression of activation markers such as , CD 107a, etc. For NK cells, increases in proliferation, cytotoxicity (ability to kill target cells and increases in CD107a, granzyme, and perforin expression), cytokine production (eg, I FNy and TNFa), and the expression of receptors on the cell surface (eg, CD25) would be indicative of immune modulation that would be consistent with increased death of cancer cells.
[0907] In one embodiment, T and S cell activation is measured, for example, by cytokine secretion or by proliferation or by changes in the expression of activation markers.
[0909] In one embodiment, Th1 cell activation is measured, for example, by cytokine secretion or by changes in the expression of activation markers.
[0911] Appropriate increases in activity or response (or decreases, as appropriate, as noted above), are increases of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60% , 70%, 80%, 90%, 95% or 98 to 99% percent on the signal either in a reference sample or in control samples, for example, test samples that do not contain an anti-PVRIG antibody of the invention . Specific increases in activity are depicted in Figures 27 to 34. For example, with respect to increases in T cell proliferation, CHA.7.518.1.H4 (S241P) shows an increase of approximately 60% and CHA.7.538.1.2.H4 (S241P) shows an increase of 47%; relevant increases are shown well in T or IFN-cell proliferation and from about 10 to 70%, also finding use of about 20 to 60%.
[0913] Similarly, increments of at least one, two, three, four, or five times compared to control or control samples show efficacy.
[0915] XIII. Examples
[0917] A. Example 1: surface plasmon resonance studies of the binding of PVR, PVRL2, and PVRL3 to PVRIG, DNAM, and TIGIT
[0919] Materials and methods
[0921] All experiments were performed using a ProteOn XPR 36 instrument at 22 ° C.
[0923] Step 1: A high-density goat anti-human fc polyclonal antibody surface (Invitrogen H10500) was prepared on six lanes of a GLC chip using a ProteOn XPR 36 biosensor. The activation step for the surface of anti-human fc occurred in the direction of horizontal flow while the immobilization stage for high-density pAb occurred in the direction of vertical flow. The blocking stage occurred in both the vertical and horizontal positions, so that the horizontal "midpoints" could be used as reference surfaces. An average of ~ 4,400 RU of goat anti-human pAbs were immobilized in each lane.
[0925] Step 2: For each cycle, three different lots of human PVRIG fusion protein (human fc, GenScript lots 451, 448, 125), human DNAM-1 fusion protein (human fc, R&D Systems), fusion protein of Human TIGIT (human fc, R&D Systems), and a human control IgG (Synagis) were each captured on a different vertical lane for two minutes at a concentration of 2 pg / mL. PVR, two batches of PVRL2, and PVRL3 were each injected in the horizontal flow direction at six different concentrations on the six captured ligands at different ligand capture cycles. The injections were for two minutes followed by 10 minutes of dissociation at a flow rate of 50pL / min. The concentration range of PVR was 1.4nM-332nM in a 3-fold dilution series, both batches of PVRL2 were injected at a concentration range of 1.3nM-322nM in a 3-fold dilution series, and PVRL3 was injected at a concentration range of 1.4nM-334nM in a 3-fold dilution series. All protein reagents were prepared in run buffer which was degassed PBS buffer with the addition of 0.05% Tween 20 and 0.01% BSA. Capture surfaces of anti-human fc were regenerated with two 30 second pulses of 146 mM phosphoric acid after each cycle.
[0927] Step 3: The sensorgram data of the binding of the analytes to each captured ligand was processed and referenced twice using ProteOn Manager version 3.1.0.6 using the midpoint reference and a pre-target injection identical to the analyte injections.
[0928] Results
[0930] a) PVR: Binds weakly to captured DNAM-1 and TIGIT and shows no binding to the three batches of PVRIG and to the control IgG. Not enough information was generated to estimate the KD of PVR interactions with DNAM-1 and TIGIT (data not shown).
[0932] b) PVRL2: both lots of PVRL2 showed binding to the three lots of PVRIG and DNAM-1, but minimal or absent binding to TIGIT and no binding to control IgG. Sensorgrams showed complex kinetics, therefore binding constants could not be estimated (data not shown).
[0934] c) PVRL3: showed minimal binding to TIGIT and did not bind to the other proteins (data not shown).
[0936] B. Example 2: effect of inactivation of PVRIG (KD) and anti-PVRIG antibody on the function of human melanoma-specific TIL
[0938] The objective of these assays is to evaluate the functional ability of PVRIG in human derived TILs, as measured by cytokine secretion and activation markers, after co-culturing with target melanoma cells.
[0939] 1. Example 2 (1):
[0941] The effect of the anti-PVRIG antibody (CP A.7.021), which has been shown to block the interaction of PVRIG and PVRL2, alone or in combination with other antibodies (eg, anti-TIGIT, anti-DNAM1), was evaluated . PD1 was used as a reference immune checkpoint for the inactivation studies (siRNA).
[0943] Materials and Methods: TIL: tumor infiltrating lymphocytes (TIL) from three patients with melanoma were used (1) TIL-412-HLA-A2 specific for Mart1, (2) TIL-F4-HLA-A2 specific for gp100, and ( 3) TIL-209- HLA-A2 specific for gp100. The TILs were thawed in complete IMDM medium (BI, 01-058-1A) supplemented with 10% human serum (Sigma, H3667) 1% Glutamax (Life technologies, 35050-038) 1% Pyruvate-Na (Biological Industries , 03-042-1B) 1% non-essential amino acids (Biological Industries, 01-340-1B) 1% Pen-Strep (Biological Industries, 03 031 -1B) 300 U / ml rhIL2 (Biolegend, 509129).
[0945] Tumor cell lines: Mel-624 human melanoma cells express the MART-1 and gp-100 antigens in the context of the MHC-I HLA-A2 halotype. Cells were cultured in complete DMEM medium supplemented with 10%, 25 mM HEPES buffer, 1%, and 1% Pen-Strep.
[0947] Inactivation in TILs: Inactivation (KD) of human PVRIG and human PD1 in TILs was done using 100 pmoles of human PVRIG siRNA Dharmacon ON-TARGETplus-SMARTpool (L-032703-02) or human PD1 siRNA-SMARTpool ( L-004435) or undirected siRNA (D-001810-01-05). The siRNAs were electroporated in the TILs (AMAXA, program X-005). Electroporation was done on resting TILs cultured in complete IMDM supplemented with IL-2 24hr after thawing. After electroporation, the TILs were seeded in a 96-well TC plate for 24hr recovery. After 24 hr, cells were harvested and stained with viability staining agent (BD Horizon; Cat. No. 562247, BD biosciences), washed with PBS, and stained with anti-human PVRIG-CPA.7.021 ( CPA.7.021 IgG2 A647, 7.5ug / ml) or with anti-human PD-1 (Biolegend, # 329910 AF647, 5ug / ml) at room temperature for 30min. The isotype control used is synagis (IgG2 A647, 7.5ug / ml) and mouse IgG1 (Biolegend # 400130 A647, 5ug / ml), respectively. All samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using FlowJo software (v10.0.8).
[0949] Coculture of TILs with 624 melanoma cells: TILs electroporated with siRNA were harvested and seeded in a 96-well TC plate at 5x104 / well. Mel-624 cells were also harvested and seeded in 1: 1/1: 3 E: T ratios in coculture. The plate was incubated overnight (18hr) at 37 ° C, 5% CO2.
[0951] To evaluate the effect of the anti-PVRIG (CPA.7.021), anti-TIGIT (Clone 10A7; from Genentech US Patent Application No. US 2009/0258013) and anti-DNAM1 (clone DX11, described first place in Shibuya et al Immunity Volume 4, Issue 6, June 1, 1996, Pages 573-581; BD Biosciences; Mouse anti-human DNAM-1 Clone DX11, Cat. No. 559787) on TIL activity Specific to melanoma, TILs (1x105 cells / well) were pre-incubated with either assayed antibodies or relevant isotype controls in monotreatment (10jg / mL) or in combination treatment (final 10jg / mL for each) before addition of target cells. melanoma 624 at a 1: 1 ratio Effector: Diana. The plate was incubated overnight (18hr) at 37 ° C, 5% CO2.
[0953] Assessment of TIL activation: 16 hours after coculture, cells were stained with viability staining agent (BD Horizon; Cat. No. 562247, BD biosciences), washed with PBS, and exposed to blocking solution of Fc (Cat. No. 309804, Biolegend), followed by surface staining with anti-CD8a (Cat. No. 301048, Biolegend) and anti-CD137 (Cat. No. 309804, Biolegend) at 4 ° C for 30min. All samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using Flow Jo software (v10.0.8). Culture supernatants were collected and analyzed for cytokine secretion by the CBA kit (Cat.
[0954] 560484, BD).
[0956] Results
[0957] PVRIG inactivation in TILs: TIL MART-1 and TIL F424 hr were cultured with IL-2. 100 pmoles of human PVRIG siRNA ON-TARGETplus-SMART pool (L-032703-02) or human PD1 siRNA-SMARTpool (L-004435) or undirected siRNA (D-001810-01-05) were electroporated in the T il (AMAXA, program X-005). Detection of PVRIG or PD-1 was performed 24 hr after electroporation (and before coculture). Cells were stained with the viability staining agent followed by a 30 min incubation at RT with anti-PVRIG or anti PD-1. The percent population KD is indicated in Figure 82 of USSN 15 / 048,967, incorporated by reference herein.
[0959] Functional assay using inactivated TILs: Human TILs, cultured for 24 hours with IL2 were electroporated with siRNA encoding human PVRIG or PD-1 or a random sequence as a control. TILs were assayed for expression of PVRIG and PD-1 24 hr after electroporation. ~ 80% inactivation of PVRIG and ~ 50% inactivation of PD-1 were observed compared to TILs electroporated with random sequences, as demonstrated in Figure 82 of USSN 15 / 048,967, incorporated by reference herein. memory.
[0960] TIL KDs were cultured with Mel-624 cells at 1: 1 or 1: 3 E: T for 18hr and stained for CD137 expression. Elevated levels of the activation marker CD137 were shown in TIL MART-1 electroporated with PVRIG siRNA, similarly to TILs electroporated with PD-1 siRNA, compared to control random sequence siRNA (as demonstrated in Figure 83A of USSN 15 / 048,967, incorporated by reference herein). The culture supernatant was collected and assayed for the presence of secreted cytokines. TILs that were electroporated with PVRIG siRNA show a significant increase in IFNy and TNF levels compared to SCR control siRNA. A similar effect was shown in TILs that were electroporated with PD-1 siRNA (as demonstrated in USSN 15 / 048,967 Figure 83B-C, incorporated by reference herein).
[0962] The same trend of increase in activation levels was observed in TIL F4. Coculture of PVRIG siRNA electroporated TIL F4 with Mel-624 at 1: 3 E: T resulted in increased levels of CD137 surface expression, as well as increased IFNy secretion in the culture supernatant as shown in Figure 84A and 84B of USSN 15 / 048,967, incorporated by reference herein. Similar trends were seen in TILs that were electroporated with PD-1 siRNA.
[0964] Functional assay using Ab blockers:
[0966] In vitro monotherapy and combination therapy with anti-PVRIG and anti-TIGIT: TIL 209 were cultured with Mel-624 cells at 1: 1 E: T for 18hr. The culture supernatant was collected and assayed for the presence of secreted cytokines. Anti-TIGIT treatment did not affect IFNy or TNF secretion levels. However, an increase in IFNy and TNF levels was observed when anti-TIGIT and anti-PVRIG were added to the coculture in combination (Figure 8A-B).
[0968] In vitro monotherapy and combination therapy with anti-PVRIG and anti-TIGIT: TIL 209 were cultured with Mel-624 cells at 1: 1 E: T for 18hr. The TILs were stained for expression on the surface of the activation marker CD 137 and showed a reduced level of expression after anti-DNAM-1 treatment. The supernatant from the cocultures was collected and assayed for the presence of secreted cytokines. Anti-DNAM-1 treatment mediated an upward trend in secreted cytokines IFNy and TNF. Treatment with anti-DNAM-1 and anti-PVRIG in combination partially reversed the effect on CD137 expression (Figure 9C) and increased the effect on IFNy and TNF cytokine secretion (Figure 9A-B). TIL MART-1 were cultured with Mel-624 cells at 1: 1 E: T for 18hr. The supernatant from the cocultures was collected and assayed for the presence of secreted cytokines. Treatment with anti-DNAM-1 reduced the surface expression of CD137 in the TILs and also the secreted cytokines IFNy and TNF. Treatment with anti-DNAM-1 and anti-PVRIG in combination partially reversed these effects (Figure 9D-F).
[0970] Summary and Conclusions: PD1 KD enhanced TIL activity as measured by IFNy secretion in TIL F4 and MART-1. An increase (~ 20%) of IFNy and TNF secretion on PVRIG KD was observed in TlL MART-1 compared to control siRNA. The same trend was observed in the expression of CD137 after coculture with melanoma 624 cells in TlL F4.
[0972] Anti-TIGIT treatment did not affect IFNy or TNF secretion levels from TlL cocultured with Mel 624, however, an increase in IFNy and TNF levels was observed when anti-TIGIT and anti-PVRIG (CPA .7.021) to combination coculture.
[0974] Treatment with anti-DNAM-1 reduced the activation of TIL-MART-1 manifested by CD137 and secretion of reduced cytokines and anti-PVRIG (CPA.7.021) could partially reverse this effect in the treatment in combination with Ab against DNAM- one. In TlL 209, IFNy and TNF secretion levels were slightly elevated (~ 10%) with anti-DNAM-1, and an increase in IFNy and TNF levels (~ 40% and 30%, respectively) was observed when anti-DNAM1 and anti-PVRIG (CPA.7.021) were added to the coculture in combination. Collectively, our results showed that PVRIG is a new co-inhibitory receptor for PVRL2.
[0976] 2. Example 2 (2):
[0977] The effect of additional anti-PVRIG antibodies (CHA.7.518.1.H4 (S241P); CHA.7.524; CHA.7.530; CHA.7.538), which has been shown to block the interaction of PVRIG and PVRL2, alone or in combination with other antibodies (eg, anti-TIGIT, PD1) on the activity of TIL-209, TIL-412, and TIL-463-F4 after co-culturing with the 624 melanoma cell line.
[0979] The functional antibodies used in this assay were anti-hPVRIG (core mIgG1 core) hybridoma Ab -CHA.7.518.1.H4 (S241 P); CHA.7,524; CHA.7,530; CHA.7.538 (ml lot # 30816); anti-hTIGIT (core mIgG1) -clone 10A7 (Genescript), anti-TIGIT clone MBSA43 (e-biosciences) and mIgG1 (cat no. 400166, clone m OpC-21, Biolegend).
[0980] TIL and mel 624 Coculture: TILs were thawed and cultured as described at 2.1 24 hr prior to coculture. The Ab tested were added in monotreatment (10ug / mL) or in combination with anti-TIGIT (20ug / mL) to seeded TIL and incubated (in total 100uL) for 1 hr at 37 ° C, 5% CO2. Mel-624 cells were harvested and seeded in a 1: 3 Efector: Target ratio in coculture with the TILs. The plate was incubated overnight (18hr) at 37 ° C, 5% CO2.
[0982] Evaluation of the functional capacity of the TILs: the activity of the T cells was evaluated on the basis of the detection of IFNy in the supernatants of the cocultures. The culture supernatants were harvested and assayed for cytokines by a CBA kit (Cat. No. 560484, BD) or by a human IFNY / TNFa MAGPIX kit. Two-tailed unpaired T-tests were calculated. P <0.05 was referred to as statistically significant.
[0983] Results
[0985] Functional assay using TIL and melanoma cells in the presence of anti-PVRIG hybridoma Ab: human TILs, cultured for 24 hours with IL2, were co-cultured with Mel-624 cells at 1: 3 E: T for 18hr and assayed for cytokine secretion. Figure 31 depicts a representative experiment of 5-6 performed. TILs were cocultured with melanoma 624 cells in the presence of anti-TIGIT or anti-PVRIG Ab (blue) or in combination of anti-TIGIT and anti-PVRIG (green) and assayed for IFNy / TNF secretion. In this experiment, the 4 monotherapy treatments with anti-PVRIG Ab increased (20-30%) the secretion of I FNy in 2 out of 3 TILs tested (TIL-209 and TIL463-F4), while in combination with anti-TIGIT, all of the Ab anti-PVRIG CHA.7.518.1.H4 (S241P), CHA.7.530, CHA.7.538 increased secretion of I FNy compared to treatment with anti-TIGIT alone.
[0986] The effect of Ab CHA.7.518.1.H4 (S241P) was found to be statistically significant throughout the experiments on TIL 463-F4-gp100 in 5 experiments as monotherapy and in combination with anti-TIGIT (Figure 9E, G ). The effect of the combination treatment of anti-PVRIG CHA.7.518.1.H4 Ab (S241P) was also statistically significant in TIL 209 (Figure 9C). The effect of anti-PVRIG CHA.7.538 Ab combination treatment was found to be statistically significant in TIL 463-F4-gp100 (Figure 9F).
[0988] Summary and Conclusions: In the experimental systems described herein, we observed an effect of anti-PVRIG on TILs in response to target melanoma cells as observed by changes in I FNy secretion. The anti-PVRIG hybridoma Ab tested mediated an increase in I FNy secretion compared to the relevant isotype control. CHA.7.518.1.H4 Ab (S241P) appears to have an advantage in mediating an increase in I FNy secretion as a monotherapy and compared to other aPVRIG Ab tested, however, the magnitude of these effects varies between different TIL. This effect is increased in combination with anti-TIGIT treatment.
[0990] 3. Example 2 (3):
[0992] The objective is to evaluate the functional activity of human anti-PVRIG antibodies (CHA.7.518.1.H4 (S241 P); CHA.7.544; 0 CHA.7.538) on the activity of human TILs after coculture with CHO-S cells pulsed with the peptide that stably co-express HLA-A2, microglobulin b2 (B2M) and PVRL2.
[0994] TIL of resected metastases from three patients with melanoma were used: TIL-412-HLA-A2 specific for Mart1 (26-35), TIL-463-F4-HLA-A2 specific for gp100 (209-217), TIL-463- F5-HLA-A2 specific for gp100 (209-217), and TIL-209-HLA-A2 specific for gp100 (209-217).
[0996] The TILs were thawed in complete IMDM medium supplemented with human serum 10% Glutamax 1% Pyruvate-Na 1% non-essential amino acids 1% Pen-Estrep 300 U / ml rhIL2 (Biolegend, 589106).
[0997] CHO-S cells (target cells) were stably transduced with a lentivirus expressing HLA-A2 / B2M (lentivirus vector cat. No. CD515B-1 -SBI, system biosciences) and grown under selection with 600ug / ml of hygromycin B in CD CHO medium (Cat. No. 10743-011) supplemented with GlutaMax 8mM 1% and Pen / Strep 1%. Cells expressing HLA-A2 / B2M were then cloned by limiting dilution. Clone 3E8 with high expression of HLA-A2 and B2M was then transduced with a lentivirus expressing human PVRL2 (lentivirus vector cat. No. CD510B-1 -SBI, system biosciences), and grown under selection with 6ug / ml of puromycin.
[0999] In the experimental system described herein (represented in Figure 35), TIL reagents for gp100 or MART-1 that endogenously express TIGIT, DNAM-1 and PVRIG Figure 37) were co-cultured with CHO-S h La - cells. A2 / B2M / PVRL2 pulsed with the peptide.
[1000] The functional antibodies used in this assay were human anti-PVRIG; Ab 461 (Aldeveron) -referred to 544 in this example, Ab anti-human PVRIG chimera (hIgG4 core) -CHA.7,538; CHA.7.518 (referred to c538 and c518 in this example, which means that the variable heavy and light regions of 7.538 and 7.518 were fused with constant regions of human IgG4 anti-human TIGIT (core mIgG1) clone MBSA43 (e-biosciences), mIgG1 (biolegend) and hIgG4 (biolegend).
[1002] The TILs were thawed and cultured as described herein for 24hr prior to co-culturing with the target cells. The tested antibodies were added in monotreatment (10ug / mL) or in combination with anti-TIGIT (total 20ug / mL) to seeded TIL and incubated (in total 100uL) for 30 min at 37 ° C, 5% CO2. The CHO-S cells were harvested and pulsed with 0.1 or 0.5ug / ml of gp-100 (gp100209-217) or with 20ug / ml of MART-126-35 peptides, for 1 hour at 37 ° C in Opti-MEM ™ medium with reduced serum. After three washes with serum-reduced Opti-MEM ™ medium, peptide pulsed target cells were co-cultured overnight (18hr) with TIL at an Efector: Target ratio of 1: 3 (33k: 100k).
[1004] Evaluation of the functional capacity of TILs: the effect of anti-PVRIG antibodies (10ug / ml) as monotherapy or as combination treatment with anti-TIGIT on the activity of TILs was evaluated using the measurement of cytokine secretion of supernatants from overnight cocultures using the Combined Bead Array (CBA) kit (Cat. No. 560484, BD). All samples were acquired on a MACSQuant analyzer (Miltenyi) and data was analyzed using Flow Jo software (v10.0.8).
[1006] Dose response of anti-PVRIG antibodies: the effect of the dose response of anti-PVRIG antibodies c518, c538 (or hIgG4 isotype control) was tested in the described assay with an antibody concentration of 30, 10, 3, 1, 0.3, 0.1 and 0.03ug / ml. Two-tailed unpaired T-tests were calculated. P <0.05 was referred to as statistically significant.
[1008] Results
[1010] Effect of anti-PVRIG antibodies on TIL activity after coculture with CHO-S HLA-A2 / B2M cells expressing PVRL2: the effect of three anti-PVRIG antibodies (544, c538 and c518) on the activity of four Different TILs (412, 463, 462 and 209) from two different experiments are summarized in Figure 37. Ab served as the non-blocking control Ab. The detailed results of the experiments are presented in Figure 39. Treatment with antibodies 544, c538 and c518 increased the levels of IFN secretion from TILs (on average 6%, 28% and 23%, respectively) in comparison with the isotype antibody treatment. Increased IFN secretion was detected in TILs treated with c538 or c518 compared to 544, the non-blocking control. No significant difference was found between treatments with Ab c538 to c518. Treatment with anti-TIGIT increased IFN secretion from TILs (on average 49%) compared to isotype. Combination treatment of c518 and c538 with anti-TIGIT induced an additive effect on IFN secretion from TILs, but the effect of the combination was not statistically significant compared to TIGIT monotherapy treatment.
[1012] Effect of the dose response of anti-PVRIG antibodies on the functional capacity of TILs: the effect of the addition of anti-PVRIG antibodies (c538 and c518) on the dose response on the activity of TILs was evaluated F4 and 209 (figure 80). The EC50 of antibodies c518 and c538 is in the single nM digit compared to the isotype control as measured by the effect of TNFα secretion from TILs.
[1014] Summary and conclusions: in the experimental system described in this report, we observed an effect of anti-PVRIG antibodies on TIL activity in response to coculture with CHO-S HLA-A2 / B2M target cells pulsed with peptides that overexpress PVRL2. . The anti-PVRIG antibodies that were tested mediated increased secretion of IFN and TNF from the TILs compared to the relevant isotype control. Antibodies c518 and c538 have a statistically significant advantage (p-0.0063 and p-0.0034, respectively) in TIL activity, as manifested by IFN secretion, compared to 544, which is an antibody non-blocking of PVRIG (based on competition experiment performed on cells expressing PVRIG). Both c518 and c538 antibodies had an additive effect with anti-TIGIT antibody (not statistically significant).
[1016] 4. Example 2 (4)
[1018] The objective of this example was to evaluate the functional capacity of PVRIG in human-derived TILs, as measured by cytokine secretion after coculture with target melanoma cells. The effect of anti-PVRIG antibodies (Ch A.7.518.1.H4 (S241P); CHA.7.524; CHA.7.530; CHA.7.538), which has been shown to block the interaction of PVRIG and PVRL2, alone, was evaluated or in combination with other antibodies (eg, anti-TIGIT, PD1).
[1019] Purified CD3 + T cells were obtained using the Rossetesep Human T Cell Enrichment Mix Kit (Stem cell technologies) in blood buffy coat samples. The cells were thawed and marked with CFSE (Moleculare probes) to be able to follow the proliferation in the coculture.
[1021] CHO-S-OKT3 cells: CHO-S cells were transduced with CD5L-OKT3-scFv-CD14 into CD710B-1 (SBI, cat no. CS965A-1, lot no. 151014-005, 1.40x108 ifus / ml). Cells were cultured in the presence of CD ChO (Gibco, life technologies Cat. No. 10743-011) with the addition of 8mM GlutaMax and 6pg / ml puromycin. The Surface levels of OKT3 by flow cytometry using PE-anti-F (ab) '2 goat mouse IgG at a 1: 200 dilution (Jackson Immunoresearch, cat no. 115-116-146). CHO-S-OKT3 cells were transiently transfected with human PVRL2 (delta isoform) or empty vector using the Amaxa electroporation system (Lonza, Walkersville, MD, USA) according to the manufacturer's instructions. 5ug of plasmid pcDNA3.1 (empty vector or hPVRL2) were used per 2X106 cells in Ingenio ™ Electroporation Solution (Mirus, Cat. No. MC-MIR-50115) and pulse program U-024. Expression of PVRL2 in transfected CHOS-S-OKT3 cells was assessed by flow cytometry using anti-PVRL2 Ab (cat. No. 337412, Biolegend).
[1023] The functional antibodies used in this assay were anti-hPVRIG (core mIgG1 core) hybridoma Ab -CHA.7.518.1.H4 (S241 P); CHA.7,524; CHA.7,530; CHA.7.538, anti-TIGIT clone MBSA43 (e-biosciences) and mIgG1 (cat # 400166, clone MOPC-21, Biolegend).
[1025] Coculture of CD3 T cells and CHO-OKT3 cells: CD3 + T cells were thawed and immediately labeled with CFSE. In parallel, CHO-S-OKT3-PVRL2 cells were harvested and treated with Mitomycin-C for 1 hr at 37 ° C, washed and added to the coculture with T cells in 1: 5 E: T (1x105 T cells and 2x104 CHO-OKT3-PVRL2 or sham). The Ab were added in monotreatment (10ug / mL) or in combination with anti-TIGIT (10ug / mL) and the coculture plates were incubated at 37 ° C, 5% CO2 for 5 days. After 5 days, the cells were harvested and the proliferation of the T cells was analyzed by FACS selecting for the CD4 and CD8 subpopulations.
[1027] Effect of anti-PVRIG antibodies in the CHOS-OKT3 coculture assay: CFSE-labeled T cells were stimulated with stimulator cells (CHO cells expressing membrane-bound anti-CD3 mAb fragments). CHOS stimulator cells expressing human PVRL2 and control stimulator cells (empty vector) were treated with mitomycin C (50ug / ml for 1h) before co-culturing with CFSE-labeled human T cells at the ratio of 1: 5. After 5 days at 37 ° C and 5.0% CO2, the effect of anti-PVRIG antibodies (10ug / ml) on T cell proliferation (CFSE dilution) and cytokine secretion ( ELISA or CBA TH1 / 2/17 kits) in the culture supernatants. All samples were acquired on a MACSQuant analyzer (Miltenyi) and data was analyzed using FlowJo software (v10.0.8). Culture supernatants were collected and analyzed for cytokine secretion by the CBA kit (Cat. No. 560484, BD).
[1029] Results
[1031] Effect of anti-PVRIG antibodies on PVRL2 overexpression in the CHOS-OKT3 assay: CHOS-OKT3 cells overexpressing PVRL2 or mock (empty vector) were co-cultured with CD3 + cells and the effect of anti-PVRIG antibodies was tested as monotherapy or in combination with anti-TIGIT in the proliferation of T cells and in the secretion of cytokines (Figure 40). After 5 days, cells were harvested and analyzed by CFSE dilution. In parallel, the supernatant from the co-cultures was collected and assayed for cytokine secretion. Figure 41 shows the effect of anti-PVRIG Ab in responding versus non-responding donor. The effect of various anti-PVRIG Ab on T cell proliferation was evaluated as a single treatment in combination with anti-TIGIT. Although some anti-PVRIG Ab increases T cell proliferation, an additive effect with anti-TIGIT antibody was not observed in this system (Figure 42). These effects were not observed when Ab were assayed in coculture of CD3 + cells with sham CHO-S cells (transfected with empty vector) (data not shown).
[1033] A total of 10 donors were tested and 5 out of 10 donors responded to anti-PVRIG Ab. Treatment of Ab CHA.7.518.1.H4 (S241P) consistently resulted in increased secretion of I FNy that varied between 20-50% in the 5 responding donors tested, while treatment with other Ab did not demonstrate an clear trend (Figure 43). Similar effects were observed on the proliferation of CD8 + cells. The effect of Ab treatment is summarized in Figure 44.
[1035] C. Example 3: effect of anti-PVRIG antibody on human melanoma-specific TIL function in combination with anti-TIGIT and anti-PD1 antibodies
[1037] 1. Example 3 (1):
[1039] Materials and methods
[1041] TIL: tumor infiltrating lymphocytes (TIL) from three patients with melanoma were used: (1) TIL-412-HLA-A2 specific for Mart1, (2) TIL-F4-HLA-A2 specific for gp10ü and (3) TIL- 209-HLA-A2 specific to gp100.
[1043] The TILs were thawed in complete IMDM medium (BI, 01-058-1A) supplemented with 10% human serum (Sigma, H3667) 1% Glutamax (Life technologies, 35050-038) 1% Pyruvate-Na (Biological Industries , 03-042-1B) 1% non-essential amino acids (Biological Industries, 01-340-1B) 1% Pen-Strep (Biological Industries, 03 031 -1B) 300 U / ml rhIL2 (Biolegend, 509129).
[1045] Tumor cell lines: Mel-624 human melanoma cells express the MART-1 and gp-100 antigens in the context of the MHC-I HLA-A2 halotype. Cells were cultured in complete DMEM medium (Biological Industries, 01-055-1A) supplemented with 10% FBS (BI, 04-127-1A), 25 mM HEPES buffer (BI, 03-025-lB), Glutamax al 1% (Life technologies, 35050-038), and Pen-Estrep 1% (Biological Industries, 03-031-1B).
[1046] Coculture of TIL with 624 melanoma cells in the presence of anti-PVRIG, anti-TIGIT and anti-PD1 blocking antibodies: to evaluate the effect of anti-PVRIG (CPA.7.021), anti-TIGIT (Clone 10A7) and anti- PD1 (mAb 1B8, Merck) in the activity of melanoma-specific TILs, TILs (3x104 cells / well) were pre-incubated with the tested antibodies or relevant isotype controls in monotreatment (10pg / mL) or in combination treatment (final 10pg / mL for each) prior to addition of 624 target melanoma cells at a 1: 3 Effector: target ratio. The plate was incubated overnight (18hr) at 37 ° C, 5% CO2.
[1048] Evaluation of TIL activation: culture supernatants were collected and analyzed for cytokine secretion by the CBA kit (Cat. No. 560484, BD).
[1050] In vitro anti-PVRIG monotherapy and combination therapy with blocking anti-TIGIT and PD1 antibodies: TIL F4 (gp100 specific) were cultured with Mel-624 cells at 1: 3 E: T for 18 hrs. The supernatant from the cocultures was collected and assayed for the presence of secreted cytokines. Anti-TIGIT or anti-PD1 treatment did not affect IFNy or TNF secretion levels. However, an increase in IFNy and TNF levels was observed when anti-TIGIT or anti-PD1 in combination with anti-PVRIG were added to the combination coculture (Figure 10A-B).
[1051] The treatment of anti-PVRIG, anti-TIGIT and PD1 alone did not affect the secretion levels of I FNy or TNF from the coculture of TIL with Mels 624, however, an increase in the levels of I FNy and TNF was observed when anti-TIGIT or anti-PD1 antibodies were added in combination with anti-PVRIG (CP A.7.021). The data presented suggest that there is a synergistic effect for combination therapy with anti-TIGIT or anti-PD1 antibodies.
[1053] 2. Example 3 (2):
[1055] Again, the ability of anti-PVRIG antibodies to increase CD4 + and CD8 + T cell function in combination with an anti-TIGIT antibody was evaluated in an in vitro primary cell-based assay.
[1057] CHO-S OKT3 Assay: The CHO-S OKT3 Assay was used to determine whether the combination of a humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), and a commercially available anti-TIGIT antibody could increase T-cell proliferation, and cytokine secretion rather than a single anti-PVRIG or anti-TIGIT antibody treatment. The target cells used in the coculture assay were the Chinese hamster ovary cell line, CHO-S (ATCC), which stably overexpressed the single chain variable fragment of the human anti-CD3 antibody Clone OKT3 (abbreviated as OKT3) , and human PVRL2 (abbreviated as hPVRL2). Parental CHO-S OKT3 cells were grown in serum-free CD-CHO medium supplemented with 40mM glutamax, penicillin / streptomycin, and 6pg / ml puromycin. CHO-S OKT3 hPVRL2 cells were grown in serum-free CD-CHO medium supplemented with 40mM glutamax, penicillin / streptomycin, 6pg / ml puromycin, and 600 pg / ml hygromycin B.
[1059] Primary CD3 + and CD8 + T cells were isolated from healthy human donors using the RosetteSep ™ human CD3 + T cell enrichment mix (Stemcell Technologies), and human CD8 + microbeads (Miltenyi Biotec), respectively, and frozen in liquid nitrogen. On the day of the coculture assay, CD3 + or CD8 + T cells were thawed, counted, and labeled with 1pM CFSE (Life Technologies) for 10 minutes at 37 ° C. After this incubation, the T cells were washed and resuspended in complete medium containing RPMI, supplemented with 10% heat inactivated FBS, glutamax, penicillin / streptomycin, non-essential amino acids, sodium pyruvate, and 50pM p-mercaptoethanol. CHO-S OKT3 hPVRL2 cells were harvested from culture, and treated with mitomycin C for 1 hour at 37 ° C with periodic mixing. After incubation, the target cells were thoroughly washed, counted, and resuspended in complete RPMI medium. The assay was established with a 5: 1 ratio of T cells (100,000) to target cells (20,000). Target cells, T cells, and 10pg / ml from each antibody treatment were added together to a 96-well U-bottom plate (Costar), and incubated well for 3 days (CD8 + T cells), or 5 days ( CD4 + T cells) at 37 ° C. Antibody treatments included human IgG4 CHA.7.518.1.H4 (S241P) alone, a human IgG4 isotype control combined with mouse anti-human TIGIT (Clone MBSA43, eBioscience), and a combination of CHA.7.518. 1.H4 (S241P) and anti-TIGIT (Clone MBSA43). Furthermore, the activity of mouse anti-human DNAM-1 IgG1 (Clone DX11, BioLegend), mouse IgG1 isotype control (Clone MOPC21, BioLegend), and a human IgG4 isotype control were also evaluated.
[1061] After the 3 or 5 day incubation period, the co-culture supernatants were analyzed for secreted cytokines, including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL- 13, IL-17A, IL-17F, IL-21, IL-22, TNFa, and IFNy, with the Cytometric Bed Array (CBA) Human Th1 / Th2 / Th17 Cytokine Kit (BD Biosciences), or with the LEGENDplex ™ Human Th Cytokine Kit (BioLegend). T cell proliferation was measured by CD4 + or CD8 + T cell staining with the LIVE / DEAD fixable aqua cell stain kit (ThermoFisher Scientific), anti-CD4 antibody (Clone RPA-T4, BioLegend), and anti-CD4 antibody. CD8 (Clone HIT8a, BioLegend), and selecting the percentage of proliferating live CD4 + or CD8 + T cells, with low CFSE. Data were acquired using a Canto II FACS (BD Biosciences), and analyzed using Flow Jo (Treestar) and Prism (Graphpad) software.
[1063] Results: Combination of CHA.7.518.1.H4 (S241P) and an Anti-TIGIT Antibody Increases Proliferation of CD4 + T Cells Compared to Single Antibody Treatments: Antibody Capability Against Humanized Hybridoma-derived PVRIG , CHA.7.518.1.H4 (S241P), to increase the proliferation of primary CD4 + T cells in vitro when combined with an anti-TIGIT antibody was evaluated with the CHO-S OKT3 assay.
[1064] Figures 33A and B show the percentage of proliferating CD4 + T cells from two different donors in response to co-culturing with target CHO-S OKT3 hPVRL2 cells, and treated with anti-PVRIG and anti-TIGIT antibodies either alone or in combination. In these two representative human donors of CD3 + T cells, the combination of CHA.7.518.1.H4 (S241 P) and anti-TIGIT antibody increases the proliferation of CD4 + T cells compared to CHA.7.518.1.H4 ( S241P) alone, or the combination of the IgG4 isotype and the anti-TIGIT antibody. The anti-DNAM-1 antibody reduces the proliferation of CD4 + T cells compared to the IgG1 isotype control in both donors.
[1066] CHA.7.518.1.H4 (S241 P) and an anti-TIGIT antibody increase CD8 + T cell proliferation and IFN-g secretion. Figure 34A illustrates the ability of the humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), to increase the proliferation of CD8 + T cells in combination with the anti-TIGIT antibody in the CHO-S OKT3 assay. In a representative human donor of CD8 + T cells, the combination of CHA.7.518.1.H4 (S241P) and anti-TIGIT antibody increases the proliferation of CD8 + T cells when T cells are co-cultured with CHO-S OKT3 cells. hPVRL2. The combination of anti-PVRIG and anti-TIGIT antibodies increases proliferation more than CHA.7.518.1.H4 (S241P) alone, or the hIgG4 isotype plus treatment with the anti-TIGIT antibody. Figure 34B shows that in the same representative human CD8 + T cell donor as described above, the humanized PVRIG antibody, CHA.7.518.1.H4 (S241P), in combination with the anti-TIGIT antibody also increases secretion. IFNy in the CHO-S OKT3 assay. The combination of anti-PVRIG and anti-TIGIT antibodies increases I FNy secretion more than CHA.7.518.1.H4 (S241P) alone, or the hIgG4 isotype plus anti-TIGIT antibody treatment. Anti-DNAM-1 antibody reduces both CD8 + T cell proliferation and I FNy production compared to the IgG1 isotype control antibody.
[1068] Summary and conclusions
[1070] Together, the humanized PVRIG antibody, CHA.7.518.1.H4 (S241 P) and the anti-TIGIT antibody had functional in vitro activity in the primary CHO-S OKT3 cell-based assay. The combination of CHA.7.518.1.H4 (S241 P) and the anti-TIGIT antibody resulted in increased proliferation of CD4 + and CD8 + T cells, as well as secretion of I FN and CD8 + T cells compared to treatment with either CHA.7.518.1.H4 (S241P) or the anti-TIGIT antibody alone. Together, these data demonstrate that coblocking of the two checkpoint receptors, PVRIG and TIGIT, increased T-cell function compared to blocking of a single receptor.
[1072] It should be noted that TIGIT does not interact with CD112 (PVRL2; see Figures 4E and 4F of Zhu et. Al., J. Exp. Med. (2016): 1-10); instead, it interacts with PVR, a different ligand. PVR is expressed in the CHO / CD112 system of Zhu et al. Therefore, our interpretation of the effect of the combination of aCD112R (anti-PVRIG antibody) and anti-TIGIT is that aCD112R / aPVRIG is blocking the interaction of human CD112R with human CD112, but the anti-TIGIT antibody is blocking the interaction of TIGIT human with human or hamster PVR (in T cells or CHO cells), Zhu et al do not really provide a hypothesis as to why the effect of the anti-CD112R / anti-TIGIT combination is occurring in the CHO CD112 assay. That is, the effect of the combination is not through the PVRL2 / CD112 ligand alone.
[1074] D. Example 4: Epitope Mapping of Human Anti-PVRIG Antibodies Based on Cynomolgus Cross-reactivity
[1076] Rational basis and objectives
[1078] The objective of this study is to identify the epitopes in the PVRIG protein that determine the cross-reactivity of human anti-PVRIG antibodies against the cynomolgus (cyno) monkey ortholog. Many of the antibodies to the human PVRIG target show varying degrees of cross-reactivity with cyno despite the fact that many of these antibodies belong to the same class of epitopes. To shed light on the molecular basis of human / cyno cross-reactivity (or the lack thereof), various cyno-to-human mutations of the recombinant PVRIG proteins were designed, expressed and purified, and assayed for binding to a panel of anti-human PVRIG antibodies in ELISA.
[1080] Methods
[1082] Design of the cyno-to-human variants of PVRIG: The alignment of human and PVRIG ECD sequences shows 90% sequence identity and 93% sequence homology between the human and cyno orthologs. Based on the nature of the mutations (conserved versus non-conserved) and the prediction of the secondary structure (spiral versus extended) of the region of the mutations, three site-directed mutants of cyno PVRIG were designed to test the cyno cross-reactivity focused on epitope mapping. These mutants include H61R, P67S, and L95R / T97I from cyno PVRIG. Wild-type human and cyno PVRIGs were also generated.
[1084] Expression and purification of cyno, human, and hybrid PVRIG variants: All PVRIG variants were expressed as ECD fusions with a C-terminal 6XHis tag in mammalian cells. Proteins were purified by affinity purification, ion exchange chromatography, and size exclusion chromatography. The purified protein buffer was exchanged to PBS buffer (pH 7.4) and stored at 4 ° C.
[1085] ELISA to determine PVRIG-antibody interaction: Functional ELISA was performed as follows: PVRIG of cyno, human, and hybrid cyno / human recombinant proteins (His-tagged) were adsorbed on an IA plate overnight at 4 ° C. The coated wells of the plate were washed twice with PBS and incubated with 300 pL of blocking buffer (5% skimmed milk powder in PBS pH 7.4) at room temperature (RT) for 1 hr. The blocking buffer was removed and the plates were washed two more times with PBS. Plate-bound PVRIG variants were incubated with anti-human PVRIG mAb (human igG1 isotype) in solution (linear range 0.1 pg / mL to 8 pg / mL in a volume of 50 pL / well) at RT for 1 hr. Plates were washed three times with PBS-T (PBS 7.4, 0.05% Tween20), then three times with PBS and 50pL / well of a HRP-conjugated secondary antibody (specific human IgG Fc domain, Jackson ImmunoResearch). This was incubated at RT for 1 hr and the plates were washed again. ELISA signals were developed in all wells by adding 50 pL of Sureblue TMB substrate (KPL Inc) and incubating for 5-20 min. The HRP reaction was stopped by adding 50 pL of H2SO42N (VWR) and the absorbance signals at 450 nm were read on a SpectraMax (Molecular Devices) or EnVision (PerkinElmer) spectrophotometer. The data was exported to Excel (Microsoft) and plotted in GraphPad Prism (GraphPad Software, Inc.).
[1087] Results
[1089] Residues S67, R95, and I97 as determinants of cvno cross-reactivity: The binding data shown in Figure 18 clearly shows that residues S67, R95, and I97 affect the cyno cross-reactivity of various antibodies. Although the cyno-to-human P67S mutation negatively impacts the binding of CPA.7.002 and CPA.7.041, the cyno-to-human L95R / T97I mutation significantly improves the binding of CPA.7.002, CPA.7.021, CPA.7.028, and CPA .7.041. On the other hand, the mutation from cyno to human H61R does not affect the binding of any of the antibodies tested.
[1091] Relative binding of cyno to human variants suggests three groups of epitopes: Relative binding of antibodies to cyno, human, and hybrid PVRIG variants suggests 3 distinct epitope groups: Group 1 binds to residues R95 / I97 (CPA.7.021 and CPA.7.028). Group 2 binds to residues S67 and R95 / I97 (CPA.7.002 and CPA.7.041). Group 3 does not bind to residues S67 or R95 / I97 (CPA.7.024 and CP A.7.050). The epitope clusters show a strong correlation in the degree of cyno cross-reactivity of these antibodies (Figure 19).
[1093] Summary and conclusions: Restricted epitope mapping based on cyno-to-human variations in the PVRIG ECD identified residues S67, R95, and I97 as determinants of cyno cross-reactivity of anti-human PVRIG antibodies. The complete restoration of the binding to PVRIG of cyno L95R / T97I for the antibodies CPA.7.021 and CPA.7.028 and the improved binding of CP A.7.002 to this mutant strongly suggests that residues R95 and I97 are critical epitopes of human PVRIG for these antibodies. These findings also suggest a possible way to predict cross-reactivity to non-human primate PVRIG orthologs in their primary amino acid sequence.
[1095] E. Example 5: humanized antibodies: Analysis of receptor-ligand binding and blocking of humanized anti-PVRIG hybridoma-derived antibodies, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P )
[1097] This experiment was performed to characterize the binding of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) to human and cynomolgus PVRIG protein in cell lines and primary leukocytes, to characterize the ability to CHA.7.518.1.H4 (S241P) and Ch A.7.538.1.2.H4 (S241P) to block the interaction between PVRIG and PVRL2 and to characterize the epitope space of CHA.7.518.1.H4 (S241P) and CHA .7.538.1.2.H4 (S241P) relative to each other, evaluating competition for binding to PVRIG antigen expressed in Jurkat cells.
[1099] FACS analysis of hPVRIG overexpressing cells: The following cell lines were used to assess the specificity of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P): parental HEK and hPVRIG overexpressing HEK cells . These cells were cultured in DMEM (Gibco) 10% fetal calf serum (Gibco) glutamax (Gibco). For HEK cells that overexpress hPVRIG, 0.5ug / ml puromycin (Gibco) was also added to the medium for positive selection. For FACS analysis, all cell lines were picked at the log phase of growth and 50,000-100,000 cells were seeded per well in 96-well plates. The binding of unconjugated CHA.7.518.1.H4 (S241 P) and c Ha .7.538.1.2.H4 (S241 P) (hIgG4) and their respective controls was evaluated in an 8-point titration series starting at 10ug / ml on ice for 30 min-1 hr. The titration series was performed as 3-fold serial dilutions. Unconjugated primary antibodies were detected with Alexa 647-conjugated anti-human Fc (Jackson Laboratories). Data was acquired using a FACS Canto II (BD Biosciences), FACS LSR Fortessa X-20 (BD Biosciences), or IntelliCyt (IntelliCyt Corporation) and analyzed using FlowJo (Treestar) and Prism (Graphpad) software.
[1101] FACS analysis of human cell lines to determine hPVRIG: The following cell lines were used to evaluate the expression and specificity of Ch A.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P): Jurkat cells and HepG2. Jurkat cells were cultured in RPMI medium 10% fetal calf serum, glutamax, non-essential amino acids (Gibco), sodium pyruvate (Gibco), and penicillin / streptomycin (Gibco). HepG2 cells were cultured in DMEM 10% glutamax fetal calf serum. For FACS analysis, all cell lines were picked at the log phase of growth and 50,000-100,000 cells were seeded per well in 96-well plates. The binding of unconjugated CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) (hIgG4) and their respective controls are evaluated in an 8-point titration series starting at 10ug / ml on ice for 30 min-1 hr. Unconjugated primary antibodies were detected with Alexa 647 conjugated anti-human Fc antibody. The titration series was performed as 3-fold serial dilutions. Data was acquired using a Canto II or IntelliCyte FACS and analyzed using FlowJo and Prism software.
[1103] FACS analysis of PVRIG in CMV-expanded CD8 T cells: CMV-reactive donors were purchased from Cellular Technology Limited (CTL). Supplied PBMCs were pulsed for 2 hours with 10uM CMV peptide 494-503 (NLVPMVATV, Anaspec). The PBMCs were subsequently washed three times, after which they were seeded in 24-well plates for 9 days in RPMI 10% human AB serum (Sigma), glutamax, penicillin / streptomycin, and a cytokine growth mixture consisting of 2ng / ml of IL-2 (R&D systems) and 10ng / ml of IL-7 (R&D systems). After 9 days, non-adherent cells were harvested, phenotyped for CD8 T cell enrichment, and stored in liquid nitrogen.
[1105] To assess expression in CMV-expanded CD8 T cells, the binding of unconjugated CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241 P) (hIgG4) and their respective controls were assessed in a 8-point titration series starting at 666nM on ice for 30 min-1 hr. The titration series was performed as a 4-fold serial dilution series. Unconjugated primary antibodies were detected with Alexa 647-conjugated anti-human Fc antibody. Data was analyzed using FlowJo and Prism software and collected on a Fortessa X-20 BD LSR.
[1106] FACS analysis of cells engineered to overexpress cynomolgus PVRIG: The following cell lines were used to assess the cross-reactivity of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) with PVRIG of cynomolgus (cPVRIG): parental expi and expi cells that overexpress cPVRIG. These cells were cultured in DMEM 10% glutamax fetal calf serum. Expi cells transiently overexpressing cPVRIG were generated by electroporating cPVRIG DNA into parental expi cells using the Neon transfection system. For FACS analysis, expi cPVRIG cells were used 1-3 days after transfection. Parental expi cells were harvested in the log phase of growth. 50,000-100,000 cells per well of each type were seeded in 96-well plates. The binding of unconjugated CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.h4 (S241P) (hIgG4) and their respective controls was assessed in an 8-point titration series starting at 10ug / ml on ice for 30 min-1 hr. The titration series was performed as a 3-fold dilution series. Unconjugated primary antibodies were detected with an Alexa 647-conjugated anti-human Fc antibody. Data were acquired using a Canto II or IntelliCyte FACS and analyzed using FlowJo and Prism software.
[1108] Cell-based receptor-ligand blocking assays: the ability of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241 P) to inhibit the interaction of PVRIG with its ligand PVRL2 in an assay cell competition performed in two orientations.
[1110] In the first orientation, PVRL2 is expressed endogenously in unmanipulated HEK cells, and the ability of CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) to block binding was measured. of soluble biotinylated PVRIG Fc to HEK cells. More specifically, the biotinylated PVRIG Fc protein (33nM) and CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241P) (1.03-198 nM, hIgG4) were added concomitantly at 100,000 He K cells and incubated for 1 hour on ice. The degree of binding of biotinylated PVRIG Fc was subsequently detected by the addition of streptavidin Alexa 647 (Jackson Laboratories) for 20-30 minutes on ice. Cells were washed twice in PBS for acquisition using a Canto II FACS. The data were analyzed using Flow Jo, Excel (Microsoft), and Prism.
[1112] In the second orientation, HEK cells were engineered to express human PVRIG (HEK hPVRIG) and the ability of CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) ( hIgG4) to inhibit soluble human PVRL2 Fc. More specifically, HEK hPVRIG cells were preincubated with CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) (0.66-66nM) for 30 min on ice, after which time it was added PVRL2 mFc (human PVRL2 with a mouse Fc) (for 1 hr on ice) and its ability to bind HEK hPVRIG was measured. The extent of PVRL2 mFc binding was detected by subsequent addition of goat mouse anti-Fc A647 (Jackson Laboratories) for 20-30 min on ice. Cells were washed twice in PBS for acquisition using a Canto II FACS. The data were analyzed using FlowJo, Excel and Prism.
[1114] Cell-based epitope spatial analysis: The epitope spacing for CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) was assessed for their ability to compete with each other for binding to Jurkat cells. Briefly, Jurkat cells were harvested in the log phase of growth and stained with □□□ g / ml of unlabeled CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) for 30 min. in ice. Jurkat cells were subsequently centrifuged, washed, and counterstained with □□ g / ml of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) labeled with Alexa 647 for 30 min on ice. . The competition of the labeled antibodies for the binding of PVRIG with unmarked antibodies in Jurkat cells was assessed by the magnitude of the Alexa 647 signal by flow cytometry. Data was acquired using a Canto II FACS and analyzed using FlowJo, Excel, and Prism.
[1116] Results
[1117] CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) Recognize PVRIG in Overexpressing Cells, Jurkat Cells, and Human T Cells: The Capability of Humanized CHA Hybridoma-Derived PVRIG Antibodies. 7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) for binding to human PVRIG was evaluated using HEK cells that overexpress human PVRIG, Jurkat cells, and primary T cells. Figure 20 illustrates the specificity of both CHA.7.518.1.H4 (S241P) (A) and CHA.7.538.1.2.H4 (S241P) (B). Both antibodies bind highly specifically to HEK hPVRIG cells, and do not bind to parent HEK cells.
[1119] Binding affinities: both CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) also bind to HEK hPVRIG cells with high affinity with their associated Kd values: 0.29 nM for CHA .7.518.1.H4 (S241P) and 0.86 nM for CHA7.538.1.2 for binding to HEK hPVRIG cells.
[1121] Figure 21 illustrates the ability of CHA.7.518.1.H4 (S241 P) (A) and CHA.7.538.1.2.H4 (S241 P) (B) to bind to Jurkat cells that endogenously express PVRIG. Both are capable of binding to Jurkat cells with comparable affinity to HEK hPVRIG cells.
[1123] The affinity of these antibodies for Jurkat cells are 0.15 nM for CHA.7.518.1.H4 (S241P) and 0.59 nM for CHA.7.538.1.2.H4 (S241 P).
[1125] Figure 22 illustrates the ability of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) to bind to CD8 T cells that were expanded by exposure to the CMV peptide (494-503, NLVPMVATV ) and endogenously expressing PVRIG.
[1127] CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) detect cynomolgus PVRIG (cPVRIG) expressed in expi cells: the ability of CHA.7.518.1.H4 (S241P) and c Ha .7.538.1.2.H4 (S241P) of binding to cPVRIG was assessed using expi cells that overexpress cPVRIG. Figure 23 illustrates the specificity of both CHA.7.518.1.H4 (S241P) (A) and CHA.7.538.1.2.H4 (S241 P) (B). Both antibodies bind highly specifically to expi cPVRIG cells, and do not bind to parent expi cells. Both CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) also exhibit high affinity binding to expi cPVRIG cells with their associated Kd values of 0.24 nM for CHA.7.518.1. H4 (S241 P) and 0.58 nM for CHA7.538.1.2.
[1129] Cell-based receptor-ligand blocking assays: The ability of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241 P) to inhibit the interaction of PVRIG with PVRL2 was evaluated in two orientations, as it is indicated in the protocols section. In the first permutation, both CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241 P) were able to completely inhibit the binding of PVRIG Fc to HEK cells (Figure 24A). The CI5u values associated with this blocking ability are 15 nM for CHA.7.518.1.H4 (S241P) and 16.1 nM for CHA.7.538.1.2.H4 (S241 P). Importantly, not all antibodies derived from the hybridoma campaign that were confirmed to bind PVRIG were able to block the binding of PVRIG Fc to HEK cells. As shown in Figure 24B, an antibody clone designated CHA.7.544 is unable to block the binding of PVRIG Fc to HEK cells.
[1131] In the second permutation, both CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) were also able to completely inhibit the binding of PVRL2 Fc to HEK hPVRIG cells (Figure 25A). The IC50 values associated with this inhibition are 1.8 nM for CHA.7.518.1.H4 (S241 P) and 2.53 nM for Ch A.7.538.1.2.H4 (S241 P). Although the ability of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.h 4 (S241P) were able to completely inhibit PVRL2 Fc binding in this permutation, consistent with their ability to inhibit PVRIG binding Fc in the first permutation, other antibodies did not show this same trend. More specifically, another humanized hybridoma-derived antibody, CHA.7.530.3, which was able to completely inhibit PVRIG Fc binding to HEK cells (first permutation, data not shown), was not able to completely inhibit PVRL2 binding. Fc to HEK hPVRIG cells (Figure 25A). Collectively, these data indicate that the second permutation of the cell-based receptor ligand blocking assay is able to distinguish the potency of receptor-ligand blocking antibodies with more sensitivity compared to the first permutation. Importantly, CHA.7.544 was shown to be unable to block the binding of PVRL2 Fc to HEK hPVRIG cells (Figure 25 B) consistent with its inability to block the binding of PVRIG Fc to HEK cells.
[1133] Cell-based epitope spatial analysis: As indicated in the protocols section, an analysis of the epitope space of CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) was performed evaluating their capacity to compete for joining PVRIG. Figure 26 shows the ability of unconjugated versions of the antibodies to inhibit the binding of Alexa 647 (A647) conjugated versions of the same antibodies. The data in Figure 26 represent the percent binding of A647-conjugated antibodies to maximum signal that yield without competition. The signal rendered by CHA.7.518.1.H4 (s 241P) A647 and CHA.7.538.1.2.H4 (S241P) A647 was not affected by preincubation of Jurkat cells with isotype control (data not shown). As expected, the signal rendered by CHA.7.518.1.H4 (S241P) A647 and CHA.7.538.1.2.H4 (S241P) A647 was significantly reduced when competing with unconjugated versions of themselves (data not shown). Interestingly, after analysis of the A647 signal from CHA.7.518.1.H4 (S241 P) and Ch A.7.538.1.2.H4 (s 241 P) in the context of preincubation with the unconjugated version of the antibody On the contrary, there was also a significant reduction. This indicates that CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (s 241 P) may share a similar epitope space in endogenously expressed PVRIG.
[1134] Summary and Conclusions: Mouse versions of anti-PVRIG antibodies designated CHA.7.518 and CHA.7.538 were successfully humanized into a human IgG4 isotype (CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P)) that retained binding properties against human PVRIG antigen. Using engineered overexpression cells, Jurkat, and CMV-expanded primary CD8 T cells, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) were shown to be highly specific for PVRIG endogenous human and bound with high affinity. In addition, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) also showed reactivity against the cyno PVRIG antigen and bound to overexpressing cells with high affinity. Functionally, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) were able to inhibit the interaction of PVRIG with PVRL2 in FACS-based assays. Finally, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) were shown to potentially share an epitope space in endogenous human PVRIG, due to their ability to compete with each other for binding to Jurkat cells.
[1136] F. Example 6: Humanized Antibodies: Functional Analysis of Humanized Antibodies
[1138] The functional activity of several humanized antibodies of the invention was validated.
[1140] CHO-S OKT3 Assay: The CHO-S OKT3 Assay was used to determine if the humanized PVRIG antibodies, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (s 241 P), could increase the proliferation of CD4 + and CD8 + T cells, and the secretion of cytokines. The target cells used in the coculture assay were the Chinese hamster ovary cell line, CHO-S (ATCC), which stably overexpress the single chain variable fragment of the human anti-CD3 antibody Clone OKT3 (abbreviated as OKT3) , or stably overexpressing both OKT3 and human PVRL2 (abbreviated as hPVRL2). Parental CHO-S OKT3 cells were grown in serum-free CD-CHO medium (Gibco) supplemented with 40mM glutamax (Gibco), penicillin / streptomycin (Gibco), and 6g / ml puromycin (Gibco). The CHO-S OKT3 hPVRL2 cells were grown in the same CD-CHO medium as the parental cells, but also supplemented with 600g / ml of hygromycin B (Gibco).
[1142] Primary CD4 + and CD8 + T cells were isolated from healthy human donors using the RosetteSep ™ human CD4 + T cell enrichment mix (Stemcell Technologies), and human CD8 + microbeads (Miltenyi Biotec), respectively, and frozen in liquid nitrogen. On the day of the coculture assay, CD3 + or CD8 + T cells were thawed, counted, and labeled with 1jM CFSE (Life Technologies) for 10 minutes at 37 ° C. After this incubation, T cells were washed and resuspended in complete medium containing RPMI (Gibco), supplemented with 10% heat inactivated FBS, glutamax, penicillin / streptomycin, non-essential amino acids (Gibco), sodium pyruvate ( Gibco), and 50jM p-mercaptoethanol (Gibco). CHO-S OKT3 and CHO-S OKT3 hPVRL2 cells were harvested from culture, and treated with mitomycin C (Sigma-Aldrich) for 1 hour at 37 ° C with periodic mixing. After incubation, the target cells were thoroughly washed, counted, and resuspended in complete RPMI medium. The assay was established with a 5: 1 ratio of T cells (100,000) to target cells (20,000). Target cells, T cells, and 10 µg / ml from each antibody treatment were added together to a 96-well U-bottom plate (Costar), and incubated well for 3 days (CD8 + T cells), or 5 days ( CD4 + T cells) at 37 ° C. Antibody treatments against PVRIG included human IgG4 CHA.7.518.1.H4 (S241 P), IgG4 CHA.7.538.1.2.H4 (S241P), human IgG4 CHA.7.530.3 (receptor / ligand partial blocking antibody) , and mouse IgG1 CHA.7.544 (non-receptor / ligand-blocking antibody). In addition to the antibodies against PVRIG, the activity of mouse anti-human DNAM-1 (Clone DX11, BioLegend), mouse IgG1 isotype control (Clone MOPC21, BioLegend), and an IgG4 isotype control were also evaluated. human. For antibody dose titrations, 3-fold dilutions of 66nM to 0.264nM of the antibodies against PVRIG, and the respective isotype control antibody were used.
[1144] After the 3 or 5 day incubation period, the co-culture supernatants were analyzed for secreted cytokines, including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL- 13, IL-17A, IL-17F, IL-21, IL-22, TNFa, and IFNy, with the Human Cytometric Bed Array (CBA) Th1 / Th2 / Th17 Cytokine Kit (BD Biosciences), or with the LEGENDplex ™ Human Th Cytokine Kit (BioLegend). T cell proliferation was measured by staining CD4 + or CD8 + T cells with the LIVE / DEAD fixable aqua stain kit (ThermoFisher Scientific), anti-CD4 antibody (Clone RPA-T4, BioLegend), and anti-CD8 antibody. (Clone HIT8a, BioLegend), and selecting the percentage of proliferating CD4 + or CD8 + T cells alive, with low CFSE. Data were acquired using a Canto II FACS (BD Biosciences), and analyzed using Flow Jo (Treestar) and Prism (Graphpad) software.
[1146] Results
[1148] CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 ('S241 P) increase proliferation of CD4 + T cells in an hPVRL2-dependent manner: the ability of hybridoma-derived antibodies to PVRIG humanized, c Ha .7.518.1.H4 (S241 P) and CHA.7.538.1.2. H4 (S241 P), to increase the proliferation of primary CD4 + T cells in vitro was evaluated with the CHO-S OKT3 assay. Figure 27A shows the percentage of proliferating CD4 + T cells from a representative donor in response to coculture with CHO-S OKT3 hPVRL2 target cells and different antibodies against PVRIG. In this donor, the humanized antibodies CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241 P) increase the proliferation of CD4 + T cells compared to the human IgG4 isotype control (dotted line ). The receptor / ligand partial blocking antibody, human IgG4 CHA.7.530.3, only weakly increases T cell proliferation, whereas the non-blocking receptor / ligand antibody, mouse IgG1 CHA.7.544 has no effect compared to isotype control antibodies. The antibody anti DNAM-1 reduces the proliferation of CD4 + T cells. Figure 27B demonstrates that the effects of the humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), and the anti-DNAM-1 antibody depend on overexpression of hPVRL2 in target cells. After treatment with the antibodies c Ha .7.518.1.H4 (S241 P) and CHA.7.538.1.1, a greater increase in the proliferation of C D4 + T cells is observed when CD4 + T cells are co-cultured with CHO cells -S OKT3 hPVRL2, compared to co-culture with parental CHO-S OKT3 cells. Similarly, anti-DNAM-1 antibody only decreases proliferation of CD4 + T cells when T cells are co-cultured with CHO-S OKT3 cells that overexpress hPVRL2.
[1149] CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) increase CD8 + T cell proliferation and IFN-g secretion: Figure 28A-B illustrates the ability of antibodies against humanized PVRIG, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), to increase the proliferation of CD8 + T cells in the CHO-S OKT3 assay. In two different human donors of CD8 + T cells, the antibodies CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) increase the proliferation of CD8 + T cells compared to the IgG4 isotype control human when T cells are co-cultured with CHO-S OKT3 hPVRL2 cells. However, mouse IgG1 CHA.7.544 has little to absent effect. As seen with CD4 + T cells, the anti-DNAM-1 antibody reduces the proliferation of CD8 + T cells. Figure 28C shows that humanized PVRIG antibodies, c Ha .7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), also increase IFNy secretion in the CHO-S OKT3 assay. In three different human donors of CD8 + T cells, the antibodies CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) increase the production of I FNy compared to the human IgG4 isotype control ( dotted line). Increases in IL-10, IL22 and TNFa were also observed after treatment with the antibodies CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) (data not shown).
[1151] CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) consistently increase proliferation of CD4 + T cells in multiple human donors: below, to demonstrate that CHA.7.518 antibodies .1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) could reproducibly increase T cell function, the effects of humanized PVRIG antibodies on T cell proliferation were examined CD4 + in 11 different donors in the CHO-S OKT3 assay. Figure 29 shows that both CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2. H4 (S241 P) consistently increased proliferation of CD4 + T cells in most donors tested compared to human IgG4 isotype control antibody when T cells were co-cultured with CHO-S OKT3 hPVRL2 cells. In addition, the receptor / ligand partial blocking antibody, CHA.7.530.3, and the receptor / ligand non-blocking antibody, CHA.7.544, do not consistently increase the proliferation of CD4 + T cells in the same donors.
[1153] CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) have a dose-dependent effect on the proliferation of CD4 + and CD8 + T cells: finally, the effect dependent on the doses of the humanized PVRIG antibodies, CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P), in the CHO-S OKT3 assay. Decreasing the dose of antibodies CHA.7.518.1.H4 (S241P) and c Ha .7.538.1.2.H4 (S241P) decreases the percentage of proliferation of CD4 + T cells (Figure 30A), and CD8 + T cells ( Figure 30B) when T cells are co-cultured with CHO-S OKT3 hPVRL2 cells. This dose-dependent effect on T cell proliferation is not observed with the CHA.7.544 antibody, nor with the IgG4 isotype control. Furthermore, a biphasic effect was not observed with dose titration, suggesting an absence of agonist activity of the antibodies against humanized PVRIG.
[1155] Summary and conclusions
[1157] Humanized PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P), had functional in vitro activity in the primary cell-based CHO-S OKT3 assay. Both CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) increased proliferation of CD4 + and CD8 + T cells in a dose-dependent manner. CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) were also able to increase I FNy secretion in the CHO-S OKT3 assay. Sr showed that the activity of the antibodies CHA.7.518.1.H4 (S241 P) and CHA.7.538.1.2.H4 (S241 P) was dependent on the overexpression of hPVRL2 in the target cells. CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P) consistently increased T-cell activity in multiple human donors, while the non-blocking antibody CHA.7.544 had little to no effect. absent.
[1159] G. Example 7: Development of rat monoclonal antibodies to mouse PVRIG
[1161] The development of rat monoclonal antibodies (mAbs) was carried out in Aldevron Freiburg (Germany). Antibodies to the mouse PVRIG protein were raised using DNA immunization technology. An immunization vector expressing mouse PVRIG was introduced into the host organism (rat). Mouse PVRIG was expressed, and a response to immunization was generated. Identification of positive antisera and hybridoma screening were analyzed in cells transiently expressing mouse PVRIG.
[1163] Generation of rat anti-mouse PVRIG pAb
[1164] Development of rat polyclonal antibodies to the mouse PVRIG protein included cloning of the mouse PVRIG extracellular domain into the proprietary Aldevron immunization vector, and cloning of the full length and extracellular domain into proprietary Aldevron screening vectors. The different expression vectors used for immunizations and for screening were confirmed by FACS in cells transiently expressing mouse PVRIG. Three rats were then immunized with the immunization vector. Immune sera were collected and diluted sera assayed by FACS using cells transiently transfected with the screening vectors. Production bleeds were collected from each rat, and purification was performed using protein-A. Generation of rat anti-mouse PVRIG mAb
[1165] Rat lymphocyte fusion and selection was performed using Aldevron assay systems. This included: the fusion of 20 x 96 well plates followed by initial screening by Cell Based ELISA (cELISA), in cells transiently transfected with mouse PVRIG or FL (full length) ECD (extracellular domain). 108 positive clones (attached to cells expressing mouse PVRIG / FL ECD) were further propagated and retested. 30 positive clones were propagated in T-25 flasks and the supernatants were tested in a cell-based ELISA. 23 hybridoma clones were selected for further subcloning. The serum-free supernatant was assayed by cELISA and by FACS. A total of 21 clones were generated and binding was confirmed in cells that overexpress the mouse PVRIG protein.
[1166] Characterization of Ab
[1167] The binding of rat mouse anti-PVRIG assay bleeds, purified pAbs, preclonal and clonal supernatants, as well as purified mAbs, was analyzed by flow cytometry, using HEK293 cells stably overexpressing mouse PVRIG. The binding of antibodies to D10.G4.1 cells that endogenously express mouse PVRIG was also tested. Cell-surface specific expression of mouse PVRIG was confirmed. Cells (1-2x105) were stained with Fixable viability stain diluted 1: 1,000 in PBS, for 10 min at R.T. followed by washing the cells with PBS. The Ab were then added to the cells (diluted in FACS buffer) followed by staining with goat anti-rat-PE (diluted 1: 100 in FACS buffer).
[1168] The specificity of the mAbs was assayed by siRNA for transfection of PVRIG from the cell line D10.G4.1 that endogenously expresses mouse PVRIG. A reduction in cell surface was observed after inactivation of mouse PVRIG.
[1169] The binding of mAbs to NK cells was also tested by FACS.
[1170] The pre-classification assay was performed to demonstrate the diversity of the mAbs.
[1171] The affinity of the purified mAbs (Kd) was determined by FACS titration in stable cells overexpressing mouse PVRIG versus cells transduced with empty vector, and in cell line D10.G4.1. Cells (1x105) were stained with Fixable viability stain diluted 1: 1,000 in PBS, for 10 min at RT followed by washing the cells with PBS. The Ab were then added to the cells (8 concentrations - serial dilution 1: 3, 10-0.01 pg / ml diluted in FACS buffer) followed by staining with goat anti-rat-PE (diluted 1: 100 in FACS buffer).
[1172] Characterization of mAbs-Summary Table
[1173] Table 7 (columns 1-10) summarizes the data generated for the characterization of mouse anti-PVRIG antibodies.
[1174] • Column 1 represents the Ab code ID
[1175] • Column 2 represents the name of the Ab provided by Aldevron
[1176] • Column 3 represents FACS data as MFI ratio in cells stably overexpressing on cells transduced with empty vector at a mAb concentration of 10pl / ml
[1177] • Column 4 represents affinity (nM) in overexpressing HEK cells
[1178] • Column 5 represents NK cell binding at a mAb concentration of 10pl / ml
[1179] • Column 6 represents the MFI binding ratio of the D10.G4.1 cell line over the isotype control
[1180] • Column 7 represents affinity (nM) to the cell line D10.G4.1
[1181] • Column 8 represents the different classes in the epitope pre-classification assay.
[1182] • Column 9 represents the Receptor-Ligand% blocking assay (binding of the mouse PVRIG-Fc fusion protein to cells that overexpress mouse PVRL2) and IC50 (nM) •
[1183] • Column 10 represents the Receptor-Ligand% blocking assay (binding of the mouse PVRL2-Fc fusion protein to cells that overexpress mouse PVRIG) and IC50 (nM)
[1184] AB-406 and AB-407 demonstrated blocking activity in both Receptor-Ligand binding assays, they have a high relative affinity, they bind to NK and D10.G4.1 cells.
[1185] These Ab were selected to determine TME expression and for in vivo studies.
[1186] Table 7. Characterization of mouse anti-PVRIG monoclonal Ab.
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[1195] H. Example 8: Combination Assay with Additional Immune Checkpoint Inhibitors Background
[1196] Although antibody blocking of the CTLA4 and PD1 pathways has emerged as an effective cancer treatment modality, most patients do not derive long-term benefit, suggesting a need to target additional immune checkpoints. By employing our unique computational algorithms to define new members of the B7 / CD28 family, we identified PVRIG, which is expressed by multiple subsets of T and NK cells. We report here its expression pattern, functional characterization, and antitumor activity of blocking antibodies directed at this molecule.
[1197] Methods
[1198] Using the Predictive Discovery platform, PVRIG was identified as a potential new immune checkpoint, after which a retroviral cell-screening library was used to identify its cognate binding counterpart. Targeted effects on T cell modulation were evaluated with primary and tumor derived T cell assays, taking advantage of overexpression, target inactivation, and antagonistic antibody strategies. Antibodies against the human protein were screened for their ability to increase T-cell activation in vitro, while antibodies directed at the mouse orthologue were evaluated in vivo for their effects on inhibiting tumor growth in syngeneic models.
[1199] Results
[1200] A PVRIG-Fc fusion protein was found to bind to PVRL2, with a binding specificity confirmed by both ELISA and flow cytometric analysis. PVRIG demonstrated unique expression kinetics in T cell activation, with target detection in memory T cells, as well as in NK cells and T and S cells. A panel of high-affinity human antibodies was generated with the ability to block the interaction of PVRIG with PVRL2, which when tested in vitro showed that they increase the activation of both primary CD4 + T cells and tumor-derived CD8 + through a dependent mechanism. by PVRL2.
[1201] Since CHA.7.518.1.H4 (S241P) is not cross-reactive with mouse, in vivo studies were performed with a surrogate blocking mouse anti-PVRIG antibody. When combined with blocking anti-PDL-1, anti-PVRIG of Mouse inhibits the growth of established tumors in the two colorectal cancer models CT26 and MC38. Testing of the combination with additional immune checkpoint inhibitors, as well as in PVRIG-inactivated mice, is ongoing.
[1202] Conclusions
[1203] The high-affinity antagonist antibody is capable of increasing human T-cell activation, and a surrogate antibody with similar characteristics shows synergy with PD-L1 in vivo in multiple syngeneic models. Overall, our data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for cancer treatment.
[1204] I. Example 9: in vivo poc study : efficacy of anti-MPVRIG mAbs in the CT26 tumor model
[1205] This example describes the efficacy of anti-mPVRIg mAb treatment in the CT26 murine colon carcinoma model as monotherapy or in combination with anti-PDL-1 treatment.
[1206] Materials and methods
[1207] Tumor pulse experiments:
[1208] Colon carcinoma CT26 was purchased from ATCC (CRL-2638). The cells were cultured in RPMI 1640 (Biological Industries, 01-100-1A) with 10% FBS (Biological Industries, 04-127-1A), and 100 µg / mL of penicillin / streptomycin (Biological Industries, 03-031 -1 B). For tumor implantation, cells were harvested and washed, counted, and suspended to 107 cells / ml in cold RPMI 1640 and placed on ice. BALB / c mice ((females, 8sem) Envigo) were anesthetized with a mixture of 10% Ketamine (Clorketam; SAGARPA Q-7090-053) and 10% Xylazine (Sedaxylan; BE-V254834) injected intraperitoneally. The backs of the mice were then shaved and disinfected with a 70% ethanol solution. Tumor cells were injected as 50 µl of 5x10 5 CT26 cells subcutaneously into the right posterior flank of the mice. Administration of mAbs started on day 4 (Monotherapy) or on day 7 (Combination treatment) after tumor inoculation when tumors had a volume of 30-50mm3 (Monotherapy) or reached the volume of 60-90mm3 (Combined treatment); and were given intraperitoneally (i.p.) in a final volume / injection of 200ul, for 3 weeks for a total of 6 administrations. Tumor growth was measured with an electronic caliper every 2-3 days and was reported as 0.5 X W2 X L mm3. Mice were euthanized with CO2 either at the end of the study or at any of the following clinical end points: tumor volume> 2250 mm3, tumor ulceration, body weight loss> 20%, or moribund appearance.
[1209] Antibodies:
[1210] The chimeric mouse anti-PVRIg antibodies (mAb 406 and mAb 407) used in this study, engineered as a rat IgG2b isotype monoclonal antibody (mAb), were shown to bind to 293HEK transfectants expressing mPVRIg and blocking the binding of mPVRL2 to these cells. The inhibitory anti-mouse PDL-1 mIgG1 used in this study was mAb YW243.55.S70. The YW243.55.S70 antibody is an anti-PD-L1 described in WO 2010/077634 (the heavy and light chain variable region sequences are shown in SEQ ID Nos. 20 and 21, respectively, of WO 2010 / 077634), and that it has a sequence described in it.
[1211] All mAbs were formulated in sterile PBS and had low endotoxin levels (<0.05 EU / mg).
[1212] Table 8. mAbs tested.
[1213] 1 mouse IgG1. k Isotype Control (MOPC-21) BP0083 BioXcell
[1214] 2 Rat IgG2b, k Isotype Control. BP0090 BioXcell
[1215] (LTF-2,)
[1216] 3 anti PDL-1 Reference YW243.55.S70 Compugen inc (mIgG1)
[1217] 4 mAb anti-CGEN PVRIG 406 BOJ-5C7-B3 ALDEVERON
[1218] (Rat IgG2b)
[1219] 5 mAb anti-CGEN PVRIG 407 BOJ-5G4-F4 ALDEVERON
[1220] (Rat IgG2b)
[1221] Study design
[1222] Monotreatment
[1223] Eight week old female BALB / c mice were purchased from Envigo and kept in an SPF animal facility for 1 week before starting the experiment. Mice were anesthetized, shaved, and inoculated subcutaneously with 50pl of 5X105 CT26 tumor cells. On day 4 after tumor inoculation, mice were randomly assigned to treatment groups of n = 10 (as described below). Mice were treated with mAbs (as detailed below) injected on day 4, 7, 11, 14, 18, and 21 after inoculation. Tumor growth was measured with a caliper every 2-3 days.
[1225] Table 9. Treatment groups.
[1230] Combined treatment
[1232] For the combinations of treatments with anti-mPVRIg and anti-mPDL-1 mAbs. Mice were treated as described in monotreatment. On day 7 after tumor inoculation, mice were randomly assigned to treatment groups of n = 10 as described below. Mice were treated with the mAbs (as detailed below) injected on day 7, 11, 14, 18, 21, and 25 after tumor inoculation.
[1234] Table 10. Treatment dosages.
[1236]
[1239] Statistic analysis:
[1241] Two-way ANOVA with repeated measures, followed by two-way ANOVA with repeated measures for selected pairs of groups using JUMP (Statistical DiscoveriesTM) software. Analyzes of tumor growth measurements were performed by comparing tumor volumes measured on the last day that the study animals were alive. Statistical differences in percentage of tumor-free mice were determined by a Mantel-Cox log rank test. Values of P <0.05 were considered significant.
[1243] * p <0.05; ** p <0.01; *** p <0.001. For each experiment, the number of replicates performed and the number of animals per group are described in the legend (s) of the corresponding figures (Figures 47-48).
[1245] Results
[1247] Activity of anti-mPVRIg and anti-mPDL-1 monotherapy in a syngeneic CT26 tumor model
[1248] We began the preclinical evaluation of anti-mPVRIg and anti-mPDL-1 monotherapy in a mouse syngeneic CT26 tumor model. We treated mice with an anti-PDL-1 mIgG1 isotype antibody (YW243.55.S70) or an anti-mPVRIg rIgG2b isotype (mAb 406 and 407).
[1250] In a CT26 colon carcinoma semi-therapeutic treatment model, monotherapy with anti-PDL-1 was significantly effective (P <0.0001), inciting 70% GIT compared to the control mIgG1 isotype, rates increased tumor rejection with rapid tumor rejection and long-lasting antitumor immunity observed in a majority of mice (Fig. 63A + B).
[1252] Groups treated well with anti-mPVRIg mAb 406, and anti-mPVRIg mAb 407 showed similar tumor growth rates with a GIT no greater than the rIgG2b isotype (Fig. 63A + B). Accordingly, treatment with anti PDL-1 mIgG1 prolonged the survival of the mice (P <0.01, Fig. 63C), with 5 out of 10 individuals demonstrating complete clearance of the tumor (Fig. 63B). No effect of anti-mPVRIg mAbs on survival rates was observed.
[1254] Activity of the combination of anti-PVRIg and anti-PDL-1 in the mouse syngeneic tumor model
[1256] Next, we evaluate the activity of anti-PVRIg and anti-PDL-1 combination therapy in the mouse syngeneic tumor model.
[1258] In a CT26 colon carcinoma therapeutic treatment model, the administration of anti-PDL-1 with rIgG2b isotype control treatment, started on day 7 after inoculation, was not effective, while the combination of anti-PVRIg mAbs 407 with anti-PDL-1 induced a significant GIT (56%, P = 0.0005), higher rates of tumor rejection with 4 out of 10 individuals demonstrating a complete clearance of the tumor (Fig, 64A + B) and promoted a better activity anti-tumor, with long-lasting anti-tumor immunity detected (P <0.01, Fig. 64C). The combination of anti-PVRIg 406 mAb with anti-PDL-1 was partially effective, resulting in 33% GIT, however, the antitumor response recorded was transient and no effect on survival rate was observed.
[1260] Conclusions
[1262] MPVRIg was predicted to play a role as a new B7-like molecule and thus as a potential target for antibody-based cancer immunotherapy. Several in vitro human experimental systems have demonstrated an immunomodulatory effect for mPVRIg. In the studies presented in this report, we have evaluated the in vivo anticancer effect of mAbs targeted against mPVRIg. In our study, treatment with 10mg / kg (200ug / mouse) of anti-mPVRIg as monotherapy in a minimal disease setting, that is, start of treatment on day 4 (tumor mean 40mm3), did not result in an advantage in GIT or survival, while the anti-PDL-1 positive control mAb exhibited significant GIT and resulted in prolonged survival.
[1264] Anti-mPVRIg mAbs were also tested in combination with anti-PDL-1 treatment. Treatment with 10mg / kg (200ug / mouse) started on day 7, when the tumors reached an average size of 75mm3. Combination therapy of anti-mPVRIg 407 mAb with anti-PDL-1 in the therapeutic CT26 model showed tumor growth inhibition and prolonged survival of the treated mice. The effect on tumor growth varied among individual mice with some individuals demonstrating full tumor clearance, while other individuals exhibited a partial response (transient TGI) and some individuals were non-responders. An in vivo effect of the anti-mPVRIg and anti-mPDL-1 combination treatment was also shown in the MC38 and B16-Db / gp100 tumor models (data not shown).
[1266] Additional in vivo studies are planned to evaluate dose dependencies and efficacy in additional syngeneic models or in combination with additional compounds or treatment regimens.
[1268] J. Example 10: Discovery of Therapeutic Antibodies to TIGIT by Phage Display
[1270] 1. Introduction
[1272] A phage display antibody discovery campaign was carried out to isolate human TIGIT binding agents from a library of unstimulated human fabs using the recombinant human TIGIT extracellular domain as the target antigen. Forty-five new human TIGIT-specific antibodies were isolated and generated as human IgG4, including an optional S241P in the hinge region as discussed herein. The resulting antibodies were screened for their ability to block TIGIT-PVR interaction and for cross-reactivity with cell-expressed cynomolgus TIGIT by flow cytometry. Two of these antibodies were further optimized for higher binding affinity to human and cynomolgus TIGIT.
[1274] 2. Protocols
[1276] Antigens for antibody discovery by phage display: Two formats of human TIGIT protein were used as antigens in phage display. The first comprised the ECD of human TIGIT (Met22-Pro 141) fused with a C-terminal polyhistidine tag (hTIGIT-HIS) and was either internally generated or obtained from commercial sources from Sino Biological Inc. The second antigen format comprised the human TIGIT ECD fused to a C-terminal human IgG1 Fc domain (hTIGIT-hFc) and was either internally generated or obtained from commercial sources from R&D Systems.
[1278] Functional QC of Antigens: Recombinant TIGIT antigens used for selection cycles were functionally validated for their ability to bind to human PVR, the human TIGIT ligand. Biotinylated antigens are assayed for PVR binding, either by ELISA or flow cytometry. Biotinylated hTIGIT-HIS was validated for its ability to bind hPVR-hFc (Sino Biological Inc.) by ELISA. Biotinylated hTIGIT-hFc was validated by flow cytometry for its ability to bind to PVR endogenously expressed on the surface in Expi293 cells.
[1279] Recognition and selection of phage from the human antibody library: Two phage campaigns were run, using either human TIGIT-HIS (campaign 1) or human TIGIT-hFc (campaign 2) as antigens. The recognition and selection reactions were carried out in solution, using streptavidin-coated magnetic beads to expose the biotinylated TIGIT antigens. Both campaigns used a human fab antibody phage display library for initial discovery. Three rounds of recognition and selection were carried out using the respective human TIGIT antigens, with higher stringency in the wash and lower antigen concentrations in each successive round of recognition and selection. Antibody CPA.9.002, generated in campaign 1, was optimized for improved binding to human TIGIT by generating a phage library by saturation mutagenesis of L-CDR3 and recognition and selection of the resulting library against human TIGIT-HIS (campaign 3). Two antibodies, CPA.9.059 and CPA.9.027, generated in campaigns 2 and 3, respectively, were also optimized for improved affinity for human TIGIT and cross-reactivity with cyno TIGIT (campaign 4). For each antibody, a phage library was generated by saturation mutagenesis of two CDRs (any combination of H-CDR1, H-CDR2, H-CDR3, L-CDR1, or L-CDR3). The resulting phage libraries were screened and screened for four rounds against human TIGIT-HIS and recombinant cyno TIGIT ECD protein C-terminally tagged with HIS in alternating rounds of screening and screening. The recognition and selection antigens used were the following: human TIGIT-HIS 1 nM in round 1, cyno-HIS TIGIT 1 nM in round 2, human TIGIT-HIS 0.1 nM in round 3, and 0.1 nM cyno-HIS in round 4.
[1281] Binding screens using antibodies expressed as fab fragments: the phagemid construct contains an amber stop codon that allows it to function as a fab expression vector. Transformation of these vectors into E. coli and induction with isopropyl p-D-1-thiogalactopyranoside (IPTG) results in periplasmic expression of soluble fab molecules. Fab proteins secreted into the E. coli periplasm were removed by osmotic shock for binding screens.
[1283] Primary ELISA Screening: fab PPE extracts were tested for binding to hTIGIT-HIS or hTIGIT-hFc screening and selection antigen by ELISA. Positive hits from the ELISA screen were sequenced using specific heavy chain and light chain primers. The sequences were assembled and analyzed. Clones were considered unique in sequence, if there was more than one non-conservative difference in heavy chain CDR3.
[1285] Secondary screening by flow cytometry: Single sequence ELISA positive fab clones were selected and analyzed for their ability to bind to Expi293 cells that overexpress human TIGIT by flow cytometry. Parental Expi293 cells were used as a negative control for each fab sample.
[1287] Reformatting of production and fab hits as human IgG4 molecules: Potential human TIGIT-binding fabs were converted to full-length human IgG4 (including a S241P mutant in the hinge, see, Aalberse et al., Immunology 202 105: 9-19, hereby incorporated by reference in its entirety, and in particular for the discussion of S241P and references 1, 2 and 3 cited in the document) for further characterization. The protein expression constructs were obtained by PCR amplification of the variable heavy and light chain sequences, which were subcloned into the pUNO3 vector (Invivogen).
[1289] 3. Results
[1291] Functional QC of recombinant human TIGIT proteins: Recombinant proteins hTIGIT-HIS and hTIGIT-hFc, generated either internally or from commercial sources, were functionally validated for their ability to bind to human PVR. Human PVR (conjugated to Fc) showed dose-dependent binding to biotinylated hTIGIT-HIS in ELISA (data not shown). Similar binding was observed in the reverse orientation when PVR was immobilized on the ELISA plate and hTIGIT-HIS was in solution (data not shown).
[1293] The hTIGIT-hFc protein was functionally validated by binding to PVR in a flow cytometry assay. In this assay, the hTIGIT-hFc protein was titrated against Expi293 cells that endogenously express human PVR. The interaction was detected using a secondary anti-hFc antibody conjugated to the fluorescence marker AF647. An irrelevant Fc protein was used as a control (data not shown).
[1295] Functional tests were done on several of the candidates as described in the Examples below.
[1297] Affinity maturation binding screens using antibodies expressed as fab fragments: Eight 96-well plates of extracted periplasmic fab clones were analyzed for de novo campaigns (1 and 2). Seventy-three unique clones were identified in campaign 1 using the hTIGIT-HIS protein as the target antigen. Secondary screening of the 73 positive clones in ELISA by flow cytometry identified 21 positive for binding to Expi293 cells that overexpress human TIGIT. A similar screen for campaign 2 (hTIGIT-hFc as target antigen) yielded 37 ELISA positive clones, 24 of which were also positive for binding to Expi293 cells that overexpress human TIGIT, by flow cytometry (Figure 52).
[1299] Two of the 96-well plates of fab clones (as PPE) were screened for optimization / affinity maturation campaigns (3 and 4). Unique ELISA positive variants were screened for binding to Expi293 cells that overexpress human and / or cynomolgus TIGIT on flow cytometry. The binding affinities of the best clones to the hTIGIT-HIS protein was also evaluated by Surface Plasmon Resonance (SPR). The first cycle of affinity maturation of the CPA.9.002 antibody yielded 5 new antibodies, CPA.9.021, CPA.9.027, CPA.9.044, CPA.9.048, and CPA.9.049, with mutations in L-CDR3 and an improvement of al minus 3-fold in binding affinity for recombinant human TIGIT. A second round of optimization of the CPA.9.027 antibody yielded 4 new antibodies with an at least 25-fold improvement in binding to recombinant human TIGIT. The new variants showed mutations in H-CDR2 and L-CDR3 (CPA.9.083 and CPA.9.086) and additionally in L-FR4 for CPA.9.089 and CPA.9.093. Optimization of c Pa .9.059 resulted in two new antibodies, CPA.9.101 and CPA.9.103, with significantly improved binding to cynomolgus TIGIT, as well as a significant improvement in human TIGIT binding to CPA.9.103. Mutations were seen in H-CDR3 and L-CDR1 for the two new variants. Additionally, minor changes were observed in L-FR1 for CPA.9.101.
[1301] Reformatting of ELISA and FACS positive fabs in hIgG4: Forty-five positive unique fabs were reformatted in ELISA and flow cytometry for binding to human TIGIT for expression as human IgG4 molecules, including an optional S241P hinge variant as shown. discussed herein. Furthermore, 11 affinity optimized variants were also reformatted as IgG4. Selected phage derived antibody sequences are shown in Figure 53. The sequences of two reference antibodies, BM26 (WO2016 / 028656, Clone 31C6) and BM29 (US2016 / 0176963, Clone 22G2) are also shown in Figure 53 for comparison. The reformatted antibodies were evaluated for their binding to Expi293 cells that overexpress human TIGIT and a binding curve was generated to calculate the equilibrium binding constant (KD). These antibodies were also evaluated for binding to Expi293 cells that overexpress cyno TIGIT, as well as their ability to block the interaction between human TIGIT and human PVR in cell-based assays. Based on this characterization, a subset of these antibodies was selected for in vitro functional assays as more fully described below.
[1303] K. Example 11: discovery of therapeutic antibodies against TIGIT by hybridoma
[1305] 1. Rational basis and objectives
[1307] Hybridoma technology was used using known and standard methods in the field to generate murine antibodies that bind to human TIGIT with high affinity, cross-react with TIGIT from non-human primates (cynomolgus macaque, Macaca fascicularis, referred to as cyno), and they block the interaction of TIGIT with its ligand, PVR (CD155).
[1309] 2. Summary
[1311] Balb / c mice were immunized with recombinant forms of cyno and human TIGIT extracellular domain proteins. Cells isolated from the spleen and lymph nodes of immunized mice were fused with the Sp2 / 0 myeloma cell line to generate murine antibody-secreting hybridomas. The supernatants from the subcloned monoclonal and polyclonal hybridomas were screened for binding to Expi293 cells that overexpress human and cyno TIGIT and for binding affinity to recombinant human and cyno TIGIT proteins using standard SPR methods. Selected hybridoma murine antibodies were purified and characterized extensively in binding and functional assays. Five functional and cross-reactive murine antibodies to cyno were humanized to contain a framework (including an optional hinge variant as indicated herein) and hIgG4 isotype. The sequences are shown in Figure 53.
[1313] L. Example 12: FACS KD Measurements of Hybridoma and Phage Derived Antibodies Binding to Cells Overexpressing Human and Cyno TIGIT
[1315] 1. Protocols
[1317] The following cell lines were prepared to estimate the binding affinities of mouse and human phage anti-TIGIT antibodies: Parental Expi293, Expi293 that overexpress human TIGIT, and Expi293 that overexpress cyno TIGIT. The following hybridoma and phage antibodies were each prepared in a 2-fold 11-point serial dilution series in a 195pM-200nM binding site concentration range:
[1318] Phage-generated antibodies: CPA.9.027, CPA.9.049, CPA.9.059.
[1320] Hybridoma-generated antibodies (pre-humanization): CHA.9.536, CHA.9.541, CHA.9.543, CHA.9.546, CHA.9.547 and CHA.9.560. Two different reference antibodies were included, BM26 (WO2016 / 028656A1, Clone 31C6 as mouse IgG1) and BM29 (US2016 / 0176963 A1, Clone 22G2 as mouse IgG1).
[1321] The 12th well of each titration contained only buffer to function as a background. Each cell type was incubated with an anti-human TIGIT mAb for 60 minutes at 4 ° C. After washing, AF647-tagged goat anti-human F (ab ') (Jackson Immunoresearch) and AF647-tagged goat anti-mouse IgG-Fc (Southern Biotech # 1030-30) were added to cells incubated with the human and mouse mAbs, respectively. A FACS Canto II HTS instrument then recorded the Geometric Mean Fluorescence Intensity (gMFI) of 5,000-10,000 events for each well. A plot of the gMFI was fitted as a function of the molecular concentration of human PVR using the Graphpad Prism model "one site, specific binding" to estimate the KD and 95% confidence intervals of each non-linear fit.
[1323] 2. Results
[1325] The two independent FACS KDs measured for each mAb differed no more than 2-fold on average. A single representative measurement for KD along with the 95% confidence interval of the binding isotherm fit is listed for each mAb for human and cyno overexpressing cells in Figure 54 and Figure 55, respectively. CPA.9.059 did not show binding to cells that overexpress cyno. It should be noted that the binding site concentrations (2X the molecular concentration) for all mAbs are used for the non-linear curve fit, meaning that the assumption is made that this KD FACS method is measuring the constant of the binding site (kD) instead of the stoichiometric or molecular binding constant.
[1327] M. Example 13: Hybridoma-derived phage-derived anti-human TIGIT mAb FACS blocking assay that inhibit PVR-Fc binding to TIGIT
[1329] 1. Introduction
[1331] The purpose of this assay is to characterize the ability of phage and hybridoma derived anti-human TIGIT antibodies to inhibit the binding of human PVR to overexpressed human TIGIT on a cell surface. First, the human TIGIT-human PVR binding affinity for FACS will be determined. The binding isotherms showed the saturating concentration of human PVR that was used for the blocking assays. Cells overexpressing human TIGIT were then titered with anti-TIGIT mAbs produced in phage and hybridoma, followed by the addition of a saturating concentration of human PVR. The binding of the anti-human TIGIT antibody in the overexpressing cells was then measured using FACS.
[1333] 2. Protocols
[1335] FACS KD Assay: Various isotypes of human PVR-Fc were assayed via FACS for optimal binding and it was determined that human PVR-h1Fc (Sino Biological # 10109-H20H) and human PVR-m2aFc (Compugen) exhibited the higher levels of binding to cells that overexpress human TIGIT. The two PVR isotypes were each serially diluted 2-fold in an 11-point titer series to a final molecular concentration range of 98pM-100nM. The 12th well of each titration contained only buffer to function as a background. Each cell type was incubated with mAb for 60 minutes at 4 ° C. After washing, AF647-tagged goat anti-human Fc (Jackson Immunoresearch # 109-606-098) and AF647-tagged goat anti-mouse IgG (SouthernBiotech # 1033-31) -labeled goat F (ab ') 2 fragment were added to wells titrated with the human and mouse anti-TIGIT mAbs, respectively. A FACS Canto II HTS instrument then recorded the Geometric Mean Fluorescence Intensity (gMFI) of 5,000-10,000 events for each well. A plot of the gMFI was fitted as a function of the molecular concentration of human PVR using the Graphpad Prism model "one site, specific binding" to estimate the KD and 95% confidence intervals of each non-linear fit. The results of human PVR-m2aFc and human PVR-h1 Fc are shown in Figure 57A and B, respectively.
[1337] Phage mAb Blocking Assay: The following phage-derived hIgG4 antibodies and reference mAbs were each prepared in a 3-point 5-fold serial dilution series at a concentration range at the 267pM-6 binding site, 7 nM: CPA.9.027, CPA.9.049 and CPA.9.059, as well as BM26 (WO2016 / 028656A1, Clone 31C6 as hIgG4) and Synagis hIgG4 (negative isotype control).
[1339] The 4th well of each titration contained only buffer to function as a background. Cells were incubated with mAb for 15 minutes at 4 ° C. Human PVR-m2aFc (Compugen) was then incubated for 1 hour at 4 ° C. After washing, AF647-tagged goat anti-mouse IgG (SouthernBiotech # 1033-31) was added. A FACS Canto II HTS instrument then recorded the Geometric Mean Fluorescence Intensity (gMFI) of 5,000-10,000 events for each well. The bound human PVR gMFI values for the cells preincubated with the mAbs were compared to the gMFI values of the cells preincubated with the blocking reference mAb and non-blocking control mAb. If the phage antibody reduced the human PVR-m2aFc binding signal as compared to the signal from the titer with the known non-blocking mAb, the antibody was characterized as blocking PVR binding at that concentration of the phage mAb. The blocking trends of the phage mAbs were similar to the blocking of PVR with the reference BM26 (Figure 58).
[1341] Hybridoma mAb Blocking Assay: The following hybridoma antibodies were each prepared in an 11-point 2.5-fold dilution series at a concentration range at the 14pM-133nM binding site: CHA.9.536, CHA .9.541, CHA.9.546, CHA.9.547, CHA.9.560, BM26 (WO2016 / 028656A1, Clone 31C6 as mouse IgG1) and BM29 (US2016 / 0176963A1, Clone 22G2 as mouse IgG1).
[1342] The 12th well of each titration contained only buffer to function as a background. Cells were incubated with mAb for 15 minutes at 4 ° C. Human PVR-h1Fc (Sino Biological # 10109-H20H) was then added, and the cells were incubated for 1 hour at 4 ° C. After washing, AF647-tagged goat anti-human Fc F (ab ') 2 fragment (Jackson Immunoresearch) was added. A FACS Canto II HTS instrument then recorded the Geometric Mean Fluorescence Intensity (gMFI) of 5,000-10,000 events for each well. A plot of gMFI was fitted as a function of concentration at the mAb binding site in a non-linear fashion using the Graphpad Prism model "log (inhibitor) versus response-variable slope (four parameters)" to estimate IC50. of each nonlinear fit. This experiment was repeated twice for two days.
[1344] 3. Results
[1346] Figures 58 and 59 demonstrate that both phage and hybridoma antibodies potently block the binding of human PVR-Fc to overexpressed human TIGIT on the cell surface of Expi293 cells. The blocking activity of the phage and hybridoma antibodies is comparable to the two reference antibodies tested, BM26 and BM29.
[1348] N. Example 14: Surface plasmon resonance (SPR) kinetic studies of the binding of nine phage and hybridoma derived mAbs to human, cyno, and mouse TIGIT
[1350] 1. Protocols
[1352] All experiments were performed using a ProteOn XPR 36 instrument at 22 ° C. First, high-density capture surfaces were prepared with polyclonal goat anti-human Fc antibody (Thermo # H10500) and rabbit anti-mouse antibodies (GE Healthcare # BR100838), respectively, immobilized on the vertical capture lanes and horizontal midpoints on separated GLC chips using standard amine coupling. Typical immobilization levels for the anti-human capture pAb and the anti-mouse capture antibody for each GLC chip were around 5,000RU. Human TIGIT was obtained from Sino Biologicals, while mouse TIGIT monomer and cyno TIGIT monomer were prepared internally. The purified mAbs studied for binding to human, mouse, and cyno TIGIT are listed below:
[1354] Phage antibodies: CPA.9.027, CPA.9.049 and CPA.9.059
[1356] Hybridoma antibodies: CHA.9.536, CHA.9.541, CHA.9.543, CHA.9.546, CHA.9.547 and CHA.9.560
[1358] Reference comparisons: BM26 (WO2016 / 028656A1, Clone 31C6 as hIgG4) and BM29 (US2016 / 0176963A1, Clone 22G2 as hIgG4).
[1360] Each mAb was diluted to ~ 0.5 pg / mL in run buffer that was 1xPBST with filtered BSA added to a final concentration of 100 pg / mL. For each cycle of "one shot kinetics" on the ProteOn instrument, a different mAb was captured on one of the six unique vertical capture lanes for approximately 1.5-2.5 minutes. After changing the flow of the ProteOn buffer to the horizontal direction, the capture surfaces were stabilized for approximately 15-20 minutes. Six concentrations of a 3-fold dilution series of human TIGIT (346pM-84.1 nM), cyno TIGIT (371pM-90.2nM), or mouse TIGIT (382pM-92.9nM) were injected for 2 minutes followed 20 minutes of dissociation at a flow rate of 50pL / min. An identical buffer injection preceded each series of injected antigen for double reference. Anti-human antibody surfaces were regenerated with two 30 second pulses of 146mM phosphoric acid and anti-mouse antibody capture surfaces were regenerated with two 30 second pulses of 10mM glycine, pH 1.7. Sensorgrams of the TIGIT antigen injected on captured mAbs were processed using a ProteOn version of Scrubber and fitted to a 1: 1 kinetic binding model including a term for mass transport.
[1362] Figure 56 shows the resulting kinetic rate constants and the equilibrium dissociation constants, where the data were reliable enough to estimate the binding constants (sensorgram data not shown). The asterisks indicate the kd values that had to be kept constant at 1.00x10-5 / sec. In cases such as the binding of clone CHA.9.560 to human TIGIT, the kinetic model was able to estimate a Kd, but it is virtually impossible to accurately estimate a Kd on the order of 1 x10-6 / sec after only 20 minutes of dissociation data, given the sensitivity of the instrumentation.
[1364] O. Example 15: functional analysis of anti-TIGIT antibodies
[1366] 1. Rational basis and objectives
[1368] To functionally characterize the ability of anti-human TIGIT antibodies to inhibit the interaction of TIGIT and its PVR ligand, and to consequently increase the activation of human T cells either as a monotherapy or in combination with an anti-human PVRIG antibody, CHA .7.518.1.H4 (S241 P).
[1370] 2. Protocols
[1372] Human TIGIT / CD155 Jurkat IL-2 Luciferase Reporter Assay: The Jurkat IL-2 Human TIGIT / PVR Luciferase Reporter Bioassay Kit (Promega) was used to assess the effect of antibody treatment anti human TIGIT in T cell activation. Jurkat T cells were stably transfected with recombinant human TIGIT and a luciferase reporter gene driven by the IL-2 response element (IL-2-RE). The stimulator cells were artificial CHO-K1 APC cells (aAPC) expressing recombinant human PVR, and an engineered cell surface protein designed to activate TCR-mediated signaling in an antigen-independent manner. Following coculture of these cells, the human TIGIT / human PVR interaction inhibits TCR signaling and IL-2-RE mediated luminescence. The addition of an anti-human TIGIT antibody that blocks the human TIGIT / human PVR interaction releases the inhibitory signal, resulting in T cell activation and IL-2-RE mediated luminescence. The test was carried out according to the manufacturer's instructions. Briefly, CHO-K1 aAPC cells with human PVR were thawed in a 37 ° C water bath and diluted in F-12 medium supplemented with 10% FBS (Promega). 25,000 cells / well were seeded in 96-well round bottom tissue culture treated white plates (Costar). The plates were then incubated overnight at 37 ° C. The next day, hybridoma and phage derived anti-human TIGIT antibodies, mouse IgG1 (mIgG1) and hIgG4 isotype control, or anti-human TIGIT reference antibodies (BM) were added either as a single dose at 10 µg / ml, or in a 2-fold dilution series of 10 points, starting at 20 µg / ml. Human Jurkat IL-2-RE luciferase TIGIT cells were thawed in a 37 ° C water bath and diluted in RPMI medium supplemented with 10% FBS (Promega). 125,000 Jurkat cells were added to each well. The plates were then incubated at 37 ° C with 5% CO2 for 6 hours. After incubation, the plates were removed from the incubator and allowed to equilibrate at room temperature for 30 minutes. 80 µL of Bio-Glo luciferase substrate (Promega) was added to each well and the mixture was allowed to equilibrate for 10 minutes at room temperature protected from light. Luminescence was quantified on an En Vision multi-label reader (Perkin Elmer) with an ultrasensitive luminescence detector. The luminescence signal was reported in relative light units (RLU).
[1374] Expansion of CMV-specific human CD8 + T cells: Human peripheral blood mononuclear cells (PBMC) reactive with CMV (CTL) were thawed, resuspended at 2x10 6 cells / ml, and stimulated with 1 μg / ml of the CMV peptide pp65 ( Anaspec) in complete RPMI medium supplemented with 2 ng / ml of recombinant human IL-2 (R&D systems) and 10 ng / ml of recombinant human IL-7 (R&D systems) at 37 ° C with. After 9 days, the cells were divided 1: 2 and left with a low dose of human IL-2 (100 IU / ml). The frequency of CMV-specific CD8 + T cells was determined with the CMV pp65 / HLA-A2 tetramer (MBL). CMV-specific CD8 + T cells that were 65-98% positive for the tetramer were used in assays between days 12 and 16 after challenge with the CMV peptide.
[1376] CMV-specific human CD8 + T-cell coculture assay with melanoma cell lines expressing human PVR: An in vitro CMV-specific human CD8 + T-cell co-culture assay was used to assess the effect of anti-human TIGIT antibodies on the antigen-specific cytokine secretion. The target cell line used in the assay was the HLA-A2 + melanoma cell line, Mel624 stably transduced with a lentivirus containing human PVR DNA (System Biosciences). A stable pool of Mel624 cells overexpressing human PVR was pulsed with the CMV peptide pp65 at 0.0033 µg / µl or 0.001 µg / µl at 37 ° C for 1 hour. Cells were then washed and plated at 50,000 cells / well. Hybridoma and phage derived anti-human TIGIT antibodies, mIgG1 or hIgG4 isotype control antibodies, or anti-human TIGIT BM antibodies were added at a concentration of 10 µg / µl. CMV-specific human CD8 + T cells from three different donors, specified as Donor 2, Donor 4, and Donor 210 were expanded according to the above protocol. 50,000 human CD8 + T cells were added to each well. The cocultures were incubated at 37 ° C with 5% CO2 for 24 hours. After incubation, the plates were centrifuged at 1,200 rpm for 1 minute and the supernatant was collected. The amount of human interferon gamma (IFNy) in the coculture supernatant was measured by flow cytometry using a bed cytometric assay (BD).
[1378] Coculture assay of CMV-specific human CD8 + T cells with melanoma cell lines expressing human PVR and human PVRL2 (CD112): the combined effect of anti-human TIGIT antibodies and CHA.7.518.1.H4 (S241 P) was evaluated , an anti-human PVRIG antibody, on antigen-specific cytokine secretion by an in vitro coculture assay with CMV-specific human CD8 + T cells similar to the assay described above. The target cell line used in the assay was the HLA-A2 + melanoma cell line, Mel624, which stably expresses human PVR and human PVRL2, the ligands for TIGIT and PVRIG, respectively, through lentiviral transduction (System Biosciences). . Mel624 cells overexpressing human PVR and human PVRL2 were pulsed with the CMV pp65 peptide at 0.0033 µg / µl or 0.001 µg / µl at 37 ° C for 1 hour. Cells were then washed and plated at 50,000 cells / well. Hybridoma and phage derived anti-human TIGIT antibodies, or an anti-human TIGIT BM antibody, were added to the culture in combination with CHA.7.518.1.H4 (S241P) or a hIgG4 isotype control antibody at 10 µg / m l. CMV-specific human CD8 + T cells from three different donors, specified as Donor 4, Donor 25, and Donor 210 were expanded according to the above protocol. 50,000 human CD8 + T cells were added to each well. The cocultures were incubated at 37 ° C for 24 hours. After incubation, the plates were centrifuged at 1,200 rpm for 1 minute and the supernatant was collected. The amount of human interferon gamma (IFNy) in the coculture supernatant was measured by flow cytometry using a bed cytometric assay (BD).
[1380] 3. Results
[1381] Anti-Human TIGIT Antibodies Increase IL-2 Signaling: The ability of hybridoma and phage-derived anti-human TIGIT antibodies to increase IL-2 signaling was evaluated with the human TIGIT luciferase / human PVR reporter assay in Jurkat. Figure 60 and Figure 62 demonstrate the effect of 10 pg / ml of phage or hybridoma derived human anti-TIGIT antibodies on IL-2 signaling, respectively. Three phage-derived antibodies, CPA.9.027, CPA.9.049, and CPA.9.059 robustly increased IL-2 signaling compared to the hIgG4 isotype control. Furthermore, all three phage antibodies induced more IL-2 signaling compared to anti-human TIGIT BM antibodies, BM26 and BM29. The five hybridoma-derived antibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, and CHA.9.560 also induced IL-2 signaling compared to the mIgG1 isotype control. It should be noted that all five hybridoma antibodies induced similar IL-2 signaling compared to BM26 and BM29. The non-blocking human anti-TIGIT antibody, CHA.9.543, did not significantly increase IL-2 signaling. To determine if the effect of the anti-TIGIT antibodies was dose dependent, the assay was carried out with a 2-fold dilution series of 10 points for each antibody starting at 20 pg / ml (Figures 61 and 63). IL-2 signaling was decreased in a dose-dependent manner with all eight anti-human TIGIT antibodies, as well as with BM26 and BM29.
[1383] Anti-human TIGIT Antibodies Increase IFNy Secretion from CMV-Specific Human CD8 + T Cells: The Ability of Hybridoma- and Phage-Derived Anti-Human TIGIT Antibodies to Modulate I FNy Secretion Was Assessed with the Cell Coculture Assay CMV / Mel624 specific tees. Figure 64 shows the effect of anti-human TIGIT antibodies on IFNy secretion. Three phage-derived antibodies, CPA.9.027, CPA.9.049, and CPA.9.059 increased IFNy secretion compared to medium alone and the hIgG4 isotype control antibody. Additionally, five hybridoma derived antibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, and CHA.9.560 also increased IFNy production compared to the mIgG1 isotype control antibody. Hybridoma and phage derived TIGIT antibodies induced IFNy in a similar manner to BM26 and BM29. As expected, the non-blocking human anti-TIGIT antibody, CHA.9.543, did not significantly affect IFNy secretion.
[1385] Figure 65 shows the combined effect of anti-human TIGIT and CHA.7.518.1.H4 (S241P) on IFNy secretion. Three phage-derived antibodies, CPA.9.027, CPA.9.049, and CPA.9.059, and five hybridoma-derived antibodies, CHA.9.536, CHA.9.541, CHA.9.546, CHA.9.547, and CHA.9.560, including BM26, all increased IFNy secretion compared to their respective isotype control antibodies, when treated alone, or in combination with CHA.7.518.1.H4 (S241P). The non-blocking anti-human TIGIT antibody, CHA.9.543, resulted in lower IFNy secretion compared to other anti-human TIGIT antibodies. The percentage increase in IFNy secretion in each antibody over the respective isotype control antibodies is summarized in Figure 66. A synergistic effect is observed in the combined treatment of anti-human TIGIT and CHA.7.518.1 antibodies. H4 (S241 P).
[1387] 4. Summary and conclusions
[1389] The addition of anti-human TIGIT antibodies to the Jurkat human TIGIT / human PVR reporter assay induced a robust dose-dependent increase in IL-2 signaling. Additionally, anti-human TIGIT antibodies increased IFNy secretion from CMV-specific human CD8 + T cells when co-cultured with Mel624 cells with human PVR. IFNy secretion was further increased by anti-human TIGIT antibodies in combination with an anti-human PVRIG antibody. Taken together, these data demonstrate that anti-human TIGIT antibodies can block TIGIT-mediated suppression of human T-cell activation, and human T-cell activation is enhanced by the coblocking of both TIGIT and PVRIG.
[1390] P. Example 16: Pre-classification analysis of anti-TIGIT antibodies
[1392] 1. Protocols
[1394] The experiments were performed by Wasatch Microfluidics Inc. (Salt Lake City, UT) using a Continuous Flow Microdoser (CFM) and an SPR IBIS MX96 Imager (MX96 SPrí). The following anti-human TIGIT mAbs and human PVR-Fc variants were each diluted to ~ 10pg / mL in 10mM sodium acetate, pH 5.0 and covalently immobilized using standard amine coupling at independent sites on a prism chip Xantec 200M biosensor for 7 minute cycles using the CFM:
[1396] 1 CPA.9.009-H4 17 CPA.9.081 -H4 32 CHA.9.555
[1398] 2 CPA.9.011-H4 18 CHA.9.519 33 CHA.9.560
[1400] 3 CPA.9.012-H4 19 CHA.9.521 34 CHA.9.525
[1402] 4 CPA.9.013-H4 20 CHA.9.522 35 CHA.9.538
[1404] 5 CPA.9.014-H4 21 CHA.9.527 36 CHA.9.543
[1406] 6 CPA.9.015-H4 22 CHA.9.528 37 CHA.9.553
[1407] 7 CPA.9.018-H4 23 CHA.9.529 38 CHA.9.556
[1409] 8 CPA.9.027-H4 24 CHA.9.535 39 CHA.9.561
[1411] 9 CPA.9.049-H4 25 CHA.9.536 40 BM8-H4
[1413] 10 CPA.9.053-H4 26 CHA.9.541 41 BM9-H4
[1415] 11 CPA.9.057-H4 27 CHA.9.546 42 BM26-H4
[1417] 12 CPA.9.059-H4 28 CHA.9.547 43 BM29-H4
[1419] 13 CPA.9.064-H4 29 CHA.9.549 44 MBSA43-M1
[1421] 14 CPA.9.069-H4 30 CHA.9.552 45 PVR-Fc M2A
[1423] 15 CPA.9.071-H4 31 CHA.9.554 46 Otherwise PVR-Fc
[1425] 16 CPA.9.077-H4
[1427] BM8-H4 and BM9-H4 refer to (US2015 / 0216970A1, Clones 10A7 and 1F4 reformatted as hIgG4), respectively. MBSA43-M1 is a mouse anti-human TIGIT IgG1 from eBioscience. The prism chip was then washed with 1X PBST for 3 minutes and then directly loaded into the SPRi MX96 imager, where excess NHS esters were neutralized with a 5 minute injection of 1M ethanolamine. Preliminary experiments included several cycles of injection of 100 nM human monomeric TIGIT (Sino Biologicals, Cat. No. 10917-H08H) on all immobilized mAbs for four minutes followed by regeneration in order to test the binding activity of the antibodies and better determine regeneration conditions by evaluating the reproducibility of the TIGIT binding. These preliminary experiments showed that the best reagent to reproducibly regenerate most of the immobilized mAbs was a 30 second pulse of 1/500 phosphoric acid. The immobilized PVR, however, did not retain activity and therefore their blocking patterns were generated and "pre-classified" as analytes in solution alone. In these preliminary experiments and the pre-classification experiments described below, all protein samples were prepared in the running buffer which was degassed HBST. A "sandwich" epitope pre-classification protocol was performed, where each mAb and PVR was injected onto TIGIT that had previously formed a complex with each immobilized mAb to determine whether or not the immobilized mAb blocks the mAb in solution preventing binding to TIGIT . For each cycle, 100 nM TIGIT was first injected onto the immobilized mAbs for 4 minutes followed immediately by a 4 minute injection of a competitor or ligand mAb at 274nM (binding site concentration). This was repeated with each mAb and PVR acting as the competitor analyte. Control cycles with running buffer instead of competitor protein were performed after every 12 cycles for a double reference. All surfaces were regenerated after each cycle with a 30 second pulse of 1/500 phosphoric acid. Sensorgram data was processed and referenced using proprietary Wasatch software. An antibody pair was classified as having a shared TIGIT-binding epitope if no binding of the competitor injection was observed on the TIGIT forming a pre-complex with the immobilized mAb. An antibody pair was classified as binding to different epitopes in TIGIT, or "sandwich", if the competitor mAb injection showed binding to TIGIT forming a previous complex. Low or minimal binding of the competitor was classified as an "intermediate" blocker. Hierarchical grouping of TIGIT blocking patterns in pairs for each mAb and ligand was performed using Wasatch's proprietary software.
[1429] 2. Results
[1431] Both the PVR-Fc and 13 mAb proteins either lost activity or could not regenerate as ligands, so their blocking patterns were determined as analytes in solution alone. The mAb CPA.9.014-H4 was not pre-classified because it did not show binding to TIGIT. Figure 67 shows dendrogram pooling based on the pairwise blocking patterns for each mAb and two PVR proteins. The vertical axis represents the factor of statistical similarity in the blocking patterns. Wasatch Microfluidics applied a cut-off factor of 5 to group mAbs as indicated by the line in Figure 67. For the strictest definition of an epitope "class", where only those mAbs (and PVRs) that show patterns of Identical locks are pre-classified together, there are a total of 12 discrete classes. If the blocking patterns showing only minor differences are pooled together, there are four closely related "communities" of mAbs and PVRs. These "communities" are indicated by different shaded blocks at the bottom of Figure 67. Figure 68 groups together the mAbs and PVRs that populate each discrete unique class, with each class indicated by a black box. The gray boxes surround all the unique classes that make up each "community" of related lock patterns. The mAbs and PVRs in Figure 68 are listed with the key number representing each protein in the dendrogram in Figure 67.
[1433] Q. Example 17: Administration of anti-PVRIG antibodies to inactivate TIGIT in mice
[1435] Rational basis and objectives
[1436] To examine whether deletion of TIGIT in combination with blocking mouse PVRIG can increase tumor growth inhibition and survival in a mouse syngeneic tumor model.
[1438] Protocols
[1440] Animals
[1442] TIGIT inactivated (KO) mice were generated at Ozgene Pty LTD (Australia). C57BL / 6 wild-type (WT) mice (Ozgene) served as controls. Eight to eleven week old female TIGIT KO and C57BL / 6 mice were used. All studies were approved by the Institutional Animal Care and Use Committee at Tel-Aviv University (Tel-Aviv, Israel).
[1444] In vivo tumor models
[1446] 1 x 105 B16 / Db-hmgp100 melanoma cells were inoculated subcutaneously (s.c.) into the right flank of C57BL / 6 WT or TIGIT KO mice. Treatment with the antibodies was started on the same day as tumor inoculation (day 0), with 7-10 mice per treatment group. The antibodies used were mouse IgG1 isotype control (Clone MOPC-21 BioXcell), and mouse anti-mouse PVRIG IgG1 (Clone 407, Compugen LTD). Antibodies were administered at 10mg / kg by intraperitoneal injection, twice a week for 3 weeks. Tumor growth was measured with an electronic caliper every 2-3 days and was reported as 0.5 X W2 X L mm3 (L is length and W is tumor width). Animals that reached a tumor size of 2,250mm3 were anesthetized.
[1448] Statistic analysis
[1450] Two-way ANOVA with repeated measures was performed, followed by two-way ANOVA with repeated measures for selected pairs of groups using JUMP (Statistical DiscoveriesTM) software. Analyzes of tumor growth measurements were performed by comparing tumor volumes measured on the last day that the study animals were alive. Statistical differences in the percentage of tumor-free mice were determined by the Mantel-Cox log rank test. Values of P <0.05 were considered significant. * p <0.05; ** p <0.01; *** p <0.001.
[1452] Results
[1454] Inhibition of tumor growth in vivo after treatment with blocking anti-mouse PVRIG antibody in mice with inactivated TIGIT
[1456] We tested the in vivo efficacy of TIGIT deletion in combination with mouse PVRIG blocking in a mouse syngeneic B16 / Db-hmgp100 subcutaneous melanoma tumor model. Treatment of tumor-bearing C57BL / 6 WT mice with a blocking anti-mouse PVRIG antibody had a minor effect on tumor growth inhibition (TGI) compared to isotype treatment (17% TGI on day 11 and 8 % TGI at the end point, day 18). The effect of TIGIT deletion on tumor growth was less compared to the C57BL / 6 WT control group (17% TGI at day 11 and 13% TGI at the end point). However, when the TIGIT deletion was combined with treatment with the mouse anti-PVRIG antibody (Clone 407), a significant GIT was evident (63% at day 11 and 49% GIT at end point) (Figure 80A and 80B). According to TGI, the TIGIT KO mice treated with the mouse anti-PVRIG antibody (Clone 407) showed increased survival compared to the C57BL / 6 WT control group, however, no statistical significance was achieved (Figure 80C) .
[1458] Summary and conclusions
[1460] The combination of TIGIT deletion and PVRIG blockade significantly reduced tumor growth in vivo, indicating that both TIGIT and PVRIG play an inhibitory role in this melanoma tumor model. These data suggest that targeting both TIGIT and PVRIG could be another combination therapy that significantly increases antitumor responses.
[1462] R. Example 18: PVRIG antagonism increases effector function of T cells and reduces tumor growth
[1463] Summary
[1465] Despite recent advances, most patients do not get long-term benefit from checkpoint inhibitors. PVRIG is a new immunosuppressive receptor of the DNAM / TIGIT family and we have here demonstrated a role for PVRIG in the regulation of antitumor responses. PVRIG binds to PVRL2 and exhibits significantly increased expression in infiltrating lymphocytes in tumors compared to lymphocytes in normal tissues. Antagonism of PVRIG increased the activation of human T cells and the combination of PVRIG with either PD-1 inhibitors or TIGIT synergistically further increased lymphocyte function. Next, we address the role of PVRIG in preclinical tumor models. PVRIG - / - mice exhibited significantly increased in vitro T cell activation and reduced MC38 tumor growth that was mediated by increased CD8 effector function. The antagonistic anti-PVRIG antibody significantly reduced tumor growth in combination with anti-PD-L1 or when tested in TIGIT - / - mice. In summary, we demonstrate that the PVRIG-PVRL2 pathway was induced in human cancers and that antagonism of PVRIG-PVRL2 interactions resulted in increased T-cell function and reduced tumor growth.
[1467] State of significance
[1469] These data demonstrate that PVRIG is a promising target for cancer treatment and provide the rationale for testing a PVRIG inhibitor, CHA.7.518.1.H4 (S241 P), as a new cancer immunotherapy agent either as monotherapy or in combination with TIGIT or PD1 lock.
[1471] Introduction
[1473] Increasing evidence shows that endogenous immune responses are critical in sculpting cancer initiation, progression, and suppression (1) (2). The immune status of patients, as well as the content of tumor infiltrating leukocytes (TIL) in the tumor microenvironment (TME), are key prognostic indicators not only of cancer survival rates, but also of how patients respond to therapy (3) (4). T cells are a key component of TILs that can elicit an antitumor response, and most antitumor immune responses ultimately rely on the functionality of effector lymphocyte cells. A CD8 T cell enrichment in a patient's tumor TME, as well as other factors that bias an immune response toward an effective CD8 T cell response, such as mutational load and a Th1-polarized TME, are all key prognostic indicators. for a favorable antitumor immune response (5) (6).
[1474] A key observation in many solid tumors is that effector T cells have an activated or 'depleted' phenotype in TME (7). This indicates that, although the T cells in the TME have initially seen the cognate antigen, have been activated, and transported to the tumor, they are not subsequently capable of eliciting an effective antitumor response. Preactivated or depleted T cells are defined by increased surface expression of co-inhibitory receptors, such as PD-1 and CTLA-4 (8). Targeting these co-inhibitory receptors with antibodies that inhibit interactions with their cognate ligands has shown remarkable clinical efficacy in patients with multiple advanced cancers (9). Mechanistically, targeting of these co-inhibitory receptors has been shown to result in the expansion of already tumor-reactive T cells that preexist in TME and the production of T-cell pools with a broader diversity of T-cell receptors. (10) (11) (12). Although checkpoint inhibitors currently in the clinic have revolutionized cancer treatment and demonstrated the power of the immune system to fight cancer, many patients still relapse and / or do not respond to treatment. Consequently, increased understanding of the immune response in cancer and targeting additional immunity-based pathways will lead to additional therapeutic treatments.
[1475] Among these new pathways, a group of receptors and ligands in the nectin family and similar to nectin are currently being investigated as new cancer immunotherapies. Receptors in this family include DNAM-1 (CD226), CD96 (TACTILE), TIGIT, and more recently, PVRIG (CD112R) (13) (14) (15). Of these molecules, DNAM is an activating receptor in this subfamily, binding to 2 ligands, PVR (CD155) and PVRL2 (CD112), to deliver an activating signal to lymphocytes (16). Two receptors in this family have been shown to inhibit human lymphocyte function, TIGIT, and more recently, PVRIG (17) (18). TIGIT is reported to have a high affinity interaction with PVR, a much weaker affinity for PVRL2, and has been shown to inhibit both T cell and NK cell responses by supplying an inhibitory signal to lymphocytes through their rest ITSM (19) (20). More recently, PVRIG was shown to bind with high affinity to PVRL2 and to deliver an inhibitory signal through its ITIM residue (15). In both cases, the affinity of TIGIT for PVR and of PVRIG for PVRL2 is greater than the affinity of DNAM for PVR or PVRL2, suggesting that TIGIT and PVRIG may outperform DNAM with respect to PVR and PVRL2, providing an indirect mechanism by the which TIGIT and PVRIG can reduce T cell function. In this family, PVR is also a ligand for CD96. CD96 function has been reported to be inhibitory in mouse lymphocytes (21) but activating in human lymphocytes (22). On the basis of these data, we postulate in human lymphocytes that 2 receptors, TIGIT and PVRIG, bind with high affinity to PVR and PVRL2, respectively, to deliver inhibitory signals that decrease T cell function.
[1477] Although human PVRIG has been shown to inhibit the T-cell response in a recent report, the role of PVRIG and PVRL2 in cancer immune surveillance is not well understood. In particular, the expression profile of this pathway in cancers and the role of PVRIG in the regulation of antitumor responses of CD8 T cells have not been reported. Furthermore, the functional characterization of the mouse PVRIG gene and the effect of disruption of the PVRIG-PVRL2 interaction in vivo in preclinical tumor models have not been reported. Herein, we elucidate the role of PVRIG in a cancerous environment by reporting the expression profile of PVRIG and PVRL2 in cancer and the effect of PVRIG antagonism in tumor cell co-culture assays and in preclinical tumor models. We show that PVRIG has a differentiated expression profile in T cell subsets compared to TIGIT or CD96 and that the expression of PVRIG and PVRL2 was induced in cancer compared to adjacent normal tissues. In multiple human in vitro assay systems, a high affinity PVRIG antagonist monoclonal antibody (CHA.7.518.1.H4 (S241 P)) increased T-cell function, particularly when combined with anti-TIGIT antibody. or anti-PD1. In addition, we reported the new characterization of mouse PVRIG using antagonist antibodies or PVRIG-deficient mice and demonstrated that inhibition of the PVRIG-PVRL2 interaction reduced tumor growth, with the most potent effects in combination with inhibition of PD-1 or TIGIT genetic deficiency. Collectively, these data show that PVRIG is a receptor critical inhibitor in the regulation of antitumor responses of T cells and support the development of CHA.7.518.1.H4 (S241 P), for clinical trials in cancer patients.
[1479] Materials and methods
[1481] Expression studies in peripheral blood and human tumors
[1483] Human PBMC were obtained from healthy donors from Stanford University according to the Declaration of Helsinki. The human tissues were provided by the Cooperative Human Tissue Network, a resource funded by the National Cancer Institute. Human cancer tissue and concordant adjacent normal tissues were dissociated into single cells according to the manufacturer's protocol (Miltenyi Biotec). Dissociated cells were analyzed by flow cytometry to determine the expression of various targets in different subsets of cells. For expression of each target in an individual cell subset, an expression fold value was calculated by taking the MFI value of the target divided by the MFI value of the isotype control. Other researchers may have received samples of these same tissue specimens. The tumor type was determined based on the review of the pathology report for each sample. For IHC studies, anti-PVRL2 antibody (HPA-012759, Sigma) and PD-L1 (Sp142, SpringBio) were used to stain tumor microarrays (Biochain institute) using the conditions as described in the supplemental methods. Scoring was performed by 2 independent reviewers on duplicate nuclei from the same tumor.
[1485] Generation and characterization of antibodies against PVRIG
[1487] Anti-human PVRIG and anti-mouse PVRIG antibodies were generated as detailed in the supplemental methods. Briefly, the specificity and binding affinity of the antibodies were assessed by selective binding to cells engineered with PVRIG without detectable binding to cells that do not express the gene. The antagonistic activity of these anti-PVRIG antibodies was determined using ELISA and FACS based assays in which the interaction of PVRIG with PVRL2 was disrupted. For characterization in cell-based assays, antibodies were tested in various T cell-target cell coculture assay systems, consisting of PVRL2-expressing target cells in culture with PBMC or tumor derived T cells. Gp100-specific T cell lines were expanded from melanoma tumors as previously described (23). T cells reactive for CMVpp65 were expanded from healthy donor PBMC (CTL immunospot) with CMVpp65 (495-503), IL-2, and IL-7 for 10 days. For combination studies, antibodies to PD-1, TIGIT, and PVRIG were used at 10 pg / ml. Cytokine concentrations in the conditioned medium were determined using Cytometric Bed Matrix (CBA) and FACS staining was performed as described in the supplemental methods.
[1489] Characterization of mouse PVRIG expression and function
[1491] Binding interactions of mouse PVRIG with mPVRL2 and mPVR were evaluated by SPR and ELISA using recombinant proteins PVRIG, PVRL2, and PVR and by FACS using engineered cell lines that ectopically overexpress PVRIG and PVRL2 or cell lines transfected with PVR siRNA or PVRL2. Mice deficient for PVRIG and TIGIT were generated as described in the supplemental methods. Expression analysis was performed to examine the expression of PVRIG in spleen, lymph node, and tumor in various subsets of cells. Cellular functional assays demonstrating T cell modulatory activity for mouse PVRIG were established using WT and PVRIG - / - T cells and target cells ectopically expressing PVRL2 Fc or PVRL2 as detailed in the supplemental materials and methods. The tumor models CT26, MC38, and B16 / Dbhmgp100 were performed as described in the supplemental methods. All studies were approved by the Institutional Animal Care and Use Committee at Tel-Aviv University (Tel-aviv, Israel) or Johns Hopkins University (Baltimore, USA).
[1493] Results
[1495] The highest expression of PVRIG occurs in peripheral blood effector T cells and tumors
[1497] The Ig superfamily (IgSF) consists of hundreds of proteins, but only a few of them are inhibitory receptors for T cells. IgSF proteins tend to evolve rapidly (24) and therefore the sequence similarity between them protein is generally low and is not optimal for identifying new immune receptors. To identify new immune checkpoints, we develop bioinformatic algorithms based on genomic and proteomic characteristics shared between known immune checkpoints, such as gene structure, protein domains, predicted cell location, and pattern of expression. Using these algorithms, PVRIG was identified as a new immune receptor. A report has also recently shown that human PVRIG (CD112R) binds to PVRL2 and inhibits T-cell function (15). However, the relevance of this pathway in regulating tumor immune surveillance has not been reported. Here, we have elucidated the expression and function of PVRIG and PVRL2 in human cancers and in preclinical tumor models. In the peripheral blood of healthy donors, PVRIG was expressed exclusively on lymphocytes, with the highest expression on CD8 T cells and NK cells (Figure 83A). Further analysis of the T-cell subsets showed the highest expression of PVRIG in CD8 or CD4 memory / effector T cell subsets compared to the Treg subset (Figure 83B, Figure 90A). The expression pattern predominantly in memory T cells differentiates PVRIG from other receptors in the family (TIGIT, CD96) that tend to have the same or greater expression in Treg in comparison with memory / effector T cells. We further compared the expression kinetics of PVRIG and TIGIT after T-cell activation in 2 assay systems (CMV recall response Figure 83C, DC-MLR Figure 83D, Figure 90B) and show that PVRIG has induction kinetics delayed and more sustained expression at the late time point compared to TIGIT. The preferential expression of PVRIG memory / effector cells compared to TIGIT suggests a unique role for PVRIG in the regulation of T cell responses.
[1499] The delayed and sustained induction of PVRIG expression in T cells after activation, suggested that it could be expressed in the tumor microenvironment. Next, we analyze the expression of PVRIG in human tumor leukocytes directly dissociated ex vivo by FACS. Expression of PVRIG was detected in CD8 T cells, CD4 T cells, and NK cells from multiple tumor types (Figure 83E-G, Figure 90C). PVRIG was co-expressed with PD-1 and TIGIT on CD4 and CD8 T cells (Figure 83F). On average, the highest expression was detected in TIL CD4 + and CD8 + from breast, endometrial, head and neck, lung, kidney, and ovarian tumors compared to bladder, colorectal, and prostate. In tumor samples in which PVRIG expression was low / not present ex vivo, activation with anti-CD3 and anti-CD28 increased PVRIG expression, suggesting that PVRIG expression in TIL may be further induced after reactivation (Figure 90D). For colon, lung, kidney, endometrial, and ovarian tumors, we were able to obtain adjacent normal tissue from the same patient and perform a comparison of PVRIG expression in lymphocytes isolated from tumor tissue versus normal. TILs showed significant induction of PVRIG in CD4 and CD8 T cells compared to cells isolated from concordant adjacent normal tissues (NAT) (Figure 90E). As with PBMCs, we further compared the expression of PVRIG, TIGIT, and PD1 in Tregs against CD8 T cells from lung, endometrial, and kidney tumors. In TIL, TIGIT expression was higher in Treg compared to CD8 T cells, while for PVRIG and PD1, a similar or higher expression was observed in CD8 T cells compared to Treg (Figure 83H). Next, we examine the co-regulation of PVRIG, TIGIT, and PD-1 in T cell populations by correlation analysis of either the magnitude of expression in TILs ex vivo or the magnitude of the fold change in expression between tumor and tumor. NAT. In both analyzes, CD4 and CD8 T cells showed a positive and significant correlation between PVRIG and PD1 or TIGIT in (Figure 90F). Taken together, these data demonstrate that PVRIG is expressed in T cells and NK cells of multiple human cancers, positioning PVRIG as a new target inhibitory receptor that may be critical in the regulation of T cell function in the tumor.
[1501] PVRL2 expression is increased in tumor tissue compared to adjacent normal tissue
[1503] As PD-L1 expression has been shown to help predict responses to PD-1 inhibitors, we examined whether the expression of PVRL2 was concomitant with the expression of its cognate receptor, PVRIG, in human cancer tissues. Using an anti-PVRL2 antibody that we validated for staining of FFPE samples (Figure 91A), we stained tumor microarrays (TMAs) composed of lung, colon, skin, breast, ovarian / endometrial, and kidney cancer tissues and scored each nucleus based on the prevalence and intensity of PVRL2 expression. PVRL2 expression was either absent or minimally expressed in most normal tissue samples from these organs. In tumor tissues, PVRL2 expression in tumor cells was detected in ~ 50-70% of lung, colon, breast, and ovarian / endometrial cancers (Figure 84A, 84F). Expression in kidney cancer samples ranged from 20-40%, while expression in melanoma was the lowest (~ 10%) (Figure 84A, 84F). The expression of PVRL2 was detected in tumor cells and immune cells on the invasive front (Figure 84B). To determine the specific immune cell subsets that express PVRL2, we performed flow cytometry on freshly dissociated tumors. Expression of PVRL2 was detected in CD45 + immune cells, particularly myeloid cells (eg, CD14 + tumor-associated macrophages (TAM) and myeloid DC) and in non-immune CD45- cells of multiple tumor types (Figure 84C, D ). No expression of PVRL2 was detected in lymphocytes (data not shown). Comparison of PVRL2 expression in CD45- and TAM cells isolated from colon, lung, kidney, endometrial, and ovarian tumors, showed a significant induction of PVRL2 in cells isolated from the tumor compared to cells isolated from NAT concordant thereof. donor (Figure 92). For the samples where we obtained expression of PVRIG and PVRL2, we examined the expression of PVRIG in lymphocytes compared to PVRL2 in myeloid and CD45 cells from multiple tumor types. Of the cancer types examined, endometrial, lung, and kidney cancers had the highest prevalence of high PVRIG lymphocytes and high PVRL2 TAM or nonimmune CD45- cells (Figure 842E, Figure 93). By integrating data from dissociated TMA and tumor studies, we demonstrate that tumors of the breast, endometrial, lung, head and neck, kidney, and ovary may be representative of a tumor type responding to PVRIG antagonism.
[1505] Compared to PD-L1, PVRL2 expression is differentially regulated and present in PD-L1- tumors.
[1506] As PVRIG and PD-1 can be co-expressed in tumor infiltrating lymphocytes (TIL), we also examined the co-expression of PVRL2 and PD-L1 in the same tumor by staining serial sections of the same TMA. Expression of PVRL2 in tumor cells was clearly detected in PD-L1- tumor samples (as defined by nonmembranous PD-L1 staining in tumor cells or immune cells) at a similar frequency and average score compared to samples PD-L1 +. (Figure 85A, Figure 84F). In immune cells, 3 out of 5 tumors in which PVRL2 expression was detected in immune cells also expressed PD-L1 (data not shown), but low sample numbers made it difficult to conclude on immune cell co-expression of PD -L1 and PVRL2. Expression of PVRL2 in tumor cells in PD-L1 negative tumors suggested that PVRL2 expression was more prevalent than PD-L1 in some tumor types and that it is targeted by this pathway it may be particularly effective in PD-L1 'tumors. While PD-L1 is induced primarily by IFN- y as an adaptive resistance mechanism (28), PVRL2 is modulated by genomic stress, DNA damage, and tumor suppressor genes (29,30). To better understand the different regulation of PD-L1 and PVR / PVRL2, we now evaluate the regulation of the expression of PVR, PVRL2 and PD-L1 in tumor cell lines and in DC derived from monocytes by exposure to various inflammatory stimuli (Figure 85D ). Treatment of DC with pro-inflammatory signals generally resulted in increased expression of PVR, PVRL2, and PD-L1, demonstrating that the expression of PVR, PVRL2, and PD-L1 is increased after DC maturation. In contrast, treatment of epithelial cells with I FN- y increased the expression of PD-L1, but had no effect on the high baseline expression of PVRL2 (Figure 85E), supporting the differential regulation of PVRL2 expression in comparison with PD-L1 by I FN-y. In summary, these findings indicate that PD-L1 and PVRL2 can be co-regulated in antigen presenting cells (APC), such as DC, but can be differentially regulated in epithelial cells. The presence of PVRL2 in PD-L1 negative tumors suggests that targeting this pathway may have potential benefit in patients who do not respond to or progress with PD-1 inhibitors.
[1508] CHA.7.518.1.H4 (S241P) is a high affinity humanized monoclonal antibody against PVRIG that disrupts the interaction of pVr IG with PVRL2
[1510] To examine the functional consequences of antagonizing human PVRIG-PVRL2 interactions, we generated a high affinity antagonistic anti-PVRIG antibody, CHA.7.518.1.H4 (S241 P), which blocks the interaction of PVRIG and PVRL2. This antibody selectively bound to HEK293 cells that ectopically express human PVRIG or cynomolgus macaque PVRIG and also bound to Jurkat cells that endogenously express PVRIG with subnanomolar affinity (Figure 86A). In biochemical assays, CHA.7.518.1.H4 (S241P) blocked the interaction of PVRIG Fc with HEK293 PVRL2 + cells (Figure 86B) and also blocked the binding of pVr L2 Fc to HEK293 PVRIG + cells (Figure 86C). Using this antibody, we observed a functional effect of an antagonist anti-PVRIG in several T-cell assays. Artificial antigen presenting cells (aAPC) that ectopically express an anti-CD3 antibody on the cell surface and human PVRL2 were generated and co-cultured with human primary CD4 T cells, either in the presence of anti-PVRIG (CHA.7.518.1.H4 (S241 P)) or isotype control. Expression of PVRIG was induced in proliferating CD4 T cells after coculture with the anti-CD3 aAPC CHO (Figure 94A). Antagonism of PVRIG with CHA.7.518.1.H4 (S241p) increased proliferation of T c D4 cells from multiple donors (Figure 86D). We also tested the effect of anti-PVRIG on 2 gp100 reactive human CD8 T cell lines that were derived from melanoma tumors. These T cell lines were individually co-cultured with aAPC expressing HLA-A2 and PVRL2 (Figure 94B) in the presence of IgG or anti-PVRIG isotype control antibodies. As observed in both lines, anti-PVRIG increased the production of I FN-y and TNF-to ~ 20-50%. In an assessment of dose response, CHA.7.518.1.H4 (S241 P) exhibited EC50 values of one nanomolar digit in multiple assays (Figure 94C, D). These data collectively demonstrate that antagonism of PVRIG-PVRL2 interactions with CHA.7.518.1.H4 (S241 P) resulted in increased activation of T cells.
[1512] CHA.7.518.1.H4 (S241P) in combination with TIGIT or PD-1 inhibitors resulted in a synergistic increase in T-cell function.
[1514] The combination of PVRIG and TIGIT blockade synergistically increased CD4 T cell function in a T cell-dendritic cell coculture assay (15), suggesting a role for this pathway in the regulation of T cell-APC interactions. . The effects of blocking PVRIG and TIGIT on CD8 T cells in a tumor cell co-culture environment have not been reported. As our tumor expression profiling demonstrated the expression of PVRL2 in CD45- immune cells, we further explored the effect of targeting this pathway in T cell-tumor cell cocultures using 2 T-cell assay systems. First, we performed a Coculture of 2 gp100 tumor antigen specific CD8 T cell lines with a melanoma cell line, MEL624, in the presence of anti-PVRIG, anti-TIGIT, or isotype control antibodies, either individually or in combination. MEL624 cells express both PVR and PVLR2 and both TIL-209 and TIL-463 expressed PVRIG, TIGIT, and PD-1 (Figure 86F). In TIL-209, we observed that anti-PVRIG or anti-TIGIT alone did not increase I FN-y and that the combination of anti-PVRIG and anti-TIGIT synergistically increased the production of I FN-y (Figure 86G). In TIL-463, we observed that anti-PVRIG or anti-TIGIT modestly increased the production of I FN-y, and that the combination of anti-PVRIG and anti-TIGIT increased I FN-y additively (Figure 86G). In an additional assay system, we used CMVpp65-reactive CD8 T cells as a model system to study human T cell responses. CD8 HLA-A2 + CMVpp65 T cells expanded in the presence of CMVpp65 (495-503) and the expression of PVRIG, TIGIT, and PD-1 was observed on day 10 (Figure 86F). PVRIG was expressed in CMVpp65-specific CD8 T cells to a similar magnitude to that seen in human cancer samples (Figure 83). As target cells, we identified a cancer cell line HLA-A2 + PD-L1 high (Panc05.04) and pD-L1low (Colo205) that both expressed similar amounts of PVR and PVRL2 (Figure 86F). Next, we co-cultured the CMVpp65-reactive T cells with HLA-A2 + tumor cell lines pulsed with the pp65 peptide (495-503) in the presence of blocking antibodies against PVRIG, TIGIT, and / or PD-1. We observed that anti-PVRIG Ab increased I FN- y by ~ 50% in the coculture with Panc05.04 cells and minimally in the coculture with Colo205 (Figure 86I). The combination of anti-TIGIT Ab with anti-PVRIG synergistically increased the production of I FN-y in both target cell lines, leading to a greater increase in I FN-y compared to the PD-1 antibody alone (Figure 86H). The combination of anti-PVRIG and anti-PD-1 also resulted in a synergistic increase in I FN- y production compared to the single antibody (Figure 86I). Taken together, these data suggest a Potent synergy of the combination of PVRIG and TIGIT or PVRIG and PD1 blockade in increasing activation of human CD8 T cells after interaction with tumor cells.
[1516] PVRIG deficiency resulted in increased proliferation of T cells and reduced tumor growth
[1517] Although the sequence for mouse PVRIG and its interaction with mouse PVRL2 have been reported, the expression profile and immune modulatory activity of mouse PVRIG is not well understood. First, we analyzed the expression of mPVRIG RNA and transcript in NK, KT and T cells (Figure 87A). Activated mouse CD8 T cells had elevated PVRIG transcripts with delayed induction kinetics compared to TIGIT (Figure 87B). We confirmed that the recombinant mPVRIG bound to the mPVRL2 protein by surface plasmon resonance (SPR) and ELISA performed in various assay orientations (Figure 95A-D). We also observed an interaction between mPVRIG and mPVR, although the affinity was approximately 10x lower than the interaction with mPVRL2 (Figure 95E). To determine whether PVR or PVRL2 is the dominant ligand for mPVRIG, we assayed the binding of mouse PVRIG Fc to B16F10 cells expressing PVR and PVRL2 (data not shown). PVRIG Fc showed dose-dependent binding to B16F10 cells that was completely abolished after inactivation by siRNA of PVRL2 in B16F10 cells (Figure 95F). In comparison, the binding of the PVRIG Fc fusion protein was slightly, but consistently, reduced after PVR inactivation (Figure 95F) suggesting that there is a very weak interaction between mPVRIG and mPVR. Taken together, these results demonstrate that, in mice, PVRL2 is the major ligand for PVRIG, as is the case in humans.
[1519] To delineate the role of PVRIG in immune responses, we generated PVRIG-deficient (- / -) mice (Figure 96). PVRIG - / - mice were born in the expected Mendelian ratios, did not show an evident phenotype until 10 months of age, and at 8 weeks of age they had similar leukocyte cellularity (peripheral and lymphoid tissue) when compared to type mice. wild (Figure 97). Wild-type CD8 T cells (WT) and NK cells express PVRIG and the absence of PVRIG expression was detected in PVRIG - / - cells (Figure 87C). To examine the role of PVRIG in the regulation of mouse T cell responses, we examined the proliferation of WT and PVRIG - / - T cells in 2 assay systems. WT or PVRIG - / - T cells were activated with immobilized anti-CD3 in the presence of soluble PVRL2 Fc or control Fc protein. The soluble PVRL2 Fc significantly inhibited the proliferation of CD4 + WT T cells but not the proliferation of CD4 + PVRIG - / - T cells (Figure 87D), suggesting that PVRIG - / - cells lack an inhibitory signal. To evaluate the role of mouse PVRIG in the interaction of CD8 + T cells with tumor cells, PVRIG - / - mice were crossed with pmeI TCR transgenic mice, which express a transgenic TCR specific for gp10025-33 (28). Activated PmeI CD8 + PVRIG - / - or WT T cells were co-cultured with B16-Db / gp100 melanoma tumor cells endogenously expressing PVRL2 (data not shown) and activation and effector function were evaluated. PMeI CD8 + PVRIG - / - T cells showed increased degranulation and effector cytokine production (IFN-y and TNF-a) compared to WT cells (Figure 87E). These data indicate that mouse PVRIG inhibits the activation and effector function of tumor-specific T cells after co-culturing with target PVRL2 + tumor cells.
[1521] Next, we study the effects of PVRIG deficiency on tumor growth in the MC38 syngeneic model. PVRIG - / - mice exhibited significantly reduced tumor growth compared to WT mice (p <0.05; Figure 88A-B). Furthermore, PD-L1 blockade began on day 14, further amplifying antitumor responses and reducing tumor growth in PVRIG - / - mice compared to WT mice treated with anti-PD-L1 (p = 0.052) ( Figure 88C-D). To assess the functional effects of PD-L1 blocking in PVRIG - / - and WT tumor microenvironments, we collected tumors and tumor-draining lymph nodes from each of the four experimental cohorts on day 18, when the groups had received 2 doses of isotype or anti-PD-L1, but no differences were observed in tumor volume, and flow cytometry was performed to determine the composition of the immune subset and intracellular cytokines. Immune cell transport (CD45 +) in PVRIG - / - tumors was moderately increased (88% compared to WT tumors) as did CD8 + T cells (92% compared to WT tumors) and IFN-producing CD8 + T cells -y (110% increase over WT tumors; Figure 88E). In combination with PD-L1 blockade, CD45 + cell infiltration was significantly increased in PVRIG - / - tumors (160% compared to anti-PD-L1 treated WT mice tumors; p = 0.032; Figure 88F) . PVRIG - / - tumors treated with anti-PD-L1 also had higher numbers of total CD8 + T cells per tumor weight (252% increase) and IFN-y producing CD8 + T cells (297% increase), compared to anti-PD-L1 treated WT tumors (Figure 88F). We also observed that PVRIG - / - mice had unchanged numbers of tumor-infiltrating effector CD4 + T cells and Foxp3 + Treg regardless of PD-L1 blockade (data not shown). Rescue of immune dysfunction in PVRIG - / - tumors, particularly after PD-L1 blockade, was reflected in tumor-draining lymph nodes that had increased frequencies of effector CD8 + T cells IFN-Y + TNF-a + relative to mice. WT treated with anti-PD-L1 (Figure 88G-H). Taken together, these data demonstrate that PVRIG ablation results in reduced tumor growth associated with an increased antitumor immune response, particularly when combined with anti-PD-L1 antibody treatment.
[1523] Anti-mPVRIG antibody inhibited tumor growth in combination with PD-1 antibody or TIGIT deficiency
[1525] After demonstrating that genetic PVRIG deficiency resulted in reduced tumor growth, we next set out to demonstrate that antibody-mediated inhibition of the PVRIG-PVRL2 interaction could enhance antitumor immunity, particularly in combination with PD1 or TIGIT inhibitors as demonstrated by our in vitro human data. To evaluate this, we generated a high affinity antagonist anti-mPVRIG antibody. FACS-determined anti-mPVRIG mAb affinity assessments showed a subnanomolar Kd (0.33 nM in HEK293 mPVRIG, 0.39 nM in D10.G4.1 cells), similar to CHA.7.518.1.H4 (S241P ) (Figure 95G-H). The specificity of this antibody was further confirmed by suppressing the majority of binding to D10.G4.1 cells after inactivation of mPVRIG (Figure 95I). Anti-mPVRIG was tested for disruption of the mPVRIG-mPVRL2 interaction by inhibiting the binding of mPVRIG Fc to B16F10 and the binding of mPVRL2 Fc to HEK293 cells that overexpress mPVRIG (Figure 89A). Complete blocking of the PVRIG-PVRL2 interaction by the anti-mPVRIG antibody was observed in both assay formats (Figure 89A, Figure 95J), demonstrating that it is an antagonistic anti-mPVRIG antibody. Next, we tested the in vivo efficacy of mPVRIG blockade in a CT26 syngeneic subcutaneous colon tumor model. Expression of PVRIG was elevated on NK and T cells in the tumor microenvironment, compared to subsets of spleen or corresponding draining lymph nodes (Figure 89B). Treatment of tumor-bearing mice with the anti-mPVRIG blocking mAb as monotherapy did not reduce tumor growth (data not shown). However, the combination of anti-PVRIG and anti-PD-L1 mAbs effectively delayed CT26 tumor growth (Figure 89C) and significantly increased survival of treated mice with a 40% rate of complete responders (Figure 89D). . Consistent with our data in the human T cell assay, these data demonstrate that the combination of PD-1 inhibitors and PVRIG can reduce tumor growth.
[1527] We also tested the effect of ablation of both PVRIG and TIGIT signaling on the regulation of antitumor responses. For these studies, we tested the efficacy of the anti-mPVRIG antibody in WT or TIGIT - / - mice inoculated with B16F10 / Db-hmgp100 melanoma cells. Treatment of tumor bearing WT mice with blocking anti-mPVRIG mAb had a lesser effect compared to isotype treatment (17% GIT on day 11 and 8% GIT on end point, day 18). The effect of TIGIT deletion on tumor growth was also less, compared to the WT control group (17% TGI at day 11 and 13% TGI at the end point). However, when TIGIT deletion was combined with anti-PVRIG mAb treatment, significant inhibition of tumor growth was observed (63% at day 11 and 49% TGI at end point (Figure 89E, F). According to the inhibition of tumor growth, the TIGIT - / - mice treated with the anti-PVRIG 407 mAb showed increased survival compared to the WT control group, however, no statistical significance was achieved in this aggressive tumor model of rapid growth (data not shown) Taken together, these data demonstrate synergistic activity of PVRIG inhibitors with PD1 or TIGIT inhibitors and are in agreement with our human functional data providing the rationale for the clinical trial of CHA.7.518 .1.H4 (S241 P) with PD1 or TIGIT inhibitors.
[1529] Discussion
[1531] Although antibodies directed at T-cell immune checkpoints, such as CTLA4 and PD-1, have increased the survival of cancer patients, the majority of cancer patients still have no clinical benefit. One possible reason for this is the presence of additional T-cell regulators that inhibit T-cell antitumor immunity. Here, we elucidate the role of PVRIG in regulating T-cell effector function and demonstrate that PVRIG antagonism increases the antitumor responses of T cells and reduces tumor growth.
[1533] PVRIG is a new member of the nectin family and similar to nectin, placing it among several known immunoregulatory receptors in the family. Understanding the interaction of receptors in this family is crucial to understanding the relevance and mechanism of action of PVRIG. Of these receptors, DNAM, TIGIT, and CD96 are the most related to PVRIG in terms of sharing the same ligands, PVR and PVRL2. DNAM binds to both PVR and PVRL2 and supplies a costimulatory signal to lymphocytes. TIGIT is reported to bind PVR and weakly PVRL2. We were unable to detect an interaction between TIGIT and PVRL2 using ELISA or SPR (data not shown), suggesting that PVR is the dominant ligand for TIGIT. Using similar methods, we and a recent report detected a high affinity interaction between PVRL2 and PVRIG, suggesting that PVRIG is the dominant inhibitory receptor for PVRL2. These data suggest that TIGIT and PVRIG comprise dual signaling nodes on this axis and that blocking of both is necessary for maximal increase in T-cell activation in this family. In addition to interacting with different ligands, we observed that PVRIG has the highest expression in effector or memory T cells, similarly to PD-1, while TIGIT has the highest expression in regulatory T cells. Furthermore, we observed that PVRIG exhibited late induction after T-cell activation compared to TIGIT. These data suggest that PVRIG has a unique role in this family, interacting with high affinity with PVRL2 and having a differentiated expression in memory cells and a late induction profile with respect to TIGIT.
[1535] Here we report the novel role of PVRIG in the regulation of T-cell antitumor responses using PVRIG-deficient mice and antagonist anti-PVRIG antibodies. Mouse PVRIG has been shown here to be expressed on T cells and NK cells, induced after lymphocyte activation, and is higher in TME compared to the periphery. Furthermore, we show that PVRIG deficiency results in increased T cell function in-vitro and reduced tumor growth in-vivo. An antagonistic antibody to PVRIG reduced tumor growth when combined with anti-PD-L1 or genetic TIGIT deficiency, demonstrating a necessary role for PVRIG in regulating T-cell responses. These new data provide proof of concept in vivo using preclinical tumor models that targeting PVRIG in combination with PD1 or TIGIT antagonism is a potential new therapy for the treatment of cancers.
[1537] Here we report a high affinity anti-human PVRIG antibody that disrupts the interaction of PVRIG and PVRL2 that we want to carry out in clinical trials. To determine potential indications in cancer that could provide information on patient selection in clinical trials, we examined the expression profile of this axis in human cancers by FACS and IHC. For PVRIG, we observed that the mean expression of PVRIG in CD4 and CD8 T cells by FACS is higher in endometrial, lung, kidney, and ovarian cancer, although this difference did not achieve a statistical difference with other types of cancer, as determined by ANOVA with a Tukey multiple comparison test with the current number of samples. Since PVRIG is induced after T-cell activation and since most infiltrating T cells in tumors are antigen-scavenged, it is perhaps not surprising that the mean PVRIG expression was similar between tumor samples and cancer types. . We observed that the expression of PVRIG correlated with the expression of PD-1 and TIGIT, which suggests that the interaction of these 3 inhibitory receptors will be important in the regulation of the antitumor response. In this report, we observed a synergistic increase in T-cell function when antibodies to PVRIG were combined with antibodies to TIGIT in a CD8 T-cell tumor cell coculture, better than PD-1 in combination with PVRIG or TIGIT inhibitors. . These data, together with a previous study that demonstrated a role for PVRIG and TIGIT in the regulation of DC-T cell interactions, show that this pathway could be involved in the regulation of T cell-APC and T-cell-tumor cell interactions. , and providing multiple mechanisms by which PVRIG targeting could increase the antitumor immune response.
[1538] As the expression of PD-L1 has been correlated with the clinical response to PD-1 inhibitors, we also analyzed the expression of PVRL2 in tumors by FACS and IHC to evaluate whether certain cancers have a higher expression. When evaluating dissociated tumor cells, we observed that the mean expression of PVRL2 in macrophages from endometrial, head and neck, kidney, lung, and ovary samples was higher compared to other tumor types. Mean PVRL2 expression in non-immune CD45- cells was higher in breast, colorectal, endometrial, lung, ovarian, and prostate cancers compared to other cancers. Based on the expression of PVRIG and PVRL2, we determined that endometrial, head and neck, lung, kidney, and ovarian cancers have a higher incidence of tumors with a high expression of PVRIG and PVRL2 and that these were potential cancers that could respond to inhibitors of this pathway.
[1540] Here it was observed that the expression of PVRL2 can be modulated in antigen-producing cells in vitro by inflammatory mediators, while the expression of PVRL2 in cancer cells was not altered. These data suggest that PVRL2 expression in antigen-presenting cells may be regulated by inflammation and could be an indicator of an inflamed tumor. In fact, we observed that all PD-L1 + tumors also expressed PVRL2, both in tumor cells and in the immune compartment. Expression of PVRL2 in myeloid cells could help predict responses to PVRIG inhibitors in a combination setting with PD-1 or TIGIT to further enhance the antitumor effect. Interestingly, a part of the PD-L1 negative tumors also expressed PVRL2, mainly in tumor cells and not in immune cells. It is reported that the expression of PVR and PVRL2 in epithelial cells is induced in tumorigenesis, as well as in the response to stress and DNA damage. These data are consistent with the in vitro findings that regulation of PVRL2 expression in tumor cells is not dependent on IFN-g. As PD-L1 is induced in an adaptive resistance environment in response to IFN-g and is associated with an inflammatory response, the expression of PVRL2 in the absence of PD-L1 suggests that the expression of PVRL2 is more prevalent than PD-L1 and that PVRL2 is expressed in non-inflamed tumors. Based on the above, it is possible that the presence of PVR and PVRL2 contributes to suppressing immune responses, independently of PD-L1, and that PVRIG and TIGIT inhibitors could be of particular importance in PD-negative patients. L1 or non-responders to / progressing with PD-1 inhibitors.
[1542] In summary, this report provides several new insights into the biology of PVRIG, including the characterization of the expression of this axis in human cancers, demonstrating a prominent role for PVRIG / TIGIT in the regulation of CD8 cell-tumor cell interaction, and showing that PVRIG antagonism in combination with PD-1 inhibition or TIGIT deficiency results in a synergistic reduction in tumor growth. These data extend our current understanding of PVRIG biology and provide the rationale for the clinical trial of CHA.7.518.1.H4 (S241P), a high-affinity anti-PVRIG antibody, in cancer patients.
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[1574] 26. Cerboni C, Fionda C, Soriani A, Zingoni A, Doria M, Cippitelli M, et al. The DNA Damage Response: A Common Pathway in the Regulation of NKG2D and DNAM-1 Ligand Expression in Normal, Infected, and Cancer Cells. Front Immunol 2014 ; 4: 508 doi 10.3389 / fimmu.2013.00508.
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[1578] 28. Overwijk WW, Tsung A, Irvine KR, Parkhurst MR, Goletz TJ, Tsung K, et al. gp100 / pmel 17 is a murine tumor rejection antigen: induction of "self-reactive, tumoricidal T cells using high-affinity, altered peptide ligand. J Exp Med 1998 ; 188 (2): 277-86.
[1580] S. Example 19: tumor cell death assay
[1582] The effect of an anti-human TIGIT antibody and CHA.7.518.1.H4 (S241P), either alone or in combination, on tumor cell death was evaluated by an in vitro co-culture assay with human CD8 + T cells specific for CMV. The HLA-A2 + target cell lines used in the assay were the melanoma cell line, Mel624, which stably expresses human PVR and PVRL2, and the pancreatic adenocarcinoma cell line, Panc05.04, which expresses endogenous levels of PVR and PVRL2. humans. Both tumor cell lines were stably transduced with a luciferase reporter gene via lentiviral transduction (System Biosciences). Mel624 and Panc05.04 cells were pulsed with the CMV pp65 peptide at 0.0033 gg / ml or 0.01 gg / ml at 37 ° C for 1 hour, respectively. Cells were then washed and plated at 20,000 cells / well. A reference anti-human TIGIT antibody and CHA.7.518.1.H4 (S241 P) were added to the culture in combination, or with a hIgG4 isotype control antibody at 10 gg / ml. CMV-specific human CD8 + T cells from three different donors, specified as Donor 4, Donor 72, and Donor 234, were added at 100,000 cells / well. The cocultures were incubated at 37 ° C for 16 hours. After incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature for 30 minutes. Bio-Glo Luciferase Substrate (Promega) was added to each well and the mixture was equilibrated for 10 minutes at room temperature protected from light. Luminescence or relative light units (RLU) was quantified in an En Vision multi-label reader (Perkin Elmer) with an ultrasensitive luminescence detector. Percent specific death was calculated by [(RLU for treatment antibody - RLU for medium alone) / RLU for medium alone] x 100.
[1584] Results
[1586] Figure 99A and B show the effect of treatment with anti-TIGIT antibody and CHA.7.518.1.H4 (S241P) on the death of Mel624 and Panc05.04 cells, respectively. When added to the coculture alone, both the anti-TIGIT antibody and CHA.7.518.1.H4 (S241P) induced significant death by T cells of the tumor cell lines compared to the isotype control antibody. For the anti-TIGIT antibody, the percentage of specific death ranged from 19-41% for Mel624 cells, and 3-44% for Panc05.04 cells in the 3 different CMV-reactive donors tested. For CHA.7.518.1.H4 (S241 P), the percentage of specific death ranged from 16-20% for Mel624 cells, and from 0.21-29% for Panc05.04 cells. In some cases, an additive effect on tumor cell death was observed in the combined treatment of anti-TIGIT antibody and CHA.7.518.1.H4 (S241 P).
[1588] To determine whether the effect of an anti-TIGIT antibody and CHA.7.518.1.H4 (S241P) on tumor cell death was dose dependent, the assay was carried out with a 2-fold dilution series of 10 points for each antibody starting at 0.5 gg / ml for anti-TIGIT antibodies, and 10 gg / ml for CHA.7.518.1.H4 (S241 P) (Figure 100). Mel624 death decreased in a dose-dependent manner when anti-TIGIT antibody, BM26 or c Pa .9.086, were combined with CHA.7.518.1.H4 (S241P). A more potent kill was observed for the combination of CPA.9.086 and CHA.7.518.1.H4 (S241P) with an EC50 of 0.40 ± 0.49 nM, compared to the combination of BM26 and CHA.7.518.1 .H4 (S241 P) with an EC50 of 2.6 ± 1.7 nM.
[1590] T. Example 20: Biophysical measurement of K ^d
[1592] KinExA equilibrium experiments were performed using a KinExA 3200 instrument (Sapidyne Instruments, Boise, ID, USA) at 22 ° C. His-tagged recombinant human TIGIT was obtained from Sino Biologicals (Beijing, China) and reconstituted in 1XPBS. All antigen and antibody samples for KinExA analysis were prepared in degassed PBST buffer (PBS with 0.05% tween 20) with 100 gg / mL filtered BSA and 0.02% sodium azide. The secondary detection antibody used was Alexa Flour 647 (H + L) -labelled goat anti-human IgG (Jackson ImmunoResearch Laboratories) diluted 400 to 700-fold in the PBST buffer (with BSA and azide) described above from a stock preparation of 0.5 mg / mL in 1X PBS, pH 7.4. For each KinExA experiment, ~ 20 gg human TIGIT was diluted in 1 mL of 50 mM sodium carbonate, pH 9.2 that was added directly to 50 mg of azlactone beads (Ultralink Support, Thermo Scientific, Rockford, IL, USA) and were gently shaken overnight at 4 ° C. After gentle shaking, the beds were washed once with 1 M Tris buffer, pH 8.5, containing 10 mg / mL BSA and gently shaken for one hour at room temperature in the same buffer. Coupled beads were added to the bead reservoir on the KinExA instrument and diluted to ~ 30 mL with 1X HBS-N (0.01M Hepes, 0.15M NaCl, GE Healthcare) containing 0.02% sodium azide containing it was also the operating buffer for the KinExA instrument. All antigen-coupled beads were used immediately after preparation.
[1594] For two replicate K ^d measurements for CPA.9.086 (Table 1), 14 concentrations of TIGIT ranging from 957 to 212 pM were equilibrated at room temperature for ~ 72 hours with CPA.9.086 binding sites 2.5 pM and CPA.9,086 1.8pM binding sites. For CPA.9.083, 14 TIGIT concentrations ranging from 478 am-196pM were equilibrated for ~ 72 hours with 1.8pM CPA.9.083 binding sites. For duplicate measurements of the reference antibody, BM26 hIgG4, 14 concentrations of TIGIT ranging from 9.6fM-3.53nM were equilibrated for ~ 72 hours with 20pM b M26 binding sites and 8.0 pM BM26 binding sites . For CHA.9.547.13, 14 concentrations of TIGIT ranging from 10.5fM-2.2nM were equilibrated for ~ 72 hours with binding sites of the CHA.9.547.138pM mAb. The volume that flowed through the bed pack for each equilibrated sample for all experiments ranged from 4mL to 11mL at a flow rate of 0.25 mL / min. Data were fitted with a 1: 1 "standard equilibrium" binding model using KinExA Pro software (Version 4.2.10; Sapidyne Instruments) to estimate K ^d and generate the 95% confidence interval (CI) of the fit. of the curve.
[1596] Results
[1598] Both CPA.9.083 and CPA.9.086 bound human TIGIT with a femtomolar binding affinity, while CHA.9.547.13 and BM26 bound with a picomolar affinity. Thus, CPA.9.083 and CPA.9.086 bound human TIGIT with the highest affinity of the four different antibodies tested.
[1600] Table 1: K ^d measurements of anti-human TIGIT hIgG4 antibodies determined by KinExA
[1602] Antibody K ^d ± 95% CI (n = 1) K ^d ± 95% CI (n = 2)
[1604] CHA.9.547.13 18.8 ± 5.8pM Not determined
[1606] CPA.9.083 694 ± 277fM Not determined
[1608] CPA.9.086 553 ± 230fM 665 ± 378fM
[1610] BM26 8.2 ± 2.8pM 11.2 ± 3.6pM
[1612] U. Example 21: development and functional characterization of CPA.9.086, a new therapeutic antibody directed at the TIGIT immune checkpoint
[1614] Background: TIGIT is a co-inhibitory receptor that is highly expressed on lymphocytes, including CD4 + effector and regulatory (Treg) T cells, CD8 + effector T cells, and NK cells, which infiltrate different types of tumors. The binding of TIGIT with its reported ligands, poliovirus receptor (PVR) and PVR-like proteins (PVRL2 and PVRL3) directly suppresses lymphocyte activation. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a dominant immune escape mechanism for cancer. We report here the biophysical and functional characterization of CPA.9.086, a therapeutic antibody directed to TIGIT. We also demonstrate that the coblocking of TIGIT and a new checkpoint inhibitor, PVRIG, increases T-cell responses.
[1616] Materials and Methods: human antibody discovery campaigns were carried out by phage display and mouse by hybridoma to generate anti-TIGIT therapeutic antibodies. The resulting antibodies were evaluated for their ability to bind with high affinity to recombinant human TIGIT and expressed on the cell surface. The cross-reactivity of the antibodies with mouse and cynomolgus macaque TIGIT was also examined. A subset of antibodies that bound human TIGIT with high affinity, and that cross-reacted with cynomolgus TIGIT, was further characterized for their ability to block the interaction between TIGIT and PVR. Blocking antibodies were screened for their ability to increase antigen-specific T-cell activation in vitro either alone, or in combination with an anti-PVRIG antibody, CHA.7.518.1.H4 (S241 P).
[1618] Results: A leading antibody, CPA.9.086, was identified that binds human TIGIT with high femtomolar affinity. This antibody bound endogenously expressed TIGIT on human CD8 + T cells with a higher affinity than the reference antibodies tested, and also cross-reacted with both cynomolgus and mouse TIGIT. When tested for activity in vitro, CPA.9.086 increased cytokine secretion and tumor cell death by CMV-specific CD8 + T cells with greater or equivalent potency than the reference antibodies tested. Combination of CPA.9.086 with an anti-PD1 or CHA.7.518.1.H4 antibody (S241P) resulted in increased activity of CMV-specific CD8 + T cells. Furthermore, we show that TIGIT is predominantly expressed on Treg and effector CD8 + T cells of solid tumors compared to peripheral blood, suggesting that these populations will likely be the target of CPA.9.086.
[1619] Conclusion: The development of a very high affinity antagonist TIGIT antibody, CPA.9.086, which is currently in preclinical development, is described. We postulate that the femtomolar affinity of CPA.9.086 could lead to a lower and less frequent dosage in patients. CPA.9.086 can increase human T-cell activation either alone or in combination with other checkpoint antibodies. Thus, these data demonstrate the utility of targeting TIGIT, PD1, and PVRIG for the treatment of cancer.

权利要求:
Claims (16)
[1]
1. A composition comprising an antigen-binding domain that binds human TIGIT (SEQ ID NO: 97) comprising:
a) a variable heavy domain comprising SEQ ID NO: 160; and
b) a variable light domain comprising SEQ ID NO: 165.
[2]
2. A composition according to claim 1 wherein said composition is an antibody comprising: a) a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said VH comprises SEQ ID NO: 160; and
b) a light chain comprising VL-VC, wherein said VL comprising SEQ ID NO: 165 and VC is either kappa or lambda.
[3]
3. A composition according to claim 2 wherein the sequence of said CH1-hinge-CH2-CH3 is selected from human IgG1, IgG2 and IgG4, and variants thereof.
[4]
4. A composition according to claim 2 or 3 wherein said heavy chain has SEQ ID NO: 164 and said light chain has SEQ ID NO: 169.
[5]
A composition according to any of claims 2 to 4 further comprising a second antibody that binds to a human checkpoint receptor protein.
[6]
6. A composition according to claim 5 wherein said second antibody binds human PD-1.
[7]
7. A composition according to claim 5 wherein said second antibody binds human PVRIG (SEQ ID NO: 2).
[8]
8. A composition according to claim 7 wherein said second antibody comprises an antigen-binding domain comprising a variable heavy domain comprising SEQ ID NO: 5 and a variable light domain comprising SEQ ID NO: 10.
[9]
9. A composition according to claim 7 wherein the heavy chain of said second antibody has SEQ ID NO: 9 and the light chain of said second antibody has SEQ ID NO: 14.
[10]
10. A nucleic acid composition comprising:
a) a first nucleic acid encoding a variable heavy domain comprising SEQ ID NO: 160; and b) a second nucleic acid encoding a variable light domain comprising SEQ ID NO: 165.
[11]
11. A nucleic acid composition according to claim 10 wherein said first nucleic acid encodes a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said VH comprises SEQ ID NO: 160; and said second nucleic acid encodes a light chain comprising VL-VC, wherein said VL comprising SEQ ID NO: 165 and VC is the lambda domain.
[12]
12. An expression vector composition comprising a first expression vector comprising said first nucleic acid according to claim 10 or 11 and a second expression vector comprising said second nucleic acid according to claim 10 or 11, respectively.
[13]
13. An expression vector composition comprising an expression vector comprising said first nucleic acid according to claim 10 or 11 and said second nucleic acid according to claim 10 or 11, respectively.
[14]
14. A host cell comprising said expression vector composition according to claim 12 or 13.
[15]
15. A method of preparing an anti-TIGIT antibody comprising:
a) culturing said host cell of claim 14 under conditions wherein said antibody is expressed; and b) recovering said antibody.
[16]
16. A composition according to any one of claims 1 to 9 for use in treating cancer by activating T cells.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201662376335P| true| 2016-08-17|2016-08-17|

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US201662376334P| true| 2016-08-17|2016-08-17|

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US201662417217P| true| 2016-11-03|2016-11-03|

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US201762477974P| true| 2017-03-28|2017-03-28|

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US201762513916P| true| 2017-06-01|2017-06-01|

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US201762513771P| true| 2017-06-01|2017-06-01|

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US201762513775P| true| 2017-06-01|2017-06-01|

---

US201762538561P| true| 2017-07-28|2017-07-28|

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PCT/IB2017/001256|WO2018033798A1|2016-08-17|2017-08-17|Anti-tigit antibodies, anti-pvrig antibodies and combinations thereof|

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