binding agent, nucleic acid, expression vector, cell, composition, methods for treating a disease an

专利摘要:
The present invention relates to new binding agents and their use in medicine. Specifically, the invention relates to binding agents, such as bispecific antibodies, which bind to human PD-L1 and which bind to human CD137. Furthermore, the invention relates to uses of the antibodies of the invention and to methods, nucleic acid constructs and host cells for producing antibodies of the invention.
公开号:BR112020001944A2
申请号:R112020001944-5
申请日:2018-08-02
公开日:2020-08-04
发明作者:Isil Altintas；David Satijn；Rik RADEMAKER；Paul Parren；Ugur Sahin；Friederike Gieseke；Alexander Muik；Christian GRUNWITZ
申请人:Genmab A/S；Biontech Ag；
IPC主号:

专利说明:

[001] [001] The present invention relates to new binding agents and their use in medicine. Specifically, the invention relates to binding agents, such as bispecific antibodies, which bind to human PD-L1 and which bind to human CD137. Furthermore, the invention relates to uses of the binding agents of the invention and to methods, nucleic acid constructs and host cells for producing antibodies of the invention. Fundamentals of the invention
[002] [002] CD137 (4-1BB, TNFRSF9) is part of the family of tumor necrosis factor (TNF) receptors (TNFR). CD137 is a co-stimulating molecule of CD8 + and CD4 + T cells, regulatory T cells (Tregs), natural killer cells (NK) and NKT, B cells and neutrophils. In T cells, CD137 is not expressed constitutively, but induced upon activation of T cell receptors (TCR). Stimulation through its natural ligand 4-1BBL or agonist antibodies leads to signaling using the factor associated with TNFR (TRAF) -2 and TRAF-1 as adapters. The initial signaling by CD137 involves poly-ubiquitination reactions of K-63 which, in the end, result in the activation of the nuclear factor (NF) -κB and mitogen-activated protein kinase (MAP) pathways. Signaling leads to increased co-stimulation and proliferation of T cells, cytokine production, maturation and prolonged survival of CD8 + T cells. Agonist antibodies against CD137 have been shown to promote antitumor control by T cells in several preclinical models (Murillo et al. 2008 Clin. Cancer Res. 14 (21): 6895-6906). Antibodies that stimulate CD137 can induce survival and
[003] [003] Programmed death ligand 1, Programmed death ligand 1, PD-L1, PDL1, CD274, B7H1) is a 33 kDa, single-pass type I membrane protein. Three isoforms of PD-L1 have been described, based on alternative splicing. PD-L1 belongs to the immunoglobulin (Ig) superfamily contains a type C2-like domain and type V Ig-like domain. Recently isolated T and B cells express minimal amounts of PD-L1 and a fraction (approximately 16%) of monocytes CD14 + constitutively express PD-L1. However, interferon-γ (IFNγ) is known to regulate PD-L1 positively in tumor cells.
[004] [004] PD-L1 obstructs anti-tumor immunity by 1) tolerating tumor reactive T cells to bind to their receptor, programmed cell death protein 1 (PD-1) (CD279) in activated T cells; 2) making tumor cells resistant to lysis, mediated by CD8 + T cells and Fas ligand, by signaling to PD-1 via PD-L1 expressed in tumor cells; 3) to induce T cell tolerance by reverse signaling through CD80 expressed in T cells (B7.1); and 4) promote the development and maintenance of induced regulatory T cells. PD-L1 is expressed in many human cancers, including melanoma, ovarian, lung and colon cancer (Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6).
[005] [005] Antibodies that block PD-L1 have demonstrated clinical activity in several cancers known to overexpress PD-L1 (including melanoma, non-small cell lung cancer (NSCLC)). For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. It is currently in clinical studies such as immunotherapy for
[006] [006] Horton et al. (J Immunother Cancer. 2015; 3 (Suppl 2): O10) disclose the combination of an agonist antibody against 4-1BB with neutralizing antibody against PD-L1.
[007] [007] The combined therapy of utomilumab and avelumab is currently being tested in the clinic (Chen et al., J Clin Oncol 35, 2017 suppl; abstr TPS7575, and clinical study NCT02554812).
[008] [008] However, despite advances in technique, there is a need for multispecific antibodies that can bind to both PD-L1 and CD137. These will provide simultaneous binding to antigen presenting cells (APCs) or tumor cells that express PD-L1 and to T cells with CD137 expression, resulting in conditioned activation of T cells (cytotoxic). The binding of PD-L1 to PD1 expressed in activated T cells will result in the inhibition of T cells. Thus, the present invention aims to
[009] [009] In a first aspect, the invention provides binding agents that comprise a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein the second antigen binding region inhibits the binding of human PD-L1 to human PD-1. Such binding agents comprising a first and a second antigen binding region have a dual effect.
[0010] [0010] Firstly, through its region of connection to PD-L1, the
[0011] [0011] Finally, the binding agent of the invention inhibits the binding of human PD-L1 to human PD-1, thereby preventing PD-L1 from obstructing tumor immunity through PD-1. In this way, the binding agent prevents T cells from receiving an inhibitory signal through the PD-1 / PD-L1 interaction, while receiving an activation signal through binding to the CD137 molecule, resulting in signaling that reinforces proliferation, activation , effector and memory functions of T cells.
[0012] The PD-L1xCD137 binding agents of the present invention are especially useful in therapeutic settings where the stimulation of an activation receptor, such as CD137, and the blocking of an inhibition signal, such as PD-1 / PD-L1 , in T cells can be achieved simultaneously. This can result in greater magnitude of proliferation, activation and survival
[0013] [0013] In one embodiment of the invention, the PD-L1xCD137 binding agent is a bispecific antibody.
[0014] [0014] In another embodiment of the invention, the binding agent is a bispecific antibody having a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein the second antigen-binding region inhibits the binding of human PD-L1 to human PD-1.
[0015] [0015] In yet another embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein a) the The first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 9, 10, 11, respectively, and a light chain variable region ( VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO: 13, GAS, 14, respectively, and b) the second antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 18, 19, 20, respectively, and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO : 22, DDN, 23, respectively.
[0016] [0016] Alternatively, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, in which a) the first binding region to the antigen comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and
[0017] [0017] In another embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137, which is derived from a humanized antibody and / or a second antigen binding region that binds to PD -L1 human, which is derived from a human antibody.
[0018] [0018] In a further aspect, the invention relates to the use of the binding agents of the invention in medicine, in particular for the treatment of cancer.
[0019] These and other aspects and modalities of the invention, including nucleic acids that encode the amino acid sequences of such linkers for example, antibodies; expression vectors comprising such nucleic acids; cells comprising such nucleic acids or expression vectors; compositions comprising such binding agents, nucleic acids, expression vectors or cells; such binding agents, nucleic acids, expression vectors, cells or compositions for use in the treatment of cancer or other diseases; methods for producing such binding agents, for example, bispecific antibodies; and diagnostic methods and kits based on such binding agents, for example, multispecific antibodies, in particular bispecific ones, are described in more detail below.
[0020] [0020] Figure 1: Sequences alignments of human CD137 and African elephant and wild boar. The amino acids in CD137 of the African elephant and wild boar that differ from those in the human sequence are highlighted in black.
[0021] [0021] Figure 2: Constructions of CD137 by shuffle, containing CD137 domains of African elephant (shuffle 5) or wild boar (shuffle 1-4, 6).
[0022] [0022] Figure 3: Expression of CD137 constructs by shuffling HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137 constructs by shuffling. The expression of the constructions on the cell surface was measured by flow cytometry, using a polyclonal anti-CD137 antibody that recognizes human, wild boar and African elephant CD137.
[0023] [0023] Figure 4: Binding of the CD137-009 antibody to the CD137 constructs by shuffling expressed in HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137 constructs by shuffling and with human CD137 (hCD137 wt), African wild boar CD137 elephant. The binding of the CD137-009 antibody to these constructs expressed in HEK293-T17 cells was measured by flow cytometry. Staining with anti-CD137 polyclonal antibody is shown as a control.
[0024] [0024] Figure 5: Effect of the monovalent antibody b12-FEALxPD- L1-547-FEAR on the PD-1 / PD-L1 interaction. The effect of b12-FEALxPD-L1- 547-FEAR was determined in a PD-1 / PD-L1 inhibition bioassay. The data shown are from the induction factor in relation to the control (no antibody added), from a representative experiment.
[0025] [0025] Figure 6: Schematic representation of the predicted mode of action of bispecific antibodies CD137xPD-L1. (A) PD-L1 is expressed
[0026] [0026] Figure 7: Release of PD-1 / PD-L1-mediated T-cell inhibition and additional co-stimulation of CD8 + T-cell proliferation by CD137-009-FEALxPD-L1-547-FEAR in an antigen-T cell assay specific with active PD-1 / PD-L1 axis. CFSE-labeled T cells electroporated with in vitro translated RNA (IVT) of TCR specific for claudin-6 and PD-1 were incubated with immature dendritic cells electroporated with RNA-IVT-claudin-6 in the presence of CD137-009- FEALxPD- L1-547-FEAR, b12-FEALxPD-L1-547-FEAR or control antibody b12, 0.1 µg / mL and 0.02 µg / mL, for five days. The proliferation of CD8 + T cells was measured by flow cytometry. The data shown are (A and C) representative CFSE histogram of two different donors and (B and D) the corresponding percentages of divided cells and proliferation index, calculated using the FlowJo software. (B) shows the analysis of data from donor 1, represented as shown in (A). (D) shows the analysis of data from donor 2 represented in (C). The error bars (SD) indicate the variation within the experiment (three replicates, using cells from a donor).
[0027] [0027] Figure 8: Analysis of the EC50 value of the bispecific antibody CD137-009-FEALxPD-L1-547-FEAR in an antigen-specific T cell assay with active PD1 / PD-L1 axis. CFSE-labeled T cells
[0028] [0028] Figure 9: Comparison of CD137-009-FEALxPD-L1-547- FEAR with a combination of the two antibodies with monovalent binding to CD137 (CD137-009-FEALxb12-FEAR + b12-FEALxPD-L1-547-FEAR) or the two original antibodies (CD137-009 + PD-L1-547) in an antigen-specific T cell assay with active PD1 / PD-L1 axis. CFSE-labeled T cells electroporated with RNA-IVT-PD-1 and TCR specific for claudin-6 were incubated with immature dendritic cells electroporated with RNA-IVT-claudin-6 in the presence of 0.25 μg / mL (i) CD137- 009- FEALxPD-L1-547-FEAR, (ii) CD137-009-FEALxb12 + b12-FEALxPD-L1- 547-FEAR, (iii) CD137-009-FEALxb12, (iv) b12-FEALxPD-L1-547-FEAR , (v) CD137-009 + PD-L1-547, (vi) CD137-009, (vii) PD-L1-547 or (viii) b12 control antibody for five days. The proliferation of CD8 + T cells was measured by flow cytometry. The data shown are (A) representative histograms with CFSE and (B and C) the corresponding mean values of the percentage of divided cells and proliferation index, calculated using the FlowJo software. Error bars (SD) indicate the variation within the experiment (three replicates, using cells from a donor)
[0029] [0029] Figure 10: Ex vivo expansion of tumor infiltrating lymphocytes
[0030] [0030] Figure 11: Effect of the mouse mCD137-3H3xmPD-L1-MPDL3280A substitute antibody on the proliferation of antigen-specific T cells in an adoptive OT-I cell transfer configuration. Oval + OTy + Thy1.1 + cytotoxic T-cells specific for ovalbumin (OVA), isolated from donor mice, were injected retro-orbitally (r.o.) into virgin (naïve) C57BL / 6 recipient mice. The day after the transfer of adoptive cells, recipient mice were injected ro with 100 µg of OVA as antigenic stimulus, followed by a ro injection of 100 µg or 20 µg of the antibody mCD137-3H3xmPD-L1-MPDL3280A, mCD137-3H3xb12 or mPD-L1-MPDL3280Axb12 per mouse. An injection of PBS (indicated as OVA only in the figure) was used as the baseline reference, and untreated animals were used as a negative control. After 6 days, 100 µL of blood was taken via the r.o. route and analyzed for Thy1.1 + CD8 + T cells. The data shown are (A) a schematic representation of the experimental design of the transfer of adoptive cells OT-I and (B)
[0031] [0031] Figure 12: Antitumor efficacy of the mouse mCD137-3H3xmPD-L1-MPDL3280A replacement antibody in a mouse model with a subcutaneous, syngeneic CT26 tumor. Female BALB / c mice with subcutaneous CT26 tumors were treated with intraperitoneal injections of 20 µg of antibody (i) mCD137-3H3xmPD-L1- MPDL3280A, (ii) mCD137-3H3xb12 or (iii) mPD-L1-MPDL3280Axb or por por (iv) PBS, after the tumors have reached a volume ≥ 30 mm3. The administration schedule was: every 2-3 days for the first eight injections, followed by an injection every 7 days until the end of the experiment. On Day 29, 100 µL of blood was taken via the r.o. route and analyzed for CD8 + gp70-specific T cells. The data shown are (A) tumor growth curves with each line representing a single mouse, (B) the Kaplan-Meier analysis of the resulting survival and (C) the CD8 + gp70-specific T cell frequencies for each treatment group in 29 days after implantation. PFS = progression-free survival.
[0032] [0032] Figure 13: Binding of monospecific, bivalent antibodies against PD-L1 and monovalent b12xPD-L1 antibodies to tumor cells. Binding of PD-L1-547 and b12-FEALxPD-L1-547-FEAR to MDA-MB-231 (A), PC-3 (B) and SK-MES-1 (C) cells. The data shown are of average fluorescence intensities (MFI), as determined by flow cytometry. The bivalent monospecific antibodies b12 were included as a negative control.
[0033] [0033] Figure 14: Binding of antibodies against CD137 to variants of
[0034] [0034] Figure 15: Comparison of PD-L1-547-FEALxCD137-009- HC7LC2-FEAR with a combination of the two monovalent controls (b12- FEALxCD137-009-HC7LC2-FEAR + b12-FEALxPD-L1-547-FEAR) or the two original antibodies (CD137-009-HC7LC2-FEAR + PD-L1-547-FEAR) in a non-antigen-specific T cell proliferation assay. CFSE-labeled PBMCs were incubated with suboptimal concentration of anti-CD3 antibody (0.03 µg / mL and 0.1 µg / mL) or without (without) anti-CD3 antibody (as a negative control for T cell activation), and grown in
[0035] [0035] Figure 16: Determination of EC50 values for the induction of T cell proliferation by PD-L1-547-FEALxCD137-009-HC7LC2-FEARx in a non-antigen-specific T cell proliferation assay. CFSE-labeled PBMCs were incubated for four days with a suboptimal concentration of anti-CD3 antibody and serial dilutions of PD-L1- 547-FEALxCD137-009-HC7LC2-FEAR (1-0,00015 µg / mL) or b12 IgG 1 µg / mL as a control antibody. The data shown are from two representative donors; PBMCs from donor 1 were stimulated with 0.03 µg / mL anti-CD3 (A, B) and donor PBMCs with 0.09 µg / mL anti-CD3 (C, D). The proliferation of CD4 + (A and C) and CD8 + (B and D) T cells was measured by flow cytometry. The data shown are the average values of the expansion index of three replicates, calculated using the FlowJo software v10.4 and adjusted with four logarithmic parameters. The error bars (SD) indicate the variation within the experiment (three replicates, using cells from a donor).
[0036] [0036] Figure 17: Effect of PD-L1-547-FEALxCD137-009- HC7LC2-FEAR on the secretion of 10 pro-inflammatory cytokines in an antigen-specific T cell assay with or without PD-1 electroporation
[0037] [0037] Figure 18: Effect of PD-L1-547-FEALxCD137-009- HC7LC2-FEAR on the secretion of 10 pro-inflammatory cytokines in a non-antigen-specific T cell assay. Human PBMCs were suboptimally stimulated with anti-CD3 antibody in the presence of different concentrations of de-L1-547-FEALxCD137-009-HC7LC2-FEAR (three-fold serial dilutions; ranging from 1 µg / mL to 0.00015 µg / ml) or the control antibody b12, b12-IgG-FEAL. The cytokine levels in the supernatants were determined 48 hours after the addition of the antibody by intercalated multiplex immunoassay using the MSD V-Plex Human Proinflammatory Panel 1 (10-Plex) kit. Each data point represents the mean ± SD of three individual wells. Detailed description of the invention Definitions
[0038] [0038] The term "immunoglobulin" refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, a pair of light chains (L) of low molecular weight and a pair of heavy chains (H), all the four interconnected by disulfide bonds. The immunoglobulin structure has been well distinguished. See, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven
[0039] [0039] The term "amino acid corresponding to position ..." in this specification refers to an amino acid position number in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by aligning with human IgG1. Thus, an amino acid or segment in a sequence that "matches" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically in standard configurations and that has at least 50%, at least 80%, at least 90%, or at least 95% identity with a human IgG1 heavy chain. It is considered well known in the art how to align a sequence or segment in a sequence and thus determine the position, in a sequence, corresponding to an amino acid position according to the present invention.
[0040] [0040] The term "binding agent", in the context of the present invention, refers to any agent capable of binding to desired antigens. In certain embodiments of the invention, the linker is an antibody, antibody fragment or construction thereof. The linker may also comprise synthetic, modified or unnatural moieties, in particular non-peptide moieties. Such portions can, for example, bind desired antigen-binding functionalities or regions such as antibodies or antibody fragments. In one embodiment, the binding agent is a synthetic construct comprising CDRs or variable regions of binding to the antigen.
[0041] [0041] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of a molecule
[0042] [0042] The term "monoclonal antibody" in this specification refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a unique specificity and binding affinity for a given epitope. Thus, the term "human monoclonal antibody" refers to antibodies that exhibit a single binding specificity and with variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be generated by a hybridoma that includes a B cell obtained from a transgenic or non-human transchromosomal animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and
[0043] [0043] The term "bispecific antibody" or "bs", in the context of the present invention, refers to an antibody having two different antigen-binding regions defined by different antibody sequences. In some embodiments, said different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, said different antigen-binding regions bind to different target antigens. A bispecific antibody can be of any shape, including any of the bispecific antibody formats described below.
[0044] [0044] When used in the present, unless contrasted by the context, the term "Fab arm" or "arm" includes a heavy-light chain pair and is used interchangeably with "half a molecule" in the present.
[0045] [0045] When a bispecific antibody is described as consisting of a half molecule antibody "derived from" a first antibody, and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the antibody bispecific was generated by recombination, by any known method, of said half molecules of each of said first and second antibodies in the resulting bispecific antibody. In this context, “recombination” is not intended to be restricted to any particular method of recombination and thus includes all of the methods for producing bispecific antibodies described below, including, for example, recombination by exchanging half a molecule, as well as recombination at the nucleic acid level and / or through the joint expression of two half molecules in the same cells.
[0046] [0046] The term "monovalent antibody" means, in the context of the present invention, that an antibody molecule is capable of binding a single antigen molecule and thus is not capable of cross-linking antigens or cells.
[0047] [0047] The term "complete", when used in the context of an antibody, indicates that the antibody is a fragment, but contains all domains of the particular isotype normally found for that isotype in nature, for example, the VH, CH1 domains , CH2, CH3, hinge, VL and CL for an IgG1 antibody.
[0048] [0048] In this specification, unless contrasted by the context, the term "Fc region" refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least one hinge region, a CH2 domain and a CH3 domain.
[0049] [0049] In this specification, the term "heterodimeric interaction between the first and the second CH3 region" refers to the interaction between the first CH3 region and the second CH3 region in a heterodimeric protein with the first CH3 / second CH3.
[0050] [0050] In this specification, the term "homodimeric interactions of the first and second CH3 regions" refers to the interaction between a first CH3 region and another first CH3 region on a homodimeric protein with the first CH3 / first CH3 and the interaction between a second CH3 region and another second CH3 region on a homodimeric protein with second CH3 / second CH3.
[0051] [0051] In this specification, the terms "to bind or" to bind ", in the context of the binding of an antibody to a predetermined antigen or epitope, is a binding with an affinity corresponding to a KD close to or less than 10-7 M, such as near or less than 10-8 M, such as near or less than 10-9 M, near or less than 10-10 M or near or even less than 10-11 M, when determined by interferometry bi-layer (BLI) or, for example, when determined using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte.
[0052] [0052] The term “kd” (s-1), in this specification, refers to the constant rate of dissociation of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
[0053] [0053] The term “KD” (M), in this specification, refers to the dissociation constant in the balance of an antibody-antigen interaction in particular o.
[0054] [0054] In a preferred embodiment, the antibody of the invention is isolated An "isolated antibody" in this specification is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities. In a preferred embodiment, an isolated bispecific antibody that specifically binds to PD-L1 and CD137 is substantially free of monospecific antibodies that specifically bind to PD-L1 or CD137. In another preferred embodiment, the antibody, or a pharmaceutical composition comprising the antibody, is substantially free of naturally developed antibodies that are unable to bind PD-L1. In yet another preferred embodiment, the antibody of the invention has a structural change in its amino acid sequence, in relation to the structure of a natural anti-PD-L1 antibody, in which said
[0055] [0055] The term "PD-L1", in this specification, refers to programmed death-ligand 1 (programmed death-ligand 1). PD-L1 is found in humans and other species and thus the term “PD-L1” is not limited to human PD-L1 unless otherwise indicated by the context. Sequences of human, monkey (cynomolgus monkey), African elephant, wild boar and mouse PD-L1 can be found through Genbank accession number NP_054862.1, XP_005581836, XP_003413533, XP_005665023 and NP_068693, respectively. The sequence of human PD-L1 is also shown in SEQ ID NO: 28, where amino acids 1-18 are predicted to be a signal peptide. The sequence of monkey PD-L1 (monkey cynomolgus) is also shown in SEQ ID NO: 29, where amino acids 1-18 are predicted to be a signal peptide.
[0056] [0056] The term "PD-L2" in this specification refers to programmed cell death ligand protein 2 1 (Genbank accession number NP_079515).
[0057] [0057] The term "PD-1" in this specification refers to human programmed cell death protein 1, also known as CD279.
[0058] [0058] The term “CD137”, in this specification, refers to the protein of the Human Differentiation Cluster (grouping) 137. CD137 (4- 1BB), also referred to as TNFRSF9, is the receptor for the TNFSF9 / 4-1BBL linker. CD137 is believed to be involved in the activation of T cells. In one embodiment, CD137 is human CD137, with UniProt access number Q07011. The human CD137 sequence is also
[0059] [0059] An "antibody against PD-L1" or "anti-PD-L1 antibody" is an antibody as described above, which specifically binds to the human PD-L1 antigen, PD-L1 in particular.
[0060] [0060] An "antibody against CD13" or "anti-CD137 antibody" is an antibody as described above, which specifically binds to CD137.
[0061] [0061] An "antibody against CD137xPD-L1", "anti-CD137xPD-L1 antibody", "antibody PD-L1xCD137" or "anti-PD-L1xCD137 antibody" is a bispecific antibody, which comprises two different antigen-binding regions , one of which specifically binds to the PD-L1 antigen and the other specifically binds to CD137.
[0062] [0062] The present invention also provides antibodies that comprise functional variants of the VL regions, VH regions or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH or CDR, used in the context of an antibody, still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95 % or more) of the "reference" or "original" antibody's affinity and / or specificity / selectivity and, in some cases, such antibody may be associated with greater affinity, selectivity and / or specificity than the original antibody.
[0063] [0063] Such functional variants typically retain significant sequence identity with the original antibody. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (ie% homology = # of identical positions / # total of positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percentage of identity between two nucleotide or amino acid sequences, for example, being determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which was incorporated in the ALIGN program (version
[0064] Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of the original antibody sequences mainly by conservative substitutions; for example, 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative substitutions for amino acid residues.
[0065] [0065] In the context of the present invention, conservative substitutions can be defined by substitutions within the classes and amino acids reflected in the following table.
[0066] [0066] Classes of amino acid residues for conservative substitutions Acid residues Asp (D) and Glu (E) Basic residues Lys (K), Arg (R), and His (H) Hydrophilic residues without charge Ser (S), Thr ( T), Asn (N) and Gln (Q) Aliphatic unloaded residues Gly (G), Ala (A), Val (V), Leu (L) and Ile (I) Non-polar unloaded residues Cys (C), Met (M) and Pro (P) Aromatic residues Phe (F), Tyr (Y) and Trp (W)
[0067] [0067] In the context of the present invention, the following notes are
[0068] [0068] In the context of the present invention, "inhibition of the binding of PD-L1 to PD-1" refers to any significant reduction that can be detected in the binding of PD-L1 to PD-1 in the presence of an antibody capable of bind to PD-L1. Typically, inhibition means at least a reduction of close to 10%, such as one of at least approximately 15%, for example, one of at least approximately 20%, such as a reduction of at least 40% in the bond between PD-L1 and PD-1, caused by the presence of an anti-PD-L1 antibody. Inhibition of PD-L1 binding to PD-1 can be determined by any suitable technique. In one embodiment, inhibition is determined as described in Example 6 below.
[0069] [0069] The term "specificity" in this specification is intended to have the following meaning unless contrasted by the context. Two antibodies exhibit "the same specificity" if they both bind to the same antigen and epitope.
[0070] [0070] The term "epitope" means a protein determinant capable of specific binding with an antibody. Epitopes typically consist of groupings on the surface of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
[0071] [0071] The term "chimeric antibody" in this specification refers to an antibody in which the variable region is derived from a non-human species (for example, derived from rodents) and the constant region is derived from a different species , such as human. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce the immunogenicity of the antibody. The terms "variable region" or "variable domain", when used in the context of chimeric antibodies, refer to a region comprising the CDRs and immunoglobulin heavy and light chain framework regions. Chimeric antibodies can be generated using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Chapter 15. The chimeric antibody may be a recombinant antibody developed by genetic engineering or enzymatically. Generating a chimeric antibody is within the skill of the art and, therefore, the generation of the chimeric antibody according to the present invention can be performed by methods other than those described herein.
[0072] [0072] The term “humanized antibody” in this specification refers to a non-human antibody obtained by genetic engineering, which contains constant domains of human antibody and variable domains not
[0073] [0073] The term "human antibody" in this specification refers to antibodies having variable and constant regions derived from immunoglobulin germline sequences. Human antibodies can include amino acid residues not encoded by immunoglobulin germline sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" in this specification is not intended to include antibodies in which CDR sequences derived from the germline of another species of mammal, such as a mouse or rat, have been grafted onto human framework sequences. Antibodies.
[0074] [0074] The term “recombinant host cell” (or simply “host cell”) in this specification is intended to refer to a cell in which an expression vector has been introduced, for example, an expression vector that encodes an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells, and lymphocyte cells.
[0075] [0075] The term "treatment" refers to the administration of an effective amount of a therapeutically active antibody of the present invention for the purpose of alleviating, ameliorating, interrupting or eradicating (curing) symptoms or disease states.
[0076] [0076] The term "effective amount" or "therapeutically effective amount" refers to an effective amount, in doses and for periods of time necessary to achieve a desired therapeutic result, A therapeutically effective amount of an antibody may vary according to factors such as the individual's disease state, age, sex and weight and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also that in which any toxic or unfavorable effects of the antibody or part of the antibody are outweighed by the therapeutically beneficial effects.
[0077] [0077] The term "anti-idiotypic antibody" refers to an antibody that recognizes unique determinants generally associated with the antigen-binding site of an antibody.
[0078] [0078] The term "compete" and "competition" refers to the competition between a first antibody and a second antibody for the same antigen. Alternatively, "compete" and "competition" may also refer to the competition between an antibody and an endogenous ligand for binding to
[0079] [0079] As described above, in a first aspect, the invention relates to a binding agent comprising a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to PD -L1 human, where the second antigen binding region inhibits the binding of human PD-L1 to human PD-1.
[0080] Such a binding agent thus comprises two different antigen-binding regions, one of which is capable of binding to PD-L1 and the other of which is capable of binding to CD137.
[0081] [0081] As shown by the inventor of the present invention, a binding agent according to the present invention can activate and / or induce proliferation in one cell by binding to CD137, while simultaneously binding to PD-L1 in another cell. In humans, CD137 is expressed in activated T cells, such as CD8 + T cells and CD4 + T cells, while PD-L1 is expressed predominantly in antigen presenting cells (APCs) such as dendritic cells or tumor cells. Thus, binding agents, such as bispecific antibodies, according to the present invention capable of binding to CD137 and PD-L1 are capable of binding simultaneously to T cells and APCs or T cells and tumor cells. Thus, binding agents such as antibodies
[0082] [0082] Thus, a binding agent, such as a bispecific antibody, of the present invention can be used for the treatment of a disease that can benefit from T cell reactivation, such as cancer.
[0083] [0083] In one embodiment of the invention, the second antigen-binding region binds to human PD-L1 as shown in SEQ ID NO: 28 or to the mature polypeptide thereof.
[0084] [0084] In one embodiment of the invention, the second antigen-binding region binds to the PD-L1 of monkey cynomolgus (Macaca fascicularis) as shown in SEQ ID NO: 29 or to the mature polypeptide thereof. Thus, binding agents with an antigen binding region that exhibits cross-specificity by human and monkey cynomolgus PD-L1 are suitable for preclinical testing in monkey cynomolgus.
[0085] [0085] Different binding agents, such as antibodies, capable of
[0086] [0086] In one embodiment of the invention, the second antigen-binding region that binds to human PD-L1 does not bind to human PD-L2.
[0087] [0087] In one embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises variable regions of an heavy and light chain of an antibody, which competes for binding to human PD-L1 with an antibody which comprises: a. a heavy chain variable region comprising a complementarity determining region 3 of the heavy chain (HCDR3) with the sequence shown in SEQ ID NO: 20 or a sequence in which even an amino acid, such as an amino acid, is modified in SEQ ID NO: 20 and b. a light chain variable region comprising a complementarity determining region 3 of the light chain (LCDR3) with the sequence shown in SEQ ID NO: 23 or a sequence in which up to two amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 23.
[0088] [0088] In one embodiment of the invention, said modified amino acids can be an amino acid substitution, such as a conservative amino acid substitution. In one embodiment of the invention, even an amino acid is modified in SEQ ID NO: 20, as is even a conservative amino acid substitution in SEQ ID NO: 20. In a
[0089] [0089] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises variable regions of an heavy and light chain of an antibody, which competes for binding to human PD-L1 with an antibody which comprises: a. a heavy chain variable region comprising a heavy chain complementarity determining region 1, 2 and 3 (HCDR1, HCDR2 and HCDR3) with the sequence shown in SEQ ID NO: 18, 19 and 20, respectively, and b. a light chain variable region comprising a light chain complementarity determining region 1, 2 and 3 (LCDR1, LCDR2 and LCDR3) with the sequence shown in SEQ ID NO: 22, DDN and 23, respectively.
[0090] [0090] In one embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises variable regions of an antibody heavy and light chain, which competes for binding to human PD-L1 with a region antigen binding pathway comprising a heavy chain variable region (VH) and a light chain variable region (VL), where VH comprises a sequence as shown in: SEQ ID NO: 17, and VL comprises a sequence as shown in: SEQ ID NO: 21. Antibodies that compete for binding to a target antigen can bind to different epitopes on the antigen, where the epitopes are so close to each other that binding a first antibody to an epitope prevents binding of a second antibody to the other epitope. In other situations, however, two different antibodies can bind to the same epitope on the antigen and would compete for binding in a competition trial for binding.
[0091] [0091] In one embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises variable regions of an antibody heavy and light chain, which exhibits the PD-L1 specificity of an antibody that comprises a heavy chain variable region comprising an HCDR3 with the sequence shown in SEQ ID NO: 20, or a sequence in which even one amino acid is modified in SEQ ID NO: 20, and a light chain variable region comprising LCDR3 with the sequence shown in SEQ ID NO: 23 or a sequence in which up to two amino acids are modified in SEQ ID NO: 23.
[0092] [0092] In one embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises variable regions of an heavy and light chain of an antibody, which binds to the same epitope on PD-L1 as a antibody comprising a heavy chain variable region comprising an HCDR1, HCDR2 and HCDR3 with the sequence shown in SEQ ID NO: 18, 19 and 20, respectively, or a sequence in which even one amino acid is modified, in total, in the HCDR sequences shown in SEQ ID NO: 18, 19 and 20 and a light chain variable region comprising an LCDR1, LCDR2 and LCDR3 with the sequences shown in SEQ ID NO: 22, DDN and 23, respectively, or a sequence in which up to two amino acids they are modified, in total, in the LCDR sequences presented in SEQ ID NO: 22, DDN and 23. Currently, modalities are described that allow the modification of up to one amino acid in the three HCDR sequences of VH and modifications of up to two amino acids in the three LC strings DR from VL.
[0093] [0093] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 binds to the same epitope of human PD-L1 as an antibody comprising a VH comprising a sequence as shown in SEQ ID NO: 17 and a
[0094] [0094] In one embodiment of the invention, said second antigen binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an HCDR3 with the sequence shown in SEQ ID NO: 20 or a sequence in which even one amino acid is modified in SEQ ID NO: 20.
[0095] [0095] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an HCDR2 with the sequence shown in SEQ ID NO: 19 or a sequence in which even one amino acid is modified in SEQ ID NO: 19.
[0096] [0096] In an embodiment of the invention, said second antigen binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an HCDR1 with the sequence shown in SEQ ID NO: 18 or a sequence in which even one amino acid is modified in SEQ ID NO: 18.
[0097] [0097] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, wherein the sequence of HCDR1, HCDR2 and HCDR3 comprises the sequence as shown in: SEQ ID NO: 18, 19 and 20,
[0098] [0098] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, wherein the sequence of HCDR1, HCDR2 and HCDR3 comprise the sequence as shown in: SEQ ID NO: 18, 19 and 20, respectively.
[0099] [0099] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a light chain variable region (VL) comprising an LCDR3 with the sequence shown in SEQ ID NO: 23 or a sequence in which up to two amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 23.
[00100] [00100] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a region
[00101] [00101] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VL) comprising an LCDR1 with the sequence shown in SEQ ID NO: 22 or a sequence in which up to two amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 22.
[00102] [00102] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 22, DDN, 23, respectively, in which up to two amino acids, such as two amino acids, such as one amino acid, are modified, in total, in the three sequences of LCDR.
[00103] [00103] In an embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprise the sequence as shown in: SEQ ID NO: 22, DDN, 23, respectively.
[00104] [00104] In an embodiment of the invention, said second antigen binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3 and a variable chain region lightweight (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, where the sequence of HCDR1, HCDR2 and HCDR3s are the sequences as shown in: SEQ
[00105] [00105] In one embodiment of the invention, said second antigen binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 17.
[00106] [00106] In one embodiment of the invention, said second antigen binding region that binds to human PD-L1 comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VL sequence as shown in: SEQ ID NO: 21.
[00107] [00107] Thus, for example, said antigen-binding region capable of binding to human PD-L1 comprises: a VH sequence that has at least 70% amino acid sequence identity with the VH sequence shown in: SEQ ID NO: 17 and a VL sequence that has at least 70% amino acid sequence identity with the VL sequence shown in: SEQ ID NO: 21 or a VH sequence that has at least 75% sequence identity of amino acids with the VH sequence shown in: SEQ ID NO: 17 and a VL sequence that has at least 75% amino acid sequence identity with the VL sequence shown in: SEQ ID NO: 21 or
[00108] [00108] In one embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH), where VH comprises the sequence as shown in: SEQ ID NO : 17.
[00109] [00109] In one embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a light chain variable region (VL), wherein the VL comprises the sequence as shown in: SEQ ID NO : 21.
[00110] [00110] In a preferred embodiment of the invention, said second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) and a variable region (VL), where VH comprises the sequence as shown in: SEQ ID NO: 17 and VL comprises the sequence as shown in: SEQ ID NO: 21.
[00111] [00111] In yet another modality, said VH and VL sequences each comprise three sequences of CDR, CDR1, CDR2 and CDR3, respectively, and four frame sequences, FR1, FR2, FR3 and FR4, respectively, and the the respective VH FR1, FR2, FR3 and FR4 combined framework sequences have at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the respective FR1, FR2, FR3 combined framework sequences and FR4 of said VH sequences and in which the VH CDR sequences do not mutate and where the respective combined FR1, FR2, FR3 and FR4 sequences of the VL have at least 90%, at least 95%,
[00112] [00112] As described, the binding agents of the invention mentioned above comprise a first binding to the antigen that binds to human CD137. Thus, the binding agent according to the invention can be a bispecific antibody having a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein the second antigen-binding region inhibits the binding of human PD-L1 to human PD-1.
[00113] [00113] In one embodiment of the invention, the first antigen binding region binds to human CD137, as shown in SEQ ID NO: 30 or a mature polypeptide thereof.
[00114] [00114] In one embodiment of the invention, the first antigen binding region binds to monkey cynomolgus CD137 (Macaca fascicularis), as shown in SEQ ID NO: 31 or a mature polypeptide thereof. Thus, binding agents having an antigen binding region that exhibits cross-specificity by human and monkey cynomolgus CD137 are suitable for preclinical testing in the cynomolgus monkey.
[00115] [00115] In one embodiment of the invention, the first antigen-binding region binds to human CD137 as shown in SEQ ID NO: 30 or a mature polypeptide of the same to a greater extent, measured by cell binding to cells transfected with the constructs below, what binds to a mutant human CD137 as shown in SEQ ID NO: 33 or a
[00116] [00116] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 binds to at least one,
[00117] [00117] In particular, and as when determined by scanning with alanine, for example, as described below and in Example 13, the binding of the antibody according to the invention to human CD137 may be dependent on one or more of the following residues amino acids: Phe (F) at position 13, Phe (F) at position 30, Thr (T) at position 38, Asp (D) at position 40 and Asn (N) at position 60 of SEQ ID NO: 41, corresponding to F36, F53, T61, D63 and N83, respectively, in SEQ ID NO: 30.
[00118] [00118] According to this modality, the binding of the antibody to a mutant CD137 in which any one or more of the amino acid residues in the positions corresponding to positions 13, 30, 38, 40 and 60 in SEQ ID NO: 41 was / were replaced by alanines, is reduced when compared to wild-type CD137 having the amino acid sequence shown in SEQ ID NO: 41. Preferably, reduced binding is determined, such as z score (relative expression) of said antibody, when lower at -1.5, where the z score (relative expression) for antibody binding to the mutant CD137 is calculated as defined in Example 13.
[00119] [00119] Phe (F) in position 13 and / or Phe (F) in position 30 can have a structural impact on the epitope, without being directly involved in the binding of the antibody. Consequently, the antibody according to the invention can bind to an epitope on human CD137, where Thr (T) at position 38, Asp (D) at position 40 and / or Asn (N) at position 60 of SEQ ID NO: 41 is / are directly involved in antibody binding.
[00120] [00120] In another embodiment, the binding of the antibody according to the invention to human CD137 may be dependent on one or more of the following amino acid residues: Leu (L) in position 1, Gln (Q) in position 2, Pro ( P) in position 4, Gly (G) in position 11, Thr (T) in position 12, Asp
[00121] [00121] According to this modality, the binding of the antibody to a mutant CD137 in which any one or more of the amino acid residues in the positions corresponding to positions 1, 2, 4, 11, 12, 15 and 20 in SEQ ID NO: 41 was / were replaced by alanines, is reduced when compared to wild-type CD137 having the amino acid sequence shown in SEQ ID NO: 41. Preferably, reduced binding is determined, such as z score (relative expression) of said antibody, when less than -1.5, where the z score (relative expression) for antibody binding to the mutant CD137 is calculated as defined in Example 13.
[00122] [00122] The procedure for comparing binding by wild-type CD137 to that of alanine-substituted CD137 may comprise the steps of: i) expressing wild-type and alanine-substituted CD137 in a suitable cell line, such as HEK293 cells; ii) harvest the cells one day after transfection and incubate, for each data point, a sample of 100,000 cells with antibody according to the invention, labeled, as with a compound according to formula I (A488), at room temperature for 30 minutes in FACS buffer (Saline solution with phosphate buffer (PBS), 1% serum bovine albumin, 0.02% sodium azide), iii) wash each sample with FACS buffer, submit the sample to analysis by cytometry flow and determine the geometric mean fluorescence intensity (gMFI) for antibody binding; and iv) Normalize the data with respect to the binding intensity of a specific CD137 cross-blocking antibody and calculate the z score (relative expression) as described in Example 13.
[00123] [00123] As described in Example 13, data can be normalized against the binding strength of a CD137-specific, non-cross-blocking control antibody, using the following equation: in which "position aa" refers to the position that has been mutated to alanine or glycine.
[00124] [00124] The z score can be determined according to the following calculation: where are the mean and standard deviation of the normalized gMFI, calculated from all mutants.
[00125] [00125] In one embodiment of the invention, the first antigen binding region binds to mutant human CD137, as shown in SEQ ID NO: 34 (shuffle 4 / wild boar), or a mature polypeptide thereof, with the same degree to which it binds to a human CD137 as shown in SEQ ID: NO 30, or a mature polypeptide thereof, when measured by cell binding to cells transfected with the constructions shown in SEQ ID NO: 34 and SEQ ID NO: 30. The mutant human CD137 as shown in SEQ ID NO: 34 (Shuffle 4 / wild boar) corresponds to the amino acid sequence of human CD137 where amino acids 89-114 have been replaced by the corresponding amino acids
[00126] [00126] In one embodiment of the invention, said first antigen binding region that binds to human CD137 competes for binding to human CD137 with an antigen binding region comprising a heavy chain variable region (VH) and a variable region light chain (VL), where VH comprises a sequence as shown in: SEQ ID NO: 8 and VL comprises a sequence as shown in: SEQ ID NO: 12.
[00127] [00127] In one embodiment of the invention, said first antigen binding region that binds to human CD137 competes for binding to human CD137 with an antigen binding region comprising a heavy chain variable region (VH) and a variable region light chain (VL), where VH comprises a sequence as shown in: SEQ ID NO: 15 and VL comprises a sequence as shown in: SEQ ID NO: 16.
[00128] [00128] In one embodiment of the invention, said first antigen binding region that binds to human CD137 competes for binding to human CD137 with an antigen binding region comprising a heavy chain variable region (VH) and a variable region light chain (VL), where VH comprises a sequence as shown in: SEQ ID NO: 49 and VL comprises a sequence as shown in: SEQ ID NO: 53.
[00129] [00129] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises variable regions of an heavy and light chain of an antibody, which competes for binding to human CD137 with an antibody comprising:
[00130] [00130] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises variable regions of an antibody heavy and light chain, which competes for binding to human CD137 with an antibody comprising: a. a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3) with the sequence shown in SEQ ID NO: 52 or a sequence in which up to three amino acids, such as three amino acids, such as two amino acids, such as an amino acid, are modified in SEQ ID NO: 52 and b. a light chain variable region comprising a complementarity determining region 3 of the light chain (LCDR3) with the sequence shown in SEQ ID NO: 55 or a sequence in which up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as an amino acid, are modified in SEQ ID NO: 55.
[00131] [00131] In an embodiment of the invention, even an amino acid, such as an amino acid, is modified in the sequence of HCDR3. In one embodiment of the invention, up to two amino acids, such as two amino acids, as
[00132] [00132] In an embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises variable regions of an antibody heavy and light chain, which competes for binding to human CD137 with an antibody comprising: a. a heavy chain variable region comprising a heavy chain complementarity determining region 1, 2 and 3 (HCDR1, HCDR2 and HCDR3) with the sequence shown in SEQ ID NO: 9, 10 and 11, respectively, and b. a light chain variable region comprising a light chain complementarity determining region 1, 2 and 3 (LCDR1, LCDR2 and LCDR3) with the sequence shown in SEQ ID NO: 13, GAS and 14, respectively.
[00133] [00133] In an embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises variable regions of an antibody heavy and light chain, which competes for binding to human CD137 with an antibody comprising: a. a heavy chain variable region comprising a
[00134] [00134] In one embodiment of the invention, said first antigen binding region that binds to human CD137 binds to the same epitope of human CD137 as an antibody comprising a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 16.
[00135] [00135] In one embodiment of the invention, said first antigen binding region that binds to human CD137 binds to the same epitope of human CD137 as an antibody comprising a VH sequence as shown in SEQ ID NO: 49 and a VL sequence as shown in SEQ ID NO: 53.
[00136] [00136] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises variable regions of an antibody heavy and light chain, which exhibits CD137 specificity of an antibody comprising a variable region heavy chain comprising an HCDR3 with the sequence shown in SEQ ID NO: 11, or a sequence in which up to three amino acids, such as three amino acids, such as two amino acids, such as one amino acid are modified in SEQ ID NO: 11, and a light chain variable region comprising an LCDR3 with the sequence shown in SEQ ID NO: 14 or a sequence in which up to four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 14.
[00137] [00137] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises variable regions of an antibody heavy and light chain, which exhibits the CD137 specificity of an antibody comprising a variable region heavy chain comprising an HCDR3 with the sequence shown in SEQ ID NO: 52, or a sequence in which up to three amino acids, such as three amino acids, such as two amino acids, such as one amino acid are modified in SEQ ID NO: 52, and a light chain variable region comprising an LCDR3 with the sequence shown in SEQ ID NO: 55 or a sequence in which up to four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 55.
[00138] [00138] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an HCDR3 with the sequence shown in SEQ ID NO: 11, or a sequence wherein up to three amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 11. In one embodiment of the invention, even one amino acid is modified in the sequence of HCDR3. In one embodiment of the invention, up to two amino acids are modified in the sequence of HCDR3. In one embodiment, up to three of the amino acids are modified in the sequence of HCDR3.
[00139] [00139] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an HCDR3 with the sequence shown in SEQ ID NO: 52 or a sequence in that up to three amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 52. In one embodiment of the invention, even one amino acid is modified in the sequence of HCDR3. In a
[00140] [00140] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an HCDR2 with the sequence shown in SEQ ID NO: 10 or a sequence in that up to three, such as three amino acids, such as two amino acids, such as one amino acid, amino acids are modified in SEQ ID NO: 10. In one embodiment of the invention, even one amino acid is modified in the sequence of HCDR2. In one embodiment of the invention, up to two amino acids are modified in the sequence of HCDR2. In one embodiment, up to three of the amino acids are modified in the sequence of HCDR2.
[00141] [00141] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an HCDR2 with the sequence shown in SEQ ID NO: 51 or a sequence in that up to three, such as three amino acids, such as two amino acids, such as an amino acid, amino acids are modified in SEQ ID NO: 51. In one embodiment of the invention, even one amino acid is modified in the sequence of HCDR2. In one embodiment of the invention, up to two amino acids are modified in the sequence of HCDR2. In one embodiment, up to three of the amino acids are modified in the sequence of HCDR2.
[00142] [00142] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an HCDR1 with the sequence shown in SEQ ID NO: 9 or a sequence in that up to three, such as three amino acids, such as two amino acids, such as one amino acid amino acids are modified in SEQ ID NO: 9. In a
[00143] [00143] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an HCDR1 with the sequence shown in SEQ ID NO: 50 or a sequence in that up to three, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 50. In one embodiment of the invention, even one amino acid is modified in the sequence of HCDR1. In one embodiment of the invention, up to two amino acids are modified in the sequence of HCDR1. In one embodiment, up to three of the amino acids are modified in the sequence of HCDR1.
[00144] [00144] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 sequence and HCDR3 comprises the sequence as shown in: SEQ ID NO: 9, 10 and 11, respectively, in which up to three amino acids are modified, in total, in the three HCDR sequences.
[00145] [00145] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising sequences of HCDR1, HCDR2 and HCDR3, wherein the sequence of HCDR1, HCDR2 and HCDR3 comprises the sequence as shown in: SEQ ID NO: 50, 51 and 52, respectively, in which up to three amino acids are modified, in total, in the three HCDR sequences.
[00146] [00146] In one embodiment of the invention, said first region of
[00147] [00147] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises a variable heavy chain (VH) region comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 sequence and HCDR3 comprises the sequence as shown in: SEQ ID NO: 50, 51 and 52, respectively.
[00148] [00148] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising an LCDR3 with the sequence shown in SEQ ID NO: 14 or a sequence in that up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 14.
[00149] [00149] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising an LCDR3 with the sequence shown in SEQ ID NO: 55 or a sequence in that up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 55.
[00150] [00150] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises a light chain variable region (VL) comprising an LCDR2 having the GAS sequence or a sequence in which up to two amino acids are modified at
[00151] [00151] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising an LCDR2 having the SAS sequence or a sequence in which up to two amino acids are modified in the SAS sequence. In one embodiment of the invention, even an amino acid is modified in the SAS sequence.
[00152] [00152] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising an LCDR1 with the sequence shown in SEQ ID NO: 13 or a sequence in that up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 13.
[00153] [00153] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising an LCDR1 with the sequence shown in SEQ ID NO: 54 or a sequence in that up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified in SEQ ID NO: 54.
[00154] [00154] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising sequences of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 13, GAS, 14, respectively, where up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are
[00155] [00155] In an embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising sequences of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 54, SAS, 55, respectively, where up to four amino acids, such as four amino acids, such as three amino acids, such as two amino acids, such as one amino acid, are modified, in total, in the three LCDR sequences.
[00156] [00156] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising sequences of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 13, GAS, 14, respectively.
[00157] [00157] In an embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising sequences of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 54, SAS, 55, respectively.
[00158] [00158] In a preferred embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3 and a variable region of light chain (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, where the sequences of HCDR1, HCDR2 and HCDR3 are the sequences as shown in: SEQ ID NO: 9, 10, 11, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 are the sequences as shown in: SEQ ID NO: 13, GAS, 14, respectively.
[00159] [00159] In a preferred embodiment of the invention, said first
[00160] [00160] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 15. In an embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 8.
[00161] [00161] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 49.
[00162] [00162] In an embodiment of the invention, said first region of
[00163] [00163] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VL sequence as shown in: SEQ ID NO: 12.
[00164] [00164] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VL sequence as shown in: SEQ ID NO: 53.
[00165] [00165] Thus, for example, said first antigen-binding region capable of binding to human CD137 comprises: a VH sequence that has at least 70% amino acid sequence identity with the VH sequence shown in: SEQ ID NO: 15 and a VL sequence that has at least 70% amino acid sequence identity with the VL sequence shown in: SEQ ID NO: 16 or a VH sequence that has at least 75% amino acid sequence identity amino acids with the VH sequence shown
[00166] [00166] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH), where VH comprises the sequence as shown in: SEQ ID NO: 15 .
[00167] [00167] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL), wherein the VL comprises the sequence as shown in: SEQ ID NO: 16 .
[00168] [00168] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a variable region (VL), wherein the VH sequence comprises the sequence as shown in: SEQ ID NO: 15 and the VL sequence comprises the sequence as shown in: SEQ ID NO: 16.
[00169] [00169] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH), where VH comprises the sequence as shown in: SEQ ID NO: 8 .
[00170] [00170] In one embodiment of the invention, said first antigen-binding region that binds to human CD137 comprises a region
[00171] [00171] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a variable region (VL), wherein the VH sequence comprises the sequence as shown in: SEQ ID NO: 8 and the VL sequence comprises the sequence as shown in: SEQ ID NO: 12.
[00172] [00172] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH), wherein VH comprises the sequence as shown in: SEQ ID NO: 49 .
[00173] [00173] In one embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a light chain variable region (VL), wherein the VL comprises the sequence as shown in: SEQ ID NO: 53 .
[00174] [00174] In an embodiment of the invention, said first antigen binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a variable region (VL), wherein the VH sequence comprises the sequence as shown in: SEQ ID NO: 49 and the VL sequence comprises the sequence as shown in: SEQ ID NO: 53. Bispecific binding agents
[00175] [00175] As described above, the binding agent according to the invention comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1. Thus, the binding agent according to the invention can be a multispecific binding agent, for example, a multispecific antibody or a bispecific antibody.
[00176] [00176] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR3, as shown in: SEQ ID NO: 11, and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the HCDR3 sequence, as shown in: SEQ ID NO: 20.
[00177] [00177] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR2, as shown in: SEQ ID NO: 10, and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR2, as shown in: SEQ ID NO: 19.
[00178] [00178] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, as shown in: SEQ ID NO: 9, and b. the second antigen-binding region comprises a heavy chain variable region (VH) comprising a sequence of
[00179] [00179] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR3, as shown in: SEQ ID NO: 52, and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the HCDR3 sequence, as shown in: SEQ ID NO: 20.
[00180] [00180] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR2, as shown in: SEQ ID NO: 51, and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR2, as shown in: SEQ ID NO: 19.
[00181] [00181] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, as shown in: SEQ ID NO: 50, and b. the second antigen-binding region comprises a
[00182] [00182] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequences of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 9, 10, 11, respectively, and b. the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 18, 19, 20, respectively.
[00183] [00183] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequences of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 50, 51, 52, respectively, and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequences of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 18, 19, 20, respectively.
[00184] [00184] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to PD-L1
[00185] [00185] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising an LCDR2 having the GAS sequence, and b. the second antigen-binding region comprises a light chain variable region (VL) comprising LCDR2 having the DDN sequence.
[00186] [00186] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising an LCDR1 sequence, as shown in: SEQ ID NO: 13, and b. the second antigen binding region comprises a light chain variable region (VL) comprising an LCDR1 sequence, as shown in: SEQ ID NO: 22.
[00187] [00187] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to CD137
[00188] [00188] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising an LCDR2 having the SAS sequence, and b. the second antigen-binding region comprises a light chain variable region (VL) comprising an LCDR2 having the DDN sequence.
[00189] [00189] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising an LCDR1 sequence, as shown in: SEQ ID NO: 54, and b. the second antigen binding region comprises a light chain variable region (VL) comprising an LCDR1 sequence, as shown in: SEQ ID NO: 22.
[00190] [00190] In one embodiment of the invention, the binding agent
[00191] [00191] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising the sequences of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO: 54, SAS and 55, respectively, and b. the second antigen-binding region comprises a light chain variable region (VL) comprising the sequences of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO: 22, DDN, 23, respectively.
[00192] [00192] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of
[00193] [00193] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 50, 51 and 52, respectively, and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO: 54, SAS and 55, respectively, and b. the second antigen-binding region comprises a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 18, 19 and 20, respectively, and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO: 22, DDN and 23, respectively.
[00194] [00194] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to PD-L1
[00195] [00195] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 15 and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 17.
[00196] [00196] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 49 and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 17.
[00197] [00197] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to CD137
[00198] [00198] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 16 and b. the second antigen binding region comprises a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 21.
[00199] [00199] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 53 and b. the second antigen binding region comprises a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 21.
[00200] [00200] In one embodiment of the invention, the binding agent
[00201] [00201] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 15 and a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 16 and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 17 and a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 21.
[00202] [00202] In one embodiment of the invention, the binding agent comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, wherein: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as
[00203] [00203] In yet another embodiment of the invention, the binding agent is a multispecific antibody such as a bispecific antibody.
[00204] [00204] In a preferred embodiment of the invention, the binding agent is a bispecific antibody.
[00205] [00205] In one embodiment of the invention, the binding agent is in the form of a complete antibody or an antibody fragment.
[00206] [00206] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises a first and a second antigen binding region, wherein each of the antigen binding regions comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein, preferably, said variable regions each comprise three CDR, CDR1, CDR2 and CDR3 sequences, respectively, and four framework sequences , FR1, FR2, FR3 and FR4, respectively. Thus, CDRs in variable regions of heavy chain can be indicated as HCDR1, HCDR2 and HCDR3, respectively, and CDRs in variable regions of light chain can be indicated as LCDR1, LCDR2 and LCDR3, respectively. In addition, the framework sequences in the heavy chain variable regions can be indicated as HFR1, HFR2, HFR3 and HFR4, respectively, and the framework sequences in the light chain variable regions can be indicated as LFR1, LFR2, LFR3 and LFR4, respectively.
[00207] [00207] Thus, in an antibody mode
[00208] [00208] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody, such as a bispecific antibody, comprises said first antigen binding region comprising a first heavy chain variable region (VH) and a first light chain variable region (VL) and said second antigen binding region comprises a second heavy chain variable region (VH) and a second light chain variable region (VL).
[00209] [00209] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises a heavy and light chain variable region, wherein each variable region comprises three determining regions of complementarity (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).
[00210] [00210] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises said complementarity determining regions and said framework regions, which are provided with the amino termination for the carboxy termination in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[00211] [00211] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as an antibody
[00212] [00212] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises a variable light chain region, in which the complementarity determining regions and the framework regions are arranged from the amino termination to the carboxy termination in the following order: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4.
[00213] [00213] In one embodiment of the invention, the linker comprises a polypeptide in which the polypeptide is a heavy chain (HC). In one embodiment of the invention, the heavy chain (HC) comprises a heavy chain variable region (VH) and a heavy chain constant region (CH).
[00214] [00214] In one embodiment of the invention, the heavy chain constant region (CH) comprises a region of domain 1 of the constant region (CH1), a hinge region, a region of domain 2 of the constant region (CH2) and a region of domain 3 of the constant region (CH3).
[00215] [00215] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises (i) a polypeptide comprising said first heavy chain variable region (VH) and further comprising a first heavy chain constant region (CH) and (ii) a polypeptide comprising said second heavy chain variable region (VH) and further comprising a second heavy chain constant region (CH).
[00216] [00216] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises (i) a polypeptide comprising said first
[00217] [00217] In one embodiment of the invention, the binding agent is an antibody, such as a multispecific, preferably bispecific, antibody comprising a first binding arm and a second binding arm, wherein: a. the first linker arm comprises i) a polypeptide comprising said first heavy chain variable region (VH) and said first heavy chain constant region (CH) and ii) a polypeptide comprising said first light chain variable region (VL) ) and said first light chain constant region (CL) e; B. the second linking arm comprises iii) a polypeptide comprising said second heavy chain variable region (VH) and said second heavy chain constant region (CH) and iv) a polypeptide comprising said second light chain variable region (VL) ) and said second light chain constant region (CL).
[00218] [00218] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises a first and second heavy chain constant (CH) region comprising one or more of a region of the domain 1 of the constant region (CH1 region), a hinge region, a CH2 region and a CH3 region, preferably at least one hinge region, a CH2 region and a CH3 region.
[00219] [00219] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In one embodiment of the invention, the isotype is
[00220] [00220] In one embodiment of the invention, the first antigen-binding region is derived from a rabbit antibody. In one embodiment of the invention, the first antigen-binding region is derived from a humanized antibody. In one embodiment of the invention, the first binding arm is derived from a complete antibody. In one embodiment of the invention, the first binding arm is derived from a monoclonal antibody. In one embodiment of the invention, the first binding arm is derived from a complete IgG1 antibody, IgG1 λ (lambda) or κ (kappa). In one embodiment of the invention, the second antigen-binding region is derived from a mouse antibody. In one embodiment of the invention, the second antigen-binding region is human. In one embodiment of the invention, the second antigen-binding region is derived from a humanized antibody. In one embodiment of the invention, the second binding arm is derived from a complete antibody. In one embodiment of the invention, the second binding arm is derived from a monoclonal antibody. In one embodiment of the invention, the second binding arm is derived from a complete IgG1 antibody, IgG1 λ (lambda) or κ (kappa). In one embodiment of the invention, the first and second antigen-binding regions are derived from humanized antibodies. In one embodiment of the invention, the first and second antigen-binding regions are human antibodies. In one embodiment of the invention, the first and second binding arms are derived from complete antibodies, such as complete IgG1 antibodies, from IgG1 λ (lambda) or κ (kappa). In one embodiment of the invention, the first and second link arms are derived from monoclonal antibodies.
[00221] [00221] In one embodiment of the invention, the first antigen-binding region is derived from an IgG1 lambda and the second antigen-binding region is derived from an IgG1 kappa.
[00222] [00222] The antibodies described herein include IgG1, IgG2, IgG3 and IgG4 antibodies and combinations thereof, wherein the heavy chains are of different isotypes and / or subclasses. In several embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, of the IgG1 kappa or lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (for example, IgG2a, κ, λ), an antibody IgG2b (for example, IgG2b, κ, λ), an IgG3 antibody (for example, IgG3, κ, λ) or an IgG4 antibody (for example, IgG4, κ, λ).
[00223] [00223] In one embodiment of the invention, the binding agent is a multispecific binding agent such as bispecific. In one embodiment of the invention, the binding agent is an antibody (in particular a multispecific antibody, for example, a bispecific antibody), such as a chimeric or humanized or human antibody. In one embodiment of the invention, the linker is in the form of a complete antibody or an antibody fragment. In one embodiment of the invention, the first antigen-binding region is derived from a monoclonal antibody. In one embodiment of the invention, the second antigen-binding region is derived from a monoclonal antibody. In one embodiment of the invention, the first antigen-binding region is derived from a monoclonal antibody and the second antigen-binding region is derived from a monoclonal antibody.
[00224] [00224] In one embodiment of the invention, the binding agent is a complete IgG1 antibody. In one embodiment of the invention, the binding agent is a complete human IgG1 antibody. In one embodiment of the invention, the binding agent is a complete human IgG1 antibody with one or more mutations in the constant region.
[00225] [00225] In one embodiment of the invention, the binding agent is a chimeric, humanized or human antibody. In embodiments of the invention, where the binding agent is a bispecific antibody, where the half molecules can be human, humanized or chimeric, or the half
[00226] [00226] In one embodiment of the invention, the binding agent comprises a first and a second antigen binding region, wherein: a. the first antigen-binding region that binds to CD137 is derived from a chimeric antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a chimeric antibody.
[00227] [00227] In one embodiment of the invention, the binding agent comprises a first and a second antigen binding region, wherein: a. the first antigen-binding region that binds to CD137 is derived from a humanized antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a humanized antibody.
[00228] [00228] In one embodiment of the invention, the binding agent comprises a first and a second antigen binding region, wherein: a. the first antigen binding region that binds to human CD137 is derived from a human antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a human antibody.
[00229] [00229] In a preferred embodiment of the invention, the binding agent comprises a first and a second antigen binding region, wherein: a. the first antigen-binding region that binds to human CD137 is derived from a humanized antibody and / or b. the second antigen-binding region that binds to PD-
[00230] [00230] In one embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody, comprises a first and a second heavy chain constant (CH) region comprising a CH3 region and wherein the two CH3 regions comprise asymmetric mutations.
[00231] [00231] In a preferred embodiment of the invention, the binding agent, in particular in the form of a multispecific antibody such as a bispecific antibody comprises a first and a second heavy chain constant region (CH), wherein each of said first and second heavy chains comprise at least one hinge region, a CH2 region and a CH3 region, wherein, in said first heavy chain constant region (CH), at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, according to the EU numbering, has been replaced and, in said second heavy chain at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, according to the EU numbering, has been replaced, and in which said first and second heavy chain are not replaced in the same positions.
[00232] [00232] More preferably, (i) the amino acid at the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, is L, in said first heavy chain constant region (CH), and the amino acid in position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, is R in said second heavy chain constant region (CH), or (ii) the amino acid in the position corresponding to K409 in an IgG1 heavy chain according to the EU numbering, is R in said first heavy chain and the amino acid in
[00233] [00233] In one embodiment of the invention, the binding agent is an antibody, such as a multispecific antibody, preferably bispecific, wherein said antibody induces an Fc-mediated effector function to a lesser extent when compared to another antibody comprising the same first and second antigen binding regions and two heavy chain constant regions (CHs) comprising hinge and human CH2 and CH3 regions. In one embodiment of the invention, said first and second heavy chain constant regions are modified so that the antibody induces an Fc-mediated effector function to a lesser extent when compared to an antibody that is identical except that it comprises first and second heavy chains not modified.
[00234] [00234] In one embodiment of the invention, said Fc-mediated effector function is measured by binding to IgG Fc (Fcγ) receptors, C1q binding or Fc-mediated cross-linking induction between FcRs.
[00235] [00235] In a preferred embodiment of the invention, said Fc-mediated effector function is measured by binding to C1q.
[00236] [00236] In one embodiment of the invention, said first and second heavy chain constant regions have been modified so that the binding of C1q to said antibody is reduced when compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, wherein binding to C1q is preferably determined by ELISA.
[00237] [00237] In one embodiment of the invention, in at least one of said first and second heavy chain constant regions, one or more amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain, according to EU numbering, they are not L, L, D, N and P, respectively. In a modality
[00238] [00238] In yet another especially preferred embodiment, the binding agent is a bispecific antibody PD-L1xCD137 comprising a first and a second heavy chain constant region, with positions corresponding to positions L234, L235 and D265 on a heavy chain of human IgG1, according to the EU numbering, of the first and second heavy chain constant regions are F, E and A, respectively, and where (i) the position corresponding to F405 in a human IgG1 heavy chain, according to EU numbering, the first heavy chain is L and the position corresponding to K409 in a human IgG1 heavy chain, according to EU numbering, the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, of the first heavy chain is R and the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, of the second heavy chain is L.
[00239] [00239] In one embodiment of the invention, the binding agent induces and / or enhances the proliferation of T cells. In one embodiment of the invention, said T cells are CD4 + and / or CD8 + T cells.
[00240] [00240] In one embodiment of the invention, the binding agent activates CD137 signaling only when the second antigen binding region binds to PD-L1.
[00241] [00241] In one embodiment of the invention, T cell proliferation is measured by co-culturing T cells that express a T cell receptor
[00242] [00242] In one embodiment, said induction or intensification of T cell proliferation is determined by a specific antigen assay, where DCs are transfected with claudin-6 antigen and T cells are transfected with a TCR that recognizes an epitope derived from claudina-6 presented in HLA-A2 in DC. This test is described in Example 7.
[00243] [00243] The bispecific binding agent of the invention may be able to mediate the expansion of tumor infiltrating lymphocytes (TILs) in an ex vivo culture of human tumor tissue. The expansion of TILs can be 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more or 10 times or more. The expansion of CD3-CD56 + natural killer (NK) cells can be at least 10 times, such as at least 20 times, at least 30 times, at least 40 times, or as at least 50 times. The expansion of CD3 + CD8 + cytotoxic T lymphocytes (CTLs) can be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times or as at least 7 times. Preferably, the expansion of TILs is determined as the expansion of TIL of a human non-small cell lung carcinoma specimen in response to incubation with a concentration of bispecific binding agent corresponding to 0.01; 0.1 and 1 µg / ml, as in response to incubation with a bispecific binding agent concentration corresponding to 0.1 µg / ml.
[00244] [00244] The expansion of TILs can be determined in any assay comprising the steps of: i) providing a resection specimen, such as a fresh resection specimen, of tumor tissue and washing the specimen in hematopoietic cell medium,
[00245] The bispecific binding agent of the invention may be able, in particular, to induce the expansion of CD40 + and CD8 + T cells in a population of peripheral blood mononuclear cells (PBMCs), in which T cells are activated, such as activated suboptimally, by incubation with an anti-CD3 antibody, such as the UCHT1) [sic] clone, preferably at a concentration between 0.03 and 0.1 µg / ml and are preferably incubated with the bispecific binding agent of according to the invention at a concentration corresponding to 0.2 μg / mL. Specifically, the process for determining T cell expansion can comprise the steps of: i) obtaining buffy coats PBMCs from healthy donors, such as by Ficoll gradient isolation, ii) labeling PBMCs with succinimidyl carboxyfluorescein ether ester (CFSE) in PBS,
[00246] [00246] As described above, various formats of antibodies have been described in the art. The binding agent of the invention can, in principle, be an antibody of any isotype. The choice of isotype will typically be guided by the desired Fc-mediated effector functions, such as ADCC induction or the requirement for an antibody lacking the Fc-mediated effector function ("inert" antibody). Exemplary isotypes are IgG1, IgG2, IgG3 and IgG4. Any of the human light chain constant regions, kappa or lambda, can be used. The effector function of the antibodies of the present invention can be modified by changing the isotype to, for example, an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody of various therapeutic uses. In one embodiment, the two heavy chains of an antibody of the present invention are of the IgG1 isotype, for example, an IgG1, κ. Optionally, the heavy chain can be modified in the hinge and / or CH3 region as described elsewhere in the present.
[00247] [00247] Preferably, each of the antigen-binding regions
[00248] [00248] In one embodiment of the invention, the binding agent is a complete antibody, such as a complete IgG1 antibody. In another embodiment, the antibody is a complete IgG4 antibody, preferably with a stabilized hinge region. Modifications that stabilize the IgG4 hinge region, such as the S228P mutation in the central hinge region, have been described in the art, see for example, Labrijn et al., 2009 Nat Biotechnol. 27 (8): 767-71.
[00249] [00249] In other embodiments of the invention, the inventive binding agent comprises an antibody fragment, such as a Fab 'fragment or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, a monovalent antibody as described in WO2007059782 (Genmab), an F (ab ') 2 fragment, an Fd fragment, an Fv fragment, an dAb, camelid or nanobodies fragment or an isolated complementarity determining region (CDR).
[00250] The binding agents of the invention are preferably human, humanized or chimeric antibodies. In embodiments, where the antibody is a bispecific antibody, the two half molecules may be human, humanized or chimeric, or the half molecules may differ in character with respect to the origin of the sequence.
[00251] [00251] For example, in one embodiment, the binding agent, for example, bispecific antibody, comprises two half molecules, each comprising an antigen binding region, in which: (i) the half (s) ) molecule (s) comprising the
[00252] [00252] For example, in another embodiment, the bispecific antibody comprises two half molecules, each comprising an antigen-binding region, wherein: (i) the half molecule (s) comprising the region of binding to the antigen capable of binding to human PD-L1 is / are humanized (s) and / or (ii) the half molecule comprising the antigen binding region capable of binding to human CD137, if present, is humanized.
[00253] [00253] For example, in yet another embodiment, the bispecific antibody comprises two half molecules, each comprising an antigen-binding region, in which: (i) the half molecule (s) comprising the region binding antigen capable of binding human PD-L1 is human (s) and / or (ii) the half molecule comprising the antigen binding region capable of binding human CD137, if present, is human.
[00254] [00254] Thus, for example, in one embodiment, the antigen-binding region (s) capable of binding to the human PD-L1 is humanized, and the binding region to the antigen capable of binding to human CD137, if present, is humanized.
[00255] [00255] In an embodiment of the invention, the antigen-binding region (s) capable of binding human PD-L1 is / are human, and the antigen-binding region capable of binding binding to human CD137, if present, is human.
[00256] [00256] In yet another embodiment, the binding agent is a bispecific antibody comprising an antigen binding region capable of
[00257] [00257] Preferably, the half molecule comprising the antigen binding region capable of binding to human PD-L1 is human and the half molecule comprising the antigen binding region capable of binding to human CD137 is humanized. Bispecific antibody formats
[00258] [00258] Many different formats and uses of bispecific antibodies are known in the art, and have been reviewed by Kontermann; Drug Discov Today, 2015 Jul; 20 (7): 838-47 e; MAbs, 2012 Mar-Apr; 4 (2): 182-97.
[00259] [00259] A bispecific antibody according to the present invention is not limited to any particular bispecific format or method of producing it.
[00260] [00260] Examples of bispecific antibody molecules that can be used in the present invention comprise (i) a single antibody with two arms comprising different regions of antigen binding; (ii) a single chain antibody with specificity for two different epitopes, for example, via two scFvs linked in tandem by an extra peptide linker; (iii) a double variable domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Imunmuglobulin ( DVD-Ig ™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically linked bispecific (Fab ') 2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a bispecific tetravalent antibody that has two
[00261] [00261] In one embodiment of the invention, the binding agent of the present invention is a diabody or a cross-body. In one embodiment, the binding agent of the invention is a bispecific antibody obtained through a controlled exchange of Fab arms (as described in WO2011131746 (Genmab)).
[00262] [00262] Examples of different classes of binding agents according to the present invention include, among others (i) IgG- like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant double-directed IgG-like molecules, where both sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which complete IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, in which single chain Fv molecules or stabilized diabodies are fused to heavy chain constant domains, Fc regions or parts thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together, fused to heavy chain constant domains, Fc regions or parts thereof; and (vi) heavy chain antibodies based on ScFv and diabody (for example, domain antibodies, nanobodies) in which different Fv molecules from
[00263] [00263] Examples of IgG-like molecules with molecules from the complementary CH3 domain include, among others, the Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), the so-called Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and the electrostatically combined molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), the LIGHT-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012. 287 (52): 43331-9, doi: 10,1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body molecules (SEEDbody) (EMD Serono, WO2007110205), the Biclonics molecules (Merus, WO2013157953), the FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), the Mercymetric frameworks , WO2012058768), the mAb-Fv molecules (Xencor, WO2011028952), anti bivalent bispecific bodies (WO2009080254) and DuoBody® molecules (Genmab, WO2011131746).
[00264] [00264] Examples of double-directed IgG-like recombinant molecules include, among others, double-directed Ig (DT) molecules (WO2009058383), two-in-one antibody (Genentech; Bostrom, et al. 2009. Science 323, 1610–1614.), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr; 5 (2): 208-18), common light chain approaches (Crucell / Merus, US 7,262,028), κλBodies (NovImmune, WO2012023053) and CovX-body (CovX / Pfizer;
[00265] [00265] Examples of IgG fusion molecules include, but are not limited to, double variable domain Ig molecules (DVD) (Abbott, US 7 612 181), antibodies with double variable domain heads (Unilever; Sanofi Aventis, WO20100226923) , bispecific IgG-like molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb; 32 (2): 191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30; 393 (3): 672-92) and BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).
[00266] [00266] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep; 42 (6): 1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538) and molecules (ScFv ) 2-Fab Double (National Research Center for Antibody Medicine - China).
[00267] [00267] Examples of bispecific Fab fusion antibodies include, but are not limited to, F (ab) 2 molecules (Medarex / AMGEN; Deo et al. J Immunol. 1998 Feb 15; 160 (4): 1677-86.), Double-Action or Bis-Fab molecules (Genentech, Bostrom, et al. 2009. Science 323, 1610–1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J Immunol. 2000 Dec 15; 165 (12): 7050-7.) And Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).
[00268] [00268] Examples of antibodies based on ScFv, diabody and domain antibodies include, but are not limited to, bispecific molecules
[00269] [00269] In one aspect, the bispecific antibody of the invention comprises a first Fc sequence, comprising a first CH3 region, and a second Fc sequence comprising a second CH3 region, in which the sequences of the first and the second CH3 region are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), the contents of which are hereby incorporated by reference.
[00270] [00270] As described in more detail below, a stable bispecific PD-L1xCD137 antibody can be obtained in high yield using a particular method based on a homodimeric antibody against initial PD-L1 and a homodimeric antibody against initial CD137 containing only a few asymmetric mutations conservative in CH3 regions. Asymmetric mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions in non-identical positions.
[00271] [00271] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at a position selected from the group consisting of: 366, 368, 370 , 399, 405, 407 and 409 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution in a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain, and where the first and second CH3 regions are not substituted at the same positions.
[00272] [00272] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 366 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution in a position selected from the group consisting of: 368, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain. In one embodiment, the amino acid at position 366 in a human IgG1 heavy chain is selected from Ala, Asp, Glu, His, Asn, Val or Gln.
[00273] [00273] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 368 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution at a position selected from the group consisting of: 366, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain.
[00274] [00274] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 370 in a human IgG1 heavy chain, and a second CH3 region with
[00275] [00275] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 399 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution in a position selected from the group consisting of: 366, 368, 370, 405, 407 and 409 in a human IgG1 heavy chain.
[00276] [00276] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 405 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409 in a human IgG1 heavy chain.
[00277] [00277] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 407 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 409 in a human IgG1 heavy chain.
[00278] [00278] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises a first CH3 region with an amino acid substitution at position 409 in a human IgG1 heavy chain, and a second CH3 region with an amino acid substitution at a position selected from the
[00279] Thus, in one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, comprises the sequences of said first and second CH3 regions containing asymmetric mutations, that is, mutations in different positions in the two CH3 regions. , for example, a mutation at position 405 in one of the CH3 regions and a mutation at position 409 in the other CH3 region.
[00280] [00280] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, the first CH3 region has an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of : 366, 368, 370, 399, 405 and 407. In such a modality, said first CH3 region has an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe , Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has an amino acid excluding Phe, for example, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys or Leu, in position 405. In yet another modality of the same, said first CH3 region has an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has an amino acid excluding Phe, Arg or Gly, for example, Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 405.
[00281] [00281] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises Phe at position 405 and an amino acid
[00282] [00282] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises Phe at position 405 and an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region comprises Leu at position 405 and Lys at position 409 In yet another embodiment thereof, said first CH3 region comprises Phe at position 405 and Arg at position 409 and said second CH3 region comprises an amino acid excluding Phe, Arg or Gly, for example, Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 405, and Lys at position 409. In another embodiment, said first CH3 region comprises Phe at position 405 and Arg at position 409 and said second CH3 region comprises Leu at position 405 and Lys at position 409.
[00283] [00283] In yet another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises an amino acid excluding Lys, Leu or
[00284] [00284] In yet another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises Lys at position 370, Phe at position 405 and Arg at position 409 and said second region CH3 comprises Lys at position 409, Thr at position 370 and Leu at position 405.
[00285] [00285] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser , Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region comprises Lys at position 409 and: a) Ile at position 350 and Leu in position 405, or b) Thr in position 370 and Leu in position 405.
[00286] [00286] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises Arg at position 409 and said second CH3 region comprises Lys at position 409 and: a) Ile at position 350 and Leu in position 405, or b) Thr in position 370 and Leu in position 405.
[00287] [00287] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409 and a said second CH3 region comprises Lys at position 409 and: a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.
[00288] [00288] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409 and a said second CH3 region comprises Ile at position 350, Thr at position 370, Leu at position 405 and Lys at position 409.
[00289] [00289] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has an amino acid excluding Lys, Leu or Met at position 409, and said second CH3 region has an amino acid excluding Phe at position 405, such as excluding Phe, Arg or Gly at position 405; or said first CH3 region has an amino acid excluding Lys, Leu or Met at position 409 and said second CH3 region has an amino acid excluding Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.
[00290] [00290] In one embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein comprises a first CH3 region with an amino acid excluding Lys, Leu or Met at position 409 and a second CH3 region with an amino acid excluding Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.
[00291] [00291] In one embodiment, bispecific antibody of the invention, as defined in any of the embodiments disclosed herein comprises a first CH3 region with Tyr at position 407 and an amino acid excluding Lys, Leu or Met at position 409 and a second CH3 region with a amino acid excluding Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and Lys at position 409.
[00292] [00292] In one embodiment of the invention, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first CH3 region with Tyr at position 407 and Arg at position 409 and a second CH3 region with an amino acid excluding Tyr, Asp, Glu , Phe,
[00293] [00293] In another embodiment of the invention, said first CH3 region has an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu , Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has an amino acid excluding Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, for example, Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp or Cys, at position 407. In another embodiment of the invention, said first CH3 region has an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has Ala, Gly, His, Ile, Leu , Met, Asn, Val or Trp at position 407.
[00294] [00294] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser , Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has Gly, Leu, Met, Asn or Trp at position 407.
[00295] [00295] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has Tyr at position 407 and an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has an amino acid excluding Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, for example, Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407, and Lys at position 409.
[00296] [00296] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has Tyr at position 407 and an amino acid excluding
[00297] [00297] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has Tyr at position 407 and an amino acid excluding Lys, Leu or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and said second CH3 region has Gly, Leu, Met, Asn or Trp in position 407 and Lys in position 409.
[00298] [00298] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has Tyr at position 407 and Arg at position 409 and said second CH3 region has an amino acid excluding Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, for example, Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, in position 407, and Lys in position 409.
[00299] [00299] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has Tyr at position 407 and Arg at position 409 and said second CH3 region has Ala, Gly, His , Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409.
[00300] [00300] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, said first CH3 region has Tyr at position 407 and Arg at position 409 and said second CH3 region has Gly, Leu, Met , Asn or Trp at position 407 and Lys at position 409.
[00301] [00301] In another embodiment, the bispecific antibody of the invention, as defined in any of the embodiments disclosed herein, the first
[00302] [00302] In one embodiment, the first CH3 region has Arg, Ala, His or Gly at position 409, and the second CH3 region has: (i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn , Arg, Ser, Thr, Val or Trp at position 368, or (ii) Trp at position 370, or (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys , Arg or Tyr at position 399, or (iv) Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.
[00303] [00303] In one embodiment, the first CH3 region has Arg at position 409, and the second CH3 region has: (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val or Trp at position 368, or ( ii) Trp at position 370, or (iii) Phe, His, Lys, Arg or Tyr at position 399, or (iv) Ala, Asp, Glu, His, Asn, Val, Gln at position 366.
[00304] [00304] In a preferred embodiment of the invention, the bispecific antibody comprises a first and a second heavy chain, wherein each of said first and second heavy chains comprises at least one hinge region, a CH2 region and a CH3 region, wherein (i) the amino acid at the position corresponding to F405 on the human IgG1 heavy chain is L on said first heavy chain, and the amino acid at the position corresponding to K409 on the human IgG1 heavy chain is R on said second heavy chain, or (ii) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in said first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in said second heavy chain.
[00305] [00305] In addition to the amino acid substitutions specified above, said first and second heavy chains may also contain substitutions, deletions or insertions of amino acids in relation to wild-type heavy chain sequences.
[00306] [00306] In an embodiment of the invention, neither said first nor said second Fc sequence comprises a Cys-Pro-Ser-Cys sequence in the (central) hinge region.
[00307] [00307] In yet another embodiment of the invention, said first and said second Fc sequence comprises a Cys-Pro-Pro-Cys sequence in the (central) hinge region. Methods for preparing bispecific antibodies
[00308] [00308] Traditional methods such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26: 649)) can be used in the preparation of the bispecific antibodies of the invention. Coexpression in a host cell of two antibodies, which consist of different heavy and light chains, leads to a mixture of possible antibodies produced in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography
[00309] [00309] Strategies that favor the formation of a bispecific, functional product, when coexpressing different antibody constructs, can also be used, for example, the method described by Lindhofer et al. (1995 J Immunol 155: 219). The fusion of rat and mouse hybridomas, which produce different antibodies, leads to a limited number of heterodimeric proteins because of the preferential pairing restricted to heavy / light chain species. Another strategy to promote the formation of heterodimers on homodimers is a “knob-into-hole” strategy in which a protuberance is introduced in a first heavy chain polypeptide and a corresponding cavity in a second heavy chain polypeptide, so that the protuberance can be positioned in the cavity at the interface of these two heavy chains in order to promote the formation of heterodimers and prevent the formation of homodimers. "Bulges" are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains. Compensating "cavities" of identical or similar size to the protrusions are created at the interface of the second polypeptide, replacing large side chains of amino acids with smaller ones (U.S. Patent 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies to favor the formation of heterodimers when coexpressing different antibody domains in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in the two CH3 domains are replaced by a charged amino acid, such that the formation of homodimers is electrostatically unfavorable and the heterodimerization is electrostatically favorable. WO2007110205 (Merck) describe yet another strategy, in which differences between CH3 domains of IgA and IgG are exploited to promote heterodimerization.
[00310] [00310] Another in vitro method for producing bispecific antibodies
[00311] A preferred method for preparing the bispecific PD-L1xCD137 antibodies of the present invention includes the methods described in WO2011131746 and WO2013060867 (Genmab) which comprise the following steps: a) providing a first antibody comprising an Fc region, said Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region, wherein the first antibody is an antibody against CD137 and the second antibody is an antibody against PD-L1, or vice versa; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody PD-L1xCD137.
[00312] [00312] Likewise, a method is provided for producing an antibody according to the invention, comprising the steps of: a) culturing a host cell that produces a first antibody comprising an antigen binding region capable of binding to CD137 human, as defined herein, and purify said first antibody of the culture;
[00313] [00313] In an embodiment of the invention, said first antibody together with said second antibody are incubated under sufficient reducing conditions to allow the cysteines in the hinge region to undergo isomerization of the disulfide bonds, wherein the interaction between said first and second antibodies in the resulting heterodimeric antibody is such that there is no exchange of Fab arm in 0.5 mM GSH after 24 hours at 37 ° C.
[00314] [00314] Without being limited to theory, in step c), the disulfide bonds of the heavy chains in the hinge regions of the original antibodies are reduced and the resulting cysteines are then able to form disulfide bonds between heavy chains with cysteine residues from the other molecule of the original antibody (originally with a different specificity). In one embodiment of this method, the reducing conditions in step c) comprise the addition of a reducing agent, for example, a reducing agent selected from the group consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and beta-mercapto-ethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. In yet another embodiment, step c) comprises restoring the conditions so that they become non-reducing or less reducing, for example, by removing a reducing agent, for example, by desalination.
[00315] [00315] For this method, any of the antibodies against CD137 and PD-L1 described above can be used including a first and second antibodies against CD137 and PD-L1, respectively, comprising a first and / or a second Fc region. Examples of such first and second Fc regions, including the combination of such first and second Fc regions can include any of those described above. In a particular embodiment, the first and second antibodies against CD137 and PD-L1, respectively, can be chosen to obtain a bispecific antibody as described herein.
[00316] [00316] In one embodiment of this method, said first and / or second antibodies are complete antibodies.
[00317] [00317] The Fc regions of the first and second antibodies can be of any isotype, including, but not limited to, IgG1, IgG2, IgG3 or IgG4. In one embodiment of this method, the Fc regions of both, said first and said second antibody, are of the IgG1 isotype. In another embodiment, one of the Fc regions of said antibodies is of the IgG1 isotype and the other of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an IgG1 Fc sequence and an IgG4 Fc sequence and may therefore have interesting intermediate properties with regard to the activation of effector functions.
[00318] [00318] In yet another embodiment, one of the starting proteins of the antibody was developed to not bind to Protein A, thus allowing to separate the heterodimeric protein from said initial homodimeric protein by passing the product along a column of protein A .
[00319] [00319] As described above, the sequences of the first and second CH3 region of the starting homodimeric antibodies are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details about these interactions and how
[00320] [00320] Specifically, a stable bispecific PD-L1xCD137 antibody can be obtained in high yield using the above method of the invention based on two homodimeric antibodies that bind to CD137 and PD-L1, respectively, and that contain only few mutations asymmetric, conservative in CH3 regions. Asymmetric mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions in non-identical positions.
[00321] The bispecific antibodies of the invention can also be obtained by coexpressing constructs that encode the first and second polypeptides in a single cell. Thus, in a further aspect, the invention relates to a method for producing a bispecific antibody, said method comprising the following steps: a) providing a first nucleic acid construct that encodes a first polypeptide comprising a first Fc sequence and a first antigen binding region of a first antibody heavy chain, said first Fc sequence comprising a first CH3 region, b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc sequence and a second binding region to the antigen of a second antibody heavy chain, said second Fc sequence comprising a second CH3 region, wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions, and where the di OK
[00322] [00322] In additional aspects, the invention relates to materials and methods for the recombinant production of antibodies according to the invention. Suitable expression vectors, including promoters, enhancers, etc., and host cells suitable for the production of antibodies are well known in the art.
[00323] [00323] Thus, in one aspect, a nucleic acid construct is provided which comprises: (i) a nucleic acid sequence that encodes a heavy chain sequence of an antibody comprising an antigen binding region capable of binding to the PD Human -L1, as defined herein, and / or (ii) a nucleic acid sequence encoding an antibody light chain sequence comprising an antigen binding region capable of binding to human PD-L1 as defined herein.
[00324] [00324] In one embodiment, the nucleic acid construct further comprises: (i) a nucleic acid sequence that encodes a
[00325] [00325] In a further aspect, the invention relates to an expression vector comprising dome nucleic acid constructs defined above.
[00326] [00326] In another aspect, the invention relates to a nucleic acid that encodes a binding agent according to any aspect or embodiment described herein or a polypeptide chain thereof. In another aspect, the invention relates to an expression vector comprising a nucleic acid.
[00327] [00327] An expression vector in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (a nucleic acid sequence comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and DNA from facts and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid encoding an antibody against PD-L1 or against CD137 is included in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for example, Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a packed nucleic acid vector (as described in, for example, US 6 077 835 and / or WO 00/70087), a plasmid vector such as pBR322 , pUC 19/18 or pUC 118/119, a minimally sized “midge” nucleic acid vector (such as
[00328] [00328] In one embodiment, the vector is suitable for expression of the antibody against PD-L1 and / or the antibody against CD137 in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison WI) and similar vectors).
[00329] [00329] An expression vector can also or alternatively be a suitable vector for expression in a yeast system. A vector suitable for expression in a yeast system can be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., Ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York ( 1987), and Grant et al., Methods in Enzymol, 153, 516-544 (1987)).
[00330] [00330] An expression vector may also or alternatively be a suitable vector for expression in mammalian cells, for example, a vector comprising glutamine synthetase as a selectable marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10: 169-175.
[00331] [00331] A nucleic acid and / or vector can also comprise a nucleic acid sequence that encodes a sequence of
[00332] [00332] The expression vector can comprise or be associated with any suitable promoter, intensifier and other elements that facilitate expression. Examples of such elements include strong expression promoters (eg, human CMV IE promoter / enhancer as well as HIV RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective poly (A) termination sequences, a origin of replication for plasmid product in E. coli, an antibiotic resistance gene as a selectable marker and / or a convenient cloning site (for example, a polylinker). Nucleic acids can also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.
[00333] [00333] In one embodiment, the expression vector encoding the antibody against PD-L1 and / or CD137 can be positioned in and / or delivered to the host cell or the host animal via a viral vector.
[00334] [00334] In yet another aspect, the invention relates to a host cell comprising one or more of the nucleic acid constructs or the expression vector specified above.
[00335] [00335] Thus, the present invention also relates to a recombinant eukaryotic or prokaryotic host cell that produces an antibody of the present invention, such as a transfectome.
[00336] [00336] Examples of host cells include yeast, bacterial, vegetable and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells or lymphocyte cells. A preferred host cell is a CHO-K1 cell.
[00337] [00337] In one embodiment of the invention, the cell is a mammalian cell, such as a Chinese hamster ovary cell.
[00338] [00338] For example, in one embodiment, the host cell can comprise a first and a second nucleic acid construct stably integrated into the cell genome. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, comid, phagemid or linear expression element, which comprises a first and a second nucleic acid construct as specified above.
[00339] [00339] In a further aspect, the invention relates to a hybridoma that produces an antibody against PD-L1 as defined herein. Fc Regions
[00340] [00340] In some embodiments of the invention, the linker according to the present invention comprises, in addition to the antigen-binding regions, an Fc region consisting of the Fc sequences of the two heavy chains.
[00341] [00341] The first and second Fc sequences can be each of any isotype, including, but not limited to, IgG1, IgG2, IgG3 and IgG4, and can comprise one or more mutations or modifications. In one embodiment, each of the first and second Fc sequences is of the IgG4 isotype or derived from it, optionally with one or more mutations or modifications. In another embodiment, each of the first and second Fc sequences is of the IgG1 isotype or derived from it, optionally with one or more mutations or modifications. In another embodiment, one of the Fc sequences is of the IgG1 isotype and the other of the IgG4 isotype, or is derived from such respective isotypes, optionally with one or more mutations or modifications.
[00342] [00342] In one embodiment of the invention, one or both of the Fc sequences are deficient in effector functions. For example, the Fc sequence (s) may be of an IgG4 isotype or of a non-IgG4 type, for example, IgG1, IgG2 or IgG3, which has been mutated in such a way that the ability to mediate functions effects, such as ADCC, have been reduced or even
[00343] [00343] The antibodies according to the present invention can comprise modifications in the Fc region. When an antibody comprises such modifications, it can become an inert, non-activating antibody. The term "inertia", "inert" or "non-activator", in this specification, refers to an Fc region that is at least unable to bind to Fcγ receptors, induce FcR-mediated cross-linking to FcRs, or induce cross-linking to target antigens, mediated by FcR, through the two Fc regions of individual antibodies, or is unable to bind to C1q. The inertia of an Fc region of a humanized or chimeric antibody against CD137 or PD-L1 is advantageously tested using the antibody in monospecific format.
[00344] [00344] Several variants can be constructed to render the Fc region of an antibody inactive for interactions with Fcγ (gamma) and C1q receptors in the development of therapeutic antibodies. Examples of such variants are described herein.
[00345] Thus, in an embodiment of the antibody of the invention, said antibody comprises a first and a second heavy chain, in which one or both heavy chains are modified so that the antibody induces an Fc-mediated effector function to a lesser extent in relation to an antibody that is identical, except that it comprises an unmodified first and second heavy chain. Said Fc-mediated effector function can be measured by binding to Fcγ receptors, by binding to C1q or by inducing FcR-mediated FcRs cross-linking.
[00346] [00346] In another of such modality, the constant sequences of the heavy and light chains were modified so that the C1q binding of said
[00347] [00347] Thus, the amino acids in the Fc region that play a dominant role in interactions with C1q and the Fcγ receptors can be modified.
[00348] [00348] Examples of amino acid positions that can be modified, for example, in an IgG1 isotype antibody include positions L234, L235 and P331. Combinations thereof, such as L234F / L235E / P331S, can cause a profound decrease in binding to human CD64, CD32, CD16 and C1q.
[00349] [00349] Consequently, in one embodiment, the amino acid in at least one position corresponding to L234, L235 and P331 can be A, A and S, respectively (Xu et al., 2000, Cell Immunol. 200 (1): 16- 26; Oganesyan et al., 2008, Acta Cryst. (D64): 700-4). In addition, amino acid substitutions L234F and L235E can result in Fc regions with canceled interactions with Fcγ and C1q receptors (Canfield et al., 1991, J. Exp. Med. (173): 1483-91; Duncan et al., 1988, Nature (332): 738-40). Consequently, in one embodiment, the amino acids at positions corresponding to L234 and L235 can be F and E, respectively. An D265A amino acid substitution can decrease binding to all Fcγ receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276): 6591-604). Consequently, in one embodiment, the amino acid at the position corresponding to D265 can be A. The binding to C1q can be canceled by mutations at positions D270, K322, P329, and P331. Mutation at these positions to D270A or K322A or P329A or P331A can render the antibody deficient in CDC activity (Idusogie EE, et al., 2000, J Immunol. 164: 4178-84). Consequently, in one embodiment, the amino acids in at least one position corresponding to D270, K322, P329 and P331 can be A,
[00350] [00350] An alternative approach to minimize the interaction of the Fc region with Fcγ and C1q receptors is by removing the glycosylation site of an antibody. The mutation at position N297 to, for example, Q, A or E removes a glycosylation site that is critical for IgG-Fc gamma receptor interactions. Consequently, in one embodiment, the amino acid in a position corresponding to N297 can be G, Q, A or E (Leabman et al., 2013, MAbs; 5 (6): 896-903). Another alternative approach to minimize the interaction of the Fc region with Fcγ receptors can be obtained by the following mutations; P238A, A327Q, P329A or E233P / L234V / L235A / G236del (Shields et al., 2001, J. Biol. Chem. (276): 6591-604).
[00351] [00351] Alternatively, the human IgG2 and IgG4 subclasses are considered naturally compromised in their interactions with C1q and Fc gamma receptors, although interactions with Fcγ receptors have been reported (Parren et al., 1992, J. Clin Invest. 90: 1537 -1546; Bruhns et al., 2009, Blood 113: 3716-3725). Mutations that cancel out these residual interactions can be made in both isotypes, resulting in a reduction in the unwanted side effects associated with binding to the FcR. For IgG2, these include L234A and G237A and, for IgG4, L235E. Consequently, in one embodiment, the amino acid at a position corresponding to L234 and G237 in the human IgG2 heavy chain can be A and A, respectively. In one embodiment, the amino acid at a position corresponding to L235 in the human IgG4 heavy chain can be E.
[00352] [00352] Other approaches to further minimize the interaction with Fcγ and C1q receptors on IgG2 antibodies include those described in WO2011066501 and Lightle, S., et al., 2010, Protein Science (19): 753-62.
[00353] [00353] The hinge region of the antibody may also be of importance with respect to interactions with Fcγ receptors and the complement (Brekke et al., 2006, J Immunol 177: 1129-1138; Dall'Acqua WF, et al., 2006 ,
[00354] [00354] Thus, in one embodiment, the antibody comprises a first and a second immunoglobulin heavy chain, wherein, in at least one of said first and second immunoglobulin heavy chains, one or more amino acids at the positions corresponding to the L234 positions, L235, D265, N297 and P331 in a human IgG1 heavy chain are not L, L, D, N and P, respectively.
[00355] [00355] In one embodiment, in both, the first and the second heavy chain, one or more amino acids in the position corresponding to the positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are not L, L, D , N and P, respectively.
[00356] [00356] In one embodiment of the invention, in both said first and second heavy chains, the amino acid at the position corresponding to the D265 position in a human IgG1 heavy chain is not D.
[00357] [00357] Thus, in one embodiment of the invention, in both said first and second heavy chains, the amino acid at the position corresponding to the D265 position in a human IgG1 heavy chain is selected from the group consisting of: A and E .
[00358] [00358] In yet another embodiment of the invention, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.
[00359] [00359] In a particular embodiment of the invention, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.
[00360] [00360] In one embodiment of the invention, in both said first and second heavy chains, the amino acids in the positions corresponding to the
[00361] [00361] In a particular embodiment of the invention, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E, and A, respectively.
[00362] [00362] In an especially preferred embodiment of the invention, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.
[00363] [00363] In a more especially preferred embodiment of the invention, the binding agent is a bispecific antibody comprising a first and a second heavy chain, wherein the positions corresponding to the L234 and L235 positions in a human IgG1 heavy chain, according to the EU numbering of both the first heavy chain and the second heavy chain are F and E, respectively, and where (i) the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, of the first heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, the first heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, the second heavy chain is L.
[00364] [00364] In a more especially preferred embodiment of the invention, the binding agent is a bispecific antibody comprising a first and a second heavy chain, where the positions corresponding to the L234, L235 and D265 positions in a human IgG1 heavy chain according to the EU numbering, of both the first chain
[00365] [00365] Variants of the antibodies containing the combination of three amino acid substitutions L234F, L235E and D265A and, in addition, the K409R or F405L mutation are here designated with the suffix "FEAR" or "FEAL", respectively.
[00366] [00366] In a preferred embodiment, the bispecific antibody of the invention comprises: (i) an antibody with a half molecule derived from IgG1- CD137-FEAL, and an antibody with a half molecule derived from IgG1- PDL1-547-FEAR or ( ii) an antibody with a half molecule derived from IgG1- CD137-FEAR, and an antibody with a half molecule derived from and an antibody with a half molecule derived from IgG1-PD-L1-547-FEAL.
[00367] [00367] In yet another embodiment of the invention, one or both antibodies that form the bispecific antibody have been developed to reduce or increase the binding to the neonatal Fc receptor (FcRn) in order to manipulate the serum half-life of the bispecific antibody. Techniques for increasing or reducing the serum half-life are well known in the field. See, for example, Dall'Acqua et al. 2006, J. Biol. Chem., 281: 23514-24; Hinton et al. 2006, J. Immunol., 176: 346-56; and Zalevsky et al. 2010 Nat. Biotechnol., 28: 157-9.
[00368] [00368] In a further aspect, the present invention provides antibodies that are attached or conjugated to one or more therapeutic moieties, such as a cytokine, an immunosuppressant, an immunostimulatory molecule and / or a radioisotope. Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins".
[00369] [00369] In one embodiment, the first and / or the second Fc sequence is conjugated to a drug or a prodrug or contains an acceptor group for it. Such an acceptor group can be, for example, an unnatural amino acid. Compositions
[00370] [00370] In one aspect, the invention relates to a composition comprising a binding agent (such as a bispecific antibody), a nucleic acid, an expression vector or a cell according to any of the modalities or aspects disclosed herein . In one embodiment of the invention, the composition is a pharmaceutical composition. In an embodiment of the invention, the composition further comprises an acceptable pharmaceutical carrier and / or excipient.
[00371] [00371] In a further aspect, the invention relates to a pharmaceutical composition comprising a binding agent (such as a multispecific antibody, such as bispecific antibody), a nucleic acid, an expression vector or a host cell, according to with any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier.
[00372] [00372] The pharmaceutical composition of the present invention may contain a binding agent (such as a multispecific antibody, preferably a bispecific one) of the present invention or a combination of different
[00373] [00373] Pharmaceutical compositions can be formulated according to conventional techniques such as those described in Remington: The Science and Practice of Pharmacy, 19th edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. A pharmaceutical composition of the present invention may, for example, include diluents, fillers, salts, buffers, detergents (for example, a non-ionic detergent, such as Tween-20 or Tween-80), stabilizers (for example, sugars or amino acids free of proteins), preservatives, tissue fixatives, solubilizers and / or other materials suitable for inclusion in a pharmaceutical composition.
[00374] [00374] Pharmaceutically acceptable vehicles include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption retardants and similar vehicles that are physiologically compatible with a binding agent (for example , a multispecific antibody such as bispecific), a nucleic acid, an expression vector or a host cell of the present invention. Examples of suitable aqueous and non-aqueous vehicles that can be employed in the pharmaceutical compositions of the present invention include water, saline, saline with phosphate buffer, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and mixtures. vegetable oils, colloidal solutions of carboxymethylcellulose, tragacanth gum and injectable organic esters, such as ethyl oleate, and / or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Adequate fluidity can be maintained, for example, using
[00375] [00375] The pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants, for example, (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
[00376] [00376] The pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
[00377] [00377] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which can enhance the shelf life or the effectiveness of the pharmaceutical composition. The binding agents (such as multispecific antibodies, such as bispecifics) of the present invention can be prepared with vehicles that will protect the binding agent against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. . Such vehicles can include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyiortester and polylactic acid alone or with a wax, or other materials well known in the art. Methods
[00378] [00378] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of ingredients, for example, as listed above, if necessary, followed by sterilization microfiltration. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients, for example, among those listed above. In the case of sterile powders to prepare sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization) which produce a powder of the active ingredient plus any desired additional ingredient from a previously filtered sterile solution thereof.
[00379] [00379] The actual dosage levels of the active ingredients in pharmaceutical compositions can be varied in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a given patient, composition and mode of administration, without being toxic for the patient. The dose level selected will depend on a variety of pharmacokinetic factors, including the activity of the compositions of the present invention specifically employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the compound specifically employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the compositions specifically employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated and similar factors well known in medical techniques.
[00380] [00380] The pharmaceutical composition can be administered by any suitable route and method. In one embodiment, a composition
[00381] [00381] In one embodiment, this pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion. Uses
[00382] [00382] In one aspect, the invention relates to the binding agent according to any of the embodiments disclosed herein, or the nucleic acid, expression vector, host cell or pharmaceutical composition described herein, for use as a medicament.
[00383] [00383] In a further aspect, the invention relates to the binding agent according to any of the embodiments disclosed herein, or the nucleic acid, expression vector, host cell or pharmaceutical composition as described herein, for use in the treatment of a disease, such as cancer.
[00384] [00384] In a further aspect, the invention relates to a method for treating a disease which comprises administering an effective amount of a binding agent according to any of the embodiments disclosed herein, or of the nucleic acid, expression vector , host cell or pharmaceutical composition as described herein, to an individual in need thereof.
[00385] [00385] In particular, the binding agents according to the invention can be useful in therapeutic contexts in which specific targeting and elimination, mediated by T cells, of cells
[00386] [00386] The binding agents of the invention also have additional utility in therapy and diagnosis of a variety of diseases related to PD-L1. For example, binding agents (in particular antibodies) can be used to elicit one or more of the following biological activities in vivo or in vitro: inhibit the growth and / or differentiation of a cell expressing PD-L1; eliminating a cell expressing PD-L1; mediating phagocytosis or ADCC of a cell expressing PD-L1 in the presence of human effector cells; mediating CDC of a cell expressing PD-L1 in the presence of the complement; mediating apoptosis of a cell expressing PD-L1; and / or induce translocation to lipid rafts upon binding to PD-L1. In another aspect, the invention relates to a method for treating a disease comprising administering a binding agent, a nucleic acid, an expression vector, a cell or a composition according to any embodiment disclosed herein to an individual with need for it. In one embodiment of the invention, the method relates to the treatment of a disease, in which the disease is cancer.
[00387] [00387] In one aspect, the invention relates to the binding agent according to any of the embodiments disclosed herein, or the nucleic acid, expression vector, host cell or pharmaceutical composition as described herein, for use in the treatment of cancer .
[00388] [00388] In a further aspect, the invention relates to the binding agent according to any of the embodiments disclosed herein, or the nucleic acid, expression vector, host cell or pharmaceutical composition as described herein, for use in the treatment of a cancerous disease distinguished by the presence of solid tumors.
[00389] [00389] In one embodiment of the invention, the binding agent, nucleic acid, expression vector, cell or composition as described herein is
[00390] [00390] In a further aspect, the invention relates to the binding agent according to any of the embodiments disclosed herein, or the nucleic acid, expression vector, host cell or pharmaceutical composition as described herein, for use in the treatment of a cancerous disease selected from the group consisting of: melanoma, ovarian cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, bladder cancer, esophageal cancer, cancer pancreas, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndromes, ovarian cancer, cancer by endometriosis, cancer prostate cancer, penis cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma and mesothelioma.
[00391] [00391] In a particular embodiment, lung cancer is non-small cell lung cancer (CPCNP).
[00392] [00392] In a further aspect, the invention relates to the use of a binding agent according to any of the embodiments disclosed herein or of the nucleic acid, expression vector, host cell or pharmaceutical composition as described herein for the production of a medicine, such as a cancer treatment medicine, for example, a cancerous disease distinguished by the presence of solid tumors or a cancerous disease selected from the group consisting of: melanoma, ovarian cancer, lung cancer, cancer of colon and head and neck cancer.
[00393] [00393] In one embodiment of the invention, the method or use of
[00394] [00394] In one aspect, the present invention relates to a method for producing a bispecific antibody according to any embodiment described herein, which comprises the steps of: a. cultivating a host cell that produces a first antibody comprising an antigen binding region that binds to human CD137 and, optionally, purifying said first antibody from the culture; B. cultivating a host cell that produces a second antibody comprising an antigen binding region that binds to human PD-L1 and, optionally, purifying said second antibody from the culture; ç. incubating said first antibody together with said second antibody under sufficient reducing conditions to allow the cysteines in the hinge region to undergo isomerization of the disulfide and d bonds. obtaining said bispecific antibody CD137xPD-L1.
[00395] [00395] The present invention also relates to a method for inhibiting the growth and / or proliferation of one or more tumor cells expressing PD-L1, and / or inducing the death and / or elimination of one or more tumor cells expressing PD -L1, which comprises administering, to an individual in need thereof, a binding agent (e.g., a bispecific antibody) of the present invention or a composition of the present invention.
[00396] [00396] The present invention also relates to a method for treating cancer, which comprises: a) selecting an individual suffering from cancer that comprises tumor cells expressing PD-L1 and
[00397] [00397] Dose schedules in treatment methods and uses are adjusted to provide the optimal dose desired (for example, a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. Parenteral compositions can be formulated in unit dose form for ease of administration and dose uniformity.
[00398] [00398] Efficient doses and dosing schedules for the binding agent depend on the disease or condition to be treated and can be determined by those skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is approximately 0.001-10 mg / kg, such as approximately 0.001-5 mg / kg, for example, approximately 0.001-2 mg / kg, such as approximately 0.001-1 mg / kg, for example, approximately 0.001, approximately 0.01, approximately 0.1, approximately 1 or approximately 10 mg / kg. Another exemplary, non-limiting range for a therapeutically effective amount of a binding agent (for example, a bispecific antibody) of the present invention is approximately 0.1-100 mg / kg, such as approximately 0.1-50 mg / kg, for example, approximately 0.1-20 mg / kg, such as approximately 0.1-10 mg / kg, for example, approximately 0.5, approximately such as 0.3, approximately 1, approximately 3, approximately 5 or approximately 8 mg / kg.
[00399] [00399] A doctor or veterinarian with common competence in the subject can quickly determine and prescribe the effective amount of
[00400] [00400] In one embodiment, the binding agent can be administered in a weekly dose calculated as a fixed dose for up to 8 times, as well as 4 to 6 times when given once a week. Such a schedule can be repeated one or more times, as needed, for example, after 6 months or 12 months. Such fixed doses may, for example, be based on the doses in mg / kg provided above, with an estimated body weight of 70 kg. The dose can be determined or adjusted by measuring the amount of the binding agent (e.g., bispecific antibody) of the present invention in the blood, upon administration, by
[00401] [00401] In one embodiment, the binding agent can be administered as maintenance therapy, such as, for example, once a week for a period of 6 months or more.
[00402] [00402] A binding agent can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of an event in the progression of the cancer and / or reduce the risk of recurrence when a cancer is in remission.
[00403] [00403] The binding agents of the invention can also be administered in combination therapy, i.e., combined with other therapeutic agents relevant to the disease or condition to be treated. Thus, in one embodiment, the drug containing the binding agent (for example, bispecific antibody) is for combination with one or more additional therapeutic agents, such as a cytotoxic, chemotherapeutic or antiangiogenic agent.
[00404] [00404] In one aspect, the invention relates to an anti-idiotypic antibody that binds to the first and / or the second antigen-binding region as defined in any of the embodiments disclosed herein.
[00405] [00405] In a further aspect, the invention relates to an anti-idiotypic antibody that binds to the CD137 binding region, as defined in any of the embodiments disclosed herein.
[00406] [00406] In a further aspect, the invention relates to an anti-idiotypic antibody that binds to the PD-L1 binding region, as defined in any of the embodiments disclosed herein.
[00407] [00407] The present invention is illustrated in more detail by the following examples, which should not be interpreted as limitations to the
[00408] [00408] Antibodies CD137-005 and CD137-009 were generated as described in Example 1 of WO2016 / 110584. Briefly, rabbits were immunized with a mixture of proteins containing a human CD137-Fc fusion protein. Single blood B cells were separated and screened for the production of specific antibody against CD137 by ELISA and flow cytometry. Of the B cells with positive screening, RNA was
[00409] [00409] Humanized rabbit anti-CD137-009 antibody sequences were generated at Antitope (Cambridge, UK). Humanized antibody sequences were generated using germline humanization technology (CDR grafts). Humanized V region genes were designed based on human germline sequences with the closest homology to the VH and Vκ amino acid sequences of the rabbit antibody. A series of seven VH V and three Vκ (VL) genes, humanized with the germ line, was designed. Structural models of the V regions of the original non-human antibody were produced using Swiss PDB and analyzed in order to identify amino acids in the frameworks of the V region that may be important for the binding properties of the antibody. These amino acids were noted for incorporation into one or more variant antibodies with grafted CDRs. The germline sequences used as the basis for humanized projects are shown in Table 2.
[00410] [00410] Variant sequences with the lowest incidence of potential epitopes on T cells were then selected using in silico technologies exclusively owned by Antitope, iTope ™ and TCED ™ (T cell epitope database) (Perry, LCA, Jones, TD and Baker, MP New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9 (6): 385-396; 20 Bryson, CJ, Jones, TD and Baker, MP Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24 (1): 1-8). Finally, the nucleotide sequences of the projected variants had the codons optimized.
[00411] [00411] The sequences of the variable region of the humanized antibody against CD137 (CD137-009-HC7LC2) are shown in SEQ ID NO: 15 and SEQ ID NO: 16 of the attached Sequence Listing. Example 3: DNA shuffling between wild boar CD137 or elephant CD137 and human CD137 to determine important domains for antibody binding against CD137
[00412] [00412] To determine important domains of antibody binding against CD137 to human CD137, DNA shuffling was performed between human CD137 and the wild boar (Sus scrofa; XP_005665023) or between human CD137 and the African elephant (Loxodonta africana; XP_003413533). The scrambling constructs were prepared from the DNA encoding human CD137, replacing human domains with wild boar (Scramble 1-4, 6) or elephant (Scramble 5 construction) domains. The amino acid sequence of the shuffling constructions are shown
[00413] [00413] If a domain in human CD137 is important for binding an anti-CD137 antibody, the binding will be lost when the domain is replaced by the wild boar or African elephant domain.
[00414] [00414] The homology between human CD137 and wild boar and between human CD137 and African elephant is 70.2 and 74.5%, respectively. The requirement for the selection of these two species was that the domain of interest in the African elephant and the wild boar was sufficiently different when compared to the human, resulting in loss of connection, while maintaining the critical structural interactions that are necessary to minimize the risk of error folding or loss of expression. Figure 1 shows the sequence alignments of human CD137, wild boar and African elephant. Figure 2 shows the constructs of human CD137 containing the wild boar CD137 or African elephant CD137 domains, as indicated.
[00415] [00415] 3 x 106 HEK293T-17 cells were seeded in T75 culture flasks (Greiner Bio-One, cat. No 658175) in 20 mL of RPMI 1640 GlutaMAX medium containing FCS 10% FCS (Biochrom, cat. No S 0115) . After overnight incubation, cells were transiently transduced with expression vectors encoding the wild boar, African elephant or human CD137 scrambling constructs downstream of the human elongation factor-1 (EF-1 alpha) promoter constitutively active, using the TransIT®-LT1 Transfection Reagent, Mirus Bio reagent (VWR International, cat. no 731-0029), according to the manufacturer's instructions. The next day, the cells were harvested with 1.5 mL of Accutase (Sigma Aldrich, cat. No A6964) (incubation at 37 ° C for 5 min.), And flow cytometry was performed, essentially as described above, for measure the expression on the surface of the scrambling constructions and of the human CD137, African elephant and wild boar and measure the bond
[00416] [00416] All shuffling constructions of CD137, as well as human, African elephant and wild boar CD137 were expressed on the cell surface with similar levels of expression (Figure 3).
[00417] [00417] Figure 4 shows that CD137-009 exhibited loss of binding to CD137 from the African elephant and the wild boar. CD137-009 also exhibited loss of attachment to the shuffling construction, compared to binding to human CD137. Example 4: Generation of antibody against PD-L1
[00418] [00418] Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). A cDNA encoding amino acid 19-238 of human PD-L1 was cloned into expression plasmids owned by Aldevron. The PD-L1-547 antibody was generated by immunizing OmniRat animals (transgenic mice expressing a diverse repertoire of antibodies with fully human idiotypes; Ligand Pharmaceuticals Inc., San Diego, USA) by intradermal application of PD-L1 cDNA coated particles using a manual device for particle bombardment (“gene gun”). Serum samples were collected after a series of immunizations and tested by cytometry of
[00419] [00419] Bispecific IgG1 antibodies were generated by changing the Fab arm under controlled reducing conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific conditions of the assay as described in WO2011 / 131746. The F405L and K409R mutations (EU numbering) were introduced into the relevant antibodies to create antibody pairs with complementary CH3 domains.
[00420] [00420] To generate bispecific antibodies, the two complementary original antibodies, each antibody at a final concentration of 0.5 mg / mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 µL of PBS at 31 ° C for 5 hours. The reduction reaction was interrupted by the removal of the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer's protocol.
[00421] [00421] Bispecific antibodies were generated by combining the following antibodies from Example 1 and 4:
[00422] [00422] The effect of the monovalent antibody against PD-L1, b12-FEALxPD-L1-547-FEAR, on the interaction of PD-1 and PD-L1 was determined in a bioassay of the inhibition of PD-1 / PD-L1, as developed by Promega (Madison, USA). This is an assay based on a bioluminescent cell that consists of two cell lines created by genetic engineering: PD-1 effector cells, which are Jurkat T cells expressing human PD-1 and a luciferase reporter directed by an NFAT response element (NFAT -RE), and PD-L1 aAPC / CHO-K1 cells, which are CHO-K1 cells expressing human PD-L1 and a modified cell surface protein, designed to activate cognate TCRs in an antigen-independent manner. When the two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. The addition of an antibody that blocks the PD-1 / PD-L1 interaction releases the inhibitory signal and results in TCR activation and NFAT-RE-mediated luminescence.
[00423] [00423] PD-L1 aAPC / CHO-K1 cells (Promega, cat. No J109A) were thawed according to the manufacturer's protocol, resuspended in Ham's F12 medium (Promega, cat. No J123A) containing 10% fetal bovine serum (FBS; Promega, cat. No J121A) and seeded in 96-well flat-bottom culture plates (CulturPlate-96, Perkin Elmer, cat. No 6005680). The plates were incubated for 16 hours at 37 ° C, 5% CO2. The supernatant was removed and serial dilutions of the antibody (final concentration ranging from 5 to 0.001 μg / mL; dilutions 4 times in RPMI 1640 [Lonza, cat. In BE12-115F] containing 1% fetal bovine serum [FBS; Promega, cat. in J121A]) have been added. PD-1 effector cells (Promega, cat. No J115A; thawed according to the manufacturer's protocol and resuspended in RPMI / FBS 1%) were added. The plates were incubated for 6 hours at 37 ° C, 5% CO2. After equilibration to room temperature, 40 μL of the Bio-Glo reagent (substrate of the Bio-Glo luciferase assay [Promega cat. No G720B], reconstituted in Bio-Glo luciferase assay buffer [Promega, cat. No G7198] according to the manufacturer's protocol) were added to each well. The plates were incubated at room temperature for 5-10 minutes and luminescence was measured using an EnVision Multilabel reader (PerkinElmer). The effect on the PD1-PD-L1 interaction in relation to the control (without added antibody) was calculated as follows: Relative induction = RLU (induced-background) / RLU (without control-background antibody), RLU is relative units of light
[00424] [00424] Figure 5 shows that the monovalent b12-FEALxPD-L1-547-FEAR antibody efficiently inhibited the PD1-PD-L1 interaction. Example 7: Antigen-specific CD8 + T cell proliferation assay to measure effects by bisecting specific antibodies to PD-L1 and CD137
[00425] [00425] The schematic representation of the predicted mode of action of the bispecific antibodies CD137xPD-L1 is shown in Figure 6.
[00426] [00426] To measure the induction of T cell proliferation by the bispecific antibody directed against PD-L1 and CD137 in an antigen-specific assay, dendritic cells (DCs) were transfected with in vitro transcribed RNA (RNA-IVT) from claudin-6 to express the claudin-6 antigen. T cells were transfected with PD-1 RNA-IVT and with the T-cell receptor (TCR), restricted to HLA-A2, specific for claudin-6. This TCR is able to recognize the claudin-6-derived epitope presented on HLA-A2 in CD. The bispecific antibody CD137xPD-L1 can cross-bind with PD-L1 endogenously expressed in monocyte-derived dendritic cells or in tumor cells and with CD137 in T cells, leading to inhibition of the inhibitory interaction PD-1 / PD-L1 and at the same time as the CD137 cluster, resulting in proliferation of T cells. The clustering of the CD137 receptor expressed in T cells leads to activation of the CD137 receptor, which in turn releases a co-stimulatory signal to the T cell.
[00427] [00427] HLA-A2 + peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs by activated cell magnetic separation technology (MACS) using anti-CD14 microspheres (Miltenyi; cat. No 130-050-201), according to the manufacturer's instructions. Peripheral blood lymphocytes (PBLs, CD14-negative fraction) were frozen for future T-cell isolation. For differentiation into immature DCs (iDCs), 1x106 monocytes / mL were cultured for five days in RPMI GlutaMAX (Life technologies GmbH, cat. 61870-044) containing 5% human serum AB (Sigma-Aldrich Chemie GmbH, cat. no H4522-100ML), sodium pyruvate (Life technologies GmbH, cat. no 11360-039), non-essential amino acids (Life technologies GmbH, No. 11140-035), penicillin-streptomycin 100 IU / mL (Life technologies GmbH, No. 15140-122), granulocyte colony stimulating factor-
[00428] [00428] One day before the start of an antigen-specific CD8 + T cell proliferation assay, frozen PBLs and iDCs from the same donor were thawed. CD8 + T cells were isolated from PBLs by MACS technology using anti-CD8 microspheres (Miltenyi, cat. No 130-045-201), according to the manufacturer's instructions. About 10-15 x 106 CD8 + T cells were electroporated with 10 µg of in vitro translated RNA (IVT) encoding the alpha chain plus 10 µg of RNA-IVT encoding the beta chain of a murine TCR specific for claudin-6 (restricted to HLA-A2; described in WO 2015150327 A1) plus 10 µg of PD-1 encoding RNA-IVT in 250 µL of X-Vivo15 (Biozym Scientific GmbH, cat. No 881026) in a 4 mm electroporation cuvette (VWR International GmbH, cat. No 732-0023) using the BTX ECM® 830 Electroporation System device (BTX; 500 V, 1 x 3 ms pulse). Immediately after electroporation, the cells were transferred to fresh IMDM medium (Life Technologies GmbH, cat. No. 12440-061) supplemented with 5% human serum AB and rested at 37 ° C, 5% CO2 for at least 1 hour. T cells were labeled with carboxyfluorescein succinimidyl ester, 1.6 µM (CFSE; Invitrogen, cat. No C34564) in PBS according to the manufacturer's instructions, and incubated in IMDM medium supplemented with 5% human serum AB, overnight .
[00429] [00429] Up to 5 x 106 defrosted iDCs were electroporated with 5 µg of complete claudin-6 encoding RNA-IVT, in 250 µL of X-Vivo15 medium, using the electroporation system as described above (300 V, 1x 12 ms pulse ) and incubated in IMDM medium supplemented with 5% human serum AB, overnight.
[00430] [00430] The next day, the cells were harvested. Cell surface expression of claudin-6 and PD-L1 in DCs and TCR and PD-1 in T cells was verified by flow cytometry. The DCs were stained with a specific antibody against CLDN6, conjugated with Alexa647 (not commercially available; in-house production) and with anti-human CD274 antibody (PD-L1, eBioscienes, cat. No. 12-5983) and T cells were stained with a mouse TCR ß-chain antibody (Becton Dickinson GmbH, cat. no 553174) and with human anti-CD279 antibody (PD-1, eBioscienes, cat. no 17-2799). 5,000 electroporated DCs were incubated with
[00431] [00431] The monovalent control antibody against PD-L1, with an irrelevant binding arm, b12-FEALxPD-L1-547-FEAR, intensified the proliferation of T cells to some extent when compared to incubation with b12 (as regular IgG1), and the bispecific antibody CD137-009- FEALxPD-L1-547-FEAR induced strong proliferation of CD8 + T cells (Figure 7). This was reflected by an increase in the percentage of cells
[00432] [00432] In addition, the EC50 value in that assay was determined for CD137-009-FEALxPD-L1-547-FEAR. For this, the bispecific antibody was analyzed in 3-fold serial dilutions from 1 to 0.00015 µg / mL (Figure 8). The percentage of cells divided and the proliferation index were determined by the FlowJo software. The curves were analyzed by non-linear regression (sigmoidal dose-response with variable inclination) using the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The EC50 values for inducing the proliferation of CD137-009-FEALxPD-L1-547-FEAR antigen-specific T cells was 0.003492 µg / mL, for “% of divided cells” and 0.005388 µg / mL for “ proliferation index ”. Example 8: Comparison of the bispecific antibody targeting PD-L1 and CD137 with a combination of two antibodies that monovalently bind to CD137 and PD-L1 or the two original antibodies (PD-L1-547 + CD137-009) in one antigen-specific T cell assay with active PD1 / PD-L1 axis
[00433] [00433] To measure induction of T cell proliferation by the bispecific antibody directed to PD-L1 and CD137, an antigen-specific T cell proliferation assay with active PD1 / PD-L1 axis was performed (general assay configuration analogous to that of Example 7). Briefly,
[00434] [00434] Neither the monovalent control antibody against CD137, nor CD137-009-FEALxb12-FEAR, having an irrelevant binding arm, nor the corresponding bivalent original antibody CD137-009 had an effect on T cell proliferation when compared to IgG1-b12 . In contrast, incubation with the monovalent control antibody against PD-L1 as well as the original bivalent antibody (b12-FEALxPD-L1-547-FEAR and PD-L1- 547, respectively) led to moderately enhanced T cell proliferation when compared to incubation with IgG1, b12 control antibody. A comparable level of T cell proliferation was detectable when incubating with the combined monovalent control antibodies (CD137- 009-FEALxb12-FEAR + b12-FEALxPD-L1-547-FEAR) and the corresponding original combined antibodies (CD137-009 + PD -L1-547). In contrast, the bispecific antibody CD137-009-FEALxPD-L1-547-FEAR induced a strong proliferation of CD8 + T cells, which was superior to both combined controls (monovalent and bivalent) (Figure 9). This was reflected by an increase in the percentage of cells divided (Figure 9B) as well as by an increase in the proliferation index (Figure 9C). Example 9: Ex vivo TIL expansion assay to assess the effects of the bispecific antibody CD137xPD-L1 on tumor infiltrating lymphocytes
[00435] [00435] To assess the effects of CD137-009-FEALxPD-L1-547- FEAR on tumor infiltrating lymphocytes (TIL), an ex vivo culture of human tumor tissue was performed as follows. Fresh specimens of human tumor tissue, obtained by resection, were washed three times by transferring the isolated tumor pieces from a cavity on a
[00436] [00436] After a total culture period of 10-14 days, TILs were harvested and analyzed by flow cytometry. The cells were stained with the following reagents, all diluted 1:50 in staining buffer (D-PBS containing 5% FCS and 5 mM EDTA), anti-human CD4-FITC (Miltenyi Biotec, cat. No 130-080-501 ), human anti-CD3-PE-Cy7 (BD Pharmingen, cat. no 563423), 7-aminoactinomycin D (7-AAD, Beckman Coulter, cat. no
[00437] [00437] Figure 10 shows the TIL expansion analysis of a non-small cell lung carcinoma tissue species. Here, the following concentrations of CD137-009-FEALxPD-L1-547-FEAR were added; 0.01; 0.1 and 1 µg / mL; a tissue specimen from the same patient with no added antibody served as a negative control. After 10 days of culture, TILs were collected and analyzed by flow cytometry. Five samples (from 5 original wells) were measured for each antibody concentration derived from different wells of the 24-well plate. In all samples cultured with the bispecific antibody, the viable TIL count was significantly increased compared to the control samples without antibody. In general, a 10-fold expansion of viable TILs was observed when CD137-009-FEALxPD-L1-547-FEAR, 0.1 µg / mL, was added to the cultures (Figure 10 A). CD3 + CD4 + helper T cells were only slightly expanded (Figure 10 C; 2.8 fold expansion), while in contrast, the most prominent expansion of TILs was seen for NK CD3-CD56 + cells (Figure 10 D; up to 64 times in
[00438] [00438] Bispecific mouse substitute antibodies mCD137-3H3xmPD-L1-MPDL3280A, mCD137-3H3xb12 and mPD-L1- MPDL3280Axb12 were generated using a method to generate murine bispecific antibodies based on the controlled exchange of Fab's arm (Labrij. , 2017 Sci Rep. 7 (1): 2476 and WO2016097300).
[00439] [00439] The monoclonal antibody 3H3, which binds to mouse 4-1BB, was obtained from BioXcell (cat. No BE0239) and the protein was sequenced at ProtTech. The inferred cDNA sequence was deduced using proprietary methods. The variable regions of the heavy and light chain have the genes synthesized and cloned into a mouse IgG2a expression vector including a constant murine IgG2a constant region containing the following amino acid mutations: L234A, L235A, F405L and R411T. Likewise, the variable regions of b12 have been cloned into this expression vector.
[00440] [00440] The MPDL3280A antibody (variable heavy and light chain sequences shown in SEQ ID NOs: 57 and 58, respectively) has been reported to bind to human and mouse PD-L1. The variable regions of the heavy and light chains of this antibody were cloned into a mouse IgG2a expression vector, including a murine IgG2a constant region containing the following amino acid mutations: L234A, L235A, T370K and K409R.
[00441] [00441] Bispecific mouse antibodies (in essence,
[00442] [00442] Female C57BL / 6JOlaHsd mice (Envigo RMS GmbH, Rossdorf, Germany), 6-8 weeks old, weighing between 17 and 24 g, were acclimated in the animal facility for at least six days before inclusion in the study. These mice were used as recipients. Female or male C57BL / 6 Thy1.1 x C57BL / 6J OT-1 mice, homozygous for both OT-1 and Thy1.1 alleles, were bred internally (crossbred C57BL / 6-Tg (TcraTcrb) 1100Mjb / Crl and mice) B6.PL-Thy1a / CyJ) and were used as donors. The mice had free access to food (ssniff M-Z autoclavable Soest, Germany) and sterile water and were housed in 12-hour light / dark cycles at 22 ° C ± 2 ° C with 55% ± 15% relative humidity.
[00443] [00443] On the day the study started, the C57BL / 6 Thy1.1 x C57BL / 6J OT-1 donor mice were sacrificed and the spleens were isolated. The spleens were mechanically dissociated and the erythrocytes were lysed by resuspension of the splenocyte pellet with red cell lysis buffer (NH4Cl 8.25 g / L, KHCO3 1 g / L, EDTA 0.1 mM, pH 7). Subsequently, the splenocytes were washed with Dulbecco's PBS (DPBS), and CD8 + T cells were isolated using mouse CD8a (Ly-2) microspheres in combination with the autoMACS Pro Separator (both from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany ). CD8 + / OT-1 + / Thy1.1 + T cells (2.5- 5x105 cells) were injected retro-orbitally in a total volume of 200 µL per C57BL / 6JOlaHsd receptor mouse. The day after the transfer of adoptive cells, the recipient mice were “vaccinated” retro-orbitally with 100 µg of ovalbumin / 200 µL of PBS as an antigenic stimulus. After 6 hours, the mice were treated retro-orbitally with the respective bispecific antibody. In detail, 100 µg or 20 µg of the mCD137-3H3xmPD-L1-MPDL3280A antibody,
[00444] [00444] Figure 11 A is a schematic representation of the design of the adoptive OT-1 T cell transfer assay. Figure 11 B shows the analysis of the frequencies of Thy1.1 + CD8 + T cells as determined by flow cytometry. For each treatment modality with bispecific antibody, n = 5 mice were used. The antigenic stimulus with ovalbumin only led to a detectable increase in the frequency of Thy1.1 + CD8 + T cells compared to untreated animals. Interestingly, the two monovalent control antibodies having an irrelevant b12 binding arm, mCD137-3H3-xb12 and mPD-L1- MPDL3280Axb12, were unable to reinforce the expansion of ovalbumin-specific OT-1 T cells compared to animals that had been treated with ovalbumin only. In contrast, the bispecific mCD137-3H3xmPD-L1-MPDL3280A antibody was able to induce a strong proliferation of OT-1 T cells, leading to T cell frequencies of 10-20% of CD8 + / OT-1 + / Thy1 T cells. 1+ (% of total T cell population) at both tested dose levels (20 and 100 µg of antibody). Example 11: Effect of a bispecific antibody substitute for
[00445] [00445] Female BALB / c Rj mice (Janvier, Genest-St.-Îsle, France), 6-8 weeks old, weighing between 17 and 24 g, were acclimated for at least six days before inclusion in the study. The mice had free access to food (ssniff M-Z autoclavable Soest, Germany) and sterile water and were housed in a 2 hour light / dark cycle at 22 ° C ± 2 ° C with 55% ± 10% relative humidity. CT26 cells were obtained from ATCC® (Cat No CRL-2638 ™) and cultured in Roswell Park Memorial Institute (RPMI) 1640, GlutaMAX ™ (Life technologies, Cat No 61870-044) medium supplemented with 10% fetal bovine serum (FBS) (Biochrom, Cat No S 0115) in 5% CO2 at 37 ° C. The cells were harvested with the StemPro® Accutase® Cell Dissociation Reagent (Life technologies, Cat No. A1110501), resuspended in DPBS (Life technologies, Cat No 14190-169), and 0.5 x 106 cells / 100 μL per mouse implanted subcutaneously (SC) on the scraped right flank of female BALB / c Rj mice. Tumor volume was assessed by caliper measurements every 2-3 days and is expressed as the product of perpendicular diameters using the following formula: a2 x b / 2, where b is the longest of the two diameters (a <b). The animals were stratified into four groups when an average tumor volume of 30 mm3 was reached. The treatment started the next day with an intraperitoneal injection of 20 µg of the bispecific antibody that binds mPD-L1 and mCD137 (mCD137-3H3xmPD-L1-MPDL3280A), with monovalent antibodies controlling against mCD137 or mPD-L1 having a binding arm negative irrelevant. The administration schedule was applied every 2-3 days of the first eight injections, followed by an injection every 7 days until the end of the experiment. On the 29th day after the tumor cells were inoculated, 100 µL of blood was taken by retro-orbital route and analyzed for gp70-specific CD8 + T cells (gp70 is an envelope protein
[00446] [00446] Figure 12 A shows the tumor growth curves for all four treatment groups with individual lines representative of a single tumor / mouse. The frequencies of progression-free survival (PFS), for the respective treatment groups, are provided at the bottom of each graph. Figure 12 B shows the corresponding Kaplan-Meier survival curves until the end of the experiment on the 71st day after tumor cell inoculation. Figure 12 C shows the analysis of the frequencies of CD8 + T cells gp70 + tetrameter, as determined by flow cytometry. For each treatment modality, all mice that were still alive on the 29th day after tumor cell implantation were analyzed. In short, the bispecific antibody that binds mPD-L1 and mCD137 (mCD137-3H3xmPD-L1-MPDL3280A) provided the most efficient tumor control with 5 out of 10 (ie 50%) animals going for complete tumor regression. In comparison, a slightly weaker but still prominent antitumor effect was observed for the control mCD137-3H3xb12; the treatment took 3 of 11 (that is, 27%) animals to be able to reject the tumors. In both cases, all mice that went into remission remained tumor-free until the end of the experiment. In striking contrast, the two cohorts treated with mPD-L1-MPDL3280Axb12, as well as that with control PBS failed to control tumor burden, and treatment with mPD-L1- MPDL3280Axb12 leading to at least some intermittent inhibition of tumor growth on 2 December. 11 (ie 18%) animals between 15 and 30 days
[00447] [00447] The binding of antibodies against PD-L1 and bispecific antibodies b12xPD-L1 to human tumor cell lines MDA-MB-231 (breast adenocarcinoma; ATCC; Cat. No HTB-26), PC-3 (prostate adenocarcinoma) ; ATCC; Cat. In CRL-1435) and SK-MES-1 (squamous cell carcinoma of the lung; ATCC; Cat. In HTB-58) was analyzed by flow cytometry.
[00448] [00448] The cells (3-5 x 104 cells / well) were incubated in 96-well round bottom polystyrene plates (Greiner bio-one, cat. No 650101) with serial dilutions of antibodies (range: 0.0001 to 10 µg / mL in 5-fold dilution steps) in 50 µL of PBS / 0.1% BSA / 0.02% azide (FACS buffer) at 4 ° C for 30 min. After washing twice with FACS buffer, the cells were incubated with a secondary antibody at 4 ° C for 30 min. As a secondary antibody, goat anti-F (ab ') 2 human IgG conjugated to R-phycoerythrin (PE) (Cat. No. 109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), diluted 1: 500 in 50 µL of FACS buffer, for all experiments. Then, the cells were washed twice with FACS buffer, resuspended in 20 μL of FACS buffer and analyzed on an iQue tracker (Intellicyt Corporation,
[00449] [00449] Quantitative flow cytometry (QIFIKIT®, Dako; cat. No K0078) was performed as described (Poncelet and Carayon, 1985, J. Immunol. Meth. 85: 65-74), using MPDL3280A (variable heavy chain sequences and light presented in SEQ ID NOs: 57 and 58, respectively) to quantify the antigen density in the plasma membrane of MDA-MB-231, PC-3 and SK-MES-1 cells. It has been determined that cell lines have the following density of the PD-L1 antigen (ABC, antibody binding capacity): MDA-MB-231: approx. 21,000 ABC / cell PC-3: approx. 6,000 ABC / cell SK-MES-1: approx. 30,000 ABC / cell Link to cells MDA-MB-231
[00450] [00450] Figure 13 (A) shows dose-dependent binding of b12-FEALxPD-L1-547-FEAR to MDA-MB-231 cells, with maximum binding greater than monospecific, bivalent PD-L1-547-FEAR. Connection to PC-3 cells
[00451] [00451] Figure 13 (B) shows dose-dependent binding of b12- FEALxPD-L1-547-FEAR to PC3 cells, with maximum binding greater than monospecific, bivalent PD-L1-547-FEAR. Connection to SK-MES-1 cells
[00452] [00452] Figure 13 (C) shows dose-dependent binding of b12- FEALxPD-L1-547-FEAR to SK-MES-1 cells, with maximum binding greater than PD-L1-547-FEAR monospecific, bivalent. Example 13. Determination of the contribution of CD137 amino acid residues to antibody binding by alanine scanning
[00453] [00453] A CD137 library with a single alanine residue (Uniprot Q07011) was synthesized (Geneart), in which all amino acid residues in the extracellular domain of human CD137 were individually mutated to alanines, except for positions that already contained alanines or cysteines . To minimize the probability of structural breakdown of the antigen, cysteines did not mutate. Alanine positions were changed to glycines by mutation. The library was cloned into the pMAC expression vector containing a CMV / TK-polyA expression cassette, an Amp resistance gene and a pBR322 origin of replication. Library production and tracking
[00454] [00454] CD137 wild-type and alanine mutants were expressed individually in FreeStyle HEK293 cells according to the manufacturer's instructions (Thermo Scientific). One day after transfection, cells were harvested. Approximately 100,000 cells were incubated with 20 µL of the antibody of interest conjugated to Alexa Fluor® 488 (A488) in FACS buffer (Table 3). The cells were incubated at room temperature for 30 min. Subsequently, the cells were washed twice by centrifugation with 150-200 µL of FACS buffer. The cells were suspended in 30 µL of FACS buffer and stored at 4 ° C until analysis by flow cytometry using an iQue tracker.
[00455] [00455] The entire experiment was carried out in triplicate. Table 3: Antibodies used to determine the contribution of CD137 amino acid residues to antibody binding using alanine scanning. The antibodies were labeled with Alexa488 (Invitrogen, cat. No A20000) according to the manufacturer's instructions, before the experiment was carried out. CD137-MOR7480-FEAR is a substitute antibody against MOR7480 that has been cloned into the human IgG1 backbone containing the
[00456] [00456] For each sample, the average amount of antibody bound per cell was determined as the geometric mean fluorescence intensity (gMFI) of the population of single, viable cells. GMFI is influenced by the antibody's affinity for the mutant CD137 and the expression level of the mutant CD137 per cell. Since specific mutations for alanine may impact the level of expression on the surface of the mutant CD137, and to correct differences in the expression of each mutant CD137 in general, the data were normalized against the binding intensity of a non-CD137-specific control antibody. cross blocker, using the following equation: In which, “aa position” refers to the position that has been mutated to alanine or glycine. To express loss or gain of antibody binding, the z score was determined according to the following calculation: Where are the mean and standard deviation of the normalized gMFI calculated from all mutants.
[00457] [00457] A z score of 0 indicates that there is no loss or gain of binding by a particular antibody, when compared to the binding of the reference antibody; a z score> 0 indicates binding gain, when compared to the reference antibody binding; a z score <0 indicates loss of binding when compared to binding of the reference antibody. In most cases, the gain in binding, as determined by the z score, is caused by the loss of binding of the reference antibody to mutants
[00458] [00458] If the gMFI of the control antibody for a given mutant CD137 was less than the gMFIposition aa mean - 2.5 x SD of the gMFIAb mean control, the data were excluded from the analysis (it is assumed that the expression levels for those CD137 mutants were not enough to draw conclusions).
[00459] [00459] Figure 14 shows the loss of binding of antibodies against CD137 to CD137 variants with alanine or glycine mutations at positions 1 to 163 (according to SEQ ID 41). The results indicate that: ● the CD137-005-FEAR antibody showed loss of binding when aa L1, Q2, P4, G11, T12, D15 or Q20 mutated to alanine. This suggests that the binding of the CD137-005-FEAR antibody is at least dependent on aa L1, Q2, P4, G11, T12, D15, Q20 of human CD137, ● the antibody b12-FEALxCD137-009-FEAR showed loss of binding when aa F13, F30, T38, D40 or N60 mutated to alanine. This suggests that the binding of the b12-FEALxCD137-009-FEAR antibody is at least dependent on aa F13, F30, T38, D40 and N60 of human CD137. As F13 and F30 are most likely to affect the interaction with the epitope, the b12-FEALxCD137-009-FEAR antibody is at least dependent on aa T38, D40 and N60, ● the CD137-MOR7048-FEAR antibody showed loss of binding when aa L72, G93, F102, N103, I109, R111, or W113 mutated to alanine. This suggests that the binding of the MOR7048 antibody is at least dependent on aa L72, G93, F102, N103, I109, R111 and W113 of human CD137. Example 14: Non-antigen-specific T cell proliferation assay to measure the effects of bispecific antibodies that bind to
[00460] [00460] A schematic representation of the predicted mode of action of the bispecific antibodies PD-L1xCD137 is shown in Figure 6.
[00461] [00461] To measure induction of T cell proliferation in activated polyclonated T cells, PBMCs were incubated with a suboptimal concentration of anti-CD3 antibody (UCHT1 clone) to activate T cells, combined with the bispecific antibody PD-L1-547 -FEALxCD137-009- HC7LC2-FEAR or control antibodies. Within the population of PBMCs, cells expressing PD-L1 can be linked by the bispecific antibody's specific PD-L1 arm, whereas T cells in the population can be linked by the CD137 specific arm. In this assay, the proliferation of T cells is a measure for the transactivation of T cells through the specific arm of CD137, induced by cross-linking with cells expressing PD-L1 through the bispecific antibody and by blocking the PD-L interaction. L1: PD-1, and is measured as the proliferation of T cells.
[00462] [00462] PBMCs were obtained from leukocyte bilayers from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany) with Ficoll gradient (VWR, cat. No 17-5446-02). PBMCs were labeled with 1.6 µM succinimidyl ester carboxyfluorescein (CFSE) (Thermo Fisher, cat. No C34564) in PBS, according to the manufacturer's instructions. 75,000 CFSE-labeled PBMCs were seeded per well in a 96-well round bottom plate (Sigma Aldrich, CLS3799-50EA) and incubated with a suboptimal concentration of anti-CD3 antibody (R&D Systems, UCHT1 clone, cat. In MAB100; final: 0.03-0.1 µg / mL) that was predetermined for each donor to induce suboptimal T cell proliferation, and bispecific antibodies or control, in 150 µL of IMDM GlutaMAX supplemented with 5% human serum AB at 37 ° C, CO2, 5% for four days. The proliferation of CD4 + and CD8 + T cells was analyzed by flow cytometry, essentially as described above. 30 µL of medium
[00463] [00463] PBMCs from three different donors were analyzed by testing two different concentrations of anti-CD3 for stimulation and as a control without anti-CD3. Figure 15 shows that the bispecific antibody PD-L1-547-FEALxCD137-009-HC7LC2-FEAR induced a strong expansion of CD4 + and CD8 + T cells. The monovalent control antibody against CD137, b12-FEALxCD137-009-HC7LC2-FEAR, having an irrelevant arm, and the corresponding bivalent original antibody CD137-009-HC7LC2-FEAR did not affect the proliferation of CD4 + (A) or CD8 + (B) T cells ) when compared to incubation with the isotype control b12 IgG antibody. The monovalent control antibody against PD-L1 as well as the original bivalent antibody (b12-FEALxPD-L1-547-FEAR and PD-L1-547-FEAR, respectively) slightly increased the proliferation of T cells
[00464] [00464] In another independent study, the EC50 values for PD-L1-547-FEALxCD137-009-HC7LC2-FEAR were determined using PBMCs obtained from two donors, which were suboptimally stimulated with 0.03 anti-CD3 and 0.09 µg / mL. PD-L1-547-FEAL xCD137- 009-HC7LC2-FEAR was analyzed using serial dilutions starting at 1 µg / mL and ending at 0.15 ng / mL, and b12-IgG-FEAL at 1 µg / mL was included as control isotype antibody. For proliferation of CD4 + and CD8 + T cells, dose-response curves were generated (Figure 16) and, for proliferation of CD8 + T cells, EC20, EC50 and EC90 values were also determined, as shown in Table 4. Table 4. Determination of EC20, EC50 and EC90 values of PD-L1-547- FEALxCD137-009-HC7LC2-FEAR based on CD8 + T cell expansion data, as measured by a non-antigen-specific T cell proliferation assay. The data shown are the values calculated based on the adjustments of four logarithmic parameters (Figure 16). anti-CD3 EC50 value Calculated EC20 slope Calculated EC90 Donor [µg / mL] [µg / mL] Hill [µg / mL] [µg / mL] 1 0.03 0.01218 1.134 0.00359 0.08455 2 0 , 09 0.00689 0.635 0.00078 0.21917
[00465] [00465] The induction of cytokine release by the bispecific antibody PD-L1-547-FEALxCD137-009-HC7LC2-FEAR directed to PD-L1 and CD137 was measured in an antigen-specific assay, performed essentially as described in Example 7.
[00466] [00466] T cells were electroporated with 10 µg of RNA encoding the α chain and 10 µg of the β chain of TCR, with or without 2 µg of RNA IVT encoding PD-1. The electroporated cells were not labeled with CFSE (as described above), but transferred to fresh IMDM medium (Life Technologies GmbH, cat. No. 12440-061) supplemented with 5% human serum AB, immediately after electroporation. iDCs were electroporated with 5 µg of claudin-6 encoding RNA (CLDN6), as described above. After overnight incubation, DCs were stained with Alexa647-conjugated CLDN6 specific antibody, and T cells with mouse TCR β chain antibody and human anti-CD279 antibody as described above.
[00467] [00467] 5,000 electroporated DCs were incubated with 50,000 electroporated T cells in the presence of different concentrations of the bispecific PD-L1-547-FEALxCD137-009-HC7LC2-FEAR or b12xIgG-FEAL control antibody in IMDM GlutaMAX, supplemented with human serum AB 5 %, in a 96-well round bottom plate. After a 48-hour incubation period, the plates were centrifuged at 500 xg for 5 minutes and the supernatant was carefully transferred from each well to a fresh 96-well round bottom plate and stored at -80 ° C until analysis of cytokines on the MSD® platform. The supernatants collected from the antigen-specific proliferation assay were analyzed for cytokine levels of 10 different cytokines by an MSD V-
[00468] [00468] The addition of PD-L1-547-FEALxCD137-009-HC7LC2-FEAR led to a concentration-dependent increase in the secretion of primarily IFN-γ, TNF-α, IL-13 and IL-8 (Figure 17). The cytokine levels of all other cytokines (IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-6) did not rise above those detected for cocultures treated with the control antibody b12- IgG-FEAL. When comparing T: DC cell cultures where T cells were not electroporated with PD-1 RNA to those where T cells were electroporated with 2 µg PD-1 RNA, slightly higher levels of cytokines were detectable for cocultures without electroporation with PD-1 RNA. This was observed not only for the PD-L1-547-FEALxCD137-009-FEAR dose-response curve, but also for the values of the control antibody b12-IgG-FEAL. Example 16: In vitro antigen-specific T cell proliferation assay to measure cytokine release induced by bispecific antibody binding to PD-L1 and CD137
[00469] [00469] The induction of cytokine release by the bispecific PD-L1-547-FEALxCD137-009-HC7LC2-FEAR targeting PD-L1 and CD137 was measured in an in vitro antigen-specific T cell proliferation assay, performed essentially as described above (Example 14). The cross-linking effect, that is, the simultaneous binding of both arms to their respective targets, on the release of cytokines from ten pro-inflammatory cytokines (IFN-γ, TNF-α, IL-13, IL-8, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-6) was analyzed by a multiplex immunoassay interspersed with the supernatants collected 48 hours after the addition of the antibody.
[00470] [00470] PBMCs were not labeled with CpSFE (as described above), but were seeded immediately after isolation and only one concentration of anti-CD3 antibody (final concentration: 0.03 µg / mL) was used.
[00471] [00471] After a 48 hour incubation period, the cells were collected by centrifugation at 500 xg for 5 minutes, and the supernatant was carefully transferred from each well to a fresh round bottom plate and 96 wells and storage at -80 °. C until cytokine analysis on the MSD® platform. The collected supernatants were analyzed for cytokine levels of 10 different cytokines by an MSD V-Plex Human Proinflammatory Panel 1 (10-Plex) kit (Meso Scale Diagnostics, LLC., Cat. No K15049D-2) on a MESO QuickPlex instrument SQ 120 (Meso Scale Diagnostics, LLC., Cat. No R31QQ-3), according to the manufacturer's instructions.
[00472] [00472] The addition of PD-L1-547-FEALxCD137-009-HC7LC2-FEAR induced a concentration-dependent increase in the secretion of primarily IFN-γ, TNF-α, IL-2 and IL-13 (Figure 18). A dose-response curve with only slightly elevated levels was also detectable for IL-10, IL-12p70, as well as for IL-4. The levels of cytokines of IL-1β, IL-6 and IL-8 remained at baseline levels and, consequently, were comparable to those levels detected for cocultures treated with the control antibody b12-IgG-FEAL.

权利要求:
Claims (88)
[1]
1. Binding agent, characterized by the fact that it comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, in which the second binding region antigen inhibits the binding of human PD-L1 to human PD-1.
[2]
2. Binding agent according to claim 1, characterized by the fact that the second antigen binding region binds to the PD cynomolgus monkey (Macaca fascicularis) PD-L1, as shown in SEQ ID NO: 29, or a mature polypeptide thereof.
[3]
Binding agent according to claim 1 or 2, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises variable regions of heavy and light chain of an antibody, which competes by binding to human PD-L1 with an antibody comprising: a. a heavy chain variable region comprising a complementarity determining region 3 of the heavy chain (HCDR3) having the sequence shown in SEQ ID NO: 20 or a sequence in which even one amino acid is modified in SEQ ID NO: 20, and b. a light chain variable region comprising a complementarity determining region 3 of the light chain (LCDR3) having the sequence shown in SEQ ID NO: 23 or a sequence in which up to two amino acids are modified in SEQ ID NO: 23.
[4]
Binding agent according to any one of claims 1 to 3, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises variable heavy and light chain regions of an antibody, the which displays the PD-L1 specificity of an antibody comprising: a. a heavy chain variable region comprising a
2/23 HCDR3 having the sequence shown in SEQ ID NO: 20 or a sequence in which even one amino acid is modified in SEQ ID NO: 20, and b. a light chain variable region comprising an LCDR3 having the sequence shown in SEQ ID NO: 23 or a sequence in which up to two amino acids are modified in SEQ ID NO: 23.
[5]
Binding agent according to any one of claims 1 to 4, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an HCDR3 having the sequence shown in SEQ ID NO: 20 or a sequence in which even one amino acid is modified in SEQ ID NO: 20.
[6]
Binding agent according to any one of claims 1 to 5, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an HCDR2 having the sequence shown in SEQ ID NO: 19 or a sequence in which even one amino acid is modified in SEQ ID NO: 19.
[7]
Binding agent according to any one of claims 1 to 6, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an HCDR1 having the sequence shown in SEQ ID NO: 18 or a sequence in which even one amino acid is modified in SEQ ID NO: 18.
[8]
Binding agent according to any one of claims 1 to 7, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence HCDR1, HCDR2 and HCDR3, where the sequence of HCDR1, HCDR2 and HCDR3 comprises the sequence as shown in: SEQ ID NO: 18, 19, 20,
3/23 respectively.
[9]
Binding agent according to any one of claims 1 to 8, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 22, DDN, 23, respectively.
[10]
Binding agent according to any one of claims 1 to 9, characterized in that said second antigen binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence HCDR1, HCDR2 and HCDR3 and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, where the sequences of HCDR1, HCDR2 and HCDR3 are the sequences as shown in: SEQ ID NO: 18, 19 , 20, respectively and the sequences of LCDR1, LCDR2 and LCDR3 are the sequences as shown in: SEQ ID NO: 22, DDN, 23, respectively.
[11]
Binding agent according to any one of claims 1 to 10, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 17.
[12]
Binding agent according to any one of claims 1 to 11, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a region
4/23 heavy chain variable (VH), where VH comprises the sequence as shown in: SEQ ID NO: 17.
[13]
Binding agent according to any one of claims 1 to 12, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VL sequence as shown in: SEQ ID NO: 21.
[14]
Binding agent according to any one of claims 1 to 13, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a light chain variable region (VL), wherein the VL comprises the sequence as shown in: SEQ ID NO: 21.
[15]
15. Binding agent according to any one of the preceding claims, characterized in that said second binding region to the antigen that binds to human PD-L1 comprises a heavy chain variable region (VH) and a variable region ( VL), where VH comprises the sequence as shown in: SEQ ID NO: 17 and VL comprises the sequence as shown in: SEQ ID NO: 21.
[16]
16. Binding agent according to any one of the preceding claims, characterized by the fact that the first antigen binding region binds to monkey cynomolgus (Macaca fascicularis) CD137, as presented in SEQ ID NO: 31, or a mature polypeptide thereof.
[17]
17. Binding agent according to any one of the preceding claims, characterized in that the first antigen binding region binds to human CD137, as shown in SEQ ID
5/23 NO: 30 or a mature polypeptide thereof, to a greater degree than it binds to a mutant human CD137 CD137 as set out in SEQ ID NO: 33 or a mature polypeptide thereof.
[18]
18. Binding agent according to any one of the preceding claims, characterized in that the first antigen binding region binds to the mutant human CD137, as presented in SEQ ID NO: 34 or a mature polypeptide thereof, with the same grade that binds to a human CD137, as shown in SEQ ID NO: 30 or a mature polypeptide thereof.
[19]
19. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises variable heavy and light chain regions of an antibody, which competes for binding to human CD137 with an antibody comprising: a. a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3) having the sequence shown in SEQ ID NO: 11 or a sequence in which up to three amino acids are modified in SEQ ID NO: 11, and a variable region light chain comprising a complementarity determining region 3 of the light chain (LCDR3) having the sequence shown in SEQ ID NO: 14 or a sequence in which up to four amino acids are modified in SEQ ID NO: 14; or b. a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3) having the sequence shown in SEQ ID NO: 52 or a sequence in which up to three amino acids are modified in SEQ ID NO: 52, and a variable region light chain comprising a complementarity determining region 3 of the light chain (LCDR3) having the sequence shown in SEQ ID NO: 55 or a sequence in which up to
6/23 four amino acids are modified in SEQ ID NO: 55.
[20]
20. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 binds to at least one of the amino acids in the amino acid sequence as presented in SEQ ID NO: 40.
[21]
21. Binding agent according to any one of the preceding claims, characterized by the fact that said first binding region to the antigen that binds to human CD137: a. it binds to the same human CD137 epitope as an antibody comprising a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 16; or b. it binds to the same human CD137 epitope as an antibody comprising a VH sequence as shown in SEQ ID NO: 49 and a VL sequence as shown in SEQ ID NO: 53.
[22]
22. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. heavy and light chain variable regions of an antibody, which exhibits CD137 specificity of an antibody comprising a heavy chain variable region comprising an HCDR3 having the sequence shown in SEQ ID NO: 11, or a sequence in which up to three amino acids are modified in SEQ ID NO: 11, and a light chain variable region comprising an LCDR3 having the sequence shown in SEQ ID NO: 14 or a sequence in which up to four amino acids are modified in SEQ ID NO: 14; or b. heavy and light chain variable regions of an antibody, which exhibits CD137 specificity of an antibody comprising a heavy chain variable region comprising an HCDR3 having the
7/23 sequence shown in SEQ ID NO: 52, or a sequence in which up to three amino acids are modified in SEQ ID NO: 52, and a light chain variable region comprising an LCDR3 having the sequence shown in SEQ ID NO: 55 or a sequence in which up to four amino acids are modified in SEQ ID NO: 55.
[23]
23. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a heavy chain variable region (VH) comprising an HCDR3 having the sequence shown in SEQ ID NO: 11 or a sequence in which up to three amino acids are modified in SEQ ID NO: 11; or b. a heavy chain variable region (VH) comprising an HCDR3 having the sequence shown in SEQ ID NO: 52 or a sequence in which up to three amino acids are modified in SEQ ID NO: 52.
[24]
24. Binding agent according to any of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises a heavy chain variable region (VH) comprising: a. an HCDR2 having the sequence shown in SEQ ID NO: 10 or a sequence in which up to two amino acids are modified in SEQ ID NO: 10; B. an HCDR2 having the sequence shown in SEQ ID NO: 52 or a sequence in which up to three amino acids are modified in SEQ ID NO: 52.
[25]
25. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a heavy chain variable region (VH) comprising
8/23 an HCDR1 having the sequence shown in SEQ ID NO: 9 or a sequence in which up to three amino acids are modified in SEQ ID NO: 9; or b. a heavy chain variable region (VH) comprising an HCDR1 having the sequence shown in SEQ ID NO: 50 or a sequence in which up to three amino acids are modified in SEQ ID NO: 50.
[26]
26. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a heavy chain variable region (VH) comprising sequences of HCDR1, HCDR2 and HCDR3, wherein the sequence of HCDR1, HCDR2 and HCDR3 comprises the sequence as shown in: SEQ ID NO: 9, 10, 11, respectively; or b. a heavy chain variable region (VH) comprising sequences of HCDR1, HCDR2 and HCDR3, wherein the sequence of HCDR1, HCDR2 and HCDR3 comprises the sequence as shown in: SEQ ID NO: 50, 51 and 52, respectively.
[27]
27. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a light chain variable region (VL) comprising sequences of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 13, GAS, 14, respectively; or b. a light chain variable region (VL) comprising sequences of LCDR1, LCDR2 and LCDR3, wherein the sequence of LCDR1, LCDR2 and LCDR3 comprises the sequence as shown in: SEQ ID NO: 54, SAS, 55, respectively.
[28]
28. Liaison officer according to any of the preceding claims, characterized by the fact that said first region
9/23 binding to the antigen that binds to human CD137 comprises: a. a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3 and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, where the sequences of HCDR1, HCDR2 and HCDR3 are the sequences as shown in: SEQ ID NO: 9, 10, 11, respectively and the sequences of LCDR1, LCDR2 and LCDR3 are the sequences as shown in: SEQ ID NO: 13, GAS, 14, respectively; or b. a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3 and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, where the sequences of HCDR1, HCDR2 and HCDR3 are the sequences as shown in: SEQ ID NO: 50, 51 and 52, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 are the sequences as shown in: SEQ ID NO: 54, SAS, 55, respectively.
[29]
29. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 15; or b. a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VH sequence as shown in: SEQ ID NO: 49.
[30]
30. Liaison officer according to any of the
10/23 previous claims, characterized by the fact that said first binding region to the antigen that binds to human CD137 comprises: a. a heavy chain variable region (VH), where VH comprises the sequence as shown in: SEQ ID NO: 15; or b. a heavy chain variable region (VH), where VH comprises the sequence as shown in: SEQ ID NO: 49.
[31]
31. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VL sequence as shown in: SEQ ID NO: 16; or b. a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence of the VL sequence as shown in: SEQ ID NO: 53.
[32]
32. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises: a. a light chain variable region (VL), where the VL comprises the sequence as shown in: SEQ ID NO: 16; or b. a light chain variable region (VL), where the VL comprises the sequence as shown in: SEQ ID NO: 53.
[33]
33. Binding agent according to any one of the preceding claims, characterized in that said first binding region to the antigen that binds to human CD137 comprises:
11/23 a. a heavy chain variable region (VH) and a variable region (VL), where the VH sequence comprises the sequence as shown in: SEQ ID NO: 15 and the VL sequence comprises the sequence as shown in: SEQ ID NO : 16; or b. a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH sequence comprises the sequence as shown in: SEQ ID NO: 49 and the VL sequence comprises the sequence as shown in: SEQ ID NO: 53.
[34]
34. Binding agent according to any one of the preceding claims, characterized in that it comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, where: a. the first antigen binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2 and CDR3, as shown in: SEQ ID NO: 9, 10, 11, respectively, or a heavy chain variable region (VH) comprising the sequences of HCDR1, HCDR2 and HCDR3, as presented in: SEQ ID NO: 50, 51, 52, respectively; and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2 and CDR3, as shown in: SEQ ID NO: 18, 19, 20, respectively.
[35]
35. Binding agent according to any one of the preceding claims, characterized in that it comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, where: a. the first antigen binding region comprises: - a heavy chain variable region (VH) comprising a sequence of CDR1, CDR2 and CDR3, as shown in: SEQ ID NO:
12/23 9, 10, 11, respectively, and a light chain variable region (VL) comprising a sequence of CDR1, CDR2 and CDR3, as shown in: SEQ ID NO: 13, GAS, 14, respectively, or - a heavy chain variable region (VH) comprising a sequence of HCDR1, HCDR2 and HCDR3, as shown in: SEQ ID NO: 50, 51 and 52, respectively, and a light chain variable region (VL) comprising a sequence of LCDR1, LCDR2 and LCDR3, as shown in: SEQ ID NO: 54, SAS and 55, respectively; and b. the second antigen-binding region comprises a heavy chain variable region (VH) comprising a sequence of CDR1, CDR2 and CDR3, as shown in: SEQ ID NO: 18, 19, 20, respectively, and a light chain variable region (VL) comprising a sequence of CDR1, CDR2 and CDR3, as shown in: SEQ ID NO: 22, DDN, 23, respectively.
[36]
36. Binding agent according to any one of the preceding claims, characterized in that it comprises a first antigen binding region that binds to human CD137 and a second antigen binding region that binds to human PD-L1, where: a. the first antigen binding region comprises: - a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 15 or - a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 49; and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 17.
[37]
37. Liaison officer according to any of the
13/23 previous claims, characterized by the fact that it comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, in which: a. the first antigen binding region comprises: - a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 15 and a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 16 or - a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 49 and a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 53; and b. the second antigen binding region comprises a heavy chain variable region (VH) comprising the sequence as shown in: SEQ ID NO: 17 and a light chain variable region (VL) comprising the sequence as shown in: SEQ ID NO: 21.
[38]
38. Binding agent according to any one of the preceding claims, characterized in that the binding agent is a multispecific antibody.
[39]
39. Binding agent according to any one of the preceding claims, characterized in that the binding agent is in the form of a complete antibody or an antibody fragment.
[40]
40. Binding agent according to any one of the preceding claims, characterized in that said first antigen binding region comprises a first heavy chain variable region (VH) and a first light chain variable region (VL) and that said second antigen binding region comprises a second heavy chain variable region (VH) and a second light chain variable region
14/23 (VL).
[41]
41. Binding agent according to any one of the preceding claims, characterized by the fact that each variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).
[42]
42. Binding agent according to claim 41, characterized by the fact that said complementarity determining regions and said framework regions are disposed of the amino termination for carboxy termination in the following order: FR1, CDR1, FR2, CDR2, FR3 , CDR3, FR4.
[43]
43. Binding agent according to any one of the preceding claims, characterized in that it comprises (i) a polypeptide comprising said first heavy chain variable region (VH) and further comprising a first heavy chain constant region (CH) and (ii) a polypeptide comprising said second heavy chain variable region (VH) and further comprising a second heavy chain constant region (CH).
[44]
44. Binding agent according to any one of the preceding claims, characterized in that it comprises (i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL) and (ii) a polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL).
[45]
45. Binding agent according to claim 43 or 44, characterized in that it is an antibody comprising a first binding arm and a second binding arm, wherein: a. the first linker comprises i) a polypeptide comprising said first heavy chain variable region (VH) and said
15/23 first heavy chain constant region (CH) and ii) a polypeptide comprising said first light chain variable region (VL) and said first light chain constant region (CL) e; B. the second linking arm comprises iii) a polypeptide comprising said second heavy chain variable region (VH) and said second heavy chain constant region (CH) and iv) a polypeptide comprising said second light chain variable region (VL) ) and said second light chain constant region (CL).
[46]
46. Binding agent according to any one of claims 43 to 45, characterized in that each of the first and second heavy chain constant (CH) regions comprises one or more of a domain 1 region of the constant region (CH1 region), a hinge region, a CH2 region and a CH3 region, preferably at least one hinge region, a CH2 region and a CH3 region.
[47]
47. Binding agent according to any one of the preceding claims, characterized in that the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.
[48]
48. Binding agent according to any one of the preceding claims, characterized in that the binding agent is a complete IgG1 antibody.
[49]
49. Liaison officer according to any one of the preceding claims, characterized by the fact that: a. the first antigen-binding region that binds to CD137 is derived from a chimeric antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a chimeric antibody.
[50]
50. Liaison officer according to any one of the preceding claims, characterized by the fact that:
16/23 a. the first antigen-binding region that binds to CD137 is derived from a humanized antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a humanized antibody.
[51]
51. Liaison officer according to any one of the preceding claims, characterized by the fact that: a. the first antigen binding region that binds to human CD137 is derived from a human antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a human antibody.
[52]
52. Liaison officer according to any of the preceding claims, characterized by the fact that: a. the first antigen-binding region that binds to human CD137 is derived from a humanized antibody and / or b. the second antigen-binding region that binds to human PD-L1 is derived from a human antibody.
[53]
53. Binding agent according to any one of the preceding claims, characterized in that each of the first and the second heavy chain constant regions (CHs) comprises a CH3 region and in which the two CH3 regions comprise asymmetric mutations.
[54]
54. Binding agent according to any one of the preceding claims, characterized in that, in said first heavy chain constant region (CH), at least one of the amino acids in a position corresponding to a position selected from the group that consists of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, according to the EU numbering, has been replaced and, in said second heavy chain constant region (CH), at least one of the amino acids in a position corresponding to a selected position the
17/23 from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, according to the EU numbering, has been replaced, and in which said first and second heavy chains are not replaced in the same positions.
[55]
55. Binding agent according to claim 54, characterized by the fact that (i) the amino acid at the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, is L in said first constant region of heavy chain (CH) and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, is R, in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, is L in said second heavy chain.
[56]
56. Binding agent according to any one of the preceding claims, characterized in that said antibody induces an Fc-mediated effector function to a lesser extent when compared to another antibody comprising the same first and second antigen binding regions and two heavy chain constant regions (CHs) comprising the hinge, CH2 and CH3 regions of human IgG1.
[57]
57. Binding agent according to claim 56, characterized in that said first and second heavy chain constant regions (CHs) are modified so that the antibody induces an Fc-mediated effector function to a lesser extent when compared to one antibody that is identical except that it comprises the first and the second unmodified heavy chain constant region (CHs).
[58]
58. Binding agent according to claim 56 or 57, characterized by the fact that said Fc-mediated effector function is measured
18/23 by binding to Fcγ receptors, binding to C1q or Fc-mediated cross-linking induction of Fcγ receptors.
[59]
59. Binding agent according to claim 58, characterized in that said Fc-mediated effector function is measured by binding to C1q.
[60]
60. Binding agent according to claim 53, characterized in that said first and second heavy chain constant regions have been modified so that the binding of C1q to said antibody is reduced when compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, wherein the C1q binding is preferably determined by ELISA.
[61]
61. Binding agent according to any one of the preceding claims, characterized by the fact that, in at least one of said first and said second heavy chain constant region (CH), one or more amino acids in the positions corresponding to the positions L234, L235, D265, N297 and P331, in a human IgG1 heavy chain, according to the EU numbering, are not L, L, D, N and P, respectively.
[62]
62. Binding agent according to claim 61, characterized by the fact that the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, according to the EU numbering, are F and E, respectively, in said first and second heavy chains.
[63]
63. Binding agent according to claim 61, characterized in that the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain, according to the EU numbering, are F, E, and A, respectively, in said first and second heavy chain constant regions (HCs).
[64]
64. Bonding agent according to claim 63, characterized in that the positions corresponding to positions L234,
19/23 L235 and D265 in a human IgG1 heavy chain, according to the EU numbering, of both, the first and the second heavy chain constant region, are F, E and A, respectively, and where (i) the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering. , of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, the first heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain, according to EU numbering, of the second heavy chain is L.
[65]
65. Binding agent according to claim 62, characterized in that the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to the EU numbering of both the first and the second constant region heavy chain, are F and E, respectively, and where (i) the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, the first heavy chain constant region is L, and the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain, according to the EU numbering, of first heavy chain constant region is R, and the position corresponding to F405 in a human IgG1 heavy chain, according to the EU numbering, of the second heavy chain is L.
[66]
66. Binding agent according to any one of the preceding claims, characterized in that the binding agent is a multispecific antibody such as a bispecific antibody.
[67]
67. Binding agent according to any of the preceding claims, characterized by the fact that the binding agent
20/23 induces and / or intensifies the proliferation of T cells.
[68]
68. Binding agent according to claim 67, characterized in that said T cells are CD4 + and / or CD8 + T cells.
[69]
69. Binding agent according to any one of the preceding claims, characterized in that the binding agent activates CD137 signaling only when the second antigen binding region binds to PD-L1.
[70]
70. Binding agent according to claim 67 or 68, characterized in that the proliferation of T cells is measured by co-culturing T cells that express a specific T cell receptor (TCR) with dendritic cells (DCs) that present the antigen corresponding to the main histocompatibility complex, which is recognized by the TCR.
[71]
71. Nucleic acid, characterized in that it encodes a binding agent as defined in any one of claims 1 to 70 or a polypeptide chain thereof.
[72]
72. Expression vector, characterized in that it comprises a nucleic acid as defined in claim 71.
[73]
73. Cell, characterized by the fact that it comprises a nucleic acid as defined in claim 71 or an expression vector as defined in claim 72.
[74]
74. Cell according to claim 73, characterized in that said cell is a mammalian cell, such as a Chinese hamster ovary cell.
[75]
75. Composition, characterized in that it comprises a binding agent as defined in any one of claims 1 to 70, a nucleic acid as defined in claim 71, an expression vector as defined in claim 72 or a cell as defined in
21/23 claim 73 or 74.
[76]
76. Composition according to claim 75, characterized in that it is a pharmaceutical composition.
[77]
77. Composition according to claim 76, characterized in that it further comprises a pharmaceutically acceptable carrier and / or excipient.
[78]
78. Binding agent according to any one of claims 1 to 70, nucleic acid according to claim 71, expression vector according to claim 72, cell according to claim 73 or 74 or composition according to any one of claims 75 to 77, characterized by the fact that it is for use as a medicine.
[79]
79. Binding agent, nucleic acid, expression vector, cell or composition according to claim 78, characterized in that it is for use in the treatment of cancer.
[80]
80. A method of treating a disease, characterized in that it comprises administering a binding agent as defined in any one of claims 1 to 70, a nucleic acid as defined in claim 71, an expression vector as defined in claim 72, a cell as defined in claim 73 or 74 or a composition as defined in any one of claims 75 to 77 to an individual in need thereof.
[81]
81. Method according to claim 80, characterized by the fact that the disease is cancer.
[82]
82. Binding agent, nucleic acid, expression vector, cell or composition, for use as defined in claim 79 or the method as defined in claim 81, characterized by the fact that cancer is characterized by the presence of solid tumors or is selected from the group consisting of: melanoma, ovarian cancer, cancer of the
22/23 lung, colon cancer and cancer of the head and neck.
[83]
83. Binding agent, nucleic acid, expression vector, cell or composition for use as defined in claim 82 or the method as defined in claim 81, characterized by the fact that the cancer is non-small cell lung cancer (CPCNP ).
[84]
84. Use of a binding agent as defined in any one of claims 1 to 70, a nucleic acid as defined in claim 71, an expression vector as defined in claim 72, a cell as defined in claim 73 or 74 or a composition as defined in any of claims 75 to 77, characterized in that it is for use in the production of a medicament, such as a medicament for the treatment of cancer, for example, a cancer distinguished by the presence of solid tumors or a cancer selected from the group consisting of: melanoma, ovarian cancer, lung cancer, colon cancer and cancer of the head and neck.
[85]
85. Use according to claim 84, characterized by the fact that lung cancer is non-small cell lung cancer (CPCNP).
[86]
86. Method according to claim 80 or 81 or use according to claim 84, characterized in that the method or use comprises a combination with one or more additional therapeutic agents, such as a chemotherapeutic agent.
[87]
87. Method for producing a bispecific antibody as defined in claim 66, characterized in that it comprises the steps of: a. cultivating a host cell that produces a first antibody comprising an antigen-binding region that binds to human CD137, as defined in any of claims 1 and 16 to 36 and optionally purifying said first culture antibody;
23/23 b. cultivating a host cell that produces a second antibody comprising an antigen binding region that binds to human PD-L1, as defined in any of claims 1 to 15 and 49 to 52, and optionally purifying said second antibody from the culture; ç. incubating said first antibody together with said second antibody under reducing conditions, sufficient to allow the cysteines in the hinge region to undergo isomerization of the disulfide bonds, and d. obtaining said bispecific antibody CD137xPD-L1.
[88]
88. Anti-idiotypic antibody, characterized by the fact that it binds to the first and / or the second antigen-binding region as defined in any one of claims 1 to 70.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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EP2017069839|2017-08-04|

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EPPCT/EP2017/069839|2017-08-04|

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EPPCT/EP2018/052946|2018-02-06|

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EP2018052946|2018-02-06|

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PCT/EP2018/071002|WO2019025545A1|2017-08-04|2018-08-02|Binding agents binding to pd-l1 and cd137 and use thereof|

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