immune cell or immune cell population of the same, immune effector cell or stem cell, cell presentin

专利摘要:
The disclosure provides compositions and a method that promote adoptive cell therapy. The disclosure provides polynucleotides, vectors, systems and cells that comprise chimeric antigen receptors (CARs), synthetic immune receptors (SIRs) and the like in combination with specific activators of NFkB activity, thereby improving cell proliferation, expression and reduced apoptosis, the which improves cell persistence in adoptive cell therapy.
公开号:BR112020005938A2
申请号:R112020005938-2
申请日:2018-09-27
公开日:2020-11-17
发明作者:Preet M. Chaudhary
申请人:University Of Southern California；
IPC主号:

专利说明:

[001] [001] The application claims the benefit provided for in Title 35 of USC § 119 (e) of the provisional application for US Serial Number 62 / 564.249, filed on September 27, 2017, the disclosures of which are incorporated into this document as a reference . TECHNICAL FIELD
[002] [002] Innovative co-stimulating modules and innovative chimeric antigen receptors are provided in this document for adoptive cell therapies for cancer, infection, allergic, degenerative and immunological disorders. INCORPORATION AS A LISTING REFERENCE OF SEQUENCES
[003] [003] Accompanying this deposit is a Sequence Listing entitled “Sequence_ST25.txt”, created on September 27, 2018 and with
[004] [004] Adoptive T-cell immunotherapy has taken the lead in cancer treatment approaches. T cells can be genetically engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor-associated antigens. CARs are genetically engineered immune receptors that can redirect T cells to selectively kill tumor cells. The general premise for its use in cancer immunotherapy is to rapidly generate T cells targeted to tumors, bypassing the barriers and incremental kinetics of active immunization and thus acting as “live drugs”. Unlike the physiological T cell receptor (TCR), which involves HLA peptide complexes, CARs involve molecules that do not require that peptide processing or HLA expression be recognized. CARs, therefore, recognize the antigen in any HLA history, in contrast to TCRs, which need to match the patient's haplotype. In addition, CARs can target tumor cells that have down-regulated HLA expression or proteasomal antigen processing, two mechanisms that contribute to the escape of tumors from TCR-mediated immunity. Another feature of the wide applicability of CARs is their ability to bind not only to proteins, but also to carbohydrate and glycolipid structures, expanding the range of potential targets again. SUMMARY
[005] [005] The disclosure provides an immune cell or a population of immune cells expressing (i) at least one non-naturally occurring immune receptor and (ii) at least one non-naturally occurring agent that selectively activates the NF- signaling pathway. κB. In one embodiment, the at least one non-naturally occurring immune receptor comprises at least one antigen-binding domain and at least one transmembrane domain. In another or another modality, the at least one
[006] [006] The disclosure also provides at least one recombinant polynucleotide that encodes at least one non-naturally occurring immune receptor, the at least one recombinant polynucleotide comprising (a) a first nucleic acid domain that encodes a partial or total transmembrane domain and / or cytoplasmic and, optionally, the extracellular domain of an endogenous protein, in which the endogenous protein is expressed on the surface of lymphocytes and triggers the activation and / or proliferation of the lymphocyte; (b) optionally, a polynucleotide
[007] [007] The disclosure also provides at least one recombinant polynucleotide that comprises a first nucleic acid that encodes an unnaturally occurring immune receptor; and a second nucleic acid encoding an accessory module comprising a selective NF-κB activator. In one embodiment, the first nucleic acid and the second nucleic acid are separated by an oligonucleotide linker that encodes a cleavable peptide linker. In another embodiment, the at least one comprises two recombinant polynucleotides, so that the first nucleic acid and the second nucleic acid are expressed from separate vectors. In another or another modality, the selective NF-κB activator is a non-naturally occurring selective NF-κB activator. In another or another modality, the non-naturally occurring immune receptor is selected from the group consisting of a CAR, an Ab-TCR, a TFP, a cTCR, a SIR and a recombinant TCR. In another or another embodiment, the unnaturally occurring immune receptor comprises (i) a specific extracellular antigen domain, (ii) a transmembrane domain, and (iii) an optional intracellular signaling domain that comprises an activation motif based on in immunoreceptor tyrosine (ITAM), where (iii) is located at the C terminal of the unnaturally occurring immune receptor. In another or another embodiment, by expressing the first and second nucleic acid sequences, the non-naturally occurring immune receptor and the selective NF-κB activator polypeptide are not physically or chemically linked. In another or another modality,
[008] [008] The disclosure also provides at least one vector comprising at least one polynucleotide from any of the polynucleotide constructs described in this document and above. In one embodiment, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, AAV vector, a retroviral vector, a baculovirus vector, a Sleeping Beauty transposition vector and a piggybac transposition vector.
[009] [009] The disclosure also provides an immune effector cell or stem cell comprising at least one recombinant polynucleotide, construct or vector described in this document and above. In one modality, the
[0010] [0010] The disclosure also provides a method for (i) prolonging the life of an immune cell in expression, (ii) stimulating the proliferation of an immune cell, (iii) stimulating the production of cytokines by an immune cell, (iv ) improve the presentation of antigens by an immune cell, (v) protect an immune cell from apoptosis, in which the method comprises transfecting or transforming the immune cells with a polynucleotide encoding a selective NF-κB activator or a specific stimulating polypeptide of NF-κB. In one embodiment, the selective NF-κB activator or a specific NF-κB stimulating polypeptide is selected from the group consisting of vFLIP K13, K13-opt, a NEMO mutant, a NEMO fusion protein, IKK1-S176E-S180E, IKK2 -S177E-S181E, RIP, IKKα, IKKβ, Tcl-1, MyD88-L265, any NF-κB activating protein or protein fragment, any inhibitor of an NF-κB pathway inhibitor, any homolog or variant thereof and any combination thereof. In another or an additional embodiment, the NF-κB-specific stimulator or selective stimulator polypeptide is expressed in a constitutive or inducible manner. In another or an additional embodiment, the selective NF-κB activator or a specific NF-κB stimulating polypeptide is controlled post-translation by contacting the T cell with a compound. In another or an additional embodiment, the selective NF-κB activator or NF-κB-specific stimulating polypeptide is expressed as a fusion construct with one or more copies of the FKBP domain. In another or an additional embodiment, the activity of the selective NF-κB activator or a specific NF-κB stimulating polypeptide is controlled at the post-translational level by
[0011] [0011] The disclosure also provides a method for producing an immune effector cell that expresses an unnaturally occurring immune receptor that comprises introducing at least one vector or at least one recombinant polynucleotide into an effector cell or a hematopoietic stem cell or progenitor cell that can give rise to an immune effector cell, under conditions such that an unnaturally occurring immune receptor is expressed and the immune effector cell comprises (i) prolonged life, (ii) improved T cell proliferation and / or ( iii) reduced apoptosis compared to a CAR-T cell that lacks a specific NFkB stimulating polypeptide In another or another embodiment, the method further comprises providing a population of immune effector cells; and removing regulatory T cells from the population, thereby providing a population of depleted regulatory T cells; wherein the steps are performed before the introduction of the recombinant vector or polynucleotide that encodes the specific stimulating polypeptide of CAR and / or NFkB to the population. In another or another embodiment, regulatory T cells are removed from the cell population with the use of an anti-CD25 antibody or an anti-GITR antibody. In another or another embodiment, the method further comprises a) providing a population of immune effector cells; and b) enriching P-glycoprotein positive cells (P-gn or Pgp; MDR1, ABCB1, CD243) from the population, thereby providing a population of P-glycoprotein enriched cells (P-gn or Pgp; MDR1, ABCB1, CD243); wherein steps a) and b) are performed before or after the introduction of the recombinant vector or polynucleotide encoding the CAR and / or NFkB-specific stimulating polypeptide. In another or an additional modality, positive P glycoprotein cells are enriched
[0012] [0012] The disclosure also provides a method of generating a population of cells genetically engineered by RNA that comprises introducing RNA or RNAs transcribed in vitro or synthetic RNA or RNAs into a cell or cell population, wherein the RNA or RNAs comprise a recombinant polynucleotides or polynucleotides of the disclosure.
[0013] [0013] The disclosure also provides a method of providing anti-disease immunity in an individual comprising administering to the individual an effective amount of the immune effector cell or a stem cell that can give rise to an immune effector cell of the disclosure, wherein the cell is a cell
[0014] [0014] The disclosure also provides a composition that comprises an immune effector cell or a stem cell that can generate immune effector cells that comprise an unnaturally occurring immune receptor and a selective NFkB activator, in which said disease-associated antigen is selected from a group consisting of: CD5, CD19; CD123; CD22; CD30; CD171; CS-1 (also called subset CD2 1, CRACC, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); tyrosine kinase 1 orphan receptor (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); Mesothelin; Alpha interleukin 11 receptor (IL-11Ra); prostate stem cell antigen (PSCA); Serine protease 21 (testin or PRSS21); growth factor receptor 2
[0015] [0015] Disclosure also provides a method for treating or
[0016] [0016] Details of one or more embodiments of the invention are presented in the attached drawings and in the description below. Other features, objects and advantages of the invention will be evident from the description and drawings and the claims. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] [0017] Figure 1 depicts a drawing of a current antibody, T cell receptor (TCR), CAR and next generation CARs and SIRs.
[0018] [0018] Figure 2 depicts a drawing comparing the biological activity and structure of second generation CAR with a modality of the present disclosure that depicts a CAR without CD28 or 41BB, but expressing an NF-κB stimulating molecule (NEMO and / or K13 , or mutants thereof).
[0019] [0019] Figure 3 shows strong activation of NF-kB by mNEMO-K270A, hNEMO-K277A and weak activation by hNEMO-K277I and hNEMO-K277G mutant.
[0020] [0020] Figure 4 shows activity of a Bispecific T-cell engager targeting MPL and using a scFv-161 targeting domain. T cells and HEL-pLenti-hGluc were previously incubated separately with the following supernatants at 4 ° C for 2 h only in Medium and pLenti-161-StreptagII-CD3-Myc-His-P02 (042517-P02-SC). After incubation, the cells were co-cultured in a 96-well U-bottom plate in the E: T ratio of 1: 1 or 5: 1 for 4h at 37 C. 50 μl cells + sup / well were transferred to a 384 wells in triplicate. The hGLuc assay was performed using 15 µl CTZ assay buffer (1: 100).
[0021] [0021] Figure 5A to C shows CRISPR / TF9 gene mediated by cas9 targeting within the TRAC site and strategies to rescue TRAC expression at, Top, TRAC site with the 5 '(gray) end of the first TRAC exon , the TRAC gRNA (blue) and the corresponding PAM sequence (see guide). The two blue arrows indicate the expected Cas9 double ribbon break. Lower integration, directed to CRISPR / Cas9 at the TRAC site. The targeting construct (AAV) contains a splicing acceptor (SA), followed by an F2A coding sequence, the TFP gene and a polyA sequence, flanked by sequences homologous to the TRAC site (LHA and RHA, left homology arm and right). Once integrated, the endogenous TCRα promoter directs TFP expression, while the TRAC site is disrupted. B) The targeting construct expresses TFP and coexpresses
[0022] [0022] Figure 6A to E shows several construction projects for targeting cassette to direct an Ab-TCR to the TRAC site.
[0023] [0023] Figure 7A to F shows several construction projects for the targeting cassette to direct a cTCR (SIR) to the TRAC site.
[0024] [0024] Figure 8A to D shows several construction projects for the targeting cassette to direct a cTCR (SIR) and a TCR to the TRAC site.
[0025] [0025] Figure 9A to D shows several construction projects for the targeting cassette to target a single chain cTCR (SIR) to the TRAC site. DETAILED DESCRIPTION
[0026] [0026] Initial first generation CARs were constructed by fusing an antigen-binding domain based on scFv (single chain fragment variable) to an inert CD8 transmembrane domain, linked to a cytoplasmic signaling domain derived from γ chains. CD3-ζ or Fc receptor (Figure 1).
[0027] [0027] Although CD3-CD chain aggregation is sufficient to allow lytic activity of T cells, they failed to obtain a robust cytokine response, including interleukin-2 (IL-2), and support the expansion of T cells after repeated exposure to the antigen. For optimal activation and proliferation, T cells require T cell receptor engagement and signaling, as well as co-stimulating signaling via
[0028] [0028] Figure 2 depicts a cartoon of a 2nd generation CAR, as described above next to a first generation CAR plus a specific stimulating molecule of NF-κB that depicts the biological activity associated with each one.
[0029] [0029] The CAR constructs in current clinical use are of artificial design, since they represent the fusion of several different proteins. In particular, the inclusion of the co-stimulating domain in the 2nd generation CAR construct results in non-physiological signaling through the receptor, which, in turn, could contribute to its toxicity. Some CARs show antigen-independent tonic signaling, which leads to unrestricted cell activation, ultimately resulting in apoptosis, excessive release of cytokines independent of cognate antigens and immune exhaustion. Tonic signaling through co-stimulating domains (eg, 41BB and CD28 domains) has been shown to prevent T cell survival. Therefore, it is necessary to improve the CAR design to obtain long-term persistence of CAR modified T cells without the risk of toxicity
[0030] [0030] To overcome some of the design limitations of conventional 2nd generation CARs, several alternative projects, collectively referred to as next generation CARs, have been described, including Ab-TCR (Document WO 2017/070608 A1 incorporated in this document for reference) , TCR or TFP receptor fusion proteins (WO 2016/187349 A1 incorporated in this document for reference), Synthetic Immune Receptors (SIRs) (see WO 2018/102795 A1, incorporated in this document for reference) , multifunctional T cell antigen coupler (Tri-TAC) (see WO 2015/117229 A1, incorporated by reference in this document). These alternative CAR projects, in general, lack a co-stimulating domain.
[0031] [0031] To overcome the limitations of AKT activation and tonic signaling, this disclosure demonstrates the use of selective NF-κB activators, such as NEMO mutants (eg, hNEMO-K277A, hNEMO-K277A- DeltaV249-K255, NEMO-K270A of mouse), K13-opt, IKK2-S177E-S181E or IKK1-S176E-S180E, to provide co-stimulating function. In contrast to the 41BB and CD28 derived co-stimulating domains, which activate various signaling pathways (see 2nd and 3rd generation CARs in Figure 1), selective NF-κB activators, such as, for example, hNEMO-K277A, hNEMO-K277A- DeltaV249-K255, mouse NEMO-K270A, K13-opt, IKK2-S177E-S181E or IKK1-S176E-S180E, selectively activate the NF-κB pathway by activating the I-kKKinas (IKK) complex. The disclosure also describes an alternative non-naturally occurring immune receptor project, for example, CAR, in which co-stimulation is provided by an accessory module comprising a selective NF-κB activator that is co-expressed with the non-naturally occurring immune receptor ( for example, a CAR). However, in contrast to the 2nd generation CAR constructs, in which the
[0032] [0032] The disclosure demonstrates that the expression of selective NF-κB activators, such as, for example, hNEMO-K277A, hNEMO-K277A- DeltaV249-K255, mouse NEMO-K270A, IKK2-S177E-S181E, IKK1-S176- S180E and K13-opt, in T cells, extend their ability to proliferate in the long term in culture without undergoing senescence, demonstrating for the first time that the activation of a single pathway (ie, NF-κB) is sufficient to postpone cell senescence T. For example, CD19-CAR constructs that coexpress hNEMO-K277A or hNEMO-K277A-DeltaV249-K255, but without a co-stimulating domain, demonstrate superior in vivo efficacy compared to the second generation CAR construct that contains the 41BB co-stimulating domain. The disclosure further demonstrates that the selective activation of NF-κB is sufficient to promote the proliferation of T cells, delay senescence and improve the performance of T cells for adoptive cell therapy, including CAR-T cell therapy. Thus, the disclosure provides composition and methods to improve survival, proliferation, cytokine secretion, delay exhaustion and senescence and improve the in vivo expansion, persistence and antitumor activity of an immune cell, for example, T cell, for example, CAR -T or TCR-T or SIR-T cell and / or an immune cell that expresses an unnaturally occurring immune receptor, by means of selective or preferential activation (that is, without AKT activation) of the pathway
[0033] [0033] The disclosure further demonstrates that selective activators of NF- κB, such as, for example, hNEMO-K277A, hNEMO-K277A-DeltaV249- K255, mouse NEMO-K270A, K13-opt, IKK2-S177E-S181E or IKK1-E S176E-S180E, can be used to improve the performance of vaccines by promoting the secretion of cytokines and the presentation of antigens by immune cells, for example, cells that have antigens, for example, dendritic cells. For example, bone marrow-derived dendritic cells (DC) that express selective NF-kB activators, such as hNEMO-K277A, hNEMO-K277A-DeltaV249-K255, mouse nemo-K270A, K13-opt, IKK2-S177E-S181 or IKK1-S176E-S180E, show superior cytokine production, antigen presentation and immune response (for example, antitumor response or anti-infective agent response) compared to control DC.
[0034] [0034] The disclosure further provides NF-κB activators, including selective NF-κB activators that are of human origin and therefore are less immunogenic.
[0035] [0035] The disclosure also provides NF-κB activators, including selective NF-κB activators that can be expressed in the cytosol. The disclosure also provides NF-κB activators, including selective NF-κB activators, which are constitutively active and do not require a stimulus, for example, treatment with a ligand, for its ability to activate NF-κB.
[0036] [0036] The disclosure also provides several antigen-binding domains that can be used in the generation of conventional CARs (eg
[0037] [0037] The disclosure further provides new methods for generating allogeneic T cells that express TCR and CARs, including next generation CARs (eg, TFP, SIR, Ab-TCR, cTCR), for the purpose of ready-to-use adoptive cell therapy .
[0038] [0038] The disclosure further provides new methods of combined therapies using autologous and allogeneic T cells that express TCR and CARs, including next generation CARs (eg TFP, SIR, Ab-TCR and cTCR). The disclosure provides methods for restoring the expression and / or activity of TFPs based on the CD3ε, CD3γ and CDδ chains in T cells without the expression of the native TCRα, TCRβ, TCRγ or TCRδ chains coexpressing the TCRα constant chains in the cells expressing the TFPs , TCRβ, TCRγ or TCRδ. The disclosure also provides methods of restoring the expression and / or activity of TFPs based on the CD3ε, CD3γ and CDδ chains in T cells without the expression of the native TCRα, TCRβ, TCRγ or TCRδ chains coexpressing in the cells expressing the SIRs TFPs or Ab-TCR encoding the full length or fragments of constant chains TCRα, TCRβ, TCRγ or TCRδ. The disclosure ensures that TFPs based on CD3ε, CD3γ and CDδ chains can be combined with SIRs or Ab-TCR that encode the constant chains of TCRα, TCRβ, TCRγ or TCRδ constant chains in T cells without the native TCRα, TCRβ, TCRγ or TCRδ for allogeneic and ready to use therapy.
[0039] [0039] As used in this document and the appended claims, the singular forms "one (a)" and "one (a)" include plural referents, unless the context clearly indicates otherwise. Thus, for example, the reference to "a cell" includes a plurality of such cells, and the reference to "polynucleotide" includes reference to one or more polynucleotides and so
[0040] [0040] In addition, the use of "or" means "and / or", unless otherwise specified. Likewise, "understand", "understand", "comprising", "include", "includes" and "including" are interchangeable and are not intended to be limiting.
[0041] [0041] It should also be understood that, when the descriptions of various modalities use the term "comprising", those skilled in the art would understand that, in some specific cases, a modality can be described alternatively using the language "consisting essentially of" or " consisting of ”.
[0042] [0042] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by an individual versed in the technique to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., Revised edition. J. Wiley & Sons (New York, NY 2006); Smith, March’s Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (28 November 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides a person skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor, NY, 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. July 1976, 6 (7): 511 to 519; Queen and
[0043] [0043] All publications contained in this document are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Any references cited do not admit that any of the information provided in this document is prior art or relevant to the currently claimed invention, or that any publication specifically or implicitly mentioned is prior art.
[0044] [0044] The term “about” when referring to a measurable value, such as a quantity, a time duration and the like, should cover variations of ± 20% or, in some cases, ± 10%, or, in some cases, ± 5%, or in some cases ± 1% or in some cases ± 0.1% of the specified value, as these variations are appropriate for performing the methods disclosed or describing the compositions in this document. In addition, any value or range (for example, less than 20 or similar terminology) explicitly includes any integer between those values or even the value.
[0045] [0045] The term “Ab-TCR” or “AbTCR” refers to a next generation CAR platform, as described in WO 2017/070608 A1, which is incorporated by reference in this document. In one embodiment, an Ab-TCR comprises an antibody fraction that specifically binds to a target antigen fused to a TCR module capable of recruiting at least one TCR signaling module. Exemplary TCR modules that can be used in the Ab-TCR construct are provided in SEQ ID NO: 959 to 964 (Table 6D) and WO 2017/070608 A1, which is incorporated by reference in this document. In the TCR module, TCRb-IAH-6MD, three amino acid residues (F133, E136 and Q139) found in the human TCRb chain (SEQ ID NO: 15,053) (see Tables 4, 5 and 6D) are mutated to the Isoleucine residues , Alanine and Histidine found in the murine chain of TCRb, respectively, in order to improve the expression of this module. Likewise, in the IgG1-CH1-TCRa-SDVP-6MD TCR module, four amino acid residues (P91, E92, S93, S94) found in the human TCRa chain (SEQ ID NO:
[0046] [0046] The term “accessory module” refers to any one or more of hNEMO-K277A (or NEMO-K277A), hNEMO-K277A-delta-V249- K555, mNEMO-K270A, K13-opt, IKK2-S177E-S181E (or IKK2-SS / EE), IKK1-S176E-S180E (or IKK1-SS / EE), MyD88-L265P, TCL-1a, MTCP-1, CMV-141, 41BBL, CD40L, vFLIP-K13, MC159, cFLIP -L / MRITα, cFLIP- p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f , PD1-4H1 scFV, PD1-5C4 scFV, PD1-4H1-Alb8-vHH, PD1- 5C4-Alb8-vHH, CTLA4-Ipilimumab-scFv, CTLA4-Ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A -scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, Brd4 targeting shRNA, IgSP- [hTRAC-opt2], IgSP-
[0047] [0047] As used in this document, "affinity" is intended to describe a measure of the binding force. Affinity, in some cases, depends on the proximity of the stereochemical adjustment between a binding agent and its target (for example, between an antibody and antigen, including epitopes specific for the binding domain), the size of the contact area between the same and the distribution of charged and hydrophobic groups. Affinity generally refers to the "ability" of the liaison officer to bind its target. There are a number of ways used in the technique to measure "affinity". For example, methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. The binding affinity can be determined using various techniques known in the art, for example, surface plasmon resonance, bi-layer interferometry, double polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA,
[0048] [0048] The “Via AKT” or “Via PI3K-AKT”, as used in this document, is a signal transduction pathway that promotes survival and growth in response to extracellular signals. The main proteins involved are PI3K (phosphatidylinositol 3-kinase) and Akt (Protein Kinase B).
[0049] [0049] The term "antibody", as used in this document, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. The antibodies can be monoclonal or polyclonal immunoglobulins, multiple or single chain or intact and can be derived from natural or recombinant sources. The antibodies can be tetramers of immunoglobulin molecules. The antibody can be "humanized", "chimeric" or non-human.
[0050] [0050] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with (for example, by binding, steric hindrance, stabilization / destabilization, spatial distribution) an epitope of an antigen . Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (ab'h, Fv fragments, scFv antibody fragments,
[0051] [0051] The term "antibody heavy chain" refers to the greater of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations and which normally determines the class to which the antibody belongs.
[0052] [0052] The term "antibody light chain" refers to the lesser of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. The Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.
[0053] [0053] "Anticancer agent" refers to agents that inhibit aberrant cell division and growth, inhibit the migration of neoplastic cells, inhibit invasion or prevent the growth and metastasis of cancer. The term includes chemotherapeutic agents, biological agent (eg, siRNA, viral vectors such as genetically engineered MLV, adenovirus, herpes viruses that deliver cytotoxic genes), antibodies and the like.
[0054] [0054] The term “anticancer effect” refers to a biological effect that
[0055] [0055] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent cells, or both. The person skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, the antigens can be derived from recombinant or genomic DNA. A person skilled in the art will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response, therefore encodes an "antigen", as that term is used in this document. In addition, a person skilled in the art will understand that an antigen does not need to be encoded by just a complete nucleotide sequence of a gene. Disclosure includes, but is not limited to, the use of partial nucleotide sequences from more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. In addition, an individual skilled in the art will understand that an antigen does not need to be encoded by a "gene". It is readily apparent that an antigen can be generated synthesized, or it can be derived from a biological sample, or it can be macromolecule in addition to a polypeptide. This biological sample may include, but is not limited to, a tissue sample, a sample of
[0056] [0056] Non-limiting examples of target antigens include: CD5; CD19; CD123; CD22; CD30; CD171; CS1 (also called subset CD2 1, CRACC, MPL, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1- 1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); tyrosine kinase 1 orphan receptor (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); Mesothelin; Alpha interleukin 11 receptor (IL-11Ra); prostate stem cell antigen (PSCA); Serine protease 21 (testin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis antigen (Y); CD24; Platelet-derived growth factor beta receptor (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Alpha folate receptor (FRa or FR1); Beta folate receptor (FRb); ERBB2 tyrosine-protein kinase receptor (Her2 / neu); Mucine 1 associated with the cell surface (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostasis; prostatic acid phosphatase (PAP); mutation elongation factor 2 (ELF2M); Ephrin B2; alpha fibroblast activation protein (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase
[0057] [0057] The term “antigen presenting cell” or “APC” refers to an immune system cell, such as an accessory cell (for example, a B cell, a dendritic cell and the like) that exhibits a foreign antigen complexed with the major histocompatibility complexes (MHCs) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.
[0058] [0058] The term “anti-infection effect” refers to a biological effect that can be manifested by various means, including, but not limited to, for example, decreased infectious agent titer, decreased infectious agent colony count, improvement of various physiological symptoms associated with the infectious condition. An “anti-infectious effect”
[0059] [0059] The term "anti-tumor effect" or "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of cells tumor cells, a decrease in tumor cell proliferation or a decrease in tumor cell survival.
[0060] [0060] An "antigen binding domain" or "antigen binding module" or "antigen binding segment" or "specific antigen domain" (ASD) refers to a polypeptide or peptide which, due to its sequence primary, secondary or tertiary, post-translational modifications and / or charge binds to an antigen with a high degree of specificity. The antigen-binding domain can be derived from different sources, for example, an antibody (full-length heavy chain, Fab fragments, Fv single chain fragments (scFv), divalent single chain antibodies or diabetes), a binding protein not immunoglobulin, a ligand or a receptor. However, there are numerous alternatives, such as linked cytokines (which lead to the recognition of cells carrying the cytokine receptor), affibodies, naturally occurring receptor ligand-binding domains, soluble protein / peptide ligand for a receptor (for example, in a tumor cell), peptides and vaccines to solicit an immune response, which can be used in various embodiments of the invention. In some embodiments, almost any molecule that binds to a particular antigen with high affinity can be used as an ASD, as will be appreciated by those skilled in the art. In some embodiments, the antigen-binding domain comprises T cell receptors (TCRs) or portions thereof. In exemplary modalities, acids
[0061] [0061] The term "association constant (Ka)" is defined as the equilibrium constant of the association of a receptor and ligand.
[0062] [0062] "Autoantibody" refers to an antibody that is produced by a specific B cell for an autoantigen.
[0063] [0063] The term "autoantigen" refers to an endogenous antigen that stimulates the production of an autoimmune response, such as the production of autoantibodies. The autoantigen also includes an autoantigen or antigen from normal tissue that is the target of a cell-mediated or antibody-mediated immune response that can result in the development of an autoimmune disease. Examples of autoantigens include, but are not limited to, desmoglein 1, desmoglein 3 and fragments thereof.
[0064] [0064] "Avidity" refers to the strength of the interaction between a binding agent and its target (for example, the strength of the interaction between an antibody and its antigen target, a receptor and its cognate and the like). Greed can be weak or strong. Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. The activity of the antibody in functional assays (for example, flow cytometry assay) also reflects the affinity of the antibody. Antibodies and affinities can be characterized phenotypically and compared using functional assays (for example, flow cytometry assay).
[0065] [0065] As used in this document, the term “main structure” refers to the specific combination of CARs (Table 1) and accessory modules, as described in Table 2. In exemplary modalities,
[0066] [0066] As used in this document, "beneficial results" may include, but are not limited to, decreasing or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the development of the disease condition disease, decrease a patient's chances of developing the disease condition, and prolong a patient's life or life expectancy. As non-limiting examples, “beneficial results” or “desired results” may be relief of one or more symptoms, decrease in the extent of the deficit, stabilized (that is, not worsening) cancer progression, delayed or slow metastasis or invasiveness and improvement or palliation of symptoms associated with cancer.
[0067] [0067] As used in this document, the term "binding domain" or "antibody molecule" refers to a protein, for example, an immunoglobulin chain or fragment thereof, ligand domain or fragment thereof (as appropriate) ), comprising at least one domain, for example, immunoglobulin variable domain sequence that can bind to a target with greater affinity than a non-specific domain. The term encompasses antibodies and antibody fragments, or ligands and ligand fragments. In another embodiment, an antibody molecule is a multispecific antibody molecule, for example, comprising a plurality of immunoglobulin variable domain sequences, wherein
[0068] [0068] "Binds the same epitope as" means the ability of an antibody, scFv or other antigen binding domain to bind to a target antigen and have the same epitope as an exemplified antibody, scFv, or another antigen binding domain . As an example, the epitopes of
[0069] [0069] As used in this document, the term "biological equivalent of the same" must be synonymous with "equivalent of the same" when referring to a reference protein, antibody or fragment thereof, polypeptide or nucleic acid, having minimal homology while maintaining the desired structure or functionality. Unless specifically stated in this document, it is contemplated that any of the above also includes their equivalents. For example, an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and, alternatively, or at least about 85%, or
[0070] [0070] As used in this document, the term "CDR" or "complementarity determining region" is intended to mean the sites of combination of non-contiguous antigens found in the variable region of the polypeptides of the heavy and light chains. These particular regions have been described by Kabat et al., J. Bioi. Chern. 252: 6,609 to 6,616 (1977); Kabat et al., US Department of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Bioi. 196: 901 to 917 (1987); and MacCallum et al., J. Mol. Bioi. 25 262: 732 to 745 (1996), in which the definitions include overlap or subsets of amino acid residues when compared to each other. However, the application of any of the definitions to refer to a CDR of an antibody or grafted antibodies or variants thereof must be within the scope of the term as defined and used in this document. As used in this document, the different CDRs of an antibody can also be defined by a combination of the different definitions. For example, vHCDR1 can be defined based on Kabat and VHCDR2 can be defined based on Chothia. The amino acid residues that comprise CDRs, as defined by each of the references cited above, are as follows:
[0071] [0071] (The waste numbers correspond to the identified reference).
[0072] [0072] The SEQ IDs of the CDRs of the different vL and vH segments that can form CAR antigen binding domains of the disclosure are provided in SEQ ID NO: 13.204 to 14.121 and SEQ ID NO: 14.122 to 15.039, respectively (Tables 6A, B) and Tables 5 to 6 in document PCT / US2017 / 064.379, which are incorporated by reference in this document.
[0073] [0073] In some embodiments, the reference to an antigen binding module (such as a Fab or Fv antigen binding module) that specifically binds to a target antigen means that the antigen binding module binds to the target antigen with (a) an affinity that is at least about 10 (e.g., about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1,000 or more) times your affinity binding to other molecules; or (b) a Kd no more than about 1/10 (e.g. 1/10, 1/20, 1/30, 1/40, 1/50, 1175, 1/100, 1/200, 1 / 300, 1/400, 1/500, 1/750, 1 / 1,000 or less) times your Kd to bind to other molecules. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell classification analysis (FACS) or radioimmunoprecipitation assay (RIA). Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay, using, for example, Biacore instruments, or clinical exclusion assay (KinExA) using, for example, Sapidyne instruments.
[0074] [0074] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, B cell lymphomas (Hodgkin's lymphomas and / or non-Hodgkins lymphomas), T-cell lymphomas, myeloma, myelodysplastic syndrome, skin cancer, brain tumor, breast cancer, colon cancer, rectal cancer, esophageal cancer, anal cancer, cancer of unknown primary site, endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, reproductive organ cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer (eg glioblastoma multiforme), prostate cancer, including, but not limitation, androgen-dependent prostate cancer and androgen-independent prostate cancer, and leukemia. Other cancers and cell proliferative disorders will be easily recognized in the art. The terms "tumor" and "cancerous" are used interchangeably in this document, for example, the two terms encompass solid and liquid tumors, for example, diffuse or circulating. As used in this document, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors. The term "cancer" should include all types of cancerous growth or oncogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the histopathological type or stage of invasion. Examples of solid tumors include malignant neoplasms, for example, adenocarcinomas, sarcomas and carcinomas, of various organ systems, such as those affecting the breast, liver, lung, brain, lymphoid, gastrointestinal (for example, colon), genitourinary tract (for example, renal, urothelial cells), prostate and pharynx. Adenocarcinomas include cancers, like most colon cancers, rectal cancer,
[0075] [0075] "Chemotherapeutic agents" are compounds that are known to be used in cancer chemotherapy. Non-limiting examples of chemotherapeutic agents can include alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene phosphorphide and triethylene thiophosphoramide; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including synthetic analogue topotecan); briostatin; calistatin; CC-1065 (including their synthetic analogues adozelesin, carzelesin and bizelesin); cryptoficina (particularly cryptoficina 1 and criptoficina 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleuterobin; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mecloretamine, meclorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enedinic antibiotics (for example, calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, for example, Agnew, Chem. Intl. Ed. Engl. Engl., 33: 183 to 186 (1994)); dinemicin A; bisphosphonates, such as clodronate; a speramycin; as well as neocarzinostatin chromophore and related chromoprotein antibiotic chromophores), aclacinomysins, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin. -diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN® (including morpholino-doxorubicin,
[0076] [0076] "Chimeric antigen receptors" (CARs) are receptors for artificial (non-naturally occurring) immune cells (eg, T cells) contemplated for use as cancer therapy, using a technique called adoptive cell transfer. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. The functions of antigen binding, signaling and stimulation of the complex were manipulated by methods of genetic recombination to a single polypeptide chain, generally called Chimeric Antigen Receptor (CAR). See, for example, Eshhar, Pat. in the US
[0077] [0077] "Codon optimization" or "codon bias control of species" refers to the preferential use of codons from a specific host cell. As will be understood by those skilled in the art, it may be advantageous to modify a coding sequence to improve its expression in a specific host. The genetic code is redundant with 64 possible codons, but most organisms generally use a subset of these codons. Codons that are used most often in a species are called ideal codons, and those that are not used very often are classified as rare or low-use codons.
[0078] [0078] Optimized coding sequences containing preferred codons by a specific prokaryotic or eukaryotic host (see also Murray et al. (1989) Nucl. Acids Res. 17: 477 to 508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts with desirable properties, such as a longer half-life, compared to transcripts produced from a non-optimized sequence. The translation stop codons can also be modified to reflect the host's preference. Those skilled in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds that
[0079] [0079] As used in this document, "coexpress" refers to the expression of two or more polynucleotides or genes. The genes can be nucleic acids that encode, for example, a single protein or a chimeric protein as a single polypeptide chain. A CAR or TCR described in this document can be encoded by a single polynucleotide chain and expressed as a single polypeptide chain, which is subsequently cleaved into different polypeptides, each representing a distinct functional unit. In some embodiments, where the CAR or a TCR consists of two or more functional polypeptide units, the different functional units are coexpressed using one or more polynucleotide chains. In one embodiment, co-stimulation is provided by an accessory module that is co-expressed with the CAR or a TCR, but is not an integral part of the CAR or TCR polypeptide. Such an accessory module that provides co-stimulation to a cell that expresses CAR or TCR or any cell, but is not an integral part of the CAR or the TCR polypeptide, is called a CAR or CICM independent co-stimulating module. In another embodiment, the different polynucleotide chains are linked by nucleic acid sequences that code for cleavable ligands (for example, T2A, F2A, P2A, E2A, etc.) (Table 6D). In another embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NO: 4,844) is also added upstream of the cleavable linker sequences to increase cleavage efficiency. Polynucleotides that encode the different units of a CAR or a TCR can be linked by IRES (Internal Ribosomal Entry Sites) sequences. Alternatively, the different functional units of a CAR or TCR are encoded by two different polynucleotides that are not linked by means of a linker, but are encoded by, for example, two different vectors. The nucleic acid and amino acid sequences of
[0080] [0080] A "conservative substitution" or "conservative sequence modifications" refers to modifications of amino acids that do not affect or significantly alter the binding characteristics or functions of the encoded protein. For example, "conservative sequence modifications" refers to amino acid modifications that do not affect or significantly alter the characteristics or binding function of a CAR construct of the disclosure (eg, a conservative change in the constant chain, antibody, antibody fragment or non-immunoglobulin binding domains). Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the disclosure can be replaced by other amino acid residues from the same family of side chains and the altered CAR can be tested using the binding and / or functional assays described in this document.
[0081] [0081] The term "alpha T cell receptor constant region" or "alpha T cell receptor constant chain" or "TCRα" or "Cα" is defined as the protein provided as SEQ ID NO: 15,041 or equivalent residues (ie, a homolog) of a non-human species, for example, mouse, rodent, monkey, simian and the like. The disclosure also provides certain mutations in the TCRα polypeptides that can be used in the SIRs and Ab-TCR construct (Tables 3 and 6D). For example, Cα mutation sites that demonstrate increased expression and decreased mismatch are located at positions 91, 92, 93 and 94 of SEQ ID NO
[0082] [0082] The human genome codes for two highly homologous TCR beta constant chains; TCR beta1 (TCRβ1 or TCRb1 or cβ1) and TCR beta 2 (TCRβ2 or TCRb2 or cβ2). The disclosure CARs can comprise either of these two chains. Likewise, the TCR beta1 or TCR beta2 chains of other mammalian species can be used in the methods of the disclosure.
[0083] [0083] The term "T-beta 1 cell receptor constant chain" or "T-beta 1 cell receptor constant region" (TCR-beta1 or TCRβ1 or TCRb1 or TCRb1 or hTCR-beta1 or Cβ1) is defined as a protein provided as SEQ ID NO: 15,051 or equivalent residues (i.e., a homolog) of a non-human species, for example, mouse, rodent, monkey, simian and the like.
[0084] [0084] The term "T-beta 2 cell receptor constant chain" or "T-beta 2 cell constant region" (TCR-beta2 or TCRβ2 or TCRb2 or Cβ2) is defined as the protein provided as SEQ ID AT THE:
[0085] [0085] The term "beta-cell receptor constant chain" or "beta-cell receptor constant region" (TCR-beta or TCRβ or TCRb or Cβ) "is defined as the protein provided as SEQ ID NO :
[0086] The protein sequences for Cβ2 (SEQ ID NO: 15,052) and Cβ1 (SEQ ID NO: 15,051) are known (Table 6D). The differences between the Cβ2 and β1 sequences are easily identified by aligning the sequences using the typical and common skill in the technique. The disclosure also provides certain mutations to TCRβ that can be used in the construction of SIRs and Ab-TCRs. For example, Cβ mutation sites that
[0087] [0087] The term "TCR-gamma constant chain" or "TCR-gamma constant region" (TCR-gamma or TCRγ or TCRg or TCR-gamma1 or TCRγ1 or TCRg1 or Cγ) is defined as the protein provided as SEQ ID NO: 15,068 or equivalent residues (ie, a homologue) of a non-human species, for example, mouse, rodent, monkey, simian and the like.
[0088] [0088] The term "TCR-delta constant chain" or "TCR-delta constant region" (TCR-delta or TCRδ or TCRd or Cδ) is defined as the proteins provided as SEQ ID NO: 15,069 or equivalent residues ( that is, a counterpart) of a non-human species, for example, mouse, rodent, monkey, simian and the like.
[0089] [0089] It will be recognized that proteins can have identity or homology to each other and retain similar or identical functions. The disclosure includes regions included in the TCR that have an identity of 85%, 90%, 95%, 97%, 98%, 98.5%, 99% or 99.9% to any of the sequences described in this document, maintaining the activity biological.
[0090] [0090] Therefore, the disclosure provides a constant T cell receptor chain having a sequence selected from the group consisting of: (a) an amino acid sequence that is at least 98% identical to SEQ ID NO: 15,041 and that can have one or more mutations at positions 61, 91, 92, 93 and / or 94; (b) an amino acid sequence that is at least 98% identical to SEQ ID NO: 15,051 and can have one or more mutations at positions 18, 22, 57, 79, 133, 136, and / or 139; (c) an amino acid sequence that is at least 98% identical to SEQ ID NO: 15,052 and can have one or more mutations at position 18, 22, 57, 79, 133, 136, and / or 139; (d) an amino acid sequence that is at least 98% identical to SEQ ID NO: 15,068; and (e) an amino acid sequence that is at least 98% identical to SEQ ID NO:
[0091] [0091] The term "constitutively active" refers to a molecule, for example, a protein, which has signaling activity without the need for a stimulus. Exemplary constitutive active proteins are NEMO-K277A and vFLIP K13 since they can activate NF-
[0092] [0092] The term "co-stimulating molecule" or "co-stimulating receptor" refers to a cognate binding partner in a T cell that specifically binds to a co-stimulating ligand, thereby mediating a co-stimulating response by the T cell, as, but without limitation, proliferation. Co-stimulating extracellular molecules are cell surface molecules that are not receptors for antigens or their ligands that contribute to an efficient immune response. Co-stimulating molecules include, but are not limited to, a MHC class I molecule, BTLA and a Toll ligand receptor, in addition to OX40, Dap10, CD27, CD28, CD2, CD5, CD8, ICAM-1, LFA-1 (CD11a / CD18 ), ICOS (CD278), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and 4-1BB (CD137). Other examples of such co-stimulating molecules include CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL2R IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29 , ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a linker that specifically binds to CD83. A co-stimulating receptor can be expressed in cells of other T cells, such as NK cells or macrophages.
[0093] [0093] A "co-stimulating intracellular signaling domain" or "co-stimulating domain" (CSD) can be the intracellular portion of a co-stimulating receptor. A co-stimulating molecule can be represented in
[0094] [0094] The term "cTCR" refers to a wild-type TCR nucleic acid coding sequence and the corresponding wild-type TCR protein linked to an antigen-binding domain. cTCRs are used in some reference modalities and controls. For example, a cTCR that has a CD19 binding domain and a CD19-CAR (comprising a mutant TCR chain and CD19 binding domain) will have different expression and / or different binding affinities to the target antigen.
[0095] [0095] The term "cytosolic" or "cytoplasmic" refers to an agent, for example, a protein, which is located in the cytoplasm of a cell in its mature form. A cytosolic protein can translocate to the nucleus, but it is not a transmembrane protein and is not secreted outside the cell. An exemplary cytosolic protein is vFLIP K13.
[0096] [0096] The term "degenerative disorders" refers to a disease that is the result of a continuous process based on degenerative cellular changes, affecting tissues or organs, which deteriorate more and more
[0097] [0097] "Derived from", as the term is used in this document, indicates a relationship between a first and a second molecule. It refers, in general, to the structural similarity between the first molecule and a second molecule and does not connote or include a process or a source of limitation in the first molecule that is derived from a second molecule. For example, in the case of an antigen binding domain that is derived from an antibody molecule, the antigen binding domain retains sufficient antibody structure so that it has the necessary function, namely, the ability to bind to an antigen. It does not connote or include a limitation on a particular process of producing the antibody, for example, this does not mean that, to obtain the antigen-binding domain, one must start with an antibody sequence and exclude unwanted sequence, or impose mutations, to reach the antigen-binding domain.
[0098] [0098] "Dimerization molecule", as the term is used in this document, refers to a molecule that promotes the association of a first exchange domain with a second exchange domain. In the modalities, the dimerization molecule does not occur naturally in the subject or does not occur in concentrations that would result in significant dimerization. In the embodiments, the dimerization molecule is a small molecule, for example, rapamycin or a rogue, for example, RAD001, Rimiducid or AP20187. Rimiducid (AP1903) is a lipid-permeable tacrolimus analogue with homodimerizing activity. Rimiducid homodimerizes an analogue of the human protein FKBP12 (Fv) that contains a single acid substitution (Phe36Val). Rimiducid is used to homodimerize domains
[0099] [0099] The phrase “disease associated with the expression of a target antigen” or “disease associated antigen, as described in this document” includes, but is not limited to, a disease associated with the expression of a target antigen, as described in this document, or a condition associated with cells that express a target antigen as described in this document, including, for example, proliferative diseases, such as cancer or malignancy or a precancerous condition, such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or a non-cancer indication associated with cells that express a target antigen as described in this document. In one aspect, a cancer associated with the expression of a tumor antigen, as described in this document, is a hematological cancer. In one aspect, a cancer associated with the expression of a tumor antigen as described in this document is a solid cancer. Other diseases associated with the expression of a tumor antigen described in this document include, but are not limited to, atypical and / or non-classical cancers, malignant diseases, precancerous conditions or proliferative diseases associated with the expression of a tumor antigen, as described in this document. Non-cancer indications associated with the expression of a target antigen, as described in this document, include, but are not limited to, for example, autoimmune disease (eg, lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, cells that express target antigen express, or have expressed at some point, mRNA that encodes the target antigen. In another embodiment, cells that express target antigen produce the target antigenic protein (eg, wild type or mutant), and the
[00100] [00100] "Disease driven by genetically modified cells", as used in this document, covers the targeting of any cell involved in any disease in any way by the genetically modified cells of the invention, regardless of whether the genetically modified cells reach diseased cells or healthy cells for effect a therapeutically beneficial outcome. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells or embryonic stem cells. Genetically modified cells express conventional CARs and innovative main structures containing conventional CARs with accessory modules of the invention, whose CARs can target any of the antigens expressed on the surface of target cells. Examples of antigens that can be targeted include, but are not limited to, antigens expressed on B cells; antigens expressed in carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors and blastomas; antigens expressed on several immune cells; and antigens expressed on cells associated with various hematological diseases, autoimmune diseases and / or inflammatory diseases. Other antigens that can be targeted will be evident to those skilled in the art and can be targeted by the CARs of the invention in connection with alternative modalities thereof.
[00101] [00101] The term "dissociation constant (K d)" is defined as the equilibrium constant of the dissociation of a receptor-ligand interaction.
[00102] [00102] As used in this document, a “diversified set
[00103] [00103] As used in this document, an "epitope" is defined as the portion of an antigen capable of eliciting an immune response or the portion of an antigen that binds to an antibody or antibody fragment. Epitopes can be a protein sequence or subsequence.
[00104] [00104] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-action elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. The expression vectors include all those known in the art, including cosmids, plasmids (for example, naked or contained in liposomes) and viruses (for example, lentivirus, retrovirus, adenovirus and adena-associated viruses) that incorporate the recombinant polynucleotide.
[00105] [00105] The term "functional portion", when used in reference to a CAR, refers to any part or fragment of the CAR, part or fragment that retains the biological activity of the CAR of which it is part (the CAR of origin). Functional portions include, for example, parts of a CAR that retain the ability to recognize target cells, or detect, treat or prevent a disease, to a similar extent, to the same extent or to a greater extent, than the CAR of In reference to the CAR of origin, the functional portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more, of the CAR source.
[00106] [00106] "Genetically modified cells", "redirected cells", "genetically engineered cells" or "modified cells", as used in this document, refer to cells that express a disclosure CAR. In some embodiments, genetically modified cells comprise vectors that encode a CAR. In some embodiments, genetically modified cells comprise vectors that encode a CAR and one or more accessory molecules in the same vector. In some embodiments, the genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes the accessory molecule. In some embodiments, genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes more than one accessory molecule. In some embodiments, the genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes the first accessory molecule and a third vector that encodes a second accessory molecule.
[00107] [00107] "Hinge region" (HR), as used in this document, refers to the hydrophilic region that is between the antigen-binding domain and the transmembrane domain. Hinge regions include, but are not limited to, Fc antibody fragments or fragments or
[00108] [00108] The term "immune disorder" refers to a disease characterized by dysfunction of the immune system. An autoimmune disease is a condition that results from an abnormal immune response to a normal part of the body. There are at least 80 types of autoimmune diseases.
[00109] [00109] "Immune cell", as used in this document, refers to cells of the mammalian immune system, including, among others, cells that have antigen, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, cells Helper T, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T cells.
[00110] [00110] "Immune effector cell", as this term is used in this document, refers to a cell that is involved in an immune response, for example, in promoting an effective immune response. Examples of immune effector cells include T cells, for example, alpha / beta T cells and
[00111] [00111] "Effective immune function" or "effective immune response", "effective function" refers to the specialized function of a differentiated cell. The effector function of a T cell, for example, can be cytolytic or auxiliary activity, including cytokine secretion. For example, an immune function or effector response refers to a property of a T or NK cell that promotes the death or inhibition of the growth or proliferation of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of an effective immune system function or response. In the case of cells that have antigens (for example, dendritic cells), the presentation of antigens and the secretion of cytokines are examples of effector functions.
[00112] [00112] "Immune response", as used in this document, refers to immunities, including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and / or hyperactive immunity .
[00113] [00113] An "intracellular signaling domain" (ISD) or "cytoplasmic domain" as the term is used in this document refers to an intracellular signaling portion of a molecule. The intracellular signaling domain generates a signal that promotes an immunological effector function of the cell. Examples of immunological effector functions include cytolytic and auxiliary activity, including secretion of cytokines. Examples of domains that effect signal transduction of the effector function include, but are not limited to, the z chain of the T cell receptor complex or any of its homologues (for example, h chain, FceR1g and b chains, MB1 (Iga) chain, chain B29 (Igb), etc.), human CD3 zeta chain, CD3 polypeptides (D, dee), tyrosine kinases of the syk family (Syk, ZAP 70,
[00114] [00114] In another embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent simulation. In another embodiment, the intracellular signaling domain may comprise a co-stimulating intracellular domain. Exemplary co-stimulating intracellular signaling domains include those derived from molecules responsible for co-stimulating signals or antigen-independent stimulation. For example, a primary intracellular signaling domain may comprise a CD3z cytoplasmic sequence, and a co-stimulating intracellular signaling domain may comprise the cytoplasmic sequence of the co-stimulant or co-stimulating molecule, such as CD28 or 41BB.
[00115] [00115] A primary intracellular signaling domain can comprise a signal motif that is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, derivatives of CD3 zeta, FeR common gamma (FCER1G), Fe gamma RIIa, FeR beta (Fe Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAPlO and DAP12.
[00116] [00116] The term "isolated", as used in this document, refers to biological or cellular molecules or products being substantially free of other materials. In one aspect, the term "isolated" refers to nucleic acid, such as DNA or RNA, or protein or
[00117] [00117] As used in this document, the term "ligand" (also "ligand domain" or "ligand region") refers to an oligo or polypeptide (or an oligo encoding the polypeptide) that joins two or more domains or regions of a CAR polynucleotide or polypeptide, respectively, disclosed in this document. The linker can be 1 to 500 amino acids in length or 3 to 1500 nucleotides in length. In some embodiments, the "linker" is cleavable or non-cleavable. Unless otherwise indicated, the term "binder" used in this document means a non-cleavable binder. Said non-cleavable linkers can be composed of flexible residues that allow freedom of movement of protein doamines adjacent to each other. Non-limiting examples of such residues include glycine and serine. In some embodiments, the binders include non-flexible residues. Examples of cleavable linkers include
[00118] [00118] In one embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 931 to 32) is also added upstream of the cleavable link sequences to increase cleavage efficiency. A potential disadvantage of cleavable ligands is the possibility that the small 2A marker left at the end of the N-terminal protein may affect the protein's function or contribute to the antigenicity of the proteins. To overcome this limitation, in some embodiments, a furin cleavage site (RAKR) (SEQ ID NO: 933 to 935) is added upstream of the SGSG motifs to facilitate cleavage of the residual 2A peptide after translation.
[00119] [00119] The term "flexible polypeptide linker", as used in this document, refers to a peptide linker consisting of amino acids, such as glycine and / or serine residues, used alone or in combination, to link the polypeptide chains (for example , heavy variable and light chain variable regions together) In one embodiment, the flexible polypeptide ligand is a Gly / Ser ligand and comprises the amino acid sequence (Gly-Gly-Gly-Ser) n, (SEQ ID NO: 4,191 to 4,192 ) where n is
[00120] [00120] The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses because they are capable of infecting cells that do not divide; they can provide a significant amount of genetic information in the host cell's DNA, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV and FIV are all examples of lenti viruses.
[00121] [00121] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector, as provided in Milone et al. Mol. Ther. 17 (8): 1,453 to 1,464 (2009). Other examples of lentivirus vectors that can be used in the clinic include, but are not limited to, for example, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX ™ vector system and the like. Non-clinical types of lentiviral vectors are also available and would be known to an individual skilled in the art. Other examples of lentivirus vectors are pLENTI-EF1α (SEQ ID NO: 3,837), pLENTI-EF1α-DWPRE (SEQ ID NO:
[00122] [00122] "Mammal", as used in this document, refers to any member of the Mammalia class, including, without limitation, humans and non-human primates, such as chimpanzees and other monkeys and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs, and the like. The term does not indicate a specific age or gender. Thus, adult and newborn individuals, as well as fetuses, male or female, should be included in the scope of this term.
[00123] [00123] "Naked DNA", as used in this document, refers to the DNA that encodes a CAR cloned into a suitable expression vector in proper orientation for expression. Viral vectors that can be used include, but are not limited to, lentiviral SIN vectors, retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, hybrid vectors and / or plasmid transposons (for example, transposon system Sleeping Beauty) or integrase-based vector systems. Other vectors that can be used in connection with alternative modalities of the invention will be evident to those skilled in the art.
[00124] [00124] "Native" or "Naturally occurring" or "endogenous", as used in this document, refers to a gene, protein, nucleic acid (for example, DNA, RNA, etc.) or a fragment thereof that is native to a cell or is naturally expressed in a cell. Thus, a native or endogenous T cell α TCRα polypeptide consists of a variable domain (Vα) linked to a constant TCRα chain. The native or endogenous TCRα chain precursor polypeptide also consists of
[00125] [00125] The Essential modulator NF-Kappa-B (NEMO) refers to a structural protein component of the IκB kinase complex necessary for the activation of NF-kB. NF-κB is a transcription factor that controls inflammation, cell proliferation and apoptosis.
[00126] [00126] "NF-κB pathway" or "NF-κB signaling pathway" refers to a signal transduction pathway that results in nuclear translocation of NF-κB subunits and transcriptional activation of genes responsive to the NF subunit -κB. NF-κB refers to the family of transcription factors involved in the regulated expression of several genes involved in the inflammatory and immune response. Five known members of that family have been characterized to date and include c-Rel, NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p105), p65 (RelA) and RelB. Although many dimeric forms of NF-κB have been described, the classic NF-κB complex is a heterodimer of p65 / RelA and p50 subunits and is found in most cells in association with a family of inhibitory proteins, called IκBs, of which the most common is IκBα. In the classic NF-kB pathway, stimulation by a series of cytokines, such as TNFα and IL-1, results in the activation of an IκB kinase multisubunit complex (IKK), which contains two catalytic subunits, IKK1 / IKKαI and IKK2 / IKKβ, and a regulatory subunit, NEMO / IKKγ. The activated IKK complex leads to the induced phosphorylation of Iκ B proteins and their subsequent degradation, thus releasing NF-κB from its inhibitory influence. Once released, NF-κB is free to migrate to the nucleus and bind to the promoter of specific genes that have their cognate binding site. The transcriptional activity of the NF-κB dimers in the nucleus is further modified by its phosphorylation. An alternative (or non-canonical) pathway for NF-κB activation, which involves proteasome-mediated processing of p100 / NF-κB2 in
[00127] [00127] "NF-κB stimulating molecule" or "NF-κB stimulating" or "NF-κB activator" refers to a subset of accessory molecules that promote the activity of the NF-κB signaling pathway or activity / expression of target genes downstream of the NF-κB signaling pathway. In some embodiments, an NF-κB activator is an unnatural NF-κB activating agent. An example of an unnatural NF-κB activating agent is hNEMO-K277A. In one embodiment, the NF-κB stimulating molecule or NF-κB stimulant is a selective NF-κB stimulant or a selective NF-κB activator. A “selective NF-kB activator” or a “selective NF-kB stimulant”, as described in this document, refers to an agent that selectively activates the NF-kB signaling pathway without or with minimal activation of the other pathways. signaling. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of one or more signaling pathways selected from the group of AKT, PI3K, JNK, p38 kinase, ERK, JAK / STAT and interferon signaling pathways. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of the AKT signaling pathway. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of the AKT signaling pathway. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of the PI3K signaling pathway. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of the ERK signaling pathway. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of the JNK signaling pathway. In one embodiment, a selective NF-κB activator activates the NF-κB signaling pathway without activation or minimal activation of the p38 kinase signaling pathway. In a
[00128] [00128] As used in this document, an "unnaturally occurring agent" or "non-native" or "exogenous" refers to an agent that is not naturally expressed in a cell. In other words, the unnaturally occurring agent is "genetically engineered" to be expressed in a cell. An unnaturally occurring agent can be a cloned version of a naturally occurring agent. Exemplary non-naturally occurring agents include CARs, SIRs, Ab-TCRs, TFPs, recombinant TCR, NEMO-K277A, vFLIP-K13 and K13-opt. An unnaturally occurring agent can be expressed in a cell using gene transfer techniques known in the art, such as lentivirus or retrovirus-mediated gene transfer. An unnaturally occurring agent can be expressed in an immune cell using an exogenous promoter (for example, EF1α promoter) or an endogenous promoter (for example, TCRα promoter). When an endogenous gene (for example, IKK1, IKK2, IKKγ / NEMO) is cloned and expressed ectopically in a cell, it represents another example of an agent that is not natural.
[00129] [00129] As used herein, an "unnaturally occurring immune receptor" or "exogenous immune receptor" refers to a receptor
[00130] [00130] As used in this document, an "unnaturally occurring TCR antigen binding domain" or "exogenous TCR antigen binding domain" refers to a binding domain operably linked to a constant region of the TCR that is chimeric and non-naturally occurring in relation to a TCR present in nature. In other words, the non-naturally occurring TCR antigen binding domain is “genetically engineered” using recombinant molecular biology techniques to be operationally linked to a TCR and, furthermore, that the antigen binding domain is obtained or derived of a molecule distinct from a TCR found in nature. An antigen-binding domain that is distinct from a TCR in nature includes vH and vL antibody fragments, humanized antibody fragments, chimeric antibody fragments, receptor ligands and the like.
[00131] [00131] As used in this document, "non-viral origin" refers to an agent (for example, a protein) that is not wholly or partially
[00132] [00132] The term "operationally linked" or "functionally linked" refers to the link or functional association between a first component and a second component, so that each component can be functional. For example, operably linked includes the association between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In the context of two polypeptides that are operationally linked, a first polypeptide works in the way it would be independent of any link and the second polypeptide functions as if there is no link between the two.
[00133] [00133] "Percent identity" in the context of two or more nucleic acids or polypeptide sequences refers to two or more equal sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of the same amino acid or nucleotide residues (for example, 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
[00134] [00134] For sequence comparison, usually a sequence acts as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the program parameters of the sequence algorithm are designated. Standard program parameters can be used, or alternative parameters can be assigned. The sequence comparison algorithm calculates the percent identities of the test sequences relative to the reference sequence, based on the program parameters. Sequence alignment methods for comparison are well known in the art. The ideal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2: 482c, by the Homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Bioi. 48: 443, by searching for the similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. EUA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular
[00135] [00135] Two examples of algorithms that can be used to determine the percentage of identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1977) Nuc. Acids Res. 25: 3,389 to 3402; and Altschul et al., (1990) J. Mol. Bioi. 215: 403 to 410, respectively. The software for performing BLAST analysis is available to the public at the National Center for Biotechnology Information.
[00136] [00136] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4: 11 to 17), which has been incorporated into the ALIGN program (see version 2.0), using a PAM120 weight residual table, a gap length penalty of 12 and a gap penalty of 4. In addition, the identity percentage between two amino acid sequences can be determined using Needleman and Wunsch (1970) J. Mol. Bioi. 48: 444 to 453), which was incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a P AM250 matrix and a weight difference of 16, 14, 12, 10, 8, 6 or 4 and a weight of length 1, 2, 3, 4, 5 or 6.
[00137] [00137] The term "polynucleotide", "nucleic acid" or "recombinant nucleic acid" refers to polymers of nucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA).
[00138] [00138] A "protein" or "polypeptide", the terms of which are used interchangeably in this document, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.
[00139] [00139] The term "retrovirus vector" refers to a vector derived from at least a portion of a retrovirus genome. Examples of retrovirus vectors include MSCVneo, MSCV-pac (or MSCV-pure), MSCV-hygro, as available from Addgene or Clontech.
[00140] [00140] The term "Sleeping Beauty Transposition" or "Sleeping Beauty Transposition Vector" refers to a vector derived from at least a portion of a Sleeping Beauty Transposon genome.
[00141] [00141] The term "single chain variable region" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, in which light and variable regions of the heavy chain are contiguously linked, for example, via a synthetic linker, for example, a short flexible polypeptide linker capable of being expressed as a single chain polypeptide, and in which scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the variable regions vL and vH in any order, for example, with respect to the ends of the N-terminus and C-terminus of the polypeptide, the scFv can comprise vL-linker-vH or you can understand vH-linker-vL. In this invention, an scFv is also described as vL-Gly-Ser-Linker-vH. Alternatively, an scFv is also described as (vL + vH) or (vH + vL).
[00142] [00142] The term "signaling domain" refers to the functional region of a protein that transmits information within the cell to regulate cellular activity through defined signaling pathways generating second messengers or functioning as effectors responding to these messengers.
[00143] [00143] The term "Synthetic Immune Receptor" or, alternatively, a "SIR" refers to a set of polypeptides, typically two in some modalities, which, when expressed in an effector cell, provide the cell with specificity for a target cell , normally a cancer cell, and with intracellular signal generation. The SIRs represent the next CAR generation platforms described in document WO 2018/102795 A1, which is incorporated in this document by way of
[00144] [00144] The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (for example, a TCR / CD3 complex) with its cognate ligand (or target antigen), thereby mediating a transduction event of signal, but, without limitation, signal transduction by means of TCR / CD3. Stimulation can mediate the altered expression of certain molecules.
[00145] [00145] The term "stimulating molecule" refers to a molecule expressed by an immune cell (eg, T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence (or sequences) that regulate immune cell activation in a stimulating manner by at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated, for example, by binding a TCR / CD3 complex to a peptide-loaded MHC molecule that leads to the mediation of a T cell response, including, but not limited to, proliferation , activation, differentiation and the like. A primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulating manner may contain a signaling motif that is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of a cytoplasmic signaling sequence containing ITAM include, but are not limited to, those derived from CD3 zeta, FeR common gamma (FCERIG), Fe gamma RIIa, FeR beta (Fe Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a , CD79b, DAPIO and DAP12.
[00146] [00146] The term "subject" is intended to include living organisms in which an immune response can be triggered (for example, any
[00147] [00147] "Exchange domain", or a "dimerization domain", as used in this document, usually refers to an entity based on polypeptide that, in the presence of a dimerization molecule, associates with another exchange domain. The association results in a functional coupling of a first entity linked to, for example, fused to, a first switching domain, and a second entity linked to, for example, fused to, a second switching domain. A first and a second switch domain are collectively called the dimming switch. In embodiments, the first and second switching domains are the same, for example, they are polypeptides with the same primary amino acid sequence and are collectively referred to as a homodimerization switch. In the modalities, the switch is intracellular. In the embodiments, the exchange domain is an entity based on polypeptide, for example, FKBP (FK506 binding protein), and the dimerization molecule is a small molecule, for example, AP20187.
[00148] [00148] The terms "T cell" and "T lymphocyte" are interchangeable and used in this document as synonyms. Examples include, but are not limited to, virgin T cells (“lymphocyte progenitors”), central memory T cells, effective memory T cells, stem memory T cells (Tscm), iPSC derived T cells, synthetic T cells or combinations thereof.
[00149] [00149] The term "TCR-associated signaling module" refers to a molecule that has a cytoplasmic immunoreceptor tyrosine-based (ITAM) activation motif that is part of the TCR-CD3 complex. The signaling modules associated with the TCR include CDγε, CDδε and CD3ζζ.
[00150] [00150] "Therapeutic agents", as used in this document, refers to agents that are used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of
[00151] [00151] "Therapeutic controls", as used in this document, refers to an element used to control the activity of a cell that expresses CAR. In some embodiments, therapeutic controls to control the activity of the CAR expressing cells of the invention comprise any one or more truncated epidermal growth factor receptors (tEGFR), truncated epidermal growth factor receptors viii (tEGFRviii), truncated CD30 (tCD30 ), Truncated BCMA (tBCMA), truncated CD19 (tCD19), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, inducible caspase 9, nucleoside purine phosphorylase, lentose / ligne / ligne / ligne / ligne , deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic (IAA), gamma-glutamylcysteine synthetase, CD20 / alphaCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV TKSR39), AP1903 / Fas system, a chimeric cytokine receptor (CCR), a selection marker and combinations thereof.
[00152] [00152] The term "therapeutic effect" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, decrease in the volume of the tumor, a decrease in the number of cancer cells, decrease in the number of metastases, an increase in life expectancy, decreased proliferation of cancer cells, decreased survival of cancer cells, decreased titer of the infectious agent, decreased colony count of the infectious agent,
[00153] [00153] The term "therapeutically effective amount", as used in this document, refers to the amount of a pharmaceutical composition comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to decrease at least one or more symptoms of the disease or disorder, and refers to a sufficient amount of pharmacological composition to provide the desired effect. The phrase "therapeutically effective amount", as used herein, means a sufficient amount of the composition to treat a disorder, with a reasonable benefit / risk ratio applicable to any medical treatment.
[00154] [00154] A therapeutic or prophylactically significant reduction of a symptom is, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50 %, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter compared to a control or untreated subject or the state of the subject prior to the administration of the oligopeptides described in this document. Measured or measurable parameters include clinically detectable disease markers, for example, elevated or depressed levels of a biological marker, as well as parameters related to a scale of symptoms or clinically accepted markers for diabetes. It will be understood, however, that the total daily use of the compositions and formulations disclosed in this document will be decided by the physician
[00155] [00155] The term "TCR or TFP receptor fusion proteins" refers to a next generation CAR platform, as described in WO 2016/187349 A1, which is incorporated by reference in this document. In one embodiment, a TFP comprises an antibody fraction that specifically binds to a target antigen fused to a TCR chain, such as CD3ε, CD3γ, CD3δ, TCRα or TCRβ. Exemplary TCR chains that can be used in the construction of TFP are represented by SEQ ID NOs: 944 to 945, 948, 949 to 950 and 958 and are provided in WO 2017/070608 A1 which is incorporated by reference in this document. A TFP incorporating the CD3ε chain is referred to as TFP CD3ε. A TFP that incorporates the CD3γ chain is referred to as TF3 CD3γ. A TFP that incorporates the CD3δ chain is referred to as TF3 CD3δ. TFP that incorporates CD3ε, CD3γ or CD3δ chains is collectively referred to as TF3 CD3ε / γ / δ. Exemplary TFPs that incorporate different antigen-binding domains (for example, vL and vH fragments, ligands, receptors, etc.) described in this disclosure and coexpressing an accessory module encoding NEMO-K277A are provided in SEQ ID NO: 1,900 to 3,123 (Table 13). The SEQ ID Nos, antigen-binding domains and target antigens of these TFPs can be determined by referring to Table 12, as these TFP constructs have antigen-binding domains identical to the first generation CAR constructs that coexpress NEMO-K277A shown in the Table 12 and are numbered in identical order. However, the accessory module encoding NEMO-K277A is optional. The TFP with the antigen-binding domains (i.e., vL and vH fragments, ligands and receptors, etc.) described in this disclosure can be constructed without NEMO-K277A. As such, this accessory module together with the
[00156] [00156] The term "transfer vector" refers to a composition of matter that comprises an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid to cells, such as, for example, a poly-lysine compound, liposome and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adena-associated virus vectors, retroviral vectors, lentiviral vectors and the like.
[00157] [00157] "Transmembrane domain" (TMD), as used in this document, refers to the CAR region that crosses the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (e.g., Type I transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains will be apparent to those skilled in the art and can be used in connection with alternative embodiments of the invention. In some embodiments, the TMD-encoded CAR comprising any of the main structures described in this
[00158] [00158] As used in this document, “Multifunctional T cell antigen coupler or Tri-TAC” refers to a next generation CAR platform described in WO 2015/117229 A1, which is incorporated by reference in this document. Tri-TAC targeting different antigens can be constructed using the antigen-binding domains (for example, fragments of vL and vH, scFv, vHH, ligands and receptors, etc.) described in this disclosure using techniques known in the art. In addition, the different accessory modules (for example, NEMO-K277A, mNEMO-K270A, etc.) described in this disclosure can be expressed in immune cells that express Tri-TAC, for example, T cells, for example, CAR-T cells .
[00159] [00159] As used in this document, the terms "treat", "treatment", "treating" or "improvement" refer to therapeutic treatments, in which the objective is to reverse, alleviate, improve, inhibit, delay or interrupt the progression or severity of a condition associated with a
[00160] [00160] "Tumor", as used in this document, refers to all growth and proliferation of neoplastic cells, malignant or benign, and all precancerous and cancerous cells and tissues.
[00161] [00161] "Vector", "cloning vector" and "expression vector", as used in this document, refer to the vehicle by which a polynucleotide sequence (for example, a foreign gene) can be introduced into a host cell, from in order to transform the host and promote expression (for example, transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.
[00162] [00162] The term "zeta" or, alternatively, "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein supplied as GenBan Ace. in BAG36664.1, or equivalent residues of a non-human species, for example, mouse, rodent, monkey, simian and the like, and a "zeta stimulating domain" or, alternatively, a "CD3-zeta stimulating domain" or a " stimulating domain TCR-zeta ”is defined as the amino acid residues of the cytoplasmic domain of the zeta chain, or their functional derivatives, which are sufficient to functionally transmit an initial signal necessary for the activation of T cells. In one aspect, the cytoplasmic domain of zeta comprises GenBank Ace residues 52 to 164. in BAG36664.1 or equivalent residues of a non-human species, for example, mouse, rodent, monkey, simian and the like, which are its functional orthologists. In one aspect, the "zeta stimulating domain" or a "CD3-zeta stimulating domain" is the sequence provided as SEQ ID NO:
[00163] [00163] The CAR binding domain is selected to bind to a desired epitope. For example, the epitope recognized by a CAR can also be determined from the epitope recognized by the scFv that comprises the CAR. For example, since the CAR antigen specific domain CD8SP-MPL-161- (vL-vH) -Myc-BBz-T2A-PAC (SEQ ID NO: 1.509 and SEQ ID NO: 5.422) is composed of MPL of scFv MPL-161- (vL-vH) (SEQ ID NO: 808 and SEQ ID NO: 4,721), CAR is expected to target the same epitope as scFv and the parental antibody from which scFv is derived. The epitope recognized by scFv MPL-161- (vL-vH) (SEQ ID NO: 808 and SEQ ID NO: 4.721) is provided in SEQ ID NO: 15.160. The epitopes recognized by various scFv and / or their parental antibodies used in the construction of the CARs and major structures of this disclosure are known in the art. Alternatively, the target epitope of a CAR (including the CARs that are present as part of major structures)
[00164] [00164] Compositions comprising a non-naturally occurring immune receptor, for example, a CAR and an accessory module (including NF-κB stimulating molecules and selective NF-κB activators) and a method for using them are also provided in this document. in the treatment of diseases, including cancer. As described in this document, specific combinations of conventional CARs (Table 1) and accessory modules are provided, as described in Table 2.
[00165] [00165] Table 1: Conventional CAR architectures. Conventional first-generation CARs (conventional CAR I) have an intracellular signaling domain (ISD) (for example, CD3z) and no co-stimulating domain. TCR fusion proteins (TFP) are another example of conventional CAR 1. Conventional second generation CARs (CAR 2 or conventional CAR II) have a co-stimulating domain (for example, 41BB or CD28) and an intracellular signaling domain (ISD) (for example, CD3z). Conventional third generation CARs (CAR 3 or conventional CAR III) have two co-stimulating domains (for example, 41BB and CD28) and an intracellular signaling (ISD) domain (for example, CD3z). Ab-TCRs are duel chain receptors and have been described in PCT / US2016 / 058305. CTCRs are single, one and a half or double chain receptors, consisting of the antigen-binding domain derived from a vL and vH fragment that are fused to one or more TCR constant chains and result in activation of T cell signaling. Receptors immune
[00166] [00166] Abbreviations; 1st Generation CAR, First Generation CAR; 2nd Generation CAR, Second Generation CAR; DC SIR, double-stranded SIR; OHC SIR, half chain SIR.
[00167] [00167] The accessory modules in the exemplary constructs above in Table 10 are optional and can be excluded or replaced by other accessory modules. TABLE 11: SEQ ID NOS OF CARS CONTAINING DIFFERENT ANTIGEN BINDING DOMAINS USING SEQ ID NOS OF CARS WITH HIV1-N49P6 AS A REFERENCE Target Antigen Domain binding to SEQ ID NO CAR SEQ ID NO of antigen CAR (DNA) ) HIV1 Env HIV1-N49P6 8,704 to 8,737 11,402 to 11,435 HIV1 Env HIV1-N49P7 8,738 to 8,771 11,436 to 11,469 HIV1 Env HIV1-N49P11 8,806 to 8,839 11,504 to 11,537 HIV1 Env HIV1-N60P1-1 8,840 to 8,871 11,538 to 11,538 -N60P25 8,942 to 8,975 11,640 to 11,673 HIV1 Env HIV1-N49P9 8,772 to 8,805 11,470 to 11,503 HIV1 Env HIV1-N60P2-1 8,874 to 8,907 11,572 to 11,605 HIV1 Env HIV1-N60P31-1 9,010 to 9,043 11,708 to 11,741 HIV1 Env1 -1 8,908 to 8,941 11,606 to 11,639 HIV1 Env HIV1-N60P38 9,146 to 9,179 11,844 to 11,877 HIV1 Env HIV1-N60P30 8,976 to 9,009 11,674 to 11,707 HIV1 Env HIV1-N60P36 9,078 to 9,111 11,776 to 11,809 HIV1 Env 9,111,978,811 HIV1 Env HIV1-N6039-1 9,316 to 9,349 12,014 to 12,047 HIV1 Env HIV1-N60P47 9,214 to 9,247 11,912 to 11,945 HIV1 Env HIV1-N60P48 9,248 to 9,281 11,946 to 11,979 HIV1 Env HIV1-N60P51 9,282 to 9,315 11,980 to 12,013 HIV1 Env HIV1-N60P35 9,044 to 9,077 11,742 to 11,775 HIV1 Env HIV1-N60P37 9,112 to 9,145 11,810 to 11,843 Lym 10,403 13,068 to 13,101 Lym2 hu-Lym2 10,404 to 10,437 13,102 to 13,135 BCMA BCMA-USC1 9,418 to 9,451 12,116 to 12,149 BCMA BCMA-USC2 9,452 to 9,485 12,150 to 12,183 BCMA BCMA-USC3 9,486 to 9,519 12,184 BC to 9,519 BC 9,554 12,218 to 12,252 BCMA BCMA-USC5 9,555 to 9,587 12,253 to 12,285 BCMA BCMA-USC6 9,588 to 9,621 12,286 to 12,319 BCMA BCMA-USC7 9,622 to 9,655 12,320 to 12,353 CD43 CD43-huJL-1-257-10 9,756 to 9,791 BCMA BCMA-huC11.D5.3L1H3 9,350 to 9,383 12,048 to 12,081 BCMA BCMA-huC13-F12 9,384 to 9,417 12,082 to 12,115 CD20 CD20-Ubli-v4 9,656 to 9,689 12,354 to 12,387 CD37 CD37-TRU-HL 12,724 to 9,724 to 9,724 CD70 CD70-1G2D 9,792 to 9,825 12,490 to 12,523 CD70 CD70-10B4 9,826 to 9,859 12,524 to 12,557 CD70 CD70-13D 9,860 to 9,893 12,558 to 12,591 CD7 0 CD70-16D 9,894 to 9,927 12,592 to 12,625 CD70 CD70-21D 9,928 to 9,961 12,626 to 12,659 CD70 CD70-24D 9,962 to 9,995 12,660 to 12,693 CD70 CD70-25D 9,996 to 10,029 12,694 to 12,727 CD70 CD70-69A7 10,030 to 10,063 12,728 CD70 CD70-hu-2H5 10,064 to 10,097 12,762 to 12,795 CD123 CD123-DART-1 10,098 to 10,131 12,796 to 12,829 CD123 CD123-DART-2 10,132 to 10,165 12,830 to 12,863
[00168] [00168] In some embodiments, the compositions comprise nucleic acids encoding conventional CARs 1 to 6 (Table 1), in which the specific CAR antigen domain targets one or more specific antigens, as described in Tables 6A to C or in Tables 5 and 6 in document PCT / US2017 / 064379, which are incorporated by reference in this document. In some embodiments, the compositions comprise nucleic acids that encode any one or more of the main structures 1 to 72 (Table 2), where the specific antigen domain of the encoded CAR targets one or more specific antigens, as described in Tables 6A to C or Tables 5 and 6 in document PCT / US2017 / 064379. In some embodiments, the compositions comprise nucleic acids encoding the main structural-1, where the specific CAR antigen domain in the main structural-1 targets one or more cancer-specific antigens, as described in this document in Tables 6A to C or Tables 5 and 6 of the document
[00169] [00169] In various embodiments, isolated nucleic acid molecules encoding the unnaturally occurring immune receptor, for example, a CAR, components of the main structures described in this
[00170] [00170] In one embodiment, each specific antigen region comprises the full-length IgG heavy chain (specific for the target antigen) with the VH, CH1, hinge and CH2 and CH3 (Fc) Ig domains, if the VH domain alone it is sufficient to confer specificity to the antigen (“single domain antibodies”). The full-length IgG heavy chain can be linked to a co-stimulating domain and an optional intracellular signaling domain via the appropriate transmembrane domain. If both the VH and VL domains are required to generate a specific fully active antigen targeting region, the unnaturally occurring immune receptor containing VH, for example, a CAR and the full length lambda light chain (IgL) are introduced in the cells to generate a specific active antigen targeting region.
[00171] [00171] In some embodiments, the antigen specific domain of the encoded unnatural immune receptor, for example, a CAR molecule comprises an antibody, an antibody fragment, a scFv, an Fv, a Fab, an (Fab ') 2, a single antibody domain (SDAB), a vH or vL domain or a camelid vHH domain. In some embodiments, the antigen-binding domain of the unnaturally occurring immune receptor, for example, a CAR, is a fragment of scFv antibody that is humanized compared to the murine scFv sequence from which it is derived.
[00172] [00172] In some cases, scFvs can be prepared according to methods known in the art (for example, Bird et al., (1988) Science 242: 423 to 426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5,879 a
[00173] [00173] In one embodiment, the specific antigen domain comprises one, two or all three heavy chain CDRs (hc), hcCDR1, hcCDR2 and hcCDR3 of an antibody or scFv listed in this document (Table 6B; SEQ ID NOs: 14,122 to 15,039) and / or one, two or all three light chain CDRs (lc), lcCDRl, lcCDR2 and lcCDR3 of an antibody or scFv listed in this document (Tables 6A; SEQ ID NOs: 13,204 to 14,121) (see also Tables 5 and 6 of document PCT / US2017 / 064379) In some modalities, the ASD comprises a fragment VL (or vL) that
[00174] [00174] In another embodiment, the specific antigen domain comprises a humanized antibody or an antibody fragment. In some respects, a non-human antibody is humanized, in which specific sequences or regions to the antibody are modified to increase similarity to an antibody produced naturally in a human or a fragment thereof. In one aspect, the antigen-binding domain is humanized. A humanized antibody can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting, coating or resurfacing and shuffling of chains.
[00175] [00175] In an additional embodiment, each specific non-naturally occurring immune receptor antigen domain, for example, a CAR, can comprise a divalent (or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs). In, for example, CARs comprising di scFVs, two scFvs specific for each antigen are linked together, producing a single peptide chain with two VH regions and two VL regions, producing tandem scFvs. (Xiong, Cheng-Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). “Development of tumor targeting anti- MUC-1 multimer: effects of di-scFv unpaired cysteine location on
[00176] [00176] In another embodiment, each ASD of the unnaturally occurring immune receptor, for example, a CAR, comprises a diabody. In a diabody, scFvs are created with peptide ligands that are too short for the two variable regions to fold, causing scFvs to dimerize. Even shorter ligands (one or two amino acids) lead to the formation of trimers, called triabodies or tribodies. Tetrabodies can also be used.
[00177] [00177] In some embodiments, the ASD of the non-naturally occurring immune receptor, for example, a CAR, comprises VHH fragments (nanobodies) as described in this document (see Tables 5 to 6 of document PCT / US2017 / 064379). In some embodiments, the ASD of the non-naturally occurring immune receptor, for example, a CAR, comprises affibodies as described in this document (see Tables 5 and 6 of document PCT / US2017 / 064379).
[00178] [00178] In another embodiment, the antigen-specific binding domain comprises a ligand for a cognate expressed in a cell
[00179] [00179] In one embodiment, a specific antigen domain of an unnaturally occurring immune receptor, for example, a CAR, against a target antigen is an antigen-binding portion, for example, CDRs, of fragments of vHH targeting this antigen (see Tables 5 and 6 of document PCT / US2017 / 064379).
[00180] [00180] In one embodiment, a specific antigen domain of a non-naturally occurring immune receptor, for example, a CAR, against a target antigen is an antigen-binding portion of a non-immunoglobulin support targeting that antigen (see Tables 5 and 6 of document PCT / US2017 / 064379).
[00181] [00181] In one embodiment, a specific antigen domain of an unnaturally occurring immune receptor, for example, a CAR, against a target antigen is an antigen binding portion of a receptor that is known to bind this target antigen (see , Tables 5 and 6 of document PCT / US2017 / 064379).
[00182] [00182] In another aspect, the antigen binding domain is a T cell receptor ("TCR"), or a fragment thereof, for example, a single chain TCR (scTCR). Methods for producing such TCRs are known in the art. See, for example, Willemsen RA et al., Gene Therapy 7: 1,369 to 1,377 (2000); Zhang T et al., Cancer Gene Ther 11: 487 to 496 (2004); Aggen et al., Gene Ther. 19 (4): 365 to 74 (2012) (references are incorporated in this document in their entirety). For example, scTCR can be genetically engineered to contain the Vα and νβ genes of a clone of T cells linked by a ligand (for example, a flexible peptide). This approach is very useful for the cancer-associated target that is intracellular; however, a fragment of this antigen (peptide) is presented on the surface of cancer cells by MHC.
[00183] [00183] In some modalities, the specific antigen domain is
[00184] [00184] In some embodiments, the specific antigen domain of an unnaturally occurring immune receptor, for example, a CAR, described in this document binds to a peptide presented by the MHC. Normally, peptides derived from endogenous proteins fill the pockets of class I molecules of the major histocompatibility complex (MHC) and are recognized by T cell receptors (TCRs) on CD8 + T lymphocytes. Class I MHC complexes are expressed constitutively by all nucleated cells. In cancer, peptide / MHC complexes specific for viruses and / or tumors represent a unique class of cell surface targets for immunotherapy. TCR-type antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA) -A1 or HLA-A2 have been described (see, for example, Sastry et al., J Viral. 2011 85 (5): 1,935 to 1,942; Sergeeva et al. Blood, 2011117 (16): 4,262 to 4,272; Verma et al., Jlmmunol2010 184 (4):
[00185] [00185] In some modalities, the specific antigens for the
[00186] [00186] In some embodiments, disease-specific antigens that can be targeted by the unnaturally occurring immune receptor, for example, a CAR, when expressed alone or with accessory modules, as described in this document include, but are not limited to, one or more from 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, lymphoma cell B, antigen C242, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19 , CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CD123, CEA, CNTO888, CTLA-4 , DR5, EGFR, EpCAM, CD3, FAP, extra B domain of fibronectin,
[00187] [00187] A CAR, when used alone or with accessory modules, as described in this document can comprise an antigen-binding domain (eg, antibody or antibody fragment) that binds to a disease-supporting antigen (eg, disease support antigen, as described in this document). In some modalities, the disease-supporting antigen is an antigen present in cells that support the survival and proliferation of disease-causing cells. In some embodiments, the disease support antigen is an antigen present in a stromal cell or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors and cytokines to promote cell proliferation in the microenvironment. MDSC cells can block the proliferation and activation of T cells. Without wishing to be limited by theory, in some embodiments, cells that express CAR destroy the disease supporting cells, thereby indirectly blocking the growth or survival of cells disease-causing.
[00188] [00188] In certain embodiments, a stromal cell antigen is selected from one or more of the following: stromal cell antigen
[00189] [00189] In another embodiment, the disclosure provides an unnaturally occurring immune receptor, for example, a CAR, which binds to the same epitope on different targets described in Tables 6A to C as any of the non-naturally occurring immune receptors of the disclosure (for example, CARs that have the ability to cross-compete for connection to different targets with any of the disclosure CARs). In some embodiments, the antigen-specific domains of these non-naturally occurring immune receptors, for example, a CAR, may be derived from vL fragments, vH fragments or scFv antibody fragments. In some embodiments, the reference antibodies for cross-competition studies to determine the target epitope recognized by an unnaturally occurring immune receptor, for example, a CAR, of the disclosure are scFvs described in Table 6C in this document having sequences as shown in SEQ ID NOs: 4,555 to 4815, 11,165 to 11,401, 15,070 to 15,132 (Table 6C) or as described in Tables 5 and 6 of PCT / US2017 / 064379. In an exemplary embodiment, the reference scFv FMC63 (vL-vH) represented by SEQ ID NO: 4,555 can be used in cross-competition studies to determine the target epitope recognized by the conventional CARs and main structures based on FMC63 in the disclosure. In some modalities, the vHH fragments of reference for studies of
[00190] [00190] In another embodiment, the reference antibodies for cross-competition studies to determine the target epitopes recognized by the MPL-targeting CARs of the disclosure are mAb-1.6, mAb-
[00191] [00191] In another embodiment, the reference scFvs for cross-competition studies to determine the target epitopes recognized by the CARs targeting the MPL of the disclosure are scFvs having sequences as shown in SEQ ID NOs: 4,720 to 4,727, in Table 6C or as described in Tables 5 and 6 of document PCT / US2017 / 064379.
[00192] [00192] In another embodiment, the reference ligands for cross-competition studies to determine the target epitopes recognized by the CARs targeting the MPL of the disclosure are TPO and mTPO ligands with
[00193] [00193] In another embodiment, the reference CARs for cross-competition studies to determine the target epitopes recognized by the CARs targeting the MPL of the disclosure are CARs having sequences as shown in SEQ ID NOs: 5.120 to 5.126.
[00194] [00194] In the preferred modality, the CARs directed to MPL of the disclosure bind to an epitope corresponding to the sequences shown in SEQ ID NO: 15.160.
[00195] [00195] In one embodiment, the reference scFvs for cross-competition studies to determine the target epitopes recognized by the CD19-directed CARs of the invention are scFvs having sequences as shown in SEQ ID NOs: 4,555 to 4,568 and in Table 6C or as described in Tables 5 and 6 of document PCT / US2017 / 064379. In another embodiment, the reference CARs for cross-competition studies to determine the target epitopes recognized by the CD19-directed CARs of the invention are CARs having sequences as shown in SEQ ID NOs:
[00196] [00196] In one embodiment, the reference scFvs for cross-competition studies to determine the target epitopes recognized by the CD20-directed CARs of the invention are CD20-directed scFvs and with SEQ IDs as listed in Table 6C or as described in Tables 5 and 6 of PCT / US2017 / 064379. In another embodiment, the reference CARs for cross-competition studies to determine the target epitopes recognized by the CD20-directed CARs of the invention are CD20-directed CARs and with SEQ IDs as listed in Tables 12.
[00197] [00197] In the preferred modality, the CARs directed to CD20 of the disclosure bind to the epitopes corresponding to one or more of the sequences shown in SEQ ID NO: 15.149 to 15.154.
[00198] [00198] In one modality, the scFvs of reference for studies of
[00199] [00199] In the preferred modality, the CARs directed to BCMA of the disclosure bind to the epitopes corresponding to one or more of the sequences shown in SEQ ID NO: 15.155 to 15.159.
[00200] [00200] In one embodiment, the reference scFvs for cross-competition studies against CARs targeting DLL3 of the invention are scFvs targeting DLL3 and with SEQ IDs, as listed in Table 6C or as described in Tables 5 and 6 of the PCT / US2017 / 064379. In another embodiment, the reference CARs for cross-competition studies against the DLL3-targeted CARs of the invention are the DLL3-targeted CARs and with SEQ IDs as listed in Table 12.
[00201] [00201] In one embodiment, the reference scFvs for cross-competition studies against LAMP1-targeted CARs of the invention are scFvs targeting LAMP1 and with SEQ IDs as listed in Table 6C or as described in Tables 5 and 6 of document PCT / US2017 / 064379. In another embodiment, the reference CARs for cross-competition studies against the LAMP1-targeted CARs of the invention are LAMP1-targeted CARs and with SEQ IDs as listed in Table 12.
[00202] [00202] In one embodiment, the reference scFvs for cross-competition studies against CARs targeting TROP2 of the invention are scFvs targeting TROP2 and with SEQ IDs as listed in Table 6C or as described in Tables 5 and 6 of document PCT / US2017 / 064379.
[00203] [00203] In one embodiment, the reference scFvs for cross-competition studies against PTK7-directed CARs of the invention are scFvs directed to PTK7 and with SEQ IDs as listed in Table 6C or as described in Tables 5 and 6 of document PCT / US2017 / 064379. In another embodiment, the reference CARs for cross-competition studies against the PTK7-directed CARs of the invention are the PTK7-directed CARs and with SEQ IDs as listed in Table 12.
[00204] [00204] In one embodiment, the reference scFvs for cross-competition studies against CD22, CD123, CD33, CD37, CD70, CD138, CS1, IL13Ra2, Folate α receptor, Folate β receptor, TCRB1, TCRB2, TCRγδ, CD30 , Mesothelin, Her2, EGFRviii and the HIV1-targeted CARs of the invention are scFvs targeting these antigens and with SEQ IDs as listed in Table 6C or as described in Tables 5 and 6 of PCT / US2017 / 064379. In another modality, the reference CARs for cross-competition studies against CD22, CD123, CD33, CD37, CD70, CD138, CS1, IL13Ra2, Folate α receptor, Folate β receptor, TCRB1, TCRB2, TCRγδ, CD30, Mesothelin, Her2, EGFRviii, and the HIV1-targeted CARs of the invention are the CARs targeting these antigens and with SEQ IDs as listed in Table 12.
[00205] [00205] In some embodiments, the CARs described in this document comprise a hinge or ligand region between the specific antigen domain and the transmembrane domain. In some embodiments, the hinge region comprises any one or more of human CD8α or an Fc fragment of an antibody or functional equivalent, fragment or derivative thereof, a human CD8α hinge region or an antibody or functional equivalent, fragment or derived from it,
[00206] [00206] In some embodiments, two or more functional domains of non-naturally occurring immune receptors, for example, CARs, as described in this document, are separated by one or more ligands. The ligands are an oligo or polypeptide region of about 1 to 100 amino acids in length, which together link any of the non-naturally occurring immune receptor domains / regions, for example, CARs, of the disclosure. In some embodiments, the linkers can be, for example, 5 to 12 amino acids in length, 5 to 15 amino acids in length or 5 to 20 amino acids in length. The ligands can be composed of flexible residues such as glycine and serine, so that the adjacent protein domains are free to move relative to each other. Longer linkers, for example those with more than 100 amino acids, can be used in connection with alternative modalities of disclosure and can be selected, for example, to ensure that two adjacent domains do not interfere stereotypically with each other.
[00207] [00207] As described in this document, the CARs described in this document comprise a transmembrane domain. The transmembrane domain can comprise the transmembrane sequence of any protein that has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR of disclosure can also comprise a
[00208] [00208] A transmembrane domain can include one or more additional amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids) at each end of the transmembrane region (for example, one or more amino acids extending extracellularly and / or one or more amino acids extending intracellularly) to the transmembrane region. In one aspect, the transmembrane domain is contiguous with one of the other domains of the CAR. In one embodiment, the transmembrane domain can be from the same protein from which the signaling domain, co-stimulating domain or hinge domain is derived. In another aspect, the transmembrane domain is not derived from the same protein from which any other CAR domain is derived.
[00209] [00209] In various embodiments, the isolated nucleic acid molecules encoding the unnaturally occurring immune receptors, for example, CAR components of the major structures described in this document, encode zero, one, two, three or more intracellular signaling domains.
[00210] [00210] As described in this document, non-naturally occurring immune receptors, for example, CARs, described in this document can optionally comprise an intracellular signaling domain. This domain can be cytoplasmic and can transduce the signal of the effector function and direct the cell to perform its specialized function. Examples of intracellular signaling domains include, but are not limited to, chain of the T cell receptor or any of its homologs (for example, η, CD3ε, CD3γ, CD3δ, FcεR1γ and β chains, MB1 (Igα), B29 chains ( chain (Igβ), etc.), CD3 polypeptides (Δ, δ and ε), tyrosine kinases of the Syk family (Syk, ZAP 70, etc.), tyrosine kinases of the src family (Lck, Fyn, Lyn, etc.) and other molecules involved in the transduction of T cells, such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain can be human CD3 zeta chain, FcγRIII, FcεRI, Fc receptor cytoplasmic tails, immunoreceptor tyrosine-based activation motif (ITAM) containing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be evident to those skilled in the art and can be used in connection with alternative embodiments of the invention. In some embodiments, the intracellular signaling domain comprises a signaling domain of one or more of a human CD3 zeta chain, FcgRIII, FceRI, a cytoplasmic tail of an Fc receptor, an activation motif based on immunoreceptor tyrosine (ITAM) containing receptors cytoplasmic and combinations thereof.
[00211] [00211] In various embodiments, the nucleic acid molecules
[00212] [00212] One or more polypeptides encoded by one or more nucleic acid molecules encoding conventional CARs 1 to 6 (Table 1) or any one or more of the main structures 1 to 72 described in this document are provided in this document (Table 2) .
[00213] [00213] Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules that encode conventional CARs 1 to 6. In some embodiments, the specific antigen domain of CARs is specific for one, two, three or more antigens on the target cells, such as cancer cells. As described in this document, each component of the CAR is contiguous and in the same reading frame with each other component of the CAR. In some modalities, the CAR comprising the main structural comprises more than one specific antigen domain, with each of the specific antigen domains contiguous and in the same reading frame as the other domains
[00214] [00214] Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules encoding main structures 1 to 10 comprising conventional CAR I and an accessory module encoding an NF-κB stimulating molecule (for example example, vFLIP-K13, hNEMO-K277A, FKBPx2-hNEMO-K277A, FKBPx2- hNEMO-L753 (251), FKBPx2-hNEMO-L600 (200), FKBPx2-RIP-ID, IKK2- S17E-S17E-E17 S180E, MyD88-L265P, TCL-1A or variants thereof) as described in this document. The accessory module in the main structures 1 to 10 can be replaced by other accessory modules that encode different molecules, including different NF-κB activators (for example, K13-opt, hNEMO-K277A-delta-V249-K255 or hNEMO-K277L, etc.). Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules encoding main structures 11 to 12 comprising conventional CAR I and an accessory module encoding IgSP- [hTRAC-opt2] and IgSP- [hTRBC -opt2]. In some embodiments, the CAR antigen-specific domain comprising major structures-1 to 12 is specific for one, two, three or more antigens on target cells, such as cancer cells. As described in this document, each component of the CAR is contiguous and in the same reading frame with each other component of the CAR comprising main structures 1 to 12. In some embodiments, the CAR comprising main structures 1 to 12 comprises more than one specific domain of antigen, each of the specific domains of the antigen is contiguous and in the same reading frame as the other specific domains of the antigen in the same CAR.
[00215] [00215] Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules that encode major structures 13 to 22 comprising CAR II
[00216] [00216] Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules encoding main structures 37 to 46 comprising Ab-TCR and an accessory module encoding an NF-κB stimulating molecule (for example example, vFLIP-K13, hNEMO-K277A, FKBPx2-hNEMO-K277A, FKBPx2- hNEMO-L753 (251), FKBPx2-hNEMO-L600 (200), FKBPx2-RIP-ID, IKK2- S17E-S17E-E17 S180E, MyD88-L265P, TCL-1A or variants thereof) as described in this document. The accessory module in the main structures 37 to 46 can be replaced by other accessory modules that encode different molecules, including different NF-κB activators (for example, K13-opt, hNEMO-K277A-delta-V249-K255 or
[00217] [00217] Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules encoding main structures 49 to 58 comprising double-stranded cTCR / SIR and an accessory module encoding an NF- stimulating molecule κB (e.g. vFLIP-K13, hNEMO-K277A, FKBPx2-hNEMO-K277A, FKBPx2-hNEMO-L753 (251), FKBPx2-hNEMO-L600 (200), FKBPx2-RIP-ID, IKK-S1, IKK2 -S176E-S180E, MyD88-L265P, TCL-1A or variants thereof) as described in this document. The accessory module in the main structures 49 to 58 can be replaced with other accessory modules that encode different molecules, including different NF-κB activators (for example, K13-opt, hNEMO-K277A- delta-V249-K255 or hNEMO-K277L, etc.).
[00218] [00218] Also provided in this document are one or more polypeptides encoded by one or more nucleic acid molecules that encode the main structures 61 to 70 comprising one and a half strand cTCR / SIR and an accessory module that encodes a stimulating molecule of NF-κB (e.g., vFLIP-K13, hNEMO-K277A, FKBPx2-hNEMO-K277A, FKBPx2-hNEMO-L753 (251), FKBPx2-hNEMO-L600 (200), FKBPx2-RIP-SE2, IK2 , IKK1-S176E-S180E, MyD88-L265P, TCL-1A or variants thereof) as described in this document. The accessory module in the main structures 61 to 70 can be replaced by other accessory modules that encode different molecules, including different NF-κB activators (for example, K13-opt, hNEMO-K277A- delta-V249-K255 or hNEMO-K277L, etc.).
[00219] [00219] In various modalities, the polypeptides encoded by the nucleic acid molecules that encode the CARs that are part of the main structures described in this document, as main structure-1, main structure-2, main structure-13, main structure-14, structure
[00220] [00220] In some embodiments, polypeptides encoded by the nucleic acid molecules encoding the CARs that are part of the conventional CARs 1 to 6 or part of the main structures described in this document, are provided as main structural-1, main structural- 2, main structural-13, main structure-14, main structure-37, main structure-38, main structure-49, main structure-50, main structure-60 or main structure-61, in which the specific antigen domain of CARs is specific for the thrombopoietin receptor, MPL. In some embodiments, polypeptides encoded by the nucleic acid molecules encoding the CARs that are part of the conventional CARs 1 to 6 or part of the main structures described in this document are provided in this document, as main structure-1, main structure-2, structure principal-13, principal structure-14, principal structure-37, principal structure-38, principal structure-49, principal structure-50, principal structure-60 or principal structure-61, in which the specific antigen domain of the CARs is specific for CD19. In some embodiments, polypeptides encoded by the nucleic acid molecules encoding the CARs that are part of the conventional CARs 1 to 6 or part of the main structures described in this document are provided in this document, as main structure-1, main structure-2, structure main-13, main structure-14, main structure-37, main structure-38, main structure-49, main structure-50, main structure-60 or main structure-61, in which the specific domain
[00221] [00221] The nucleic acid sequences that code for the desired components of the unnaturally occurring immune receptors, for example, CARs and / or a selective coding sequence for the NF-κB activator described in this document can be obtained using known recombinant methods in the art, such as, for example, searching libraries of cells that express the nucleic acid molecule by deriving the nucleic acid molecule from a vector known to include the same or by isolating directly from cells and tissues containing it, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically, rather than cloned.
[00222] [00222] In some embodiments, the nucleic acid molecule encoding non-naturally occurring immune receptors, for example, CARs and / or accessory molecules (for example, an NF-κB activating sequence) described in this document is provided as a messenger RNA (mRNA) transcript. In another embodiment, the nucleic acid molecule encoding non-naturally occurring immune receptors, for example, CARs and / or accessory molecules (for example, a selective NF-κB activator coding sequence) described in this document is provided as a DNA construct.
[00223] [00223] The methods of cloning and expression will be evident to an individual skilled in the art and can be as described in WO
[00224] [00224] The disclosure provides a recombinant nucleic acid construct comprising a nucleic acid molecule that encodes an unnaturally occurring immune receptor, for example, CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence that encodes one or more more antigen-binding domains, where the nucleotide sequences encoding each of the antigen-binding domains are contiguous with and within the same reading frame as the nucleic acid sequences encoding: (i) hinge / ligand
[00225] [00225] The disclosure also provides a vector or vectors comprising a sequence or sequences of nucleic acids that encode an unnaturally occurring immune receptor, for example, CAR, described in this document and an accessory module. In some embodiments, the accessory module encodes an NF-κB activator, for example, a selective NF-κB activator. In some modality, the selective NF-κB activator is an unnatural NF-κB activator. In one embodiment, the non-naturally occurring immune receptor, for example, CAR, and the accessory module, for example, an accessory module that encodes an NF-κB activator, are encoded by a single vector. In another embodiment, the non-naturally occurring immune receptor, for example, CAR, and the accessory module, for example, an accessory module that encodes an NF-κB activator, are encoded by more than one vector. In yet another embodiment, an unnaturally occurring immune receptor, for example, CAR, and the accessory module, for example, an accessory module that encodes an NF-κB activator, are each encoded by a separate vector or by separate nucleic acids. In one embodiment, the two functional polypeptide units (for example, CAR and accessory module) are encoded by a single vector
[00226] [00226] A retroviral vector can also be, for example, a gamma-retroviral vector. A gamma-retroviral vector can include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (for example, two) long terminal repeats (LTR) and a transgene of interest , for example, a gene that encodes an unnaturally occurring immune receptor, for example, CAR. A gamma-retroviral vector may lack viral structural genes, such as gag, pol and env. Gamma-retroviral vectors include the murine leukemia virus (MLV), the spleen-forming virus (SFFV) and the myeloproliferative sarcoma virus (MPSV) and vectors derived from it. Other gamma-retroviral vectors are described, for example, in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” virus. June 2011; 3 (6): 677 a
[00227] [00227] In some modalities, a dissemination vector may also comprise a promoter. Non-limiting examples of a promoter include, for example, an MNDU3 promoter, a CMV IE gene promoter, an EF-la promoter, a ubiquitin C promoter, a nucleus promoter or a phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is an EF-1 promoter. In some embodiments, the vector comprises a poly (A) tail. In some embodiments, the vector comprises a 3'UTR.
[00228] [00228] The disclosure also includes an RNA construct that can be directly transfected in a cell. One method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a model with specially designed primers, followed by addition of poly A, to produce a construct containing 3 'and 5' untranslated sequence ('RTU') (for example, a 3 'and / or 5' RTU described in this document), a 5 'cap (for example, a 5' cap described in this document) and / or Internal Ribosome Entry Site (IRES) ( for example, an IRES described in this document), the nucleic acid to be expressed and a poly A tail, typically 50 to 2,000 bases in length (SEQ ID NO: 3,855). The RNA thus produced can efficiently transfect different types of cells. In one embodiment, the model includes sequences for the unnaturally occurring immune receptor, for example, CAR, and / or the NF-kB stimulating molecule. In one embodiment, a CAR-NFκB RNA vector is transduced into a cell, for example, a T cell or an NK cell, by electroporation. In another embodiment, a CAR RNA vector and / or an NF-κB activator vector is transduced into a cell, for example, a T cell or an NK cell, causing transient cell membrane disturbances using a microfluidic device. Different chains (or functional polypeptide units) can also be introduced into a cell
[00229] [00229] RNA can be introduced into target cells using any of several different methods, for example, commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts) or Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection or particle delivery systems such as “gene weapons” (see, for example, Nishikawa, et al. Hum Gene Ther., 12 (8): 861 to 870 (2001) or causing transient disturbances in cell membranes using a microfluidic device ( see, for example, patent applications WO 2013/059343 A1 and PCT / US2012 / 060646).
[00230] [00230] In some modalities, the non-viral method includes the use of a transposon (also called a transposon element). In some modalities, a transposon is a piece of DNA that can be inserted into a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome or a piece of DNA that can be spliced from a longer nucleic acid and inserted elsewhere in a genome. For example, a transposon comprises a DNA sequence composed of flanking genes of inverted repeats for transposition.
[00231] [00231] Exemplary methods of delivering nucleic acid using a transposon include a Sleeping Beauty transposition system (SBTS) and a piggyBac transposition system (PB). See, for example, Aronovich et al. Hum. Mol. Genet. 20.R1 (2011): R14-20; Singh et al. Cancer Res. 15 (2008): 2,961 to 2,971; Huang et al. Mol. Ther. 16 (2008): 580 to 589; Grabundzija et al. Mol. Ther. 18 (2010): 1,200 to 1,209; Kebriaei et al. Blood. 122.21 (2013): 166; Williams. Molecular Therapy 16.9 (2008): 1,515 to 1,516; Bell et al. Nat. Protoc. 2.12 (2007): 3,153 to 3,165; and Ding et al. Cell.
[00232] [00232] The SBTS includes two components: 1) a transposon containing a transgene and 2) a source of the transposase enzyme. Transposase can transpose the transposition of a carrier plasmid (or other donor DNA) to a target DNA, such as a chromosome / genome of the host cell. For example, the transposase binds to the carrier / donor DNA plasmid, cuts off the transposon (including transgene (or transgenes)) from the plasmid and inserts it into the host cell genome. See, for example, Aronovich et al. supra.
[00233] [00233] Exemplary transposons include a pT2-based transposon. See, for example, Grabundzija et al. Nucleic Acids Res. 41.3 (2013): 1,829 to 1,847; and Singh et al. Cancer Res. 68.8 (2008): 2,961 to 2,971,
[00234] [00234] The use of SBTS allows efficient integration and expression of a transgene, for example, a nucleic acid encoding a CAR and / or an NF-κB activator described in this document. Methods of generating a cell, for example, T cell or NKT or stem cell or iPSC or synthetic T cell, which stably expresses a CAR and / or an NF-κB activator described in this document, are provided in this document. example, using a transposition system like SBTS.
[00235] [00235] According to the methods described in this document, in some embodiments, one or more nucleic acids, for example, plasmids, containing the SBTS components are delivered to a cell (for example, T or NKT cell or stem cell or iPSC or synthetic T cell). For example, nucleic acids are delivered by standard nucleic acid delivery methods (for example, plasmid DNA), for example, methods described in this document, for example, electroporation, transfection or lipofection. In some embodiments, the nucleic acid contains a transposon that comprises a transgene, for example, a nucleic acid that encodes an unnaturally occurring receptor, for example, CAR, and / or an NF-kB activator described in this document. In some embodiments, the nucleic acid contains a transposon that comprises a transgene (for example, a nucleic acid that encodes an unnaturally occurring receptor, for example, CAR, and / or an NF-kB activator described in this document), as well as as a sequence of nucleic acids encoding a transposase enzyme. In other modalities, a system with two
[00236] [00236] As described above and elsewhere in this document, the disclosure demonstrates that the coexpression of an immune receptor (for example, a CAR, an endogenous TCR or a recombinant TCR) of the disclosure with an NF-κB stimulating molecule (for example, example, a selective NF-κB activator, eg an unnaturally occurring NF-κB activating agent, eg hNEMO-K277A) improves immune cell functions, such as survival, expansion, proliferation, activation, persistence, cytokine production and in vivo activity. In some embodiments, the immune receptor is an unnaturally occurring immune receptor (for example, recombinant CAR or TCR). In some embodiments, the immune receptor is a naturally occurring immune receptor (for example, a native TCR). In one embodiment, an NF-κB stimulating molecule is coexpressed with a first generation, second generation, third generation CAR, TFP, AbTCR or SIR. As mentioned above, the NF-κB stimulating molecule may be, but preferably is not, linked to a major CAR, TCR or SIR structure. In addition, in certain embodiments, a disclosure CAR does not include a CD28 or 41BB domain and, optionally, includes a CD3 domain.
[00237] [00237] In one embodiment, the disclosure demonstrates that the expression of a selective NF-κB activator improves the functions of immune cells (for example, T cells, dendritic cells, CAR-T cells or TCR-T cells, etc.), such as survival, expansion, proliferation, activation, persistence, cytokine production and in vivo activity. A selective NF-κB activator, as described in this document, refers to an agent that selectively activates the NF-κB signaling pathway without or with minimal activation of the others
[00238] [00238] In one embodiment, a selective NF-κB activator induces more than 20% (for example, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of increase in NF-κB activity as measured using the Phospho-NF-κB p65 antibody (Ser536) (Cell Signaling Technology in Danvers, MA), but less than 20% increase in AKT pathway activity as measured using the Phospho antibody -Akt (Ser473) (Cell Signaling Technology in Danvers, MA) when exposed or expressed in a test human T cell compared to a control human T cell. In some modalities, a selective NF-κB activator induces more than 20% (for example, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of increased activity of NF-κB as measured using the Phospho-NF-κB p65 antibody (Ser536) (Cell Signaling Technology in Danvers, MA), but less than a 20% increase in JNK pathway activity, as measured using the Phospho-SAPK antibody / JNK (Thr183 / Tyr185) (eg, G9 clone; Cell Signaling Technology in Danvers, MA) when exposed to or expressed in a test human T cell compared to a control human T cell. In some modalities, a selective NF-κB activator induces more than 20% (for example, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of increased activity of NF-κB as measured using the Phospho-NF-κB p65 antibody (Ser536) (Cell Signaling Technology in Danvers, MA), but
[00239] [00239] In one embodiment, a selective NF-κB activator induces more than 20% (for example, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of increase in NF-κB activity as measured using the Phospho-IκBα (Ser32) antibody (eg, 14D4, Cell Cloning Signaling Technology; in Danvers, MA), but with less than a 20% increase in AKT pathway activity as measured using the Phospho-Akt antibody (Ser473) (Cell Signaling Technology in Danvers, MA) when exposed or expressed in a test human T cell compared to a control human T cell. In some embodiments, a selective NF-κB activator induces more than 20% (for example, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of increase
[00240] [00240] Alternative methods for measuring the activation of the NF-κB, AKT, JNK, p38, ERK, JAK / STAT and interferon signaling pathways are known in the art and can be used to identify selective NF-κB signaling pathway activator . For example, a selective NF-κB activator induces a greater increase in NF-κB DNA-binding activity when exposed or expressed in a target cell (eg, a T cell or 293FT cell) compared to an increase in DNA binding of c- Jun, c-Fos, JunD, ATF2, STAT3, NFAT1c, ELK-1, CREB, IRF3 or IRF7. Kits are commercially available to measure DNA binding activities of different transcription factors belonging to different signaling pathways (eg, TransAM® Transcription Factor Assays; Active Motive) and can be used to identify selective activator of the signaling pathway of NF-κB.
[00241] [00241] In one embodiment, a selective NF-κB activator induces a greater increase in the rate of increase in IκBα phosphorylation to increase AKT phosphorylation compared to CD28 when both are expressed in human T cells or when signaling through both it is activated in human T cells under comparable conditions. In one embodiment, a selective NF-κB activator induces a greater increase in the ratio of increased IκBα phosphorylation to increased AKT phosphorylation compared to 41BB when both are expressed in human T cells under comparable conditions or when signaling through both are activated in human T cells under comparable conditions. In one embodiment, a selective NF-kB activator when coexpressed with a 1st generation CAR without a co-stimulating domain induces a greater increase in the ratio of increase in IκBα phosphorylation to increase in Akt phosphorylation in comparison
[00242] [00242] In one embodiment, a selective NF-κB activator induces a greater increase in the ratio of increased phosphorylation of p65 / RelA to increase phosphorylation of AKT compared to CD28 when both are expressed in human T cells or when signaling through both are activated in human T cells under comparable conditions. In one embodiment, a selective NF-κB activator induces a greater increase in the ratio of increase in phosphorylation of p65 / RelA to increase in phosphorylation of AKT compared to 41BB when both are expressed in human T cells under comparable conditions or when signaling through both is activated on human T cells under comparable conditions. In one embodiment, a selective NF-kB activator when coexpressed with a 1st generation CAR without a co-stimulating domain induces a greater increase in the ratio of increase in phosphorylation of p65 / RelA to increase in phosphorylation of Akt when compared to a 2nd generation CAR containing a co-stimulating domain of CD28 when both are expressed in human T cells and exposed to cells containing target antigen under comparable conditions. In one embodiment, a selective NF-kB activator when coexpressed with a 1st generation CAR without a co-stimulating domain (for example, a CAR represented by SEQ ID NO: 1.016) induces a greater increase in the rate of increase in p65 / RelA phosphorylation for increasing Akt phosphorylation when compared to a 2nd generation CAR containing a 41BB co-stimulating domain (for example, a CAR represented by SEQ ID NO:
[00243] [00243] In one embodiment, a selective NF-kB activator when coexpressed with a 1st generation CAR without a co-stimulating domain (for example, a CAR represented by SEQ ID NO: 1.016) induces a greater increase in the rate of increase in phosphorylation of IκBα for increased phosphorylation of JNK, ERK or p38 kinase compared to a 2nd generation CAR containing a 41BB co-stimulating domain (for example, a CAR represented by SEQ ID NO: 1,318) when both are expressed in human T cells and exposed to the target antigen containing cells (for example, RAJI) for an appropriate period of time (for example, 1 to 24 hours) under comparable conditions. In one embodiment, a selective NF-kB activator when coexpressed with a 1st generation CAR without a co-stimulating domain (for example, a CAR represented by SEQ ID NO: 1.016)
[00244] [00244] In one embodiment, an NF-κB activator, including a selective NF-κB activator, is an unnaturally occurring agent and is expressed exogenously in the cell. In one embodiment, the selective NF-kB activator is of viral origin, that is, it is encoded by a virus or is derived from a virus-encoded protein or has a domain of more than 10 amino acid residues (for example, more of 15 amino acid residues, 20 amino acid residues, 30 amino acid residues or 50 amino acid residues) with more than 80% (for example, more than 85%, 90%, 95%, or 99%) of identity with one or more more viral proteins. An exemplary selective NF-κB activator of viral origin is vFLIP K13 (SEQ ID NO :) which is derived from the herpesvirus associated with Kaposi's sarcoma. In another embodiment, the selective NF-κB activator is of cellular or mammalian origin. Exemplary selective NF-kB activators of mammalian origin are human NEMO-K277A mutant, human NEMO-K277- deltaV249-K255 mutant, mouse NEMO-K270A mutant, IKK2- S177E-S181E and IKK1-S176E-S180. In another modality, the selective NF-κB activator is of human origin; that is, it has a domain with more than 10 amino acid residues (for example, more than 15 amino acid residues, 20 amino acid residues, 30 amino acid residues or 50 amino acid residues) with more than 80% (for example, more than 85%, 90%, 95% or 99%) identity with one or more human proteins. In some embodiments, a selective NF-κB activator is composed of two or
[00245] [00245] In some embodiments, the selective NF-κB activator is encoded by the wild-type nucleic acid sequence, while in other embodiments, the selective NF-κB activator is encoded by the codon-optimized nucleic acid sequence or a mutant sequence . In an exemplary embodiment, vFLIP K13 is encoded by the nucleic acid sequence optimized for human codon, for example, K13-opt (SEQ ID NO: 7.768).
[00246] [00246] In some embodiments, immune cells express a single selective NF-κB activator, while in other embodiments, immune cells express more than one selective NF-κB activator (for example, NEMO-K277A plus K13-opt or IKK2 -S177E-S181E plus IKK1-S176E- S180E).
[00247] [00247] In some embodiments, the selective NF-κB activator is expressed in an immune cell in a constitutive manner. In other embodiments, the selective NF-κB activator is expressed in an immune cell in an inducible manner. In an exemplary embodiment, the inducible expression of a selective NF-κB activator can be achieved through the use of an inducible promoter. Examples of inducible promoters include, but are not limited to, a metallothionine-inducible promoter, a glucocorticoid-inducible promoter, a progesterone-inducible promoter and a tetracycline-inducible promoter. The RheoSwitch® system represents another regulatory transcription platform to control the expression of a protein.
[00248] [00248] Methods for controlling protein activity are known in the art and can be used to control protein activity.
[00249] [00249] In some embodiments, the selective NF-κB activator is expressed in immune cells by altering its genomic copy using gene editing techniques known in the art. In an exemplary embodiment, a gene editing system (for example, TALON, Zn finger nuclease or CRISP / Cas9) is used to convert one or both of the human NEMO alleles into a mutant form of human NEMO-K277A. In another exemplary embodiment, a gene editing system is used to convert one or both of the human NEMO alleles to the mutant form
[00250] [00250] In another modality or additional modality of any of the previous embodiments described in this document, immune effector cells that express an accessory module encoding a selective NF-κB activator (for example, hNEMO-K277A, hNEMO-K277A-deltaV249- K555, mNEMO-K270A, K13-opt, IKK2-S177E-S181E or IKK1-S176E-S180E) show improvement in in vitro activity (eg target antigen induced IL-2 production, proliferation, expansion and delay in terminal differentiation, senescence delay, etc.) against a cell that expresses the target antigen compared to a corresponding immune effector cell that lacks the accessory module when compared under similar conditions. Activation of NF-κB in immune effector cells is measured using techniques known in the art, including, but not limited to, measurement of phosphorylated IκBα, phosphorylated p65, nuclear IκBα translocation, total p65, increasing regulation of
[00251] [00251] In another modality or additional modality of any of the previous modalities described in this document, immune effector cells that express an accessory module that encodes a selective NF-kB activator show greater activity in vivo (for example, in vivo expansion , in vivo persistence, tumor reduction, reduction in the bioluminescence value obtained from a tumor that expresses luciferase or animal survival) against a cell that expresses target antigen compared to control immune effector cells that do not express the
[00252] [00252] In another or additional modality of any of the previous modalities described in this document, immune effector cells that express an accessory module, for example, hNEMO-K277A, hNEMO-K277A-deltaV249-K555, mNEMO-K270A, K13- opt, IKK2-S177E- S181E, IKK1-S176E-S180E, or MYD88-L265P, show increased activity in vivo (for example, in vivo expansion, persistence in vivo, tumor reduction, reduction in the bioluminescence value obtained from a tumor that expresses Fluc or animal survival) against a cell that expresses target antigen compared to a corresponding immune effector cell that lacks the accessory module when compared under similar conditions.
[00253] [00253] The disclosure also guarantees that the expression of a selective NF-κB activator can be used to improve cytokine secretion, antigen presentation and immune response generated by cells that have antigen, including dendritic cells. The disclosure also provides a method to improve the vaccine's effectiveness, including cancer vaccines, by expressing a selective NF-κB activator in cells that have antigen ex vivo or in vivo. In one embodiment, the use of selective NF-κB activators increases the production of cytokines (eg, TNFα) by cells that have antigen (eg, dendritic cells) by more than at least 15%.
[00254] [00254] The disclosure further guarantees that an accessory module encoding CMV-141 (SEQ ID NO: 7.770) can be expressed in immune effector cells, for example, T cells, for example, CAR-T cells or TCR-T cells, to delay your exhaustion and improve your long-term persistence. CMV-141 can be expressed in immune effector cells in an inducible or constitutive manner.
[00255] [00255] In some embodiments, cells, for example, stem cells, or T or NKT cells, or IPSC, or synthetic T cells, are generated that express an unnaturally occurring receptor, for example, CAR, and / or an NF-κB stimulating molecule described in this document using
[00256] [00256] In another embodiment, disclosure provides a method for producing a cell (for example, an immune effector cell or population thereof) comprising introducing (for example, transducing) a cell, for example, a T cell, a NKT cell or stem cell or iPSC or synthetic T cell described in this document, with a vector comprising a nucleic acid encoding an unnaturally occurring immune receptor, for example, CAR, and / or an NF stimulating molecule -κB.
[00257] [00257] In various embodiments, cells for modifications with an unnatural immune receptor and / or NF-κB stimulating molecule described herein, including T cells or NK cells, can be obtained from a subject who desires therapy. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue and tumors. T cells can be tissue-resident gamma-delta T cells, which can be cultured and expanded in vitro before expression of the unnaturally occurring immune receptor, for example, CAR and / or NF-kB stimulating molecule.
[00258] [00258] In one embodiment, the disclosure provides numerous chimeric antigen (CAR) receptors comprising an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) genetically engineered for specific binding to an associated antigen to the disease, for example, a tumor antigen described in this document. In one embodiment, disclosure provides a cell
[00259] [00259] The disclosure provides immune effector cells (eg, T cells, NK cells) that are designed to contain one or more non-naturally occurring immune receptors (eg, CARs / TCRs) and / or NF-κB stimulating molecules that direct immune effector cells to sick cells or disease-associated cells, such as cancer cells. This is achieved through an antigen-binding domain at the immune receptor that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the disclosure CARs: (1) cancer-associated antigens that are expressed on the surface of cancer cells; and (2) cancer-associated antigens that are intracellular, however, a fragment of that antigen (peptide) is presented on the surface of cancer cells by MHC (main histocompatibility complex). The disclosure also provides immune effector cells (eg, T cells, NK cells) that contain endogenous and / or genetically engineered TCRs to express one or more NF-κB stimulating molecules that target immune effector cells to sick cells or cells associated with diseases such as cancer cells.
[00260] [00260] In addition, the disclosure provides CARs, TCRs and cells that express CAR / TCR that also express an NF-κB stimulating molecule and its use in drugs or methods for treatment, among other diseases, cancer or any malignant or autoimmune disease or infectious or degenerative disease or allergic disease involving cells or
[00261] [00261] In one embodiment, the disclosure provides an immune effector cell (eg, T cell, NK cell) genetically engineered to express an unnaturally occurring immune receptor, for example, CAR and / or TCR, and a stimulating molecule of NF-κB, in which the genetically engineered immune effector cells exhibit an anti-disease property, such as the anti-tumor property. One type of antigen is a cancer-associated antigen (ie, tumor antigen) described in this document. In one aspect, the antigen-binding domain of the unnaturally occurring immune receptor, for example, CAR, comprises a partially humanized antibody fragment. In one embodiment, the non-naturally occurring immune receptor antigen-binding domain, for example, CAR, comprises a partially humanized scFv. Accordingly, the disclosure provides non-naturally occurring immune receptors, for example, CARs, which comprise a humanized antigen binding domain and are genetically manipulated in a cell, for example, a T cell or an NK cell, in which the cell it also expresses a stimulating NF-κB molecule and methods for using them for adoptive therapy.
[00262] [00262] In one embodiment, the disclosure provides an immune effector cell (eg, T cell, NK cell) with its endogenous immune receptor (eg, a TCR) that is genetically engineered to express an NF-κB stimulating molecule, wherein the genetically engineered immune effector cell exhibits an anti-disease property, such as anti-tumor property or anti-HIV-1 property.
[00263] [00263] Genetically modified cells are also provided in this document, comprising the polynucleotides and / or non-naturally occurring immune receptors described in this document. In some
[00264] [00264] Stimulation of T cells by an antigen under appropriate conditions results in cell proliferation (expansion) and / or production of IL-2. The cells comprising the non-naturally occurring immune receptors (for example, CARs and / or TCRs) and / or the NF-κB stimulating molecule of the disclosure will expand in number in response to the binding of one or more antigens to the specific targeting regions of antigen from non-naturally occurring immune receptors (for example, CARs and / or TCRs). The disclosure also provides a method of producing and expanding cells that express a non-naturally occurring immune receptor (for example, CAR and / or TCR). The method comprises transfecting or transducing cells with the vector (or vectors) that expresses the unnaturally occurring immune receptor (for example, CAR and / or TCR) and / or the NF-κB stimulating molecule and stimulating cells with cells that express the target antigens, recombinant target antigens or an antibody against the receptor causing cell proliferation, in order to produce and expand T cells. In one embodiment, the cells can be any one or more T lymphocytes (T cells), killer cells natural (NK), hematopoietic stem cells (HSCs) or pluripotent embryonic / induced stem cells capable of giving rise to therapeutically relevant offspring. In one embodiment, the molecule
[00265] [00265] Immune effector cells, such as T cells and NK cells, comprising non-naturally occurring immune receptors (for example, CARs and / or TCRs) and / or NF-κB stimulating molecule, as described in this document, can be activated and expanded generally using methods such as those described, for example, in US patents 6,352,694;
[00266] [00266] In one embodiment, the genetically modified cells comprise nucleic acid molecules that encode the conventional CARs 1 to 6 or the conventional CARs 1 to 6 that are part of the main structures described in this document, as main structure-1, main structure- 2, main structure-13, main structure-14, main structure-37, main structure-38, main structure-49, main structure-50, main structure-60 or main structure-61 and an NF-κB stimulating molecule in whereas the specific antigen domain of CARs is specific for MPL, CD19, CD20, BCMA, CD22, CD30, CD33, CD123, CD138, CLL1, TCR-beta 1 constant chain, TCR-beta2 constant chain, TCR gamma / delta, mesothelin, IL13Ra2, ALK, PTK7, DLL3, TROP2, Tim1, LAMP1, CS1, Lym1, Lym2, TSHR, NY-ESO-1 / MHC class i complex, WT1 / MHC class I complex, class i complex Ras / MHC complex, AFP / MHC class i complex, HPV-E6 / MHC casse i complex, HPV-E7 / MHC class i, CD179a, CLD18A2, CD43 epitope or HIV1 envelope gp120 protein.
[00267] [00267] In one embodiment, the cell is a T cell and the T cell is deficient in one or more chains of endogenous T cell receptors. T cells that lack stable expression of a functional TCR according to the disclosure can be produced using a variety of approaches, such as the use of Zn finger nucleases (ZFN), CRISP / Cas9 and shRNA targeting the endogenous T cell receptor chains. An exemplary non-limiting method related to shRNAs is described in US 2012 / 0321667A1, which is incorporated herein by reference. Another exemplary non-limiting method related to elimination of endogenous TCR expression using ZFNs targeting constant regions of α and β chains of TCRs is described in Torikai H et al. (Blood, 119 (24), June 14, 2012).
[00268] [00268] A T cell without a functional endogenous TCR can be, for example, genetically engineered to not express any functional endogenous TCR on its surface, genetically engineered so that it does not express one or more subunits (eg, TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ or pre-TCRα) that comprise a functional endogenous TCR or genetically engineered to produce very little functional endogenous TCR on its surface. Alternatively, the T cell can express an endogenous TCR substantially impaired, for example, by the expression of mutated or truncated forms of one or more of the TCR subunits. The term "substantially compromised TCR" means that this TCR will not cause an adverse immune reaction in a host. In a further alternative, an unnaturally occurring immune receptor (eg, CAR and / or TCR) and / or an NF-κB stimulating molecule can be cloned into a TCR site in a T cell genome and thus , non-naturally occurring immune receptors (for example, CARs and / or TCRs) and / or NF-κB stimulating molecule would be under the control of the endogenous T cell expression system.
[00269] [00269] The disclosure shows that, in contrast to the situation with the 1st or 2nd generation CAR constructs, TFPs based on CD3ε, CD3γ and CD3δ chains (designated as CD3ε / γ / δ TFPs) have a weak expression and activity when expressed in αβ T cells that lack or have native or endogenous functional TCRα chain polypeptide on their surface. For example, it is observed that CD3ε / γ / δ TFPs show impaired expression and activity (for example, T cell activation, proliferation, cytokine production and cytotoxicity, etc.) in the αβ T cell in which the endogenous TRAC genomic site was interrupted by the TFP expression cassette. The disclosure provides a solution to this problem by re-expressing a TCRα constant chain polypeptide (TRAC chain) or a fragment thereof in T cells in which the expression of the native full-length TCRα chain polypeptide has been reduced or eliminated. In one embodiment, the TCRα constant chain polypeptide or the TCRα constant chain fragment expressed again allows the reconstitution of a functional TFP signaling complex CD3ε / γ / δ-TCR-CD3 in a T cell α in which the expression of the constant chain Native TCRα is impaired, reduced or eliminated. In one embodiment, the TCRα constant chain or TCRα constant chain fragment expressed again improves by more than 15% (for example, more than 20%, 50%, 75% or 100%, etc.) antigen-induced cytokine production target (for example, IL2, TNFα, IFNγ), proliferation and / or cytotoxic activity of the αβ T cell that expresses TFP CD3ε / γ / δ in which the expression of the native TCRα constant chain is impaired, reduced or eliminated. In one embodiment, the TCRα constant chain or the TCRα constant chain fragment expressed again allows for enhanced expression of the TFP CD3ε / γ / δ in an αβ cell in which the expression of the native TCRα constant chain is impaired, reduced or eliminated. In one embodiment, the TCRα constant chain or the TCRα constant chain fragment expressed again allows more than 15% (for
[00270] [00270] In an alternative embodiment, the expression of the TCRα constant-chain polypeptide can be restored in αβ T cells that lack
[00271] [00271] In an exemplary embodiment, the expression of the TCRα constant chain polypeptide can be restored in αβ T cells in which the endogenous or native TCRα chain gene has been interrupted by the targeted integration of a cassette encoding a TFP when designing the cassette of targeting so that the TFP cassette is followed in the frame by a cleavable linker 2A, a signal peptide (for example, a CD8 signal peptide or an IgH signal peptide) and the first exon of the TCRα constant chain (TRAC) (Figure 5C) . An example of such a bleaching construct is
[00272] [00272] The disclosure also demonstrates that, in contrast to the situation with the 1st or 2nd generation CAR constructs, TF3 CD3ε / γ / δ lose their activity when expressed in αβ T cells (that is, T cells that express the chains TCRα AND TCRβ) that do not have or have functional endogenous or native TCRβ1 and TCRβ2 polypeptides on their surface. For example, the disclosure ensures that TF3 CD3ε / γ / δ exhibit impaired expression and activity (eg, T cell activation, proliferation, cytokine production and cytotoxicity, etc.) in αβ T cells in which the endogenous genomic sites of TCRβ1 and TCRβ2 were discontinued. The disclosure provides a solution to this problem by re-expressing TCRβ1 or TCRβ2 constant-chain polypeptides or a fragment thereof in T cells in which the expression of full-length TCRβ1 and TCRβ2 native polypeptides has been reduced or eliminated. In one embodiment, the TCRβ1 / β2 constant chain polypeptide or the TCRβ1 / β2 constant chain fragment expressed again allows the reconstitution of a TFP-
[00273] [00273] In an alternative embodiment, the expression of the TCRβ1 or β2 constant chain polypeptide can be restored in αβ T cells that do not have or have impaired functional endogenous or native TCRα chain polypeptide on their surface using the TCRβ1 constant chain gene or endogenous β2. In an exemplary embodiment, the expression of the TCRβ1 / β2 constant-chain polypeptide and the coexpressed TFP can be restored in αβ T cells that do not have or have functional endogenous or native TCRβ chain polypeptide on their surface operationally linking a sequence of acids nucleic acids encoding a signal peptide to at least one copy of the endogenous TCRβ1 or TCRβ2 constant chain gene using gene editing techniques known in the art. In an exemplary embodiment, the nucleic acid sequence encoding a signal peptide is operationally linked in the frame to the first exon of at least one of the endogenous constant chain genes TCRβ1 or TCRβ2, from
[00274] [00274] In an exemplary embodiment, the expression of the TCRβ1 / β2 constant chain polypeptide can be restored in αβ T cells in which the endogenous or native TCRβ1 and TCRβ2 chain genes have been disrupted by the targeted integration of cassettes encoding a TFP when designing the targeting cassette so that the TFP cassette is followed in frame by a 2A cleavable linker, a signal peptide (for example, a CD8 signal peptide or an IgH signal peptide) and the first exon of the TCRβ1 / β2 constant chain (TRBC ).
[00275] [00275] It has been observed that directing the CAR cassette to the TRAC site results in approximately 95% of T cells becoming negative for TCR. Such TCR negative T cells can be used in an allogeneic environment, as they are less likely to cause graft versus host disease (GVHD). However, the new expression of the TRAC chain in T cells in which the TRAC site was targeted by a TFP cassette would potentially lead to the expression of the full-length TCRβ chain, including the Vβ region. Such T cells, even without the MHC recognition provided by the Vα region, would potentially be able to recognize alloantigens presented by the MHC complex through its TCRβ chains and, therefore, potentially cause GVHD. In modalities
[00276] [00276] In the example above, an exogenous TRAC or TRBC is coexpressed with a construct that expresses TFP to restore the expression and / or activity of TFP CD3ε / γ / δ in α / β T cells in which the expression of TCRα and / chains or endogenous TCRβ have been downgraded or eliminated by, for example, targeting their genomic sites. In an alternative embodiment of the invention, the expression of exogenous constant chains TCRα and / or TCRβ1 / β2 is used to restore the expression of the TCR / CD3 complex in any T a / β cell, including a wild type T a / β cell or a a / β T cell that expresses a chimeric antigen receptor, a chimeric T cell receptor (cTCR), an AbTCR or a synthetic immune receptor. Finally, a similar approach can be used to restore the expression of CAR / TFP and / or TCR / CD3 in T γ / δ cells in which the expression of endogenous TCRγ and / or TCRδ chains has been down-regulated or eliminated. Exemplary TCRγ (TRGC) and TCRδ (TRDC) constant chains that can be expressed in γ / δ T cells in which the expression of endogenous TCRγ and / or TCRδ chains has been regulated or decreased or eliminated are represented by SEQ ID NO: 3,912 and 3,913.
[00277] [00277] The disclosure also ensures that the expression and activity of TFP CD3ε / γ / δ can be restored in T cells with deficiency or lack of expression of native TCRα / β / γ or δ chains by the new expression of fragments or variants of constant chains TCRα / β / γ or δ. The fragments / variants of TCRα / β / γ and δ constant chains that can be used to restore TFP CD3ε / γ / δ expression in cells without native TCRα / β / γ or δ chains are provided in SEQ ID Nos: 15.141 a 15,144
[00278] [00278] The disclosure further provides that the expression and activity of TFP CD3ε / γ / δ can be restored in T cells with impaired or native TCRα / β / γ or δ chains by coexpression of a SIR or an Ab -TCR comprising the missing TCRα / β / γ or δ constant chains. Thus, in T aβ cells with deficiency or lack of expression of the native TCRα chain, the expression and activity of a CD3ε / y / δ TFP can be rescued by the expression of a SIR comprising a TCRα constant chain. In an exemplary embodiment, in an αβ T cell with a deficiency or lack of expression of the native TCRα chain, the expression and activity of a CD3ε / γ / δ TFP (for example, a TFP encoded by SEQ ID NOs: 8,708 to 8,714) can be rescued by the expression of a CRS (for example, a CRS represented by SEQ ID NO: 9,668, 9,669 or 9,684, etc.) comprising a constant chain TCRα. In another exemplary modality, in T aβ cells with deficiency or lack of expression of the native TCRα chain, the expression and activity of a CD3ε / γ / δ TFP (for example, a TFP encoded by SEQ ID NOs: 8,708 to 8,714) can be rescued by the expression of an Ab-TCR (for example, an Ab-TCR represented by SEQ ID NO: 9,677 or 9,678, etc.) comprising a portion of the TCRα constant chain. The disclosure ensures that, for therapies combined with allogeneic T cells involving two CARs, a TF3 CD3α / γ / δ is preferably combined with a SIR and / or an Ab-TCR that incorporate the TCR constant chain or TCR constant chain fragment whose expression it is reduced or absent in allogeneic T cells.
[00279] [00279] The disclosure also guarantees that, in T aβ cells with deficiency or lack of expression of native TCRβ chains, the expression and activity of a CD3ε / γ / δ TFP can be rescued by the expression of a
[00280] [00280] The disclosure also guarantees that, in γδ T cells with deficiency or lack of expression of the native TCRγ chain, the expression and activity of a CD3ε / γ / δ TFP can be rescued by the expression of a CRS comprising a TCRγ constant chain . In an exemplary embodiment, in γδ T cells with deficiency or lack of expression of the native TCRγ chain, the expression and activity of a CD3ε / γ / δ TFP (for example, a TFP encoded by SEQ ID NOs: 8,708 to 8,714) can be rescued by the expression of a CRS (for example, a CRS represented by SEQ ID NO: 9,689) comprising a constant chain TCRγ. In another exemplary modality, in γδ T cells with deficiency or lack of expression of the native TCRγ chain, the expression and activity of a CD3ε / γ / δ TFP (for example, a TFP encoded by SEQ ID NOs: 8,708 a
[00281] [00281] The disclosure also guarantees that, in γδ T cells with deficiency or lack of expression of the native TCRδ chain, the expression and
[00282] [00282] Disclosure also provides methods and constructs that allow a next generation CAR (for example, SIR and AbTCR), cTCR and TCR to be expressed under the physiological regulation mechanisms provided by endogenous TCR genes. The disclosure also provides methods and constructs that allow a next generation CAR (for example, SIR and AbTCR), cTCR and TCR to be expressed under the 3 'promoter and regulatory mechanisms provided by endogenous TCR genes. In one embodiment, the disclosure provides methods for an expression cassette that encodes an SIR / cTCR / Ab-TCR / TCR to be directed to the endogenous TCRα, TCRβ1 / β2, TCRγ or TCRδ gene site. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRα gene (TRAC), so that the TCRα constant chain of the SIR / cTCR / Ab-TCR / TCR is expressed totally or partially at from the native endogenous TCRα constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRα gene (TRAC) so that the TCRα constant chain of the
[00283] [00283] In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRβ gene (TRBC) so that the TCRβ constant chain of the SIR / cTCR / Ab-TCR / TCR is expressed fully or partially from the endogenous native TCRβ1 or TCRβ2 constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is targeted to the site of the endogenous TCRβ1 / β2 (TRBC) gene so that the TCRβ constant chain of the SIR / cTCR / Ab-TCR / TCR is either fully encoded or in part by at least one of the exons of the endogenous TCRβ constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRβ1 / β2 (TRBC) gene, so that the SCR / cTCR / Ab-TCR / TCR constant TCRβ chain shares completely or in part of the 3 'untranslated region and the polyadenylation sequence of the native / endogenous TCRβ constant chain gene.
[00284] [00284] In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRγ gene (TRGC), so that the TCRγ constant chain of the SIR / cTCR / Ab-TCR / TCR is expressed fully or partially from the native endogenous TCRγ constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRγ gene (TRGC), so that the TCRγ constant chain of the SIR / cTCR / Ab-TCR / TCR is encoded in whole or in part by at least one of the exons of the endogenous TCRγ constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the
[00285] [00285] In one embodiment, the SIR / cTCR / Ab-TCR / TCR is targeted to the site of the endogenous TCRδ gene (TRDC), so that the TCRδ constant chain of the SIR / cTCR / Ab-TCR / TCR is expressed totally or partially from the endogenous native TCRδ constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the site of the endogenous TCRδ gene (TRGC), so that the TCRδ constant chain of the SIR / cTCR / Ab-TCR / TCR is encoded completely or in part by at least one of the exons of the endogenous TCRδ constant chain gene. In one embodiment, the SIR / cTCR / Ab-TCR / TCR is directed to the endogenous TCRδ gene (TRDC) site, so that the SIR / cTCR / Ab-TCR / TCR constant TCRδ chain shares all or part of 3 'untranslated region and polyadenylation sequence of the native / endogenous TCRδ constant chain gene.
[00286] [00286] T cells or natural killer (NK) or stem cells can be obtained from a subject. The term "subject" is intended to include living organisms in which an immune response can be elicited (for example, mammals). Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue and tumors. T cells can be tissue-resident gamma-delta T cells, which can be cultured and expanded in vitro prior to the expression of CAR / TCR and / or an NF-κB stimulating molecule.
[00287] [00287] In certain disclosure modalities, immune effector cells, for example, T cells, can be obtained from a blood unit collected from a subject using any number of techniques known to the specialist, such as Ficoll ™ separation. In a preferred aspect, an individual's circulating blood cells are obtained by apheresis. The apheresis product usually contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and, optionally, to place the cells in a buffer or medium appropriate for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution has no calcium and may not have magnesium or may not have many, if not all, divalent cations.
[00288] [00288] Initial activation steps in the absence of calcium can lead to increased activation. As those skilled in the art would readily observe, a washing step can be performed by methods known to those skilled in the art, such as the use of a “continuous flow” semi-automatic centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, PBS without Ca, PBS without Mg, PlasmaLyte A or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed and the cells resuspended directly in the culture medium.
[00289] [00289] It is recognized that the methods of application may use conditions of culture media comprising 5% or less, for example 2%, of human AB serum, and employ conditions and media composition.
[00290] [00290] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by counterflow centrifugal elutriation or centrifugation through a PERCOLL ™ gradient.
[00291] [00291] In one embodiment, disclosure provides methods of treating or preventing a disease, providing the subject in need of the same immune effector cells (eg, T cells) or stem cells that can give rise to immune effector cells that are manipulated genetically to express an X-CAR or an X-TCR and an NF-κB stimulating molecule, where X represents a disease-associated antigen as described in this document, and in which the disease-causing or associated cells express said X antigen Table 9 provides a list of different antigens and the exemplary diseases that can be prevented, inhibited or treated using immune effector cells that express the CARs targeting these antigens.
[00292] [00292] In another modality, the disclosure provides methods of treatment or prevention of cancer, infection, autoimmune or allergic diseases providing the subject in need of the same immune effector cells (for example, T cells) or stem cells that can give rise to immune effector cells that are genetically engineered to express an unnaturally occurring immune receptor (eg, CAR and / or TCR) of the disclosure and / or an NF-kB stimulating molecule. In one embodiment, the NF-κB stimulating molecule is a selective NF-κB activator. In one embodiment, the NF-κB activator, for example, a selective NF-κB activator, is a non-viral NF-κB activator. In one mode, the
[00293] [00293] In another modality, the disclosure provides methods of treatment or prevention of cancer, infection, autoimmune or allergic diseases providing the subject in need of the same immune effector cells (for example, T cells) or stem cells that can give rise to immune effector cells that are genetically engineered to express a naturally occurring immune receptor (for example, a native TCR) and an NF-kB stimulating molecule. In one embodiment, the NF-κB stimulating molecule is a selective NF-κB activator. In one embodiment, the NF-κB activator, for example, a selective NF-κB activator, is a non-viral NF-κB activator. In one embodiment, the NF-κB activator, for example, a selective NF-κB activator, is not a transmembrane protein and is expressed in the cytosol or is preferably present in the cytosol. In one embodiment, the NF-κB activator, for example, a selective NF-κB activator, is constitutively active. In one embodiment, the NF-κB activator, for example, a selective NF-κB activator, is not constitutively active. In one embodiment, the selective NF-κB activator is activated by administration
[00294] [00294] In another modality, the disclosure provides methods of treatment or prevention of cancer, infection, autoimmune or allergic diseases providing the subject in need of the same immune effector cells (for example, T cells) or stem cells that can give rise to immune effector cells that are genetically engineered to express a naturally occurring immune receptor (eg, native TCR) or a non-naturally occurring immune receptor (eg, CAR and / or recombinant TCR) of the disclosure and / or a stimulating molecule of NF-κB. In some embodiments, the activity of CAR-T or TCR-T cells can be controlled using a water-soluble salt from Dasatinib.
[00295] [00295] In another aspect, a method of treating a subject, for example, reducing or ameliorating a hyperproliferative disorder or condition (for example, a cancer), for example, solid tumor, soft tissue tumor, blood cancer or metastatic injury in a subject is provided.
[00296] [00296] In yet another modality, the disclosure refers to a method of treating a disease in a subject. The method comprises administering to the subject a cell expressing a naturally occurring immune receptor and / or an unnaturally occurring immune receptor (for example, recombinant CAR and / or TCR) of the disclosure and / or an NF- stimulating molecule kB of disclosure so that the disease is treated in the subject. In one aspect, the method comprises administering to the subject a cell that expresses its endogenous (or native) TCR and a molecule
[00297] [00297] In some embodiments, the unnaturally occurring immune receptor (eg, CAR and / or TCR) specifically binds to an HIV antigen. In some embodiments, the HIV antigen is an HIV-
[00298] [00298] Disclosure includes a type of cell therapy in which cells
[00299] [00299] Disclosure also includes a type of cell therapy in which stem cells (for example, hematopoietic stem cells or lymphoid stem cells or embryonic stem cells or induced pluripotent stem cells) that are capable of giving rise to cells immune effectors (eg, T cells) are modified to express an unnaturally occurring immune receptor (eg, CAR and / or TCR) of the disclosure and / or a
[00300] [00300] Disclosure includes a type of cell therapy in which immune effector cells (for example, T cells or stem cells that give rise to T cells) are genetically modified to express CARs targeting two or more different antigens on the same cell, and that T cell or stem cell is infused into a recipient in need of it. In one embodiment, at least one of the CARs targets an antigen expressed on hematopoietic cells. In one embodiment, at least one of the CARs targets an antigen selected from the group of CD19, CD20, CD22, BCMA, CS1, CD138, Lym1, Lym2, CD33 and CD123. In one embodiment, at least one of the CARs targets an antigen expressed on hematopoietic cells and at least one of the CARs targets an antigen expressed on solid tumors. In one embodiment, at least one of the CARs targets an antigen selected from the group of CD19, CD20, CD22, BCMA, CS1, CD138, Lym1, Lym2, CD33 or CD123 and at least one other CAR targets an antigen selected from the group of Mesothelin, Her2, Folate Receptor 1, ROR1, IL13Ra2, AFP, WT1, Ras, NY-ESO-1, DLL3, CD70 and PTK7. In one embodiment, at least one of the CARs is a CRS. In one embodiment, at least one of the CARs is an Ab-TCR. In one embodiment, at least one of the CARs is a SIR and the other CAR is a TFP CD3ε / γ / δ. In one embodiment, at least one of the CARs is an Ab-TCR and the other CAR is a TFP CD3ε / γ / δ. In one embodiment, the cells have impaired expression of at least one of the native TCR chains. The disclosure also includes a type of cell therapy in which immune effector cells (for example, T cells or stem cells that give rise to T cells) are genetically modified to express CARs targeting two different antigens and an NF-κB stimulating molecule, and these cells are infused to a recipient in need of it. At
[00301] [00301] With regard to ex vivo immunization, at least one of the following occurs in vitro before administration of the cell to a mammal: i) expansion of the cells, ii) introduction of a nucleic acid encoding a non-occurring immune receptor natural (e.g., CAR and / or TCR) of the disclosure and / or an NF-κB stimulating molecule for the cells or iii) cell cryopreservation.
[00302] [00302] Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal (for example, a human) and genetically modified (that is, transduced or transfected in vitro) with one or more vectors that express a non-naturally occurring immune receptor (for example, CAR and / or TCR) of the disclosure and / or an NF-κB stimulating molecule disclosed in this document. The unnaturally occurring immune receptor (e.g., CAR and / or TCR) and the NF-kB activator-modified cell can be administered to a mammalian receptor to provide a therapeutic benefit. The mammalian receptor can be a human and non-naturally occurring immune receptor (e.g., CAR and / or TCR), and the cell modified by NF-κB activator can be autologous to the receptor. Alternatively, the cells can be allogeneic, syngeneic or xenogenic in relation to the receptor.
[00303] [00303] The procedure for ex vivo expansion of hematopoietic stem cells and progenitor cells is described in US Patent 5,199,942, incorporated herein by reference, and can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore, the present invention is not limited to any method
[00304] [00304] Generally, the activated and expanded cells as described in this document can be used in the treatment and prevention of diseases that arise in immunocompromised individuals. In certain aspects, the disclosure cells are used to treat patients at risk of developing diseases, disorders and conditions associated with the expression of a disease-associated antigen, as described in this document. Thus, the disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with the expression of a disease-associated antigen, as described in this document, comprising administering to a subject in need of therapeutically effective amount of stem cells or immune effector cells modified by CAR / TCR / NF-κB stimulating molecules (e.g., T cells) that are capable of generating immune effector cells of the disclosure.
[00305] [00305] In one aspect, cells expressing CAR / TCR / NF-κB stimulating molecules of the disclosures can be used to treat a proliferative disease such as cancer or malignancy or is a precancerous condition, such as myelodysplasia, a syndrome myelodysplastic or pre-leukemia. In addition, a disease associated with a cancer-associated antigen, as described in this document, includes, but is not limited to, for example, atypical and / or non-classical cancers,
[00306] [00306] The immune effector cells modified by the CAR / TCR / NF-κB stimulating molecule (e.g., T cells) of the disclosure can be administered alone or as a pharmaceutical composition in combination with diluents and / or with other components such as IL- 2 or other cytokines or cell populations.
[00307] [00307] Hematological cancer or blood cancer disorders are the types of cancer, such as leukemia, lymphoma and malignant lymphoproliferative disorders that affect the blood, bone marrow and the lymphatic system.
[00308] [00308] Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can also be classified as acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphoid leukemia (LLC). Other related conditions include myelodysplastic syndromes (SMD, formerly known as “pre-leukemia”), which are a diverse collection of hematological disorders linked by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation into AML.
[00309] [00309] Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin's lymphoma and Hodgkin's lymphoma.
[00310] [00310] The disclosure provides compositions and methods for the treatment and prevention of cancer. In one respect, cancer is cancer
[00311] [00311] Disclosure provides a method for administering to a subject an effective amount of a cell, for example, an immune effector cell or a population thereof, wherein each cell comprises an unnaturally occurring immune receptor (for example, CAR and / or TCR) and / or
[00312] [00312] In some modalities, the disease to be treated or prevented is hematological cancer. In other modalities, hematological cancer is leukemia. Non-limiting examples of acute leukemias include acute B-cell lymphoid leukemia (“LLAB”), acute T-cell lymphoid leukemia (“LLAT”), acute lymphoid leukemia (ALL); one or more chronic leukemias including, but not limited to, for example, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (LLC); additional haematological cancers or haematological disorders, including, but not limited to, for example, B cell prolinocytic leukemia, blast plasmacytoid cell dendritic neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, follicular lymphoma of
[00313] [00313] In some modalities, the tumor antigen associated with the disease is selected from: CD5; CD19; CD123; CD22; CD30; CD171; CS1 (also called subset CD2 1, CRACC, MPL, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); tyrosine kinase 1 orphan receptor (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin receptor alpha-2 subunit
[00314] [00314] In some embodiments, the disease to be treated is an infectious disease including, but not limited to, infection with HIV1, HIV2, HTLV1, Epstein Barr virus (EBV), cytomegalovirus (CMV), adenovirus, adeno-associated virus, virus BK, Human herpesvirus 6, Human herpesvirus 8, influenza virus, parainfluenza virus, avian influenza virus, MERS and SARS coronavirus, Democratic Republic of Congo Crimean hemorrhagic fever virus, rhinoceros virus, enterovirus, dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa fever virus, Zika virus, RSV, measles virus, mumps virus, rhinoceros virus, chickenpox virus, herpes simplex virus 1 and 2, varicella zoster, HIV-1, HTLV1, hepatitis virus, enterovirus, hepatitis B virus, hepatitis C virus, Nipah and Rift valley virus, Japanese encephalitis virus, mycobacterium tuberculosis, atypical mycobacterial species, Pneumocystis jirovecii, toxoplasmosis, rickettsia, nocardia, aspergillus, mucor or candida. In such diseases, the target antigen associated with the disease is selected from: HIV1 envelope glycoprotein, HIV1-gag, HTLV1- Tax, CMV pp65, EBV-EBNA3c, influenza A (HA) hemagglutinin and GAD.
[00315] [00315] The disease to be treated or prevented by the methods and compositions of the disclosure can be an immune or degenerative disease, for example, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, pemphigus vulgaris, ankylosing spondylitis, Hoshimoto's thyroiditis, SLE, sarcoidosis, scleroderma, mixed connective tissue disease, graft versus host disease or Alzheimer's disease. In such modalities, the target antigen associated with the disease is an autoantibody.
[00316] [00316] Additional non-limiting examples of diseases associated with the expression of a target antigen include any of the following cancers or related conditions: colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer,
[00317] [00317] In certain modalities of the methods or uses described in this document, the cell expressing CAR / TCR that comprises an unnaturally occurring immune receptor (eg, CAR and / or TCR) and / or NF-kB stimulating molecule is administered in combination with an agent that increases the effectiveness of the immune effector cell, for example, one or more protein phosphatase inhibitors, kinase inhibitor, cytokine, chemokine, scFV fragment, bispecific antibody, inhibitor of an immune inhibitory molecule ; a cell signaling protein, a viral signaling protein, or an agent that decreases the level or activity of a TREG cell. Non-limiting examples of protein phosphatase inhibitors include an SHP-1 inhibitor and / or an SHP-2 inhibitor. Non-limiting examples of kinase inhibitors include a CDK4 inhibitor, a CDK4 / 6 inhibitor (eg palbocyclib), a BTK inhibitor (eg
[00318] [00318] In other embodiments, cells expressing an unnaturally occurring immune receptor (eg, CAR and / or TCR) and / or an NF-κB stimulating molecule are administered in combination with an agent that improves one or more side effects associated with the administration of a cell that expresses a CAR / TCR and / or an NF-κB stimulating molecule. The side effects associated with the CAR / TCR expression cell and / or NF-κB stimulating molecule can be chosen from cytokine release syndrome (CRS), hemophagocytic lymphohistiocytosis (HLH) or neurological complications. Examples of such agents include steroids (eg, prednisone, dexamethasone), IL6R antagonists (eg, tocilizumab), IL1R antagonists (eg, anakinra), src kinase inhibitors (eg, dasatinib or a water-soluble salt dasatinib), a kinase inhibitor (eg Ibrutinib), calcineurin inhibitors (eg tacrolimus or cyclosporine A) or chemotherapy drugs (eg cyclophosphamide, methotrexate or vincristine).
[00319] [00319] In one embodiment, cells expressing an unnaturally occurring immune receptor (eg, CAR and / or TCR) and / or NF-κB stimulating molecule are administered in combination with a low dose and immune-enhancing dose of an mTOR inhibitor. Although it is not desired to be bound by theory, it is believed that treatment with a low dose and immune enhancement (for example, one dose does not completely suppress the immune system, but is sufficient to improve immune function) is accompanied by a reduction in PD-1 positive T cells or an increase in PD-1 negative cells. PD-1 positive T cells, but not PD-1 negative T cells, can be
[00320] [00320] The pharmaceutical compositions of the disclosure may comprise a cell that expresses an unnaturally occurring immune receptor (e.g., CAR and / or TCR) and / or NF-kB stimulating molecule, e.g., a plurality of cells that express CAR / TCR and / or NF-kB stimulating molecules, as described herein, in combination with one or more pharmaceutically or physiologically acceptable vehicles, diluents or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids like glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. The compositions of the disclosure can be formulated for intravenous administration. The composition may additionally comprise a secondary active agent (for example, an anticancer, antiviral or antibiotic agent).
[00321] [00321] The pharmaceutical compositions of the disclosure can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's illness. When "an immunologically effective amount", "an antitumor effective amount", "a tumor inhibiting effective amount" or "therapeutic amount" or "anti-infectious" is indicated, the amount of the disclosure compositions to be administered can be determined by a doctor, considering the individual differences in age, weight, tumor size, extent of infection or metastasis and condition of the patient (subject), as appropriate. It can generally be said that a pharmaceutical composition comprising immune effector cells (for example, T cells,
[00322] [00322] In certain respects, it may be desirable to administer activated immune effector cells (for example, T cells, NK cells) to a subject and then collect blood again (or perform an apheresis), activate immune effector cells (for example, T cells, NK cells), according to the disclosure, and reinfuse the patient with these activated and expanded immune effector cells (eg, T cells, NK cells). This process can be performed several times every few weeks. In certain respects, immune effector cells (for example, T cells, NK cells) can be activated by collecting blood from 10 cm³ to 400 cm³. In some respects, immune effector cells (eg, T cells, NK cells) are activated from blood collection of 20 cm³, 30 cm³, 40 cm³, 50 cm³, 60 cm³, 70 cm³, 80 cm³, 90 cm³ or 100 cm³.
[00323] [00323] In some embodiments, individuals may experience leukapheresis, in which leukocytes are collected, enriched or depleted ex vivo to select and / or isolate the cells of interest, for example, T cells. These T cell isolates can be expanded by methods known in the art and treated and / or transformed so that one or more constructs of the disclosure can be introduced, thus creating a CAR-T or TCR-T cell of the disclosure that coexpresses an accessory module that encodes an NF activator -κB. Needy individuals can subsequently undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain respects, after
[00324] [00324] Kits are also provided to practice dissemination. For example, kits for treating cancer in a subject or producing a cell that expresses an unnaturally occurring immune receptor (for example, CAR and / or TCR) and / or NF-κB stimulating molecule disclosed in this document. The kits can include at least one nucleic acid molecule or vector that encodes an unnaturally occurring immune receptor (for example, CAR and / or TCR) and / or NF-κB stimulating molecule along with a method for introducing the nucleic acid in immune effector cells. The kit may include a virus comprising a nucleic acid that encodes a non-naturally occurring immune receptor (for example, CAR and / or TCR) and / or stimulating molecule and NF-κB chemicals, such as polybrene, to improve virus transduction . The kit may contain components for the isolation of T cells for the expression of a non-naturally occurring immune receptor (for example, CAR and / or TCR). Alternatively, the kit may contain immune effector cells (for example, T cells or NK cells) or stem cells that express an unnaturally occurring immune receptor (for example, CAR and / or TCR) and / or NF stimulating molecule -κB. More than one of the immune receptors does not occur naturally (for example, CAR and / or TCR) and / or the disclosed NF-kB stimulating molecules can be included in the kit. The kit may include a container and a label or label on the container or associated therewith.
[00325] [00325] Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from a variety of materials, such as glass or plastic. The container typically
[00326] [00326] The disclosure is further described by reference to the following experimental examples. These examples are provided for illustrative purposes only and should not be limiting, unless otherwise specified. Thus, disclosure should in no way be interpreted as limited to the following examples, but should be interpreted in such a way as to cover any and all variations that become evident as
[00327] [00327] Cell lines genetically engineered to express luciferases (eg, GLuc or NLuc) to measure the cytotoxicity of different constructs targeting different cell surface and intracellular antigens are provided in Table A. The cell lines used in these experiments, the target antigens in cell lines and growth media are shown in Table A below. The cells were cultured at 37 ° C, in an incubator humidified with 5% CO2. Cell lines were obtained from the ATCC, NIH AIDS reagent program or were available in the laboratory. TABLE A: Cell line Culture Conditions Exemplary CAR Target Antigens Expressed BC-1 RPMI, 20% FCS BCMA, GPRC, CD138 BC-3 RPMI, 20% FCMA BCMA, GPRC, CD138 BCBL-1 RPMI, FCS at 20 % GPRC, CD138 JSC-1 RPMI, 20% FCS GPRC, CD138 MM1S RPMI, 10% FC38 CD38, GPRC, CD44, CD200R U266 RPMI, 10% BCMA, WT1 / HLA-A2 +, CS1, CLL1, CD138, c-MET, IL6R, CD179b, NY-ESO / HLA-A2, NYBR, LAMP1 L363 RPMI, 10% FCS BCMA, GPRC, WT1 / HLA-A2 +, CS1, CLL1, CD138, NY- ESO / HLA -A2, NYBR, LAMP1 K562 RPMI, 10% FCS CD33, IL1Ra, TnAg BV173 RPMI, 10% FCS CD123, CD179b, IL1Ra, WT1 / HLA-A2 +, CXCR4, FLT3, CD179a Nalm6 RPMI, FCS 10% CD19 , CD20, CD22, CD179b, CD179a HL60 RPMI, 10% FCS CD33, CD34, CLL1, IL6R, CD32, CD179 U937 RPMI, 10% FCS CD4, CLL1 RS: 411 RPMI, 20% FCS CD19, Beta Receiver Folate (FRbeta), TGFbeta, CD179b, NKG2DNKG2D, FLT3, CD179a MV: 411 RPMI, 10% FCS FLT3, CD123, FRbeta Raji RPMI, 10% CD19, CD20, CD22, BCMA, CD38, CD70, CD79, Receiver Folate Beta, CLL1 HEL-92.1.7 RP MI, FCS 10% MPL, CD33, CD32, CD200R (HEL) Jurkat RPMI, FCS 10% TnAg, TSLRP, TSHR, CD4, CD38 Daudi RPMI, FCS 10% BCMA, FRbeta REC-1 RPMI, FCS 10 % NKG2DNKG2D, ROR1 KG-1 RPMI, FCS 20% CD33, CD34, CD123, TSLRP CEM RPMI, FCS 10% CD5, CD43 U937 RPMI, FCS 10% CD4, CLL1 LAMA5 RPMI, FCS 10% WT1 / HLA -A2 A549 DMEM, 10% FCS ROR1, CD22, TIM1, CDH17 HT29 DMEM, 10% FCS EGFR, SLEA, c-MET Molm-13 RPMI, 20% FCS FLT3, IL6R, LAMP1, TSLRP, CD4, CSF2RA , CXCR4, IL6R, CSF2RA, GPC3 A431 DMEM, 10% FCS EGFR, Alpha Folate Receptor, Her3
[00328] [00328] The Jurkat cell line (clone E6-1) genetically engineered with an NFAT-dependent EGFP (or GFP) reporter gene was a gift from Dr. Arthur Weiss from the University of California at San Francisco and was described to study signaling from CAR ((Wu, CY et al., Science 350: 293 to 302,2015). Jurkat cells were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin. GENERATION OF LENTIVIRAL VECTORS THAT CODE CHEMICAL ANTIGEN RECEPTORS AGAINST MPL
[00329] [00329] The pLENTI-Blast vector was derived from the pLenti6v5gw_lacz vector (Invitrogen; ThermoFisher Scientific) by removing the LacZ gene. PLenti-MP2 was a gift from Pantelis Tsoulfas (plasmid Addgene # 36097) and was used to generate the lentiviral vector pLenti-EF1a or pLenti-EF1α [SEQ ID NO: 3,837] by replacing the CMV promoter with the human EF1α promoter using techniques standard molecular biology. PLenti-EF1a-DWPRE [SEQ ID NO: 3,838] was derived from the pLENTI-EF1α vector by deleting the WPRE sequence. An internal Sac II fragment was excluded from the EF1α promoter to generate the EF1alpha (EF1a) -D-SACII promoter (SEQ ID NO: 3,842). The psPAX2 vector was a gift from Didier Throne (Addgene plasmid # 12260). The pLP / VSVG envelope plasmid and 293FT cells were obtained from Invitrogen (ThermoFisher Scientific). The retroviral transfer vector MSCVneo, MSCVhygro and MSCVpac and the packaging vector pKAT were obtained in Dr. Robert Illaria's laboratory. The plasmid phRGTK Renilla Luciferase was from Promega.
[00330] [00330] The generation of chimeric antigen receptor containing vectors with main structures BBz, CD28z and z-K13, the generation and use of GGS-NLuc fusion proteins and the generation and use of luciferase reporter cell lines (e.g. GLuc) for cell cytotoxicity measurement using the Matador assays have been described (documents PCT / US2017 / 024843, PCT / US2017 / 025602 and PCT / US2017 / 052344). LENTIVIRUS AND RETROVIRUS VECTORS
[00331] [00331] Lentiviruses were generated by transfection based on calcium phosphate in 293FT cells, essentially as previously described (Matta H et al., Cancer biology and therapy. 2 (2): 206 to 210. 2003). 293FT cells were cultured in DMEM with 4 mM L-glutamine in 10% FCS, non-essential amino acids MEM at 0.1 mM and sodium pyruvate MEM at 1 mM (referred to herein as DMEM-10). For the generation of lentivirus, 293FT cells were plated in 10 ml of medium
[00332] [00332] Leukocyte cream cells were obtained from healthy, unidentified adult donors from the Blood Bank of Children's Hospital in Los Angeles and used to isolate peripheral blood mononuclear cells (PBMC) by Ficoll-Hypaque gradient centrifugation. PBMCs were used as such or used to isolate T cells using CD3 magnetic microspheres (Miltenyi Biotech) and following the manufacturer's instructions. The isolated PBMC or T cells were resuspended in XVIVO medium (Lonza) supplemented with 10 ng / ml of CD3 antibody, 10ng / ml of CD28 antibody and 100 IU of recombinant human IL2. The cells were cultured at 37 ° C, in an incubator humidified with 5% CO2. At
[00333] [00333] Essentially, a procedure similar to that described above for the production of lentivirus vectors was used for generation of retroviral vectors, with the exception that 293FT cells were generally transfected in 10 cm tissue culture plates in 10 ml of DMEM-10 medium using 10 μg of retroviral construct, 4 μg of pKAT and 2 μg of plasmid VSVG. Virus collection and infection of target cells was performed essentially as described above for lentiviral vectors.
[00334] [00334] Blinatumomab was obtained from Amgen. Digitonin was purchased from Sigma (Cat. D141) and a 100 mg / ml stock solution was produced in DMSO. A diluted stock of 1 mg / ml was made in PBS. The final concentration of digitonin used for cell lysis was 30 μg / ml, unless otherwise indicated. ELISA
[00335] [00335] Human IL2, IFNγ, IL6 and TNFα were measured in the supernatant of Jurkat-NFAT-GFP effector cell culture or T cells expressing CAR that were co-cultured with specific cell lines for 24 to 96 hours using ELISA kits commercially available from R&D systems (Minneapolis, MN) and BD Biosciences and following the manufacturer's recommendations. FACS ANALYSIS TO DETECT CAR EXPRESSION
[00336] [00336] Monoclonal Antibody conjugated to mouse anti-human APC c-Myc (catalog number IC3696A) was from R&D Systems (Minneapolis, MN). Biotinylated protein L was purchased from GeneScript (Piscataway, NJ), reconstituted in phosphate buffered saline (PBS) at 1 mg / ml and stored at 4 ° C. Streptavidin-APC (SA1005) was purchased from ThermoFisher Scientific.
[00337] [00337] For the detection of CARs using Myc staining, 1 x 106 cells were harvested and washed three times with 3 ml of 1 x cold PBS containing 4% bovine serum albumin (BSA) wash buffer. After washing, the cells were resuspended in 0.1 ml of the cold wash buffer containing 10 ml of Myc antibody conjugated to APC and incubated in the dark for 1 hour, followed by two washes with cold wash buffer.
[00338] [00338] For the detection of CARs using Protein L staining, 1 x 106 cells were harvested and washed three times with 3 ml of 1 x cold PBS
[00339] [00339] To measure cell extermination, an innovative assay based on the ectopic cytosolic expression of Gluc, NLuc and other luciferases was used as described in PCT / US2017 / 052344 "A Non-Radioactive Cytotoxicity Assay". The method involves the expression of a reporter in the target cells in a way that is preferentially retained within healthy cells, but is released from exterminated and exterminating cells or whose activity can be preferably measured in exterminated and exterminated cells. The preferred reporter for this essay are 1) non-secreted forms of copepod luciferases, such as Gaussia princeps, 2) luciferase reporters genetically engineered from deep-sea shrimp, such as NanoLuc. The sequence of several of these exemplary reporter vectors is provided in SEQ ID NO: 3,845 to SEQ ID NO:
[00340] [00340] A 100X stock solution of native coelenterazine (CTZ; Nanolight, catalog number 303) was produced by dissolving 1 mg of lyophilized CTZ powder in 1.1 ml of 100% methanol supplemented with 30 µl of 6N HCl to prevent oxidation of CTZ over time. To produce the CTZ assay buffer, the 100X CTZ stock solution was diluted to a concentration of 0.5X in PBS. Unless otherwise indicated, a total volume of 15 μl of the CTZ assay buffer (as prepared above) was added to each well of a 384-well white plate (Greiner, 384-well white plate, catalog number 781075) containing cells that express the non-secretory form of luciferase in approximately 50 to 60 μl volume of medium and the plates were read for luminescence using the BioTek synergyH4 plate reader. For 96-well plates, cells were plated in 200 μl of medium and approximately 50 μl of 0.5X CTZ assay buffer was added. Unless otherwise indicated, the 0.5X CTZ assay buffer was used to assess the activity of GLuc, TurboLuc16 and MLuc7. The CTZ assay buffer (diluted to a
[00341] [00341] The expression of antigens in target cells was determined by bioinformatics approaches in combination with immunostaining with specific antibodies for antigens or a highly sensitive antigen detection assay, as described in document PCT / US2017 / 025602 and incorporated in this document in its as a reference. This assay involves fusing a GLuc or NLuc reporter fragment to the antigen-binding domain of an antibody, a scFv, a vHH or any other antigen-binding fragment or any receptor and ligand. The resulting fusion protein is incubated with the target cells that express the test antigen and the binding of the fusion protein is determined by the addition of coelentrazine or another suitable substrate from the luciferase reporter. GENERATION OF A DIVERSIFIED SET OF T CAR CELLS
[00342] [00342] The above assays were used to screen the different antigen binding modules (eg scFv, vHH, receptors, ligands) used in the construction of the CARs of this invention, and the antigen binding modules that showed specific binding activity were
[00343] [00343] It is possible that different CARs or a subset of CARs are ideally suited for different disease conditions, depending on several factors, including, among others, the prevalence and level of expression of the target antigen in the disease-causing and associated cells , disease burden and rate of disease progression. Different CARs may be perfectly adequate, even for a single disease condition in different patients, depending on their efficacy and toxicity profile and the patient's condition. Disclosure provides a solution to significant technical and logistical obstacles to generating a diverse adoptive immune response.
[00344] [00344] The normal diversity of the TCR is produced by genetic rearrangement. The rigorous positive and negative selection processes in the thymus ensure that only T cells that express the αβ TCR restricted to autopeptide / MHC recognition within a low affinity range can fill the periphery. Thus, the thymic environment allows the generation of a set of αβ T cells that are self-restricted, but not autoreactive.
[00345] [00345] The generation of a diverse set of CAR-T cells from different domains of antigen binding is limited by the technical and financial obstacles to generating and testing various domains of antigen binding. More importantly, since each of the antigen-binding domains (for example, fragments of vL and vH from an antibody) has potential for binding to other antigens and causes off-target toxicity, a diverse set of CARs based only on a plurality of antigen-binding domains potentially has an increased risk of toxicity. Therefore, the potential diversity of such a set would have to be
[00346] [00346] This diverse set of CARs can be used to provide a diverse immune response against disease-causing cells or those associated with diseases that express said antigen. Alternatively, the diverse set of CARs can optionally be encoded in DNA code using techniques known in the art and subsequently used to select a single or a subgroup of CARs with ideal clinical and biological characteristics. These characteristics may include, but are not limited to, performance in in vitro biological assays (eg, cytotoxicity, cytokine secretion, binding affinity, cell surface expression, off-target effects, T cell proliferation, expression of exhaustion markers and terminal differentiation, etc.), performance in in vivo assays (eg survival, tumor shrinkage, T cell persistence, T cell expansion, etc.) and clinical experience (eg disease remission, recurrence rate, toxicities, etc.). The CARs of the disclosure can be used alone or in combination with other CARs and other natural and synthetic immune receptors known in the art to generate a diverse set of immune effector cells for the prevention and treatment of various disease conditions caused by or associated with cells expressing your target antigens.
[00347] [00347] Use of in vitro and live selection to select CARs with the desired properties. A set of CARs targeted to CD19 (SEQ
[00348] [00348] Mouse NEMO-K270A (SEQ ID NO: 992) is known to activate NF-κB constitutively. To demonstrate the ability of this mutant to provide costimulation of T cells, CD3 + ve T cells were cultured in XVIVO medium (Lonza) supplemented with 10 ng / ml soluble anti-CD3, 10 ng / ml soluble anti-CD28 and 100 IU of recombinant human IL2. The cells were cultured at 37 ° C, in an incubator atmosphere humidified with 5% CO2, and after 1 day infected with a lentiviral vector (pLENTI-EGFP-Blasticidin) expressing EGFP and lentiviral vectors that express NEMO-K270A mutants mouse (pLENTI-mNEMO-K270A-FLAG-Blasticidin and pLENTI-mNEMO-K270A- HA-Blasticidin) or mouse NEMO-wt (pLENTI-mNEMO-FLAG- Blasticidin). The sequences of mNEMO-K270A and mNEMO-wt are provided in SEQ ID NOs: 992 and 991, respectively. Approximately 1 day after infection, cells were selected with blasticidine and the numbers of
[00349] [00349] The human NEMO is longer than the mouse NEMO and the human mutant NEMO-K277A (hNEMO-K277A; SEQ ID NO: 979) corresponds to the mouse mutant NEMO-K270A (mNEMO-K270A). To test whether the hNEMO-K277A mutant also activates NF-κB, the expression vector (pCDNA3) encoding this mutant was generated. In addition, expression constructs that encode several other hNEMO mutants in which Lys (K) at amino acid residue 277 has been replaced by different amino acid residues (for example, K277Q, K277T, K277I, K277N, K277S, K277M, K277G, K277R ). The different constructs were transfected into 293FT cells together with an NF-κB-Luciferase reporter construct and an RSV-LacZ (normalization control) reporter construct and tested for their ability to activate NF-κB using the assay described above. Figure 3 shows strong activation of NF-κB by mNEMO-K270A, hNEMO-K277A and weak activation by hNEMO-K277I and hNEMO-K277G. In a similar experiment, the hNEMO-K277L and hNEMO-K277A-DeltaV249-K255 mutants also showed activation of NF-κB when transfected into 293FT cells. The hNEMO-K277A- DeltaV249-K255 mutant lacks the human NEMO V249-K255 amino acid residues and also carries the K277A mutation. These results suggest that active mutants constituting NEMO can be quickly generated and identified by mutating the mouse NEMO K270 residue and the human NEMO K277 residue. A similar approach can be used to generate mutants in other NEMO residues that have the capacity to
[00350] [00350] An FMC63-based CAR CAR CD19 construct was generated that coexpressed with the full length hNEMO-K277A or hNEMO-L753 mutant (encoding amino acids 1 to 251) in fusion with an N-terminal FKBPx2 dimerizer domain . The constructs were transfected into 293FT cells together with an NF-kB-Luciferase reporter construct and an RSV-LacZ reporter construct. Approximately 8 hours after transfection, the cells were either left untreated or treated with AP20187 (100 nM). After approximately 72 hours, cell lysates were prepared and analyzed for NF-κB luciferase and LacZ activities, as previously described. NF-κB-Luc activity was normalized to LacZ activity to control the difference in transfection efficiency. The results showed that treatment with AP20187 led to increased NF-κB activity in 293FT cells transfected with CAR coding constructs that coexpress FKBPx2-hNEMO-K277A mutants (SEQ ID NO: 1.006) and FKBPx2- hNEMO-L753 (SEQ ID NO: 1.007). These results demonstrate the ability to induce NF-κB inducibly in a chimeric CAR or TCR or TCR construct by coexpression of full-length NEMO or its exclusion mutants in fusion with a dimerizing domain followed by the addition of a dimerizer.
[00351] [00351] J-N-G cells are infected with 1st generation CARs based on FMC63 directed to CD19 that coexpress FKBPx2-hNEMO-K277A or FKBPx2-hNEMO-L753. The cells are co-cultured with RAJI target cells in the absence and presence of compound AP20187 and have been shown to induce EGFP expression, demonstrating that FKBPx2-hNEMO-K277A or FKBPx2-hNEMO-L753 can be coexpressed with a CAR without interfering with its activity.
[00352] [00352] In addition to NEMO, it is known that several other cellular proteins
[00353] [00353] The expression of these co-stimulating proteins can be controlled by expressing them using inducible promoters known in the art, such as Tet-inducible promoter or RheoGene system. In one embodiment, the hNEMO-K277A and hNEMO-K277A-DeltaV249-K255 mutants are cloned into the pSLIK-Tet-On vector (Gopalakrishnan et al, Clinical Cancer Res; 19 (18), 2013) and the resulting virus is used to infect T cells. Treatment of T cells with doxycycline has been shown to induce hNEMO-K277A and hNEMO-K277A-DeltaV249-K255 expression and NF-κB activity. NF-κB activity is measured by the Fosfo-IkBα antibody conjugated to AlexaFlour and flow cytometry.
[00354] [00354] In alternative embodiments of the invention, other proteins
[00355] [00355] NF-κB activators for adoptive T cell therapy.
[00356] [00356] Leukocyte cream cells are obtained from healthy de-identified adult donors from a Blood Bank and used to isolate peripheral blood mononuclear cells (PBMC) by centrifugation in Ficoll-Hypaque gradient. T cells are isolated using CD3 microspheres (Miltenyi), grown in XVIVO 15 supplemented medium with CD3 / CD28 Dynabeads and 50 IU / ml recombinant IL-2. Alternatively, T cells are resuspended in XVIVO medium (Lonza) supplemented with 10 ng / ml CD3 antibody, 10 ng / ml CD28 antibody and 100 IU of recombinant human IL-2.
[00357] [00357] The next day, T cells are infected with vectors
[00358] [00358] The next day, the spin infection is repeated and the cells are transferred to T-75 cell culture flasks with XVIVO 15 medium supplemented with CD3 / CD28 Dynabeads, 50 IU / ml IL2 and 5% FBS.
[00359] [00359] After 4 days of expansion, CAR-T cells are checked for CAR expression using L protein staining, CD19 binding, cytokine production (IL2, IFNγ, TNFa) and cytotoxicity (Matador assay).
[00360] [00360] After 10 days of expansion, CAR / SIR-T cells are used for in vivo experiment. To that end, NSG mice are injected with 106 Nalm-6-Luc cells by injection into the caudal vein. Two days later, injected with 3 x 106 CAR / SIR-T cells. The mice are subjected to bioluminescence imaging every week after administration of D-Luciferin and followed for survival.
[00361] [00361] It is observed that the T cells expressing the first generation CARs together with hNEMO-K277A (SEQ ID NO: 1.594 a
[00362] [00362] T cells expressing the CAR constructs corresponding to SEQ ID NO: 1,900 to 1,913, 2,206 to 2,219, 2,512 to 2,525,
[00363] [00363] There is also a difference between the different constructs that contain the same main structure, but with different domains of antigen binding. Thus, among the first-generation CAR constructs that coexpress hNEMO-K277A constructs (Main Structure 2) containing the scFV-derived antigen-binding domain 4G7 (for example, SEQ ID NO: 1,599), huBly3 (for example, SEQ ID NO:
[00364] [00364] In previous experiments, the hNEMO-K277A module is coexpressed with the CAR module in T cells using a single vector. The experiment is repeated in which the two modules are expressed using two separate lentiviral vectors. The SEQ ID of the nucleic acid construct encoding an exemplary CD20 CAR without an hNEMO-K277A module is shown in SEQ ID: 9,668. T cells are co-infected with the two lentiviral vectors at a multiplicity of infection of 5 and the ratio between the two vectors (ie CAR: hNEMO-K277A) ranges from 1: 1 to 1:10. T cells are
[00365] [00365] In an alternative embodiment, homologous recombination using gene editing techniques known in the art (eg, CRISP / Cas9 finger nucleases, TALON, Zn, etc.) is used to induce the K277A mutation in one or both copies of the endogenous human NEMO gene in T cells. The resulting T cells that carry the hNEMO-K277A mutation are then used for adoptive cell therapy, including to express the CD19-targeted CAR constructs and NY-ESO- 1. T cells carrying the hNEMO-K277A mutations demonstrate increased proliferation, cytokine production, expansion, long-term persistence in vivo and antitumor activity compared to control T cells without the hNEMO-K277A mutation.
[00366] [00366] The experiments described in the previous paragraphs are repeated using CAR constructs in which the accessory module hNEMO-K277A is replaced by accessory modules encoding FKBPx2- hNEMO, FKBPx2-hNEMO-K277A (SEQ ID NO: 1.006), FKBPx2-hNEMO - L753 (251) (SEQ ID NO: 1007), FKBPx2-hNEMO-L600 (200) (SEQ ID NO:
[00367] [00367] The experiments described in the previous paragraphs are repeated using constructs in which the accessory module hNEMO-K277A is replaced by accessory modules encoding hNEMO-K277A- DeltaV249-K255, IKK2-S177E-S181E, IKK1-S176E-S180E, MYD88- L265P, TCL-1A and MTCP-1. CAR-T cells expressing the accessory modules hNEMO-K277A-DeltaV249-K255, IKK2-S177E-S181E, IKK1- S176E-S180E, MYD88-L265P demonstrate increased cytokine production, proliferation, in vivo expansion and antitumor activity in
[00368] [00368] Use of human NEMO-K277A, human NEMO-K277A-deltaV249- K255, mouse NEMO-K270A and IKK2-S177E-S181E in vaccination
[00369] [00369] Lentiviral vectors are generated expressing human NEMO-K277A, human NEMO-K277A-deltaV249-K255, mouse NEMO-K270A and IKK2-S177E-S181E. Lentiviral vectors are also generating amino acid residues of chicken amine ovalbumin expressed 242 to 353 and the C-terminus of the class II invariant chain of the major histocompatibility complex (MHC) (II-OVA), as described in Rowe HM et al., Molecular Therapy, 13, 2, 2006. Finally, lentiviral vectors are generated that coexpress a cassette encoding human NEMO-K277A, human NEMO-K277A-deltaV249-K255, mouse NEMO-K270A or IKK2- S177E-S181E with a cassette that encodes II-OVA, in which the two cassettes are separated by a 2A cleavage sequence.
[00370] [00370] DC transduction and flow cytometry. Dendritic cells (DCs) derived from murine bone marrow are prepared as described previously. Immature DCs undergo transduction on day 4 in an MOI of 20 with lentiviral vectors, as described (Rowe HM et al., Molecular Therapy, 13, 2, 2006) and fed every 4 days with fresh medium containing stimulating factor of colonies of granulocytes and macrophages (50 ng / ml; from Peprotech). On day 5 post-transduction, DCs are collected, washed and blocked to the Fc receptors before staining the surface of the maturation markers with the following Abs conjugated to biotin: anti-CD11c, anti-CD86 and anti-I-Ab ( MHC class II) (all from BD Pharmingen); anti-CD40 (from Serotec); and anti-CD80, anti-ICAM-1 and anti-Kb (MHC class I) (all from eBioscience). A hamster isotype control
[00371] [00371] ELISA. Culture supernatants are collected from plate DCs at 5 x 105 cells per well (in 1.5 ml). IL-12p70 and tumor necrosis factor alpha (TNF-α) are detected by the enzyme-linked immunosorbent assay (ELISA), using kits from eBioscience, according to the manufacturer's guidelines.
[00372] [00372] Purification of DC from lymph nodes. C57 / BL6 mice (Harlan) are injected subcutaneously (sc) at the base of the tail with 1 × 108 infectious units (i.u.) of lentivector. Six days later, the lymph nodes (para-aortic and inguinal) are harvested (cells from mice in each group were pooled), incubated with collagenase CLS-4 (Worthington) and crushed to obtain single cell suspensions. Fc receptors are blocked before CD11c positive cells are selected using MACS microspheres (Miltenyi Biotec).
[00373] [00373] Pentamer coloring. One million splenocytes per sample are incubated with 10 μl of SIINFEKL / Kb pentamer or tetramer conjugated to phytoerythrin (Proimmune) for 12 minutes at room temperature. The cells are then washed and incubated on ice with anti-CD8 conjugated with biotin (Serotec) for 15 minutes before being washed and incubated with streptavidin-allophicocyanine (eBioscience) for 15 minutes. The samples are washed and purchased in a BD LSR machine using the Cell-Quest software (BD Biosciences).
[00374] [00374] Intracellular staining of cytokines. Splenocytes are incubated overnight with or without OVA257-264 peptide. Solution
[00375] [00375] ELISPOT assay. Enzyme-linked immunospot plates (ELISPOT) (Millipore) are coated overnight at 4 ° C with purified anti-IFN-γ (BD Pharmingen). Ex vivo ELISPOT assays are performed with serial dilutions of total splenocytes in triplicate with or without class I peptide OVA257-264 (Proimune). The plates are grown overnight and developed according to the manufacturer's instructions. Points are counted using a counter and AID ELISPOT software.
[00376] [00376] Tumor therapy. EG7.OVA tumor cells are cultured in RPMI plus 0.4 mg / ml G418 (Invitrogen). C57BL / 6 mice are challenged with 2 x 106 tumor cells injected s.c. in the flank and then vaccinated. Animals are killed when the tumor reaches a diameter> 15 mm.
[00377] [00377] Dendritic cells (DCs) derived from mouse BM are infected with lentivectors encoding human NEMO-K277A, human NEMO-K277A-deltaV249-K255, NEMO-K270A and IKK2-S177E-S181E in isolation combination with II-OVA. The expression of human NEMO-K277A, human NEMO-K277A-deltaV249-K255, NEMO-K270A and mouse IKK2-S177E-S181E is shown as a result of nuclear p65 translocation (RelA) in the DC cores at a level similar to that in the DCs DCs treated with LPS, but not in control vector DCs or untreated, in which the level of cytoplasmic p65 is higher. The increased nuclear NF-kB binding activity is also
[00378] [00378] After the transduction of BM-derived DCs with human NEMO-K277A, human NEMO-K277A-deltaV249-K255, NEMO-K270A and mouse IKK2-S177E-S181E, maturation markers are analyzed in the transduced or non-transduced cells. CD86, CD40, ICAM-1 and CD80 are increasingly regulated in DCs expressing human NEMO-K277A, human NEMO-K277A-deltaV249-K255, human NEMO-K270A and IKK2-S177E-S181E compared to transduced DCs in the vector group of control. In addition, DCs transduced with human NEMO-K277A-, NEMO-K277A-deltaV249-K255- human, NEMO-K270A- and mouse IKK2-S177E-S181E have been shown to retain their increasingly regulated CD86 for several weeks in culture. The secretion of IL-12p70 and TNF-α was found to be upregulated in the culture of DCs transduced with human NEMO-K277A-, human NEMO-K277A-deltaV249- K255-, NEMO-K270A- and IKK2-S177E-S181E from mouse.
[00379] [00379] After s.c. injection of lentivector, transduced DCs are detected in the drainage lymph nodes. A similar percentage of lymph node DCs (CD11c + / MHC + class II) is transduced after sc injection with the human NEMO-K277A-, NEMO-K277A- deltaV249-K255-, NEMO-K270A- and IKK2- control Mouse S177E-S181E. However, there is increasing regulation of CD86 in DCs in animals injected with human NEMO-K277A-, NEMO-K277A-deltaV249- K255- human, NEMO-K270A- and mouse IKK2-S177E-S181E compared to animals injected with control vector.
[00380] [00380] The capacity of vectors encoding human NEMO-K277A-2A- II-OVA, human NEMO-K277A-deltaV249-K255-2A-II-OVA,
[00381] [00381] Mice are inoculated with a lethal dose of EG7.OVA tumor cells before being vaccinated with transduced DCs or with human NEMO-K277A-, NEMO-K277A-deltaV249-K255- human, NEMO-K270A- e IKK2-S177E-S181E- from mouse directly. All mice are shown to develop tumors. After transduced DC injection or direct lentivector injection, the number of tumor-free mice in the human NEMO-K277A-, NEMO- K277A-deltaV249-K255- human, NEMO-K270A- and IKK2-S177E-S181E- group is greater than that of the control group. The effectiveness of NEMO-K277A-2A-II-OVA, human NEMO-K277A-deltaV249-K255-2A-II-OVA, mouse NEMO-K270A-2A-II-OVA mouse, IKK2-S177E-S181E- 2A- Ii-OVA is tested on a parasite protection model (Polley R et al., Infect. Immun. 74: 773 to 776, 2016) using ovalbumin that expresses L. donovani.
[00382] [00382] Use of human NEMO-K277A, NEMO-K277A-deltaV249- human K255, mouse NEMO-K270A and IKK2-S177E-S181E in vaccination
[00383] [00383] The cells that present antigen collected in a single leukapheresis are transduced with the adenoviral vector that encodes NEMO-
[00384] [00384] Murine monoclonal antibody 161 targets human MPL (thrombopoietin receptor or TPO-R). To generate a CAR directed to MPL, but with reduced immunogenicity, the sequence of the scFV fragment was humanized comprising the antigen binding domain of the murine antibody 161. The humanized scFv fragment 161 (SEQ ID NO: 891), designated Hu-161 -2, was cloned into the 2nd generation CAR main structural containing the 41BB co-stimulating domain and CD3z activation domain (SEQ ID NO: 1.582). Jurkat-NFAT-EGFP (J-N-G) cells were transduced in a stable manner with the lentivirus encoding the humanized CAR MPL-hu-161-2 construct. The parental and CAR expressing Jurkats were subsequently co-cultured with HEL cells and the induction of EGFP expression monitored by FACS analysis after 4 h. Co-culture of Jurkat cells expressing the CAR MPL-hu-161-2 construct with HEL cells led to an increase in EGFP expression compared to cells that had not been exposed to HEL cells, indicating the capacity of the CAR MPL-hu construct -161-2 humanized to recognize the target antigen and activate signaling. Essentially similar results are obtained when the experiment is repeated with first generation CARs
[00385] [00385] Murine monoclonal 175 and 111 also bind to human MPL. Therefore, the sequence of scFv fragments comprising the antigen-binding domain of these antibodies is humanized and used to produce the corresponding 2nd generation CAR constructs (CAR II) (SEQ ID NOS: 1,583 and 1,584), as well as main structures 1 and 2 of coexpressing vFLIP K13 (SEQ ID NO: 1,287, 1,288) and hNEMO-K277A (SEQ ID NOs: 1,896 and 1,897). The experiment is repeated as in the previous example. Co-culture of Jurkat cells expressing CAR MPL-hu-175-2 and hu-111-2 constructs with HEL cells led to an increase in EGFP expression compared to cells that had not been exposed to HEL cells, indicating the ability of humanized CAR MPL-hu-175-2 and hu-111-2 constructs to recognize the target antigen and activate signaling. CONSTRUCTION AND TESTING OF CARS DIRECTED TO CD70
[00386] [00386] Several constructs are constructed targeting CD70 (SEQ ID NO: 9,781 to 10,086; and 7,783 to 7,789). The constructs are expressed in J-N-G and T cells and tested for T cell activation and cytotoxicity against target cell lines expressing CD70 RAJI and THP-1 using in vitro and in vivo assays.
[00387] [00387] Construction and testing of CARs targeting CD70, PTK7, kappa light chain, Claudin18A2, Ras / HLA-A2 complex, NY-ESO / HLA-A2 complex, Streptag and a CD43 epitope expressed in leukemia cells.
[00388] [00388] CAR constructs are generated by targeting PTK7, kappa light chain, Claudin18A2, Ras / HLA-A2 complex, NY-ESO / HLA-
[00389] [00389] Various TFP-based CARs are constructed by targeting MPL based on hu-161-2 scFV as the antigen binding domain. The sequence of these TFP CAR constructs is shown in SEQ ID NO: 3,526 to
[00390] [00390] Next, TFP CARs are built, targeting several different antigens. To provide costimulation, the constructs also coexpress hNEMO-K277A. Constructs are expressed in J-N-G cells
[00391] [00391] Several Ab-TCR are constructed targeting MPL based on murine scFV 161 as the antigen binding domain. To improve the expression of Ab-TCR based on TCRα and TCRβ, specific mutations are introduced in their TCR receptor modules. The sequence of TCRγ / TCRd, wild-type TCRα / TCRβ (marked as wt-op2) and mutant TCRα / TCRβ (marked as SDVP-IAH) containing Ab-TCR constructs are shown in SEQ ID NO: 959 to 964. The Jurkat-NFAT-EGFP (JNG) cells are transduced with lentiviruses encoding MPL-directed Ab-TCRs (SEQ ID NO: 2.091, 2,397, 2,703) and selected with
[00392] [00392] Next, Ab-TCR are constructed targeting several different antigens. To provide costimulation, the constructs also coexpress hNEMO-K277A. The constructs are expressed in primary J-N-G and T cells and tested for their ability to recognize cells that express their target antigen using the assays described above. Ab-TCRs that express J-N-G cells have been shown to induce EGFP expression after co-culture with the target cell expressing its cognate antigen. T cells expressing these Ab-TCRs targeting different antigens have been shown to induce cytotoxicity of target cells expressing the corresponding antigen using the GLuc-based cytotoxicity assay described above. Table A shows the target cell lines that express the different target antigens that are used in the assay. Additional cell lines that express the different target antigens are known in the art or can be genetically modified to express a desired antigen by techniques known in the art. In the example above, the Ab-TCR constructs contain an accessory module that coexpresses the hNEMO-K277A mutant to provide costimulation. In alternative modalities, also
[00393] [00393] Several CARs targeting the HIV1 envelope glycoprotein are generated and are represented by SEQ ID NO: 8.704 to 9.349. The following assays are used to test their anti-HIV1 activity in vitro. The active constructs are used individually or in combination for the treatment of patients with HIV1 and AIDS.
[00394] [00394] T2 cells infected with HIV-1, which have low MHC class I due to an exclusion in the processing associated carrier (TAP) (Salter, et al. (1986) EMBO J 5: 943 to 949) and previously demonstrated being target cells suitable for a specific HIV-1 CAR (Severino et al. (2003) Virology 306: 371 to 375), served as target cells. These are infected with an excess of NL4-3 HIV-1-based reporter virus containing a murine CD24 gene (mCD24) at the vpr site (Ali, et al. (2003) J Virol Methods 110: 137 to 142) to produce > 90% of infected cells for 3 or 4 days after infection, as previously described (Bennett, et al. (2007) J Virol 81: 4,973 to 4,980; Yang, et al. (1996) J Virol 70: 5,799 to 5,806 and Yang et al (1997) J. Virol 71: 3,120 to 3,128). These are irradiated immediately before use with 10,000 rads in a cesium irradiator, as well as peripheral blood mononuclear cells from a healthy 3,000 rad donor (feeding PBMCs). CD1 + T lymphocytes transduced by HIV1-CAR are marked with CellTrace Violet and washed according to the manufacturer's instructions (ThermoFisher Scientific, Grand
[00395] [00395] The ability of CD1 + T lymphocytes transduced by HIV1- CAR and expanded and enriched clones to suppress HIV-1 replication is tested as previously described (Yang, et al. (1997) PNAS USA 94: 11,478 to 11,483 ; Yang, et al. (1997) J. Virol 71:
[00396] [00396] Effector cells that express HIV1-CAR are also
[00397] [00397] T2-gluc cells infected with HIV-1 strain NL4-3 as above are used as target cells for primary CD8 + T lymphocytes transduced by HIV-1-CAR in the Matador assay or using standard 51 Cr release assays as previously described (Bennett et al. (2007) J Virol 81: 4,973 to 4,980; Yang, et al. (1996) J Virol 70: 5,799 to
[00398] [00398] Bispecific Antibodies, such as Bispecific T Cell Engager (BiTE) and Dual Affinity Redirect (DART),
[00399] [00399] A targeting MPL based on bispecific T agent based on scFV 161 as the antigen binding domain is constructed. The sequence of this bispecific construct is shown in SEQ ID NO: 3,736. Bispecific constructs contain a GGGSG-Streptagx2-Tag linker (SEQ ID NO: 287), but alternative linkers (eg, SGGGS) can be used.
[00400] [00400] The bispecific construct was transfected into 293FT cells, and the supernatant containing the fusion protein collected after 48 to 96 hours. HEL-GLuc cells cultured with T cells in the presence of the bispecific fusion protein MPL-161 have been shown to undergo cell lysis, as determined by the GLuc assay, compared to cells cultured with the bispecific fusion protein alone or only T cells.
[00401] [00401] Bispecific antibodies encoding constructs based on scFv 175, 111, hu-161-2, hu-175-2 and hu-111 are then constructed and observed to be active in the HEL-GLuc cytotoxicity assay. Finally, bispecific antibodies targeting several other antigens, including PTK7, DLL3, TROP2, CD179a, CD179b, CD23, LAMP1, CDH1, CDH17, CD32, CDH19, HIV1-gp120 envelope glycoprotein, etc., are constructed in a similar manner and are seen to have activity when co-cultured with target cell lines that express their cognate antigen.
[00402] [00402] Figure 4. Activity of a Bispecific T-cell engager directed to MPL and using a 161-scFv targeting domain. T cells and HEL-pLenti-hGluc were previously incubated separately with the following supernatants at 4 ° C for 2 h in Medium and pLenti-161- StreptagII-CD3-Myc-His-P02 (042517-P02-SC) only. After incubation, cells were co-cultured in a 96-well U-bottom plate in the E: T ratio of 1: 1
[00403] [00403] TFP expression and activity in Jurkat cells without TCRα and TCRβ expression.
[00404] [00404] Jurkat-NFAT-GFP (J-N-G) cells (T-cell lymphoma) are infected with lentiviral vectors that express gRNAs targeting TCRα and TCRβ1 / β2 constant chains and coexpressing Cas9Streptococcus Pyogenes. Exemplary gRNA target sequences for TCRα chains are given in SEQ ID NO: 7,754 and 7,755. Exemplary gRNA target sequences for TCRβ1 / β2 chains are given in SEQ ID NO: 7,756 to
[00405] [00405] The next lentiviral vectors that express the codon-optimized TCRα constant chain (IgSP- [hTRAC-opt2]; SEQ ID NO: 1010) or the TCRβ constant chain (IgSP- [hTRBC-opt2]; SEQ ID NO: 1,011 ) under the EF1α promoter are used to infect different populations of JNG cells. Expression of the TCRα constant chain in JNG cells that express MPL-specific TFP, in which the TCRα chain was interrupted by gRNA-mediated genetic knockout, results in increased expression of TFP on the cell surface and induction of GFP expression and IL2 secretion in coculture with target cells HEL.92.1.7. Likewise, the expression of the TCRβ constant chain in JNG cells that express MPL-specific TFP, in which the TCRβ1 / β2 chain was interrupted by gRNA-mediated knockout, results in increased expression of TFP on the cell surface and induction of expression of GFP and IL2 secretion after coculture with HEL target cells.92.1.7
[00406] [00406] Ab-TCR and cTCR / SIRs expression and activity in Jurkat cells without TCRα and TCRβ expression.
[00407] [00407] The above experiment is repeated with the exception that expression cassettes encoding Ab-TCRs and SIRs targeting human CD19 are used in place of TFPs targeting CD19. Ab-TCRs targeting CD19 are represented by SEQ ID NO: 3,124. The cTCR / SIRs
[00408] [00408] Allogeneic and ready-to-use T cells that express CAR, TFP and Ab-TCR of disclosure
[00409] [00409] Allogeneic or ready-to-use CAR-T cells are generated by decreasing or eliminating the expression of the endogenous TCRα and / or TCRβ chain using TALON, CRISP / Cas9 or other nucleases.
[00410] [00410] MPL-specific TFP cassettes (SEQ ID Nos: 3,527,
[00411] [00411] The MPL-specific TFP constructs (SEQ ID Nos:
[00412] [00412] The cells expressing the constructs are exposed to the target cells HEL.92.1.7-GLuc and tested in functional assays as described above. T cells in which the TFP-TRAC constructs are directed to the TRAC site show better expression of TFP on the cell surface compared to the T cells in which the TFP constructs alone (that is, without coexpression of the exogenous TRAC chain) are directed to the TRAC site. In addition, T cells in which the TFP-TRAC constructs are directed to the TRAC site show increased proliferation, activation, cytokine production (eg, IL-2 and TNFa), cytotoxicity, in vivo expansion, anti-tumor activity in live against target cells compared to T cells in which the TFP constructs alone (i.e., without coexpression of the exogenous TRAC chain) are directed to the TRAC site.
[00413] [00413] TFPs expression and activity are also restored in T cells in which the endogenous TRAC site has been disrupted by projecting the
[00414] [00414] Alloreactivity of T cells expressing TFP-TRAC that have no expression of the native TCRα chain, but in which the expression and cell surface activity of TFP are rescued by the expression of the TCRα constant chain, are tested using the reaction of mixed lymphocyte culture using T cells irradiated from an allogeneic donor. T cells expressing TFP-TRAC that lack native TCRα expression show a marked reduction in the absence of alloreactivity, as measured by the proliferative response, compared to T cells in which TFPs are expressed using lentiviral vectors. The ability of human T cells that express TFP-TRAC and lack native TCRα expression to induce graft versus host disease (GVHD) is examined by administering 5 million TCRα-deficient T cells that express TFP-TRAC by animal in immunodeficient NSG mice (Jackson Lab). The animals are observed for more than 90 days. The human T cells in which the TFP-TRAC cassettes are directed to the TRAC site are markedly reduced to the absence of Graft-versus-host disease (GVHD) when infused in immunodeficient NSG mice (Jackson Lab) while GVHD is observed in animals that receive T cells in which PFT is expressed using lentiviral vectors. The ability of human T cells deficient in TCRα and expressing TFP-TRAC to induce graft versus host disease (GVHD)
[00415] [00415] Essentially similar results are obtained when the experiment is repeated with T cells in which the TFPs are directed to the TRBC site. The target sequence of the TRBC-directed gRNA is shown in SEQ ID NO: 7,756 to 7,758. These gRNAs are used in combination with Cas9 Streptococcuc Pyogenes using methods known in the art. Targeting the TFP expression cassettes to the TRBC genomic site has been shown to result in impaired activity of MPL-specific TFPs. However, TFP activity is restored by coexpression of the exogenous TCRβ constant chain (TRBC). The nucleotide sequence of exemplary exogenous TRBC chains that can be used to restore complex TCR / CD3 signaling function is shown in SEQ ID NO: 3,899 to 3,910. Exogenous TRBC can be expressed in T cells alone (SEQ ID NO: 1.011) or can be coexpressed with the TFP expression cassettes from a single vector. The nucleotide sequence of exemplary constructs that coexpress a TRBC chain with TFP constructs targeting MPL is shown in SEQ ID NO: 3,537, 3,539 and 3,541. These TFP expression constructs can be cloned into vectors targeting suitable TRBC, using techniques known in the art. In an alternative embodiment, TRBC expression can be restored in T cells in which the endogenous TRBC site has been disrupted by designing the targeting cassette so that the TFP cassette is followed in the frame by a cleavable ligand 2A, a signal peptide ( for example, a CD8 signal peptide or IgH signal peptide) and the first
[00416] [00416] Two Ab-TCR constructs targeting CD19 based on scFv FMC63 are generated in the lentiviral vector (SEQ ID NO: 3,837) conducted by the EF1α promoter. The nucleotide sequences of these constructs, CD8SP-FMC63-vL- [IgCL-TCRb-IAH-6MD] -F-P2A-SP-FMC63- vH- [IgG1-CH1-TCRa-SDVP-6MD] and CD8SP-FMC63-vL- [IgCL-TCRg-6MD] -F-P2A-SP-FMC63-vH- [IgG1-CH1-TCRd-6MD], are represented by the nucleotide sequences encoding the Ab-TCR component of SEQ ID NO: 3,124 and 3,324. Primary human T cells are infected with the corresponding lentiviral supernatants and analyzed for cell surface expression of Ab-TCRs using the FLAG-CD19-ECD-GGSG-NLuc-AcV5 supernatant (SEQ ID NO: 1.014) and for cytotoxicity against RAJI-GLuc cells. T cells that express Ab-TCRs show modest expression and activity. The expression of Ab-TCRs is directed to the TRAC site essentially as described by Eyquem J et al (Nature, 543 (7643): 113 to 117, 2017) using gene targeting constructs (see Figure 6) and represented by SEQ ID NO: 3,861 to 3,864. The targeting construct contains a splicing acceptor (SA), followed by an F2A coding sequence, the Ab-TCR cassette, flanked by sequences homologous to the TRAC site (LHA and RHA, left and right homology arm). In cassettes A and B (SEQ ID NO: 3,861 to 3,862), the nucleotide sequence encoding the Ab-TCR expression cassettes is followed by a stop codon, polyA, Exon 1 sequences from TRAC and the sequence homologous to the site of TRAC (RHA: right homology arm). In cassette C, the nucleotide sequence encoding the Ab-TCR expression cassette is followed by a stop codon, TRAC Exon 1, and the sequence homologous to the TRAC site (RHA: right homology arm),
[00417] [00417] Three cTCRs (or SIR) constructs directed to CD19 based on scFv FMC63 are generated in the lentiviral vector (SEQ ID NO: 3,837) conducted by the EF1α promoter. The nucleotide sequences of these constructs are represented by SEQ ID NO: 3,878, 3,879 and 3,880, respectively. They all have the same vL and vH regions. While SEQ ID NO: 3,880 has the wild-type nucleotide sequence of the constant chains TCRα and TCRβ, SEQ ID NO: 3,878 and 3,879 have optimized codon sequences. SEQ ID NO: 3,878 also carries several amino acid substitutions to improve expression and base pairing of the constant chains TCRα and TCRβ. Primary human T cells are infected with the corresponding lentiviral supernatants and analyzed for SIR cell surface expression using the FLAG-CD19-ECD-GGSG-NLuc-AcV5 supernatant (SEQ ID NO: 1.014) and for cytotoxicity against RAJI cells -GLuc. The cTCR / SIR construct with SEQ ID NO: 3,880 is not seen to express well or induce lysis of the target cell. CTCR / SIR are also directed to the TRAC site
[00418] [00418] A TCR construct and a cTCR construct (or SIR) targeting the NY-ESO-1 / HLA-A2 complex are generated in the lentiviral vector (SEQ ID NO: 3,837) and are based on the NYESO-1G4 TCR and mimetic TCR NYESO-35-15 antibody. The nucleotide sequences of these constructs are represented by SEQ ID NO: 3,883 and 3,882, respectively. The two constructs are also directed to the TRAC site using the targeting constructs represented by SEQ ID NO:
[00419] [00419] The single-stranded cTCR / SIRs in which the FMC63-scFv is linked to the codon-optimized TCRa constant chain or the murine-optimized murineized TCRa constant chain (SEQ ID NO: 3881) are expressed in T cells using the vector lentiviral and show poor expression and activity. The same constructs are directed to the TRAC site using the targeting constructs shown in Figure 9 and represented by SEQ ID NO: 3,869 to 3,872. The T cells in which
[00420] [00420] In the examples above, CAR / TFP / Ab-TCR / TCR / cTCRs are directed to the TRAC site. Essentially, a similar procedure can be used to target the CAR / TFP / Ab-TCR / TCR / cTCR or an accessory module to the TCBC, CD3ε, CD3δ, CD3γ and CD3ζ sites using techniques known in the art.
[00421] [00421] A shorter EF1α promoter maintains strong promoter activity in T cells and is suitable for adoptive cell therapy
[00422] [00422] The use of strong viral promoters in adoptive cell therapy applications carries the risk of activation of oncogenes downstream and development of cancer. As such, the human elongation factor 1α (EF1α) promoter is often used in adoptive cell therapy applications, as it provides strong expression and is of human origin. A limitation of the EF1α promoter, however, is its relatively large size. Although a mini-EF1α promoter has been described, it is much weaker compared to the EF1α promoter. To determine whether an internal deletion in the EF1α promoter would allow to shorten its length while preserving its promoter strength, a SacII fragment was excluded from the EF1α promoter. The nucleotide sequence of the resulting EF1α-D-SacII promoter is shown in SEQ ID NO: 3,842. Lentiviral vectors encoding a CD19-directed FMC63-BBz CAR were constructed in the vectors with the wild-type EF1α promoter (SEQ ID NO: 3,840) or EF1α-D-SacII promoter
[00423] [00423] Dasatinib is a drug poorly soluble in water and commercial Dasatinib is a monohydrate and reported to have a solubility of 8 μg / ml at 24 ° C. As patients with CRS and neurological complications have difficulty taking the oral form of Dasatinib, the water-soluble form of Dasatinib is desirable. Dasatinib water-soluble salts have been described in WO2015107545 A1. Injectable compositions comprising soluble salts of Dasatinib methanesulfonate monohydrate can be prepared according to the method of WO2015107545 A1 and used to treat patients with CRS and neurological complications associated with the administration of CAR-T and Blinatumomab cells. The dose of Dasatinib
[00424] [00424] Patients with many different diseases, including infectious diseases (for example, HIV1, EBB, CMV, HTLV1, etc.), degenerative diseases (for example, Alzheimer's disease), allergic diseases (for example, chronic idiopathic urticaria) and several cancers will be included in the IRB-approved phase I clinical trial of immunotherapy with autologously transferred autologous CAR-T cells that coexpress NEMO-K277A (main structure 2) targeting different disease-causing or disease-associated antigens. The CAR for different diseases will be selected based on the known expression of its target antigen in the disease-causing or associated cells. When possible, the expression of the CAR target in the cells causing or associated with the disease will be confirmed by the link with the
[00425] [00425] Patients with many different diseases, including infectious diseases (for example, HIV1, EBB, CMV, HTLV1, etc.), degenerative diseases (for example, Alzheimer's disease), allergic diseases (for example, chronic idiopathic urticaria) and several cancers will be included in a IRB-approved phase I clinical trial of immunotherapy with allogeneic CART-transferred cells targeted at different disease-causing or disease-associated antigens. The CAR for different diseases will be selected based on the known expression of its target antigen in the disease-causing or associated cells. Whenever possible, the expression of the CAR target in the cells causing or associated with the disease will be
[00426] [00426] In addition to intravenous infusion, T cells expressing the CARs and conventional main structures 1 to 72 described in this invention can be infused intra-arterially to provide high concentration of CAR-T cells in a local area or organ involved with a disease. In the following example, this approach is used in the case of a patient with liver metastases from gastrointestinal cancer that expresses the Receptor
[00427] [00427] A mapping angiogram will be performed by approaching the right common femoral artery at baseline. The right gastroduodenal and gastric arteries, in addition to other potential sources of extrahepatic perfusion, will be embolized with microbobins. The same arterial access procedure will be performed to administer T cells that express the construct CD8SP-FR1-huMov19- (vL-vH) -Myc-z-P2A- hNEMO-K277A-T2A-PAC (SEQ ID NO: 1.727). T cells will be collected from the patient on day 0 and will be infected with lentiviruses that encode the construct CD8SP-FR1-huMov19- (vL-vH) -Myc-z-P2A-hNEMO-K277A-T2A-PAC and expanded as described in the examples previous ones. CAR-T cells will be administered in order to increase the dose on day 14 (107 CAR-T cells), day 28 (108 CAR-T cells) and day 44 (109 CAR-T cells). CAR-T cells will be injected manually through a 60 cm3 syringe at a rate of <2 cm3 / second. The total volume of infusion will be approximately 100 cm3. Calibrated contrast rate angiography will be performed after the first 50 cm3 infusion and at the end of the CAR-T infusion to confirm the preserved arterial flow. The infusions will be delivered to the appropriate hepatic artery when possible. Certain patients may have aberrant hepatic arterial anatomy, in which the right or left hepatic artery does not arise from the appropriate hepatic artery. In such cases, the dose of CAR-T cells will be divided based on the lobar volume calculations. In such cases, divided doses will be delivered separately to the right and left hepatic arteries to ensure proportional delivery of CAR-T to both hepatic lobes. INTRAPERITONEAL ADMINISTRATION OF CAR-T CELLS
[00428] [00428] CAR-T cells can also be administered intraperitoneally, essentially as described in Koneru M et al (Journal of Translational Medicine; 2015; 13: 102). In the following example, this approach is used in patients with peritoneal involvement with ovarian cancer that expresses Folate Alpha Receptor (FR1). Essentially, a similar approach can be used for intraperitoneal infusion of CAR-T cells targeting other tumor antigens described in this disclosure.
[00429] [00429] An informed screening consent will be offered to patients with recurrent high-grade serous ovarian cancer to test their cancer for the expression of FR1. After FR1 expression is confirmed by immunohistochemistry, patients will have a leukapheresis product obtained from peripheral blood. Excess contamination by platelets and red blood cells will be removed from the leukapheresis product, and the product will be frozen. In the treatment phase of the study, the leukapheresis product will be thawed and washed. Subsequently, CD3 + T cells will be isolated from the thawed leukapheresis product by magnetic separation using CD3 / CD28 microspheres. The activated T cells will be transduced slowly with the construct CD8SP-FR1-huMov19- (vL-vH) -Myc-z-P2A- hNEMO-K277A-T2A-PAC and further expanded using the CD3 / CD28 microsphere expansion protocol.
[00430] [00430] Patients with fallopian tube, peritoneal or ovarian carcinoma of high recurrent serous grade demonstrating to express FR1 antigen confirmed by immunohistochemical analysis (IHC) of deposited (paraffin) tumor or recently biopsied will be potentially eligible for the study.
[00431] [00431] Phase I dose escalation dosage will be used in the study. Cohorts of 3 to 6 patients will receive increasing doses of modified T cells to establish the maximum tolerated dose (BAT). There will be four levels of planned dose: 3 x 105, 1 x 106, 3 x 106 and 1 x 107 CAR-T cells / kg.
[00432] [00432] An IP catheter will be placed before the infusion of T cells. Patients will be admitted to the hospital inpatient unit before the first infusion of T CAR cells and will remain hospitalized until at least 3 days after the second infusion of T T cells. The first cohort of patients to be treated and the first patient treated in each subsequent cohort will be admitted to the intensive care unit (ICU); Subsequent patients may be admitted to the medical oncology inpatient service (subject to the clinical judgment of the attending physician).
[00433] [00433] Patients will receive a single dose of chemotherapy with lymphodepletion cyclophosphamide (750 mg / m2 IV) 2 to 4 days before starting treatment with T cells modified with CAR. The transduced T cells will be tested for quality for number, purity, viability and sterility before infusion. All patients will receive 50% of the dose of genetically modified T cells intravenously. Patients will be monitored closely for toxicities. One to three days later, the remaining dose of T cells will be administered as an IP infusion. At least 3 patients will be treated at dose level 1, with an accumulation of no more than 2 patients per month within each dose level. All patients treated in the previous cohort will be observed for a minimum of 4 weeks from the day of the initial T cell infusion before escalation to the next cohort occurs. Blood samples will be obtained from all patients before and after treatment to assess the toxicity, therapeutic effects and survival of genetically modified T cells.
[00434] [00434] CAR-T cells can also be administered intra-tumorally, essentially as described in Brown CE, et al., Clin Cancer Res. 2015 September 15; 21 (18): 4,062 to 4,072. In the following example, this approach will be used for patients with recurrent glioblastoma (GBM) that expresses IL13Ra2. Essentially, a similar approach can be used for intra-tumor injection of T cells that express conventional CARs or conventional CARs that express accessory modules (main structures 1 to 72) targeting other tumor antigens.
[00435] [00435] A pilot safety and feasibility study will be conducted to test CD8SP-IL13Ra2-Hu108- (vL-vH) -Myc-z-P2A- hNEMO-K277A-T2A-PAC T cells (SEQ ID NO: 1.769) that express T cells in recurrent GBM. All participating patients must give written informed consent. The clinical protocol will be approved by the Institutional Review Board of the University of Southern California and conducted under a New Drug Investigation Request, and registered with ClinicalTrials.gov. Eligible patients will include adults (18 to 70 years old) with recurrent or refractory grade III or IV glioma whose tumors do not communicate with the ventricular / CSF pathways and are amenable to resection. Participation in this study will be independent of the status of the tumor antigen IL13Rα2 (or IL13Ra2). Patients will be enrolled after the initial diagnosis of high-grade glioma (grade III or IV according to WHO), when they will undergo leukapheresis to collect peripheral blood mononuclear cells (PBMC). These cells will be used to design T cells to express the construct CD8SP-IL13Ra2- Hu108- (vL-vH) -Myc-z-P2A-hNEMO-K277A-T2A-PAC containing the puromycin resistance gene (PAC) after infection by corresponding lentiviral vector, as described in the previous examples. Alternatively,
[00436] [00436] T CAR cells can be used to purge the bone marrow or the preparation of stem cells from peripheral blood from cancer cells prior to stem cell transplantation. In the following example, T cells expressing CD8SP-CS1-HuLuc64- (vL-vH) -Myc-z-P2A- hNEMO-K277A-T2A-PAC (SEQ ID NO: 1,699) will be used to purge stem cells from bone marrow or peripheral blood obtained from a patient with multiple myeloma prior to autologous stem cell (or bone marrow) transplantation.
[00437] [00437] The patient will undergo leukopheresis to collect peripheral blood mononuclear cells (PBMC). The T cells will be purified using CD3 microspheres. These cells will be used to genetically manipulate T cells to express CAR CD8SP-CS1-HuLuc64- (vL- vH) -Myc-z-P2A-hNEMO-K277A-T2A-PAC after infection with the corresponding lentiviral vector, as described in the examples previous ones. Thereafter, the therapeutic CAR-T cells tested for release will be cryopreserved and stored for later use or used fresh. Bone marrow cells and peripheral blood progenitor cell products will be collected from a patient with multiple myeloma following standard procedures. For peripheral blood stem cell mobilization, patients will receive cyclophosphamide, 3 gm / m2 followed by G-CSF, 10 μg / kg subcutaneously each day, starting 24 h after cyclophosphamide until the feresis is complete. The peripheral blood stem cells will be collected when the CD34 + cell count in the peripheral blood is 15 cells / µl. The purpose of the collection will be to process three volumes of blood per day until at least 2.0 times 106 cells
[00438] [00438] Proteins encoded by Bispecific T cell engagers are expressed in Hela cells using the constructs that have the SEQ ID Nos listed in Table 13. The proteins are purified using the metal affinity tag or StrepTag II columns using standard techniques of protein purification. The purified proteins are tested in phase I clinical trials. Patients are selected based on the expression of the target antigens of the bispecific antibodies using different assays known in the art. Bispecific antibodies are administered by a 24-hour infusion. The guidelines provided in the NCI Common Toxicity Criteria version 2.0 (http [s: //] ctep.ifo.nih.gov/l) are followed for monitoring notification of toxicity and adverse events.
[00439] [00439] Patients with mesothelioma and glioblastomas receive lentivirus-infected T cells that encode the following combination of CARs targeting mesothelin (expressed in mesothelioma), IL13Ra2 (expressed in glioblastomas) and hematopoietic markers (CD19, CD20, CD22, BCMA). T cells are either one of the wild type TCR chains or have the TCRα chain knocked out by the CRISP / Cas9 approach. It is observed that coexpression in the same T cells with the wild-type TCR chains of a CAR targeting mesothelin with a CAR targeting CD19, CD20, CD22 or BCMA results in increased T cell expansion in vivo compared to expression of isolated mesothelin. Essentially similar results are obtained with the CAR targeting glioblastoma. However, in defective T cells in the TCR chains, the coexpression of TFP-based CARs targeting CD20 (SEQ ID NO: 9,660) does not induce expansion in vivo while the coexpression of SIR (SEQ ID NO: 9,668) or Ab-TCR (SEQ ID NO: 9.676) based on CARs successfully induces T cell expansion. TABLE 15. EFFECT OF CAR COMBINATION ON THE EXPANSION OF
[00440] [00440] The various methods and techniques described above provide several ways to carry out the application. Obviously, it must be understood that not all the objectives or advantages described can be achieved according to any specific modality described in this document. Thus, for example, those skilled in the art will recognize that the methods can be executed in a way that achieves or optimizes an advantage or group of advantages as taught in this document, without necessarily achieving other objectives or advantages as taught or suggested in this document. A variety of alternatives are mentioned in this document. It should be understood that some preferential modalities specifically include one, another or several resources, while others specifically exclude one, another or several resources, while others still mitigate a specific resource by including one, another or several advantageous resources.
[00441] [00441] Furthermore, the person skilled in the art will recognize the applicability of various characteristics of different modalities. Likewise, the various elements, characteristics and steps discussed above, as well as other known equivalents for each of these elements, characteristics or steps, can be used in various combinations by an individual skilled in the art to perform methods according to the principles described in this document. Among the various elements, resources and steps, some will be specifically included and others specifically excluded in different ways.
[00442] [00442] A number of modalities have been established above to illustrate the disclosure. The following claims further establish what the Claimants consider to be their invention.

权利要求:
Claims (69)
[1]
1. Immune cell or immune cell population of the same, characterized by the fact that it expresses (i) at least one non-naturally occurring immune receptor and (ii) at least one non-naturally occurring agent that selectively activates the signaling pathway of NF-κB.
[2]
2. Immune cell or immune cell population thereof according to claim 1, characterized by the fact that the at least one non-naturally occurring immune receptor comprises at least one antigen-binding domain and at least one transmembrane domain.
[3]
3. Immune cell or immune cell population of the same according to claim 1, characterized by the fact that at least one non-naturally occurring immune receptor has the capacity to recruit at least one signaling module associated with the TCR.
[4]
4. Immune cell or immune cell population thereof according to claim 1, characterized by the fact that at least one non-naturally occurring immune receptor is a chimeric antigen (CAR) receptor or a recombinant TCR.
[5]
5. Immune cell or immune cell population according to claim 2, characterized by the fact that at least one antigen-binding domain of at least one non-naturally occurring immune receptor binds to an antigen selected from a group consisting of CD5; CD19; CD123; CD22; CD30; CD171; CS1 (also called CD2 subset 1, CRACC, MPL, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2- 3) bDGalp (1-4) bDGlcp (1-1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receiver
2/30 tyrosine kinase 1 orphan (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); Mesothelin; Alpha interleukin 11 receptor (IL-11Ra); prostate stem cell antigen (PSCA); Serine protease 21 (testin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis antigen (Y); CD24; Platelet-derived growth factor beta receptor (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Alpha folate receptor (FRa or FR1); Beta folate receptor (FRb); ERBB2 tyrosine-protein kinase receptor (Her2 / neu); Mucine 1 associated with the cell surface (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostasis; prostatic acid phosphatase (PAP); mutation elongation factor 2 (ELF2M); Ephrin B2; alpha fibroblast activation protein (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAlX); Proteasome subunit (Prosome, Macropain), Type Beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster (BCR) region and Abelson murine leukemia viral oncogene 1 homologue (Abl) (bcr-abl); tyrosinase; type A 2 ephrin receptor (EphA2); Lewis sialyl adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac (2-3) bDClalp (1-4) bDGlcp (1-1) Cer); transglutaminase 5 (TGS5); antigen associated with high molecular weight melanoma (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); related to tumor endothelial marker 7 (TEM7R); claudina 6
3/30
(CLDN6); thyroid stimulating hormone receptor (TSHR); group 5, class C receptor coupled to protein G, member D (GPRC5D); X 61 chromosome open reading frame (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; 1 placenta specific (PLAC1); hexassaccharide portion of glucoceramide globoH (GloboH); mammary gland differentiating antigen (NY-BR-1); uroplaquin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); panexin 3 (PANX3); Receptor 20 coupled to G protein (GPR20); lymphocyte 6 antigen complex, K9 locus (LY6K); Olfactory receptor 51E2 (OR51E2); Gamma TCR alternating frame protein (TARP); Wilms' tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Antigen 1 associated with melanoma (MAGE-A1); Gene 6 of the ETS translocation variant, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); Antigen Family X, Member lA (XAGEl); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma testosterone cancer antigen 1 (MAD-CT-1); melanoma testis cancer antigen 2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen 1 (PCT A-1 or Galectin 8), melanoma antigen recognized by T 1 cells (MelanA or MARTI); Mutant of rat sarcoma (Ras); human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, ETS serine 2 fusion gene (TMPRSS2)); N-acetyl-glucosaminyl-transferase V (NA17); Pax-3 (PAX3) paired box protein; Androgen receptor; Cyclin B1; homologous derived from viral oncogene neuroblastoma of avian myelocytomatosis v-myc (MYCN); Ras Homologous Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1);
4/30
Type (BORIS or Brother of the Regulator of Imprinted Sites) of Binding Factor to CCCTC (Zinc Finger Protein), squamous cell carcinoma antigen recognized by T 3 cells (SART3); Pax-5 paired box protein (PAX5); proachrosine binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); An anchor protein kinase 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receiver for advanced glycation end products (RAGE-1); 1 ubiquitous renal (RU1); ubiquitous renal 2 (RU2); legumaine; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; mutated 70-2 heat shock protein (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin type 1 receptor (LAIR1); IgA receptor Fc fragment (FCAR or CD89); Leukocyte immunoglobulin receptor (LILRA2) member 2 of subfamily A; F member of the CD300 molecule family (CD300LF); A member of family 12 of type C lectin domain (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF type module that contains mucin type 2 hormone receptor (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Type 5 Fc receiver (FCRL5); and immunoglobulin lamina-type polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalfa4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Lewis Sialila Antigen ); Fucosil-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1, TCRgama-delta, NKG2D, CD32 (FCGR2A), Tn ag, Tim1- / HVCR1 CSFRRA (GMV-CSFRRA) alpha), TGFbetaR2, Lews Ag, TCR-beta1 chain, TCR-beta2 chain, TCR gamma chain, TCR-delta chain, FITC, leutenizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), hormone receptor stimulating follicle (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein,
5/30 HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A (HA) hemagglutinin, GAD, PDL1, guanylyl cyclase C (GCC), autoantibody for desmooglein 3 (Dsg3), autoantibody for desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR , HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, Claudina18.2 (CLD18A2 or CLDN18A.2), Glycoprotein p, STEAP1, Liv1, Nectin-4, Crypto, gpA33, BST1 / CD157, low conductance chloride channel and an antigen recognized by the TNT antibody.
[6]
6. Immune cell or immune cell population according to claim 1, characterized by the fact that at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway is selected from the group that consists of vFLIP K13, K13-opt, a NEMO mutant, a NEMO fusion protein, IKK1-S176E-S180E, IKK2-S177E-S181E, RIP, IKKα, IKKβ, Tcl-1, MyD88-L265, any protein or fragment of NF-κB activation protein, any inhibitor of an inhibitor of the NF-κB pathway, any gene editing system capable of selectively activating NF-κB, any homolog or variant thereof and any combination thereof.
[7]
7. Immune cell or immune cell population according to claim 1, characterized by the fact that at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway is of non-viral origin.
[8]
8. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that the at least one non-naturally occurring agent capable of activating the NF-κB pathway is a gene editing system.
[9]
9. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that at least one
6/30 non-naturally occurring agent with the ability to selectively activate the NF-κB pathway induces the oligomerization of NEMO / IKKγ.
[10]
10. Immune cell or immune cell population according to claim 1, characterized by the fact that at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway induces the activation of the IKK complex.
[11]
11. Immune cell or immune cell population according to claim 1, characterized by the fact that at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway does not activate the AKT pathway.
[12]
12. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that at least one non-naturally occurring agent capable of selectively activating the NF-κB pathway is expressed in a constitutive or inducible manner .
[13]
13. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that at least one non-naturally occurring agent capable of selectively activating the NF-κB pathway is transiently expressed.
[14]
14. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway is expressed in a stable manner.
[15]
15. Immune cell or immune cell population according to claim 1, characterized by the fact that the activity of at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway is controlled post-translation through contact of the cell with a compound.
[16]
16. Immune cell or immune cell population according to claim 1, characterized by the fact that at least one
7/30 non-naturally occurring agent with the ability to selectively activate the NF-κB pathway is expressed as a fusion construct with one or more copies of an exchange domain.
[17]
17. Immune cell or immune cell population according to claim 9, characterized by the fact that the activity of at least one non-naturally occurring agent with the ability to selectively activate the NF-κB pathway is controlled at the post level -translation by administering a therapeutically effective amount of a compound that induces the dimerization of the exchange domain.
[18]
18. Immune cell or immune cell population thereof according to claim 16 or 17, characterized by the fact that the exchange domain comprises one or more copies of an FKBP12 domain.
[19]
19. Immune cell or population of immune cells thereof according to claim 15, characterized by the fact that the compound is AP20187 or Rimiducid or a homologue thereof.
[20]
20. Immune cell or immune cell population thereof according to claim 1, characterized by the fact that the immune cell is a T lymphocyte (T cell), a CAR-T cell, a T cell that expresses TCR, a lymphocyte infiltrated in the tumor (TIL), a tissue resident lymphocyte, a stem cell, an induced pluripotent stem cell or a Natural Exterminating Cell (NK).
[21]
21. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that the immune cell has been manipulated to not have a functional native T cell receptor (TCR) signaling complex and / or microglobulin β2.
[22]
22. Immune cell or immune cell population of the same according to claims 1 and 21, characterized by the fact that at least one non-naturally occurring immune receptor and / or at least one agent with the ability to selectively activate the pathway NF- signaling
8/30 κB are cloned into an endogenous TCR gene, so that the expression of at least one non-naturally occurring immune receptor and / or at least one agent capable of selectively activating the NF- κB signaling pathway is under control of endogenous regulatory / promoter elements for the TCR gene.
[23]
23. Immune cell or immune cell population thereof according to claim 1, which is used for the prevention and treatment of a disease selected from the group of cancer, infectious diseases, immune diseases, and allergic diseases.
[24]
24. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that at least one polynucleotide encodes at least one unnaturally occurring immune receptor and at least one unnaturally occurring agent with ability to selectively activate the NF-κB pathway are expressed from a single promoter.
[25]
25. Immune cell or population of immune cells thereof according to claim 1, characterized by the fact that at least one polynucleotide encodes at least one unnaturally occurring immune receptor and at least one unnaturally occurring agent with the ability to selectively activate the NF-κB pathway are expressed using two or more separate promoters.
[26]
26. Immune cell or population of immune cells thereof according to claim 24 or 25, characterized in that at least one polynucleotide comprises a first nucleic acid coding sequence that encodes at least one non-naturally occurring immune receptor separated from a second nucleic acid sequence encoding the non-naturally occurring agent capable of selectively activating NF-κB, so that, upon expression of the first and second nucleic acid coding sequences, the immune receptor for
9/30 unnatural occurrence and the unnaturally occurring agent with the ability to selectively activate NF-κB are not physically or chemically linked.
[27]
27. Immune cell or immune cell population thereof according to claims 24 and 25, characterized by the fact that at least one non-naturally occurring immune receptor and / or at least one agent with the ability to selectively activate the polynucleotide encoding NF-κB (or NF-κB encoding polynucleotides) are cloned into an endogenous TCR gene, so that at least one non-naturally occurring immune receptor and / or at least one non-naturally occurring agent with ability to selectively activate NF-κB are under the control of endogenous regulatory / promoter elements for the TCR gene.
[28]
28. Immune cell or immune cell population thereof according to claims 21 or 27, characterized by the fact that one or more constant chains of the TCR genes are functionally re-expressed.
[29]
29. Recombinant polynucleotide encoding at least one non-naturally occurring immune receptor, the at least one recombinant polynucleotide being characterized by the fact that it comprises: (a) a first nucleic acid domain that encodes a partial or total transmembrane domain and / or cytoplasmic and, optionally, the extracellular domain of an endogenous protein, in which the endogenous protein is expressed on the surface of lymphocytes and triggers the activation and / or proliferation of the lymphocyte; (b) optionally, a polynucleotide is a linker; (c) a second nucleic acid domain operably linked to the first nucleic acid domain, wherein the second nucleic acid domain encodes one or more non-natural TCR antigen binding domain (s);
10/30 (d) an optional third nucleic acid domain that encodes a co-stimulating domain; and (e) an optional additional nucleic acid domain that encodes an accessory module.
[30]
30. Recombinant polynucleotide characterized by the fact that it comprises: a) a first nucleic acid that encodes a non-naturally occurring immune receptor; and b) a second nucleic acid that encodes an accessory module comprising a selective NF-κB activator.
[31]
31. Recombinant polynucleotide according to claim 30, characterized in that the first nucleic acid and the second nucleic acid are separated by an oligonucleotide linker that encodes a cleavable peptide linker.
[32]
32. Recombinant polynucleotide according to claim 30, characterized in that it comprises two recombinant polynucleotides, so that the first nucleic acid and the second nucleic acid are expressed from separate vectors.
[33]
33. Recombinant polynucleotide according to claim 30, characterized in that the selective NF-κB activator is a non-naturally occurring selective NF-κB activator.
[34]
34. Polynucleotide according to claim 30, characterized by the fact that the unnaturally occurring immune receptor is selected from the group consisting of a CAR, an Ab-TCR, a TFP, a cTCR, a SIR and a TCR recombinant.
[35]
35. Polynucleotide according to claim 30, characterized in that the non-naturally occurring immune receptor comprises (i) a specific extracellular antigen domain, (ii) a transmembrane domain, and (iii) an intracellular signaling domain
11/30 optional that comprises an immunoreceptor tyrosine-based activation motif (ITAM), where (iii) is located at the C-terminus of the unnaturally occurring immune receptor.
[36]
36. Polynucleotide according to claim 30, characterized in that, upon expression of the first and second nucleic acid sequences, the non-naturally occurring immune receptor and the selective NF-κB activator polypeptide are not physically or chemically linked .
[37]
37. Polynucleotide according to claim 35, characterized by the fact that the specific extracellular antigen domain binds to any one or more among CD5; CD19; CD123; CD22; CD30; CD171; CS1 (also called subset CD2 1, CRACC, MPL, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); tyrosine kinase 1 orphan receptor (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); Mesothelin; Alpha interleukin 11 receptor (IL-11Ra); prostate stem cell antigen (PSCA); Serine protease 21 (testin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis antigen (Y); CD24; Beta factor receptor
12/30 platelet-derived growth (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Alpha folate receptor (FRa or FR1); Beta folate receptor (FRb); ERBB2 tyrosine-protein kinase receptor (Her2 / neu); Mucine 1 associated with the cell surface (MUC1); AFP / MHC complex; epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostasis; prostatic acid phosphatase (PAP); mutation elongation factor 2 (ELF2M); Ephrin B2; alpha fibroblast activation protein (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAlX); Proteasome subunit (Prosome, Macropain), Type Beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster (BCR) region and Abelson murine leukemia viral oncogene 1 homologue (Abl) (bcr-abl); tyrosinase; type A 2 ephrin receptor (EphA2); Lewis sialyl adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac (2-3) bDClalp (1-4) bDGlcp (1-1) Cer); transglutaminase 5 (TGS5); antigen associated with high molecular weight melanoma (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); related to tumor endothelial marker 7 (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); group 5, class C receptor coupled to protein G, member D (GPRC5D); X 61 chromosome open reading frame (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; 1 placenta specific (PLAC1); hexassaccharide portion of glucoceramide globoH (GloboH); mammary gland differentiating antigen (NY-BR-1); uroplaquin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); panexin 3 (PANX3); Receptor 20 coupled to G protein (GPR20); lymphocyte 6 antigen complex, K9 locus (LY6K); Olfactory receptor 51E2 (OR51E2); Gamma TCR alternating frame protein (TARP); Wilms' tumor protein (WT1);
13/30 WT1 / MHC I complex; Cancer / testis antigen 1 (NY-ESO-1); NY-ESO-1 / MHC I complex; Cancer / testis antigen 2 (LAGE-1a); Antigen 1 associated with melanoma (MAGE-A1); Gene 6 of the ETS translocation variant, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); Antigen Family X, Member lA (XAGEl); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma testosterone cancer antigen 1 (MAD-CT-1); melanoma testis cancer antigen 2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen 1 (PCT A-1 or Galectin 8), melanoma antigen recognized by T 1 cells (MelanA or MARTI); Mutant of rat sarcoma (Ras); human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, ETS serine 2 fusion gene (TMPRSS2)); N-acetyl-glucosaminyl-transferase V (NA17); Pax-3 (PAX3) paired box protein; Androgen receptor; Cyclin B1; homologous derived from viral oncogene neuroblastoma of avian myelocytomatosis v-myc (MYCN); Ras Homologous Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1); Type (BORIS or Brother of the Regulator of Imprinted Sites) of Binding Factor to CCCTC (Zinc Finger Protein), squamous cell carcinoma antigen recognized by T 3 cells (SART3); Pax-5 paired box protein (PAX5); proachrosine binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); An anchor protein kinase 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receiver for advanced glycation end products (RAGE-1); 1 ubiquitous renal (RU1); ubiquitous renal 2 (RU2); legumaine; human papilloma virus E6 (HPV E6); HPV E6 / MHC I complex; human papilloma virus E7 (HPV E7); HPV E7 / MHC I complex; complex of
14/30
AFP / MHC I; Ras / MHC I complex; intestinal carboxyl esterase; mutated 70-2 heat shock protein (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin type 1 receptor (LAIR1); IgA receptor Fc fragment (FCAR or CD89); Leukocyte immunoglobulin receptor (LILRA2) member 2 of subfamily A; F member of the CD300 molecule family (CD300LF); A member of family 12 of type C lectin domain (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF type module that contains mucin type 2 hormone receptor (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Type 5 Fc receiver (FCRL5); and immunoglobulin lamina-type polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalfa4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Lewis Sialila Antigen ); Fucosil-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1, TCRgama-delta, NKG2D, CD32 (FCGR2A), Tn ag, Tim1- / HVCR1 CSFRRA (GMV-CSFRRA) alpha), TGFbetaR2, Lews Ag, TCR-beta1 chain, TCR-beta2 chain, TCR gamma chain, TCR-delta chain, FITC, leutenizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), hormone receptor stimulating follicle (FSHR), gonadotrophin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A (HA) hemagglutinin, GAD, PDL1, guanylyl cyclase C (GCC), autoantibody for desmoglein 3 (Dsg3), autoantibody for desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D , Claudina18.2 (CLD18A2 or CLDN18A2), Glycoprotein p, STEAP1, Liv1, Nectin-4, Crypto, gpA33, BST1 / CD157, chloret channel the one with low conductance and an antigen recognized by the TNT antibody.
15/30
[38]
38. Polynucleotide according to claim 30, characterized in that the selective NF-κB activator is selected from the group consisting of vFLIP K13, a NEMO mutant, a NEMO fusion protein, IKK1-S176E-S180E, IKK2-S177E-S181E, RIP, FKBPx2-RIP-ID, IKK1, FKBPx2-IKKa, IKK2, FKBPx2-IKK2, Tcl-1, MyD88-L265, any NF-κB-activating protein or protein fragment, any inhibitor an inhibitor of the NF-κB pathway, a gene editing system with the ability to selectively activate NF-κB, an RNA interference system that selectively activates NF-κB and any combination thereof.
[39]
39. Polynucleotide according to claim 37, characterized in that the selective NF-κB activator is expressed as a fusion construct with one or more copies of the FKBP domain.
[40]
40. Polynucleotide according to claim 35, characterized by the fact that the specific extracellular antigen domain is selected from the group consisting of: - the variable region of the heavy chain (vH) of an antibody or a fragment of the same specific for a predefined target antigen; - the variable region of the light chain (vL) of an antibody or a fragment thereof specific to a predefined target antigen; - a single-stranded variable fragment (scFv) or a fragment thereof specific for a predefined target antigen; - an antibody fragment (for example, Fv, a Fab, a (Fab ') 2) specific for a predefined target antigen; - a single domain antibody (SDAB) fragment specific for a predefined target antigen; - a camelid vHH domain specific for a predefined target antigen; - non-immunoglobulin antigen binding frameworks
16/30 specific for a predefined target antigen; - a specific receptor or fragment thereof for a predefined target antigen; - a linker or fragment thereof specific for a predefined target antigen; - a bispecific antibody, -antibody fragment, -scFV, -VHH, -SDAB, -non-immunoglobulin antigen binding framework, - specific receptor or -linker for one or more predefined target antigens; and - a self-antigen or a fragment thereof.
[41]
41. Vector, characterized by the fact that it comprises at least one polynucleotide according to any one of claims 30 to 40.
[42]
42. Vector according to claim 41, the vector being characterized by the fact that it is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenoviral vector, AAV vector, retroviral vector, baculovirus vector, sleep beauty transposon vector and piggybac transposon vector.
[43]
43. Immune effector cell or stem cell, characterized by the fact that it comprises at least one recombinant polynucleotide, from any one of claims 30 to 40.
[44]
44. Immune effector cell or stem cell characterized by the fact that it comprises at least one vector according to the claim
41.
[45]
45. Immune effector cell or stem cell characterized by the fact that it comprises at least one vector according to claim 42.
[46]
46. Antigen-presenting cell, characterized by the fact that it comprises at least one vector according to claim 41.
17/30
[47]
47. Immune effector cell or stem cell according to any of claims 43 to 45, characterized in that the immune effector cell is a human T cell, a human NKT cell or a synthetic T cell, NK cell or a cell - stem that can generate an immune effector cell, optionally, in which the T cell is deficient in diaglycerol kinase (DGK) and / or Ikaros and / or Brd4.
[48]
48. Method, characterized by the fact that it is to (i) prolong the life of an immune cell in expression, (ii) stimulate the proliferation of an immune cell, (iii) stimulate the production of cytokines by an immune cell, ( iv) improving the presentation of antigens by an immune cell, (v) protecting an immune cell from apoptosis, in which the method comprises transfecting or transforming the immune cells with a polynucleotide encoding a selective NF-κB activator or a stimulating polypeptide specific NF-κB.
[49]
49. Method according to claim 48, characterized in that the selective NF-κB activator or a specific NF-κB stimulating polypeptide is selected from the group consisting of vFLIP K13, K13-opt, a NEMO mutant, a protein fusion protein, IKK1-S176E- S180E, IKK2-S177E-S181E, RIP, IKKα, IKKβ, Tcl-1, MyD88-L265, any NF-κB-activating protein or protein fragment, any inhibitor of a pathway inhibitor of NF-κB, any homologous or variant thereof and any combination thereof.
[50]
50. The method of claim 49, characterized in that the selective NF-κB activator or a specific NF-κB stimulating polypeptide is expressed in a constitutive or inducible manner.
[51]
51. The method of claim 50, characterized in that the selective NF-κB activator or a specific NF-κB stimulating polypeptide is controlled post-translation by contacting the T cell with a compound.
18/30
[52]
52. The method of claim 50, characterized in that the selective NF-κB activator or NF-κB specific stimulating polypeptide is expressed as a fusion construct with one or more copies of the FKBP domain.
[53]
53. The method of claim 52, characterized in that the activity of the selective NF-κB activator or a specific NF-κB stimulating polypeptide is controlled at the post-translational level by administering a therapeutically effective amount of a compound that induces the dimerization of the FKBP domain.
[54]
54. Method according to claim 51, characterized in that the compound is AP20187 or rimiducid.
[55]
55. Method for producing an immune effector cell that expresses an unnaturally occurring immune receptor, characterized in that it comprises the introduction of at least one vector according to claim 41, or at least one recombinant polynucleotide according to claim 29 , in an effector cell or hematopoietic stem cell or progenitor cell that can give rise to an immune effector cell, under conditions such that an unnaturally occurring immune receptor is expressed and the immune effector cell comprises (i) prolonged life , (ii) improved T cell proliferation and / or (iii) reduced apoptosis compared to a CAR-T cell that lacks an NFkB specific stimulating polypeptide.
[56]
56. Method according to claim 55, characterized in that it further comprises: a) providing a population of immune effector cells; and b) removing regulatory T cells from the population, thereby providing a population of depleted regulatory T cells; wherein steps a) and b) are performed before the introduction of the recombinant vector or polynucleotide encoding the polypeptide
19/30 specific stimulant of CAR and / or NFkB to the population.
[57]
57. The method of claim 56, characterized in that regulatory T cells are removed from the cell population with the use of an anti-CD25 antibody or an anti-GITR antibody.
[58]
58. Method according to claim 55, characterized in that it further comprises: a) providing a population of immune effector cells; and b) enriching P-glycoprotein positive cells (gp-Pp or Pgp; MDR1, ABCB1, CD243) of the population, thus providing a population of P-glycoprotein enriched cells (gp-P or Pgp; MDR1, ABCB1, CD243); wherein steps a) and b) are performed before or after the introduction of the recombinant vector or polynucleotide encoding the CAR and / or NFkB-specific stimulating polypeptide.
[59]
59. Method according to claim 58, characterized in that the positive P glycoprotein cells are enriched with the use of any one or more of the methods selected from the group consisting of: i) immunoselection using a or a cocktail of specific glycoprotein P antibodies, ii) staining with one or more fluorescent dyes that are substrates of glycoprotein P, methyl tetramethylrodamine ester (TMRM), adriamycin and actinomycin-D) under conditions in which glycoprotein P is active as pump and enrichment for cells that stain less with the dye, iii) selection of cells that are resistant to phototoxic compounds that are substrates of glycoprotein P, such as any one or more of TH9402, 2- (4,5-methyl acid methyl ester hydrochloride) -dibromo-6-amino-3-imino-3H-xanten-9-yl) -benzoic acid 2- (4,5-
20/30 dibromo-6-amino-3-imino-3H-xanten-9-yl) -benzoic, octyl ester hydrochloride 2- (4,5-dibromo-6-amino-3-imino-3H-xanten-9 -yl) -benzoic acid 2- (4,5-dibromo-6-amino-3-imino-3H-xanten-9-yl) -benzoic acid n-butyl ester hydrochloride, n-butyl acid hydrochloride 2- (6-ethyl amino-3-ethyl imino-3H-xanten-9-yl) -benzoic, or derivatives thereof or combinations thereof, and iv) selection of cells that are resistant to cytotoxic compounds that are glycoprotein substrates P, such as vincristine, vinblastine, taxol, paclitaxel, mitoxantrone, etoposide, adriamycin, daunorubicin and actinomycin-D.
[60]
60. Method for generating a population of cells manipulated by RNA, characterized by the fact that it comprises the introduction of RNA or RNAs transcribed in vitro or synthetic RNA or RNAs into a cell or population of cells, wherein the RNA or RNAs comprise a polynucleotide or recombinant polynucleotides according to claim 30.
[61]
61. Method for providing anti-disease immunity in an individual, characterized in that it comprises administering to the individual an effective amount of the immune effector cell or a stem cell that can give rise to an immune effector cell according to any of claims 43 to 47, where the cell is an autologous T cell or an allogeneic T cell or an autologous NKT cell or an allogeneic NKT cell or an autologous or allogeneic hematopoietic stem cell or an autologous or allogeneic iPSC that can give rise to an immune effector cell .
[62]
62. The method of claim 61, characterized by the fact that the allogeneic T cell or allogeneic NKT cell or hematopoietic stem cell or iPSC has no or low expression of a functional TCR or a functional HLA.
21/30
[63]
63. A composition comprising an effector immune cell or a stem cell that can generate immune effector cells that comprise an unnaturally occurring immune receptor and a selective NfkB activator, characterized by the fact that the unnaturally occurring immune receptor comprises a domain of binding to the antigen that binds to a disease-associated antigen, in which said disease-associated antigen is selected from a group consisting of: CD5, CD19; CD123; CD22; CD30; CD171; CS-1 (also called subset CD2 1, CRACC, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2- 3) bDGalp (1-4) bDGlcp (1-1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); tyrosine kinase 1 orphan receptor (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); Mesothelin; Alpha interleukin 11 receptor (IL-11Ra); prostate stem cell antigen (PSCA); Serine protease 21 (testin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis antigen (Y); CD24; Platelet-derived growth factor beta receptor (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Alpha folate receptor; ERBB2 tyrosine-protein kinase receptor (Her2 / neu); Mucine 1 associated with the cell surface (MUC1); epidermal growth factor receptor
22/30
(EGFR); neural cell adhesion molecule (NCAM); Prostasis; prostatic acid phosphatase (PAP); mutation elongation factor 2 (ELF2M); Ephrin B2; alpha fibroblast activation protein (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAlX); Proteasome subunit (Prosome, Macropain), Type Beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster (BCR) region and Abelson murine leukemia viral oncogene 1 homologue (Abl) (bcr-abl); tyrosinase; type A 2 ephrin receptor (EphA2); Fucosyl GM1; Lewis sialyl adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac (2-3) bDClalp (1-4) bDGlcp (1-1) Cer); transglutaminase 5 (TGS5); antigen associated with high molecular weight melanoma (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); related to tumor endothelial marker 7 (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); group 5, class C receptor coupled to protein G, member D (GPRC5D); X 61 chromosome open reading frame (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; 1 placenta specific (PLAC1); hexassaccharide portion of glucoceramide globoH (GloboH); mammary gland differentiating antigen (NY-BR-1); uroplaquin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); panexin 3 (PANX3); Receptor 20 coupled to G protein (GPR20); lymphocyte 6 antigen complex, K9 locus (LY6K); Olfactory receptor 51E2 (OR51E2); Gamma TCR alternating frame protein (TARP); Wilms' tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Antigen 1 associated with melanoma (MAGE-A1); Gene 6 of the ETS translocation variant, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); Antigen Family X, Member lA (XAGEl);
23/30 angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma testosterone cancer antigen 1 (MAD-CT-1); melanoma testis cancer antigen 2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen 1 (PCT A-1 or Galectin 8), melanoma antigen recognized by T 1 cells (MelanA or MARTI); Mutant of rat sarcoma (Ras); human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, ETS serine 2 fusion gene (TMPRSS2)); N-acetyl-glucosaminyl-transferase V (NA17); Pax-3 (PAX3) paired box protein; Androgen receptor; Cyclin B1; homologous derived from viral oncogene neuroblastoma of avian myelocytomatosis v-myc (MYCN); Ras Homologous Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1); Type (BORIS or Brother of the Regulator of Imprinted Sites) of Binding Factor to CCCTC (Zinc Finger Protein), squamous cell carcinoma antigen recognized by T 3 cells (SART3); Pax-5 paired box protein (PAX5); proachrosine binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); An anchor protein kinase 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receiver for advanced glycation end products (RAGE-1); 1 ubiquitous renal (RU1); ubiquitous renal 2 (RU2); legumaine; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; mutated 70-2 heat shock protein (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin type 1 receptor (LAIR1); IgA receptor Fc fragment (FCAR or CD89); Leukocyte immunoglobulin receptor (LILRA2) member 2 of subfamily A; F member of the CD300 molecule family (CD300LF); A member of family 12 of type C lectin domain (CLEC12A);
24/30 bone marrow stromal cell antigen 2 (BST2); EGF type module that contains mucin type 2 hormone receptor (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Type 5 Fc receiver (FCRL5); and immunoglobulin lamina-type polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalfa4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Lewis Sialila Antigen ); Fucosil-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1, ALK TCRgama-delta, NKG2D, CD32 (FCGR2A), CSPG4-HMW-MAA, Tim2 GM-CSFR-alpha), TGFbetaR2, VEGFR2 / KDR, Lews Ag, TCR-beta1 chain, TCR-beta2 chain, TCR gamma chain, TCR-delta chain, FITC, leutenizing hormone receptor (LHR), stimulating hormone receptor follicle (FSHR), follicle stimulating hormone receptor (FSHR), chronic gonadotropin hormone receptor (CGHR), CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, autoantibody for desmoglein 3 (Dsg3), autoantibody for desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, claudina18.2 (CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1, LIV1, NECTIN-4, CRI PTO, GPA33, BST1 / CD157, low conductance chloride channel and antigen recognized by TNT antibody.
[64]
64. Method for treating or preventing a disease associated with the expression of an antigen associated with the disease in an individual, characterized by the fact that it comprises administering to the individual an effective amount of an immune effector cell comprising an unnaturally occurring immune receptor and a selective NfkB activator, in which the unnaturally occurring immune receptor comprises a
25/30 antigen that binds to an antigen associated with the disease, in which the said antigen associated with the disease is selected from a group consisting of: CD5, CD19; CD123; CD22; CD30; CD171; CS-1 (also called subset CD2 1, CRACC, SLAMF7, CD319 and 19A24); type C lectin-type molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1- 1) Cer); maturation of member cell of the TNF receptor family (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); tyrosine kinase 1 orphan receptor (ROR1); Fms like tyrosine kinase of type Fms 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in leukemia or acute lymphoma, but not in hematopoietic parents, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); Mesothelin; Alpha interleukin 11 receptor (IL-11Ra); prostate stem cell antigen (PSCA); Serine protease 21 (testin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis antigen (Y); CD24; Platelet-derived growth factor beta receptor (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Alpha folate receptor; ERBB2 tyrosine-protein kinase receptor (Her2 / neu); Mucine 1 associated with the cell surface (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostasis; prostatic acid phosphatase (PAP); mutation elongation factor 2 (ELF2M); Ephrin B2; alpha fibroblast activation protein (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAlX); Proteasome subunit (Prosome,
26/30
Macropain), Type Beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster (BCR) region and Abelson murine leukemia viral oncogene 1 homologue (Abl) (bcr-abl); tyrosinase; type A 2 ephrin receptor (EphA2); Fucosyl GM1; Lewis sialyl adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac (2-3) bDClalp (1-4) bDGlcp (1-1) Cer); transglutaminase 5 (TGS5); antigen associated with high molecular weight melanoma (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); related to tumor endothelial marker 7 (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); group 5, class C receptor coupled to protein G, member D (GPRC5D); X 61 chromosome open reading frame (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; 1 placenta specific (PLAC1); hexassaccharide portion of glucoceramide globoH (GloboH); mammary gland differentiating antigen (NY-BR-1); uroplaquin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); panexin 3 (PANX3); Receptor 20 coupled to G protein (GPR20); lymphocyte 6 antigen complex, K9 locus (LY6K); Olfactory receptor 51E2 (OR51E2); Gamma TCR alternating frame protein (TARP); Wilms' tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Antigen 1 associated with melanoma (MAGE-A1); Gene 6 of the ETS translocation variant, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); Antigen Family X, Member lA (XAGEl); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma testosterone cancer antigen 1 (MAD-CT-1); melanoma testis cancer antigen 2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; antigen 1 of the prostate carcinoma tumor (PCT A-1 or
27/30
Galectin 8), melanoma antigen recognized by T 1 cells (MelanA or MARTI); Mutant of rat sarcoma (Ras); human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, ETS serine 2 fusion gene (TMPRSS2)); N-acetylglucosaminyl transferase V (NA17); Pax-3 (PAX3) paired box protein; Androgen receptor; Cyclin B1; homologous derived from viral oncogene neuroblastoma of avian myelocytomatosis v-myc (MYCN); Ras Homologous Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1); Type (BORIS or Brother of the Regulator of Imprinted Sites) of Binding Factor to CCCTC (Zinc Finger Protein), squamous cell carcinoma antigen recognized by T 3 cells (SART3); Pax-5 paired box protein (PAX5); proachrosine binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); An anchor protein kinase 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receiver for advanced glycation end products (RAGE-1); 1 ubiquitous renal (RU1); ubiquitous renal 2 (RU2); legumaine; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; mutated 70-2 heat shock protein (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin type 1 receptor (LAIR1); IgA receptor Fc fragment (FCAR or CD89); Leukocyte immunoglobulin receptor (LILRA2) member 2 of subfamily A; F member of the CD300 molecule family (CD300LF); A member of family 12 of type C lectin domain (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF type module that contains mucin type 2 hormone receptor (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Type 5 Fc receiver (FCRL5); and immunoglobulin lamina-type polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope tag, CD34,
28/30 LAMP1 TROP2, GFRalfa4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Lewis Sialyl Antigen); Fucosil-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1, ALK TCRgama-delta, NKG2D, CD32 (FCGR2A), CSPG4-HMW-MAA, Tim2 GM-CSFR-alpha), TGFbetaR2, VEGFR2 / KDR, Lews Ag, TCR-beta1 chain, TCR-beta2 chain, TCR gamma chain, TCR-delta chain, FITC, leutenizing hormone receptor (LHR), stimulating hormone receptor follicle (FSHR), follicle stimulating hormone receptor (FSHR), chronic gonadotropin hormone receptor (CGHR), CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1- Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, autoantibody for desmoglein 3 (Dsg3), autoantibody for desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, claudina18.2 (CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1, LIV1, NECTIN-4, CRI PTO, GPA33, BST1 / CD157, low conductance chloride channel and antigen recognized by TNT antibody, thus treating the individual or preventing a disease in the individual.
[65]
65. Use or method according to claim 63 or 64, characterized in that the disease associated with the expression of the antigen associated with the disease is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer and an indication related to non-cancer associated with the expression of the antigen associated with the disease.
[66]
66. Use or method according to claim 65, characterized by the fact that cancer is a hematological cancer chosen over
29/30 from one or more between chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), acute B-cell lymphoid leukemia (B-ALL), acute T-cell lymphoid leukemia (T-ALL), chronic myeloid leukemia (CML), B cell prolinocytic leukemia, blast plasmocytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, primary effusion lymphoma, follicular lymphoma, hair cell leukemia, small cell follicular lymphoma large, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytic dendritic cell neoplasm pre-leukemia.
[67]
67. Use or method according to claim 65, characterized by the fact that the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vagina carcinoma, vulva carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine cancer, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, solid childhood tumors, bladder cancer, kidney or ureter cancer, carcinoma renal pelvis, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, Merkel cell cancer, cancer
30/30 epidermoid, squamous cell cancer, T-cell lymphoma, environment-induced cancers, combinations of so-called cancers and metastatic lesions of said cancers.
[68]
68. Use or method according to claim 65, characterized by the fact that the disease is associated with infection by a virus, including, but not limited to, HIV1, HIV2, HTLV1, Epstein Barr virus (EBV), cytomegalovirus (CMV) , adenovirus, adenoassociated virus, BK virus, Human Herpesvirus 6, Human Herpesvirus 8, influenza virus, parainfluenza virus, avian influenza virus, MERS and SARS coronavirus, Congo hemorrhagic fever virus, rhinovirus, enterovirus, dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa fever virus, Zika virus, RSV, measles virus, mumps virus, rhinoceros virus, chickenpox virus, herpes simplex virus 1 and 2, varicella zoster virus, HIV-1, HTLV1, hepatitis virus, enterovirus, hepatitis B virus, hepatitis C virus, Nipah virus and Rift valley fever, Japanese encephalitis virus, Merkel cell polyomavirus or is associated infection by mycobacterium tuberculosis, species atypical of mycobacteria, Pneumocystis jirovecii, toxoplasmosis, rickettsia, nocardia, aspergillus, mucor or candida.
[69]
69. Use or method according to claim 65, characterized by the fact that the disease is an immune or degenerative disease, including, but not limited to, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, pemphigus vulgaris, ankylosing spondylitis, Hoshimoto's thyroiditis , SLE, sarcoidosis, scleroderma, mixed connective tissue disease, graft versus host disease or Alzheimer's disease.

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