Engineered cells and uses thereof

Abstract

The present application relates to engineered cells and uses thereof, in particular to a system for inducing immune cell activity comprising a chimeric antigen receptor, a T cell receptor, and various combinations thereof.

Description

[0001] This application is a divisional application of the invention application with application number 201980040256.6, application date June 19, 2019, and invention name “Engineered cells and their uses”.

[0002] Cross-references

[0003] This application claims priority to Patent Cooperation Treaty Application No. PCT / CN2018 / 091789, filed on June 19, 2018, the entire contents of which are incorporated herein by reference for all purposes. Background of the Invention

[0005] Effector cell activity can involve ligand binding to a membrane-bound receptor that contains an extracellular antigen binding domain and an intracellular signaling domain. The formation of a complex between the antigen binding domain and its corresponding target can lead to conformational and / or chemical modifications of the receptor itself, which in turn can produce a series of signals that are transduced within the cell. Attempts to exploit this interaction to develop immune cell therapies have shown promising efficacy, but off-target toxicity has also led to adverse side effects in the treated subjects, including cytokine release syndrome. This and other side effects can further exacerbate inflammatory responses, organ failure, and even lead to death in extreme cases.

[0006] The development and function of all T cells depend on their antigen receptors. The T cell receptor (TCR) is a multiprotein complex that consists of two modules with different functions: a ligand binding module and a signal transmission module. The ligand binding module consists of two variable polypeptide chains, TCRα and TCRβ, which form covalently linked heterodimers and are responsible for the ligand specificity of the TCR. The signal transmission module of the TCR complex consists of unchanged polypeptide chains, including CD3e, CD3g, CD3d and z. Among them, CD3e, CD3g and CD3d form non-covalently linked CD3eg and CD3ed heterodimers, and z forms covalently linked zz homodimers. The surface expression of the TCR complex requires a complete assembly of the complex subunit set. Assembly begins with the formation of CD3ed and CD3eg heterodimers in the endoplasmic reticulum. Then, they associate with TCRα and TCRβ, respectively, to produce an intermediate complex. The zz homodimer is the last subunit to engage, and following its incorporation, the entire TCR complex is transported to the plasma membrane (Klausner et al., (1990); Exley et al., (1991); Dave et al., (1997); Marie-Cardine and Schraven, (1999); Kane et al., (2000); Matthew et al., (2004)).

[0007] The pMHC bound to TCRαβ is delivered to the cell through the CD3 signal transduction unit, involving TCR-CD3 aggregation and conformational changes. Many experiments have shown that T cell activation involves a series of signals mediated by TCR, which are regulated by three different intracellular signal transduction motifs located in the cytoplasmic tail of the CD3 chain (CD3zz, CD3eg and CD3ed) (Sun et al., JImmunol (185), (2010)). Studies using chimeric molecules have shown that the cytoplasmic tails of all signal transduction chains of TCR can independently transduce signals that cause cytotoxicity and / or cytokine production, thereby bypassing the αβ recognition mode of TCR. However, based on experimental results, it was previously reported that signaling through the CD3ζ chain alone is not sufficient to trigger resting T lymphocytes (Thomas et al., J. Exp. Med., (1995)), and that mutated CD3e signaling domains in mice display incomplete T cell function (Matthew et al., J Immunol (193), (2014).) CD3eg, CD3ed, and zz chains play complementary roles and even synergistic effects in contributing to T cell function (Borroto et al., J Immunol (163), (1999)).

[0008] Chimeric antigen receptor (CAR) is a modular fusion protein comprising a binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain containing CD3z connected to one or two costimulatory molecules. The CAR structure has evolved significantly from the initial composition involving only the CD3ζ signaling domain, which is called the "first generation CAR". Since then, in order to increase the persistence and proliferation of T cells, the costimulatory terminal domain has been added, resulting in the second generation (e.g., CD3ζ plus 4-1BB or CD28 signaling domain) and the third generation (e.g., CD3ζ plus 4-1BB and CD28 signaling domain) CAR.

[0009] Adoptive transfer of CAR T cells has demonstrated remarkable success in treating hematogenous tumors; notably, the use of CD19 CARs is being explored for indications in leukemias (Gill, S et al., Blood Rev, (2015)) and patients with lymphomas and myeloma. An increasing number of clinical trials are focused on solid tumors. Unfortunately, clinical results are far less encouraging. To date, the two most active trials reported have used GD2 CARs to target neuroblastoma (complete remission in 3 of 11 patients) (Louis et al., Blood (118), (2011)), and HER2 CARs for sarcomas (4 of 17 patients showed stable disease) (Ahmed et al., J Clin Oncol (33), (2015)).

[0010] Poor trafficking, limited persistence, and T cell suppressive activity in patient serum have been proposed to contribute to the observed lack of efficacy (Kershaw et al., Clin. Cancer Res (12), (2006)). There remains an unmet need for new designs for improving the comprehensive functionality of genetically modified T cells with better cell killing effects, in vivo persistence, and better tolerance to the tumor microenvironment. Summary of the invention

[0011] In view of the foregoing, there is a great need for alternative compositions and methods for performing immunotherapy. The compositions and methods of the present disclosure address this need and also provide additional advantages. Various aspects of the present disclosure provide systems, compositions and methods for inducing the activity of immune cells.

[0012] In one aspect, a system for inducing the activity of immune cells and / or target cells is provided, the system comprising: (a) a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain that exhibits specific binding to a first epitope, a transmembrane domain, and an intracellular signaling domain; and (b) a modified T cell receptor (TCR) complex, the modified TCR complex comprising a second antigen binding domain that exhibits specific binding to a second epitope, wherein the second antigen binding domain is connected to: (i) at least one TCR chain, the at least one TCR chain being selected from the α chain, β chain, γ chain, and δ chain of the T cell receptor; (ii) the ε chain, δ chain, and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0013] In some embodiments, binding of the first antigen binding domain to the first epitope and / or binding of the second antigen binding domain to the second epitope activates immune cell activity of an immune cell expressing the system.

[0014] In some embodiments, two or more antigen binding domains are optionally linked in series to (i) at least one TCR chain selected from the group consisting of the α chain, β chain, γ chain, and δ chain of the T cell receptor; (ii) the ε chain, δ chain, and / or γ chain of cluster of differentiation 3 (CD3); (iii) the CD3 ζ chain, and wherein binding of the two or more antigen binding domains to their corresponding epitopes activates the immune cell activity of an immune cell expressing the system.

[0015] In some embodiments, the immune cell activity is selected from the group consisting of: clonal expansion of the immune cells; cytokine release by the immune cells; cytotoxicity of the immune cells; proliferation of the immune cells; differentiation, dedifferentiation or transdifferentiation of the immune cells; movement and / or trafficking of the immune cells; exhaustion and / or reactivation of the immune cells; and release of other intercellular molecules, metabolites, compounds or combinations thereof by the immune cells.

[0016] In some embodiments, binding of the first antigen binding domain to the first epitope and binding of the second antigen binding domain to the second epitope activates cytotoxicity of an immune cell expressing the system, and the cytotoxicity is enhanced compared to binding of only the first antigen binding domain to the first epitope or binding of only the second antigen binding domain to the second epitope.

[0017] In some embodiments, binding of the first antigen binding domain to the first epitope and binding of the second antigen binding domain to the second epitope activates cytotoxicity of immune cells expressing the system and increases the persistence of the cytotoxicity compared to binding of only the first antigen binding domain to the first epitope or binding of only the second antigen binding domain to the second epitope.

[0018] In some embodiments, when the system is expressed in an immune cell of a subject, binding of the two or more antigen binding domains to their corresponding epitopes activates cytotoxicity of the immune cell expressing the system and increases the persistence of the cytotoxicity compared to binding of only the first antigen binding domain to the first epitope.

[0019] In some embodiments, the modified TCR comprises a third antigen binding domain, which is connected to: (i) the second antigen binding domain; (ii) the at least one TCR chain, which is selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (iii) the ε chain, the δ chain and / or the γ chain of cluster of differentiation 3 (CD3); or (iv) the CD3ζ chain.

[0020] In some embodiments, the CAR comprises one or more additional antigen binding domains. In some embodiments, the one or more additional antigen binding domains exhibit specific binding to one or more additional epitopes. In some embodiments, the one or more additional epitopes are identical to the first epitope or the second epitope. In some embodiments, the one or more additional epitopes are different from the first epitope and the second epitope. In some embodiments, the one or more additional antigen binding domains and the first antigen binding domain are connected in series.

[0021] In some embodiments, the intracellular signaling domain of the CAR comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain of the CAR comprises an immunoreceptor tyrosine-based inhibition motif (ITIM).

[0022] In some embodiments, the intracellular signaling domain of the CAR includes a signaling domain of Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d and Zap70. In some embodiments, the intracellular signaling domain includes a signaling domain of CD3ζ.

[0023] In some embodiments, the CAR further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain includes an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activation NK cell receptor or a Toll ligand receptor signaling domain. In some embodiments, the co-stimulatory domain comprises a signaling domain of a molecule selected from the following: 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF-R / TNFRSF13C, BAFF / BLyS / TNFSF13 B. BLAME / SLAMF8, BTLA / CD272, CD100(SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160(BY 55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SL AMF9, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLA MF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLAClass I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9(CD229), lymphocyte function-associated antigen-1(LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG(CD162 ), SLAM(SLAMF1), SLAM / CD150, SLAMF4(CD244), SLAMF6(NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1 and VLA-6.

[0024] In some embodiments, the first antigen binding domain and / or the second antigen binding domain comprises a Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), a minibody, a diabody, a single domain antibody, a light chain variable domain (VL) or a variable domain (VL) of a camelid antibody. H H).

[0025] In some embodiments, at least one of the antigen binding domains comprises a receptor. In some embodiments, at least one of the antigen binding domains comprises a ligand for a receptor.

[0026] In some embodiments, the first epitope and the second epitope are present on different antigens. In some embodiments, the first epitope and the second epitope are present on a common antigen.

[0027] In some embodiments, at least one epitope is present on one or more cell surface antigens. In some embodiments, the one or more cell surface antigens are tumor associated antigens, tyrosine kinase receptors, serine kinase receptors, and G protein coupled receptors.

[0028] In some embodiments, the first epitope and / or the second epitope is present on a universal antigen.

[0029] In some embodiments, the first epitope and / or the second epitope is present on a neoantigen. In some embodiments, the first epitope and / or the second epitope is a neoepitope.

[0030] In some embodiments, the first epitope and / or the second epitope is present on a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of: 707-AP, biotinylated molecules, α-actinin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipose differentiation-related protein, AFP, AIM-2, annexin II, ART-4, BAGE, BCMA, b-catenin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-abl p210(b3a2), BING-4, CA-125, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD4, CD23, CD24, CD30, CD33, CD38, CD44v7 / 8, CD70, CD123, CD133, CDC27, CDK-4, CEA, CLCA2, CLL-1, CTAG1B, Cyp-B, DAM-10, DAM-6, DEK-CAN, DLL3, EGFR, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FAP, FBP, fetal acetylcholine receptor, FGF-5, FN, FR-α, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, GPC3, GPC-2, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, LewisY, L1-CAM, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1 , OA1, OGT, carcinoembryonic antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survivin, survivin-2B, SYT / SSX, TAG-72, TEL / AML1, TGFαRII, TGFβRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ light chain.

[0031] In some embodiments, at least one epitope is present on an immune checkpoint receptor or immune checkpoint receptor ligand. In some embodiments, the immune checkpoint receptor or immune checkpoint receptor ligand is PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, TIGIT, BLTA, CD47 or CD40.

[0032] In some embodiments, at least one epitope is present on a cytokine or cytokine receptor. In some embodiments, the cytokine or cytokine receptor is CCR2b, CXCR2 (CXCL1 receptor), CCR4 (CCL17 receptor), Gro-a, IL-2, IL-7, IL-15, IL-21, IL-12, heparanase, CD137L, LEM, Bcl-2, CCL17, CCL19 or CCL2.

[0033] In some embodiments, at least one epitope is present on an antigen presented by a major histocompatibility complex (MHC). In some embodiments, the MHC is HLA class 1. In some embodiments, the MHC is HLA class 2.

[0034] In another aspect, an isolated host cell expressing a system of the present disclosure is provided.

[0035] In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the lymphocyte is an α / β T cell and / or a γ / δ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell.

[0036] In some embodiments, the host cell exhibits specific binding to two antigens simultaneously present in the target cell.

[0037] In another aspect, an antigen-specific immune cell is provided, which comprises at least two exogenously introduced antigen binding domains, one of the at least two exogenously introduced antigen binding domains is linked to a T cell receptor (TCR) complex, and the other is linked to a chimeric antigen receptor (CAR), wherein the immune cell specifically binds to a target cell expressing one or more antigens recognized by the at least two exogenously introduced antigen binding domains.

[0038] In some embodiments, the antigen binding domain attached to the CAR primarily mediates the interaction between the immune cell and the target cell, and when the interaction between the immune cell and the target cell occurs, the antigen binding domain attached to the TCR complex primarily mediates immune cell activity.

[0039] In some embodiments, the immune cell activity is selected from the group consisting of: clonal expansion of the immune cells; cytokine release by the immune cells; cytotoxicity of the immune cells; proliferation of the immune cells; differentiation, dedifferentiation or transdifferentiation of the immune cells; movement and / or trafficking of the immune cells; exhaustion and / or reactivation of the immune cells; and release of other intercellular molecules, metabolites, compounds or combinations thereof by the immune cells.

[0040] In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the lymphocyte is an α / βT cell and / or a γ / δT cell. In some embodiments, the T cell is a CD4+T cell or a CD8+T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell. In some embodiments, two or more antigen binding domains are optionally connected in series to (i) at least one TCR chain, the at least one TCR chain being selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); (iii) CD3ζ chain.

[0041] In another aspect, a population of immune cells is provided, wherein a single immune cell expresses a system of the present disclosure, and wherein the population of immune cells is characterized in that upon exposure of the population of immune cells to a population of target cells in a subject, the population of immune cells induces death of at least 5% of the target cells within about 2 days.

[0042] In some embodiments, the immune cell population includes at most about 10 11 cells.

[0043] In some embodiments, the immune cells include lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the lymphocytes are α / β T cells and / or γ / δ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the lymphocytes are natural killer (NK) cells.

[0044] In another aspect, a method for inducing the activity of an immune cell and / or a target cell is provided, the method comprising: (a) expressing a system in an immune cell; and (b) contacting the target cell with the immune cell under conditions that induce the activity of the immune cell and / or the target cell, wherein the system expressed in the immune cell comprises: a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain having binding specificity for a first epitope, a transmembrane domain, and an intracellular signaling domain; and a modified T cell receptor (TCR) complex, the modified TCR complex comprising a second antigen binding domain, the second antigen binding domain being connected to: (i) at least one TCR chain, the at least one TCR chain being selected from the α chain, β chain, γ chain, and δ chain of the T cell receptor; (ii) the ε chain, δ chain, and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0045] In some embodiments, binding of the first antigen binding domain to the first epitope and / or binding of the second antigen binding domain to the second epitope activates cytotoxicity of the immune cell.

[0046] In some embodiments, two or more antigen binding domains are optionally linked in series to (i) at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); (iii) the CD3 ζ chain.

[0047] In some embodiments, binding of the first antigen binding domain to the first epitope and binding of the second antigen binding domain to the second epitope activates cytotoxicity of the immune cell, and the cytotoxicity is enhanced compared to binding of only the first antigen binding domain to the first epitope or binding of only the second antigen binding domain to the second epitope.

[0048] In some embodiments, binding of the first antigen binding domain to the first epitope or binding of the second antigen binding domain to the second epitope activates the cytotoxicity of the immune cell and increases the persistence of the cytotoxicity compared to binding of only the first antigen binding domain to the first epitope or binding of only the second antigen binding domain to the second epitope.

[0049] In some embodiments, the cytotoxicity of immune cells induces the death of target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the target cells are hematopoietic cells. In some embodiments, the target cells are solid tumor cells. In some embodiments, the target cells are cells identified in one or more of the heart, blood vessels, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, anus, endocrine glands, adrenal glands, kidneys, ureters, bladders, lymph nodes, tonsils, adenoids, thymus, spleen, skin, muscle, brain, spinal cord, nerves, ovaries, fallopian tubes, uterus, vagina, breast, testicles, prostate, penis, pharynx, larynx, trachea, bronchi, lungs, diaphragms, cartilage, ligaments and tendons.

[0050] In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the lymphocyte is an α / β T cell and / or a γ / δ T cell. In some embodiments, the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell.

[0051] In some embodiments, when the system is expressed in an immune cell of a subject, binding of the two or more antigen binding domains to their corresponding epitopes activates cytotoxicity of the immune cell expressing the system and increases the persistence of the cytotoxicity compared to binding of only the first antigen binding domain to the first epitope.

[0052] In another aspect, a composition is provided, the composition comprising one or more polynucleotides, the one or more polynucleotides encoding: (a) a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain having binding specificity to a first epitope, a transmembrane domain, and an intracellular signaling domain; and (b) a modified T cell receptor (TCR) complex, the modified TCR complex comprising a second antigen binding domain exhibiting specific binding to a second epitope, wherein the second antigen binding domain is connected to: (i) at least one TCR chain, the at least one TCR chain being selected from the α chain, β chain, γ chain, and δ chain of the T cell receptor; (ii) ε chain, δ chain, and / or γ chain of cluster of differentiation 3 (CD3); or (iii) CD3 ζ chain. In some embodiments, the one or more polynucleotides comprise a promoter operably connected thereto.

[0053] In another aspect, a method of producing a modified immune cell is provided, the method comprising genetically modifying the immune cell by expressing a composition of the disclosure in the immune cell, thereby producing the modified immune cell.

[0054] In another aspect, a method for treating cancer in a subject is provided, the method comprising: (a) administering to the subject an antigen-specific immune cell comprising a chimeric antigen receptor (CAR) comprising a first antigen binding domain and a modified T cell receptor (TCR) complex comprising a second antigen binding domain, wherein target cells of the subject's cancer express one or more antigens recognized by the first and / or second antigen binding domains, and wherein the immune cell specifically binds to the target cell, and (b) contacting the target cell with the antigen-specific immune cell through the first and / or second antigen binding domains under conditions that induce immune cell activity of the immune cell against the target cell, thereby inducing death of the target cell of the cancer.

[0055] In another aspect, a method of treating cancer in a subject is provided, the method comprising: (a) administering antigen-specific immune cells to the subject, wherein the antigen-specific immune cells are genetically modified immune cells that express the system of the present disclosure; and (b) contacting the target cells with the antigen-specific immune cells under conditions that induce the immune cell activity of the immune cells against target cells of the subject's cancer, thereby inducing death of the target cells of the cancer.

[0056] In some embodiments, the method further comprises genetically modifying an immune cell to produce the antigen-specific immune cell.

[0057] In some embodiments, the immune cell activity is selected from the group consisting of: clonal expansion of the immune cells; cytokine release by the immune cells; cytotoxicity of the immune cells; proliferation of the immune cells; differentiation, dedifferentiation or transdifferentiation of the immune cells; movement and / or trafficking of the immune cells; exhaustion and / or reactivation of the immune cells; and release of other intercellular molecules, metabolites, compounds or combinations thereof by the immune cells.

[0058] In some embodiments, the immune cell activity is the cytotoxicity of the immune cell. In some embodiments, the cytotoxicity of the immune cell against the target cell results in at least a 10% reduction in the cancer of the subject. In some embodiments, the immune cell activity is cytokine release by the immune cell. In some embodiments, when both the first and second antigen binding domains bind to their corresponding epitopes, the persistence of the immune cell activity is increased compared to the binding of only the first antigen binding domain alone or the binding of only the second antigen binding domain.

[0059] In some embodiments, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

[0060] In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the lymphocyte is an α / β T cell and / or a γ / δ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell.

[0061] In another aspect, an antigen binding molecule of formula AXBYCZD is provided, wherein A comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 47 to SEQ ID NO: 56; B comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 57 to SEQ ID NO: 66; C comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 67 to SEQ ID NO: 76; D comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 77 to SEQ ID NO: 86; X comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 87 to SEQ ID NO: 96; Y comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 97 to SEQ ID NO: 106; and Z comprises a sequence having at least 80% or 90% identity to any one of the group consisting of SEQ ID NO: 106. Any one of the group consisting of SEQ ID NO: 107 to SEQ ID NO: 116 has a sequence with at least 80% or 90% identity.

[0062] In some embodiments, the antigen binding molecule exhibits a binding affinity (K) for human BCMA of 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, or 1 nm or less as determined by surface plasmon resonance at 37°C. D ).

[0063] In some embodiments, the antigen binding molecule comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 23. In some embodiments, the antigen binding molecule comprises a sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 23.

[0064] In another aspect, a modified T cell receptor (TCR) complex is provided, comprising one or more antigen binding domains, wherein the one or more antigen binding domains are linked to: (iv) at least one TCR chain selected from the group consisting of the α chain, β chain, γ chain and δ chain of the T cell receptor; (v) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (vi) the CD3 ζ chain; and wherein at least one of the one or more antigen binding domains comprises an antigen binding molecule of the present disclosure.

[0065] In some embodiments, at least one or both of the one or more antigen binding domains comprise a sequence at least 80% or 90% identical to any one selected from the group consisting of SEQ ID NO:3 to SEQ ID NO:23 and SEQ ID NO:38 to SEQ ID NO:46.

[0066] In some embodiments, the modified TCR complex comprises two or more antigen binding domains. In some embodiments, the two or more antigen binding domains are connected to separate chains of the TCR complex. In some embodiments, the two or more antigen binding domains are connected to a chain of the TCR complex. In some embodiments, the two or more antigen binding domains are connected in series to a chain of the TCR complex. In some embodiments, the modified TCR complex further comprises one or more antigen binding domains connected to another chain of the TCR complex.

[0067] In another aspect, a modified T cell receptor (TCR) complex is provided, comprising two or more antigen binding domains that exhibit specific binding to two or more epitopes, wherein the two or more antigen binding domains are linked to: (i) at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0068] In some embodiments, the two or more antigen binding domains are connected to separate chains of the TCR complex. In some embodiments, the two or more antigen binding domains are connected to one chain of the TCR complex. In some embodiments, the two or more antigen binding domains are connected in series to one chain of the TCR complex.

[0069] In some embodiments, the modified TCR complex further comprises one or more antigen binding domains linked to another chain of the TCR complex.

[0070] In some embodiments, the two or more antigen binding domains bind to BCMA. In some embodiments, the two or more antigen binding domains bind to the same epitope of BCMA. In some embodiments, the two or more antigen binding domains are anti-BCMA sdAbs. In some embodiments, the two or more antigen binding domains are selected from sequences having at least 80% sequence identity with any one of SEQ ID NO: 3 to SEQ ID NO: 23.

[0071] In some embodiments, the two or more antigen binding domains are linked in tandem on the epsilon chain, the delta chain and / or the gamma chain of cluster of differentiation 3 (CD3).

[0072] In another aspect, an antigen-specific immune cell comprising the modified TCR complex of the present disclosure is provided.

[0073] In some embodiments, the antigen-specific immune cell further comprises a chimeric antigen receptor (CAR), and the CAR further comprises one or more antigen binding domains, transmembrane domains and intracellular signaling domains showing specific binding to their corresponding epitopes. In some embodiments, the one or more antigen binding domains of CAR are arranged in series.

[0074] In some embodiments, the antigen-specific immune cell further comprises two or more chimeric antigen receptors (CARs), each CAR comprising one or more antigen binding domains that exhibit specific binding to their corresponding epitopes, a transmembrane domain, and an intracellular signaling domain.

[0075] The methods disclosed herein can be used to treat a variety of cancers, including, but not limited to, a cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

[0076] Incorporation by Reference

[0077] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The novel features of the invention are particularly set forth in the appended claims. The features and advantages of the invention may be better understood by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the invention are utilized, and in the accompanying drawings:

[0079] Figure 1 A schematic diagram of a CAR-TCR-T system is shown, which includes an antigen binding domain as shown by a black and white striped oval and a black oval, wherein the antigen binding domain is capable of binding an antigen, such as a tumor-associated antigen.

[0080] Figure 2A Shown is a modified TCR complex comprising an antigen binding domain fused to the epsilon chain. Figure 2B Shown is a modified TCR complex comprising an antigen binding domain fused to a delta chain. Figure 2C Shown is a modified TCR complex comprising an antigen binding domain fused to a gamma chain. Figure 2D Shown is a modified TCR complex comprising an antigen binding domain fused to an alpha chain. Figure 2E Shown is a modified TCR complex comprising an antigen binding domain fused to a beta chain. Figure 2F A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the first epsilon chain, and the second antigen binding domain is fused to the second epsilon chain. Figure 2G A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the epsilon chain and the second antigen binding domain is fused to the gamma chain. Figure 2H Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to the epsilon chain. Fig.2I A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the α chain and the second antigen binding domain is fused to the β chain. Figure 2J A modified TCR complex comprising two identical antigen binding domains is shown. The first antigen binding domain is fused to the α chain and the second antigen binding domain is fused to the β chain. Figure 2K Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a delta chain. Figure 2L Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a gamma chain. Figure 2M Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to an alpha chain. Figure 2N Shown is a modified TCR complex comprising a TCR comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a beta chain. Fig.2O A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the epsilon chain and the second antigen binding domain is fused to the delta chain. Figure 2P A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the delta chain and the second antigen binding domain is fused to the gamma chain. Figure 2Q A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the α chain and the second antigen binding domain is fused to the ε chain. Figure 2R A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the beta chain and the second antigen binding domain is fused to the epsilon chain. Figure 2S A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the α chain and the second antigen binding domain is fused to the γ chain. Figure 2T A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the beta chain and the second antigen binding domain is fused to the gamma chain. Figure 2U A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the alpha chain and the second antigen binding domain is fused to the delta chain. Figure 2V A modified TCR complex comprising two different antigen binding domains is shown. The first antigen binding domain is fused to the beta chain and the second antigen binding domain is fused to the delta chain.

[0081] Figure 3 A CAR is shown that comprises an antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain).

[0082] Figure 4A Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to the epsilon chain. Figure 4B Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a delta chain. Figure 4C Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a gamma chain. Figure 4DShown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which is fused to a third antigen binding domain, which in turn is fused to the epsilon chain. Figure 4E Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which is fused to a third antigen binding domain, which in turn is fused to a delta chain. Figure 4F Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which is fused to a third antigen binding domain, which in turn is fused to a gamma chain. Figure 4G A modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which is in turn fused to the epsilon chain, and further comprising a third antigen binding domain fused to a fourth antigen binding domain, which is in turn fused to the delta chain. Figure 4H A modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which is in turn fused to the epsilon chain, and further comprising a third antigen binding domain fused to a fourth antigen binding domain, which is in turn fused to the gamma chain. Fig. 4I A modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a δ chain, and further comprising a third antigen binding domain fused to a fourth antigen binding domain, which in turn is fused to a γ chain. Figure 4J A modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to the epsilon chain, and the modified TCR complex also includes a third antigen binding domain fused to the gamma chain. Figure 4KA modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which is fused to a third antigen binding domain, which is in turn fused to an epsilon chain, and further comprising a fourth antigen binding domain fused to a fifth antigen binding domain, which is fused to a sixth antigen binding domain, which is in turn fused to a delta chain. Figure 4L A modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which is fused to a third antigen binding domain, which is in turn fused to the epsilon chain, and further comprising a fourth antigen binding domain fused to a fifth antigen binding domain, which is fused to a sixth antigen binding domain, which is in turn fused to the gamma chain. Figure 4M A modified TCR complex is shown, comprising a first antigen binding domain fused to a second antigen binding domain, which is fused to a third antigen binding domain, which is in turn fused to a delta chain, and further comprising a fourth antigen binding domain fused to a fifth antigen binding domain, which is fused to a sixth antigen binding domain, which is in turn fused to a gamma chain.

[0083] Figure 5A Shown is a modified TCR complex comprising a first antigen binding domain fused to an epsilon chain and a CAR comprising a second antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain). Figure 5B Shown is a modified TCR complex comprising a first antigen binding domain fused to a delta chain and a CAR comprising a second antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain). Figure 5C Shown is a modified TCR complex comprising a first antigen binding domain fused to a gamma chain and a CAR comprising a second antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain). Figure 5DShown is a modified TCR complex comprising a first antigen binding domain fused to an epsilon chain, a second antigen binding domain fused to a delta chain, and a CAR comprising a third antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain). Figure 5E Shown is a modified TCR complex comprising a first antigen binding domain fused to an epsilon chain, a second antigen binding domain fused to a gamma chain, and a CAR comprising a third antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain). Fig. 5F Shown is a modified TCR complex comprising a first antigen binding domain fused to a δ chain, a second antigen binding domain fused to a γ chain, and a CAR comprising a third antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-ζ signaling chain). Figure 5G Shown is a modified TCR complex comprising a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to the epsilon chain; and a CAR comprising a third antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain). Figure 5H Shown is a modified TCR complex comprising a first antigen binding domain fused to an epsilon chain, and a CAR comprising a second antigen binding domain fused to a third antigen binding domain fused to a transmembrane domain and an intracellular signaling domain (e.g., a CD3-zeta signaling chain).

[0084] Fig. 6A Vector constructs of anti-BCMAε TCR are shown. Figure 6B The vector construct of the anti-BCMA-4-1BB-CD3ζCAR is shown. Figure 6C Vector constructs of anti-BCMA or CD19-ε TCR and anti-CD19 or BCMA-4-1BB-CD3ζ CAR are shown. Fig.6D Vector constructs of anti-BCMA or CD19γ or δ TCR and anti-CD19 or BCMA-4-1BB-CD3ζ CAR are shown. Fig. 6E Vector constructs of tandem anti-BCMAε TCRs are shown. Fig. 6F Vector constructs of tandem anti-BCMAε TCR and tandem anti-CD19 or BCMA-4-1BB-CD3ζ CAR are shown.

[0085] Figure 7 CD19 and BCMA expression levels on different tumor cells and engineered cell lines are shown.

[0086] Fig. 8A , Figure 8B and Figure 8C Shown are the results of cytotoxicity assays at day 3 or day 6 post-transduction, where anti-BCMA antibodies (BCMA1-12) were fused to ε-TCR at effector to target cell ratios (E:T) of 0.5:1, 1.5:1, and 3:1. Fig.8D , Fig. 8E and 8F shows that by using HTRF Fig. 8A , Figure 8B and Figure 8C The amount of IFNγ in the supernatant collected in the cytotoxicity assay.

[0087] Fig. 9 Shown are the results of cytotoxicity assays on day 6 post-transfection, where the anti-BCMA systems: anti-BCMA3-ε-TCR (BCMA3 eTCR), anti-BCMA2-ε-TCR (BCMA2 eTCR), anti-BCMA2-anti-BCMA3-εTCR (tandem BCMA2&3eTCR), and anti-BCMA1-anti-BCMA2-anti-BCMA3-γTCR (tandem BCMA1&2&3gTCR), as well as control untransduced cells were co-cultured with RPMI-8226 cells (BCMA+) at an effector to target cell ratio (E:T) of 0.33:1.

[0088] Fig.10 The results of cytotoxicity assays on day 6 post-transfection are shown, where the anti-BCMA system, anti-BCMA2-anti-BCMA3ε-TCR (tandem BCMA 2-3eTCR), anti-BCMA4-anti-BCMA5ε-TCR (tandem BCMA 4-5eTCR), anti-BCMA2-anti-BCMA3-anti-BCMA4ε-TCR (tandem BCMA 2-3-4eTCR), and control untransduced cells were co-cultured with CHO / BCMA / CD19 cells (BCMA+CD19+) at effector to target cell ratios (E:T) of 1.5:1 and 0.5:1.

[0089] Fig.11AShown are the results of cytotoxicity assays at day 11 post-transfection, where anti-BCMA and / or anti-CD19 systems: anti-BCMA1ε-TCR (BCMA1 eTCR), anti-BCMA1 4-1BB-CD3ζ-CAR (BCMA1 BBzCAR), anti-CD19ε-TCR (CD19 eTCR), and anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), as well as control untransduced cells were co-cultured with NCI-H929 cells (BCMA+) at effector to target cell ratios (E:T) of 10:1 and 5:1. Fig. 11B shows that by using HTRF Fig.11A The amount of IFNγ in the supernatant collected in the cytotoxicity assay.

[0090] Fig. 12A The results of cytotoxicity assays on day 6 post-transfection are shown, in which anti-BCMA and / or anti-CD19 systems: anti-BCMA1-ε-TCR (BCMA eTCR), anti-BCMA1-4-1BB-CD3ζ-CAR (BCMA BBzCAR), anti-CD19-4-1BB-CD3ζCAR (CD19 BBzCAR), anti-CD19-εTCR (CD19 eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA BBzCAR) and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA eTCR / CD19BBzCAR) as well as control untransduced cells were co-cultured with CHO / BCMA / CD19 cells (BCMA+ and CD19+) at effector to target cell ratios (E:T) of 20:1, 10:1 and 5:1. Fig. 12B shows that by using HTRF Fig. 12A The amount of IFNγ in the supernatant collected in the cytotoxicity assay.

[0091] Fig.13AShown are the results of cytotoxicity assays at day 5 post-transfection, where anti-BCMA and / or anti-CD19 systems: anti-CD19ε-TCR (CD19 eTCR), anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19eTCR / BCMA1 BBzCAR) and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1 eTCR / CD19 BBzCAR), as well as control untransduced cells were co-cultured with CHO-BCMA-CD19 cells (BCMA+ and CD19+) at effector to target cell ratios (E:T) of 10:1 and 5:1. Fig. 13B Shown are the results of cytotoxicity assays on day 5 post-transfection, where anti-BCMA and / or anti-CD19 systems: anti-BCMA1-εTCR (BCMA1 eTCR), anti-BCMA1-4-1BB-CD3ζCAR (BCMA1 BBzCAR), anti-BCMA1 / anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19eTCR / BCMA1 BBzCAR), and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1eTCR / CD19BBzCAR), as well as control untransduced cells were co-cultured with NCI-H929 cells (BCMA+) at effector to target cell ratios (E:T) of 2.5:1 and 5:1.

[0092] Fig.14A Shown are the results of cytotoxicity assays on day 6 post-transfection, where anti-BCMA and / or anti-CD19 systems: anti-BCMA1ε-TCR (BCMA1 eTCR), anti-CD19-εTCR / anti-BCMA1-γTCR (CD19eTCR / BCMA1 gTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1 BBzCAR), and control untransduced cells were co-cultured with CHO-BCMA1-CD19 cells (BCMA+ and CD19+) at an effector to target cell ratio (E:T) of 1.3:1. Fig. 14B and Fig. 14C shows that by using HTRF Fig.14AThe cytotoxicity was determined by measuring the amount of IFNγ and TNFα in the collected supernatants.

[0093] Fig.15A The results of cytotoxicity assays on day 6 post-transfection are shown, in which anti-BCMA and / or anti-CD19 systems: anti-BCMAε-TCR (BCMA eTCR), anti-CD19-εTCR / anti-BCMA-4-1BB-CD3ζCAR (CD19eTCR / BCMABBzCAR), anti-BCMA and anti-CD19-ε-TCR (tandem BCMA / CD19 dTCR), and control untransduced cells were co-cultured with CHO-BCMA-CD19 cells (BCMA+ and CD19+) at an effector to target cell ratio (E:T) of 1.3:1. Fig. 15B and Fig. 15C shows that by using HTRF Fig.15A The cytotoxicity was determined by measuring the amount of IFNγ and TNFα in the collected supernatants.

[0094] Fig.16A The results of cytotoxicity assays on day 4 post-transfection are shown, where the anti-BCMA systems: anti-BCMA2ε-TCR (BCMA2 eTCR), anti-BCMA2-εTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2eTCR / BCMA3 BBzCAR), anti-BCMA2-γTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2gTCR / BCMA3 BBzCAR), anti-BCMA2-δTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2dTCR / BCMA3 BBzCAR), and control untransduced cells were co-cultured with RPMI-8226 cells (BCMA+) at an effector to target cell ratio (E:T) of 0.5:1. Fig. 16B shows that by using HTRF Fig.16A The amount of IFNγ in the collected supernatant was determined for cytotoxicity.

[0095] Fig.17A The results of cytotoxicity assays on day 6 post-transfection are shown, where the anti-BCMA system: anti-BCMA2-anti-BCMA3ε-TCR / anti-BCMA2-anti-BCMA3γ-TCR (tandem BCMA2&3eTCR / gTCR), anti-BCMA2-anti-BCMA3γ-TCR / anti-BCMA2-anti-BCMA3 4-1BB-CD3ζCAR (tandem BCMA2&3gTCR / BBzCAR), and control untransduced cells were co-cultured with RPMI-8226 cells (BCMA+) at an effector to target cell ratio (E:T) of 0.33:1. Fig. 17Bshows that by using HTRF Fig.17A The amount of IFNγ in the collected supernatant was determined for cytotoxicity.

[0096] Fig.18A and Fig.18B Shown are the in vivo anti-tumor efficacy of triple-specific BCMA CAR-T cells, triple-specific BCMA TCR-T cells, and triple-specific BCMA CAR-TCR-T cells evaluated in the NCG mouse model with multiple myeloma tumor xenografts (NOD_Prkdcem26Cd52 / NjuCrl).

[0097] Fig.19 Figure 2 shows the in vivo anti-tumor efficacy of anti-MSLN / FSHR dual CAR-T (MSLN CAR+FSHR CAR), anti-MSLN / FSHR dual eTCR-T (MSLN eTCR+FSHR eTCR), and anti-MSLN CAR / FSHR eTCR-T (MSLN CAR+FSHR eTCR) evaluated in the OVCAR-8 xenograft model. 6 OVCAR-8 cells were implanted subcutaneously into NODscidγ (NSG) mice. Once the tumor was 150-200 mm 3 The mice were randomly divided into treatment groups. A 200 μl dose of 0.33×10 6 The mice and their tumors were monitored for approximately 60 days after tumor cell implantation. DETAILED DESCRIPTION

[0098] Unless otherwise indicated, the practice of some methods disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, Fourth Edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel et al., eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds., (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition (RI Freshney, ed. (2010)).

[0099] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "antigen binding domain" includes a plurality of antigen binding domains.

[0100] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. Where a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range for that particular value unless otherwise stated.

[0101] As used herein, "cell" may generally refer to a biological cell. A cell may be the basic structural, functional and / or biological unit of a living organism. A cell may be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkins, hay, potatoes, cotton, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopods, hornworts, liverworts, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum splayleaf variants), patensC.Agardh, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes, the cell is not derived from a natural organism (e.g., the cell can be synthetically prepared, sometimes also referred to as an artificial cell).

[0102] As used herein, the term "T cell" or "T lymphocyte" refers to a type of lymphocyte that plays a key role in cell-mediated immunity. T cells can be distinguished from other lymphocytes (such as B cells and natural killer cells) by the presence of T cell receptors on the cell surface.

[0103] As used herein, the term "T cell receptor" or "TCR" refers to a molecule responsible for identifying antigens on the surface of a T cell or T lymphocyte. TCR is a heterodimer consisting of two different protein chains. In some embodiments, the TCR of the present disclosure is composed of an alpha (α) chain and a beta (β) chain, and is referred to as an αβTCR. αβTCR recognizes antigenic peptides degraded from proteins bound to the major histocompatibility complex molecule (MHC) at the cell surface. In some embodiments, the TCR of the present disclosure is composed of a gamma (γ) chain and a delta (δ) chain, and is referred to as a γδTCR. γδTCR recognizes peptides and non-peptide antigens in a non-MHC-dependent manner. γδT cells have been shown to play an important role in identifying lipid antigens. Specifically, the γ chain of TCR includes but is not limited to Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10, their functional variants, and combinations thereof; and the δ chain of TCR includes but is not limited to δ1, δ2, δ3, their functional variants, and combinations thereof. In some embodiments, the γδTCR can be a Vγ2 / Vδ1TCR, a Vγ2 / Vδ2TCR, a Vγ2 / Vδ3TCR, a Vγ3 / Vδ1TCR, a Vγ3 / Vδ2TCR, a Vγ3 / Vδ3TCR, a Vγ4 / Vδ1TCR, a Vγ4 / Vδ2TCR, a Vγ4 / Vδ3TCR, a Vγ5 / Vδ1TCR, a Vγ5 / Vδ2TCR, a Vγ5 / Vδ3TCR, a Vγ8 / Vδ1TCR, a Vγ8 / Vδ2TCR, a Vγ8 / Vδ3TCR, a Vγ9 / Vδ1TCR, a Vγ9 / Vδ2TCR, a Vγ9 / Vδ3TCR, a Vγ10 / Vδ1TCR, a Vγ10 / Vδ2TCR, and / or a Vγ10 / Vδ3TCR. In some examples, the γδ TCR can be a Vγ9 / Vδ2 TCR, a Vγ10 / Vδ2 TCR, and / or a Vγ2 / Vδ2 TCR.

[0104] As used herein, the terms "alpha beta T cells," "αβ T cells," and "AB T cells" are used interchangeably and refer to T cells (T lymphocytes) containing an αβ TCR or a variant or fragment thereof, and the terms "gamma delta T cells," "γδ T cells," and "GD T cells" are used interchangeably and refer to T cells (T lymphocytes) containing a γδ TCR or a variant or fragment thereof, e.g., Vγ9δ2 T cells, Vδ1 T cells, Vδ3 T cells, or Vδ5 T cells. In some embodiments, the γδT cells can be Vγ2 / Vδ1T cells, Vγ2 / Vδ2T cells, Vγ2 / Vδ3T cells, Vγ3 / Vδ1T cells, Vγ3 / Vδ2T cells, Vγ3 / Vδ3T cells, Vγ4 / Vδ1T cells, Vγ4 / Vδ2T cells, Vγ4 / Vδ3T cells, Vγ5 / Vδ1T cells, Vγ5 / Vδ2T cells, Vγ5 / Vδ3T cells, Vγ8 / Vδ1T cells, Vγ8 / Vδ2T cells, Vγ8 / Vδ3T cells, Vγ9 / Vδ1T cells, Vγ9 / Vδ2T cells, Vγ9 / Vδ3T cells, Vγ10 / Vδ1T cells, Vγ10 / Vδ2T cells and / or Vγ10 / Vδ3T cells. In some examples, the γδ T cells may be Vγ9 / Vδ2 T cells, Vγ10 / Vδ2 T cells, and / or Vγ2 / Vδ2 T cells.

[0105] As used herein, the term "activation" and its grammatical equivalents may refer to the process by which a cell is transformed from a quiescent state to an active state. The process may include a response to an antigen, migration, and / or a phenotypic or genetic change to a functionally active state. For example, the term "activation" may refer to the stepwise process of T cell activation. In some cases, T cells may require at least two signals to become fully activated. The first signal may occur after the antigen-MHC complex binds to the TCR, and the second signal may occur by the combination of co-stimulatory molecules. In some cases, anti-CD3 may simulate the first signal and anti-CD28 may simulate the second signal in vitro.

[0106] As used herein, the term "antigen" refers to a molecule or fragment thereof that can be bound by a selective binding agent. For example, an antigen can be a ligand that can be bound by a selective binding agent, such as a receptor. In some cases, a receptor can serve as an antigen, and a ligand can serve as a selective binding agent. As another example, an antigen can be an antigen molecule that can be bound by a selective binding agent, such as an immune protein (e.g., an antibody). In some cases, an immune protein can serve as an antigen, and an antigen molecule can serve as a selective binding agent. Antigen can also refer to a molecule or fragment thereof that can be used in an animal to produce an antibody that can bind to the antigen.

[0107] As used herein, the term "epitope" and its grammatical equivalents may refer to a portion of an antigen that can be recognized by an antigen binding domain. The antigen binding domain may include, for example, a protein (e.g., an antibody, an antibody fragment) present on a surface, such as a cell (e.g., a B cell, a T cell, a CAR-T cell, or an engineered cell). For example, an epitope may be a cancer epitope recognized by a TCR. Multiple epitopes within an antigen may also be recognized. An epitope may also be mutated.

[0108] As used herein, the term "antigen binding molecule" refers to a molecule that specifically binds to an antigen or epitope. Examples of antigen binding molecules include, but are not limited to, antibodies and their derivatives, such as fragments thereof. "Specific binding" means that the binding is selective for the antigen or epitope and can be distinguished from unwanted or non-specific interactions.

[0109] The term "binding affinity" as used herein refers to the strength of the binding interaction between members of a binding pair (eg, an antigen binding molecule and its antigen, or a receptor and its ligand).

[0110] The binding affinity of a subject antibody to its partner can be measured by k on , k off or K D As used herein, the term "k on " refers to the rate constant for the association of an antibody with an antigen. As used herein, the term "k off " refers to the rate constant for the dissociation of an antibody from an antibody / antigen complex. As used herein, the term "K D " refers to the equilibrium dissociation constant of the antibody-antigen interaction. For the purposes of this disclosure, K D Defined as two kinetic rate constants k on / k off The smaller the equilibrium dissociation constant, the more tightly the subject antibody and its partner bind to each other.

[0111] As used herein, the term "antibody" refers to a protein binding molecule with an immunoglobulin-like function. The term antibody includes antibodies (e.g., monoclonal antibodies and polyclonal antibodies), as well as derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc.). Its derivatives, variants or fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., completely and / or partially) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), mini antibodies, double antibodies and single domain antibodies ("sdAb" or "nanobody" or "camelids" or VH H). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically conjugated. Examples of optimized antibodies include affinity-matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies).

[0112] As used herein, the term "antigen binding domain" refers to a protein or fragment thereof that is capable of binding an antigen or epitope. As an example, an antigen binding domain can be a cell receptor. As an example, an antigen binding domain can be an engineered cell receptor. As an example, an antigen binding domain can be a soluble receptor. In some cases, an antigen binding domain can be a ligand that binds to a cell receptor, an engineered cell receptor, and / or a soluble receptor.

[0113] As used herein, the term "autologous" and its grammatical equivalents can refer to originating from the same source. For example, an autologous sample (e.g., a cell) can refer to a sample that is removed at a later time, processed, and then given back to the same subject (e.g., a patient). In terms of the process, autologous can be distinguished from an allogeneic process in which the donor (e.g., cell) of the sample is not the same subject as the recipient of the sample.

[0114] As used herein, the terms "cancer neoantigens," "neoantigens," and "neo-epitopes," and their grammatical equivalents, may refer to antigens that are not encoded in a normal, non-mutated host genome. In some cases, "neoantigens" may represent oncogenic viral proteins or abnormal proteins produced due to somatic mutations. For example, neoantigens may be produced by the activity of viral proteins that disrupt cellular machinery. As another example, neoantigens may be produced due to exposure to carcinogenic compounds, which in some cases may result in somatic mutations. Such somatic mutations may lead to the formation of tumors / cancers.

[0115] As used herein, the term "cytotoxicity" refers to an unexpected or undesirable change in the normal state of a cell. The normal state of a cell may refer to a state that is present or present before the cell is exposed to a cytotoxic composition, agent, and / or condition. A cell in a normal state may be in a steady state. An unexpected or undesirable change in the normal state of a cell may be manifested, for example, in the form of cell death (e.g., programmed cell death), reduced replication potential, reduced cell integrity (such as membrane integrity), reduced metabolic activity, reduced developmental capacity, or any of the cytotoxic effects disclosed herein.

[0116] As used herein, the phrases "reduce cytotoxicity" and "reduce cytotoxicity" refer to a decrease in the degree or frequency of unexpected or undesirable changes in the normal state of a cell when exposed to a cytotoxic composition, agent, and / or condition. The phrase can refer to a decrease in the degree of cytotoxicity of a single cell exposed to a cytotoxic composition, agent, and / or condition, or a decrease in the number of cells in a population that exhibit cytotoxicity when a population of cells is exposed to a cytotoxic composition, agent, and / or condition.

[0117] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0118] When used herein with respect to polypeptides, the terms "derivatives", "variants" and "fragments" refer to polypeptides related to wild-type polypeptides, such as polypeptides related to wild-type polypeptides by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Derivatives, variants and fragments of polypeptides may comprise one or more amino acid variants (e.g., mutations, insertions and deletions), truncations, modifications or combinations thereof compared to wild-type polypeptides.

[0119] As used herein, the term "percent identity (%)" refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be ignored for comparison purposes). To determine the percent identity, alignment may be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. The percent identity of two sequences may be calculated by the following method: aligning a test sequence with a comparison sequence using BLAST; determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acid or nucleotide at the same position in the comparison sequence; and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0120] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, competition animals and pets. It also includes tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

[0121] As used herein, the terms "treat" and "treating" refer to methods for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. For example, treatment can include administering a system or cell population disclosed herein. Therapeutic benefit can refer to any therapeutically relevant improvement or effect on one or more diseases, disorders, or symptoms being treated. For prophylactic benefit, the composition can be administered to a subject at risk for developing a particular disease, disorder, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, disorder, or symptom may not yet be manifested.

[0122] If the condition being treated changes, a "therapeutic effect" may occur. The change can be positive or negative. For example, a "positive effect" can correspond to an increase in the number of activated T cells in a subject. In another example, a "negative effect" can correspond to a decrease in the number or size of tumors in a subject. A "change" in the condition being treated can refer to a change of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75%, or 100% of the condition. The change can be based on an improvement in the severity of the individual condition being treated, or a difference in the frequency of improved conditions in a population of individuals with and without the administration of therapy. Similarly, the methods of the present disclosure may include administering a "therapeutically effective" amount of cells to a subject. The term "therapeutically effective" should be understood to have a definition corresponding to "having a therapeutic effect".

[0123] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition sufficient to produce a desired activity when administered to a subject in need thereof, such as an amount of a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells). The term "therapeutically effective" may refer to an amount of a composition sufficient to delay the manifestation of a disorder treated by the methods of the present disclosure, prevent its progression, alleviate or mitigate at least one symptom thereof.

[0124] As used herein, the term "TIL" or tumor infiltrating lymphocytes and grammatical equivalents thereof may refer to cells isolated from a tumor. TIL may be any cell found within a tumor. For example, TIL may be a cell that has migrated to a tumor. TIL may be a cell that has infiltrated a tumor. TIL may be a T cell, a B cell, a monocyte, a natural killer (NK) cell, or any combination thereof. TIL may be a mixed population of cells. A TIL population may include cells of different phenotypes, cells of different degrees of differentiation, cells of different lineages, or any combination thereof.

[0125] The term "B cell maturation antigen (BCMA or BCM)", also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), refers to the protein encoded by the TNFRSF17 gene in humans. BCMA is preferentially expressed in mature B lymphocytes and has been shown to play an important role in B cell development and autoimmune responses. BCMA is also considered a tumor-associated antigen, and abnormal expression of BCMA is also associated with many cancers, as well as autoimmune disorders and infectious diseases.

[0126] In one aspect, provided herein are antigen binding molecules having the formula AXBYCZD. In some embodiments, the disclosure provides antigen binding molecules having the formula AXBYCZD, and the A comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the group consisting of SEQ ID NO: 47 to SEQ ID NO: 56. In some embodiments, the disclosure provides antigen binding molecules having the formula AXBYCZD, and the B comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the group consisting of SEQ ID NO: 57 to SEQ ID NO: 66. In some embodiments, the present disclosure provides an antigen binding molecule having the formula AXBYCZD, and the C comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of the group consisting of SEQ ID NO: 67 to SEQ ID NO: 76. In some embodiments, the present disclosure provides an antigen binding molecule having the formula AXBYCZD, and the D comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of the group consisting of SEQ ID NO: 77 to SEQ ID NO: 86. In some embodiments, the present disclosure provides an antigen binding molecule having the formula AXBYCZD, and the X comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of the group consisting of SEQ ID NO: 87 to SEQ ID NO: 96. In some embodiments, the present disclosure provides an antigen binding molecule having the formula AXBYCZD, and the Y comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of the group consisting of SEQ ID NO: 97 to SEQ ID NO: 106. In some embodiments, the present disclosure provides an antigen binding molecule having the formula AXBYCZD, and Z comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one selected from the group consisting of SEQ ID NO: 107 to SEQ ID NO: 116.

[0127] Table 1 Sequences of A of antigen-binding molecules

[0128] SEQ ID NO A 47 QVQLVESGGGSVQAGGSLRLSCKAS 48 QVQLEESGGGSVQAGGSLRLSCAYT 49 QMQLVESGGGSVQAGGSLRLSCTAS 50 QVHLMESGGGSVQSGGSLRLSCAAS 51 QVQLVESGGGSVQAGGSLRLSCAAS 52 QVQLVESGGGSVQAGGSLRLSCKSS 53 QVQLAESGGGLVQPGGSLRLSCAGS 54 QVQLVESGGGVVQPGGSLRLSCAAS 55 QVHLVESGGGSVQAGGSLRLSCKSS 56 QVHLVESGGGSVQAGGSLRLSCKAS

[0129] Table 2 Sequences of antigen-binding molecules B

[0130]

[0131]

[0132] Table 3 Sequences of C of antigen-binding molecules

[0133] SEQ ID NO C 67 RFTISRDNAKNTMYLQMNSLEPEDTAMYYCAA 68 RFTISKDNAKNTLYLQMNSLKPEDSAMYRCAA 69 RFTISQDNAKNTMYLQMNSLKPEDTAVYSCAA 70 RFTISQDNAKNTLYLQMNSLKPEDTAMYYCGA 71 RFTISQDTAQNTLYLQMNSLKPEDTAMYYCAA 72 RFTISRDNAKNTMYLQMNSLKPEDTAMYYCAA 73 RFTASRDKAKNTLYLQMNSLKTEDTAVYYCAA 74 RFTISRDNAKNTLYLQLNNLKSEDTAVYYCSE 75 RFTISRDNAKNTMYLQMSGLRPEDTALYYCAA 76 RFTISRDNAKNTMYLQMNSLKPEDTAMYYCAA

[0134] Table 4 Sequences of D of antigen-binding molecules

[0135]

[0136]

[0137] Table 5 Sequences of X in antigen-binding molecules

[0138] SEQ ID NO X 87 GAIYDTNCMA 88 YSTYSNYYMG 89 GYTFDDSAMG 90 GYTYSSYCMA 91 GGTRSWNYMA 92 GAPYSSNCMA 93 GFTFSSYDMN 94 GFAFSNYAMT 95 GATYSSNCMA 96 GAIYDTNCMA

[0139] Table 6 Sequences of Y in antigen-binding molecules

[0140] SEQ ID NO Y 97 TIDLGNPITYYADSVKG 98 IISSDTTITYKDAVKG 99 SISSDGSTYYSDSVKG 100 AIASDGSTYYTDSVKG 101 IIDNVGSTRYADSVKG 102 TIDLASHDTYYADSVKG 103 TTFNGDDGTNYADSVLG 104 TIDSGGGSTTYSDSVKG 105 TIDLASHGTYYADSVKG 106 TIDLGNPITYYADSVKG

[0141] Table 7 Sequences of Z in antigen-binding molecules

[0142]

[0143]

[0144] In some embodiments, the present disclosure provides an antigen binding molecule having the formula AXBYCZD, wherein A comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 47 to SEQ ID NO: 56, B comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 66, C comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 67 to SEQ ID NO: 76, D comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 77 to SEQ ID NO: 86, X comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 87 to SEQ ID NO: 96, Y comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 97 to SEQ ID NO: 106, and Z comprises a sequence having at least 80% or 90% identity to any one selected from the group consisting of SEQ ID NO: 106. Any one of the group consisting of SEQ ID NO: 107 to SEQ ID NO: 116 has a sequence with at least 80% or 90% identity.

[0145] In some embodiments, the antigen binding molecule exhibits a binding affinity (K) for human BCMA D In some embodiments, K is as measured by surface plasmon resonance at 37°C. D Less than 100nm, 90nm, 80nm, 70nm, 60nm, 50nm, 40nm, 30nm, 20nm, 10nm, or 1nm or less.

[0146] In some embodiments, the antigen binding molecule comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the group consisting of SEQ ID NO: 14 to SEQ ID NO: 23. In some embodiments, the antigen binding molecule comprises a sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 23.

[0147] In one aspect, the present disclosure provides a modified T cell receptor (TCR) complex, which comprises an antigen binding domain that exhibits specific binding to an epitope, wherein the antigen binding domain is connected to: (i) at least one TCR chain, which is selected from the α chain, β chain, γ chain and δ chain of TCR; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0148] In some embodiments, an antigen binding domain may comprise one member of an interaction pair. For example, an antigen binding domain may be one member of an interaction pair comprising a receptor and a ligand, or a fragment thereof. A receptor or a ligand, or a fragment thereof, may be referred to as an antigen binding domain. Another member not referred to as an antigen binding domain may comprise an epitope to which the antigen binding domain specifically binds.

[0149] Non-limiting examples of antigen-binding domains of TCR complexes include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or functional derivatives, variants or fragments thereof, including, but not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), miniantibodies, diabodies, and single-domain antibodies (such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VL) of camelid-derived nanoantibodies). H H)). In some embodiments, the antigen binding domain of the TCR complex comprises at least one of Fab, Fab', F(ab')2, Fv and scFv. In some embodiments, the antigen binding domain of the TCR complex comprises an antibody mimetic. Antibody mimetic refers to a molecule that can bind to a target molecule with an affinity comparable to that of an antibody, and includes single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), lipocalin scaffolds, calixarene skeletons, A domains, and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant, or fragment thereof. For example, the antigen binding domain may comprise at least the ligand binding domain of a transmembrane receptor.

[0150] In some embodiments, provided herein is a modified T cell receptor (TCR) complex comprising one or more antigen binding domains, wherein the one or more antigen binding domains are linked to: (i) at least one TCR chain, wherein the at least one TCR chain is selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain; and wherein at least one or two of the one or more antigen binding domains comprise an antigen binding molecule described herein.

[0151] In some embodiments, at least one antigen binding domain of the modified TCR complex comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the group consisting of SEQ ID NO:3 to SEQ ID NO:23 and SEQ ID NO:38 to SEQ ID NO:46.

[0152] In some embodiments, the antigen binding domain of the TCR complex comprises a single domain antibody. In some embodiments, the single domain antibody is an anti-BCMA sdAb disclosed herein. In some embodiments, the anti-BCMA sdAb comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the group consisting of SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the anti-BCMA sdAb comprises a sequence selected from any one of SEQ ID NO: 3 to SEQ ID NO: 23.

[0153] The antigen binding domain of the modified TCR complex can be connected to any member of the TCR complex. In some embodiments, the antigen binding domain may be connected to at least one of the TCR chain, CD3 chain or CD3 ζ chain. In some embodiments, the antigen binding domain may be connected to the transmembrane receptor of the TCR, such as TCR-ε, TCR-δ, TCR-γ, TCR-α or TCR-β. In some embodiments, the antigen binding domain may be connected to a CD3 chain, such as CD3-ε, CD3-δ or CD3-γ. In some embodiments, the antigen binding domain may be connected to a CD3 ζ chain.

[0154] The modified T cell receptor (TCR) complex of the present disclosure may include a second antigen binding domain that exhibits binding to a second epitope. The second antigen binding domain may include any protein or molecule capable of binding to an epitope. Non-limiting examples of the second antigen binding domain of the TCR complex include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or functional derivatives, variants, or fragments thereof, including, but not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), miniantibodies, double antibodies, and single-domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VL) of nanoantibodies of camelid origin). HH)). In some embodiments, the second antigen binding domain of the TCR complex comprises at least one of Fab, Fab', F(ab')2, Fv and scFv. In some embodiments, the second antigen binding domain of the TCR complex comprises an antibody mimetic. Antibody mimetic refers to a molecule that can bind to a target molecule with an affinity comparable to that of an antibody, and includes single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), lipocalin scaffolds, calixarene backbones, A domains, and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant, or fragment thereof. For example, the antigen binding domain may comprise at least the ligand binding domain of a transmembrane receptor.

[0155] In some embodiments, the second antigen binding domain of the TCR complex comprises an antigen binding molecule disclosed herein. In some embodiments, the second antigen binding domain of the TCR complex comprises a single domain antibody. In some embodiments, the single domain antibody is an anti-BCMA sdAb. In some embodiments, the anti-BCMA sdAb comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the groups consisting of SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the anti-BCMA sdAb comprises a sequence selected from any one of SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the second antigen binding domain of the TCR complex comprises a sequence having at least 80% or 90% identity to any one of the groups consisting of SEQ ID NO: 38 to SEQ ID NO: 46.

[0156] The second antigen binding domain can be connected to any member of the TCR complex. In some embodiments, the second antigen binding domain can be connected to at least one of the TCR chain, differentiation 3 (CD3) chain or CD3 ζ chain. The second antigen binding domain can be connected to the transmembrane receptor of TCR, such as TCR-ε, TCR-δ, TCR-γ, TCR-α or TCR-β. The second antigen binding domain can be connected to CD3 chain, such as CD3-ε, CD3-δ or CD3-γ. The second antigen binding domain can be connected to CD3 ζ chain.

[0157] In some embodiments, the two or more antigen binding domains are connected to separate chains of the TCR complex. In some embodiments, the two or more antigen binding domains are connected to one chain of the TCR complex. Any number of antigen binding domains can be used in the modified TCR complex of the present disclosure, and the number of antigen binding domains is not limited to one, two or three.

[0158] In some embodiments, two or more antigen binding domains can be the same antigen binding domain. For example, two or more antigen binding domains can be the same molecule capable of binding the same ligand. In some embodiments, two or more antigen binding domains can be different antigen binding domains. For example, two or more antigen binding domains can be different molecules capable of binding the same ligand or different ligands.

[0159] In some embodiments, two or more antigen binding domains are connected in series to (i) at least one TCR chain, the at least one TCR chain selected from the alpha chain, beta chain, gamma chain and delta chain of the T cell receptor; (ii) the epsilon chain, delta chain and / or gamma chain of cluster of differentiation 3 (CD3); or (iii) CD3 zeta chain. In some embodiments, two or more antigen binding domains are connected in series to at least one of the epsilon chain, delta chain and / or gamma chain of cluster of differentiation 3 (CD3). In some embodiments, two or more antigen binding domains are connected in series to separate chains of the TCR complex. In some embodiments, two or more antigen binding domains are connected in series to a chain of a modified TCR complex. In some embodiments, two or more antigen binding domains are connected in series to two or more chains of a modified TCR complex.

[0160] In some embodiments, the modified TCR complex of the present disclosure comprises two or more sdAbs, which are connected to: (i) at least one TCR chain, the at least one TCR chain is selected from the α chain, β chain, γ chain and δ chain of the T cell receptor, (ii) the ε chain, δ chain and / or γ chain of the cluster of differentiation 3 (CD3); or (iii) CD3ζ chain. In some embodiments, the modified TCR complex of the present disclosure comprises two or more sdAbs, which are connected in series to: (i) at least one TCR chain, the at least one TCR chain is selected from the α chain, β chain, γ chain and δ chain of the T cell receptor, (ii) the ε chain, δ chain and / or γ chain of the cluster of differentiation 3 (CD3); or (iii) CD3ζ chain. In some embodiments, the modified TCR complex of the present disclosure comprises two or more sdAbs connected in series to one chain of the modified TCR complex. In some embodiments, a modified TCR complex of the present disclosure comprises two or more sdAbs linked in tandem to two or more chains of the modified TCR complex.

[0161] In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA sdAbs, which are connected in series to: (i) at least one TCR chain, the at least one TCR chain is selected from the α chain, β chain, γ chain and δ chain of the T cell receptor, (ii) the ε chain, δ chain and / or γ chain of the cluster of differentiation 3 (CD3); or (iii) CD3ζ chain. In some embodiments, the two or more anti-BCMA sdAbs have the same sequence. In some embodiments, the two or more anti-BCMA sdAbs have different sequences. In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA sdAbs connected in series to one chain of the modified TCR complex. In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA sdAbs connected in series to two or more chains of the TCR complex.

[0162] In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA antigen-binding molecules disclosed herein, which are tandemly linked to: (i) at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor, (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain. In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA sdAbs, which are tandemly linked to: (i) at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor, (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain, and the anti-BCMA sdAb comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one selected from SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the two or more anti-BCMA sdAbs have the same sequence, and the sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one selected from SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the two or more anti-BCMA sdAbs have different sequences, and the sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one selected from SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA sdAbs, which are linked in series to one chain of the TCR complex, and the anti-BCMA sdAb comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one selected from SEQ ID NO: 3 to SEQ ID NO: 23. In some embodiments, the modified TCR complex of the present disclosure comprises two or more anti-BCMA sdAbs, which are linked in series to two or more chains of the TCR complex, and the anti-BCMA sdAb comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one selected from SEQ ID NO: 3 to SEQ ID NO: 23.

[0163] In some embodiments, two or more antigen binding domains of a modified TCR complex can bind to epitopes present on different antigens. In some embodiments, two or more antigen binding domains of a modified TCR complex can bind to epitopes present on a common antigen. In some embodiments, two or more antigen binding domains exhibit specific binding to two or more epitopes. In some embodiments, two or more antigen binding domains exhibit specific binding to the same epitope.

[0164] Therefore, the present invention also provides a modified T cell receptor (TCR) complex, the modified TCR complex comprising two or more antigen binding domains that exhibit specific binding to two or more epitopes, wherein the two or more antigen binding domains are connected to: (i) at least one TCR chain, the at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of the cluster of differentiation 3 (CD3); or (iii) CD3 ζ chain. In some embodiments, at least one or two of the two or more antigen binding domains are selected from the antigen binding domains or antigen binding molecules disclosed herein.

[0165] In some embodiments, the epitope to which the antigen binding domain of the modified TCR complex binds may be present on one or more cell surface antigens. The one or more cell surface antigens may be tyrosine kinase receptors, serine kinase receptors, histidine kinase receptors, G protein coupled receptors (GPCRs), etc.

[0166] In some embodiments, the epitope bound by the antigen binding domain of the modified TCR complex can be present on an immune checkpoint receptor or an immune checkpoint receptor ligand. In some embodiments, the immune checkpoint receptor or immune checkpoint receptor ligand can be PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, TIGIT, BLTA, CD47 or CD40.

[0167] In some embodiments, the epitope bound by the antigen binding domain of the modified TCR complex can be present on a cytokine or cytokine receptor. The cytokine receptor can be, for example, CCR2b, CXCR2 (CXCL1 receptor), CCR4 (CCL17 receptor), Gro-a, IL-2, IL-7, IL-15, IL-21, IL-12, heparanase, CD137L, LEM, Bcl-2, CCL17, CCL19 or CCL2.

[0168] In some embodiments, the epitope bound by the antigen binding domain of the modified TCR complex can be present on a tumor-associated antigen. The epitope can be, for example, a tumor epitope. The tumor-associated antigen can be selected from the group consisting of: 707-AP, biotinylated molecules, a-actinin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipose differentiation-related protein, AFP, AIM-2, annexin II, ART-4, BAGE, BCMA, b-catenin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-abl p210(b3a2), BING-4, CA-125, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7 / 8, CD70, CD123, CD133, CDC27, CDK-4, CEA, CLCA2, CLL-1, CTAG1B, Cyp-B, DAM-10, DAM-6, DEK-CAN, DLL3, EGFR, EGFRvIII, EGP-2, EGP-40, ELF2, Ep -CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FAP, FBP, fetal acetylcholine receptor, FGF-5, FN, FR-α, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, GPC3, GPC-2, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, LewisY, L1-CAM, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1 , OA1, OGT, carcinoembryonic antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survivin, survivin-2B, SYT / SSX, TAG-72, TEL / AML1, TGFαRII, TGFβRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ light chain. In some embodiments, the epitope bound by two or more antigen binding domains of the modified TCR complex can be EGFR, EGFRvIII, GPC3, GPC-2, DLL3, CD19, CD20, CD22, CD123, CLL-1, CD30, CD33, HER2, MSLN, PSMA, CEA, GD2, IL13Rα2, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, FR-α, CD70, CD171, ROR1, and any combination thereof.

[0169] In some embodiments, at least one antigen binding domain of the modified TCR complex binds to an epitope present on BCMA. In some embodiments, two or more antigen binding domains of the modified TCR complex bind to an epitope present on BCMA. In some embodiments, two or more antigen binding domains of the modified TCR complex bind to the same epitope of BCMA. In some embodiments, two or more antigen binding domains of the modified TCR complex bind to different epitopes of BCMA.

[0170] In some embodiments, two or more antigen binding domains of the modified TCR complex are connected in series to (i) at least one TCR chain selected from the group consisting of the alpha chain, beta chain, gamma chain, and delta chain of the T cell receptor; (ii) the epsilon chain, delta chain, and / or gamma chain of cluster of differentiation 3 (CD3); or (iii) the CD3 zeta chain, and at least one of the binding domains can bind to BCMA. In some embodiments, two or more antigen binding domains of the modified TCR complex are connected in series to (i) at least one TCR chain selected from the group consisting of the alpha chain, beta chain, gamma chain, and delta chain of the T cell receptor; (ii) the epsilon chain, delta chain, and / or gamma chain of cluster of differentiation 3 (CD3); or (iii) the CD3 zeta chain, and two or more of the binding domains can bind to BCMA. In some embodiments, two or more antigen binding domains of the modified TCR complex are connected in series to (i) at least one TCR chain, the at least one TCR chain is selected from the group consisting of the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain, and the two or more antigen binding domains can bind to the same epitope of BCMA. In some embodiments, two or more antigen binding domains of the TCR complex are connected in series to (i) at least one TCR chain, the at least one TCR chain is selected from the group consisting of the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain, and the two or more antigen binding domains can bind to different epitopes of BCMA.

[0171] In some embodiments, the epitope to which the antigen binding domain of the modified TCR complex binds can be present on a neoantigen. For example, the epitope can be a neoepitope.

[0172] Neoantigens and neoepitopes generally refer to tumor-specific mutations that trigger anti-tumor T cell responses in some cases. For example, whole genome sequencing methods can be used to identify these endogenous mutations. Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+T cells in a patient with epithelial cancer," Science 344:641-644 (2014). Antigen binding domains, such as the antigen binding domains of the subject CAR or modified TCR complexes, can exhibit specific binding to tumor-specific neoantigens. The neoantigens bound by the antigen binding domains of the modified TCR complexes can be expressed on target cells, and, for example, are neoantigens and neoepitopes encoded by mutations in any endogenous genes. In some cases, two or more antigen binding domains bind to neoantigens or neoepitopes encoded by mutant genes. The gene can be selected from the group consisting of: ABL1, ACO11997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2Μ, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, NY-ESO, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

[0173] In some embodiments, the epitope bound by the antigen binding domain of the modified TCR complex may be present on a matrix. The matrix generally refers to a tissue that provides connection and functional support, especially for biological cells, tissues or organs. The matrix can be the matrix of the tumor microenvironment. The epitope can be present on a matrix antigen. Such antigens can be on the matrix of the tumor microenvironment. For example, new antigens and new epitopes can be present on tumor endothelial cells, tumor vasculature, tumor fibroblasts, tumor pericytes, tumor stroma and / or tumor mesenchymal cells. Exemplary antigens include, but are not limited to, CD34, MCSP, FAP, CD31, PCNA, CD117, CD40, MMP4 and tenascin.

[0174] In some embodiments, the epitope can be present on an antigen presented by a major histocompatibility complex (MHC). The MHC can be a class I or class II human leukocyte antigen (HLA). The HLA can be HLA-A, HLA-B, HLA-C, HLA-HLA-E, HLA-F, HLA-G, HLA-DP, HLA-DQ, HLA-DR, HLA-DM, or HLA-DO. In some embodiments, the epitope can be present on HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-A*25, HLA-A*26, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*32, HLA-A*33, or HLA-A*24, HLA-B*27, HLA-B*35, HLA-B*48, HLA-B*55, etc.

[0175] In some embodiments, the epitope can be soluble (e.g., not bound to cells). In some cases, the antigen can be soluble, such as a soluble antigen. The epitope can be present on a universal antigen. In some cases, the antigen binding domain of the modified TCR complex can bind multiple epitopes, such as multiple specificities.

[0176] In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a CD3-epsilon chain, Figure 2A In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a CD3-delta chain, Figure 2B In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a CD3-γ chain, Figure 2C In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a TCR-α chain, Figure 2D In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a TCR-β chain, Figure 2E In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a TCR-γ chain. In some embodiments, the modified TCR complex comprises an antigen binding domain fused to a TCR-δ chain.

[0177] The modified TCR complex disclosed herein may include more than one antigen binding domain, such as at least 2 antigen binding domains (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10 antigen binding domains). In some embodiments, the modified TCR complex of the subject system includes at least two antigen binding domains. At least two antigen binding domains may be the same antigen binding domain. For example, two antigen binding domains may be the same molecule capable of binding the same ligand. At least two antigen binding domains may be different antigen binding domains. For example, two antigen binding domains may be different molecules capable of binding the same or different ligands. In some cases, the modified TCR includes a third antigen binding domain, which is connected to (i) a second antigen binding domain, (ii) any one of the α chain, β chain, γ chain and δ chain of TCR, (iii) ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3), or (iv) CD3 ζ chain.

[0178] In some embodiments, the first antigen binding domain is fused to the first CD3-epsilon chain of the TCR complex and the second antigen binding domain is fused to the second CD3-epsilon chain of the TCR complex, Figure 2F In some embodiments, the first antigen binding domain is fused to the CD3-epsilon chain and the second antigen binding domain is fused to the CD3-gamma chain, Figure 2G In some embodiments, the first and second antigen binding domains are linked to the same chain. For example, a modified TCR complex disclosed herein may comprise a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a CD3-ε chain, Figure 2H In some embodiments, the first antigen binding domain is fused to the TCR-α chain and the second antigen binding domain is fused to the TCR-β chain. The first antigen binding domain and the second antigen binding domain can be different antigen binding domains, as shown by the black ellipse and the black and white striped ellipse ( Fig.2I ). The first and second antigen binding domains may be the same antigen binding domain, as indicated by the similarly shaded ellipse ( Figure 2J ).

[0179] In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a CD3-delta chain, Figure 2K In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a CD3-γ chain, Figure 2L In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a TCR-α chain, Figure 2M In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a second antigen binding domain, which in turn is fused to a TCR-β chain, Figure 2N The first and second antigen binding domains may be different antigen binding domains. The first and second antigen binding domains may be the same antigen binding domain.

[0180] In some embodiments, a modified TCR complex disclosed herein comprises a first antigen binding domain fused to a CD3-epsilon chain and a second antigen binding domain fused to a CD3-delta chain, Fig.2O In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a CD3-δ chain and a second antigen binding domain fused to a CD3-γ chain, Figure 2P In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a TCR-α chain and a second antigen binding domain fused to a CD3-ε chain, Figure 2Q In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a TCR-β chain and a second antigen binding domain fused to a CD3-ε chain, Figure 2R In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to an alpha chain and a second antigen binding domain fused to a CD3-gamma chain, Figure 2S In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a TCR-β chain and a second antigen binding domain fused to a CD3-γ chain, Figure 2T In some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a TCR-α chain and a second antigen binding domain fused to a CD3-δ chain, Figure 2UIn some embodiments, the modified TCR complex disclosed herein comprises a first antigen binding domain fused to a β chain and a second antigen binding domain fused to a δ chain, Figure 2V .

[0181] In various embodiments of various aspects of this article, the modified TCR complex includes a previously identified TCR. In some cases, whole genome sequencing can be used to identify TCR. For example, TCR can target new antigens or new epitopes identified by whole genome sequencing of target cells. Alternatively, TCR can be identified from autologous, allogeneic or xenogeneic libraries. Autologous and allogeneic identification may require a multi-step process. In autologous and allogeneic identification, dendritic cells (DC) can be generated from monocytes selected from CD14, and pulsed or transfected with specific peptides after maturation. Peptide-pulsed DC can be used to stimulate autologous or allogeneic immune cells, such as T cells. Single cell peptide-specific T cell clones can be isolated from these peptide-pulsed T cell lines by limiting dilution. Interested subject TCR can be identified and separated. The α, β, γ and δ chains of the TCR of interest can be cloned, codon optimized and encoded in a vector (e.g., a lentiviral vector). In some embodiments, each part of TCR can be replaced. For example, the constant region of human TCR can be replaced by the corresponding mouse region. It can be performed to replace the human constant region with the corresponding mouse region to increase TCR stability. TCR can also have a higher or super-physiological in vitro affinity. In some cases, the method for identifying TCR may include immunizing transgenic mice expressing human leukocyte antigen (HLA) system with human tumor protein to produce T cells expressing TCR for human antigens (see, for example, Stanislawski et al., Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer, Nature Immunology 2, 962-970 (2001)). Another method can be allogeneic TCR gene transfer, wherein tumor-specific T cells are isolated from subjects experiencing tumor remission, and then reactive TCR sequences can be transferred to T cells from another subject who has the disease but may be unresponsive (de Witte, MA et al., Targeting self-antigens through allogeneic TCR gene transfer, Blood 108, 870–877 (2006)). In some cases, in vitro techniques can be used to change the sequence of TCRs to enhance their tumor-killing activity by increasing the strength of the interaction (affinity) between weakly reactive tumor-specific TCRs and target antigens (Schmid, DA et al., Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function. J. Immunol. 184, 4936-4946 (2010)).

[0182] In another aspect, the present disclosure provides a system for inducing the activity of immune cells and / or target cells. The system comprises: (a) a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain, a transmembrane domain, and an intracellular signaling domain that exhibit specific binding to a first epitope; and (b) a modified T cell receptor (TCR) complex disclosed herein.

[0183] In some embodiments, the system comprises (a) a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain that exhibits specific binding to a first epitope, a transmembrane domain, and an intracellular signaling domain; and (b) a modified T cell receptor (TCR) complex, the modified TCR complex comprising a second antigen binding domain that exhibits specific binding to a second epitope, wherein the second antigen binding domain is linked to at least one of the following: (i) at least one TCR chain, the at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of TCR; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0184] The chimeric antigen receptor (CAR) of the subject system may include a first antigen binding domain that exhibits specific binding to a first epitope. The first antigen binding domain may include any protein or molecule that can bind to an epitope. Non-limiting examples of the first antigen binding domain include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, mouse antibodies, or functional derivatives, variants, or fragments thereof, including, but not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), miniantibodies, double antibodies, and single-domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VL) of nanoantibodies of camelid origin). H H)). In some embodiments, the first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, Fv and scFv. In some embodiments, the first antigen binding domain comprises an antibody mimetic. Antibody mimetic refers to a molecule that can bind to a target molecule with an affinity comparable to that of an antibody, and includes single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), lipocalin scaffolds, calixarene backbones, A domains, and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant, or fragment thereof. For example, the antigen binding domain may comprise at least a ligand binding domain of a transmembrane receptor.

[0185] In some embodiments, the antigen binding domain may include scFv. scFv can be derived from antibodies with known variable region sequences. In some embodiments, scFv can be derived from antibody sequences obtained from available mouse hybridomas. scFv can be obtained from full exon sequencing of tumor cells or primary cells. In some embodiments, scFv can be changed. For example, scFv can be modified in a variety of ways. In some cases, scFv may mutate so that scFv may have a higher affinity for its target. In some cases, the affinity of scFv to its target can be optimized for targets expressed at low levels on normal tissues. This optimization can be performed to minimize potential toxicity, such as cytokine release syndrome. In other cases, cloning a scFv with a higher affinity for the membrane-bound form of the target may be superior to its soluble form counterpart. If some soluble forms of targets of different levels can also be detected and their targeting may cause unexpected toxicity (such as cytokine release syndrome), this modification can be performed.

[0186] In some embodiments, the first antigen binding domain of CAR comprises an antigen binding molecule disclosed herein. In some embodiments, the first antigen binding domain comprises a single domain antibody. In some embodiments, the single domain antibody is an anti-BCMA sdAb. In some embodiments, the first antigen binding domain comprises a sequence selected from any one of the groups consisting of SEQ ID NO: 3 to SEQ ID NO: 23 and SEQ ID NO: 38 to SEQ ID NO: 46 with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity. In some embodiments, the anti-BCMA sdAb comprises a sequence selected from any one of SEQ ID NO: 3 to SEQ ID NO: 23.

[0187] In some embodiments, the antigen binding domain may include a member of an interaction pair. For example, the antigen binding domain may be a member or a fragment thereof of an interaction pair comprising a receptor and a ligand. A receptor or a ligand or a fragment thereof may be referred to as an antigen binding domain. Another member not referred to as an antigen binding domain may include an epitope that is specifically bound by an antigen binding domain. In some embodiments, the first antigen binding domain and / or the second antigen binding domain include a receptor that specifically binds to a ligand. Receptors may include G protein-coupled receptors (GPCRs); integrin receptors; cadherin receptors; catalytic receptors, including receptors with enzymatic activity and receptors that do not have intrinsic enzymatic activity but act by stimulating non-covalently associated enzymes (e.g., kinases); death receptors, such as members of the tumor necrosis factor receptor (TNFR) superfamily; cytokine receptors; immune receptors; and the like. In some embodiments, the first antigen binding domain and / or the second antigen binding domain include a ligand that binds to a receptor.

[0188] The antigen binding domain of the CAR of the subject system can be connected to the intracellular signaling domain through a transmembrane domain. The transmembrane domain can be a transmembrane segment. The transmembrane domain of the subject CAR can anchor CAR to the plasma membrane of a cell (eg, an immune cell). In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide connecting the antigen binding domain of CAR and the intracellular signaling domain can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least 1 (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, deletions, and insertions compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as a sequence of a polypeptide linker. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits the signal from the extracellular region of the cell to the intracellular region through the antigen binding domain. The native transmembrane portion of CD28 can be used in CAR. In other cases, the native transmembrane portion of CD8α can also be used in CAR.

[0189] The intracellular signaling domain of the CAR of the subject system may include a signaling domain or any derivative, variant or fragment thereof involved in immune cell signaling. The intracellular signaling domain of CAR can induce the activity of immune cells containing CAR. The intracellular signaling domain can transduce effector function signals and instruct cells to perform specialized functions. The signaling domain may include the signaling domain of other molecules. Although the signaling domain of another molecule can usually be used in CAR, it is not necessary to use the entire chain in many cases. In some cases, a truncated portion of the signaling domain is used in CAR.

[0190] In some embodiments, the intracellular signaling domain comprises a plurality of signaling domains involved in immune cell signaling, or any derivative, variant or fragment thereof. For example, the intracellular signaling domain may comprise at least 2 immune cell signaling domains, such as at least 2, 3, 4, 5, 7, 8, 9 or 10 immune cell signaling domains. The immune cell signaling domain may participate in regulating the primary activation of the TCR complex in a stimulatory or inhibitory manner. The intracellular signaling domain may be an intracellular signaling domain of a T cell receptor (TCR) complex. The intracellular signaling domain of the subject CAR can include a signaling domain of Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d and Zap70. In some embodiments, the signaling domain includes an immunoreceptor tyrosine-based activation motif or ITAM. The ITAM-containing signaling domain may comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, thereby generating the conserved motif YxxL / Ix (6-8)YxxL / I. When the antigen binding domain binds to the epitope, the signaling domain comprising the ITAM can be modified, for example, by phosphorylation. The phosphorylated ITAM can serve as a docking site for other proteins (e.g., proteins involved in various signaling pathways). In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated, truncated and / or optimized ITAM domain that has an altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0191] In some embodiments, the intracellular signaling domain of the subject CAR comprises an FcγR signaling domain (e.g., ITAM). The FcγR signaling domain may be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the intracellular signaling domain comprises an FcεR signaling domain (e.g., ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain comprises an FcαR signaling domain (e.g., ITAM). The FcαR signaling domain may be selected from FcαRI (CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the primary signaling domain comprises an ITAM of CD3ζ.

[0192] In some embodiments, the intracellular signaling domain of the subject CAR comprises an inhibitory motif or ITIM based on immunoreceptor tyrosine. The signaling domain comprising ITIM may be included in a conservative amino acid sequence (S / I / V / LxYxxI / V / L) found in the cytoplasmic tail region of some inhibitory receptors of the immune system. The main signaling domain comprising ITIM can be modified (e.g., phosphorylated) by enzymes such as Src kinase family members (e.g., Lck). After phosphorylation, other proteins (including enzymes) can be recruited to ITIM. These other proteins include, but are not limited to, enzymes (such as phosphotyrosine phosphatases SHP-1 and SHP-2, inositol phosphatases known as SHIP) and proteins (e.g., ZAP70) with one or more SH2 domains.The intracellular signaling domain may include the following signaling domains (e.g., ITIMs): BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), Fc receptor-like protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor transport-associated 1 (IRTA1), immunoglobulin superfamily receptor transport-associated Guan2 (IRTA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LIR2), leukocyte immunoglobulin-like receptor subfamily B member 3 (LIR3), leukocyte immunoglobulin-like receptor subfamily B member 5 (LIR5), leukocyte immunoglobulin-like receptor subfamily B member 8 (LIR8), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural cytotoxicity triggering receptor 2 (NKp44), NTB-A, programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid-binding Ig-like lectin 2 (SIGLEC2 or CD22), sialic acid-binding Ig-like lectin 3 (SIG LEC3 or CD33), sialic acid-binding Ig-like lectin 5 (SIGLEC5 or CD170), sialic acid-binding Ig-like lectin 6 (SIGLEC6), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 10 (SIGLEC10), sialic acid-binding Ig-like lectin 11 (SIGLEC11), sialic acid-binding Ig-like lectin 4 (SIGLEC4), sialic acid-binding Ig-like lectin 8 (SIGLEC8), sialic acid-binding Ig-like lectin 9 (SIGLEC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein 2 (SIRP 2), and signaling threshold regulating transmembrane adaptor 1 (SIT).In some embodiments, the intracellular signaling domain includes a modified ITIM domain, e.g., a mutated, truncated, and / or optimized ITIM domain, which has an altered (e.g., increased or decreased) activity compared to a native ITIM domain.

[0193] In some embodiments, the intracellular signaling domain comprises at least 2 ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains). In some embodiments, the intracellular signaling domain comprises at least 2 ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITIM domains) (e.g., at least 2 primary signaling domains). In some embodiments, the intracellular signaling domain includes an ITAM domain and an ITIM domain.

[0194] In some cases, the intracellular signaling domain of the subject CAR may include a co-stimulatory domain. In some embodiments, for example, a co-stimulatory domain from a co-stimulatory molecule can provide a co-stimulatory signal for immune cell signaling (such as signaling from ITAM and / or ITIM domains), for example, for activating immune cell activity and / or inactivating immune cell activity. In some embodiments, the co-stimulatory domain is operable to regulate proliferation and / or survival signals in immune cells. In some embodiments, the co-stimulatory signaling domain includes MHC class I proteins, MHC class II proteins, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA or Toll ligand receptor signaling domains. In some embodiments, the costimulatory domain comprises a signaling domain of a molecule selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100(SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160(BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNF RSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAM F7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLAClass I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9(CD229), lymphocyte function-associated antigen-1(LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG(CD162 ), SLAM(SLAMF1), SLAM / CD150, SLAMF4(CD244), SLAMF6(NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1 and VLA-6. In some embodiments, the intracellular signaling domain includes multiple costimulatory domains, such as at least two, such as at least 3, 4 or 5 costimulatory domains. The costimulatory signaling region can provide a signal that cooperates with the primary effector activation signal and can complete the requirements for activating T cells. In some embodiments, adding a costimulatory domain to CAR can enhance the efficacy and persistence of immune cells provided herein.

[0195] Examples of costimulatory signaling domains are provided in Table 8.

[0196] Table 8: Intracellular co-stimulatory signaling domains

[0197]

[0198]

[0199]

[0200] As an example, CAR may include CD3 ζ chains (sometimes referred to as first generation CAR). As another example, CAR may include CD-3 ζ chains and a single costimulatory domain (e.g., CD28 or 4-1BB) (sometimes referred to as second generation CAR). As another example, CAR may include CD-3 ζ chains and two costimulatory domains (CD28 / OX40 or CD28 / 4-1BB) (sometimes referred to as third generation CAR). These signal transduction moieties can produce downstream activation of kinase pathways together with co-receptors (such as CD8), which supports gene transcription and functional cell responses.

[0201] In some embodiments, the subject CAR may include a hinge or a spacer. A hinge or a spacer may refer to a segment between an antigen binding domain and a transmembrane domain. In some embodiments, a hinge may be used to provide flexibility to an antigen binding domain (e.g., scFv). In some embodiments, for example, when an antibody for detecting scFv is ineffective or unavailable, a hinge may be used to detect the expression of CAR on the cell surface. In some cases, the hinge is derived from an immunoglobulin molecule and may need to be optimized depending on the position of the first epitope or the second epitope on the target. In some cases, the hinge may not belong to an immunoglobulin molecule, but instead belongs to another molecule, such as a natural hinge of a CD8α molecule. The CD8α hinge may contain cysteine ​​and proline residues, which may play a role in the interaction between CD8 co-receptors and MHC molecules. In some embodiments, cysteine ​​and proline residues may affect the performance of CAR, and therefore may be engineered to affect the performance of CAR.

[0202] The hinge can have any suitable length. In some embodiments, the hinge of the CAR can be adjustable in size and can be compensated to a certain extent when normalizing the orthogonal synaptic distance between the CAR-expressing cell and the target cell. This topological structure of the immune synapse between the CAR-expressing cell and the target cell can also define a distance that cannot be functionally bridged by the CAR due to the membrane-distal epitope on the cell surface target molecule, and the membrane-distal epitope cannot make the synaptic distance close to signal transduction even with a short hinge CAR. Similarly, a membrane-proximal CAR target antigen epitope has been described, and its signaling output is only observed in the case of a long hinge CAR. The hinge disclosed herein can be tuned according to the single-chain variable fragment region that can be used.

[0203] As an example, a CAR may comprise an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. Figure 3As shown in. CAR may generally include an antigen binding domain derived from a single-chain antibody, a hinge domain (H) or a spacer, a transmembrane domain (TM) provided to anchor the plasma membrane, and a signaling domain responsible for T cell activation. CAR may include an immune cell signaling domain, such as a CD3ζ chain. CAR may include an immune cell signaling domain and a first co-stimulatory domain, such as a CD3ζ chain and 4-1BB. CAR may include an immune cell signaling domain and at least two co-stimulatory domains, such as a CD3ζ chain, 4-1BB and OX40. In some embodiments, a universal CAR may also be included in the system. Universal CAR may include an intracellular signaling domain fused to a protein domain that binds to a label (e.g., fluorescein isothiocyanate or biotin) on a monoclonal antibody. Various combinations of immune cell signaling domains and co-stimulatory domains can be used in subject CARs. In some embodiments, the immune cell signaling domain may be from CD3, CD4 and / or CD8. The co-stimulatory domain may be from 4-1BB, OX40, CD28, etc.

[0204] In some embodiments, the CAR of the subject system of the present disclosure may include one or more additional antigen binding domains, and the one or more additional antigen binding domains show specific binding to one or more additional epitopes. For example, the CAR of the subject system may include at least two antigen binding domains (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10 antigen binding domains). In some embodiments, the at least two antigen binding domains of CAR are connected in series. In some embodiments, the at least two antigen binding domains may be the same antigen binding domains. For example, the at least two antigen binding domains may be the same molecule capable of binding to the same epitope. In some embodiments, the at least two antigen binding domains may be different antigen binding domains. For example, the at least two antigen binding domains may be different molecules capable of binding to different epitopes on one or more antigens.

[0205] The antigen binding domain of the subject CAR and the modified TCR complex of the subject system can bind to epitopes present on different antigens. In some cases, the antigen binding domain of the CAR and the modified TCR complex of the subject system bind to epitopes present on a common antigen. In some embodiments, the first epitope and the second epitope can be the same epitope. In some embodiments, the first epitope and the second epitope can be different epitopes.

[0206] The first epitope and / or the second epitope may be present on one or more cell surface antigens. The one or more cell surface antigens may be tyrosine kinase receptors, serine kinase receptors, histidine kinase receptors, G protein coupled receptors (GPCRs), and the like.

[0207] The first epitope and / or the second epitope can be present on an immune checkpoint receptor or an immune checkpoint receptor ligand. In some embodiments, the immune checkpoint receptor or immune checkpoint receptor ligand can be PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, TIGIT, BLTA, CD47 or CD40.

[0208] The first epitope and / or the second epitope can be present on a cytokine or a cytokine receptor. The cytokine receptor can be, for example, CCR2b, CXCR2 (CXCL1 receptor), CCR4 (CCL17 receptor), Gro-a, IL-2, IL-7, IL-15, IL-21, IL-12, heparanase, CD137L, LEM, Bcl-2, CCL17, CCL19 or CCL2.

[0209] The first epitope and / or the second epitope may be present on a tumor-associated antigen. The epitope may be, for example, a tumor epitope. The tumor-associated antigen may be selected from the group consisting of: 707-AP, biotinylated molecules, α-actinin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipose differentiation-related protein, AFP, AIM-2, annexin II, ART-4, BAGE, BCMA, b-catenin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-abl p210(b3a2), BING-4, CA-125, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7 / 8, CD70, CD123, CD133, CDC27, CDK-4, CEA, CLCA2, CLL-1, CTAG1B, Cyp-B, DAM-10, DAM-6, DEK-CAN, DLL3, EGFR, EGFRvIII, EGP-2, EGP-40, ELF2, Ep -CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FAP, FBP, fetal acetylcholine receptor, FGF-5, FN, FR-α, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, GPC3, GPC-2, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, LewisY, L1-CAM, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1 , OA1, OGT, carcinoembryonic antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survivin, survivin-2B, SYT / SSX, TAG-72, TEL / AML1, TGFαRII, TGFβRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ light chain. In some embodiments, the first epitope and / or the second epitope can be EGFR, EGFRvIII, GPC3, GPC-2, DLL3, BCMA, CD19, CD20, CD22, CD123, CLL-1, CD30, CD33, HER2, MSLN, PSMA, CEA, GD2, IL13Rα2, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, FR-α, CD70, CD171, ROR1, and any combination thereof.

[0210] In some embodiments, the first epitope or the second epitope is present on BCMA. In some embodiments, both the first epitope and the second epitope are present on BCMA. In some embodiments, the first epitope and the second epitope are the same epitope of BCMA. In some embodiments, the first epitope and the second epitope are different epitopes of BCMA.

[0211] The first epitope and / or the second epitope may be present on a neoantigen. The first epitope and / or the second epitope may be a neoepitope.

[0212] Neoantigens and neoepitopes generally refer to tumor-specific mutations that trigger anti-tumor T cell responses in some cases. For example, whole genome sequencing methods can be used to identify these endogenous mutations. Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+T cells in a patient with epithelial cancer," Science 344:641-644 (2014). Antigen binding domains, such as the antigen binding domains of the subject CAR or modified TCR complexes, can exhibit specific binding to tumor-specific neoantigens. The neoantigens bound by the antigen binding domains of the CAR or modified TCR complexes can be expressed on target cells, and, for example, are neoantigens and neoepitopes encoded by mutations in any endogenous genes. In some cases, the first and / or second antigen binding domains bind to neoantigens or neoepitopes encoded by mutant genes. The gene can be selected from the group consisting of: ABL1, ACO1 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2Μ, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, NY-ESO, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

[0213] In some embodiments, the first epitope and / or the second epitope that can be bound by the first and / or second antigen binding domain can be present on a matrix. A matrix generally refers to a tissue that provides connection and functional support, especially for biological cells, tissues or organs. The matrix can be the matrix of a tumor microenvironment. The first epitope and / or the second epitope can be present on a matrix antigen. Such antigens can be on the matrix of a tumor microenvironment. For example, new antigens and new epitopes can be present on tumor endothelial cells, tumor vasculature, tumor fibroblasts, tumor pericytes, tumor stroma and / or tumor mesenchymal cells. Exemplary antigens include, but are not limited to, CD34, MCSP, FAP, CD31, PCNA, CD117, CD40, MMP4 and tenascin.

[0214] In some embodiments, the first epitope and / or the second epitope can be present on an antigen presented by a major histocompatibility complex (MHC). The MHC can be a class I or class II human leukocyte antigen (HLA). The HLA can be HLA-A, HLA-B, HLA-C, HLA-HLA-E, HLA-F, HLA-G, HLA-DP, HLA-DQ, HLA-DR, HLA-DM, or HLA-DO. In some embodiments, the first epitope and / or the second epitope can be present on HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-A*25, HLA-A*26, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*32, HLA-A*33, or HLA-A*24, HLA-B*27, HLA-B*35, HLA-B*48, HLA-B*55, etc.

[0215] In some embodiments, the first epitope and / or the second epitope can be soluble (e.g., not bound to a cell). In some cases, the antigen can be soluble, such as a soluble antigen. The first epitope and / or the second epitope can be present on a universal antigen. In some cases, the antigen binding domains of the subject CAR and / or modified TCR complex can each bind to multiple epitopes, such as multiple specificities.

[0216] In some embodiments, the first epitope and the second epitope can be the same epitope.

[0217] In some embodiments, the combination of at least one antigen binding domain and its epitope can activate the immune cell activity of immune cells expressing the modified TCR complex of the present disclosure. In some embodiments, the combination of two or more antigen binding domains and its epitope can activate the immune cell activity of immune cells expressing the modified TCR complex of the present disclosure. In some embodiments, the combination of the first antigen binding domain and the first epitope or the combination of the second antigen binding domain and the second epitope can activate the immune cell activity of immune cells expressing the subject system. In some cases, the combination of the first antigen binding domain and the first epitope and the combination of the second antigen binding domain and the second epitope activate the immune cell activity of immune cells expressing the system.

[0218] In some embodiments, the system for inducing the activity of immune cells and / or target cells may include more than two antigen binding domains. For example, the system may include the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth or more antigen binding domains. In some embodiments, the combination of the third antigen binding domain and the third epitope activates the immune cell activity of the immune cells expressing the system. In some embodiments, the combination of the first antigen binding domain and the first epitope, the combination of the second antigen binding domain and the second epitope, and the combination of the third antigen binding domain and the third epitope activates the immune cell activity of the immune cells expressing the system. Any number of antigen binding domains can be used in the system of the present disclosure, and the number of antigen binding domains is not limited to one, two or three.

[0219] In some embodiments, two or more antigen binding domains of the subject system are optionally connected in series to (i) at least one TCR chain, the at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); (iii) CD3ζ chain, and wherein the binding of the two or more antigen binding domains to their corresponding epitopes activates the immune cell activity of immune cells expressing the system. When necessary, the two or more antigen binding domains are connected to separate chains of the TCR complex. Alternatively, the two or more antigen binding domains are connected to a chain of the TCR complex. In some embodiments of the subject system, the two or more antigen binding domains are connected in series to the ε chain, the δ chain and / or the γ chain of cluster of differentiation 3 (CD3). In some embodiments, two or more antigen binding domains of the subject system are connected to, optionally connected in series to the CAR of the subject system.

[0220] The immune cell activity activated in the immune cell expressing the modified TCR complex and / or system of the present disclosure can be any of a variety of cell activities. In some embodiments, the immune cell activity is selected from the group consisting of: clonal expansion of the immune cell; cytokine release by the immune cell; cytotoxicity of the immune cell; proliferation of the immune cell; differentiation, dedifferentiation or transdifferentiation of the immune cell; movement and / or transport of the immune cell; exhaustion and / or reactivation of the immune cell; and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the immune cell.

[0221] In some embodiments, immune cell activity includes clonal expansion of immune cells. Clonal expansion may include generation of daughter cells by immune cells. In clonal expansion, the offspring of immune cells may include a modified TCR complex and / or system provided herein. In clonal expansion, the offspring of immune cells may include a CAR provided herein. In clonal expansion, the offspring of immune cells may include a modified TCR complex provided herein. In clonal expansion, the offspring of immune cells may include a CAR and TCR provided herein. The clonal expansion of immune cells comprising a modified TCR complex and / or system provided herein may be greater than comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or comparable immune cells wherein only one of the first and second antigen binding domains is bound to their corresponding epitopes. The clonal expansion of immune cells comprising the modified TCR complexes and / or systems provided herein can be about 5-fold to about 1-fold greater than the clonal expansion of comparable immune cells lacking the modified TCR complexes and / or systems, comparable immune cells lacking one or more components of the modified TCR complexes and / or systems (e.g., CAR, modified TCR complexes), and / or comparable immune cells in which only one of the first and second antigen binding domains binds to their corresponding epitopes. In some embodiments, clonal expansion can include quantifying the number of immune cells. Quantifying the number of immune cells can include flow cytometry, trypan blue exclusion and / or hemocytometer.

[0222] In some embodiments, immune cell activity includes cytokine release by immune cells.In some embodiments, immune cell activity includes the release of intercellular molecules, metabolites, compounds or combinations thereof.Cytokine release performed by immune cells may include the release of IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, IFNγ, TNFα, CSF, TGFβ, granzymes, etc. In some embodiments, HTRF, flow cytometry, Western blot, etc. can be used to quantify cytokine release.Cytokine release of immune cells comprising the modified TCR complex and / or system provided herein can be greater than comparable immune cells lacking the modified TCR complex and / or system, lacking the modified TCR complex and / or system One or more components (e.g., CAR, modified TCR complex) of comparable immune cells and / or wherein only one of the first and second antigen binding domains is combined with their corresponding epitopes Comparable immune cells release greater cytokine. Compared with comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or comparable immune cells wherein only one of the first and second antigen binding domains is bound to their corresponding epitopes, immune cells comprising the modified TCR complex and / or system provided herein can generate about 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, 250 times, or more than 300 times large cytokine release. In some embodiments, cytokine release can be quantified in vitro or in vivo.

[0223] In some embodiments, immune cell activity includes cytotoxicity of immune cells. In some instances, when expressed in immune cells, the modified TCR complex, subject system and composition of the present disclosure can be used to kill target cells. Immune cells or immune cell groups expressing modified TCR complexes and / or subject systems can induce the death of target cells. Killing of target cells can be used for a variety of applications, including but not limited to treating diseases or disorders that expect to eliminate cell groups or expect to inhibit the proliferation of cell groups. Cytotoxicity can refer to killing target cells. Cytotoxicity can also refer to the release of cytotoxic cytokines, such as IFNγ or granzymes, by immune cells. In some cases, the modified TCR complex and / or subject system expressed in immune cells can change (i) the release of cytotoxins such as perforin, granzymes and granulysin, and / or (ii) apoptosis induction performed by Fas-Fas ligand interaction between T cells and target cells, thereby triggering the destruction of target cells. In some embodiments, cytotoxicity can be quantified by cytotoxicity assays, and the cytotoxicity assays include co-culture assays, ELISPOT, chromium release cytotoxicity assays, etc. An immune cell comprising a modified TCR complex and / or system provided herein can be more cytotoxic than a comparable immune cell lacking the modified TCR complex and / or system, a comparable immune cell lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or a comparable immune cell in which only one of the first and second antigen binding domains binds to their corresponding epitopes. An immune cell comprising a modified TCR complex and / or system provided herein may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175% or 200% greater cytotoxicity to a target cell compared to a comparable immune cell lacking the modified TCR complex and / or system, a comparable immune cell lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or a comparable immune cell in which only one of the first and second antigen binding domains binds to their corresponding epitopes.Compared with comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or comparable immune cells wherein only one of the first and second antigen binding domains is bound to their corresponding epitopes, immune cells comprising the modified TCR complex and / or system provided herein can induce at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175% or 200% greater target cell death. In some embodiments, immune cells expressing modified TCR complexes and / or subject systems can induce apoptosis of target cells displaying target epitopes on their surfaces. In some embodiments, cytotoxicity can be determined in vitro or in vivo. In some embodiments, determining cytotoxicity may include determining the disease level after administering the cells comprising the modified TCR complex and / or system provided herein compared to the disease level before administration.In some embodiments, determining cytotoxicity may include determining the disease level after administering the cells comprising the modified TCR complex and / or system provided herein, and administering comparable immune cells lacking the modified TCR complex and / or system, lacking the comparable immune cells of one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or wherein only one of the first and second antigen binding domains is combined with their corresponding epitopes.Disease level after comparable immune cells. In some embodiments, the level of disease on the target lesions can be measured as a complete response (CR); disappearance of target lesions, a partial response (PR); a decrease of at least 30% in the sum of the longest diameters (LD) of the target lesions, based on the baseline LD sum, progression (PD); an increase of at least 20% in the sum of the LD of the target lesions, based on the minimum sum of the LD recorded since the start of treatment or the appearance of one or more new lesions, stable disease (SD); or, based on the minimum sum of the LD, neither sufficient shrinkage to meet PR nor sufficient growth to meet PD. In some embodiments, non-target lesions can be measured. The level of disease in non-target lesions can be a complete response (CR); disappearance of all non-target lesions and normalization of tumor marker levels, incomplete response; persistent presence of one or more non-target lesions, progression (PD); or appearance of one or more new lesions.

[0224] In some embodiments, immune cell activity is the proliferation of immune cells. The proliferation of immune cells may refer to the expansion of immune cells. The proliferation of immune cells may refer to the phenotypic changes of immune cells. The proliferation of immune cells comprising the modified TCR complex and / or system provided herein may be greater than the proliferation of comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or wherein only one of the first and second antigen binding domains binds to their corresponding epitopes. The proliferation of immune cells comprising the modified TCR complex and / or system provided herein may be about 5 times to about 1 times the proliferation of comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system provided herein, and / or wherein only one of the first and second antigen binding domains binds to their corresponding epitopes. In some embodiments, the number of immune cells can be quantified. The number of immune cells can be quantified and can include flow cytometry, trypan blue exclusion and / or hemocytometer. Propagation can also be determined by phenotypic analysis of immune cells. For example, the mass of immune cells in the culture can represent the proliferation of immune cells compared with comparable immune cells lacking modified TCR complexes and / or systems.

[0225] In some embodiments, immune cell activity can be differentiation, dedifferentiation or transdifferentiation.Differentiation, dedifferentiation or transdifferentiation of immune cells can be determined by evaluating the phenotypic expression of differentiation, dedifferentiation or transdifferentiation markers on the cell surface with flow cytometry.In some embodiments, compared with the comparable immune cells lacking the modified TCR complex and / or system, lacking the comparable immune cells of one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or wherein only one of the first and second antigen binding domains is combined with their corresponding epitopes, the immune cells comprising the modified TCR complex and / or system provided herein have enhanced differentiation ability. In some embodiments, compared with comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or comparable immune cells wherein only one of the first and second antigen binding domains is bound to their corresponding epitopes, immune cells comprising the modified TCR complex and / or system provided herein have enhanced dedifferentiation ability. In some embodiments, compared with comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or comparable immune cells wherein only one of the first and second antigen binding domains is bound to their corresponding epitopes, immune cells comprising the modified TCR complex and / or system provided herein have greater transdifferentiation ability.

[0226] In some embodiments, immune cell activity can be the movement and / or transportation of immune cells comprising modified TCR complexes and / or systems.In some embodiments, movement can be determined by quantitatively locating immune cells to target sites.For example, immune cells comprising modified TCR complexes and / or subject systems can be quantified at target sites after administration, for example, at sites of non-target sites.Quantitative can be performed by separating lesions and quantifying the number of immune cells (such as tumor infiltrating lymphocytes) comprising modified TCR complexes and / or systems.Movement and / or transportation of immune cells comprising modified TCR complexes and / or systems provided herein can be greater than comparable immune cells lacking the modified TCR complexes and / or systems, comparable immune cells lacking one or more components (e.g., CAR, modified TCR complexes) of the modified TCR complexes and / or systems, and / or comparable immune cells wherein only one of the first and second antigen binding domains is combined with their corresponding epitopes. In some embodiments, the number of immune cells comprising a modified TCR complex and / or system at a target site (e.g., a tumor lesion) can be about 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, or 40 times the number of comparable immune cells lacking the modified TCR complex and / or system, lacking one or more components (e.g., CAR, modified TCR complex) of the modified TCR complex and / or system, and / or wherein only one of the first and second antigen binding domains binds to their corresponding epitopes. Transport can also be determined in vitro using a transwell migration assay. In some embodiments, for example in a transplate migration assay, the number of immune cells comprising a modified TCR complex and / or system at the target site can be about 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, or 40 times the number of comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or comparable immune cells in which only one of the first and second antigen binding domains binds to their corresponding epitopes.

[0227] In some embodiments, immune cell activity can be exhaustion and / or activation of immune cells. Exhaustion and / or activation of immune cells can be determined by phenotypic analysis using flow cytometry or microscopic analysis. For example, the expression level of exhaustion markers (such as programmed cell death protein 1 (PD1), lymphocyte activation gene 3 protein (LAG3), 2B4, CD160, Tim3, and T cell immune receptor (TIGIT) with immunoglobulin and ITIM domains) can be quantitatively and / or qualitatively determined. In some cases, immune cells (such as T cells) may lose effector functions in a hierarchical manner and become exhausted. As a result of exhaustion, functions such as IL-2 production and cytokine expression and high proliferation capacity may be lost. Exhaustion may also be accompanied by defects in the production of IFNγ, TNFα and chemokines and in degranulation. The depletion and / or activation of immune cells comprising the modified TCR complexes and / or systems provided herein can be greater than the depletion and / or activation of comparable immune cells lacking the modified TCR complexes and / or systems, comparable immune cells lacking one or more components of the modified TCR complexes and / or systems (e.g., CAR, modified TCR complex), and / or comparable immune cells in which only one of the first and second antigen binding domains binds to their corresponding epitopes. In some embodiments, immune cells comprising a modified TCR complex and / or system provided herein can experience at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or more than 300-fold increase in exhaustion or activation compared to comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or comparable immune cells in which only one of the first and second antigen binding domains binds to their corresponding epitopes.In some embodiments, immune cells comprising a modified TCR complex and / or system provided herein can experience at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or more than 300-fold reduction in exhaustion or activation compared to comparable immune cells lacking the modified TCR complex and / or system, comparable immune cells lacking one or more components of the modified TCR complex and / or system (e.g., CAR, modified TCR complex), and / or comparable immune cells in which only one of the first and second antigen binding domains binds to their corresponding epitopes.

[0228] In some embodiments, the binding of the first antigen binding domain to the first epitope and the binding of the second antigen domain to the second epitope activate the cytotoxicity of the subject immune cells expressing the system. Cytotoxicity can be increased compared to (i) only the first antigen binding domain binding to the first epitope or (ii) only the second antigen binding domain binding to the second epitope. Cytotoxicity can be increased as measured by the percentage of killing in a cytotoxicity assay, compared to (i) only the first antigen binding domain binding to the first epitope or (ii) only the second antigen binding domain binding to the second epitope. The percentage of killing after contacting can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of the target cells compared to (i) only the first antigen binding domain binding to the first epitope or (ii) only the second antigen binding domain binding to the second epitope.

[0229] In some embodiments, the binding of the first antigen binding domain to the first epitope and the binding of the second antigen binding domain to the second epitope activate the cytotoxicity of immune cells expressing the system and reduce the side effects associated with the cytotoxicity compared to (i) only the first antigen binding domain binds to the first epitope, or (ii) only the second antigen binding domain binds to the second epitope. In some embodiments, the side effect associated with cytotoxicity is cytokine release syndrome. Compared to (i) only the first antigen binding domain binds to the first epitope or (ii) only the second antigen binding domain binds to the second epitope, the reduction in side effects, such as the reduction in cytokine release syndrome, can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% reduction.

[0230] In some embodiments, the binding of the first antigen binding domain to the first epitope and the binding of the second antigen binding domain to the second epitope activate the cytotoxicity of immune cells expressing the system and increase the persistence of the cytotoxicity, compared to the binding of only the first antigen binding domain to the first epitope or the binding of only the second antigen binding domain to the second epitope. When the system is expressed in immune cells in a subject, the binding of the first antigen binding domain to the first epitope and the binding of the second antigen binding domain to the second epitope can activate the cytotoxicity of immune cells expressing the system and increase the persistence of the cytotoxicity, compared to the binding of only the first antigen binding domain to the first epitope or the binding of only the second antigen binding domain to the second epitope. The increase in persistence can be determined by quantifying the level of immune cells containing the system after administration. Increased persistence can mean that an immune cell comprising the system provided herein is present for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30 days, 35 days, 40 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 year or more after administration compared to a comparable immune cell lacking the system, a comparable immune cell lacking one or more components of the system (e.g., CAR, modified TCR complex), and / or a comparable immune cell in which only one of the first and second antigen binding domains binds to their corresponding epitopes.

[0231] In one aspect, the present disclosure provides a host cell expressing any modified TCR complex and / or system (e.g., CAR, modified TCR complex) of each embodiment herein. The separated host cell may include a host cell population. The host cell may be any suitable cell for expressing a modified TCR complex and / or a subject system. In some cases, the host cell is an immune cell. The immune cell may be a lymphocyte, such as a T cell. Non-limiting examples of T cells include CD8+T cells and CD4+T cells, αβT cells, γδT cells, Vγ9δ2T cells, Vδ1T cells, Vδ3T cells, and Vδ5T cells. In some cases, lymphocytes expressing modified TCR complexes and / or subject systems are natural killer (NK) cells, effector T cells, memory T cells, cytotoxic T cells, NKT, and / or T helper cells. In some cases, lymphocytes expressing modified TCR complexes and / or subject systems are KHYG cells, such as KHYG-1 cells or derivatives thereof.

[0232] In one aspect, the present disclosure provides an antigen-specific immune cell, which includes at least two exogenously introduced antigen binding domains, one of which is connected to a T cell receptor (TCR) complex, and the other is connected to a chimeric antigen receptor (CAR). Antigen-specific immune cells can specifically bind to target cells expressing one or more antigens recognized by at least two exogenously introduced antigen binding domains. Immune cells can be lymphocytes, such as T cells. Non-limiting examples of T cells include CD8+T cells and CD4+T cells, αβT cells, γδT cells, Vγ9δ2T cells, Vδ1T cells, Vδ3T cells, and Vδ5T cells. In some cases, lymphocytes expressing modified TCR complexes and / or subject systems are natural killer (NK) cells, effector T cells, memory T cells, cytotoxic T cells, NKT, and / or T helper cells. In some cases, lymphocytes expressing subject systems are KHYG cells, such as KHYG-1 cells or derivatives thereof.

[0233] In one aspect, the present disclosure provides a population of immune cells, individual immune cells expressing any of the modified TCR complexes and / or systems of the various embodiments herein, and wherein the population of immune cells is characterized in that when the population of immune cells is exposed to a population of target cells in a subject, the population of immune cells induces death of the target cells. The population of immune cells can induce death of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of the target cells within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 year, or more after exposure.

[0234] The immune cell group can include any of a variety of immune cells. In some cases, the immune cell group includes lymphocytes. Lymphocytes can be T cells. Non-limiting examples of T cells include CD8+T cells and CD4+T cells, αβT cells, γδT cells, Vγ9δ2T cells, Vδ1T cells, Vδ3T cells and Vδ5T cells. In some cases, lymphocytes are natural killer (NK) cells, effector T cells, memory T cells, cytotoxic T cells, NKT and / or T helper cells. In some embodiments, lymphocytes expressing modified TCR complexes and / or subject systems are KHYG cells, such as KHYG-1 cells or derivatives thereof.

[0235] The immune cell population may include any suitable number of cells. The number of immune cells may be determined as the number of cells used in an in vitro assay. The number of immune cells may be determined as the number of cells administered to a subject. The number of immune cells may be determined as the number of cells prior to activation of any immune cell activity (such as proliferation and / or expansion). The number of immune cells may include at least about 1 x 10 6 cells, at least about 2x10 6 cells, at least about 3x10 6 cells, at least about 4x10 6 cells, at least about 5x10 6 cells, at least about 6x10 6 cells, at least about 7x10 6 cells, at least about 8x10 6 cells, at least about 9x10 6 cells, 1x10 7 cells, at least about 2x107 cells, at least about 3x10 7 cells, at least about 4x10 7 cells, at least about 5x10 7 cells, at least about 6x10 7 cells, at least about 7x10 7 cells, at least about 8x10 7 cells, at least about 9x10 7 cells, at least about 1x10 8 cells, at least about 2x10 8 cells, at least about 3x10 8 cells, at least about 4x10 8 cells, at least about 5x10 8 cells, at least about 6x10 8 cells, at least about 7x10 8 cells, at least about 8x10 8 cells, at least about 9x10 8 cells, at least about 1x10 9 cells, at least about 2x10 9 cells, at least about 3x10 9 cells, at least about 4x10 9 cells, at least about 5x10 9 cells, at least about 6x10 9 cells, at least about 7x10 9 cells, at least about 8x10 9 cells, at least about 9x10 9 cells, at least about 1x10 10 cells, at least about 2x10 10 cells, at least about 3x10 10 cells, at least about 4x10 10 cells, at least about 5x10 10 cells, at least about 6x10 10 cells, at least about 7x10 10 cells, at least about 8x10 10 cells, at least about 9x10 10 cells, at least about 1x10 11 cells, at least about 2x10 11 cells, at least about 3x10 11 cells, at least about 4x10 11 cells, at least about 5x10 11 cells, at least about 6x10 11 cells, at least about 7x10 11cells, at least about 8x10 11 cells, at least about 9x10 11 cells, or at least about 1x10 12 In some embodiments, the immune cell population may include up to about 5x10 10 cells, up to about 4x10 10 cells, up to about 3x10 10 cells, up to about 2x10 10 cells, up to about 1x10 10 cells, up to about 9x10 9 cells, up to about 8x10 9 cells, up to about 7x10 9 cells, up to about 6x10 9 cells, up to about 5x10 9 cells, up to about 4x10 9 cells, up to about 3x10 9 cells, up to about 2x10 9 cells, up to about 1x10 9 cells, up to about 9x10 8 cells, up to about 8x10 8 cells, up to about 7x10 8 cells, up to about 6x10 8 cells, up to about 5x10 8 cells, up to about 4x10 8 cells, up to about 3x10 8 cells, up to about 2x10 8 cells, up to about 1x10 8 cells, up to about 9x10 7 cells, up to about 8x10 7 cells, up to about 7x10 7 cells, up to about 6x10 7 cells, up to about 5x10 7 cells, up to about 4x10 7 cells, up to about 3x10 7 cells, up to about 2x10 7 cells, up to about 1x10 7 cells, up to about 9x10 6 cells, up to about 8x10 6 cells, up to about 7x10 6 cells, up to about 6x10 6 cells, up to about 5x10 6 cells, up to about 4x106 cells, up to about 3x10 6 cells, up to about 2x10 6 cells, up to about 1x10 6 cells, up to about 9x10 5 cells, up to about 8x10 5 cells, up to about 7x10 5 cells, up to about 6x10 5 cells, up to about 5x10 5 cells, up to about 4x10 5 cells, up to about 3x10 5 cells, up to about 2x10 5 cells, or up to about 1x10 5 The immune cell population may be administered to a subject in need thereof. For example, about 5 x 10 10 In some cases, the cell population can be expanded to a number sufficient for treatment. For example, 5x10 7 The cells can be rapidly expanded to produce sufficient numbers for therapeutic use. Any number of cells can be administered to a subject for therapeutic use, for example, by infusion. For example, a patient can be infused with between about 1×10 6 to 5x10 12 The patient may be infused with as many cells as can be produced for him or her.

[0236] In any cell of the various aspects of the present invention, the cell can show specific binding to two antigens simultaneously present in the target cell. The antigen can be present on the surface of the target cell, or in some cases can be an intracellular protein of the target cell displayed by another cell, such as in the case of MHC.

[0237] In various embodiments of various aspects of this article, the antigen binding domain connected to the CAR can mainly mediate the interaction between the immune cell and the target cell, and the antigen binding domain connected to the modified TCR complex can mainly mediate the activity of the immune cell when the interaction between the immune cell and the target cell occurs. As previously described herein, immune cell activity may include clonal expansion of the immune cell; cytokine release by the immune cell; cytotoxicity of the immune cell; proliferation of the immune cell; differentiation, dedifferentiation or transdifferentiation of the immune cell; movement and / or transportation of the immune cell; exhaustion and / or reactivation of the immune cell; and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the immune cell.

[0238] In one aspect, a method for inducing the activity of an immune cell and / or a target cell is provided herein, the method comprising: (a) expressing a modified TCR complex and / or system disclosed herein in an immune cell; and (b) contacting a target cell with an immune cell under conditions that induce the activity of an immune cell and / or a target cell. In some embodiments, the system expressed in an immune cell comprises a modified T cell receptor (TCR) complex, the modified TCR complex comprising two or more antigen binding domains, the two or more antigen binding domains being optionally connected in series to (i) at least one TCR chain, the at least one TCR chain being selected from the α chain, β chain, γ chain and δ chain of the TCR, (ii) ε chain, δ chain and / or γ chain of a cluster of differentiation 3 (CD3); or (iii) CD3 ζ chain. In some embodiments, the system expressed in an immune cell comprises a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain with binding specificity for a first epitope, a transmembrane domain, and an intracellular signaling domain; a modified T cell receptor (TCR) complex, the modified TCR complex comprising a second antigen binding domain, which is connected to at least one of the following: (i) at least one TCR chain, wherein the at least one TCR chain is selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0239] When the target cell is contacted with the immune cell expressing the system, the first antigen binding domain and / or the second antigen binding domain can be combined with their corresponding epitopes.These epitopes are, for example, present on the target cell.The combination of the first antigen binding domain and / or the second antigen binding domain with their corresponding epitopes can activate the cytotoxicity of immune cells.In some cases, compared with comparable immune cells lacking the system, comparable immune cells lacking one or more components of the system (e.g., CAR, modified TCR complex), and / or expressing the system and wherein only one of the first and second antigen binding domains is combined with the corresponding epitope, when the first antigen binding domain and the second antigen binding domain, the cytotoxicity activated in the immune cell is enhanced.The combination of the first antigen binding domain with its corresponding epitope and / or the combination of the second antigen binding domain with its corresponding epitope can activate the cytotoxicity of immune cells and reduce the side effects related to the cytotoxicity. In some cases, compared with the comparable immune cells lacking the system, lacking one or more components of the system (e.g., CAR, modified TCR complexes) and / or expressing the system and wherein only one of the first and second antigen binding domains is combined with the corresponding epitope, the reduction of the side effect associated with cytotoxicity is greater. In some cases, the side effect reduced is cytokine release syndrome. The combination of the first antigen binding domain and its corresponding epitope and / or the combination of the second antigen binding domain and its corresponding epitope can activate the cytotoxicity of immune cells and increase the persistence of the cytotoxicity. In some cases, compared with the comparable immune cells lacking the system, lacking one or more components of the system (e.g., CAR, modified TCR complexes) and / or expressing the system and wherein only one of the first and second antigen binding domains is combined with the corresponding epitope, the persistence of cytotoxicity increases. In some cases, the cytotoxicity of immune cells induces the death of target cells.

[0240] In various embodiments of the method for inducing the activity of immune cells and / or target cells, immune cells can be any of a variety of immune cells. In some cases, immune cells include lymphocytes. Lymphocytes can be T cells. Non-limiting examples of T cells include CD8+T cells and CD4+T cells, αβT cells, γδT cells, Vγ9δ2T cells, Vδ1T cells, Vδ3T cells and Vδ5T cells. In some cases, lymphocytes are natural killer (NK) cells, effector T cells, memory T cells, cytotoxic T cells, NKT and / or T helper cells. In some cases, lymphocytes expressing modified TCR complexes and / or subject systems are KHYG cells, such as KHYG-1 cells or derivatives thereof.

[0241] In various embodiments of the method for inducing the activity of immune cells and / or target cells, target cells can be any of a variety of cell types. Target cells can be, for example, cancer cells, hematopoietic cells or solid tumor cells. In some cases, target cells can be cells identified in one or more of heart, blood vessel, salivary gland, esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, anus, endocrine gland, adrenal gland, kidney, ureter, bladder, lymph node, tonsil, adenoids, thymus, spleen, skin, muscle, brain, spinal cord, nerve, ovary, fallopian tube, uterus, vagina, breast, testis, prostate, penis, pharynx, larynx, trachea, bronchus, lung, diaphragm, cartilage, ligament and tendon. Target cells can be diseased cells.

[0242] In one aspect, the present disclosure provides a method for treating cancer in a subject. In some embodiments, the method includes administering to the subject an antigen-specific immune cell comprising a modified TCR complex or system disclosed herein. In some embodiments, the antigen-specific immune cell comprises a modified T cell receptor (TCR) complex, the modified TCR complex comprising two or more antigen binding domains, the two or more antigen binding domains are optionally connected in series to (i) at least one TCR chain, the at least one TCR chain is selected from the α chain, β chain, γ chain and δ chain of TCR, (ii) ε chain, δ chain and / or γ chain of cluster 3 (CD3); or (iii) CD3ζ chain. In some embodiments, the antigen-specific immune cell comprises a chimeric antigen receptor (CAR) containing a first antigen binding domain and a modified T cell receptor (TCR) complex containing a second antigen binding domain. In some embodiments, the method comprises (a) administering to a subject an antigen-specific immune cell comprising a chimeric antigen receptor (CAR) comprising a first antigen binding domain and a modified T cell receptor (TCR) complex comprising a second antigen binding domain, wherein target cells of the subject's cancer express one or more antigens recognized by the first and / or second antigen binding domains, and wherein the immune cell specifically binds to the target cell, and (b) contacting the target cell with the antigen-specific immune cell through the first and / or second antigen binding domains under conditions that induce immune cell activity of the immune cell against the target cell, thereby inducing death of the target cell of the cancer.

[0243] In one aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising (a) administering antigen-specific immune cells to the subject, wherein the antigen-specific immune cells are genetically modified immune cells that express any modified TCR complex and / or system of the embodiments provided herein; and (b) contacting the target cells with the antigen-specific immune cells under conditions that induce the immune cell activity of the immune cells against target cells of the subject's cancer, thereby inducing death of the target cells of the cancer.

[0244] In some embodiments, a method of treating cancer in a subject comprises genetically modifying an immune cell to generate an antigen-specific immune cell.

[0245] After contacting the target cell with the antigen-specific immune cell, the immune cell activity of the target cell against the cancer of the subject can induce the death of the target cell. The immune cell activity can be selected from the group consisting of: clonal expansion of the immune cell; cytokine release by the immune cell; cytotoxicity of the immune cell; proliferation of the immune cell; differentiation, dedifferentiation or transdifferentiation of the immune cell; movement and / or transport of the immune cell; exhaustion and / or reactivation of the immune cell; and release of other intercellular molecules, metabolites, compounds or combinations thereof by the immune cell. In some cases, the immune cell activity is the cytotoxicity of the immune cell. The cytotoxicity of the immune cell to the target cell can produce at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or up to about 100% reduction of the cancer of the subject. In some embodiments, the immune cell activity can be cytokine release by the immune cell. In some cases, cytokines are released by immune cells.Comparable immune cells lacking the modified TCR complex and / or system, lacking the modified TCR complex and / or system one or more components (e.g., CAR, modified TCR) and / or wherein only one of the first and second antigen-binding domains is combined with their corresponding epitopes. The amount of cytokines released by the immune cells can be at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or up to about 100% less.In some cases, when the first and second antigen-binding domains are combined with their corresponding epitopes, the persistence of the immune cell activity is greater than the combination of the first antigen-binding domain alone or the second antigen-binding domain alone.

[0246] In various embodiments of the method for treating a subject's cancer, the immune cell can be any of a variety of immune cells. In some cases, the immune cell includes a lymphocyte. The lymphocyte can be a T cell. Non-limiting examples of T cells include CD8+T cells and CD4+T cells. In some cases, the lymphocyte is a natural killer (NK) cell. In some cases, the lymphocyte expressing the modified TCR complex and / or the subject system is a KHYG cell, such as a KHYG-1 cell or a derivative thereof.

[0247] In various embodiments of the method for treating a subject's cancer, the cancer can be any of a variety of cancers.Cancer is, for example, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer or vulvar cancer. In one aspect, the present disclosure provides a composition.In some embodiments, the composition comprises a modified T cell receptor (TCR) complex and / or system disclosed herein. In some embodiments, the composition comprises a modified T cell receptor (TCR) complex comprising two or more binding domains that exhibit specific binding to two or more epitopes, wherein the antigen binding domains are optionally linked in series to: (i) at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; (ii) the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain. In some embodiments, the composition comprises one or more polynucleotides encoding: (a) a chimeric antigen receptor (CAR), the CAR comprising a first antigen binding domain having binding specificity for a first epitope, a transmembrane domain, and an intracellular signaling domain; and (b) a modified T cell receptor (TCR) complex, the modified TCR complex comprising a second antigen binding domain that exhibits specific binding to a second epitope, wherein the second antigen binding domain is connected to: at least one TCR chain, the at least one TCR chain selected from the α chain, β chain, γ chain and δ chain of the T cell receptor; the ε chain, δ chain and / or γ chain of cluster of differentiation 3 (CD3); or the CD3 ζ chain. The composition may include one or more polynucleotides encoding (a) a chimeric antigen receptor (CAR) comprising a first antigen binding domain, a transmembrane domain, and an intracellular signaling domain having binding specificity to a first epitope; and (b) a second antigen binding domain, the second antigen binding domain being connected to: the alpha chain, beta chain, gamma chain, and delta chain of a T cell receptor; the epsilon chain, delta chain, and / or gamma chain of cluster of differentiation 3 (CD3); or the CD3 ζ chain. In some embodiments, the one or more polynucleotides include a promoter operably connected thereto. The one or more polynucleotides may include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). In some embodiments, one or more components of a modified T cell receptor (TCR) complex or system encoded by one or more polynucleotides are connected by a linker separating two or more nucleic acid coding regions.The linker can be a 2A sequence, furin-V5-SGSGF2A, etc.

[0248] In one aspect, the present disclosure provides a method of producing a modified immune cell, the method comprising genetically modifying the immune cell by expressing a composition provided herein in the immune cell, thereby producing the modified immune cell.

[0249] In various embodiments of various aspects of the present invention, immune cells comprising the modified TCR complexes and / or systems provided herein can be used to induce the death of target cells. Using the modified TCR complexes and / or systems and methods disclosed herein, a variety of target cells can be killed. Target cells to which this method can be applied include a variety of cell types. The target cell can be in vitro. The target cell can be in vivo. The target cell can be ex vivo. The target cell can be a separated cell. The target cell can be a cell in an organism. The target cell can be an organism. The target cell can be a cell in a cell culture. The target cell can be a cell in a collection of cells. The target cell can be a mammalian cell or derived from a mammalian cell. The target cell can be a rodent cell or derived from a rodent cell. The target cell can be a human cell or derived from a human cell. The target cell can be a prokaryotic cell or derived from a prokaryotic cell. The target cell can be a bacterial cell or can be derived from a bacterial cell. The target cell can be an archaeal cell or derived from an archaeal cell. The target cell can be a eukaryotic cell or derived from a eukaryotic cell. The target cell can be a pluripotent stem cell. The target cell can be a plant cell or derived from a plant cell. The target cell can be an animal cell or derived from an animal cell. The target cell can be an invertebrate cell or derived from an invertebrate cell. The target cell can be a vertebrate cell or derived from a vertebrate cell. The target cell can be a microbial cell or derived from a microbial cell. The target cell can be a fungal cell or derived from a fungal cell. The target cell can be from a specific organ or tissue.

[0250] The target cell may be a stem cell or a progenitor cell. The target cell may include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). The target cell may include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. The cloned cell may include the progeny of the cell. The target cell may be in a living organism. The target cell may be a genetically modified cell.

[0251] The target cell can be a primary cell. For example, a culture of a primary cell can be passaged 0, 1, 2, 4, 5, 10, 15 or more times. The cell can be a unicellular organism. The cell can be grown in culture.

[0252] The target cell can be a diseased cell. The diseased cell may have altered metabolism, gene expression and / or morphological characteristics. The diseased cell can be a cancer cell, a diabetic cell and / or an apoptotic cell. The diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancer, metabolic diseases, eye disorders, organ disorders, musculoskeletal disorders, heart disease, etc.

[0253] If the target cell is a primary cell, then, for example, in an in vitro experiment, they can be harvested from an individual by any method. For example, leukocytes can be harvested by apheresis, leukocytapheresis / density gradient separation, etc. Cells from tissues (such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc.) can be harvested by biopsy. An appropriate solution can be used to disperse or suspend the harvested cells. Such solutions can generally be balanced salt solutions (e.g., physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution (Hank's balanced salt solution), etc.), which are conveniently supplemented with fetal bovine serum or other naturally occurring factors and acceptable low concentration buffers. Buffers can include HEPES, phosphate buffer, lactate buffer, etc. Cells can be used immediately, or they can be stored (e.g., by freezing). Frozen cells can be thawed and revived, and can be reused. Cells may be frozen in DMSO, serum, media buffer (eg, 10% DMSO, 50% serum, 40% buffered media), and / or some other such common solution for preserving cells at freezing temperatures.

[0254] Target cells can be identified in one or more of the heart, blood vessels, salivary glands, esophagus, stomach, liver, gall bladder, pancreas, intestines, colon, rectum, anus, endocrine glands, adrenal glands, kidneys, ureters, bladder, lymph nodes, tonsils, adenoids, thymus, spleen, skin, muscle, brain, spinal cord, nerves, ovaries, fallopian tubes, uterus, vagina, breast, testicles, prostate, penis, pharynx, larynx, trachea, bronchi, lungs, diaphragm, cartilage, ligaments, and tendons.

[0255] Non-limiting examples of cells that can be used as target cells include, but are not limited to, hematopoietic cells, lymphoid cells (such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells)), tumor infiltrating lymphocytes (TIL), natural killer cells, cytokine-induced killer cells (CIK); myeloid cells, such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophils, granulocyte / hypersegmentedneutrophil), monocyte / macrophage, erythrocyte (reticulocyte), mast cell, platelet / megakaryocyte, dendritic cell; cells from endocrine system, including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal (chromaffin cells), pineal (pinealocytes) cells; cells of nervous system, including glial cells (astroglia, microglia), magnocellular neurosecretory cells, stellate cells, Boettcher cells cells) and pituitary (gonadotropin cells, corticotropin cells, thyrotropin cells, somatotropin cells, prolactin cells); cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust phagocytes; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including stomach (principal gastric cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells, including osteoblasts, osteoclasts, and teeth (cementoblasts and ameloblasts); cartilage cells (cartilage cell, including chondroblasts and chondrocytes; skin cells, including tricocytes, keratinocytes, and melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, Kidney proximal tubule brush border cells, and macula densa cells; reproductive system cells, including sperm, sertoli cells, Leydig cells, and eggs; and other cells,These include adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hairshaft cells, cortical hair shaft cells, cuticular hair shaft cells, epidermal hair root sheath cells, hair root sheath cells of Huxley's layer, hair root sheath cells of Henle's layer. layer), outer root sheath cells, hair matrix cells (stem cells), wet stratified barrier epithelial cells, surface epithelial cells of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cells (stem cells) of epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urothelial cells (lining bladder and urethra), exocrine epithelial cells, salivary mucous cells (polysaccharide-rich secretions), salivary serous cells (glycoprotein-rich secretions), Von Ebner's gland cells in tongue (wash taste buds) buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), cerumen gland cells in the ear (ear wax secretion), eccrine sweat gland dark cells (glycoprotein secretion), eccrine sweat gland light cells (small molecule secretion), apocrine sweat gland cells (smellous secretion, sensitive to sex hormones), molar gland cells in the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (wash the olfactory epithelium), duodenal gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secrete semen components,including fructose for sperm motility), prostate cells (secretion of semen components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricating secretion), Littre's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), goblet cells of the respiratory and digestive tracts (mucus secretion), gastric mucosal mucus cells (mucus secretion), gastric gland zymogen cells (pepsinogen secretion), gastric gland acid secretory cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), small intestinal Paneth cells (lysozyme secretion), lung type II pneumocytes (surfactant secretion), lung Clara cells, hormone-secreting cells, anterior pituitary cells, somatotropin cells, pituitary lactotroph cells, thyrotropin cells, gonadotropin cells, stimulating hormone cells Adrenal cortical hormone cells, pituitary cells, large cell neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells, testicular Leydig cells, follicular endometrial cells, luteal cells of ruptured follicles, granulosa-lutein cells, theca-lutein cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney, type I pneumocytes (lining air interlayer of lung), pancreatic duct cells (alveolar heart cells), non-shedding duct cells (sweat glands, salivary glands, breast, etc.), duct cells (spermatic vesicles, prostate, etc.), epithelial cells lining closed internal body cavities (Epithelial cells lining closed internal body cavities), ciliated cells with propulsion function, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, autoantibodies Natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitor cells of the blood and immune system (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transduction cells, autonomous neuronal cells, sensory organs and peripheral neuronal supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, sperm cells, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, trophoblast cells, follicle cells, podocytes (in the testis), thymic epithelial cells, interstitial cells and interstitial kidney cells.

[0256] Of particular interest are cancer cells. In some embodiments, the target cell is a cancer cell. The cancer may be a solid tumor or a blood tumor. The cancer may be metastatic. The cancer may be a recurrent cancer. Non-limiting examples of cancer cells include cells of cancers including acanthoma, acinar cell carcinoma, acoustic neuroma, acromelanoma, acrohidroma, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloblastic leukemia with leukemia ... maturation), acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantoma, malignant adenoma, adenoid cystic carcinoma, adenoma, odontogenic adenomatous tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor (Atypical teratoid rhabdoid tumor), basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma (Carcinoma), carcinoma in situ, penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman's diseaseDisease), embryonic tumors of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, bile duct cancer, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloproliferative disease, chronic neutrophilic leukemia, bright cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibroma protuberans sarcoma, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelioma, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoma, ependymoma, epithelioid sarcoma, erythrocytic leukemia, esophageal cancer, nasal glioma, Ewing family tumor, Ewing family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, ectopic gonadal Germinoma, Extrahepatic bile duct cancer, Paget's disease of the breast, Fallopian tube cancer, Fetus in fetus, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germ tissue tumor, Gestational choriocarcinoma, Pregnancy Trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, glioma cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, hemangioendothelioma, malignant blood tumor, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma Lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanomamelanoma), Leukemia, Leukemia, Lip and Oral Cancer, Liposarcoma, Lung Cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphatic Leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant Peripheral Nerve Sheath Tumor, Malignant Rhabdoid Tumor, Malignant Salamander Tumor, MALT Lymphoma, Mantle Cell Lymphoma, Mast Cell Leukemia, Mediastinal Germ Cell Tumor, Mediastinal Tumor, Medullary Thyroid Cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary Metastatic Squamous Neck Cancer with Occult Primary Primary), metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal tumor, neoplasm, schwannoma, neuroblastoma, neuro Blastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumors, oligoastrocytoma, oligodendroglioma, large oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor Tumor, Paget's disease of the breast, superior pulmonary sulcus tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pinealoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, polyembryonic tumor, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumortumor), sex cord-stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestinal cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphoma In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, such as a cancer stem cell. In some embodiments, the cancer is of hematopoietic lineage, such as a lymphoma. The first and / or second antigen binding domains may bind to an epitope present on an antigen of a cancer cell.

[0257] In some embodiments, the target cells can form tumors. Tumors treated with the methods herein can result in stabilized tumor growth (e.g., one or more tumors increase in size by no more than 1%, 5%, 10%, 15%, or 20%, and / or do not metastasize). In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. In some embodiments, as a result of treatment according to the methods provided herein, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated, or reduced to below the detection level. In some embodiments, the subject remains tumor-free (e.g., alleviated) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more after treatment.

[0258] Target cell death can be determined by any suitable method, including but not limited to counting cells before and after treatment, or measuring the levels of markers associated with live or dead cells (eg, live or dead target cells).

[0259] The degree of cell death can be determined by any suitable method. In some embodiments, the degree of cell death is determined with respect to the initial conditions. For example, an individual can have a known starting amount of target cells, such as a starting cell mass of known size or a circulating target cell of known concentration. In this case, the degree of cell death can be expressed as a ratio of cells surviving after treatment to the starting cell population. In some embodiments, the degree of cell death can be determined by a suitable cell death assay. A variety of cell death assays can be used, and a variety of detection methods can be utilized. Examples of detection methods include, but are not limited to, the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations of these.

[0260] When the tumor is surgically removed after the treatment period is completed, the efficacy of the treatment in reducing the size of the tumor can be determined by measuring the percentage of necrotic tissue (i.e., dead tissue) removed. In some embodiments, the treatment is therapeutically effective if the percentage of necrosis of the removed tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the percentage of necrosis of the removed tissue is 100%, that is, viable tumor tissue is absent or undetectable.

[0261] In various embodiments of various aspects provided herein, target cell is exposed to immune cell or immune cell group or target cell is contacted with immune cell or immune cell group and can be carried out in vitro or in vivo. Target cell is exposed to immune cell or immune cell group and generally refers to target cell is contacted with immune cell and / or close enough, so that the antigen of target cell (for example, comprising epitope) (for example, membrane-bound or non-membrane-bound antigen) can be bound to the antigen-binding domain of the first antigen-binding domain and / or the second antigen-binding domain. It is possible to realize that target cell is exposed to immune cell or immune cell group in vitro by co-cultivation of target cell and immune cell. Target cell and immune cell can be co-cultivated, for example, as adherent cell or alternatively suspension. Target cell and immune cell can be co-cultivated in various cell culture mediums of suitable types, for example, co-cultivated in culture medium with supplement, growth factor, ion etc. In some cases, it is possible to realize that target cell is exposed to immune cell or immune cell group in vivo by the following: immune cell is applied to subject (for example, human subject), and the immune cell is positioned to target cell by circulatory system. In some cases, immune cells can be delivered, for example by direct injection, to the immediate area where target cells are localized.

[0262] The exposure or contacting can be performed for any suitable length of time, e.g., at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, or longer.

[0263] In various embodiments of various aspects herein, the modified TCR complexes and / or systems provided herein are expressed in host cells (e.g., immune cells, such as antigen-specific immune cells). The host cell can be a human cell. The host cell can be a non-human cell. The host cell can be autologous or allogeneic for a subject in need. In some cases, the host cell can be xenogeneic. The host cell can be an immune cell, such as a lymphocyte or a bone marrow cell. The host cell can be a T cell, a B cell, a NK cell, etc. In some embodiments, the host cell can be a CD3+ cell, a CD3- cell, a CD5+ cell, a CD5- cell, a CD7+ cell, a CD7- cell, a CD14+ cell, a CD14- cell, a CD8+ cell, a CD8- cell, a CD103+ cell, a CD103- cell, a CD11b+ cell, a CD11b- cell, a BDCA1+ cell, a BDCA1- cell, a L-selectin+ cell, a L-selectin- cell, a CD25+ cell, a CD25- cell, a CD27+ cell, a CD27- cell, a CD28+ cell, a CD28- cell, a CD In some embodiments, the host cell may be positive for two or more factors. For example, the host cell may be CD4+ and CD8+. In some embodiments, the host cell may be negative for two or more factors. For example, the host cell may be CD25-, CD44- and CD69-. In some embodiments, the host cell may be positive for one or more factors and negative for one or more factors. For example, the cells can be CD4+ and CD8-.In some embodiments, host cells can be selected for the presence or absence of one or more given factors (eg, cells can be separated based on the presence or absence of one or more markers described herein).

[0264] In some embodiments, the selected host cells can also be expanded in vitro. The selected and / or expanded host cells can be administered to a subject in need. It should be understood that the cells used in any method disclosed herein can be a mixture of any cells disclosed herein (e.g., two or more different cells). For example, the composition may include a mixture of different cells (e.g., T cells and B cells). The mixture may include, for example, a stem memory T cell comprising CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+. SCM The mixture may include, for example, central memory T cells that contain L-selectin and CCR7. CM Cells, wherein the central memory cells can secrete, for example, IL-2, but not IFNγ or IL-4. The mixture can include, for example, effector memory T cells containing L-selectin or CCR7 EM cells, and produce, for example, effector cytokines, such as IFNγ and IL-4.

[0265] Host cells can be obtained from a subject. In some cases, host cells can be a population of T cells, NK cells, B cells, etc. obtained from a subject. T cells can be obtained from a variety of sources including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, a variety of techniques can be used, such as Ficoll. TM The invention relates to a method for obtaining T cells from a unit of blood collected from a subject by separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The product of apheresis generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps.

[0266] In some embodiments, the immune cell population provided herein can be heterogeneous. In some embodiments, the cells used can be composed of a heterogeneous mixture of CD4 T cells and CD8 T cells. The CD4 cells and CD8 cells can have the phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells can also have the phenotypic characteristics of effector memory cells. In some embodiments, the cells can be central memory cells.

[0267] In some embodiments, host cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subsets, such as but not limited to T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells. In some cases, the cell can be any immune cell, including any T cell, such as tumor infiltrating cells (TIL), such as CD3+T cells, CD4+T cells, CD8+T cells or any other type of T cells. T cells can also include memory T cells, memory stem T cells or effector T cells. T cells can also be selected from a large number of populations, such as selecting T cells from whole blood. T cells can also be expanded from a large number of populations. T cells can also be biased towards specific populations and phenotypes. For example, T cells can be phenotypically biased to include the following: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and / or IL-7Rα (+). Suitable cells can be selected, and the suitable cells contain one or more markers selected from the list including the following: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and / or IL-7Rα (+). Host cells also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells. The host cell may contain any number of primary cells, such as human cells, non-human cells and / or mouse cells. The host cell may be a progenitor cell. The host cell may be derived from a subject to be treated (e.g., a patient). The host cell may be derived from a human donor. The host cell may be a stem memory TSCM cell comprising CD45RO (-), CCR7 (+), CD45RA (+), CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, and the stem memory cell may also express CD95, IL-2Rβ, CXCR3 and LFA-1, and show many functional attributes unique to the stem memory cell. The host cell can be a central memory TCM cell comprising L-selectin and CCR7, which can secrete, for example, IL-2, but not IFNγ or IL-4. The host cell can also be an effector memory TEM cell comprising L-selectin or CCR7, and produces, for example, effector cytokines such as IFNγ and IL-4.

[0268] Many virus-based systems have been developed to transfer genes into mammalian cells. For example, retrovirus, slow virus and adenovirus provide a convenient platform for gene delivery systems. The subject system can be inserted into a vector and packaged in a retroviral particle using techniques known in the art. The vector derived from a retrovirus (such as a slow virus) is a suitable tool for realizing long-term gene transfer, because they allow long-term, stable integration of transgenics and its propagation in daughter cells. The advantage of the increase compared to the vector derived from a tumor retrovirus (such as a murine leukemia virus) is that they can transduce non-proliferating cells. They also have the additional advantage of low immunogenicity.

[0269] In one aspect, nucleic acids encoding systems comprising modified TCR complexes and / or CARs can be delivered virally or non-virally. Viral delivery systems (e.g., viruses comprising a pharmaceutical composition of the present invention) can be administered by direct injection, stereotactic injection, intraventricularly, by a micropump infusion system, by convection, by catheter, intravenous, parenteral, intraperitoneal and / or subcutaneous injection to cells, tissues or organs of subjects in need. In some cases, cells can be transduced in vitro or ex vivo with a viral delivery system. Transduced cells can be administered to subjects with diseases. For example, stem cells can be transduced with a viral delivery system comprising a pharmaceutical composition, and the stem cells can be implanted in a patient to treat a disease. In some cases, the dose of transduced cells given to a subject can be about 1×10 in a single dose. 5 cells / kg, about 5×10 5 cells / kg, about 1×10 6 cells / kg, about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 5×10 7 cells / kg, about 1×10 8 cells / kg, or more.

[0270] Packaging cell lines can be used to generate virus particles comprising modified TCR complexes and / or subject systems provided herein. Packaging cell lines can also be used to perform methods provided herein. Packaging cells that can be used include but are not limited to HEK 293 cells, HeLa cells, and Vero cells, etc. In some cases, the supernatant of the packaging cell line is treated with PEG precipitation to concentrate virus particles. In other cases, a centrifugation step can be used to concentrate virus particles. For example, a column can be used to concentrate viruses during centrifugation. In some cases, precipitation occurs at no more than about 4°C (e.g., about 3°C, about 2°C, about 1°C, or about 1°C) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 24 hours. In some cases, virus particles can be separated from the supernatant of PEG precipitation by performing low-speed centrifugation and then using a CsCl gradient. Low speed centrifugation can be about 4000rpm, about 4500rpm, about 5000rpm or about 6000rpm for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes. In some cases, viral particles are separated from the supernatant of PEG precipitation by centrifugation at about 5000rpm for about 30 minutes, followed by a CsCl gradient.

[0271] It can be about, roughly, at least about or at most about 1-3 hours, 3-6 hours, 6-9 hours, 9-12 hours, 12-15 hours, 15-18 hours, 18-21 hours, 21-23 hours, 23-26 hours, 26-29 hours, 29-31 hours, 31-33 hours, 33-35 hours, 35-37 hours, 37-39 hours, 39-41 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 16 days, 20 days or longer than 20 days by virus (for example, slow virus) introduced into subject cell or subject cell group.In some cases, virus vector encodes modified TCR complex and / or system, such as CAR-T, modified TCR complex or their combination.In some cases, virus vector encodes CAR-T. In some cases, the viral vector encodes a modified TCR complex. Immune cells can be transduced with viral particles encoding both CAR and modified TCR complexes. Immune cells can be transduced with viral particles encoding CAR. Immune cells can be transduced with viral particles encoding modified TCR complexes. The nucleic acid encoding the modified TCR complex and / or the subject system can be randomly inserted into the genome of the cell. The nucleic acid encoding the modified TCR complex and / or the system can encode its own promoter, or can be inserted into a position under the control of the cell's endogenous promoter. Alternatively, the nucleic acid encoding the modified TCR complex and / or the system can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter or a non-coding region. The expression of the modified TCR complex and / or the system can be verified by expression assays (e.g., qPCR) or by measuring the level of RNA in the transduced cell. Expression levels can also indicate copy number. For example, if the expression level is high, this can indicate that more than one copy of the nucleic acid encoding the modified TCR complex and / or the system is integrated into the genome of the cell. Alternatively, high expression may indicate that the nucleic acid encoding the modified TCR complex and / or system is integrated in a highly transcribed region, such as near a highly expressed promoter. Expression may also be verified by measuring protein levels, such as by Western blotting.

[0272] The cell viability of subject cells or subject colonies can be measured by fluorescence activated cell sorting (FACS). In some cases, after a virus or non-viral vector comprising a nucleic acid encoding a modified TCR complex and / or subject system is introduced into a cell or cell group, cell viability is measured. In some cases, after a viral vector is introduced into a cell or cell group, at least about or at most about or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% of the cell is vigorous. In some cases, cell viability is measured at about, at least about, or at most about 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, 180 hours, 192 hours, 204 hours, 216 hours, 228 hours, 240 hours, or longer than 240 hours after the viral vector is introduced into the cells and / or cell population. In some cases, cell viability is measured at about, at least about, or at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 45 days, 50 days, 60 days, 70 days, 90 days, or longer than 90 days after the viral vector is introduced into the cells and / or cell population. In some cases, cytotoxicity is measured at about, at least about, or at most about 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, 102 hours, 108 hours, 114 hours, 120 hours, 126 hours, 132 hours, 138 hours, 144 hours, 150 hours, 156 hours, 168 hours, 180 hours, 192 hours, 204 hours, 216 hours, 228 hours, 240 hours, or longer than 240 hours after the viral vector is introduced into the cell or cell population.

[0273] In some embodiments, one or more nucleic acids encoding modified TCR complexes and / or systems comprising modified TCR complexes and / or CARs can be delivered by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0274] In some embodiments, immune cells expressing the modified TCR complex and / or system provided herein are administered. Immune cells can be administered before, during or after the occurrence of a disease or condition, and the timing of administering immune cells can vary. For example, immune cells expressing modified TCR complexes and / or subject systems can be used as prophylactics, and can be continuously administered to subjects with a condition or disease tendency to prevent the occurrence of the disease or condition. Immune cells can be administered to subjects as quickly as possible during or after the onset of symptoms. Administration can begin within the first 48 hours of symptom onset, within the first 24 hours of symptom onset, within the first 6 hours of symptom onset, or within 3 hours of symptom onset. Initial administration can use any preparation described herein, by any suitable route, such as by any route described herein. It is feasible that after the onset of a disease or condition is detected or suspected, immune cells can be administered as soon as possible to treat the required duration of the disease, for example, from about 1 month to about 3 months. The treatment duration of each subject may vary.

[0275] The compositions comprising immune cells expressing modified TCR complexes and / or subject systems provided herein can be administered to subjects using known modes and techniques. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, but are not limited to, intratumoral, intradermal, subcutaneous (SC, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal and intrathecal (cerebrospinal fluid). Any known device for parenteral injection or infusion of preparations can be used to achieve such administration. Preparations comprising subject compositions can be administered to subjects in an amount that effectively treats and / or prevents specific indications or diseases. Doctors can determine the appropriate dose to be used. Compositions comprising immune cells expressing modified TCR complexes and / or subject systems can be administered 4 times, 3 times, 2 times or once independently every day, every other day, every three days, every four days, every five days, every six days, every week, every eight days, every nine days, every ten days, every two weeks, every month and every two months.

[0276] The compositions and methods provided herein can be combined with adjuvant therapy comprising cytotoxic / antitumor agents and anti-angiogenic agents.Cytotoxic / antitumor agents can be defined as agents that attack and kill cancer cells.Some cytotoxic / antitumor agents can be alkylating agents, which can alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin and dacarbazine.Other cytotoxic / antitumor agents can be antimetabolites of tumor cells, such as cytarabine, fluorouracil, methotrexate, mercaptopurine, thiopurine and procarbazine.Other cytotoxic / antitumor agents can be antibiotics, such as doxorubicin, bleomycin, actinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C and daunomycin.Other cytotoxic / antitumor agents can be mitotic inhibitors (vinca alkaloids). These mitotic inhibitors include vincristine, vinblastine and etoposide. Other cytotoxic / antitumor agents include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone and vindesine. Antiangiogenic agents may also be used. Suitable antiangiogenic agents for use in the disclosed methods and compositions include anti-VEGF antibodies, including humanized antibodies and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and metalloproteinase-2. (TIMP-1 and TIMP-2). Small molecules may also be used, including topoisomerases, such as razoxane, a topoisomerase II inhibitor with antiangiogenic activity.

[0277] Other anticancer agents that can be used in combination include, but are not limited to, acivicin; aclarubicin; acordazol hydrochloride; aclonine; adolesin; aldesleukin; hexamethylmelamine; ambomycin; ametrine acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; triclindac; avastin; azacitidine; azadepa; azomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; binefad; Bleomycin sulfate; Bleomycin sodium; Bropiridamine; Busulfan; Actinomycin C; Carotene; Carbamide; Carbetim; Carboplatin; Carmustine; Carrubicin hydrochloride; Carzeresin; Cedifenol; Chlorambucil; Ciromycin; Cisplatin; Cladribine; Clinatoyl mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Actinomycin D; Daunorubicin hydrochloride; Decitabine; Dextroplatin; Dezaguanine; Mesylate Dezaguanine acid; diazaquinone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drolostanone propionate; dazomicin; edatrexamate; eflornithine hydrochloride; elsamitrucin; enloplatin; enprobamate; epipipridine; epirubicin hydrochloride; erbulazole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprin; fadroxil hydrochloride ; Fazarabine; Fenretinide; Floxuridine; Fludarabine phosphate; Fluorouracil; Flucitabine; Fosquinone; Fostricin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Imofosine; Interleukin II (including recombinant interleukin II or rIL2); Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-I; Interferon γ-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; lirazole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprodol; maytansine; nitrogen mustard hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menolipir; mercaptopurine; methotrexate; methotrexate sodium; chlorpheniramine; metodepa; mibutidamide; mitocarcin; mitocromin; mitocrinol; mitomacin; mitomycin C; mitospe; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogamycin; ormaplatin; oxysulen; taxane alcohol; pegaspargase; pelimycin; pentamidine; pelimycin sulfate; perfosfamide; piperobroman; piperosulfan; pyroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfimer; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofuracin; liboadenosine; roglulimide; safingol; safingol hydrochloride; semustine; sintrazine; sodium phosphoacetyl aspartate; sparamycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptomelanocin; streptozotocin; sulfochlorophenylurea; talithromycin; tecogalan sodium; tegafur; tiloxantrone hydrochloride; temoporfin; teniposide; tiroxilon; testolactone; thiabendazole;Thioguanine; thiotepa; thiazofurin; tirapazamine; toremifene citrate; triptolon acetate; tretolide phosphate; trimetrexate; trimetrexate glucuronide; triptorelin; tobradazole hydrochloride; uramustine; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinpitidine sulfate; vincoside sulfate; vinrosin sulfate; vinorelbine tartrate; vinprodine sulfate; vinorelbine sulfate; vorozole; zeniplatin; ninstatin; daunorubicin hydrochloride. Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adenosine; adolesin; aldesleukin; ALL-TK antagonists; hexamethylmelamine; aminostine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; anrelix; anti-dorsalizing morphogenetic protein-1 (anti-dorsalizing morphogenetic protein-1) protein-1); antiandrogen; prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; glycine aphidicolin; apoptosis gene regulator; apoptosis regulator; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azamycin; diazotyrosine; baccatin III derivatives; balanol; batimastat; CAR / ABL antagonists; benzodihydropyrrol; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; diaziridinylspermine; binefad; bistratene A A); bizolexin; breflate; bropiridamine; budotetan; buthionine sulfoximine; calcipotriol; calciphosphotin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamide triazole; CaRest M3; CARN 700; cartilage-derived inhibitors; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorin; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; collismycin A; collismycin B; conpretin A4; conpretin analogs; conagenin; crambescidin 816; crestol; candidin 8; candidin A derivatives;euracin A; cyclopentanthraquinone; cycloplatam; cypemycin; cytarabine sodium octadecyl phosphate; cytolytic factor; estradiol phosphate (cytostatin); dacillin; decitabine; dehydroepiphyrin B; deserelin; dexamethasone; ifosfamide; dexrazoxane; dexverapamil; diazurone; epiphyrin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycin; diphenylspiromustine; docetaxel; diol; dolasetron; doxifluridine; droloxifene; dronabinol; dukamycin SA; ebselen; ethomustine; edilfosine; edrecolomab; eflornithine; elemene; ethidium bromide; epirubicin; aprelet; estramustine analogs; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; fluasterone; fludarabine; fluorodaunorunicin hydrochloride hydrochloride); fofolmek; formestane; fostramcin; fostramustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; regulatory proteins; hexamethylenebisacetamide; hyperforin; ibandronic acid; idarubicin; idoxifene; idemenone; imofosine; ilomastat; imidazoacridone; imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; interferon agonists; interferons; interleukins; iodobenzylguanidine; iododexorubicin; 4-adenosine; iropra; irsogladine; isobengazole; isohomohalicondrin B B); itasetron; jasplakinolide; kahalalide F; spirochetin triacetate-N; lanreotide; ranamycin; levograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibitory factor; interferon-alpha; leuprolide + estrogen + progesterone; leuprolide; levamisole; lirazole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; earthworm phospholipids; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; luteotecan; lutetium texaphyrin; lysofylline; lytic peptides; metansine;Manostatin A; Marimastat; Masoprofen; Masospin; Matrix lytic factor inhibitor; Matrix metalloproteinase inhibitor; Menolil; Merbarone; Avorelin; Methioninase; Metoclopramide; MIF inhibitor; Mifepristone; Miltefosine; Miriprastine; Mitoxantrone ... B); mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; narostem; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidants; nitrulline lyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; oramaplatin; oxalitone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoyl rhizoxin; pamidronate; panaxytriol; panomifen; parabactin ); pozenipdine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflurane; perphosphamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; pirtrexine; placetin A; placetin B; plasminogen activator inhibitors; platinum complexes; platinum compounds; platinum-triamine complexes; porfimer sodium ; porfibrinocin; prednisone; propyl diacridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; microalgae protein kinase C inhibitors; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurin; pyrazoline acridine; pyridoxal hemoglobin polyethylene oxide conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated reteptine; etidronate rhenium Re 186; gentamicin; ribozymes; RII isotretinoin;Roglulimide; Roxitocin; Romotide; Roquimec; Rubiginone B1; Ruboxyl; Safengo; Saintopin; SarCNU; Myophytol A; Sargramostim; Sdi 1 mimetic; Semustine; Senescence-derived inhibitor 1; Sense oligonucleotide; Signal transduction inhibitor; Signal transduction regulator; Single-chain antigen binding protein; Sizoran; Sobuzoxane; Borca sodium; Sodium phenylacetate; Solferol; Somatomedin binding protein; Sonamin; Fospartate; Spicamycin D; Spiromustine; Spongistatin 1 1); squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; potent vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycan; tamustine; tamoxifen methyl iodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitor; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetamine razomine; thrombopoietin; thrombopoietin mimetics; thymofasin; thymopoietin receptor agonist; thymosin; thyrotropin; ethylprotirizine; tirapazamine; titanocenyl dichloride; tosentin; toremifene; totipotent stem cell factor; translation inhibitor; retinoic acid; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; torosterol; tyrosine kinase inhibitor; tyrosine phosphorylation inhibitor; UBC inhibitor; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonist; vapreotide; variolin B (variolin B); erythrocyte gene therapy vector system; vilaresol; veratramine; verdins; verteporfin; vinorelbine; vinxaltine; integrin antibody (vitaxin); vorozole; zanoteron; zeniplatin; benzyl retinoid C; and statins ester. ;

[0278] Immune cells comprising any of the modified TCR complexes and / or systems provided herein can be administered to a subject in combination with any number of related treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2 or cytarabine (also known as ARA-C), e.g., before, simultaneously with, or after the related treatment modality. In some cases, the subject immune cells can be used in combination with chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies) or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and radiation. The engineered cell composition can also be administered to the patient in combination (e.g., before, at the same time, or after) with bone marrow transplantation, T cell ablation therapy using any chemotherapeutic agent (such as fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (such as OKT3 or CAMPATH). In some cases, the subject immune cell composition can be administered after B cell ablation therapy (such as reagents reacting with CD20, such as rituximab). For example, the subject can undergo standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject can receive an infusion of immune cells (e.g., immune cells amplified comprising modified TCR complexes and / or subject systems). In addition, amplified immune cells can be administered before or after surgery.

[0279] In some cases, for example, in compositions, formulations and methods for treating cancer, the unit dose of the composition or formulation administered can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg. In some cases, the total amount of the composition or formulation administered can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 g.

[0280] Example

[0281] The following examples are intended only to be exemplary of the present invention, and therefore should not be construed as limiting the present invention in any way.The following examples and detailed description are provided by way of illustration and not limitation.

[0282]

[00136] Various aspects of the present disclosure are further illustrated by the following non-limiting examples.

[0283] Example 1: Generation of anti-BCMA sdAb

[0284] immunity

[0285] Two camels were immunized with recombinant BCMA ECD protein (ACRO Biosystems, catalog number: BCA-H522y, SEQ ID NO: 1) in accordance with all current animal welfare regulations. For immunization, the antigen was formulated as an emulsion with CFA (primary immunization) or IFA (boosting immunization). The antigen was administered intramuscularly by double-point injection in the neck. Each animal received two injections of an emulsion containing 100 μg of BCMA ECD at weekly intervals, and four subsequent injections containing 50 μg of antigen. At different time points during the immunization, 10 ml blood samples were collected from the animals and serum was prepared. In an ELISA-based experiment using immobilized BCMA ECD protein, serum samples were used to verify the induction of antigen-specific humoral immune responses. Five days after the last immunization, 150 ml blood samples were collected from each animal. Peripheral blood lymphocytes (PBL) were isolated from 300 ml blood samples using a Ficoll-Paque gradient (Amersham Biosciences) to obtain approximately 1 × 10 9 The maximum diversity of antibodies is expected to be equal to the number of B lymphocytes sampled, which is about 10% of the number of PBLs (1×10 8 ). The fraction of heavy chain antibodies in camels is at most 20% of the number of B lymphocytes. Therefore, the maximum diversity of HCAbs in a 300 ml blood sample is estimated to be about 2 × 10 7 different molecules.

[0286] Library construction

[0287] RNA extracted from PBL was used as starting material for RT-PCR to amplify gene fragments encoding sdAbs. These fragments were cloned into in-house phagemid vectors. In frame with the sdAb coding sequence, the vector also encoded a C-terminal (His)6 tag. The library size was greater than 1×10 9 Library phages were prepared according to standard protocols and stored at 4 °C after filter sterilization for future use.

[0288] Binder Isolation and High Throughput Screening

[0289] Binding agents were separated from the above libraries using solid phase panning and cell-based panning. One round of panning was performed in both cases. The total number of output clones, BCMA positive clone percentage (by ELISA) and sequence diversity of BCMA specific binding agents of each selection output were analyzed. Based on these parameters, the best panning output was selected for high-throughput screening. To this end, selected output phages were used to infect exponentially growing Escherichia coli (E.coli) cells. The double-stranded DNA library of the output was extracted, the sdAb insert was cut out from the phagemid vector, and inserted into a soluble expression vector for high-throughput screening. In the frame with the sdAb coding sequence, the vector also encodes a C-terminal (His) 6 tag. The colony was picked and grown in a 96 deep-well plate containing 1ml2YT culture medium. The expression of sdAb was induced by adding 1mM IPTG in the supernatant.

[0290] The ability of sdAb in the supernatant to bind to BCMA ECD protein was analyzed by ELISA, and its ability to bind to BCMA stable cell lines was analyzed by FACS. All binding agents were sequenced, and some binding agents were further characterized, including ranking affinity by surface plasmon resonance (SPR) on a BIAcore T200 instrument. The experiment was performed as follows: the crude sdAb protein was captured on a sensor chip (sensorchip) by an affinity tag. The amount of antibody captured depends on the concentration of crude protein in the supernatant. High concentrations (100nM) of antigen protein, i.e., His-tagged human BCMA (ACRO Biosystems, catalog number: BCA-H522y) and Fc-fused cynomolgus monkey BCMA (ACRO Biosystems, catalog number: BCA-C5253, SEQ ID NO: 2) were flowed over the sensor chip surface and combined with sdAb. The association rate (k on ) and dissociation rate (k off ) was roughly calculated based on the association and dissociation at an antigen concentration of 100 nM and used to estimate the equilibrium dissociation constant (K D ).

[0291]

[0292]

[0293] Example 2: Viral transfection and viral particle production

[0294] To generate viral particles containing polynucleic acids encoding any system disclosed herein, a lentiviral packaging plasmid mixture containing pMDLg / pRRE (Addgene No. 12251), pRSV-Rev (Addgene No. 12253) and pMD2.G (Addgene No. 12259) was appropriately pre-mixed with a PLVX-EF1A (including a target system) vector and polyetherimide (PEI) at a pre-optimized ratio and incubated at room temperature for 5 minutes. The transfection mixture was added dropwise to 293-T cells and then gently mixed with the cells. The transfected 293-T cells were incubated overnight at 37°C and 5% CO2. 24 hours after transfection, the supernatant was collected and centrifuged at 4°C, 500g for 10 minutes to remove any cell debris, followed by an ultracentrifugation step. After ultracentrifugation, the centrifuged supernatant was filtered through a 0.45 μm PES filter to concentrate the viral supernatant. After centrifugation, the supernatant was carefully discarded and the virus pellet was rinsed with pre-chilled DPBS. The virus concentration was measured. The virus was aliquoted and stored at -80°C. The virus titer was determined by functional transduction on T cell lines.

[0295] Briefly, pLVX-Puro (Clontech No. 632164) was used to modify the lentiviral vector by replacing the original promoter with the human elongation factor 1α promoter (hEF1α) and removing the puromycin resistance gene with EcoRI and BamHI by GenScript.

[0296] PLVX-EF1A was further subjected to the lentiviral packaging procedure as described above.

[0297] Example 3: Immune cell preparation

[0298] Leukocytes were collected in R10 medium and then mixed with 0.9% NaCl solution at a ratio of 1:1 (v / v). 3mL lymphoprep medium was added to a 15mL centrifuge tube. Lymphoprep medium was slowly layered to form a 6mL diluted lymphocyte mixture. The lymphocyte mixture was centrifuged at 800g for 30 minutes at 20°C without brake. Lymphocyte buffycoat was then collected with a 200 μL pipette. The harvested fractions were diluted at least 6 times with 0.9% NaCl or R10 to reduce the density of the solution. The harvested fractions were then centrifuged at 250g for 10 minutes at 20°C. The supernatant was completely aspirated and 10mL R10 was then added to the cell pellet. The mixture was further centrifuged at 250g for 10 minutes at 20°C. The supernatant was then aspirated. 2 mL of R10 preheated to 37°C containing 100 IU / mL IL-2 was added to the cell pellet, and the cell pellet was gently resuspended. The cells were quantified and the PBMC samples were ready for experiments. Human T cells were purified from PBMC using the Miltenyi Pan T cell isolation kit (Cat. No. 130-096-535).

[0299] The prepared T cells were then preactivated for 48 hours using the Human T Cell Activation / Expansion Kit (Milteny No. 130-091-441) by using one loaded anti-biotin MACSiBead particle per two cells (bead to cell ratio 1:2).

[0300] Example 4: T cell transfection

[0301] Pre-activated T cells were collected, suspended and resuspended in 1640 medium containing 300 IU / mL IL-2. The lentiviral vector encoding the system was diluted to MOI = 5 with the same medium and infected with 1E + 06 activated T cells. Pre-activated T cells were transduced with lentiviral stock in the presence of 8 μg / ml polybrene by centrifugation at 1000g for 1 hour at 32 ° C. The transduced cells were then transferred to a cell culture incubator to express the gene under appropriate conditions. On the second day, the transduced cells were centrifuged and fresh medium was replaced, the cell concentration was measured every 2 days, and fresh medium was added to continue amplification.

[0302] Example 5: Quantification of receptor expression

[0303] On the 3rd day after transduction and later (usually the 3rd to the 8th day), the expression of the system by the cells was evaluated by flow cytometry. An aliquot of the cells was collected from the culture, washed, precipitated, and resuspended in 100ul PBS supplemented with 0.5% FBS and a dilution of the binding antibody or antigen protein 1 / 100. The cells were resuspended in about 100ul of Ab. The cells were incubated at 4°C for 30 minutes. Viability dye eFluor780 or SYTOX Blue viability dye was also added according to the manufacturer's instructions. After incubation, the cells were washed twice in PBS and resuspended in 100 to 200ul PBS for analysis. The average fluorescence of the system was quantified by flow cytometry.

[0304] For anti-BCMA staining, cells were stained with polyclonal biotinylated goat anti-human BCMA antibody (R&D, catalog number BAF 193) followed by staining with streptavidin (BD). Flow cytometric analysis of all experiments was performed using FlowJo (Tree Star, Inc.).

[0305] Example 6: Cytotoxicity Assay

[0306] BCMA Antibody Screening on the εTCR Platform

[0307] Anti-BCMA antibodies (BCMA1-12) were fused to ε-TCR alone to evaluate the cytotoxic effects on RPMI-8226 cells. On the 3rd or 6th day after transduction, the effector cells were co-cultured in 96-well plates at different effector-to-target ratios (0.5: 1, 1.5: 1, and 3: 1) at 37°C for 20 hours. Other wells contained only assay buffer (1640 phenol red-free medium plus 2% hiFBS), only target cells (T), only effector cells (E), and maximum release of target cells (target cells using 1% solution of triton-X 100). Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged, and the supernatant was collected and transferred to a new 96-well plate. According to the manufacturer's manual, the supernatant plate was diluted with an equal volume of LDH assay reagent. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492 nm and 650 nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0308] The results showed that effector cells expressing different BCMA sdAbs had different cell killing effects, such as BCMA 1, BCMA 2, BCMA 5, BCMA 6, BCMA 8, BCMA 9 and BCMA 12 antibodies showed better cell killing effects, such as in Fig. 8A , Figure 8B and Figure 8C As seen in.

[0309] IFN-γ expression was determined by HTRF as in Fig.8D , Fig. 8E and Fig.8F 384-well low volume white plates were used in the assay for IFN-γ detection (Human IFNγ Kit, Gisbio). The amount of secreted IFN-γ in the cytotoxicity assay showed a similar trend to the cell killing effect.

[0310] Multi-component system

[0311] Anti-BCMA3-ε-TCR (BCMA3 eTCR), anti-BCMA2-ε-TCR (BCMA2eTCR), anti-BCMA2-anti-BCMA3-εTCR (tandem BCMA 2&3eTCR) and anti-BCMA1-anti-BCMA2-anti-BCMA3-γTCR (tandem BCMA 1&2&3gTCR) and control untransduced cells were determined in a 20-hour co-culture assay, in which RPMI-8226 cells (BCMA+) were co-cultured with an effector to target cell ratio (E:T) of 0.33:1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by the LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged, and the supernatant was collected and transferred to a new 96-well plate. According to the manufacturer's manual, the supernatant plate was diluted with an equal volume of LDH assay reagent. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492 nm and 650 nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0312] The results showed that compared with eTCR fused with a single antibody, the tandem BCMA antibody on the TCR subunit provided a significantly better cell killing effect, indicating that the tandem BCMA antibody on the TCR is a better choice for cell killing effects (such as Fig. 9 shown).

[0313] The cytotoxicity of anti-BCMA1-anti-BCMA2-anti-BCMA3ε-TCR (tandem BCMA 1-2-3eTCR), anti-BCMA2-anti-BCMA3ε-TCR (tandem BCMA 2-3eTCR), anti-BCMA4-anti-BCMA5ε-TCR (tandem BCMA4-5eTCR), anti-BCMA2-anti-BCMA3-anti-BCMA4ε-TCR (tandem BCMA 2-3-4eTCR), anti-BCMA1-anti-BCMA4-anti-BCMA5ε-TCR (tandem BCMA 1-4-5eTCR) and control untransduced cells was determined in a 20-hour co-culture assay, in which CHO / BCMA / CD19 cells (BCMA+CD19+) were co-cultured at an effector to target cell ratio (E:T) of 0.5:1 and 1.5:1. Each condition was performed in triplicate and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-cultivation, the assay plate was centrifuged, the supernatant was collected and transferred to a new 96-well plate. The supernatant plate was diluted with an equal volume of LDH assay reagent according to the manufacturer's manual. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0314] The results showed that constructs with five combinations of selected BCMA antibodies tandemly linked to eTCR all had excellent in vitro cell killing effects (e.g. Fig.10 shown).

[0315] Anti-BCMA1ε-TCR (BCMA1 eTCR), anti-BCMA1 4-1BB-CD3ζ-CAR (BCMA1 BBzCAR), anti-CD19ε-TCR (CD19 eTCR) and anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19eTCR) and untransduced control immune cells were determined in a 20-hour co-culture assay. In the experiment, the effector cells were collected by centrifugation and then diluted to the desired concentration with 1640 phenol red-free medium (Invitrogen) supplemented with 2% heat-inactivated FBS (Invitrogen). Target cell NCI-H929 showed strong expression of the target antigen BCMA. The effector cells were co-cultured in 96-well plates at 37°C for 20 hours with different effector to target ratios (E:T=5:1 and 10:1). The other wells contained assay buffer only (1640 phenol red-free medium plus 2% hiFBS), target cells only (T), effector cells only (E), and maximum release of target cells (target cells using a 1% solution of triton-X 100). Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by the LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged, and the supernatant was collected and transferred to a new 96-well plate. According to the manufacturer's manual, the supernatant plate was diluted with an equal volume of LDH assay reagent. The assay plate was incubated at 15°C to 25°C for approximately 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0316] The results showed that effector cells expressing a BCMA binding domain (e.g., anti-BCMA1), such as BCMA1 eTCR, BCMA1 BBzCAR, and tandem BCMA1 / CD19 eTCR, had greater cell killing activity compared to untransduced cell controls and CD19 eTCR. Tandem BCMA1 / CD19 eTCR showed greater cell killing activity (e.g., anti-BCMA1) compared to BCMA1 eTCR or BCMA1 BBzCAR. Fig.11A as shown, day 11 after transfection).

[0317] like Fig. 11B IFN-γ expression was determined by HTRF as shown. 384-well low volume white plates were used in the assay for IFN-γ detection (Human IFNγ Kit, Gisbio).

[0318] In the second multi-component cytotoxicity assay, anti-BCMA1-ε-TCR (BCMA1 eTCR), anti-BCMA1-4-1BB-CD3ζ-CAR (BCMA1 BBzCAR), anti-CD19-4-1BB-CD3ζCAR (CD19 BBzCAR), anti-CD19-εTCR (CD19eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1BBzCAR) and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1 eTCR / CD19 BBzCAR) and control untransduced cells were co-cultured with CHO-BCMA-CD19 cells (BCMA+ and CD19+) at effector to target cell ratios of 5: 1, 10: 1 and 20: 1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-cultivation, the assay plate was centrifuged, the supernatant was collected and transferred to a new 96-well plate. The supernatant plate was diluted with an equal volume of LDH assay reagent according to the manufacturer's manual. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0319] The results showed that the anti-BCMA and / or anti-CD-19 systems with an E:T ratio of 20:1: anti-BCMA1ε-TCR (BCMA eTCR), anti-BCMA1-4-1BB-CD3ζ-CAR (BCMA BBzCAR), anti-CD19-4-1BB-CD3ζCAR (CD19 BBzCAR), anti-CD19-εTCR (CD19 eTCR), anti-CD19-εTCR-anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA BBzCAR), anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA eTCR / CD19BBzCAR) had greater cell killing activity compared with untransduced control cells. Compared with single antibody fused CAR or TCR, anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA BBzCAR) and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA eTCR / CD19 BBzCAR) showed greater cell killing activity at lower E:T ratios (especially 5:1) (e.g. Fig. 12A shown).

[0320] like Fig. 12B IFN-γ expression was determined by HTRF as shown. 384-well low volume white plates were used in the assay for IFN-γ detection (Human IFNγ Kit, Gisbio).

[0321] In the third multi-component cytotoxicity assay, anti-CD19ε-TCR (CD19 eTCR), anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1dTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1 BBzCAR) and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1 eTCR / CD19 BBzCAR) and control untransduced cells were co-cultured with CHO-BCMA-CD19 cells (BCMA+ and CD19+) at effector to target cell ratios of 10:1 and 5:1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-cultivation, the assay plate was centrifuged, the supernatant was collected and transferred to a new 96-well plate. According to the manufacturer's manual, the supernatant plate was diluted with an equal volume of LDH assay reagent. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0322] The results showed that the anti-BCMA and anti-CD19 systems: anti-CD19ε-TCR (CD19eTCR), anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19eTCR / BCMA1BBzCAR), and anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1eTCR / CD19 BBzCAR) had anti-tumor activity compared with untransduced control cells. At a higher E:T ratio (10:1), anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1eTCR / CD19 BBzCAR) exhibited similar cell killing activities greater than anti-CD19ε-TCR (CD19eTCR) and anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR). At a lower E:T ratio (5:1), anti-CD19ε-TCR (CD19 eTCR) and anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR) showed almost no cell-killing activity, whereas anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19eTCR), anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1 eTCR / CD19 BBzCAR) still showed a cell-killing effect and lysed approximately 40% of target cells (e.g., Fig.13A shown).

[0323] In the fourth multi-component cytotoxicity assay, anti-BCMA and / or anti-CD19 systems: anti-BCMA1-εTCR (BCMA1eTCR), anti-BCMA1-4-1BB-CD3ζCAR (BCMA1 BBzCAR), anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19eTCR / BCMA1 BBzCAR), anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1eTCR / CD19 BBzCAR) were co-cultured with NCI-H929 cells (BCMA+) at effector to target cell ratios of 2.5: 1 and 5: 1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-cultivation, the assay plate was centrifuged, the supernatant was collected and transferred to a new 96-well plate. The supernatant plate was diluted with an equal volume of LDH assay reagent according to the manufacturer's manual. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0324] The results showed that the anti-BCMA and anti-CD19 systems: anti-BCMA1-εTCR (BCMA1eTCR), anti-BCMA1-4-1BB-CD3ζCAR (BCMA1 BBzCAR), anti-BCMA1-anti-CD19-εTCR (tandem BCMA1 / CD19 eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1BBzCAR), anti-BCMA1-εTCR / anti-CD19-4-1BB-CD3ζCAR (BCMA1 eTCR / CD19 BBzCAR) had greater cell killing activity compared with the untransduced control (e.g. Fig. 13B shown).

[0325] In the fifth multi-component cytotoxicity assay, anti-BCMA and anti-CD19 systems: anti-BCMA1ε-TCR (BCMA1eTCR), anti-CD19-εTCR / anti-BCMA1-γTCR (CD19 eTCR / BCMA1 gTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1BBzCAR) and untransduced control cells were co-cultured with CHO-BCMA-CD19 cells (BCMA+CD19+) at an effector to target cell ratio of 1.3:1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged, the supernatant was collected and transferred to a new 96-well plate. The supernatant plate was diluted with an equal volume of LDH assay reagent according to the manufacturer's manual. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0326] The results showed that anti-BCMA1ε-TCR (BCMA1 eTCR), anti-CD19-εTCR / anti-BCMA1-γTCR (CD19 eTCR / BCMA1 gTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19eTCR / BCMA1dTCR), and anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1BBzCAR) had greater cell killing activity compared with the untransduced control. Compared with anti-BCMA1ε-TCR (BCMA1 eTCR), anti-CD19-εTCR / anti-BCMA1-γTCR (CD19 eTCR / BCMA1 gTCR), anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR), and anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19eTCR / BCMA1 BBzCAR) have greater cell killing activity. Compared with anti-CD19-εTCR / anti-BCMA1-γTCR (CD19 eTCR / BCMA1 gTCR) and anti-CD19-εTCR / anti-BCMA1-δTCR (CD19 eTCR / BCMA1 dTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1 BBzCAR) has greater cell killing activity (e.g. Fig.14A shown). Fig. 14B and Fig. 14C The results showed that the amounts of IFNγ and TNFα secreted from T cells in the co-culture system had a similar trend to the cell-killing effect.

[0327] In the sixth multi-component cytotoxicity assay, anti-BCMA and anti-CD19 systems: anti-BCMA1ε-TCR (BCMA1eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1BBzCAR), anti-BCMA1 and anti-CD19-ε-TCR (tandem BCMA1 / CD19 dTCR) and control untransduced cells were co-cultured with CHO-BCMA-CD19 cells (BCMA+ and CD19+) at an effector to target cell ratio of 1.3: 1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged, and the supernatant was collected and transferred to a new 96-well plate. According to the manufacturer's manual, the supernatant plate was diluted with an equal volume of LDH assay reagent. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492 nm and 650 nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0328] The results showed that anti-BCMA1ε-TCR (BCMA1 eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1 BBzCAR) and anti-BCMA1-anti-CD19-ε-TCR (tandem BCMA1 / CD19 dTCR) had greater cell killing activity compared with the untransduced control. Compared with the anti-BCMA1ε-TCR (BCMA1eTCR), anti-CD19-εTCR / anti-BCMA1-4-1BB-CD3ζCAR (CD19 eTCR / BCMA1 BBzCAR) and anti-BCMA1-anti-CD19-ε-TCR (tandem BCMA1 / CD19 dTCR) had greater cell killing activity (as shown in Figure 2). Fig.15A shown). Fig. 15B and Fig. 15C The results showed that the amounts of IFNγ and TNFα secreted from T cells in the co-culture system had a similar trend to the cell-killing effect.

[0329] In the seventh multi-component cytotoxicity assay, the anti-BCMA system: anti-BCMA2ε-TCR (BCMA eTCR), anti-BCMA2-εTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 eTCR / BCMA3 BBzCAR), anti-BCMA2-γTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 gTCR / BCMA3 BBzCAR), anti-BCMA2-δTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 dTCR / BCMA3 BBzCAR) and control untransduced cells were co-cultured with RPMI-8226 cells (BCMA+) at an effector to target cell ratio of 0.5: 1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged and the supernatant was collected in a new 96-well plate. The supernatant plate was diluted with an equal volume of LDH assay reagent according to the manufacturer's manual. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492nm and 650nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0330] The results showed that anti-BCMA2ε-TCR (BCMA2 eTCR), anti-BCMA2-εTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 eTCR / BCMA3 BBzCAR), anti-BCMA2-γTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 gTCR / BCMA3 BBzCAR), and anti-BCMA2-δTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 dTCR / BCMA3 BBzCAR) had greater cell killing activity compared with the untransduced control. Compared with anti-BCMA2ε-TCR (BCMA2 eTCR), anti-BCMA2-εTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2 eTCR / BCMA3 BBzCAR), anti-BCMA2-γTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2gTCR / BCMA3 BBzCAR), and anti-BCMA2-δTCR / anti-BCMA3-4-1BB-CD3ζCAR (BCMA2dTCR / BCMA3 BBzCAR) have significantly greater cell killing activity, such as Fig.16A shown. Fig. 16B Shown is the amount of IFNγ secreted from T cells in the co-culture system.

[0331] In the eighth multi-component cytotoxicity assay, the anti-BCMA system: anti-BCMA2-anti-BCMA3ε-TCR / anti-BCMA2-anti-BCMA3γ-TCR (tandem BCMA2&3eTCR / gTCR), anti-BCMA2-anti-BCMA3γ-TCR / anti-BCMA2-anti-BCMA3 4-1BB-CD3ζCAR (tandem BCMA2&3gTCR / BBzCAR) and control untransduced cells were co-cultured with RPMI-8226 cells (BCMA+) at an effector to target cell ratio of 0.33:1. Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche). After 20 hours of co-culture, the assay plate was centrifuged, and the supernatant was collected and transferred to a new 96-well plate. According to the manufacturer's manual, the supernatant plate was diluted with an equal volume of LDH assay reagent. The assay plate was incubated at 15°C to 25°C for about 30 minutes. The absorbance of the plate at 492 nm and 650 nm was measured using a Flexstation reader (Molecular Devices) and calculated.

[0332] The results showed that compared with the untransduced control, anti-BCMA2-anti-BCMA3ε-TCR / anti-BCMA2-anti-BCMA3γ-TCR (tandem BCMA2&3eTCR / gTCR), anti-BCMA2-anti-BCMA3γ-TCR / anti-BCMA2-anti-BCMA34-1BB-CD3ζCAR (tandem BCMA2&3gTCR / BBzCAR) had greater cell killing activity. Compared with anti-BCMA2-anti-BCMA3ε-TCR / anti-BCMA2-anti-BCMA3γ-TCR (tandem BCMA2&3eTCR / gTCR), anti-BCMA2-anti-BCMA3γ-TCR / anti-BCMA2-anti-BCMA3 4-1BB-CD3ζCAR (tandem BCMA2&3gTCR / BBzCAR) had significantly greater cell killing activity, as shown in Figure 2. Fig.17A shown. Fig. 17B Shown is the amount of IFNγ secreted from T cells in the co-culture system.

[0333] Example 7: Cytotoxicity assay (luciferase assay)

[0334] In order to evaluate the cytotoxicity of modified immune cells expressing any system provided herein, CAR-T cells, TCR-T cells and untransfected T cells (UnT) were collected by centrifugation and diluted to the desired concentration using 1640 phenol red-free culture media (Invitrogen) supplemented with 2% heat-inactivated FBS (Invitrogen). Tumor cells showing strong expression of BCMA and luciferase were used as target cells. TCR-T cells or CAR-T cells were co-cultured with target cells in 96-well plates at 37 ° C for 20 hours with different effector to target ratios (E: T). Other wells included control conditions: maximum release of target cells (T) and target cells only (1% solution of triton-X 100). Each condition was performed in triplicate, and the cytotoxicity of CAR-T cells was detected using One-Glo assay kit (Promega).

[0335] After 20 hours of co-culture, the assay plate was centrifuged and an equal volume of One-Glo assay reagent was added according to the manufacturer's instructions. The plate was incubated at room temperature for about 3 minutes. After incubation, the luciferase signal was measured using a PheraStarplus reader (BMGlabtech). The percentage of tumor cell lysis was calculated using the following formula:

[0336] Target cell lysis %=(1-(RLUE:T-RLUMax release) / (RLUT-RLUMax release))*100.

[0337] Example 8: Cytokine release detection (IFNγ & TNFα)

[0338] Supernatants from cytotoxicity assay plates were collected for cytokine release analysis (human IFNγ kit, Cisbio, catalog number 62HIFNGPEH; human TNFα kit, Cisbio, catalog number 62HTNFAPEH); human IL6 kit, Cisbio, catalog number 62HIL06PEG, and human IL2 kit, Cisbio, catalog number 62HIL02PEH). Cell supernatants and standards were dispensed directly into the assay plates to utilize Reagents are used for cytokine detection. Antibodies labeled with HTRF donors and acceptors are premixed and added in a single dispensing step. HTRF standard curves are generated using a 4-parameter Logistic (4PL) curve. Compared with linear analysis, standard curve regression enables accurate measurement of the concentration of unknown samples over a wider concentration range, making standard curve regression suitable for analyzing biological systems such as cytokine release. Applicable assay kits include human IFNγ kits, Cisbio, catalog number 62HIFNGPEH; human TNFα kits, Cisbio, catalog number 62HTNFAPEH; human IL6 kits, Cisbio, catalog number 62HIL06PEG; and human IL2 kits, Cisbio, catalog number 62HIL02PEH.

[0339] Example 9: In vivo efficacy

[0340] In vivo efficacy of BCMACAR-TCR-T via multiple myeloma tumor xenografts

[0341] The in vivo antitumor efficacy of CAR-TCR-T cells was evaluated in the NCG mouse model with multiple myeloma tumor xenografts (NOD_Prkdcem26Cd52 / NjuCrl).

[0342] The NCG mouse model was generated by sequential CRISPR / Cas9 editing of the Prkdc and I12rg loci in NOD / Nju mice, providing mice homologous to NOD / Nju. NOD / Nju mice carry a mutation in the Sirpa (SIRPα) gene that allows transplantation of foreign hematopoietic stem cells. Prkdc gene knockout generates a SCID-like phenotype lacking proper T-cell and B-cell formation. Knockout of the I12rg gene further exacerbates the SCID-like phenotype while additionally resulting in a reduction in NK cell production. Therefore, the NCG mouse is a "triple immunodeficient" mouse strain that is more immunocompromised than commonly used immunodeficient mouse strains including SCID mice and nude mice. Prkdc and Il2rg are part of the SCID (severe combined immunodeficiency) family of genes that affect the maturation and formation of T cells, B cells, NK cells, and to a lesser extent, dendritic cells. Prkdc encodes the catalytic subunit of a DNA-dependent protein kinase that is required for V(D)J recombination, which is an essential process for propagating antibody diversity in mature T cells and B cells. Il2rg encodes a common γ subunit found in the IL-2 receptor and multiple IL receptors (IL-4, IL-7, IL-9, IL-15, and IL-21), which are required for cytokine-mediated signaling for the maturation of immature lymphocytes (such as T cells, B cells, and NK cells) and other leukocytes. BCM ACAR-T cells were prepared using T cells from various donors to screen T cell sources, thereby producing CAR-T with the highest efficacy of killing RPMI8226-Luc cells in vitro. CAR-T cells were prepared using T cells from selected donors for in vivo animal assays. To produce tumor xenografts, RPMI8226-Luc cells were injected intravenously into NCG mice. Fourteen days later, tumor-transplanted mice were treated with BCMACAR-T cells (1.5e5 positive cells) or non-transduced T cells, followed by in vivo bioluminescence imaging (BLI).

[0343] In the NCG mouse model with multiple myeloma tumor xenografts (NOD_Prkdc em26Cd52Anti-BCMA1-anti-BCMA2-anti-BCMA3 BBzCAR (triple specific BCMACAR-T) cells, anti-BCMA1-anti-BCMA2-anti-BCMA3 eTCR (triple specific BCMA TCR-T) cells, and anti-BCMA2 eTCR / anti-BCMA1-anti-BCMA3BBzCAR (triple specific BCMACAR-TCR-T) cells were evaluated in a mouse model tumor suppressor cell line (NjuCrl). Anti-BCMA2 eTCR / anti-BCMA1-anti-BCMA3 BBzCAR (triple specific BCMACAR-TCR-T) showed great anti-tumor activity at low doses, such as Fig.18A and Fig.18B shown.

[0344] In vivo efficacy of MSLN FSHR CAR-TCR-T via OVCAR-8 xenograft model in NSG mice

[0345] In vivo evaluation of the anti-tumor activity of anti-mesothelin CAR-T in the OVCAR-8 xenograft model. 6 OVCAR-8 cells were implanted subcutaneously into NOD scidγ (NSG) mice. Once the tumor was 150-200 mm 3 , mice were randomized into treatment groups. 1e5 CAR-positive T cells were administered intravenously at a dose of 200 μl. Mice and tumors were monitored for approximately 60 days after tumor cell implantation.

[0346] The results showed that anti-MSLN / FSHR dual CAR-T (MSLN CAR+FSHR CAR), anti-MSLN / FSHR dual eTCR-T (MSLN eTCR+FSHR eTCR), and anti-FSHR eTCR / MSLN CAR-T (FSHR eTCR+MSLN CAR) had different in vivo anti-tumor activities, and at low doses, anti-FSHR eTCR / MSLN CAR-T (FSHR eTCR+MSLNCAR) showed greater anti-tumor activity than anti-MSLN / FSHR dual CAR-T (MSLN CAR+FSHR CAR) and anti-MSLN / FSHR dual eTCR-T (MSLN eTCR+FSHR eTCR). Fig.19 shown).

[0347] Example 10: Rapid Amplification Protocol

[0348] To generate large numbers of transduced cells, T cell proliferation was induced using the rapid expansion protocol (REP). T cells were initially cultured with anti-CD3, anti-CD28, and IL-2 before use in the REP and transduced the next day. Cells were cultured at 75 cm 2 The cells were cultured in flasks at 37°C and 5% CO2. The cells were counted every two days and 0.5×10 6 The cells are then suspended at a concentration of 10 cells / mL in fresh T cell culture medium supplemented with 300 IU / mL IL-2 and the cells will be maintained in culture for the remainder of the time.

[0349] A variety of antigen binding domain sequences can be used to construct the vector constructs and systems disclosed herein, see, for example, WO2017 / 025038, the entire contents of which are incorporated herein by reference (BCMA2 to BCMA4, BCMA 14 to BCMA21).

[0350] Non-limiting exemplary sequences are shown in Tables 10 and 11 below:

[0351] Table 10 Exemplary sequences

[0352]

[0353]

[0354]

[0355]

[0356] Table 11 Anti-CD19 V H Sequence of H

[0357]

[0358] Table 12 Sequences of anti-MSLN scFv and FSHβ33-53

[0359]

[0360]

[0361] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only by way of example. Without departing from the present invention, those skilled in the art will now expect many variations, changes and replacements. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used to practice the present invention. The appended claims are intended to define the scope of the present invention, and thus encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. An antigen-binding molecule, comprising an anti-BCMA sdAb, comprising: (1) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 15; (2) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 14; (3) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 16; (4) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 17; (5) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 18; (6) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 19; (7) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO:20; (8) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO:21; (9) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO:22; or (10) CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO:

23.

2. The antigen-binding molecule according to claim 1, wherein the CDR1, CDR2 and CDR3 comprise the following amino acid sequences respectively: (1) SEQ ID NO: 88, 98 and 108; (2) SEQ ID NO: 87, 97 and 107; (3) SEQ ID NO: 89, 99 and 109; (4) SEQ ID NO:90, 100 and 110; (5) SEQ ID NO:91, 101 and 111; (6) SEQ ID NO:92, 102 and 112; (7) SEQ ID NO:93, 103 and 113; (8) SEQ ID NO:94, 104 and 114; (9) SEQ ID NO:95, 105 and 115; or (10) SEQ ID NO:96, 106 and 116.

3. An antigen binding molecule as described in claim 1 or 2, wherein the antigen binding molecule comprises a sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 23; or the antigen binding molecule comprises a sequence having at least 80% or 90% identity with any one of the group consisting of SEQ ID NO: 14 to SEQ ID NO:

23.

4. A chimeric antigen receptor (CAR), comprising an antigen binding domain that exhibits specific binding to BCMA, a transmembrane domain, and an intracellular signaling domain; The antigen binding domain is an antigen binding molecule as described in any one of claims 1 to 3.

5. The chimeric antigen receptor of claim 4, wherein the CAR comprises one or more additional antigen binding domains.

6. The chimeric antigen receptor of claim 4 or 5, wherein the CAR comprises at least two antigen binding domains.

7. The chimeric antigen receptor of claim 6, wherein the CAR comprises at least two antigen binding domains connected in tandem.

8. A chimeric antigen receptor as described in any one of claims 4-7, wherein the intracellular signaling domain of the CAR includes an immunoreceptor tyrosine-based activation motif (ITAM), an immunoreceptor tyrosine-based inhibition motif (ITIM), an Fcγ receptor (FcγR), an Fcε receptor (FcεR), an Fcα receptor (FcαR), a neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, C Signaling domains of D21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70.

9. A chimeric antigen receptor as described in any one of claims 4-8, wherein the CAR further comprises a co-stimulatory domain; optionally, the co-stimulatory domain comprises an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), a signaling domain of an activated NK cell receptor or a Toll ligand receptor, or the co-stimulatory domain comprises a signaling domain of a molecule selected from the following: 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF-R / TNFRSF13C, BAFF / BLyS / T NFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100(SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160(BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNF SF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, C D96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA Class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT,LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L 2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG(CD162), SLAM(SLAMF1), SLAM / CD150, SLAMF4(CD244), SLAMF6( NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1 and VLA-6. , 10. A polynucleotide encoding the antigen-binding molecule according to any one of claims 1 to 3, or the chimeric antigen receptor according to any one of claims 4 to 9. A vector comprising the polynucleotide according to claim 10.

12. An isolated host cell, said host cell expressing the chimeric antigen receptor according to any one of claims 4 to 9.

13. The host cell of claim 12, wherein the host cell is an immune cell, preferably, the immune cell is selected from T cells, natural killer (NK) cells and NKT cells; more preferably, the T cell is a CD8+T cell, a CD4+T cell, an α / βT cell and / or a γ / δT cell.

14. A composition comprising the antigen binding molecule of any one of claims 1 to 3, or the host cell of any one of claims 12 to 13.

15. Use of the antigen binding molecule according to any one of claims 1 to 3, the chimeric antigen receptor according to any one of claims 4 to 9, or the host cell according to any one of claims 12 to 13 in the preparation of a medicament for treating cancer in a subject, wherein: Preferably, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer and vulvar cancer.

Images