Chimeric antigen receptor therapy for treatment of solid tumors

Abstract

Provided herein are novel anti-effector moiety antibodies or antigen binding domains thereof, and CARs comprising such effector moiety antigen binding domains with or without one or more enhancer elements, as well as host cells expressing the receptors and nucleic acid molecules encoding the receptors, and methods of using the same in patient-specific immunotherapy useful for the treatment of solid tumor cancers and other diseases and disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 393,088 filed on July 28, 2022 and U.S. Provisional Patent Application No. 63 / 433,632 filed on December 19, 2022, the entire contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of cancer, and in particular to compositions encoding functional chimeric antigen receptors and methods of using the same in patient-specific immunotherapy to treat solid tumors.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on [[XXX]], 2022, is named SequenceListing.txt, and is [[XXX]] kilobytes in size. Background of the Invention

[0007] Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after cardiovascular disease, accounting for about a quarter of deaths. Solid tumors are responsible for most of these deaths. Despite significant advances in the medical treatment of some cancers, the overall 5-year survival rate for all cancers has only increased by about 10% in the past 20 years. Cancer or malignant tumors grow and metastasize rapidly in an uncontrolled manner, making treatment extremely difficult.

[0008] Chimeric antigen receptors are hybrid molecules containing three basic units: (1) an extracellular antigen binding motif, (2) a linker / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM. Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2(4): e23621). The antigen binding motif of a CAR is usually formed after the single chain variable fragment (scFv), the minimal binding domain of an immunoglobulin (Ig) molecule. Alternative antigen binding motifs have also been engineered, such as receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Series, even triple or quadruple targeting domains can be constructed by sequentially connecting multiple antigen binding motifs and connecting them to CAR hinges, transmembrane domains and intracellular sequences. Alternative cell types (such as NK, NKT, iNKT or γ-δ T cells) for CAR expression are also under development (Brown CE et al. Clin Cancer Res. 2012; 18 (8): 2199-209; Lehner M et al. PLoS One. 2012; 7 (2): e31210). There is still important work on limiting the most active T cell populations to be transduced with CAR vectors, determining the best culture and expansion techniques, and limiting the molecular details of the CAR protein structure itself.

[0009] The connecting motif of CAR can be a relatively stable domain, such as the constant domain of IgG, or a flexible linker designed to be extended. Structural motifs (such as those derived from IgG constant domains) can be used to extend the scFv binding domain away from the T cell plasma membrane surface. This can be important for some tumor targets in which the binding domain is particularly close to the surface membrane of tumor cells (for example, for disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motif used in CAR always includes the CD3-ζ chain because the core motif is a key signal for T cell activation. The first reported second-generation CAR is characterized by a CD28 signaling domain and a CD28 transmembrane sequence. This motif is also used in a third-generation CAR containing a CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; 183 (9): 5563-74). With the advent of new technologies that utilize beads connected to anti-CD3 and anti-CD28 antibodies to activate T cells, the presence of the classic "signal 2" from CD28 no longer needs to be encoded by the CAR itself. Using bead activation, it was found that the third-generation vectors were not superior to the second-generation vectors in in vitro assays, and they did not provide a clear benefit over the second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013; 121 (7): 1165-74; Kochenderfer JN et al. Blood. 2012; 119 (12): 2709-20). This is demonstrated by the clinical success of CD19-specific CARs in the second-generation CD28 / CD3-ζ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans. LA; December 7-10, 2013) and CD137 / CD3-ζ signaling formats (Porter DL et al. N Engl J Med. 2011; 365(8): 725-33).In addition to CD137, other tumor necrosis factor receptor superfamily members (such as OX40) can also provide important persistent signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15(18): 5852-60). The culture conditions for culturing CAR T cell populations are also important.

[0010] Chimeric antigen receptor (CAR) T cell therapy is a promising approach for treating both hematological tumors and solid tumors, however, the expected therapeutic benefits have not been achieved in solid tumors, while the treatment of hematological malignancies has been shown to be highly effective, resulting in several CAR T products approved by the U.S. Food and Drug Administration (FDA) for B-cell malignancies and multiple myeloma (Gill S, et al., Blood Rev. 2016; 30(3): 157-1671; Victor E. et al., J Immunol April 1, 2021, 206(7)1561-1568; Wagner J, et al., Mol Ther. 2020 Nov 4; 28(11): 2320-2339; He C, et al., Cancers. 2020; 12(7): 196). CAR cells are recombinant antigen receptors that redirect the specificity and function of T lymphocytes and other immune cells to the intended tumor target (Sadelain M, et al., Cancer Discov. 2013; 3: 388-98). The modified CAR T molecules redirect immune activity to the desired antigen, and can have a lasting desired effect on tumor cells depending on the amount and quality of this interaction. Solid tumors present a challenge to current CAR T targeting methods. The challenges of this treatment approach include tumor antigen escape, insufficient persistence of modified CAR molecules, and reduced effectiveness in a solid tumor environment. In addition, CAR T cell-mediated toxicity leads to cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity (Immune Effector Cell Associated Neurotoxicity, ICANS), as well as off-target and on-target tumor CAR reactions, which hinder the further development of CAR therapy in solid tumors. Optimization of CAR design is still largely empirical, and small modifications to the modular design can have a significant impact on specific treatments (Guedan S, et al., Mol Ther Methods Clin Dev. 2018 Dec 31; 12: 145-156). Co-expression of multiple CAR molecules in the same effector cell and optimization of CAR structure and co-stimulatory domains are feasible and can improve CAR T effector function and persistence (Schneider D, et al., Sci Transl Med. 2021 Mar 24; 13 (586)).In addition, third-generation CAR T cells that combine the signaling potential of two co-stimulatory domains can lead to improved CAR survival, expansion, and efficacy (Subklewe M, et al., Transfus Med Hemother. 2019; 46(1): 15-24; Maria-Luisa Schubert, MD et al., Blood. 2019.134(Supplement_1)).

[0011] Tumor antigen escape and tumor target heterogeneity are common causes of CAR therapy failure, and recent studies have shown that this may be particularly important factors in the treatment of solid tumors (Majzner RG, and Mackall CL., Cancer Discov. 2018 Oct; 8(10): 1219-1226). Single targeted CAR therapy has been shown to be effective in treating a variety of cancers, including B-ALL and multiple myeloma, but relapse rates are as high as 60% in some cases (Walsh Z, et al., Curr Hematol Malig Rep 14, 451-459 (2019). By simultaneously targeting multiple antigens, the risk of relapse and resistance is reduced (Schneider D, et al., Sci Transl Med. 2021 Mar 24; 13 (586)). Several options have been tested clinically to avoid this outcome, including sequential single CAR therapy or co-infusion with CD19 and CD22 CARs, but ultimately proved to be ineffective (Shalabi H, et al., Haematologica Italy. 2018; 103: e215-8). Ongoing or completed multi-targeted CAR studies focus on various combinations of CD19, CD20, CD22, HER2, TSLPR, IL-13Rα2, etc. (Walsh, Z. et al., Curr Hematol Malig Rep 14, 451-459 (2019); Shalabi H, et al., Haematologica Italy. 2018; 103: e215-8; Bielamowicz K, et al., Neuro-Oncology. 2018; 20(4): 506-18; Han X, et al., J Hematol Oncol 12, 128 (2019)). Further clinical evidence is needed to determine which multi-targeted CAR therapy will prove to be the most effective.

[0012] As mentioned above, the efficacy of CAR therapy can be weakened by short CAR T cell persistence and ultimately overwhelmed by rebound tumor burden, and this is particularly realistic in solid tumors. The use of interleukin (IL)-7 and CCR2b in in vivo experiments has been shown to be effective, showing both improved persistence and improved anti-tumor activity in neuroblastoma and melanoma models (Guangchao Li, et al., Frontiers in Oncology. 2021; 11: 2021). In studies on hematological cancers, multi-targeting and co-stimulation through a combination of CAR and chimeric costimulatory receptor (CCR) has been shown to be an effective approach that prevents tumor clone recurrence and ultimately improves the clinical outcomes of CAR T cell therapy by enhancing cytotoxic potency and persistence (Katsarou A, et al., Sci Transl Med. 2021Dec8; 13(623); Pietrobon, V., et al., Int. J. Mol. Sci. 2021, 22, 10828).

[0013] Therefore, although it can be considered that CAR can trigger T cell activation in a manner similar to endogenous T cell receptors, the main obstacles to the clinical application of CAR-based technology to date are limited by the in vivo expansion of CAR+T cells, the rapid disappearance of cells after infusion, disappointing clinical activity, and recurrence of underlying medical diseases or conditions. Many of these problems are due to tumor target heterogeneity and tumor-mediated resistance to treatment, including the influence of tumor microenvironment and tumor matrix factors, and can be solved by CAR T cell modification.

[0014] Solid tumors provide a challenging environment for CAR, including an immunosuppressive environment characterized by physical, functional, and dynamic barriers that hinder T cell function. The tumor microenvironment (TME) can prove difficult to successfully achieve CAR function and targeting. Tumors can adopt strategies to resist the targeting effects of CAR by increasing the production of inhibitory cytokines (Lindo L, et al., Front Immunol. 2021 Feb 10; 11: 618387). In order to resist this increasingly unfavorable environment, research on "armed" CARs is already under development. Alabanza et al. (Front Immunol. 2022 Feb 9; 13: 832645) used this approach, which co-expressed a dominant negative form of TGFβ receptor II ("armed") on CAR T cells targeting BCMA to resist the inhibitory effects of TGFβ in the bone marrow niche of multiple myeloma. This resulted in functional persistence despite continued exposure to TGFβ in animal models of multiple myeloma. TME contains multiple cell populations that have proven difficult to target. Studies on ovarian cancer by Yeku et al. (2017) showed that CAR T cell therapy for ovarian cancer previously used for folate receptors was mostly unsuccessful in clinical trials, partly because of the effects of immunosuppressive cytokines such as IL-4, IL-6, LIF, IL-10, TGFβ, myeloid-derived suppressor cells, tumor-associated macrophages (TAM) and regulatory T cells, which inhibited the effect of targeting CAR. By creating IL-12-armed CAR T cells, they showed that treatment can overcome the inhibitory microenvironment, change ascites cytokines and TAM microenvironment, and overcome PD-L1-mediated inhibition (Yeku OO, et al., Sci Rep 7, 10541 (2017)).

[0015] In addition to the challenging TME, the tumor matrix barrier also shows challenges to effective CAR penetration. Solid tumors have a dense extracellular matrix (extracellular matrix, ECM) formed by cancer-associated fibroblasts (cancer-associated fibroblast, CAF), which inhibits T cells from infiltrating the deep area of ​​the tumor, thereby negating the continuous contact between tumor cells and CAR-T cells (Zhang, BL et al., Sci.China Life Sci.2016, 59 (4), 340-348). One method is to promote the degradation of ECM, so that CAR-T cells are effectively infiltrated into the matrix of solid tumors. The checkpoint blocking antibody α-PDL1 on the surface of modified hyaluronidase (HAase) and CAR-T cells shows enhanced tumor infiltration and anti-tumor efficacy in solid tumors (Yangyang Zhao, et al., ACS Central Science 20228 (5), 603-614). Similarly, the method of engineering CAR to express the enzyme heparanase (HPSE) showed an improvement in the ability to degrade ECM (Caruana I, et al., Nat Med. 2015 May; 21(5): 524-529).

[0016] In addition to the various strategies discussed above to improve the persistence and effectiveness of CAR therapy, measures continue to be developed to improve the safety of CAR T therapy. Due to many challenges, including tumor lysis syndrome, neurotoxic syndrome, and cytokine release syndrome, the widespread adoption and application of CAR-T therapy has been limited. Cytokine release syndrome (CRS) is a systemic inflammatory response triggered by T cell activation. CRS is mainly caused by the following: activated CAR-T cells cause a significant increase in proinflammatory factors (such as IL-6, IFN-γ, and TNF-α) secreted by immune cells, thereby disrupting the balance between proinflammatory and anti-inflammatory responses (Hay KA, et al., Blood. 2017; 130: 2295-306). It is becoming increasingly important to use suicide genes to prevent inappropriate off-target activity and improve the safety of CAR T cells. Suicide genes are control genes that are co-expressed with CAR constructs and can induce cell death when activated by another agent (such as a drug or antibody). By design, the best possible agent for activating suicide genes will be bioinert, have adequate bioavailability and biodistribution profile, and be characterized by negligible or non-existent toxicity (Jones BS, et aL., Front Pharmacol. 2014; 5: 254). Proof of this concept was shown in a study by Kao et al. (2019), which used a truncated epidermal growth factor receptor (EGFRt) as a suicide gene system co-delivered with anti-CD19 CAR. Both in vitro and in vivo analyses showed positive results (Kao RoyL, et al. Human Gene Therapy. Apr 2019; 413-428). Clinical evaluation of these strategies is ongoing, but has the prospect of unlocking a variety of safer CAR-T treatment strategies.

[0017] Thus, there is an urgent and long-felt need in the art to discover compositions and methods for treating cancer using CAR-based therapies that can exhibit the desired therapeutic properties that are cancer-specific without the above-mentioned disadvantages.

[0018] The present invention addresses these continuing unmet needs by providing enhanced CAR compositions and therapeutic methods using such enhanced CARs that can be used to treat solid tumors and other diseases and / or conditions expressing CAR-associated targets, wherein the enhanced CAR compositions exhibit one or more of the following characteristics: i) high surface expression on transduced T cells; ii) high cytolysis and in vivo expansion and persistence of transduced T cells; iii) multi-targeting to overcome antigen escape; iv) arming to overcome immunosuppression in the TME; v) cytokine stimulatory elements to promote cytokine stimulation of autonomous T cells, resulting in increased anti-tumor cytotoxicity, expansion, memory formation, cytokine secretion, persistence; vi) digestive enzymes to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and enable CAR T tumor penetration; and vii) turning on or off a switch to control the expression of CAR, or a co-expressed functional "boosting" element; the solid tumors include tumors expressing targetable antigens, such as CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GM1, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM, folate binding protein, folate receptor α (FOLR1), folate receptor β (FOLR2), A33, G250, prostate-specific membrane antigen (prostate-specific membraneantigen, PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, p185, IL-2 receptor, interleukin 1 receptor accessory protein,ILlRAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, αvβ3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYP1B 1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES 1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 3, PAGE4, ​​VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL 1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorf186, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endothelin, CD90, CD326, CD70, SSEA 4. CD318, CLA, TSPAN8, GPRC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15 or any combination thereof or a fragment thereof or other antigen or any combination thereof is provided, wherein the antibody or its fragment comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a nanobody, a VHH, a ligand peptide, and a single-chain Fv (ScFv), or a fragment of any of the foregoing,Or a molecule that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any of the foregoing, or any combination thereof. Summary of the invention

[0019] Provided herein are new anti-effector part antibodies or their antigen binding domains and chimeric antigen receptors (CARs) comprising such effector part antigen binding domains, as well as host cells (e.g., T cells) expressing the receptors and nucleic acid molecules encoding the receptors.CAR may consist of a single molecule expressed on the surface of effector cells, or CAR may comprise a signal transduction module and a soluble targeting module expressed by effector cells, such as when a soluble targeting module is combined with a signal transduction module expressed by a cell, to form a complete functional CAR.CAR shows high surface expression on transduced T cells, with a high degree of cell lysis and transduced T cells in vivo amplification and persistence.Also provided are methods for treating cancer in an object using disclosed CAR, host cells, and nucleic acid molecules, for example.

[0020] In its broadest aspect, provided herein is a novel chimeric antigen receptor (CAR) comprising a boosted CAR containing a CAR construct having a major effector portion molecule followed by one or more 2A sequences in frame with one or more additional "boosting" elements for improving function including enhancing tumor penetration to improve the therapeutic effect of CAR-T cells in solid tumors, hematological tumors, autoimmune diseases, genetic diseases or other related indications.

[0021] In another broad aspect, a new chimeric antigen receptor (CAR) is provided herein, comprising a reinforced CAR, wherein the functionally co-expressed reinforced CAR element is expressed by a single polycistronic vector with high transduction efficiency, thereby simplifying the manufacture and release of CAR and reducing the cost of market implementation. In one aspect, the reinforced CAR composition comprises one or more of the following features: i) high surface expression on transduced T cells; ii) multi-targeting to overcome antigen escape; iii) one or more armed elements to overcome immunosuppression in TME; iv) one or more cytokine stimulating elements (including, for example, but not limited to chemotactic receptors and / or chemotactic molecule secretions) to promote cytokine stimulation of autonomous T cells, resulting in improved anti-tumor cytotoxicity, amplification, memory formation, cytokine secretion, persistence; v) one or more digestive enzymes to overcome the physical barrier of tumor stroma / extracellular matrix (ECM) and enable CAR T tumor penetration; vi) one or more pro-inflammatory immune activators; and vii) one or more on switches or off switches to control the expression of CAR, wherein the enhanced CAR achieves a high degree of cell lysis and transduced T cell in vivo expansion and persistence in a patient-specific manner, thereby promoting the in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, regression, elimination, relief of cancer or autoimmune disease, alloimmune disease or autoinvasive disease, or prevention or improvement of the recurrence of cancer or autoimmune disease, alloimmune disease or autoinvasive disease, or a combination thereof.

[0022] In another broad aspect, provided herein are novel chimeric antigen receptors (CARs) that may comprise single, tandem or multiple targeting CAR constructs (including those in the form of DuoCARs), or any combination thereof.

[0023] In certain aspects, the new enhanced CAR is under the control of one or more constitutive promoters, tissue-specific promoters or inducible promoters or any combination thereof.

[0024] In certain aspects, the one or more switches include a tag, a kill switch, an on switch, an off switch, and / or an adapter switch, or any combination thereof.

[0025] In certain embodiments, single, tandem, multi-targeted CARs and DuoCARs (with or without one or more enhancer elements) are provided as novel chimeric antigen receptors (CARs) for transducing effector cells to treat solid tumors and hematological tumors and other diseases by targeting antigens, such as but not limited to CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosylGMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, Ep CAM, folate binding protein, folate receptor α (FOLR1), folate receptor β (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, p185, IL-2 receptor, interleukin-1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, αvβ3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYP1B 1. PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie3, PAGE4, ​​VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL 1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endothelin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPR C5D, EpCAM, Thyl, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15 or any combination thereof or a fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the following: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a nanobody, a VHH, a ligand peptide, and a single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof.

[0026] In certain aspects, effector cells include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant Natural Killer T (iNKT) cells, dendritic cells (DC), γδT cells, monocytes, macrophages, stem cells and induced pluripotent stem (iPS) cells.

[0027] In another broad aspect, one or more of the above-identified novel enhanced chimeric antigen receptors (CARs) provided above with respect to SEQ ID NOs: 151 to 256 may comprise a single, tandem or multi-targeting CAR construct (including those in the form of DuoCAR), or any combination thereof.

[0028] For each of the various aspects and embodiments of single, tandem, multi-targeting CAR and DuoCAR (with or without one or more enhancer elements) CAR constructs specifically contemplated herein, the nucleotide sequence encoding a functional CAR (with or without one or more enhancer elements) comprises the following nucleotide sequence:

[0029] SEQ ID NO: 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 245, 247, 249, 251, 253, or 255

[0030] or any combination thereof.

[0031] For each of the various aspects and embodiments of single, tandem, multi-targeting CAR and DuoCAR (with or without one or more enhancer elements) CAR constructs specifically contemplated herein, each vector encodes a functional CAR (with or without one or more enhancer elements) comprising the amino acid sequence of:

[0032] SEQ ID NO: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256

[0033] or any combination thereof.

[0034] In another broad aspect, one or more of the above-identified novel enhanced chimeric antigen receptors (CARs) provided above with respect to SEQ ID NOs: 127 to 149 may comprise a single, tandem or multi-targeting CAR construct (including those in the form of DuoCAR), or any combination thereof.

[0035] For each of the various aspects and embodiments, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN antibody or fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147 and 149.

[0036] For each of the various aspects and embodiments, an isolated polynucleotide encoding a fully human anti-HER2, FOLR1, MUC16, CD276, EGFR, GD2, NKGD2 antibody or a fragment thereof is provided, comprising a nucleic acid sequence selected from SEQ ID NO: 127, 129, 131, 133, 135, 137, 139 and 141.

[0037] For each of the multiple aspects and embodiments, provided herein is a novel single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), comprising a single, tandem, DuoCAR or multi-targeted CAR molecule (with or without one or more enhancer elements), the molecule comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-ROR1 and / or anti-MSLN antigen binding domain comprising a nucleic acid sequence selected from SEQ ID NO: 143, 145, 147 and 149.

[0038] For each of the multiple aspects and embodiments, provided herein is a novel single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), comprising a single, tandem, DuoCAR or multi-targeted CAR molecule (with or without one or more enhancer elements), the molecule comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-HER2, FOLR1, MUC16, CD276, EGFR, GD2 and / or NKGD2 antigen binding domain comprising a nucleic acid sequence selected from SEQ ID NO: 127, 129, 131, 133, 135, 137, 139 and 141. For each of the multiple aspects and embodiments, provided herein is a novel single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), comprising a single, tandem, DuoCAR or multi-targeted CAR molecule (with or without one or more enhancer elements), the molecule comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-ROR1 and / or anti-MSLN antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150.

[0039] For each of the multiple aspects and embodiments, provided herein is a novel single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), comprising a single, tandem, DuoCAR or multi-targeted CAR molecule (with or without one or more enhancer elements), the molecule comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-HER2, FOLR1, MUC16, CD276, EGFR, GD2 and / or NKGD2 antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 128, 130, 132, 134, 136, 138, 140 and 142.

[0040] In one embodiment, an isolated polynucleotide is provided that encodes a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or fragment thereof, wherein the antibody or fragment thereof comprises a nucleic acid sequence selected from SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 and 149.

[0041] In one embodiment, an isolated polynucleotide is provided that encodes a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148 and 150.

[0042] In one aspect, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted chimeric antigen receptor (CAR) (with or without one or more booster elements) is provided, which comprises from N-terminus to C-terminus at least one anti-ROR1 and / or anti-MSLN antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one anti-ROR1 and / or anti-MSLN antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 143, 145, 147 and 149.

[0043] In one aspect, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted chimeric antigen receptor (CAR) (with or without one or more booster elements) is provided, which comprises from N-terminus to C-terminus at least one anti-ROR1 and / or anti-MSLN antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one anti-ROR1 and / or anti-MSLN antigen binding domain is encoded by a nucleotide sequence comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150.

[0044] In one embodiment, the targeting domain of a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is expressed alone in the form of a monoclonal antibody, ScFv Fab, Fab'2, and comprises an antigen targeting domain coupled to an additional binding tag or epitope, the antigen targeting domain comprising a sequence selected from SEQ ID NO: 143, 145, 147 and 149, and the effector cell expression component of single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancing elements) comprises a binding domain, which specifically directs to bind to a tag or epitope expressed on a soluble single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancing elements) module, such as a soluble component of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancing elements) and a specific binding of a cell binding component of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancing elements) to form a complete functional single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancing elements) structure.

[0045] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, Duo or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the encoded single, tandem, DuoCRA or multi-targeted CAR (with or without one or more booster elements) extracellular anti-ROR1 and / or anti-MSLN antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) that binds to ROR1 and / or MSLN.

[0046] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular anti-ROR1 and / or anti-MSLN antigen binding domain is linked to a transmembrane domain via a linker domain.

[0047] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded anti-ROR1 and / or anti-MSLN extracellular antigen binding domain is preceded by a sequence encoding a leader peptide or signal peptide.

[0048] In one aspect, the single, tandem, DuoCAR or multi-targeting CAR provided herein (with or without one or more booster elements) further comprises a linker or spacer domain.

[0049] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the extracellular anti-ROR1 and / or anti-MSLN antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0050] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the encoded linker domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19 or CD28 and is connected to the transmembrane domain.

[0051] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the encoded single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) further comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0052] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a single, tandem or multi-targeted chimeric antigen receptor (CAR) construct, wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-MSLN and / or anti-ROR1 antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150, wherein the T cell is a T cell of a person suffering from cancer or an autoimmune disease, an alloimmune disease or an autoinvasive disease. Cancers particularly include hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0053] In another aspect, a method for preparing a T cell (hereinafter referred to as "CAR-T cell") containing a single, tandem, DuoCAR or multi-targeted CAR construct (with or without one or more reinforcing sub-elements) is provided. The method includes transducing T cells with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds to MSLN and / or ROR1, thereby preparing a CAR-T cell.

[0054] In another aspect, a method for producing an RNA-engineered cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding a disclosed single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) into the cells of a subject, thereby producing single, tandem, DuoCAR or multi-targeted CAR cells (with or without one or more enhancer elements).

[0055] In another aspect, a method for diagnosing a disease, disorder or condition associated with expression of MLSN and / or ROR1 on a cell is provided, comprising: a) contacting the cell with a human anti-MSLN and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150; and b) detecting the presence of MSLN and / or ROR1, wherein the presence of MSLN and / or ROR1 is diagnostic of a disease, disorder or condition associated with expression of MSLN and / or ROR1.

[0056] In another embodiment, a method of inhibiting MSLN and / or ROR1-dependent T cell suppression is provided, comprising contacting a cell with a human anti-MSLN and / or anti-ROR1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150. In one embodiment, the cell is selected from a tumor cell expressing MSLN and / or ROR1, a tumor-associated macrophage, and any combination thereof.

[0057] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed single, tandem or multi-targeting CAR (with or without one or more enhancer elements).

[0058] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering to a mammal one or more of the disclosed single, tandem or multi-targeted CARs (with or without one or more reinforcing sub-elements) in an amount effective for treating or preventing cancer in a mammal. The method includes administering to a subject a therapeutically effective amount of a host cell expressing a disclosed single, tandem or multi-targeted CAR (with or without one or more reinforcing sub-elements) that specifically binds to MSLN and / or ROR1 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex of an antigen binding domain on a single, tandem or multi-targeted CAR (with or without one or more reinforcing sub-elements) in the subject.

[0059] In another embodiment, a method for producing a persistent genetically modified T cell population in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express a single, tandem or multi-targeted CAR (with or without one or more enhancer elements), wherein the single, tandem or multi-targeted CAR (with or without one or more enhancer elements) comprises at least one MSLN and / or ROR1 antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein at least one MSLN and / or ROR1 antigen binding domain comprises SEQ ID NO: 144, 146, 148 and 150 amino acid sequences or any combination thereof, wherein after administration, the persistent genetically modified T cell population or the population of the descendants of the T cells lasts in the person for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years or three years.

[0060] In another aspect, a kit is provided for preparing a chimeric antigen receptor T cell as described above, or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression in a subject as described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells or compositions disclosed above, or any combination thereof, and instructions for using the kit.

[0061] In one aspect of the present invention, an immunotherapeutic composition is provided, which comprises a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), which can be used to transduce autologous lymphocytes in a patient-specific manner to produce an active patient-specific anti-tumor lymphocyte cell population, which can be directly infused back into the patient to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, reduction, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0062] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0063] It should be understood that single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), host cells, nucleic acids and methods are available outside the specific aspects and embodiments described in detail herein. From the detailed description below with reference to the accompanying drawings, the foregoing features and advantages of the present disclosure will become more apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The following detailed description of some preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, some currently preferred embodiments are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the accompanying drawings.

[0065] Figure 1 The structure of the enhanced CAR is depicted. The enhanced CAR consists of a CAR molecule followed by a 2A sequence in the same frame as the enhancement element. The CAR molecule represents a single CAR and a multi-targeted tandem or dual CAR. The enhancement elements are diverse and come from cytokines (membrane-bound IL7), arming (TGFβRIIdn), suicide tags (tEGFR), extracellular matrix enzymes (ECM), chemokine receptors (CXCL8, CCL2), matrix targeting molecules (FAP), etc.

[0066] Figures 2A to 2E depict the ROR1 and / or MSLN CAR structures armed with mIL7 and surface expression on transduced primary T cells. (Fig. 2A) Single CAR armed with membrane-bound IL7 (mIL7) includes ROR1 or MSLN scFv binding domain, IgG4 or CD8 hinge domain, CD8 transmembrane domain, 41BB or CD28 costimulatory domain, CD3ζ activation domain, followed by 2A peptide, and in the same frame with membrane-bound IL7. The CAR construct enhanced in series is composed of MSLN-ROR1 tandem scFv targeting domain, IgG4 short hinge, CD8 or CD28 transmembrane domain, single 4-1BB or tandem CD284-1BB costimulatory domain, CD3ζ activation domain and 2A sequence connected to mIL7. The DuoCAR construct contains a single ROR1 CAR, followed by a 2A sequence, a single MSLN CAR with different co-stimulatory domains or transmembrane domains, in frame with the 2A peptide connected to mIL7. Single ROR or MSLN CAR and tandem CAR are included as control constructs. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding ROR1 and / or MSLN CAR constructs. Staining with ROR1 Fc and / or MSLN His, followed by flow cytometry with anti-Fc-AF647 or anti-His-APC to determine the CAR surface expression of transduced T cells. (Figure 2B) The percentage of ROR1 CAR expression in T cells transduced with constructed CARs encoding ROR1 conjugates is plotted. (Figure 2C) The percentage of MSLN CAR expression in T cells transduced with CARs containing MSLN conjugates is quantified. Mean fluorescence intensity of ROR1 binder expression (Fig. 2D) and MSLN binder expression (Fig. 2E) are presented as bar graphs. Data are representative of one independent experiment from two different donors.

[0067] Figures 3A to 3C depict the cytotoxicity of ROR1 and / or MSLN CAR constructs in vitro. + MSLN + Tumor lines OVCAR3 and ROR1 - MSLN -Tumor line (Figure 3C) HL-60 was subjected to a luciferase-based cytotoxicity assay. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight with 10 series of effector to target (E: T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. Curve fitting was performed using a nonlinear EC50 offset and x as logarithmic concentration. The data represent an independent experiment from two different donors.

[0068] Figures 4A to 4C depict the relative potency of ROR1 and / or MSLN CAR constructs in vitro. + MSLN + Tumor lines were subjected to luciferase-based cytotoxicity assays. CAR T cells and tumor cells were co-cultured overnight at 10 different effector to target (E:T) ratios. Percent specific target lysis was assessed by luminescence analysis and normalized to percent CAR expression. Relative potency relative to the ROR1 CAR LTG2529 was calculated using a nonlinear EC50 shift in GraphPad Prism with x as a function of log concentration. Targeting ROR1 + MSLN + Tumor lines: (FIG. 4A) OVCAR-3, (FIG. 4B) NCI-H226, (FIG. 4C) CAPAN-1 The relative potency of each construct is plotted as a bar graph. Data are representative of one independent experiment from one to two different donors.

[0069] Figures 5A to 5C depict CAR T cell factor release in response to the NCI-H226 lung cancer cell line. + After incubation of MSLN+NCI-H226 target cells overnight with 10 different E:T ratios, the culture supernatant of CAR T cells was evaluated. Cytokine production of (Fig. 5A) IFNγ and (Fig. 5B) TNFα, (Fig. 5C) IL-2 was analyzed by ELISA. Mean ± SEM of two technical repeats. The data show an experiment performed in triplicate with technology from one donor, representing the results from three independent experiments in different donors.

[0070] Figures 6A to 6C depict membrane-bound IL7 expression and its function of maintaining CAR T cell growth after IL-2 withdrawal (withdrawal). (Figure 6A) The expression of membrane-bound IL7 was determined by western using IL7 antibody and then a goat anti-mouse HPR conjugated secondary antibody. GAPDH measured by anti-GAPDH and goat anti-rabbit secondary antibodies was included as a loading control. With or without mIL7, CAR T cells were transduced with a lentivirus encoding ROR1 and / or MSLN CAR constructs at MOI 20. The transduced CAR T cells were washed and cultured with 1e6 / ml using TexMACS culture medium without IL-2 supplementation. Long-term target cell stimulation was performed. Cell expansion (Figure 6B) and T cell size (Figure 6C) were monitored weekly until no cell expansion was observed for 2 to 3 consecutive weeks. The data represent an independent experiment from two different donors.

[0071] Figures 7A and 7B depict the 50% target cell killing (KT50) time (Figure 7A) and relative efficacy (Figure 7B) of MLSN CAR T cells before and after IL-2 withdrawal. MSLN CAR D0245 with mIL7 and ROR2 / MSLN DuoCAR D0282 with mIL7 were cultured in TexMACS medium without IL-2 supplementation for 69 days. + MSLN + The pancreatic cancer cell line AsPC-1 was measured by the xCELLigence RTCA instrument for the cytotoxicity of CAR D0245 and D0282. MSLN CAR D0181, CAR D0245, and D0282 without IL-2 withdrawal were included as controls. CAR T cells and target cells were co-cultured at an ET ratio of 2:1. The percentage of specific target lysis was assessed by blocked electron flow. KT50 represents the co-incubation time required to achieve cell lysis of 50% target cells. Relative potency was calculated based on the KT50 of MSLN CAR D0181 without IL-2 withdrawal. The data represent an independent experiment from two different donors.

[0072] Figures 8A to 8E depict the in vitro characteristics of the MSLN CAR enhanced by TGFβRIIdn. (Fig. 8A) MSLN CAR D0181 is composed of MSLN scFv binding domain, CD8 hinge domain and transmembrane domain, 41BB costimulatory domain and CD3ζ activation domain. The enhanced CAR D0211 is composed of a single MSLN CAR, a 2A peptide linker and a dominant negative TGFβ receptor II (dominant negative TGFβreceptor II, TGFβRIIdn) in the same frame. Primary T cells from healthy donors are activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding MSLN CAR D0181 and enhanced MSLN CAR D0211 constructs. (Fig. 8B) CAR surface expression is assessed by flow cytometry using MSLN-His followed by anti-His-APC staining. TGFβRIIdn expression was determined by biotinylated TGFβR and streptavidin PE staining. Histogram overlays of UTD, CAR D0181 and D0211 are shown on the right. (FIG. 8C) Luciferase-based cytotoxicity assays were performed using MSLN+ tumor lines NCI-H226, A431-MSLN and MSLN-A431. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight with 10 series of effectors to targets (E: T) ratios. The percentage of specific target lysis was assessed by luminescence analysis and normalized to the percentage of CAR expression. Curve fitting was performed using nonlinear EC50 offset and x as logarithmic concentration. (FIG. 8D) After incubation overnight with MSLN+NCI-H226 target cells at 10 different E: T ratios, the culture supernatant of CAR T cells was evaluated. Cytokine production of IFNγ and TNFα was analyzed by ELISA. Mean ± SEM of two technical replicates. Data represent one independent experiment from four different donors. (Figure 8E) Kinetic killing assay testing the function of MSLN and ROR1CAR T cells armed with TGFβRIIdn against AsPc-1 tumor cell line in the presence or absence of TGFβ.

[0073] Figures 9A to 9D depict the expression and cytotoxicity of ROR1CAR with TGFβRIIdn at a range of effector to target cell ratios in an overnight endpoint killing assay. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding ROR1CAR LTG2529 and a ROR1CAR D0228 construct armed with enhanced TGFβRIIdn. CAR surface expression was assessed by flow cytometry using ROR1 Fc followed by anti-Fc-AF647 staining. The percentage of CAR expression is plotted in Figure (Figure 9A). ROR1+ target lines, OVAR3 (Figure 9B), CAPAN-2 (Figure 9C) and NCI-H226 (Figure 9D) were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight with different effector to target (E: T) ratios. Specific target cleavage percentages were assessed by luminescence analysis and normalized to CAR expression percentages. Curve fitting was performed using nonlinear EC50 shift in Prism with x as a function of log concentration. Data are representative of one independent experiment from 1 different donor.

[0074] Figures 10A and 10B depict the structure of MSLN and ROR1 CAR with ECM enhancers and their surface expression in human primary T cells. (Figure 10A) MSLN targeting CAR is composed of all-human MSLN scFv targeting domains, CD8 hinge and transmembrane domains, 4-1BB costimulatory domains and CD3ζ activation domains. ROR1 targeting CAR is composed of all-human ROR1 scFv9 targeting domains, IgG4 short hinges, CD8 transmembrane domains, 4-1BB costimulatory domains and CD3ζ activation domains. Enhancer CAR includes single-target CAR, followed by 2A peptides in the same frame as ECM molecules. Heparanase (HPSE), metalloproteinase (MMP2), hyaluronidase PH-20 are selected as enhancer molecules. (Figure 10B) Primary T cells from healthy donors are activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding a CAR construct. Transduced T cells were assayed for CAR surface expression by flow cytometry using ROR1 Fc or MSLN-His staining followed by anti-Fc-AF647 or anti-His APC, respectively. CD4 staining was included to identify CD4+ and CD8+ populations. CAR positive percentages are listed above the graph. UTD - untransduced control.

[0075] Figures 11A to 11D depict the cytotoxicity of MSLN and ROR1 CAR constructs in vitro. + MSLN + Tumor line: (Figure 11A) MEC-1ROR1Hi MSLN Hi , , (Figure 11C) NCI H226 and ROR1 - MSLN - Tumor lines, (FIG. 11B) MEC-1 and (FIG. 11D) HL-60 were subjected to luciferase-based cytotoxicity assays. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at the specified effector to target (E: T) ratios: 1.25: 1, 5: 1, or 10: 1. The percentage of specific target lysis was assessed by luminescence analysis. The data represent an independent experiment from two different donors. Mean ± SEM of three technical replicates. A representative experiment from one donor is shown in the figure.

[0076] Figures 12A and 12B depict the expression of HPSE in the enhancer CAR and its ability to promote CAR T cell migration in vitro. (Figure 12A) HPSE secreted by CAR D0344 and CAR D0347 was measured by ELISA, and single CAR D0181, CAR D0290 and untransduced T cells (UTD) from the same donor were included as controls. The culture supernatant of CAR T cells was evaluated after overnight incubation. (Figure 12B) HPSE function was evaluated by migration assay using transwell coated with 0, 2.5 or 5 mg / ml Cultrex. One million thawed CAR T cells were inoculated into pre-coated transwells. After 24 hours, the total CAR T cells migrating to the bottom chamber were quantified by flow cytometry using absolute counting beads.

[0077] Figures 13A and 13B depict the in vivo activity of CAR T constructs in the JeKo-1 xenograft model. On day 0, NSG mice were implanted via the tail vein with 5×10 5 JeKo-1 cells. Tumor burden was determined using bioluminescent imaging. Mice with comparable mean tumor burden were randomly assigned to each group and injected with 5 × 10 6 / CAR+T cells or UTD in mice. Tumor kinetics were measured on days 13, 20, 27, 34, 41, and 48. (Figure 13A) Representative mouse bioluminescence images at the indicated time points are shown. (Figure 13B) The time course of tumor growth based on mouse whole-body bioluminescence (radiance) was quantified as photons / second / cm 2 / steradian. TA - tumor only, UTD - untransduced T cell control. N = 6, mean ± SEM.

[0078] Fig.14Figure 3. Body weight changes of mice during the JeKo-1 xenograft study. NSG mice bearing JeKo-1 mantle cell lymphoma were treated with 5×10 6 CART+ cells / mouse were treated and mouse body weights were recorded three times a week. Body weight changes were calculated as percentages relative to the start of the study. Mean ± SEM. TA-tumor only, UTD-untransduced T cell control. N = 6 mice / group.

[0079] Figures 15A and 15B depict the in vivo activity of CAR T constructs in the OVCAR-3 xenograft model. On day 0, NSG mice were injected intraperitoneally with 1×10 7 OVCAR-3-luciferase cells. Tumor burden was measured using bioluminescent imaging with an IVIS-S5 instrument. Mice with comparable tumor burden were randomly assigned to each group and treated with 5 × 10 6 / mice were treated with CAR+T cells or UTD. The kinetics of tumorigenesis were measured on days 10, 17, 24, 31, 38, 45, and 52. (Figure 15A) shows bioluminescent images of mice at the indicated time points. (Figure 15B) The time course of tumor growth based on whole-body bioluminescence (radiance) of mice was quantified as photons / second / cm 2 / steradian and plotted. TA - tumor only, UTD - untransduced T cell control. N = 4 to 5, mean ± SEM.

[0080] Fig.16 Figure 3. Body weight changes of mice during the OVCAR-3 study. NSG mice bearing disseminated OVCAR-3 tumors were treated with 5×10 6 CAR T-positive (CAR T+) cells / mouse were treated and mouse body weights were recorded three times a week. Body weight changes were calculated as percentages relative to the start of the study. Mean ± SEM. TA-tumor only, UTD-untransduced T cell control. N = 4 to 5 mice / group.

[0081] Figure 17 depicts the structure of ROR1 and FolR1 CAR containing ECM enhancers, and their surface expression in human primary T cells. A) ROR1 targeting CAR is composed of all-human ROR1 scFv9 targeting domain, IgG4 short hinge, CD8 transmembrane domain, 4-1BB costimulatory domain and CD3ζ activation domain. FolR1 targeting CAR is composed of all-human Farle scFv targeting domain, CD8 hinge and transmembrane domain, 4-1BB costimulatory domain and CD3ζ activation domain under PGK or EF1α promoter. Enhancer CAR includes single-target CAR, followed by 2A peptide in the same frame with ECM molecules. Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), hyaluronidase (PH-20) and heparanase (HPSE) are selected as enhancer molecules. For the ROR1 CAR group expressing hyaluronidase, PH-20 was expressed under natural or tPA signaling peptide in the presence, absence or retention of 7 amino acids of the GPI anchor. B) Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding a CAR construct. CAR surface expression of transduced T cells was determined by flow cytometry using ROR1-Fc or FolR1-Fc staining followed by anti-Fc-AF647. CD4 staining was included to identify CD4+ and CD8+ populations. CAR positive percentages are listed above the chart. UTD-untransduced control.

[0082] Figures 18A to 18E depict the cytotoxicity and cytokine release of ROR1 and FolR1 CAR constructs in vitro. A, B) Luciferase-based cytotoxicity assays were performed using antigen-specific tumor lines. ROR1CAR was tested for ROR1+ lines NCI-H226 and MEC-1ROR1Hi, and MEC-1 was used as a negative control line expressing basal levels of ROR1. CAR-T cells and tumor cells were co-cultured overnight with a specified effector to target (E: T) ratio: 10: 1, 5: 1 or 1.25: 1. Specific target lysis percentages were assessed by luminescence analysis. (Figure 18A) Data represent an independent experiment from 3 different donors. The mean ± SD of three technical repeats. A representative experiment from one donor is shown in the figure. (Figure 18B) Data represent an independent experiment from 1 donor. The mean ± SD of three technical repeats. (FIG. 18C) FolR1 CAR was tested against FolR1+ line OVCAR3, and HL-60 was used as a negative control for nonspecific killing. CAR-T cells and tumor cells were co-cultured overnight at the specified effector to target (E: T) ratios: 10: 1, 2.5: 1, or 1.25: 1. The percentage of specific target lysis was assessed by luminescence analysis. The data represent an independent experiment from 3 different donors. The mean ± SD of three technical replicates. A representative experiment from one donor is shown in the figure. All target lines were stably transduced with firefly luciferase. (FIG. 18D, FIG. 18E) Cytokine production of IFNγ and TNFα was analyzed by ELISA. (FIG. 18D) After incubation with ROR1+NCI-H226 target cells at E: T ratios of 10: 1, 5: 1, and 1.25: 1 overnight, the culture supernatant of CAR-T cells was evaluated. The mean ± SD of three technical replicates. Data represent 3 independent experiments from 3 different donors. (FIG. 18E) Culture supernatants of CAR-T cells were evaluated after overnight incubation with FolR1+OVCAR3 target cells at E:T ratios of 10:1, 2.5:1, and 1.25:1. Mean ± SD of three technical replicates. Data represent 3 independent experiments from 3 different donors.

[0083] Figures 19A to 19D depict the expression of enzymes in the enhancer CAR and their ability to promote CAR-T cell migration in vitro. (Figure 19A) Left: The concentration of MMP-9 secreted by ROR1 (D0373) co-expressing MMP-9. Untransduced and CAR D0290 were also measured by MMP-9 ELISA. The data represent an independent experiment in 2 different donors tested. Right: HPSE of CAR D0368 and D0369 was measured by ELISA, and CAR D0351 and untransduced T cells (UTD) from the same donor were included as controls. The culture supernatant of CAR-T cells was evaluated from the last day of CAR-T production. (Figure 19B) The culture supernatant of CAR-T cells was evaluated using O or 5mg / ml Cultrex TM The coated transwell was used to evaluate the function of MMP-2 and MMP-9 by migration assay. 500,000 thawed CAR-T cells were inoculated into the pre-coated transwell. After 24 hours, the total CAR-T cells that migrated to the bottom chamber were quantified by flow cytometry using absolute counting beads. (Figure 19C) The cells were treated with 0 or 5 mg / ml Cultrex TM The function of HPSE and PH-20 was evaluated by migration assay in transwells coated with hyaluronic acid (Figure 19C) or hyaluronic acid (Figure 19D). 500,000 thawed CAR-T cells were seeded into pre-coated transwells. After 24 hours, the cells were cultured against Cultrex TM After coating, the total CAR-T cells that migrated to the bottom chamber were quantified by flow cytometry using absolute counting beads.

[0084] Figures 20A to 20C depict the in vivo activity of FolR1 CAR-T co-expressing HPSE or PH-20 in the OVCAR3 xenograft model. NSG mice were implanted with 1×10 cells stably transduced with luciferase by intraperitoneal injection. 7 OVCAR3 cells. Tumor burden was determined using bioluminescent imaging with an IVIS-S5 instrument. Mice with comparable average tumor burden were randomly assigned to each group and injected with 5 × 10 6 / CAR+T cells or UTD of mice. Tumor dynamics were measured on days 11, 18, 25, 32, and 39. (FIG. 20A) Representative bioluminescent images of mice at the indicated time points are shown. (FIG. 20B) The time course of tumor growth based on whole-body bioluminescence (radiance, photons / second / cm2 / sr) of mice was quantified and plotted as shown. TA-tumor only, UTD-untransduced T cell control. N=4, mean ± SD. (FIG. 20C) Weight changes of mice during the OVCAR3 xenograft study. OVCAR3-bearing mice were treated with CAR-T cells and weights were recorded three times a week. Weight changes were calculated as a percentage of change relative to the start of the study. Mean ± SEM. TA-tumor only, UTD-untransduced T cell control. N=4 mice / group.

[0085] Figures 21A and 2B depict the in vivo characteristics of Farle CAR-T with enhanced ECM enzymes HPSE or PH-20. (Figure 21A) At the end of the life study, CAR-T infiltration, CAR expression (percentage and gMFI), and CD4:CD8 ratio were measured in the bone marrow (top) and spleen (middle). All samples were normalized by volume and absolute counting beads. There was no significant difference in spleen weight between treatment groups. (Figure 21B) Memory phenotype of CAR-T cells in the bone marrow (top) and spleen (bottom). Initial, central memory, effector memory and effector cells of CAR-T+ cells (left), CAR+CD4+ (middle) and CAR+CD8+ (right) were measured. Mean ± SD. Statistical differences were calculated using a one-way ANOVA in Prism software. For B, statistical differences in effector groups between different treatment groups were measured. TA-tumor only, UTD-untransduced T cell control. N = 4 mice / group.

[0086] Figures 22A to 22C depict the structure and surface expression of ROR1 and CD276 CAR on transduced primary T cells. (Figure 22A) ROR1 or CD276 CAR includes ROR1 or D276 scFv binding domain, IgG4 or CD8 hinge domain, CD8 transmembrane domain, 41BB costimulatory domain, CD3ζ activation domain. (Figure 22B) Representative flow diagram of CAR expression on transduced T cells. CAR and CAR / CCR T cells were stained with ROR1-Fc followed by anti-Fc AF647 for ROR1 CAR detection and stained with CD276-His for CD276CCR detection. (Figure 22C) Average CAR expression in T cells from three healthy donors. Error bars represent mean ± SEM.

[0087] Figures 23A to 23C depict the cytotoxicity of ROR1 or CD276 CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using ROR1+CD276+ tumor lines (Figure 23A) OVCAR3, (Figure 23B) AsPC-1, (Figure 23C) NCI-H226. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight with 10 series of effectors and targets (E: T) ratios. The specific target cleavage percentage was assessed by luminescence analysis and normalized to the percentage of CAR expression. Curve fitting was performed using a nonlinear EC50 offset in which x was the logarithmic concentration. The data represent an independent experiment in three experiments in T cells from different donors. Error bars represent mean ± SEM.

[0088] Figure 24A to 24C depicts the structure and primary human T cell surface expression of ROR1 CAR strengthened with CD276 CCR. (Figure 24A) ROR1 CAR strengthened by CD276 CCR includes ROR1 CAR in the same frame as CD276 CCR, which is connected by P2A ribosome skipping elements. (Figure 24B) Primary T cells transduced are gated based on forward and side scatter, doublet exclusion and viability dye negative. Surface CAR expression of ROR1 targeting or CD276 targeting domains of each conjugate is detected by co-staining ROR1-Fc and CD276-His followed by anti-Fc and anti-His FL conjugates. Representative flow cytometry of ROR1 CAR and CD276 CCR co-expression is shown. (Figure 24C) CAR and CCR co-expression on T cell surface is quantified. The mean value of the transduction results of T cells from three healthy donors is shown, and the error bar indicates ± SEM.

[0089] Figure 25A to 25D depicts the cytotoxicity of a single ROR1 CAR construct without CD276 CCR in vitro. Using (Figure 25A) ROR1+CD276+ tumor line OVCAR3, (Figure 25B) ROR1-CD276- tumor line RS4; 11 and single target positive cell line (Figure 25C) ROR1+CD276-RS4; 11-ROR1, (Figure 25D) ROR1-CD276+RS4; 11-CD276 for luciferase-based cytotoxicity assay. All target lines are stably transduced with firefly luciferase. CAR T cells and tumor cells are co-cultured overnight with a series of 10 effectors and target ratios. Specific target lysis percentages were assessed by luminescence analysis and normalized to CAR expression percentages. Curve fitting was performed using a nonlinear EC50 offset where x is the logarithm of the concentration. Data represent an independent experiment in three experiments performed on T cells from 3 different donors. Error bars = mean ± SEM.

[0090] Figures 26A and 26B depict the relative efficacy of ROR1 CAR / CD276 CCR constructs in vitro. CAR T cells and ROR1+ tumor cells were co-cultured overnight with 10 different effector to target ratios. The specific target lysis percentage was assessed by luminescence analysis and normalized to the percentage of ROR1 CAR expression. The relative efficacy relative to ROR1 CAR LTG2529 was calculated using the nonlinear EC50 offset function in GraphPad Prism where x is the logarithmic concentration. The relative efficacy of each construct targeting ROR1+ in tumor lines: (Figure 26A) OVCAR-3, (Figure 26B) RS4; 11-ROR1 is plotted as a bar graph. The data represent the mean ± SEM of independent experiments using T cells from 3 different donors.

[0091] Figures 27A to 27K depict that the new anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion compared to LTG2527 (with control R12 binder), but exhibits comparable cytotoxic potency in vitro and potency against hematological tumors in vivo. (Figure 27A) Schematic diagram of CAR constructs. (Figure 27B) Left: Flow cytometry example of CAR+T cell percentage; Middle: CAR+T cell percentage (n=3 donors); Right: CAR density (n=3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematological cell lines, the experiment was performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR-1 Ab from Miltenyi Biotec and R&D systems, with similar results. (FIG. 27D) In ​​vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 and LTG2527 (n=3 donors). (FIG. 27E) Cytokines secreted when CAR T was co-cultured with the Jeko-1 cell line for 18 hours were quantified by ELISA, and representative data from 3 donors are shown. (FIG. 27F to 27K): NSG mice were implanted with Jeko-1 cells (iv, 0.5e6 cells / mouse; 6 mice / group) on day #-6, followed by staging on day #-1, and administration of CAR T cells (iv, 3e6 CAR T cells) on day #0. +T cells / mouse) (Figure 27F); tumor progression was quantified by bioluminescence imaging (Figure 27G, Figure 27H), body weight was monitored (Figure 27I), blood was collected at the indicated time points, and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p < 0.05; **: p < 0.01; n / s: not significant.

[0092] Figures 28A to 28I depict that LTG2529, but not LTG2527, effectively inhibits solid tumor progression in an in vivo ovarian cancer OVCAR-3 xenograft model, despite exhibiting comparable in vitro cytotoxic activity (with higher cytokine production). (A) Quantification of ROR1 expression on the surface of multiple solid tumor cancer cell lines; the experiment was performed in duplicate using anti-ROR1 Ab from BD Biosciences; a separate experiment was also performed in duplicate using anti-ROR-1Ab from Miltenyi Biotec and R&D systems, with similar results. (B) Representative killing curves of CAR-T against multiple solid cancer cell lines in 18-hour co-culture, with the right figure showing the relative cytotoxic potency of LTG2529 and LTG2527 (n=3 donors). (C) Quantification of cytokines secreted when CAR-T was co-cultured with OVCAR-3 cell lines for 18 hours by ELISA, showing representative data, 3 independent experiments were performed using 3 donors, with similar results. (D to I): Efficacy of CAR-T in the in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with OVCAR-3 cell line (10e6 cells / mouse) on day 7, followed by staging on day 1; CAR-T (5e6 CAR + T cells / mouse) (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); blood was collected at designated time points to quantify CAR+ T cells in both CD8 and CD4 subsets (H), as well as memory T cells (I).

[0093] Figures 29A to 29K depict that dominant negative TGFbRII (DN) blocked TGFb1 signaling in vitro and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in T cells transduced with LTG2529. (Figure 29A) Schematic diagram of the constructs of LTG2529 alone and LTG2529 armed with DN (i.e., D0228). (Figure 29B) CD8 T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry on day 8 of transduction. + and CD4 +CAR expression (left: flow cytometry, middle: chart from flow cytometry) and memory phenotype (right) of both T cells; 3 independent experiments were performed with 3 donors, with similar results. (Figure 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; 3 independent experiments were performed with 3 donors, with similar results. (Figure 29D) CAR-T was starved of IL-2 for 22 hours to synchronize cells, and then treated with TGFb1 (10ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper figure: flow cytometry; lower figure: chart from the upper figure; data are representative of 3 independent experiments performed on 3 donors. (Figure 29E) Expression of ROR1 on AsPC-1 cell line was evaluated by flow cytometry. (FIG. 29F) AsPC-1 was co-cultured with CAR-T cells in the absence or presence of TGFb1 (1 or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; middle: time for 50% tumor cells to be killed (KT 50 ); right: relative potency of cytotoxicity of CAR-T treated with TGFb1 versus untreated; 2 independent experiments were performed in triplicate with 2 donors with similar results. (FIG. 29G) Cytokine production from the experiment in (FIG. 29E) was quantified by ELISA; 2 independent experiments were performed in triplicate with 2 donors with similar results. (FIG. 29H, FIG. 29I): Active or latent forms of TGFb1 produced by various solid tumor cell lines (FIG. 29H) or by AsPC-1 ectopically overexpressing TGF1 (FIG. 29I) were evaluated by ELISA; data are representative of 2 independent experiments with similar results. (FIG. 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 control were co-cultured with CAR-T, showing % cell lysis of tumor cells. (FIG. 29K) Cytokine production from the experiment in (FIG. 29E) was quantified by ELISA; 2 independent experiments were performed in triplicate with 2 donors with similar results. Notes: *: p<0.05; **: P<0.01; ***: P<0.001.

[0094] Figures 30A to 30J depict that TGFbRIIDN exhibits a higher frequency of CAR+ T cells in a pancreatic cancer xenograft model using AsPC-1 that produces low levels of TGFb1. (Figures 30A to 30D): Efficacy of CAR-T in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day 17, followed by staging and CAR-T infusion (iv, 5e6 CAR + T cells / mouse) (Figure 30A); measuring tumor volume (Figure 30B) (left: tumor volume from mice in all groups; right: tumor volume from mice treated with armed CAR and unarmed CAR starting on day 10 after T cell administration); monitoring body weight (Figure 30C); sampling the blood of mice and quantifying the CD8 subset of CAR+T cells (Figure 30D). (Figure 30E to J): On day 73 after T cell administration, mice (4 from the unarmed CAR-treated group and 3 from the armed CAR-treated group; Note: 1 mouse from the armed group was euthanized due to excessive weight loss on day 60 after T cell infusion) were re-challenged with AsPC-1 cells (1e6 cells / mouse, on the left flank; because the first attack was on the right flank) (Figure 30E); tumor volume on both flanks (Figure 30F) and survival rate (Figure 30G) were monitored; blood of mice was sampled at designated time points to quantify T cell memory phenotype (Figure 30H), CAR+ cell percentage (Figure 30I); T cells isolated from spleen and bone marrow at the termination time point were also analyzed by flow cytometry for CAR+T cell components (Figure 30J).

[0095] Figures 31A to 31G depict the attenuation of TGFb inhibition by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using AsPC-1 overexpressing TGFb. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day 15, followed by staging and CAR-T infusion (iv, 5e6 CAR +T cells / mouse) (Figure 31A); monitor tumor volume (Figure 31B); sample the blood of mice on the 5th and 15th days after T cell infusion, and quantify cytokines (Figure 31C), including TGFb1 (left), IFNg (middle) and GM-CSF (right); T cells separated from blood at a specified time point are quantified relative to total cell number (Figure 31D), CAR+ components (Figure 31E) in both CD8 and CD4 subgroups are quantified. At the termination time point (49th day after T cell infusion), T cells from blood, spleen and bone marrow are harvested, and CAR+ components (Figure 31F) and memory phenotypes (Figure 31G) in both CD4 and CD8 subgroups are quantified. DETAILED DESCRIPTION

[0096] definition

[0097] Unless the context clearly indicates otherwise, the nouns used in this article without quantifier modification represent one / kind or more / kind. For example, the term "antigen" includes single / kind or multiple / kind antigens, and can be considered to be equivalent to the phrase "at least one / kind antigen". The term "comprising" used in this article means "including". Therefore, "comprising antigens" means "comprising antigens" without excluding other elements. The phrase "and / or" means "and" or "or / or". It should be further understood that, unless otherwise indicated, any and all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acids or polypeptides are approximate, and are provided for the purpose of description. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described below. In the event of a conflict, this specification (including the explanation of the term) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. For ease of access to multiple embodiments, the following explanation of terms is provided:

[0098] When referring to a measurable value (e.g., an amount, a temporal duration, etc.), the term "about" is meant to encompass variations of + / - 20%, + / - 10%, or more preferably + / - 5%, or + / - 1%, or still more preferably + / - 0.1% relative to the particular value, as such variations are suitable for performing the disclosed methods.

[0099] Unless otherwise indicated, technical terms herein are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0100] Provided herein are new anti-effector part antibodies or their antigen binding domains and chimeric antigen receptors (CARs) comprising such effector part antigen binding domains, as well as host cells (e.g., T cells) expressing the receptors and nucleic acid molecules encoding the receptors.CAR may consist of a single molecule expressed on the surface of effector cells, or CAR may consist of a signal transduction module and a soluble targeting module expressed by effector cells, such as when a soluble targeting module is combined with a signal transduction module expressed by a cell, to form a complete functional CAR.CAR shows high surface expression on transduced T cells, with a high degree of cell lysis and transduced T cells in vivo amplification and persistence.Also provided are methods for treating cancer in an object using disclosed CAR, host cells, and nucleic acid molecules, for example.

[0101] In its broadest aspect, provided herein is a novel chimeric antigen receptor (CAR) comprising a boosted CAR containing a CAR construct having a major effector portion molecule followed by one or more 2A sequences in frame with one or more additional "boosting" elements for improving function, including enhancing tumor penetration, thereby improving the therapeutic effect of CAR-T cells in solid tumors, hematological tumors, autoimmune diseases, genetic diseases or other related indications.

[0102] In another broad aspect, provided herein are novel chimeric antigen receptors (CARs) comprising boosted CARs, wherein functionally co-expressed boosted CAR elements are expressed from a single polycistronic vector with high transduction efficiency, thereby simplifying the manufacture and release of the CAR and reducing the cost of market implementation. In one aspect, the enhanced CAR composition comprises one or more of the following features: i) high surface expression on transduced T cells; ii) multi-targeting to overcome antigen escape; iii) one or more armed elements to overcome immunosuppression in the TME; iv) one or more cytokine stimulating elements to promote cytokine stimulation of autonomous T cells, thereby improving anti-tumor cytotoxicity, expansion, memory formation, cytokine secretion, and persistence; v) one or more digestive enzymes to overcome the physical barrier of tumor stroma / extracellular matrix (ECM) and enable CAR T tumor penetration; vi) one or more pro-inflammatory immune activators; and vii) one or more on switches or off switches to control the expression of CAR; or any combination thereof, wherein the enhanced CAR achieves a high degree of cell lysis and in vivo expansion and persistence of transduced T cells in a patient-specific manner to promote in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0103] In another broad aspect, the novel chimeric antigen receptors (CARs) provided herein may comprise single, tandem or multiple targeting CAR constructs (including those in the form of DuoCARs), or any combination thereof.

[0104] In certain aspects, the new enhanced CAR is under the control of one or more constitutive promoters, tissue-specific promoters or inducible promoters or any combination thereof.

[0105] In certain aspects, the new enhanced CAR may comprise one or more pro-inflammatory immune activators.

[0106] In certain aspects, one or more pro-inflammatory immune activators may comprise enhancers that convert a "cold" immune environment to "hot," such as neutrophil-activating protein (NAP) from bacteria (e.g., Helicobacter pylori); bacterial lipopolysaccharide (LPS) components; or polyinosinic polycytidylic acid (poly(I:C)); or soluble inflammatory factors, such as FLT3 ligand; or oncolytic viruses; or TNF family cytokines, including CD40 ligand (CD40L), tumor necrosis factor (TNF), and receptor activator of nuclear factor-KB (RANKL) / TRANCE, which can trigger or enhance an exogenous bystander response to solid cancers.

[0107] In one aspect, such elements, when used as a booster for CAR T cell therapy, can reduce or eliminate tumor growth, and / or improve survival, regardless of the target antigen, tumor type, and host haplotype. Such a booster can act by supporting dendritic cell maturation and bystander responses, leading to epitope spreading and infiltration of CD8+ cells targeting tumor-associated antigens other than the CAR T-targeted antigen.

[0108] In certain aspects, the one or more switches include a tag, a kill switch, an on switch, an off switch, and / or an adapter switch, or any combination thereof.

[0109] In certain aspects, the new enhanced CAR switch can be a tag (CD19, CD34, CD22, EGFR), or a kill switch (iCAS9), or an [ON] switch, or an [OFF] switch, or an adapter switch, or any combination thereof.

[0110] In certain embodiments, single, tandem, multi-targeting, DuoCAR (with or without one or more enhancer elements) novel chimeric antigen receptors (CARs) are provided for transducing effector cells to treat solid tumors and hematological tumors and other diseases by targeting antigens, such as but not limited to CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosylGMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, Ep CAM, folate binding protein, folate receptor α (FOLR1), folate receptor β (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, p185, IL-2 receptor, interleukin-1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, αvβ3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYPlB 1. PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie3, PAGE4, ​​VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL 1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endothelin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GP RC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15 or any combination thereof or a fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the following: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a nanobody, a VHH, a ligand peptide and a single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof.

[0111] In one embodiment, an isolated polynucleotide is provided, which encodes a fully human anti-ROR1 and / or anti-MSLN and / or anti-Fo1R1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2, and / or anti-CD276, and / or anti-GPC2, and / or anti-FGFR2, and / or anti-PSMA, and / or anti-MUC1, and / or anti-MUC16, and / or anti-IL13Rα antibody, or any combination thereof or a fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab') 2 Fragments, Fv fragments, Nanobodies, VHHs, ligand peptides and single-chain Fv (ScFv).

[0112] In one embodiment, an isolated polynucleotide is provided that encodes anti-GD2, anti-GD3, anti-GM2, anti-Ley, anti-polysialic acid, anti-fucosyl GM1, anti-GM3, anti-Tn, anti-STn, anti-sLe (animal), anti-GloboH, anti-CD5, anti-CD7, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CD37, anti-CD30, anti-CD33, anti-CD38, anti-CD123, anti-CD45, anti-CAMPATH-1, anti-BCMA, anti-CS-1, anti-PD-L1, anti-CD276 / B7-H3, anti-B7-H4, anti-B7-DC, anti-HLA-DR carcinoembryonic antigen (CEA ), anti-TAG-72, anti-EpCAM, anti-folate binding protein, anti-folate receptor α (FOLR1), anti-folate receptor β (FOLR2), anti-A33, anti-G250, anti-prostate specific membrane antigen (PSMA), anti-ferritin, anti-CA-125, anti-CA19-9, anti-CD44v6, anti-epidermal growth factor, anti-p185, anti-IL-2 receptor, anti-interleukin 1 receptor accessory protein (IL1RAP), anti-EGFRvIII (de2-7), anti-fibroblast activation protein, anti-tenascin, anti-metalloproteinase, anti-endosialin, anti-vascular endothelial growth factor, anti-αvβ3, anti-WT1, anti-LMP2, anti-HPV E6, anti-HPV E7, anti-Her-2 / neu, anti-p53 non-mutant, anti-NY-ESO-1, anti-MelanA / MART1, anti-Ras mutant, anti-gp100, anti-FGFR1, anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-GPC1, anti-GPC2, anti-GPC3, anti-p53 mutant, anti-PR1, anti-bcr-abl, anti-tyrosinase, anti-survivin, anti-PSA, anti-hTERT, anti-sarcoma translocation breakpoint fusion protein, anti-EphA2, anti-PAP, anti-ML-IAP, anti-AFP, anti-ERG, anti-NA17, anti-PAX3, anti-ALK, anti-androgen receptor, anti-cyclin B1, anti-MYCN, anti-RhoC, anti-TRP-2, anti-mesothelin, anti-PSCA, anti-MAGE A1, anti-MAGE A3, anti-CYP1B 1. Anti-PLAV1, anti-BORIS, anti-ETV6-AML, anti-NY-BR-1, anti-RGS5, anti-SART3, anti-carbonic anhydrase IX, anti-PAX5, anti-OY-TES1, anti-sperm protein 17, anti-LCK, anti-HMWMAA, anti-AKAP-4, anti-SSX2, anti-XAGE 1, anti-B7H3, anti-bean protein, anti-Tie 3, anti-PAGE4, ​​anti-VEGFR2, anti-MAD-CT-1, anti-PDGFR-B, anti-MAD-CT-2, anti-TRAIL 1, anti-MUC1, anti-MUC16 / CA125, anti-MAGE A4, anti-MAGEC2, anti-GAGE, anti-EGFR, anti-EGFR1, anti-EGFR2 / Her2, anti-CMET, anti-HER3, anti-CA6, anti-NAPI2B, anti-TROP2, anti-TEM1, anti-TEM7, anti-TEM8, anti-FAP, anti-LAP, anti-CLDN6, anti-CLDN8, anti-CLDN16, anti-CLDN18.2, anti-RON, anti-LY6E, anti-DLL3, anti-PTK7, anti-UPK1B, anti-STRA6, anti-TMPRSS3, anti-TMRRSS4, anti-TMEM238, anti-Clorfl86, anti-LIV1, anti-ROR1, anti-ROR2, anti-Fos-related antigen 1, anti-VEGF R1, anti-endothelin, anti-CD90, anti-CD326, anti-CD70, anti-SSEA4, anti-CD318, anti-CLA, anti-TSPAN8, anti-GPRC5D, anti-EpCAM, anti-Thy1, anti-IL13Ra2, anti-BDCA1, anti-BDCA2, anti-BDCA3, anti-GD2, anti-PSMA, anti-FAP, anti-CLL1, anti-SLAMF7 / CS1, anti-CD147, anti-DPPA5, anti-GRP78, anti-CD66c, VISTA, LRRC5, LRRCl5 antibody, or any combination thereof or fragments thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of: Fab fragment, F(ab'), 2 Fragments, Fv fragments, nanobodies, VHHs, ligand peptides and single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof.

[0113] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded extracellular ROR1 and / or MSLN antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to ROR1 and / or MSLN.

[0114] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded extracellular ROR1 and / or MSLN antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to ROR1 and / or MSLN.

[0115] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded extracellular ROR1 and / or MSLN antigen binding domain comprises a ScFv.

[0116] In another broad aspect, one or more of the above-identified novel enhanced chimeric antigen receptors (CARs) provided above with respect to SEQ ID NOs: 151 to 256 may comprise a single, tandem or multi-targeting CAR construct (including those in the form of DuoCAR), or any combination thereof.

[0117] For each of the various aspects and embodiments of single, tandem, multi-targeting, DuoCAR (with or without one or more enhancer elements) CAR constructs specifically contemplated herein, the nucleotide sequence encoding a functional CAR (with or without one or more enhancer elements) comprises the following nucleotide sequence:

[0118] SEQ ID NO: 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 245, 247, 249, 251, 253, or 255,

[0119] or any combination thereof.

[0120] For each of the various aspects and embodiments of single, tandem, multi-targeting, DuoCAR (with or without one or more booster elements) CAR constructs specifically contemplated herein, each vector encodes a functional CAR (with or without one or more booster elements) comprising the amino acid sequence of:

[0121] SEQ ID NO: 152, 154: 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256,

[0122] or any combination thereof.

[0123] For each of the multiple aspects and embodiments, an isolated polynucleotide is provided, which encodes a fully human anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or a fragment thereof, which comprises a nucleic acid sequence selected from SEQ ID NO: 143, 145, 147, 149.

[0124] For each of the multiple aspects and embodiments, provided herein is a novel single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), comprising a single, tandem, DuoCAR or multi-targeted CAR molecule (with or without one or more enhancer elements): comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-ROR1 and / or anti-MSLN antigen binding domain comprising a nucleic acid sequence selected from SEQ ID NO: 143, 145, 147 and 149.

[0125] For each of the multiple aspects and embodiments, provided herein is a novel single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), comprising a single, tandem, DuoCAR or multi-targeted CAR molecule (with or without one or more enhancer elements): comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an anti-ROR1 and / or anti-MSLN antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150.

[0126] In one embodiment, an isolated polynucleotide is provided, which encodes a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a nucleic acid sequence selected from SEQ ID NO: 143, 145, 147 and 149.

[0127] In one embodiment, an isolated polynucleotide is provided that encodes a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150.

[0128] In one aspect, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted chimeric antigen receptor (CAR) (with or without one or more booster elements) is provided, which comprises from N-terminus to C-terminus at least one ROR1 and / or MSLN antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one ROR1 and / or MSLN antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 143, 145, 147 and 149.

[0129] In one aspect, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted chimeric antigen receptor (CAR) (with or without one or more booster elements) is provided, which comprises from N-terminus to C-terminus at least one ROR1 and / or MSLN antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one ROR1 and / or MSLN antigen binding domain is encoded by a nucleotide sequence comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150.

[0130] In one embodiment, the targeting domain of a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is expressed alone in the form of a monoclonal antibody, ScFv Fab, Fab'2, and comprises an antigen targeting domain coupled to an additional binding tag or epitope, the antigen targeting domain comprising a sequence selected from SEQ ID NO: 143, 145, 147 and 149, and the effector cell expression component of single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) comprises a binding domain, and the binding domain specifically directs to bind to a tag or epitope expressed on a soluble single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) module, such as a soluble component of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) and a cell binding component of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) The specific binding forms a complete functional single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) structure.

[0131] In another embodiment, the targeting domain of a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) is expressed alone in the form of a monoclonal antibody, ScFv Fab, Fab'2, and comprises an antigen targeting domain and an additional ScFv, wherein the antigen targeting domain comprises a nucleic acid sequence selected from SEQ ID NOs: 143, 145, 147 and 149, and the effector cell expression component of the single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) comprises a tag or epitope that specifically reacts with an additional ScFv expressed on a soluble single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) module, such as a soluble component of a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) specifically binds to a cell binding component of a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) to form a complete functional single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) structure.

[0132] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) is provided, wherein the encoded single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) extracellular ROR1 and / or MSLN antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) that binds to ROR1 and / or MSLN.

[0133] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 and / or MSLN antigen binding domain is connected to a transmembrane domain via a linker domain.

[0134] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded ROR1 and / or MSLN extracellular antigen binding domain is preceded by a sequence encoding a leader peptide or signal peptide.

[0135] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the CAR comprises at least one ROR1 and / or MSLN antigen binding domain, the ROR1 and / or MSLN antigen binding domain being encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NOs: 143, 145, 147 and 149, and wherein the single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) additionally encodes an extracellular antigen binding domain of a targeted antigen,The antigens include but are not limited to CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpC AM, folate binding protein, folate receptor α (FOLR1), folate receptor β (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, p185, IL-2 receptor, interleukin 1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, αvβ3, WTl, LMP2, HPV E6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART 1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-ab1, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGEA3, CYP1B 1. PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, pod protein,Tie 3, PAGE4, ​​VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL 1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDNl8.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, CLorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endothelin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GP RC5D, EpCAM, Thyl, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15 or any combination thereof or a fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the following: Fab fragment, F(ab')2 fragment, Fv fragment, nanobody, VHH, ligand peptide and single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof. ,

[0136] In certain embodiments, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the additionally encoded extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-BCMA ScFv antigen binding domain, an anti-CD5 ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PSMA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain. ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2 ScFv antigen binding domain, anti-NY-ESO-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0137] In one aspect, the single, tandem, DuoCAR or multi-targeting CAR provided herein (with or without one or more booster elements) further comprises a linker or spacer domain.

[0138] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the extracellular ROR1 and / or MSLN antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0139] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the encoded linker domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19 or CD28 and is connected to the transmembrane domain.

[0140] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the encoded single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) further comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, FcεR, or a combination thereof.

[0141] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded intracellular signaling domain further comprises a CD3ζ intracellular domain.

[0142] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

[0143] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain or a combination thereof.

[0144] In another embodiment, an immunotherapeutic composition is provided, wherein the at least one co-stimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), PD-1, GITR, CTLA-4, or any combination thereof.

[0145] In one embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the CAR further comprises a leader sequence or a signal peptide, wherein the leader or signal peptide nucleotide sequence comprises a nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID ID: 41 or SEQ ID NO: 43.

[0146] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID ID: 42 or SEQ ID NO: 44.

[0147] In one aspect, provided herein are single, tandem, DuoCAR or multi-targeting chimeric antigen receptors (CARs) (with or without one or more booster elements) comprising from N-terminus to C-terminus at least one ROR1 and / or MSLN antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain.

[0148] In one embodiment, a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided, wherein the extracellular ROR1 and / or MSLN antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0149] In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0150] In some embodiments, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) further encodes an extracellular antigen binding domain comprising: anti-CD19, anti-CD20, anti-CD22, anti-CD33, anti-CD38, anti-CD123 (IL3RA), anti-CD138, anti-GPC2, anti-GPC3, anti-FGFR4, anti-c-Met, anti-PSMA, anti-glycolipid F77, anti-EGFRvIII, anti-GD-2, anti-NY-ESO-1 TCR, anti-MAGE A3 TCR, anti-GD2, anti-GD3, anti-GM2, anti-Ley, anti-polysialic acid, anti-fucosyl GM1, anti-GM3, anti-Tn, anti-STn, anti-sLe (animal), anti-GloboH, anti-CD5, anti-CD7, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CD37, anti-CD30, anti-CD33, anti-CD38, anti-CD123, anti-CD45, anti-CAMPATH-1, anti-BCMA, anti-CS-1, anti-PD-L1, anti-CD276 / B7-H3, anti-B7-H4, anti-B7-DC, anti-HLA-DR carcinoembryonic antigen (CEA), anti-TAG-72 , anti-EpCAM, anti-folate binding protein, anti-folate receptor α (FOLR1), anti-folate receptor β (FOLR2), anti-A33, anti-G250, anti-prostate specific membrane antigen (PSMA), anti-ferritin, anti-CA-125, anti-CA19-9, anti-CD44v6, anti-epidermal growth factor, anti-p185, anti-IL-2 receptor, anti-interleukin 1 receptor accessory protein (IL1RAP), anti-EGFRvIII (de2-7), anti-fibroblast activation protein, anti-tenascin, anti-metalloproteinase, anti-endosialin, anti-vascular endothelial growth factor, anti-αvβ3, anti-WT1, anti-LMP2, anti-HPV E6, anti-HPV E7, anti-Her-2 / neu, anti-p53 non-mutant, anti-NY-ESO-1, anti-MelanA / MART 1, anti-Ras mutant, anti-gp100, anti-GPRC5D, anti-FGFR1, anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-GPC1, anti-GPC2, anti-GPC3, anti-p53 mutant, anti-PR1, anti-bcr-abl, anti-tyrosinase, anti-survivin, anti-PSA, anti-hTERT, anti-sarcoma translocation breakpoint fusion protein, anti-EphA2, anti-PAP, anti-ML-IAP, anti-AFP, anti-ERG, anti-NA17, anti-PAX3, anti-ALK, anti-androgen receptor, anti-cyclin B1, anti-MYCN, anti-RhoC, anti-TRP-2, anti-mesothelin, anti-PSCA, anti-MAGE A1, anti-MAGE A3, anti-CYP1B1, anti-PLAV1, anti-BORIS, anti-ETV6-AML, anti-NY-BR-1, anti-RGS5, anti-SART3, anti-carbonic anhydrase IX, anti-PAX5, anti-OY-TES1, anti-sperm protein 17, anti-LCK, anti-HMWMAA, anti-AKAP-4, anti-SSX2, anti-XAGE 1, anti-B7H3, anti-beta-glutamic acid, anti-Tie 3, anti-PAGE4, ​​anti-VEGFR2, anti-MAD-CT-1, anti-PDGFR-B, anti-MAD-CT-2, anti-TRAIL 1, anti-MUC1, anti-MUC16 / CA125, anti-MAGE A4, anti-MAGE, anti-C2, anti-GAGE, anti-EGFR, anti-EGFR1, anti-EGFR2 / Her2, anti-CMET, anti-HER3, anti-CA6, anti-NAPI2B, anti-TROP2, anti-TEM1, anti-TEM7, anti-TEM8, anti-FAP, anti-LAP, anti-CLDN6, anti-CLDN8, anti-CLDN16, anti-CLDN18.2, anti-RON, anti-LY6E, anti-DLL3, anti-PTK7, anti-UPK1B, anti-STRA6, anti-TMPRSS3, anti-TMRRSS4, anti-TMEM238, anti-Clorfl86, anti-LIV1, anti-ROR1, anti-ROR2, anti-Fos-related The invention relates to an anti-VEGFR1, anti-endothelin, anti-CD90, anti-CD326, anti-CD70, anti-SSEA4, anti-CD318, anti-CLA, anti-TSPAN8, GPRC5D, EpCAM, Thyl, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, anti-CD147, anti-DPPA5, anti-GRP78, anti-CD66c, anti-VISTA, anti-LRRC5, anti-LRRC15 antibody or any combination thereof or a fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F(ab') fragment, a F( 2 Fragments, Fv fragments, nanobodies, VHHs, ligand peptides and single-chain Fv (ScFv) fragments, or fragments of any of the foregoing, or molecules having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0151] In one embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the extracellular antigen binding domain further comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, an anti-GD-2 ScFv antigen binding domain, an anti-NY-ESo-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0152] In one embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the extracellular antigen binding domain alternatively comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESo-1TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0153] In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the extracellular antigen binding domain further comprises an immunoglobulin variable heavy chain (VH) only anti-CD19 antigen binding domain, an anti-CD20 VH antigen binding domain, an anti-CD22 VH antigen binding domain, an anti-CD33 VH antigen binding domain, an anti-CD38 VH antigen binding domain, an anti-CD123 (IL3RA) VH antigen binding domain, an anti-CD138 VH antigen binding domain, an anti-GPC2 VH antigen binding domain, an anti-GPC3 VH antigen binding domain, an anti-FGFR4 VH antigen binding domain, an anti-c-Met VH antigen binding domain, an anti-PMSA VH antigen binding domain, an anti-glycolipid F77 VH antigen binding domain, an anti-EGFRvIII VH antigen binding domain, an anti-GD-2 VH antigen binding domain, an anti-NY-ESO-1 TCR VH antigen binding domain, an anti-MAGE A3 TCR VH antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0154] In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the extracellular antigen binding domain alternatively comprises only an immunoglobulin variable heavy chain (VH) anti-CD19 antigen binding domain, an anti-CD20 VH antigen binding domain, an anti-CD22 VH antigen binding domain, an anti-CD33 VH antigen binding domain, an anti-CD38 VH antigen binding domain, an anti-CD123 (IL3RA) VH antigen binding domain, an anti-CD138 VH antigen binding domain, an anti-GPC2 VH antigen binding domain, an anti-GPC3 VH antigen binding domain, an anti-FGFR4 VH antigen binding domain, an anti-c-Met VH antigen binding domain, an anti-PMSA VH antigen binding domain, an anti-glycolipid F77 VH antigen binding domain, an anti-EGFRvIII VH antigen binding domain, an anti-GD-2 VH antigen binding domain, an anti-NY-ESO-1 TCR VH antigen binding domain, an anti-MAGE A3 TCR VH antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0155] In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the extracellular antigen binding domain further comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising: an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSA P antigen binding domain, an anti-glycolipid F77 P antigen binding domain, anti-EGFRvIIIP antigen binding domain, anti-GD-2P antigen binding domain, anti-NY-ESO-1TCR P antigen binding domain, anti-MAGE A3 TCR P antigen binding domain, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof. In another embodiment, a single, tandem, DuoCAR or multi-target CAR (with or without one or more strengthening elements) is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0156] In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the extracellular antigen binding domain alternatively comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising: an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSA P antigen binding domain, an anti-glycolipid F77 P antigen binding domain, anti-EGFRvIIIP antigen binding domain, anti-GD-2P antigen binding domain, anti-NY-ESO-1TCR P antigen binding domain, anti-MAGE A3 TCR P antigen binding domain, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof. In another embodiment, a single, tandem, DuoCAR or multi-target CAR (with or without one or more strengthening elements) is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0157] In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or a combination thereof.

[0158] In one embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 151. In one embodiment, the nucleic acid sequence encodes a boosted CAR comprising the amino acid sequence of SEQ ID NO:152.

[0159] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 153. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:154.

[0160] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 155. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:156.

[0161] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 157. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:158.

[0162] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 159. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:160.

[0163] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 161. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:162.

[0164] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 163. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 164.

[0165] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 165. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 166.

[0166] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 167. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:168.

[0167] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 179. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:180.

[0168] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 181. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 182.

[0169] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 183. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 184.

[0170] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 185. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 186.

[0171] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 187. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:188.

[0172] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 189. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:190.

[0173] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 191. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 192.

[0174] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 193. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 194.

[0175] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 195. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 196.

[0176] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 197. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:198.

[0177] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 226. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 225.

[0178] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 228. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 227.

[0179] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 230. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 229.

[0180] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 232. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 231.

[0181] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 234. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 233.

[0182] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 236. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 235.

[0183] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 238. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 237.

[0184] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 240. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 239.

[0185] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 242. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 241.

[0186] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 244. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 243.

[0187] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 245. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 246.

[0188] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 247. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 248.

[0189] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 249. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 250.

[0190] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 251. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 252.

[0191] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 253. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 254.

[0192] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 255. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 256.

[0193] In one aspect, a single, tandem, DuoCAR or multi-targeting CAR disclosed herein (with or without one or more booster elements) is modified to express or comprise a detectable marker for diagnosis, monitoring and / or prediction of treatment outcome (e.g., progression-free survival of a cancer patient) or for monitoring the progress of such treatment.

[0194] In one embodiment, the nucleic acid molecule encoding the disclosed single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) may be included in a vector, such as a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

[0195] In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0196] In another embodiment, the vector expressing single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) may also be modified to include one or more manipulators to control the expression of single, tandem, DuoCAR or multi-targeted CAR T cells (with or without one or more reinforcing elements) or eliminate single, tandem, DuoCAR or multi-targeted CAR T cells (with or without one or more reinforcing elements) cells by suicide switch. The suicide switch may include, for example, a drug that induces apoptosis-inducing signaling cascades or induces cell death. In a preferred embodiment, the vector expressing single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) may also be modified to express enzymes, such as thymidine kinase (thymidine kinase, TK) or cytosine deaminase (cytosine deaminase, CD).

[0197] In another aspect, a host cell comprising a nucleic acid molecule encoding a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) is also provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.

[0198] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a single, tandem or multi-targeted chimeric antigen receptor (CAR) construct, wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one extracellular antigen binding domain comprises an MSLN and / or ROR1 antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150, wherein the T cell is a T cell of a person suffering from cancer. The cancer particularly includes hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma or Hodgkin lymphoma) or multiple myeloma, or a combination thereof.

[0199] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population and a pharmaceutically acceptable excipient is provided, wherein the T cell comprises a nucleic acid sequence encoding a single, tandem or multi-targeted boosted chimeric antigen receptor (CAR) construct, wherein the boosted CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, followed by one or more 2A sequences, which are in frame with one or more armed molecules, one or more extracellular matrix enzymes, one or more chemokine receptors, one or more matrix targeting molecules, one or more tumor microenvironment (TME) digestion elements, one or more switch tag elements, one or more chemotaxis receptors, one or more chemotactic molecule secretions, one or more switches and / or one or more cytokines, or any combination thereof, wherein the at least one extracellular antigen binding domain comprises a sequence selected from SEQ ID NO: MSLN and / or ROR1 antigen binding domains of the amino acid sequences of 144, 146, 148 and 150, wherein the enhanced CAR is used to genetically modify one or more human T cell lymphocyte populations, wherein the T cells are T cells of people with cancer. Cancers particularly include hematological cancers, such as leukemias (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myeloid leukemia (CML), lymphomas (e.g., mantle cell lymphoma, non-Hodgkin lymphoma or Hodgkin lymphoma) or multiple myeloma, or a combination thereof.

[0200] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR (with or without one or more enhancer elements) comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0201] In another embodiment, a pharmaceutical composition is provided wherein the human cancer comprises adult carcinoma, including: oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

[0202] In another embodiment, a pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells from a human suffering from cancer is provided, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancers include hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, smoldering multiple myeloma (SMM), monoclonal gammopathy of undetermined significance (MGUS), adult and pediatric hematological malignancies (including acute lymphoblastic leukemia (ALL), CLL (chronic lymphocytic leukemia)), non-Hodgkin lymphoma (NHL) including follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML), minimal residual disease in multiple myeloma (MM), and leukemia. disease, MRD), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0203] In another aspect, a method for preparing a T cell (hereinafter referred to as "CAR-T cell") comprising a single, tandem, DuoCAR or multi-targeted CAR construct (with or without one or more enhancer elements) is provided. The method includes transducing T cells with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds to MSLN and / or ROR1, thereby preparing a CAR-T cell.

[0204] In another aspect, a method for producing an RNA-engineered cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding a disclosed single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements) into the cells of a subject, thereby producing single, tandem, DuoCAR or multi-targeted CAR cells (with or without one or more enhancer elements).

[0205] In another aspect, a method for diagnosing a disease, disorder or condition associated with expression of MLSN and / or ROR1 on a cell is provided, comprising: a) contacting the cell with a human anti-MLSN and / or ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150; and b) detecting the presence of MSLN and / or ROR1, wherein the presence of MSLN and / or ROR1 is diagnostic of a disease, disorder or condition associated with expression of MSLN and / or ROR1.

[0206] In one embodiment, the disease, disorder or condition associated with MSLN and / or ROR1 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute myeloid leukemia (AML), adult B-cell malignancies including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), childhood B-cell malignancies including B-lineage ALL (acute lymphocytic leukemia), multiple myeloma, minimal residual disease (MRD) in acute lymphocytic leukemia (ALL), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0207] In another embodiment, a method for diagnosing, prognosing or determining the risk of an MSLN and / or ROR1-related disease in a mammal is provided, which comprises detecting the expression of MSLN or ROR1 in a sample derived from the mammal, comprising: a) contacting the sample with a human anti-MSLN or anti-ROR1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150; and b) detecting the presence of MSLN and / or ROR1, wherein the presence of MSLN and / or ROR1 is diagnostic of an MSLN and / or ROR1-related disease in the mammal.

[0208] In another embodiment, a method of inhibiting MSLN and / or ROR1-dependent T cell suppression is provided, comprising contacting a cell with a human anti-MSLN and / or ROR1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150. In one embodiment, the cell is selected from a tumor cell expressing MSLN and / or ROR1, a tumor-associated macrophage, and any combination thereof.

[0209] In another embodiment, a method for blocking T cell suppression mediated by cells expressing MSLN and / or ROR1 and altering the tumor microenvironment to inhibit tumor growth in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-MSLN and / or anti-ROR1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150. In one embodiment, the cell is selected from a tumor cell expressing MSLN and / or ROR1, a tumor-associated macrophage, and any combination thereof.

[0210] In another embodiment, a method of inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-MSLN and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from a tumor cell expressing MSLN and / or ROR1, a tumor-associated macrophage, and any combination thereof.

[0211] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed single, tandem or multi-targeting CAR (with or without one or more enhancer elements).

[0212] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering to a mammal one or more of the disclosed single, tandem or multi-targeted CARs (with or without one or more reinforcing sub-elements) in an amount effective for treating or preventing cancer in a mammal. The method includes administering to a subject a therapeutically effective amount of a host cell expressing a disclosed single, tandem or multi-targeted CAR (with or without one or more reinforcing sub-elements) under conditions sufficient to form an immune complex of an antigen binding domain on a single, tandem or multi-targeted CAR (with or without one or more reinforcing sub-elements) and an extracellular domain of MSLN and / or ROR1 and / or one or more of the above antigens, wherein the CAR specifically binds to MSLN and / or ROR1 and / or one or more of the above antigens.

[0213] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem or multi-targeted chimeric antigen receptor (CAR) (with or without one or more enhancer elements), wherein the single, tandem or multi-targeted CAR (with or without one or more enhancer elements) comprises at least one extracellular MSLN and / or ROR1 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the at least one extracellular MSLN and / or ROR1 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 144, 146, 148 and 150 or any combination thereof, and wherein the T cells are T cells of a subject suffering from cancer.

[0214] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cell comprises a nucleic acid sequence encoding a single, tandem or multi-targeting chimeric antigen receptor (CAR) (with or without one or more enhancer elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more enhancer elements) comprises at least one MSLN and / or ROR1 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the at least one MSLN and / or ROR1 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 144, 146, 148 and 150 or any combination thereof, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises a transmembrane of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0215] In another embodiment, a method for treating a mammal suffering from an autoimmune, alloimmune, or autoinvasive disease, disorder, or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeting chimeric antigen receptor (CAR) (with or without one or more enhancer elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more enhancer elements) comprises at least one extracellular MSLN and / or ROR1 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the at least one extracellular MSLN and / or ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 144, 146, 148 and 150, or any combination thereof, and wherein the T cells are T cells of a subject suffering from an autoimmune, alloimmune, or autoinvasive disease, disorder, or condition.

[0216] In another embodiment, a method for treating an autoimmune disease, an alloimmune disease, or an autoinvasive disease in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem or multi-targeting chimeric antigen receptor (CAR) (with or without one or more enhancer elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more enhancer elements) comprises at least one MSLN and / or ROR1 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the at least one MSLN and / or ROR1 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 144, 146, 148 and 150, or any combination thereof, wherein the T cells are T cells of a subject suffering from an autoimmune, alloimmune or autoinvasive disease, disorder or condition. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises a transmembrane of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0217] In another embodiment, a method for treating a mammal suffering from an autoimmune, alloimmune or autoinvasive disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem or multi-targeting chimeric antigen receptor (CAR) (with or without one or more enhancer elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more enhancer elements) comprises at least one extracellular MSLN and / or ROR1 antigen binding domain comprising the following amino acid sequence:

[0218] SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256,

[0219] or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cell is a T cell of a subject having an autoimmune, alloimmune or autoinvasive disease, disorder or condition.

[0220] In another embodiment, a method for treating an autoimmune disease, an alloimmune disease, or an autoinvasive disease in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem or multi-targeting chimeric antigen receptor (CAR) (with or without one or more enhancer elements), wherein the single, tandem or multi-targeting CAR (with or without one or more enhancer elements) comprises at least one MSLN and / or ROR1 antigen binding domain comprising the following amino acid sequence:

[0221] SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cell is a T cell of a subject having an autoimmune, alloimmune or autoinvasive disease, disorder or condition. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises a transmembrane of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0222] For each of the various aspects and embodiments of the method for treating an autoimmune disease, an alloimmune disease, or an autoinvasive disease in a subject in need thereof, the single, tandem, multi-targeting, DuoCAR (with or without one or more enhancer elements) CAR constructs specifically contemplated above and / or below, the nucleotide sequences encoding any of the aforementioned functional CARs (with or without one or more enhancer elements) mentioned above and / or below, can be used to treat an autoimmune, alloimmune or autoinvasive disease, disorder or condition.

[0223] For each of the various aspects and embodiments of the method of treating an autoimmune disease, an alloimmune disease, or an autoinvasive disease in a subject in need thereof, the single, tandem, multi-targeting, DuoCAR (with or without one or more enhancer elements) CAR constructs specifically contemplated above and / or below, the amino acid sequences encoding any of the aforementioned functional CARs (with or without one or more enhancer elements) mentioned above and / or below, can be used to treat an autoimmune, alloimmune or autoinvasive disease, disorder or condition.

[0224] For the various aspects and embodiments of the methods described herein for treating an autoimmune disease, an alloimmune disease, or an autoinvasive disease, some illustrative, non-limiting examples of autoimmune diseases include chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, Goodpasture's, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, pemphigus vulgaris, Graves' disease, autoimmune hemolytic anemia, hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purpura, neuromyelitis optica, leukopenia ... optica), Evan's syndrome, IgM-mediated neuropathy, cyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticaria, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasia; while some illustrative non-limiting examples of alloimmune diseases include allosensitization from hematopoietic or solid organ transplantation, blood transfusion, pregnancy with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn (see, e.g., Blazar et al., 2015, Am. J. Transplant., 15(4):931-41) or xenosensitization, sensitization to foreign antigens, such as may occur under alternative regimens for treating genetic or acquired deficiency disorders with enzyme or protein replacement therapy, blood products, and gene therapy. The antigen binding domains having specificity for the ligands on B cells, plasma cells or plasmablasts can be used for treating the methods of autoimmune diseases, alloimmune diseases or autoinvasive diseases described herein. For example, the CAR construct may include an antigen binding domain, which has specificity for CD19, CD20, CD22, CD138, BCMA, CD319, CD10, CD24, CD27, CD38 or CD45R, but is not limited thereto. In addition, the CAR construct may include an antigen binding domain, which has specificity for autoimmune specific antigens, but is not limited thereto.Autoimmune specific antigens include, for example, antigens that cause systemic lupus erythematosus (SLE), Graves' disease, celiac disease, type 1 diabetes, rheumatoid arthritis (RA), sarcoidosis, Sjögren's syndrome, polymyositis (PM) and dermatomyositis (DM), mucocutaneous pemphigus vulgaris, myasthenia gravis. See, for example, Ellebrecht et al., 2016, Science, 353: 179-84.

[0225] In another embodiment, a method for producing a persistent genetically modified T cell population in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express a single, tandem or multi-targeted CAR (with or without one or more enhancer elements), wherein the single, tandem or multi-targeted CAR (with or without one or more enhancer elements) comprises at least one MSLN and / or ROR1 antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein at least one MSLN and / or ROR1 antigen binding domain comprises SEQ ID NO: 144, 146, 148 and 150 amino acid sequences or any combination thereof, wherein after administration, the persistent genetically modified T cell population or the progeny population of the T cell lasts in the person for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years or three years.

[0226] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0227] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders or conditions associated with elevated tumor antigen expression can be treated or prevented or improved using one or more of the single, tandem or multi-targeting CARs disclosed herein (with or without one or more enhancer elements).

[0228] In another aspect, a kit for preparing a chimeric antigen receptor T cell as described above or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression in a subject as described above is provided, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells or compositions disclosed above or any combination thereof, and instructions for using the kit.

[0229] In one aspect of the present invention, an immunotherapeutic composition is provided, which comprises a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), which can be used to transduce autologous lymphocytes to produce active patient-specific anti-tumor lymphocyte cell populations, which can be directly infused back into the patient in a patient-specific manner to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0230] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and at least one enhancer element, wherein the at least one extracellular antigen binding domain comprises an anti-ROR1 and / or anti-MSLN antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 144, 146, 148 and 150, and the at least one enhancer element comprises one or more armed molecules (TGFβRIIdn, truncated PD-1 (decoy), PD-1 dominant negative (PD-1dn), synthetic PD-1 activating receptor, truncated CTLA-4, truncated Tim-3, truncated TIGIT, TIGIT neutralizing antibody, TIGIT intracellular antibody, TIGIT shRNA), one or more extracellular matrix enzymes (ECM), one or more chemokine receptors (CXCL8, CCL2), one or more matrix targeting molecules (FAP, LRRC15, CD276 / B7-H3, TEM7, TEM8, TEM1), one or more TME digestion elements (heparanase (HPSE), MMP (MMP-1, MMP-2, MMP-9, MMP-12, MMP-13) and hyaluronidase 1, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4, PH-20 and hyaluronoglucosaminidase pseudogene 1 (HYALP1), tissue inhibitors of metalloproteinases ( metalloproteinase, TIMP) (TIMP-1, TIMP-2, TIMP-3, TIMP-4), hyaluronidase), one or more switches (tag, kill switch, on switch, off switch, adapter switch, truncated EGF receptor, truncated CD19, truncated CD20, CD20 mimotope, truncated CD34, truncated LNGF receptor), chimeric costimulatory receptor (CCR) and / or one or more cytokines (membrane-bound or soluble IL-2, IL-4 , IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ (each of the foregoing may or may not have an FC (crystallizable antibody fragment) element)), or a combination of membrane-bound receptors and tethered cytokine ligands (mbIL15, mbIL7, mbIL-21), innate system inducing ligands (TLR ligands, LPS, bacterial products), or any combination thereof, wherein the T cell is a T cell of a person suffering from cancer.Cancers particularly include hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myeloid leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0231] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0232] It should be understood that single, tandem, DuoCAR or multi-targeted CAR (with or without one or more enhancer elements), host cells, nucleic acids and methods are available outside the specific aspects and embodiments described in detail herein. From the detailed description below with reference to the accompanying drawings, the foregoing features and advantages of the present disclosure will become more apparent.

[0233] In one aspect of the above invention, the DuoCAR disclosed herein (with or without one or more enhancers) comprises at least two vectors, at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain and at least one intracellular domain, each vector encoding a functional CAR (with or without one or more enhancers), so that the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector herein is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs.

[0234] In certain aspects of the enhanced CAR of the present invention, an immunotherapy composition is provided, which comprises one or more separated nucleic acid molecules encoding at least two vectors, each vector encoding a functional DuoCAR (with or without one or more reinforcing sub-elements), so that the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs, and the immunotherapy composition can be used to transduce autologous lymphocytes to produce active patient-specific anti-tumor lymphocyte cell populations, which can be directly infused back into the patient in a patient-specific manner to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof. Provided herein are new adoptive immunotherapy compositions comprising lymphocytes transduced by two or more vectors, and methods for using the compositions in patient-specific combined immunotherapy that can be used to treat cancer and other diseases and conditions.

[0235] Therefore, in one aspect, provided herein are lentiviral vectors expressing Duo chimeric antigen receptors (DuoCAR) (with or without one or more enhancer elements), and nucleic acid molecules encoding lentiviral vectors expressing DuoCAR (with or without one or more enhancer elements). Also provided are methods of using disclosed lentiviral vectors expressing DuoCAR (with or without one or more enhancer elements), host cells, and nucleic acid molecules, for example, for treating cancer in a subject.

[0236] In one aspect, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCAR) (with or without one or more enhancer elements), each vector encoding a functional CAR (with or without one or more enhancer elements), wherein at least one binding domain in one vector is different, and thus the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs.

[0237] In one embodiment, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least three vectors (TrioCAR) (with or without one or more enhancer elements), each vector encoding a functional CAR (with or without one or more enhancer elements), whereby the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs.

[0238] In one embodiment, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least four vectors (QuatroCAR) (with or without one or more enhancer elements), each vector encoding a functional CAR (with or without one or more enhancer elements), whereby the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0239] In another embodiment, an immunotherapeutic composition is provided, comprising one or more isolated nucleic acid molecules encoding at least two, three, four, five, six, seven, eight, nine or ten vectors (e.g., "nCARs") (with or without one or more enhancer elements), each vector encoding a functional CAR (with or without one or more enhancer elements), such that the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs, wherein each unique member of the nCAR group, when assembled into a CAR product, constitutes a unique CAR composition referred to herein as "nCAR" (with or without one or more enhancer elements) (e.g., DuoCAR, TrioCAR, QuatroCAR, PentaCAR, HexaCAR, HeptaCAR, OctaCAR, NonaCAR, DecaCAR, etc.).

[0240] In another aspect, DuoCAR (with or without one or more boosters) is used to enhance the immune response to the tumor mediated by the therapeutic T cell population. The immune response is enhanced in a variety of ways.

[0241] First, DuoCAR is able to achieve multi-targeting of tumor cells, reduce the risk of tumor antigen escape, and can effectively eliminate antigen heterogeneous tumors. This feature is particularly important in targeting solid tumors, which often show antigen heterogeneity and antigen loss. Table 1 below illustrates CARs with dual targeting capabilities for solid tumor antigens mesothelin and ROR1.

[0242] In addition, the DuoCAR format allows the introduction of multiple costimulatory domains in the CAR structure, so that a stronger overall stimulation can be provided for CAR T cell effector function, differentiation and memory formation and persistence. For example, the same CAR T cell can benefit from the CD28 stimulation required for effective CAR T cell activation, amplification and cytokine production, as well as the 4-1BB stimulation that prolongs CART cell survival and persistence in patients. Each DuoCAR chain can be a second-generation or third-generation DuoCAR, and one or two costimulatory domains can be incorporated. By providing a third T cell activation sequence on a separate carrier CAR construct (with or without one or more enhancers), the inventors can regain the advantages of expressing two or more targeting domains, improving costimulation and enhancer payloads without causing the disadvantage of reduced expression (i.e., CAR% levels) of CAR on the surface of T cells.

[0243] In a second aspect, the DuoCAR of the present invention (with or without one or more boosters) can target cell types other than tumors that mediate immunosuppression. For example, if immunosuppressive cells expressing one of the targeted antigens are present in tumor lesions and also suppress anti-tumor immunity by producing IL-10 or other mediators, the second benefit of using a tumor-specific T cell population expressing DuoCAR (with or without one or more boosters) is that the immunosuppressive cell population is also removed.

[0244] For example, if there are immunosuppressive B cells within a solid tumor lesion, these cells can be eliminated by using a B cell-specific DuoCAR (e.g., a CD19-specific DuoCAR, with or without one or more boosters). If there are immunosuppressive fibroblast-like cells, these cells can be removed by a matrix-specific DuoCAR (with or without one or more boosters), such as by targeting fibroblast activating protein α (FAP). If malformed vasculature is the cause of the lack of an effective immune response, a DuoCAR specific for these types of vascular or lymphatic vessel-specific targets (e.g., anti-VEGFR) can also improve treatment outcomes.

[0245] In a third aspect, the DuoCAR of the present invention (with or without one or more enhancers) targets an immunosuppressive population distal to the tumor (i.e., present in another compartment in the body). For example, DuoCAR (with or without one or more enhancers) is used to target myeloid-derived suppressor cells (MDSC), which may be present in the tumor lesion itself or in regional lymph nodes or bone marrow. It is well established that tumor-draining lymph nodes can be sites of immune activation or immunosuppression. This depends on the overall inflammatory condition of the lymph nodes and the differentiation of distal dendritic cells before migrating to the lymph nodes. If the tumor-draining lymph nodes are filled with myeloid-derived suppressor cells (MDSC) or poorly differentiated antigen-presenting cells (e.g., dendritic cells), DuoCAR (with or without one or more enhancers) targeting these cell types distal to the tumor itself can also improve treatment outcomes. In addition to cancer-specific DuoCAR (with or without one or more enhancers) immunotherapy applications, the second application of DuoCAR (with or without one or more enhancers) will be to prevent or treat autoimmune diseases, alloimmune diseases, autoinvasive and / or inflammatory diseases. The difference from oncology-based applications is that T regulatory cells (Treg) or induced T regulatory cells (iTreg) or other cells cultured under conditions that promote Th-2-like immune responses will be cell substrates. For oncology applications, Th-1-like cells are cell substrates. In various therapeutic applications, such as graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT), allergic airway, intestinal or other mucosal inflammation or skin allergy, the presence of CAR-modified lymphocytes that produce immunosuppressive cytokines (e.g., transforming growth factor β (TFGβ)) can be used to apply a wide range of tolerogenic signals that improve autoimmunity, alloimmunity, autoinvasiveness or inflammation-driven diseases.This method includes neuroinflammatory disorders of the peripheral or central nervous system (CNS), such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, Parkinson's disease, and CTE (chronic traumatic encephalopathy due to repeated concussion or microconcussion), or connective tissue diseases, such as rheumatoid arthritis, scleroderma, granulomatosis with polyangiitis, Churg-Strauss syndrome, lupus, microscopic polyangiitis, polymyositis / dermatomyositis, Marfan syndrome, or acquired epidermolysis bullosa. This method also includes progressive scarring diseases, such as COPD (chronic obstructive pulmonary disease) or fibrotic diseases of the lungs, heart, kidneys, or liver. For example, systemic scleroderma is a progressive rare disease that causes fibrosis not only in the skin, but also in systemic tissues including the heart, lungs, and kidneys.

[0246] In the treatment of inflammatory diseases, by producing a DuoCAR (with or without one or more enhancers) expression vector specific for tissue antigens, distress markers on the surface of inflammatory cells, or misfolded proteins (e.g., tau protein or amyloid beta), lymphocytes specific for these targets can be produced. Single antibody-based treatments for Alzheimer's disease are already in clinical development (i.e., Solanezumab of Eli Lilly and Company and Aducanumab of Biogen, Inc.). In Alzheimer's disease, antibodies against monomeric or aggregated amyloid beta can be used in the form of CAR (with or without one or more enhancers) to replace the conjugate of cell surface proteins. The conjugate of tau protein or tau peptide bound by MHC molecules can also be used as a binding motif for CAR (with or without one or more enhancers). Receptors that mediate the homing of lymphocytes to specific peripheral tissues may also be included in the form of CAR (with or without one or more enhancers) to produce regional specificity for Treg groups expressing CAR (with or without one or more enhancers). Adhesion receptor domains that are known to drive lymphocyte infiltration into specific tissues, as well as cytokine sequences or cytokine or chemokine receptors or conjugates can be used as part of the CAR (with or without one or more enhancers) domain. Adhesion molecules (such as CD44 and integrin α-4) are known to target lymphocytes to the CNS, so domains including adhesion molecules from known mediating CNS migration behavior of lymphocyte groups can also be used to target lymphocytes expressing CAR (with or without one or more enhancers) to disease areas. This also applies to the intestine (i.e., a conjugate with MAdCAm-1, expression of CCR9, or anti-CCL25, etc.), lungs (i.e., P-selectin or mesothelin), skin (i.e., a conjugate with E-selectin), or other mucosal surfaces.

[0247] In order to use this method, patients with inflammatory conditions or their diseases that can be treated by relieving inflammatory pathological conditions (such as Alzheimer's disease) will be sent to the clinic and harvest peripheral blood.Treg cells can be directly selected by immunomagnetic beads (regulatory T cell separation kit, Miltenyi Biotec), or induced by culturing in an appropriate cytokine environment.Then, these Treg or iTreg are transduced with DuoCAR (with or without one or more enhancers) vectors, and if necessary, amplified in vitro (Treg amplification kit, Miltenyi Biotec).DuoCAR (with or without one or more enhancers) binding domains will be derived from antibodies or receptors mediating tissue-specific homing and disease-related conjugates (such as anti-β-amyloid protein).The modified immune effector cells thus produced will target appropriate sites, and produce cytokines consistent with their Th2 or Treg differentiation patterns.It is also known that CAR-T cells can be modified to secrete specific genetic payloads after CAR receptor (with or without one or more enhancers) activation. In addition to the DuoCAR (with or without one or more enhancers) payload expressed from the vector, additional therapeutic proteins or peptides may be expressed or secreted by the engineered T cell population to control the expression of the CAR, such as: i) one or more A-βDP (amyloid β degrading protease), ii) one or more matrix proteases (e.g., MMP-9 and MMP9), iii) one or more peptides or soluble antibody-like binders that interfere with plaque formation, iv) one or more cytokines (e.g., TGF-β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21), v) one or more arming elements to overcome immunosuppression in the TME, vi) one or more digestive enzymes to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and enable the CAR T tumor penetration, vii) one or more pro-inflammatory immune activators, and viii) one or more on switches or off switches, or any combination thereof, wherein the enhanced CAR achieves high surface expression on transduced T cells, multi-targeting activity to overcome antigen escape, high cell lysis and in vivo expansion and persistence of transduced T cells in a patient-specific manner to promote in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, regression, elimination, remission of cancer or autoimmune disease, alloimmune disease or autoinvasive disease, or prevention or improvement of the recurrence of cancer or autoimmune disease, alloimmune disease or autoinvasive disease, or a combination thereof.With respect to one or more enhancer elements of a maternal single, tandem, DuoCAR or multi-targeting CAR, the functional enhancer element portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of one or more enhancer elements of a maternal single, tandem, DuoCAR or multi-targeting CAR.

[0248] MiRNA can also be expressed in cells to regulate T cell function. Some examples of miRNA are miR-92a, miR-21, miR-155, miR-146a, miR-3162, miR-1202, miR-1246 and miR-4281, miR-142, miR-17-92. ShRNAs targeting miRNAs can also be developed. Some examples are shRNAs targeting miR-28, miR-150, and miR-107, which usually bind to PD1 and increase its expression.

[0249] In addition to applications based on oncology and based on inflammatory diseases and autoimmune diseases, alloimmune diseases or auto-invasive diseases, the third application of DuoCAR (with or without one or more boosters) technology is to generate therapeutic lymphocyte populations specific for viral, bacterial or fungal antigens. Therefore, for oncological applications described for B cell malignancies, targeting infectious diseases will allow DuoCAR (with or without one or more boosters) products to mediate immune protection or immunotherapeutic activity against infectious agents or diseased tissues where they are located based on recognition of microbial antigens. Unlike T cell receptor (TCR)-based methods in which the T cell receptor itself mediates recognition of pathogen-encoded peptides, the DuoCAR (with or without one or more boosters) method will utilize binding proteins expressed in the form of CAR (with or without one or more boosters) vectors, which provide antibody-like recognition (i.e., no antigen processing is required) for transduced T cell populations. Activation of the therapeutic T cell population will result in immune activation sites capable of eliminating infected cells and, if the microbial antigen is not cell associated, release of soluble mediators such as interferon gamma that will enable an effective immune response against the infectious agent.

[0250] For example, it is known that HIV is highly variable, but specific branches (clade) or families can still be classified, and antibodies against branch-specific viral envelope proteins (env, gp120) are produced. Using DuoCAR (with or without one or more enhancers) method, three or more branch-specific antibody-like conjugates are included in CAR (with or without one or more enhancers) constructs, resulting in extensive anti-HIV immune activity. In addition to viral proteins, bacterial proteins can also be targeted. Current medical challenges are to process antibiotic-resistant bacterial strains that usually occur in medical and health care sites. These include VRE (vancomycin-resistant enterococci), MRSA (methicillin-resistant staphylococcus aureus), KPC (Klebsiella pneumoniae carbapenemase producing gram-negative bacteria, also referred to as CRKP) etc. Klebsiella cell surface antigens include O antigens (9 variants) and K antigens (about 80 variants). The O antigen spectrum can be easily covered with a small DuoCAR (with or without one or more enhancers) library, as can many K antigens. For use, a CAR construct (with or without one or more enhancers) will be produced, characterized by antibodies bound to different K or O serotypes, and these CAR vectors (with or without one or more enhancers) are used to transduce Th1-like effector cell populations separated and activated as for oncology applications. In fungal diseases, the work of L. Cooper et al. (Kumasesan, PR, 2014, PNAS USA, 111: 10660) shows that the fungal binding protein dectin-1, which is usually expressed on human cells, can be reconstructed into CAR (with or without one or more enhancers) and used to control fungal growth in vitro. The human disease Aspergillosis occurs in severely immunosuppressed individuals and is caused by the fungus Aspergillus fumigatus (A. fumigatus). Multiple groups have generated monoclonal antibodies specific for components of Aspergillus cell surface antigens, opening the door to adoptive immunotherapy with DuoCARs (with or without one or more boosters) targeting three or more Aspergillus antigens on the fungal surface. Thus, in all of these infectious disease applications, the ability to generate immunoglobulin-like binders to microbial antigens allows multiple antigens to be targeted by effector lymphocyte populations expressing CARs (with or without one or more boosters).

[0251] Next is a detailed description of the DuoCAR (with or without one or more enhancers) disclosed herein that can be used for patient-specific autologous anti-tumor lymphocyte cell populations, including descriptions of its extracellular domain, transmembrane domain, and intracellular domain, as well as DuoCAR (with or without one or more enhancers), antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors and host cells, treatment methods, compositions, and kits using the disclosed DuoCAR (with or without one or more enhancers). Although the compositions and methods of the present invention have been exemplified with reference to the production and use of DuoCAR (with or without one or more enhancers), it is contemplated herein that the compositions and methods are particularly intended to include the production and use of TrioCAR (with or without one or more enhancers) and QuatroCAR (with or without one or more enhancers).

[0252] In one embodiment, an immunotherapeutic composition is provided, comprising: (a) at least two vectors, each comprising a nucleic acid sequence that is functional in a cell; (b) wherein each vector encodes a functional CAR (with or without one or more enhancer elements); (c) wherein each CAR (with or without one or more enhancer elements) comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) wherein at least one binding domain in one of the vectors is different; and (e) wherein in each of the vectors at least one binding domain, a single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked, wherein the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0253] In another embodiment, an immunotherapeutic composition is provided, comprising: (a) at least two vectors, each comprising a nucleic acid sequence that is functional in a cell; (b) wherein each vector encodes a functional CAR (with or without one or more enhancer elements); (c) wherein each CAR (with or without one or more enhancer elements) comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) wherein at least one binding domain in each vector is different; (e) wherein at least one signaling motif combination is different between each vector; and (f) wherein in each of said vectors at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif are covalently linked, wherein a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0254] In another embodiment, an immunotherapeutic composition is provided, wherein the linker or spacer domain of the CAR (with or without one or more enhancer elements) is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19 or CD28 and is connected to the transmembrane domain.

[0255] In another embodiment, an immunotherapeutic composition is provided, wherein the CAR (with or without one or more enhancer elements) further comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, FcεR, or any combination thereof.

[0256] In another embodiment, an immunotherapeutic composition is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

[0257] In another embodiment, an immunotherapeutic composition is provided, wherein at least one co-stimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), PD-1, GITR, CTLA-4, or any combination thereof.

[0258] In another embodiment, an immunotherapeutic composition is provided in which a single vector encoding all chimeric antigen receptors is used (e.g., retrovirus, adenovirus, SV40, herpes virus vector, POX vector, RNA, plasmid, cosmid, or any viral vector or non-viral vector) which is combined with a CRISPR system for integration.

[0259] In another embodiment, an immunotherapeutic composition is provided wherein each vector is an RNA vector or a DNA vector, alone or in combination with a transfection agent or a method of delivering RNA or DNA into cells (one non-limiting example is electroporation).

[0260] In another embodiment, an immunotherapeutic composition is provided wherein at least one vector expresses a nucleic acid molecule that modulates expression of a nucleic acid in a cell.

[0261] In another embodiment, an immunotherapeutic composition is provided wherein the nucleic acid molecule inhibits or deletes the expression of an endogenous gene.

[0262] In certain embodiments, an immunotherapeutic composition is provided, wherein an active patient-specific autologous anti-tumor lymphocyte cell population is generated within one day, two days, three days, four days, five days, seven days, ten days, twelve days, fourteen days, twenty-one days, or one month of lymphocyte harvest or tumor biopsy, and wherein the active patient-specific autologous anti-tumor lymphocyte cell population can be infused back into a patient suffering from cancer and is capable of promoting the in vivo expansion and persistence of the patient-specific anti-tumor lymphocytes in a patient-specific manner, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0263] In one aspect, provided herein are isolated nucleic acid molecules encoding the above-described chimeric antigen receptors (including the DuoCAR described above).

[0264] In one aspect, CAR (with or without one or more reinforcing sub-elements) is used for the patient-specific autologous lymphocyte population of the immunotherapy composition of the present invention, and CAR (with or without one or more reinforcing sub-elements) is modified to express or include a detectable marker for diagnosis, monitoring and / or prediction of treatment outcomes (e.g., progression-free survival of cancer patients) or for monitoring the progress of such treatment. In one embodiment of CAR (with or without one or more reinforcing sub-elements) for patient-specific autologous anti-tumor lymphocyte cell populations, a nucleic acid molecule encoding the disclosed CAR (with or without one or more reinforcing sub-elements) may be included in a vector, such as a virus or non-viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector, or a retroviral vector, or a combination thereof.

[0265] In certain embodiments of CARs for patient-specific autologous anti-tumor lymphocyte cell populations (with or without one or more enhancer elements), two or more lentiviral vectors are pseudotyped with different viral glycoproteins (GP), including, for example but not limited to, amphotropic murine leukemia virus [MLV-A], baboon endogenous virus (BaEV), GP164, gibbon ape leukemia virus [GALV], RD114, feline endogenous virus retrovirus-derived GP, and vesicularstomatitis virus [VSV], measles virus, fowl plague virus [FPV], Ebola virus [EboV], lymphocytic choriomeningitis virus [LYM], and 1ymphocytic choriomeningitis virus [LYM]. choriomeningitisvirus, LCMV]) GP of non-retroviral origin, and chimeric variants thereof, including, for example, but not limited to, chimeric GP encoding the extracellular domain and transmembrane domain of GALV or RD114 GP fused to the cytoplasmic tail region (designated TR) of MLV-A GP.

[0266] In certain embodiments of the CAR for a patient-specific autologous anti-tumor lymphocyte cell population (with or without one or more enhancer elements), the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0267] In another embodiment of a CAR (with or without one or more reinforcing sub-elements) for a patient-specific autologous anti-tumor lymphocyte cell population, the vector expressing CAR (with or without one or more reinforcing sub-elements) may also be modified to include one or more operating elements to control the expression of CAR T cells, or to eliminate CAR-T cells by means of a suicide switch. The suicide switch may include, for example, a drug that induces apoptosis-inducing signaling cascades or induces cell death. In a preferred embodiment, the vector expressing CAR (with or without one or more reinforcing sub-elements) may also be modified to express an enzyme, such as thymidine kinase (TK) or cytosine deaminase (CD).

[0268] In another aspect of the CAR (with or without one or more reinforcing sub-elements) for patient-specific autologous anti-tumor lymphocyte cell populations, a host cell comprising a nucleic acid molecule encoding CAR (with or without one or more reinforcing sub-elements) is also provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell. In one embodiment, the host cell is a CD4+ T cell. In one embodiment, the host cell is a selected CD4+ and CD8+ lymphocyte purified directly from a patient product, without considering the ratio. In another embodiment, the number of CD4+ and CD8+ T cells in the product is specific. In another embodiment, a specific subset of T cells as identified by phenotypic markers is used, which includes T initial cells (T T cell (Tn), T effector memory cell (Tem), T central memory cell (Tcm), T regulatory cell (Treg), induced T regulatory cell (iTreg), T suppressor cell (Ts), T stem cell memory cell (Tscm), natural killer (NK) cells, invariant natural killer T (iNKT) cells and lymphokine activated killer (LAK) cells.

[0269] In one embodiment, the constant natural killer T cells used herein are small αβT lymphocyte groups that are highly conserved from mice to humans. iNKT cells are believed to play an important role in regulating many diseases including cancer, infection, allergy and autoimmunity. When stimulated, iNKT cells rapidly release a large amount of effector cytokines such as IFN-γ and IL-4, not only as a cell group but also at the single cell level. These cytokines then activate a variety of immune effector cells, such as natural killer (NK) cells and dendritic cells (DC) of the innate immune system, and CD4 auxiliary and CD8 cytotoxic conventional αβT cells of the adaptive immune system of activated DC. Due to its unique activation mechanism, iNKT cells can attack a variety of diseases without relying on antigen and MHC restrictions, which makes it an attractive universal therapeutic agent. It is worth noting that due to the ability of effector NK cells and conventional αβT cells to specifically recognize diseased tissue cells, the immune response induced by iNKT cells leads to limited off-target side effects.

[0270] In one aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population and a pharmaceutically acceptable excipient is provided, wherein the human T cell comprises a novel single, tandem or multi-targeting CAR construct or any combination thereof, wherein the CAR construct comprises a CAR molecule followed by one or more 2A sequences, which are in frame with one or more armed molecules, one or more extracellular matrix enzymes, one or more chemokine receptors, one or more matrix targeting molecules, one or more tumor microenvironment (TME) digestion elements, one or more switch tag elements, one or more chemotaxis receptors, one or more chemotactic molecule secretions, one or more switches, and / or one or more cytokines or any combination thereof, wherein the enhanced CAR is used to genetically modify one or more human T cell lymphocyte populations.

[0271] In another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of an immunotherapeutic composition, the immunotherapeutic composition comprising a population of patient-specific autologous anti-tumor lymphocyte cells of a human suffering from cancer, wherein the cells of the population comprise cells containing: a nucleic acid molecule encoding at least two vectors, each vector encoding a functional CAR (with or without one or more enhancer elements), whereby the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs.

[0272] In another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of an immunotherapeutic composition, the immunotherapeutic composition comprising a population of patient-specific autologous anti-tumor lymphocyte cell populations of a human suffering from cancer, wherein the cells of the population comprise cells containing: (a) a nucleic acid molecule encoding two or more vectors; (b) wherein each vector encodes a functional CAR (with or without one or more enhancer elements); (c) wherein each CAR (with or without one or more enhancer elements) comprises at least one binding domain, at least one transmembrane domain, at least one linker domain and at least one intracellular signaling motif; (d) wherein at least one binding domain in one of the vectors is different; and (e) wherein in each of the vectors at least one binding domain, a single transmembrane domain, at least one linker domain and at least one intracellular signaling motif are covalently linked, wherein the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0273] In another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of an immunotherapeutic composition, the immunotherapeutic composition comprising a population of patient-specific autologous anti-tumor lymphocyte cell populations of a human suffering from cancer, wherein the cells of the population comprise cells containing: (a) a nucleic acid molecule encoding two or more vectors; (b) wherein each vector encodes a functional CAR (with or without one or more enhancer elements); (c) wherein each CAR (with or without one or more enhancer elements) comprises at least one binding domain, at least one transmembrane domain, at least one linker domain and at least one intracellular signaling motif; (d) wherein at least one binding domain in each vector is different; (e) wherein at least one signaling motif combination is different between each vector; and (f) wherein in each of the vectors at least one binding domain, a single transmembrane domain, at least one linker domain and at least one intracellular signaling motif are covalently linked, wherein a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0274] In one embodiment, cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents.Cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) in trace residual disease (MRD), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.In another embodiment, cancer includes hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or any combination thereof.

[0275] In another embodiment, cancers include adult epithelial cancers, including oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinomas), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

[0276] In another aspect, a pharmaceutical composition is provided, comprising an autologous lymphocyte cell population transduced with two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCAR) (with or without one or more enhancer elements), thereby producing a patient-specific autologous anti-tumor lymphocyte cell population that is capable of promoting the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0277] In another aspect, a pharmaceutical composition is provided, comprising an autologous T cell population transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCAR) (with or without one or more enhancer elements) to produce a patient-specific autologous anti-tumor lymphocyte cell population that can promote the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0278] In another aspect, a method for preparing active patient-specific autologous anti-tumor lymphocytes containing Duo (with or without one or more enhancer elements) CAR is provided. The method includes transducing lymphocytes with nucleic acid molecules encoding two or more chimeric antigen receptors (DuoCAR) (with or without one or more enhancer elements) that specifically bind to antigens or two or more vectors, thereby preparing active patient-specific autologous anti-tumor lymphocytes containing DuoCAR.

[0279] In another aspect, a method for producing an RNA-modified lymphocyte cell population is provided, which comprises introducing in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding two or more chimeric antigen receptors (DuoCAR) (with or without one or more enhancer elements) into a subject's cell population, thereby producing a patient-specific autologous anti-tumor lymphocyte cell population that can promote the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0280] In another aspect, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of an autologous lymphocyte cell population, wherein the autologous lymphocyte cell population is transduced with one or more lentiviral vectors encoding a single or multiple chimeric antigen receptor (DuoCAR) (with or without one or more enhancer elements), thereby producing a patient-specific autologous anti-tumor lymphocyte cell population that can promote the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, resulting in tumor stabilization, regression, elimination, cancer remission, or prevention or improvement of cancer recurrence, or a combination thereof.

[0281] In another aspect, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of an autologous lymphocyte cell population, wherein the autologous lymphocyte cell population is transduced with two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCAR) (with or without one or more enhancer elements) to produce a patient-specific autologous anti-tumor lymphocyte cell population, which can be directly infused back into the patient to promote the in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, resulting in tumor stabilization, regression, elimination, or cancer remission, or prevention or improvement of cancer recurrence, or any combination thereof.

[0282] In one embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising at least two vectors and a pharmaceutically acceptable excipient, each vector encoding a functional CAR (DuoCAR) (with or without one or more enhancer elements), whereby the combination of vectors results in the expression of two or more different binding domains, wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more different intracellular signaling motifs, wherein the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0283] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising: (a) a nucleic acid molecule encoding two or more vectors; (b) wherein each vector encodes a functional DuoCAR (with or without one or more enhancer elements); (c) wherein each CAR (with or without one or more enhancer elements) comprises at least one binding domain, at least one transmembrane domain and at least one intracellular signaling motif; (d) wherein at least one binding domain in one vector is different; and (e) wherein in each of the vectors at least one binding domain, a single transmembrane domain and at least one intracellular signaling motif are covalently linked, wherein the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0284] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising: (a) a nucleic acid molecule encoding two or more vectors; (b) wherein each vector encodes a functional DuoCAR (with or without one or more enhancer elements); (c) wherein each CAR (with or without one or more enhancer elements) comprises at least one binding domain, at least one transmembrane domain and at least one intracellular signaling motif; (d) wherein at least one binding domain in each vector is different; (e) wherein at least one signaling motif combination is different between each vector; and (f) wherein at least one binding domain, a single transmembrane domain and at least one intracellular signaling motif are covalently linked in each of the vectors, wherein the combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0285] In certain embodiments, the genetically modified lymphocytes are autologous T cell lymphocytes, and wherein the autologous or allogeneic T cell lymphocytes are infused directly back into the patient in order to prevent or ameliorate recurrence of the malignant disease.

[0286] In certain additional embodiments, the genetically modified lymphocytes are autologous T cell lymphocytes, and wherein the autologous lymphocytes are infused directly back into the patient to promote in vivo expansion, persistence of patient-specific anti-tumor T cell lymphocytes in a patient-specific manner, resulting in tumor stabilization, regression, elimination, or cancer remission, or prevention or amelioration of cancer recurrence, or any combination thereof.

[0287] In another embodiment, T cells have been preselected by expressing specific activation or memory-associated surface markers.

[0288] In another embodiment, the T cells are derived from a hematopoietic stem cell donor, and wherein the process is performed in the context of hematopoietic stem cell transplantation.

[0289] In certain embodiments, methods are provided wherein lymphocytes have been preselected by expression of specific activation or memory-associated surface markers.

[0290] In certain embodiments, provided herein are methods wherein the lymphocytes are T cells and are derived from a hematopoietic stem cell donor, and wherein the process is performed in the context of hematopoietic stem cell transplantation.

[0291] In another aspect, a method for generating a population of a persistent genetically engineered patient-specific autologous anti-tumor lymphocyte cell population in a human diagnosed with cancer is provided. In one embodiment, the method comprises administering to a human patient in need thereof one or more patient-specific autologous anti-tumor lymphocyte cell populations described herein, wherein after administration, the population of the persistent patient-specific autologous anti-tumor lymphocyte cell population or the population of the progeny of the lymphocytes persists in the human for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, or three years.

[0292] In one embodiment, the progeny lymphocytes in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0293] In all aspects and embodiments of the methods described herein, any of the above-mentioned cancers, diseases, disorders or conditions associated with elevated tumor antigen expression can be treated, prevented or ameliorated using a patient-specific autologous anti-tumor lymphocyte cell population comprising one or more DuoCAR (with or without one or more enhancer elements) immunotherapeutic compositions disclosed herein.

[0294] In another aspect, a kit is provided for preparing a DuoCAR immunotherapy composition comprising a patient-specific autologous anti-tumor lymphocyte cell population as described above (with or without one or more enhancer elements), or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression in a subject as described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells or compositions disclosed above or any combination thereof, and instructions for using the kit.

[0295] Although the compositions and methods of the present invention have been exemplified with reference to the generation and use of DuoCAR (with or without one or more enhancer elements), it is contemplated herein that the compositions and methods are specifically intended to include the generation and use of TrioCAR and QuatroCAR (with or without one or more enhancer elements).

[0296] In another aspect, an immunotherapeutic composition comprises one or more isolated nucleic acids encoding at least one vector, wherein the vector comprises a nucleic acid sequence that produces at least one messenger RNA (i.e., a polycistronic nucleic acid or a nucleic acid that produces more than one transcript) encoding a DuoCAR (with or without one or more enhancer elements), resulting in the ability to bind to two or more different antigen targets, thereby generating multi-antigen specificity present in a single cell expressing the vector.

[0297] In another aspect, an immunotherapeutic composition comprises an isolated nucleic acid encoding one or more of at least two vectors as described above, wherein each vector also encodes a functional tag or anti-tag binding portion (AT-CAR) (with or without one or more enhancer elements) that reconstitutes a functional chimeric antigen receptor following co-incubation or co-administration of a soluble binder (e.g., a labeled scFv or an scFv linked to an anti-tag binder), whereby the combination of the two vectors results in the ability to bind to two or more different antigen binding domains, resulting in multiple antigen specificities present in cells expressing both vectors.

[0298] In another aspect, an immunotherapeutic composition comprises one or more isolated nucleic acids encoding at least two vectors as described above, wherein each vector encodes a functional tag or anti-tag binding portion (AT-CAR) (with or without one or more enhancer elements) that reconstitutes a functional chimeric antigen receptor after co-incubation or co-administration of a soluble binder (e.g., a labeled scFv or an scFv linked to an anti-tag binder), wherein each vector expresses a unique tag (or anti-tag) that can bind to a soluble protein or protein modification structure, resulting in multi-antigen specificity, or wherein each vector expresses a unique tag (or anti-tag) that binds only to one soluble binding domain, resulting in specific linkage of the intracellular signaling motif of the encoded AT-CAR (with or without one or more enhancer elements) to the antigen binding domain of the labeled (or anti-tag) binder.

[0299] In a non-limiting embodiment for making a DuoCAR vector (with or without one or more reinforcing sub-elements), each of the compositions and methods disclosed in the above-mentioned embodiments and aspects, two vectors can be prepared separately and then added to T cells sequentially or simultaneously. In another non-limiting embodiment, the plasmid DNA of two or more vectors can be combined before or during the transfection of the production cell, or integrated into the production cell genome, to produce a mixture of viral vectors containing multiple DuoCAR (with or without one or more reinforcing sub-elements) vector particles, which are then used for transduction and genetic modification of patient T cells.

[0300] In each of the foregoing aspects and embodiments described above, for example, as disclosed in U.S. Pat. No. 10,183,993, entitled Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy, assigned to Lentigen Technology, Inc., with event number LEN_017, an scFv binder for mesothelin has been produced, which is a scFv antigen with a nucleotide sequence of SEQ ID NO: 149 and an amino acid sequence of SEQ ID NO: 150, respectively, which can be incorporated into a functional CAR with a nucleotide sequence of SEQ ID NO: 39 and an amino acid sequence of SEQ ID NO: 40, respectively, and thus can be incorporated into DuoCAR therapy.

[0301] In each of the foregoing aspects and embodiments described above, in addition to the scFv sequence, a single-chain antigen binder (as opposed to scFv) can be incorporated into a single, tandem, DuoCAR or multi-targeted CAR application. For example, as disclosed in U.S. Pat. No. 10,426,797 entitled Compositions and Methods For Treating Cancer With Anti-CD33 Immunotherapy and assigned to Lentigen Technology, Inc., event number LEN_018, the CD33-specific heavy chain-only binder, which is the nucleotide sequence SEQ ID NO: 41 and the amino acid sequence SEQ ID NO: 42, respectively, can be incorporated into the functional CARLTG1906 of the nucleotide sequence SEQ ID NO: 43 and the amino acid sequence SEQ ID NO: 44, respectively, to target CD33-expressing malignancies.

[0302] In each of the aforementioned aspects and embodiments described above, an example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is the treatment of leukemias expressing CD19, CD20 and TSLPR antigens. In this case, LTG1496 or LTG1497 (SEQ ID NO: 35, 26, respectively) can be combined with a TSLPR-specific CAR (LTG1789) (SEQ ID NO: 47 and amino acid sequence SEQ ID NO: 48, respectively), which has been generated from a TSLPR-specific scFV domain of nucleotide sequence SEQ ID NO: 45 and amino acid sequence SEQ ID NO: 46.

[0303] In each of the aforementioned aspects and embodiments described above, another example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is to treat cancer expressing CD38 antigen. For example, as the applicant has authorized the title Compositions and Methods For Treating Cancer With Anti-CD38 Immunotherapy filed on November 30, 2018 and transferred to Lentigen Technology, Inc., the CD38-specific binder disclosed in the event number LEN_026 of U.S. Patent No. 11,103,533 can be incorporated into one or more functional CARs, targeting CD38 malignancies, as disclosed in the applicant's authorized U.S. Patent No. 11,103,533, which is incorporated herein by reference in its entirety.

[0304] In each of the aforementioned aspects and embodiments described above, another example of single, tandem, DuoCAR or multi-targeted CAR therapeutic applications is to treat cancer expressing CD123 antigens. For example, as the applicant has authorized the title Compositions and Methods For Treating Cancer With Anti-CD123Immunotherapy submitted on September 20, 2019 and transferred to Lentigen Technology, Inc. event number LEN_024 and requiring the provisional patent application No. 62 / 734,106 filed on September 20, 2018 The U.S. Patent No. 10,844,128 disclosed in priority CD123 specific binding substances can be incorporated into one or more functional CARs, targeting CD123 malignancies, as disclosed in the U.S. Patent No. 10,844,128 authorized by the applicant, which is incorporated herein by reference as a whole.

[0305] In each of the aforementioned aspects and embodiments described above, another example of single, tandem, DuoCAR or multi-targeted CAR therapeutic applications is to treat cancer expressing CD123 antigens. For example, as the applicant's titled Compositions and Methods For Treating Cancer With Anti-CD123 Immunotherapy submitted on March 2, 2022 and transferred to Lentigen Technology, Inc., the co-pending U.S. patent application No. 17 / 685,132 disclosed in the event number MBG_99 CD123 specific binders can be incorporated into one or more functional CARs, targeting CD123 malignancies, such as the applicant's co-pending U.S. patent application No. 17 / 685,132 disclosed, which is incorporated herein by reference as a whole.

[0306] In each of the aforementioned aspects and embodiments described above, another example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is the treatment of cancers expressing BCMA antigens. For example, a BCMA-specific binder disclosed in U.S. Patent No. 11,052,112 entitled Fully Human BCMA CAR T Cells for the Treatment of Multiple Myeloma and Other BCMA-Positive Malignancies and assigned to Lentigen Technology, Inc., event number MBG_13, filed on May 30, 2019, as authorized by the applicant, can be incorporated into one or more functional CARs, targeting malignancies expressing BCMA, as disclosed in U.S. Patent No. 11,052,112, which is authorized by the applicant, and is incorporated herein by reference in its entirety.

[0307] In each of the aforementioned aspects and embodiments described above, some examples of the tandem CAR (comprising two scFv domains, as described in nucleotide sequence SEQ ID: 23 and amino acid sequence SEQ ID: 24) on which the technology is based include CD20_CD19 CAR LTG1497, which is a nucleotide sequence SEQ ID NO: 25 and an amino acid sequence SEQ ID NO: 26. In some cases, reversing the order of the two binders can provide better DuoCAR expression in target cells. Therefore, LTG1497, in which the CD19 scFv is closer to the membrane as shown in nucleotide sequence SEQ ID NO: 25 and amino acid sequence SEQ ID NO: 26, and LTG1496, in which the CD19 scFV is further away from the membrane as shown in nucleotide sequence SEQ ID NO: 33 and amino acid sequence SEQ ID NO: 34, can both be used as one of the members of the DuoSet containing DuoCAR.

[0308] In each of the aforementioned aspects and embodiments described above, one or more of the above-identified novel enhanced chimeric antigen receptors (CARs) provided above in each of SEQ ID NOs: 23, 24, 25, 26, 33, 34, 35, 41, 42, 43, 44, 45, 46, 47 and 48 in the above-mentioned co-pending patent applications or issued patents of the applicant may comprise single, tandem or multi-targeting CAR constructs (including those in the form of DuoCAR), or any combination thereof.

[0309] In each of the foregoing aspects and embodiments described above, applicant's co-pending patent applications and / or issued patents demonstrate one or more additional features of the DuoCAR constructs, including, for example, i) despite the reduced MFI of the larger payload construct, the multi-targeting of the DuoCAR format is superior in tumor cell killing compared to single CAR targeting; ii) compared to the unboosted mesothelin CAR, the mesothelin-boosted CAR with mbIL7 exhibits superior antigen-dependent target cell killing; iii) in addition, compared to the unboosted CAR counterpart, the mIL7-boosted DuoCAR and tandem CARs exhibited superior targeted killing; iv) additionally, mIL7-enhanced DuoCARs and tandem CARs exhibited superior cytokine elaboration in response to tumor antigens, greater long-term persistence and expansion in the absence of cytokines, and better protection of effector function; v) mesothelin CARs armed with TGFBRIIdn exhibited robust transduction and expansion in culture, and robust killing of tumor lines expressing high, medium, or low levels of mesothelin, despite armed payload; and / or vi) mesothelin and ROR1 CARs with HPSE enhancers efficiently digested ECM in a transwell migration assay, and / or any combination thereof.

[0310] A. Chimeric Antigen Receptor (as present in single, tandem, DuoCAR, multi-targeted CAR with or without one or more boosters)

[0311] CAR is an artificially constructed hybrid protein or polypeptide that contains an antigen binding domain (e.g., a single-chain variable fragment (scFv)) of an antibody connected to a T cell signaling domain via a transmembrane domain. The characteristics of DuoCAR include its ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC restricted manner, as well as the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives T cells expressing DuoCAR the ability to recognize antigens independently of antigen processing, thus bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, DuoCAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0312] As disclosed herein, the intracellular T cell signaling domain of DuoCAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a CAR comprising an intracellular domain of a T cell receptor (e.g., such as, but not limited to, the intracellular portion of a CD3ζ protein). A co-stimulatory signaling domain refers to a portion of a CAR comprising an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an effective response of lymphocytes to an antigen. In some cases, the activation domain can be weakened by a mutation of an ITAM motif in a specific site of phosphorylation, i.e., a CD3ζ chain, thereby carefully regulating the extent of signal transduction mediated by the domain.

[0313] 1. Extracellular domain

[0314] In one embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) for a patient-specific autologous anti-tumor lymphocyte cell population as disclosed herein comprises a targeting specific binding element, which is also referred to as an antigen binding domain or portion. The selection of the domain depends on the type and number of ligands that define the surface of the target cell. For example, an antigen binding domain may be selected to identify a ligand that acts as a cell surface marker on a target cell associated with a specific disease state. Therefore, some examples of cell surface markers that may serve as ligands for antigen binding domains in single, tandem, DuoCAR or multi-targeted CARs (with or without one or more reinforcing elements) include those associated with viruses, bacteria and parasitic infections, autoimmune diseases, alloimmune diseases, autoinvasive diseases and cancer cells.

[0315] In one embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) can be transformed by transforming the desired antigen binding domain that specifically binds to the antigen on the tumor cell to target the tumor antigen of interest. Tumor antigens are proteins that cause immune responses (especially T cell-mediated immune responses) produced by tumor cells. The selection of antigen binding domains will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF-1), ... factor, IGF)-I receptor, IGF-II receptor, IGF-I receptor, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GM1, GM3, Tn, STn, sLe (animal), GlOboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM, Folate binding protein, folate receptor α (FOLR1), folate receptor β (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, p185, IL-2 receptor, interleukin 1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, αvβ3, WT1, LMP2, HPVE6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART 1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYP1B 1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 3, PAGE4, ​​VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL 1, MUC1, MUC16 / CA125, MAGE A4, MAGEC2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endothelin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPRC 5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15, or any combination thereof, or a fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the following: Fab fragment, F(ab')2 fragment, Fv fragment, nanobody, VHH, ligand peptide and single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof. The tumor antigens disclosed herein are included only by way of example. This list is not intended to be exclusive, and other examples will be apparent to those skilled in the art.

[0316] In one embodiment, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can serve as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as oncogenes HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigen (onco-fetal antigen), such as carcinoembryonic antigen (CEA). In B cell lymphoma, tumor-specific idiotypic immunoglobulins constitute the unique real tumor-specific immunoglobulin antigens of individual tumors. B cell differentiation antigens (such as CD19, CD20, CD22 and CD37) are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0317] The type of tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. Expression of an antigen on a tumor may occur under conditions that enable the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0318] Some non-limiting examples of TSAs or TAAs include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens caused by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0319] In a preferred embodiment, the antigen binding domain portion of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycosyl F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosylGMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM , folate binding protein, folate receptor α (FOLR1), folate receptor β (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, pL85, IL-2 receptor, interleukin 1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, αvβ3, WT1, LMP2, HPVE6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPCl, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYPlB 1. PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie3, PAGE4, ​​VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL 1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIVl, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endothelin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPRC 5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15 or any combination thereof or provide a fragment thereof, wherein the antibody or its fragment comprises a fragment selected from the following: Fab fragment, F(ab')2 fragment, Fv fragment, nano antibody, VHH, ligand peptide and single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any of the foregoing, or any combination thereof. In another embodiment, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) is provided herein, which comprises a Tag or anti-Tag binding domain.

[0320] Depending on the desired antigen to be targeted, single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) can be modified to include an appropriate antigen binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody or scFv subfragment thereof specific for CD19 can be used as an antigen binding domain incorporated into a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements).

[0321] In an exemplary embodiment, the antigen binding domain portion of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) targets CD19. Preferably, the antigen binding domain in a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is an anti-CD19 scFV, wherein the nucleic acid sequence of the anti-CD19 scFV comprises the sequence shown in SEQ ID NO: 27. In one embodiment, the anti-CD19 scFV comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the anti-CD19 scFV portion of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) comprises the amino acid sequence shown in SEQ ID NO: 28. In a second exemplary embodiment, the antigen binding domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) targets CD20. Preferably, the antigen binding domain in a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is an anti-CD20 scFv, wherein the nucleic acid sequence of the anti-CD20 scFv comprises the sequence shown in SEQ ID NO: 1. In another embodiment, the anti-CD20 scFV portion of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) comprises the amino acid sequence shown in SEQ ID NO: 2. In a third exemplary embodiment, the antigen binding domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) targets CD22. Preferably, the antigen binding domain in a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is an anti-CD22 scFv, wherein the nucleic acid sequence of the anti-CD22 scFv comprises the sequence shown in SEQ ID NO: 7. In another embodiment, the anti-CD22 scFV portion of a single, tandem, DuoCAR, or multi-targeting CAR (with or without one or more booster elements) comprises the amino acid sequence set forth in SEQ ID NO:8.

[0322] In one aspect of the invention, a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) capable of binding to a non-TSA or non-TAA is provided, wherein the non-TSA or non-TAA includes, for example, but not limited to, antigens derived from the following: Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus and echovirus), Rubella virus, Coronavirus, Vesicular stomatitis virus, Rabies virus, Ebola virus, Parainfluenza virus, Mumps virus, Measles virus, Respiratory syncytial virus, Influenza virus, Hepatitis B virus, Parvovirus, Adenoviridae, Herpesviridae [e.g., Herpes simplex virus type 1 and type 2 (herpes simplex virus type 2)], Herpes simplex virus type 3 (herpes simplex virus type 3), Herpes simplex virus type 4 (herpes simplex virus type 5), Herpes simplex virus type 6 (herpes simplex virus type 7), Herpes simplex virus type 8 (herpes simplex virus type 9), Herpes simplex virus type 10 (herpes simplex virus type 2), Herpes simplex virus type 2 (herpes simplex virus type 11), Herpes simplex virus type 2 (herpes simplex virus type 2), Herpes simplex virus type 3 (herpes simplex virus type 3), Herpes simplex virus type 4 (herpes simplex virus type 2), Herpes simplex virus type 3 (herpes simplex virus type 3), Herpes simplex virus type 4 (herpes simplex virus type 3), Herpes simplex virus type 5 (herpes simplex virus type 2), Herpes simplex virus virus, HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus], poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0323] In another aspect of the invention, a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more booster elements) is provided that is capable of binding to an antigen derived from the following bacterial strains: Staphylococci, Streptococcus, Escherichia coli, Pseudomonas or Salmonella. In particular, single, tandem, DuoCAR or multi-targeting CARs (with or without one or more booster elements) capable of binding to antigens derived from infectious bacteria are provided, such as Helicobacter pylori, Legionella pneumophilia, bacterial strains of the genus Mycobacteria sp. (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii or Mycobacterium gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, and Streptococcus pyogenes. A Streptococcus), Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae or Clostridium tetani, or a combination thereof.

[0324] 2. Transmembrane domain

[0325] In the single, tandem, DuoCAR, multi-targeting CAR (with or without one or more boosting elements) for patient-specific autologous anti-tumor lymphocyte populations as disclosed herein, the single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) comprises one or more transmembrane domains fused to the extracellular domain of the single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements).

[0326] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19 or CD28 and is linked to the transmembrane domain.

[0327] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of the transmembrane domain and is linked to the transmembrane domain.

[0328] In some cases, transmembrane domains may be selected or chosen by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0329] The transmembrane domain can be derived from a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. The transmembrane region particularly used in the present invention can be derived from (i.e., at least comprising its transmembrane region): α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, FcεR, or any combination thereof. Alternatively, the transmembrane domain can be synthetic, in which case it will mainly comprise hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection between the transmembrane domain and the cytoplasmic signaling domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements). Glycine-serine doublets, or triplet alanine motifs provide particularly suitable linkers.

[0330] In one embodiment, the transmembrane domain in a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 12. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 12.

[0331] In some cases, the transmembrane domain of a single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements) comprises a CD8.α. hinge domain. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence of SEQ ID NO: 13. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 14. In another embodiment, the CD8 hinge domain comprises an amino acid sequence of SEQ ID NO: 14.

[0332] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary single, tandem, DuoCAR or multi-targeted CARs (with or without one or more booster elements) used in the patient-specific autologous anti-tumor lymphocyte populations disclosed herein of the present invention are believed to include, for example, but are not limited to: a) improved lateral movement within the plasma membrane allowing more efficient signal transduction, b) superior location within the plasma membrane microdomains (e.g., lipid rafts), and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior location within the plasma membrane by preferentially moving away from inhibitory (dampening) or downregulatory interactions (e.g., lower proximity or interaction with phosphatases (e.g., CD45)), and d) superior assembly into T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0333] In one embodiment of the patient-specific autologous anti-tumor lymphocyte population as disclosed herein, non-limiting exemplary transmembrane domains for use in the single, tandem, DuoCAR, multi-targeted CARs disclosed herein (with or without one or more booster elements) include TNFRSF16 and TNFRSF19 transmembrane domains, which can be used to obtain TNFRSF transmembrane domains and / or linker or spacer domains as disclosed in U.S. Patent No. 10,421,810, entitled CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE, filed on October 9, 2015, and assigned to Lentigen Technology, Inc., with event number LEN_015PRO, granted to the applicant, particularly including those other TNFRSF members listed within the tumor necrosis factor receptor superfamily as listed in Table I.

[0334] 3. Spacer domain

[0335] In a single, tandem, DuoCAR, multi-targeted CAR (with or without one or more booster elements) for a patient-specific autologous anti-tumor lymphocyte population as disclosed herein, the spacer domain may be arranged between the extracellular domain and the TNFRSF transmembrane domain, or between the intracellular domain and the TNFRSF transmembrane domain. The spacer domain means any oligopeptide or polypeptide for connecting the TNFRSF transmembrane domain to the extracellular domain and / or the TNFRSF transmembrane domain to the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0336] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill and include those listed in the following: U.S. Patent Nos. 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,4 83, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414,

[0337] and U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0338] The spacer domain preferably has a sequence that promotes single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) to bind to the antigen and enhance signal transduction into the cell. Some examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0339] As the spacer domain, all or part of the following can be used: amino acids 137 to 206 (including the hinge region) of CD8.α (NCBI RefSeq: NP.sub.--001759.3) (SEQ ID NO: 15), amino acids 135 to 195 of CD8.β (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP.sub.--000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP.sub.--006130.1). In addition, as the spacer domain, a part of the constant region of the antibody H chain or L chain (CH1 region or CL region, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 16) can be used. In addition, the spacer domain may be an artificially synthesized sequence.

[0340] In addition, in single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements), the signal peptide sequence may be connected to the N-terminus. The signal peptide sequence is present at the N-terminus of many secreted proteins and membrane proteins and is 15 to 30 amino acids in length. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements). In one embodiment, the signal peptide comprises the nucleotide sequence of the leader (signal peptide) sequence shown in SEQ ID NO: 5. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 6.

[0341] 4. Intracellular domain

[0342] The cytoplasmic domain or other intracellular signaling domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) is responsible for activating at least one normal effector function of an immune cell into which a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and instructs the cell to perform a specialized function. Although the entire intracellular signaling domain can generally be used, the entire chain does not have to be used in many cases. In terms of using a truncated portion of an intracellular signaling domain, as long as it transduces an effector function signal, such a truncated portion can be used instead of a complete chain. Therefore, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0343] Some preferred examples of intracellular signaling domains for single, tandem, DuoCAR or multi-targeted CARs (with or without one or more booster elements) include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which function together to initiate signal transduction following antigen receptor engagement; and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0344] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0345] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0346] Some examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the single, tandem, DuoCAR, multi-targeting CARs disclosed herein (with or without one or more booster elements) include those derived from: TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3, CD5, CD22, CD79a, CD79b, and CD66d. Some specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3.ζ. (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of Fc.ε.RI.γ. (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of Fc.ε.RI.B. (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ. (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3.δ. (NCBI RefSeq: NP.sub.--000723.1), amino acids 140 to 151 of CD3.ε. (NCBI RefSeq: NP.sub.--000723.1), amino acids 141 to 153 of CD3. RefSeq: NP.sub.--000724.1), amino acids 153 to 207 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1), and amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), and variants thereof having the same functions as these peptides. The amino acid number based on the amino acid sequence information of NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor of each protein (including signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecules in single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) include cytoplasmic signaling sequences derived from CD3ζ. In another embodiment, one, two or three ITAM motifs in CD3ζ are weakened by mutation or replacement of tyrosine residues by another amino acid.

[0347] In a preferred embodiment, the intracellular domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) can be designed to contain a CD3-ζ signaling domain by itself, or it can be combined with any other desired cytoplasmic domain available in the case of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements). For example, the intracellular domain of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) may include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) comprising an intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Some examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, 276 / B7-H3, and ligands that specifically bind to CD83, etc. Some specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8.α. (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--006130.1), CD137 (4-1BB, NCBI RefSeq: NP.sub.--001552.2), amino acids 214 to 255 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and variants thereof having the same functions as these peptides.Therefore, although the disclosure herein is primarily exemplified by 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the disclosure.

[0348] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) can be interconnected in a random or specific order. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0349] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28 and 4-1BB.

[0350] In one embodiment, the intracellular domain in a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more booster elements) is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence shown in SEQ ID NO: 17, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO: 19.

[0351] In one embodiment, the intracellular domain in a single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements) is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0352] In one embodiment, the intracellular domain in a single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements) is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises the amino acid sequence shown in SEQ ID NO: 18, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO: 20.

[0353] 5. Additional description of single, tandem, DuoCAR, multi-targeted CAR (with or without one or more booster elements)

[0354] The present invention also explicitly includes within the scope of the single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements) functional parts for patient-specific autologous anti-tumor lymphocyte populations as disclosed herein.When referring to single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) use, the term "functional part" refers to any part or fragment of one or more of the single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements) disclosed herein, the part or fragment retains the biological activity of the single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) (parental single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements)) as part thereof. Functional part covers, for example, retaining the ability to recognize target cells or detect, treat or prevent diseases to a similar degree or to the same degree or to a higher degree with the parent single, tandem, DuoCAR, multi-target CAR (with or without one or more reinforcing elements) single, tandem, DuoCAR or multi-target CAR (with or without one or more reinforcing elements) parts. According to the parent single, tandem, DuoCAR or multi-target CAR (with or without one or more reinforcing elements), the functional part may include, for example, parent single, tandem, DuoCAR or multi-target CAR (with or without one or more reinforcing elements) about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more.

[0355] The functional part may include additional amino acids at the amino or carboxyl end of the part or at both ends, and the additional amino acids are not present in the amino acid sequence of the parent single, tandem, DuoCAR or multi-target CAR (with or without one or more reinforcing elements). Desirably, the additional amino acids do not interfere with the biological functions of the functional part, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent single, tandem, DuoCAR or multi-target CAR (with or without one or more reinforcing elements).

[0356] The scope of the present disclosure includes the functional variants of single, series, DuoCAR, multi-target CAR (with or without one or more reinforcing elements) disclosed herein.The term "functional variant" used herein refers to single, series, DuoCAR or multi-target CAR (with or without one or more reinforcing elements) with substantial or significant sequence identity or similarity, polypeptide or protein, and the functional variant is retained in the biological activity of its single, series, DuoCAR or multi-target CAR (with or without one or more reinforcing elements) as a variant. Functional variants cover, for example, retaining the ability to identify target cells in a similar degree, the same degree or a higher degree with the parent single, series, DuoCAR or multi-target CAR (with or without one or more reinforcing elements) described herein, single, series, DuoCAR or multi-target CAR (with or without one or more reinforcing elements) (parent single, series, DuoCAR or multi-target CAR (with or without one or more reinforcing elements)). Depending on the parent single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements), the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more identity in amino acid sequence with the parent single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements).

[0357] Functional variants may, for example, include an amino acid sequence of a parent single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) having at least one conservative amino acid substitution. As an alternative or supplement, functional variants may include an amino acid sequence of a parent single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) having at least one non-conservative amino acid substitution. In this case, it is preferred that non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of functional variants. Non-conservative amino acid substitutions can enhance the biological activity of functional variants, so that compared with parent single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements), the biological activity of functional variants is improved.

[0358] The amino acid replacement of single, tandem, DuoCAR, multi-target CAR (with or without one or more reinforcing elements) is preferably a conservative amino acid replacement. Conservative amino acid replacement is known in the art, and includes an amino acid replacement in which an amino acid with certain physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be acidic / negatively charged polar amino acids replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu), amino acids having non-polar side chains replacing another amino acid having non-polar side chains (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), basic / positively charged polar amino acids replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), uncharged amino acids having polar side chains replacing another uncharged amino acids having polar side chains (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having β-branched side chains replacing another amino acid having β-branched side chains (e.g., He, Thr and Val), amino acids having aromatic side chains replacing another amino acid having aromatic side chains (e.g., His, Phe, Trp and Tyr), etc.

[0359] A single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) may consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0360] Single, tandem, DuoCAR, multi-targeting CAR (with or without one or more boosting elements) (including functional parts and functional variants) can have any length, that is, can contain any number of amino acids, provided that single, tandem, DuoCAR, multi-targeting CAR (with or without one or more boosting elements) (or functional parts or functional variants thereof) retains its biological activity, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, a single, tandem, DuoCAR or multi-targeting CAR (with or without one or more boosting elements) can be about 50 to about 5000 amino acids in length, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0361] Single, tandem, DuoCAR, multi-target CAR (with or without one or more strengthening elements) (including functional parts and functional variants of the present invention) may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0362] Single, tandem, DuoCAR, multi-targeting CAR (with or without one or more booster elements) (including functional parts and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, a disulfide bridge) or converted into an acid addition salt and / or optionally dimerized or multimerized or conjugated.

[0363] Single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements) (including functional parts and functional variants thereof) can be obtained by methods known in the art. Single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements) can be prepared by any suitable method for preparing polypeptides or proteins. Suitable methods for synthesizing polypeptides and proteins from scratch are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Methods for generating chimeric antigen receptors, T cells comprising such receptors, and their uses (e.g., for treating cancer) are known in the art and are further described herein (see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online Feb. 23, 2010, pp. 1-9; Till et al., 2008, Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; Tumaini et al., 2014, pp. 1-13). al., Cytotherapy, 15, 1406-1417, 2013; Haso et al., (2013) Blood, 121, 1165-1174; PCT Publications WO2012 / 079000, WO2013 / 126726; and US Publication 2012 / 0213783, each of which is incorporated herein by reference in its entirety).For example, a nucleic acid molecule encoding a disclosed chimeric antigen binding receptor may be included in an expression vector (e.g., a lentiviral vector) for transducing a host cell (e.g., a T cell) to prepare a disclosed single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements). In some embodiments, the method of using a chimeric antigen receptor includes isolating T cells from a subject, transducing T cells with an expression vector (e.g., a lentiviral vector) encoding a chimeric antigen receptor, and administering to the subject T cells expressing a single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements) for treatment, for example, for treating a tumor in a subject.

[0364] 6. Description of the reinforcing element (reinforcer)

[0365] In addition to the above description provided above, the enhancer elements of single, tandem, DuoCAR, multi-targeting CARs that can be used for patient-specific autologous or allogeneic anti-tumor, anti-autoimmune, anti-allogeneic or anti-self-invasive lymphocyte populations may further comprise their functional percentage identity variants, as described below.

[0366] In a specific embodiment, also explicitly included within the scope of the present invention are functional booster element portions of single, tandem, DuoCAR, multi-targeting CARs (with or without one or more booster elements) for patient-specific autologous or allogeneic anti-tumor lymphocyte populations as disclosed herein. The boosting element encompasses, for example, additional therapeutic proteins or peptides expressed or secreted by the engineered T cell population, such as: i) one or more A-βDP (amyloid β-degrading protease), ii) one or more matrix proteases (e.g., MMP-9 and MMP9), iii) one or more peptides or soluble antibody-like conjugates that interfere with plaque formation, iv) one or more cytokines (e.g., TGF-β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21), v) one or more arming elements to overcome immunosuppression in the TME, vi) one or more digestive enzymes to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and enable CAR T tumor penetration, vii) one or more pro-inflammatory immune activators, and viii) one or more on switches or off switches, or any combination thereof, to control the expression of CAR, wherein the enhanced CAR achieves high surface expression on transduced T cells, multi-targeted activity to overcome antigen escape, high cell lysis, and in vivo expansion and persistence of transduced T cells to promote in vivo expansion and persistence of patient-specific anti-tumor T cells in a patient-specific manner, resulting in tumor stabilization, reduction, elimination, relief of cancer or autoimmune disease, alloimmune disease or self-invasive disease, or prevention or improvement of the recurrence of cancer or autoimmune disease, alloimmune disease or self-invasive disease, or a combination thereof. According to one or more enhancing elements of the parent of a single, tandem, DuoCAR or multi-targeted CAR, the functional enhancing element portion may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of one or more enhancing elements of the parent of a single, tandem, DuoCAR or multi-targeted CAR.

[0367] The functional parent one or more reinforcing elements of a single, tandem, DuoCAR or multi-targeted CAR may include additional amino acids at the amino or carboxyl terminus of the part or at both ends, and the additional amino acids are not present in the amino acid sequence of the parent one or more reinforcing elements of a single, tandem, DuoCAR or multi-targeted CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional part, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent one or more reinforcing elements of a single, tandem, DuoCAR or multi-targeted CAR.

[0368] Included within the scope of the present disclosure are functional variants of single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements) disclosed herein. The term "functional variant" used herein refers to a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) with substantial or significant sequence identity or similarity to one or more reinforcing elements of a parent of a single, tandem, DuoCAR or multi-targeted CAR, polypeptide or protein, wherein the functional variant is retained in the biological activity of a single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) as a variant thereof. Functional variants encompass, for example, those variants of single, tandem, DuoCAR or multi-targeted CAR (with or without one or more reinforcing elements) described herein (one or more reinforcing elements of a parent of a single, tandem, DuoCAR or multi-targeted CAR) that retain the ability to recognize target cells to a similar degree, the same degree or a higher degree with one or more reinforcing elements of a parent of a single, tandem, DuoCAR or multi-targeted CAR). Depending on the parent one or more boosting elements of the single, tandem, DuoCAR or multi-targeting CAR, the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more identity in amino acid sequence with the parent one or more boosting elements of the single, tandem, DuoCAR or multi-targeting CAR.

[0369] Functional variants may, for example, comprise the amino acid sequence of one or more reinforcing elements of a parent of a single, tandem, DuoCAR or multi-targeted CAR having at least one conservative amino acid substitution. As an alternative or supplement, functional variants may comprise the amino acid sequence of one or more reinforcing elements of a parent of a single, tandem, DuoCAR or multi-targeted CAR having at least one non-conservative amino acid substitution. In this case, it is preferred that non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of functional variants. Non-conservative amino acid substitutions can enhance the biological activity of functional variants, so that the biological activity of functional variants is improved compared to one or more reinforcing elements of a parent of a single, tandem, DuoCAR or multi-targeted CAR.

[0370] The amino acid replacement of one or more reinforcing elements of the parent of single, tandem, DuoCAR or multi-targeted CAR is preferably a conservative amino acid replacement. Conservative amino acid replacement is known in the art, and includes an amino acid replacement in which an amino acid with certain physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be acidic / negatively charged polar amino acids replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu), amino acids having non-polar side chains replacing another amino acid having non-polar side chains (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), basic / positively charged polar amino acids replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), uncharged amino acids having polar side chains replacing another uncharged amino acids having polar side chains (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having β-branched side chains replacing another amino acid having β-branched side chains (e.g., He, Thr and Val), amino acids having aromatic side chains replacing another amino acid having aromatic side chains (e.g., His, Phe, Trp and Tyr), etc.

[0371] The parent one or more boosting elements of a single, tandem, DuoCAR or multi-targeting CAR may consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0372] The parent one or more booster elements (including functional parts and functional variants) of single, tandem, DuoCAR or multi-targeting CAR can have any length, that is, can contain any number of amino acids, provided that the parent one or more booster elements of single, tandem, DuoCAR or multi-targeting CAR (or its functional part or functional variant) retain its biological activity, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, the parent one or more booster elements of single, tandem, DuoCAR or multi-targeting CAR can be about 50 to about 5000 amino acids in length, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0373] Single, tandem, DuoCAR or multi-target CAR parent one or more strengthening elements (including functional parts and functional variants of the present invention) may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0374] One or more of the parental enhancing elements (including functional parts and functional variants) of a single, tandem, DuoCAR or multi-targeting CAR may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, a disulfide bridge) or converted into an acid addition salt and / or optionally dimerized or multimerized or conjugated.

[0375] One or more reinforcing elements (including functional parts and functional variants thereof) of the parent of a single, tandem, DuoCAR or multi-targeted CAR can be obtained by methods known in the art. One or more reinforcing elements of the parent of a single, tandem, DuoCAR or multi-targeted CAR can be prepared by any suitable method for preparing a polypeptide or protein. Suitable methods for synthesizing polypeptides and proteins from scratch are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Methods for generating chimeric antigen receptors, T cells comprising such receptors, and their uses (e.g., for treating cancer) are known in the art and are further described herein (see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online Feb. 23, 2010, pp. 1-9; Till et al., 2008, Blood, 112:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; Tumaini et al., 2003, pp. 1-13 ... al., Cytotherapy, 15, 1406-1417, 2013; Haso et al., (2013) Blood, 121, 1165-1174; PCT Publication WO2012 / 079000, WO2013 / 126726; and U.S. Publication 2012 / 0213783, each of which is incorporated herein by reference in its entirety). For example, a nucleic acid molecule encoding the disclosed chimeric antigen binding receptor may be included in an expression vector (e.g., a lentiviral vector) for transducing a host cell (e.g., a T cell) to prepare a parental one or more booster elements of the disclosed single, tandem, DuoCAR or multi-targeted CAR.In some embodiments, the method of using a chimeric antigen receptor comprises isolating T cells from a subject, transducing the T cells with an expression vector (e.g., a lentiviral vector) encoding the chimeric antigen receptor, and administering to the subject T cells expressing one or more booster elements of the parent of a single, tandem, DuoCAR, or multi-targeting CAR for treatment, e.g., for treating a tumor in the subject.

[0376] B. Antibodies and Antigen-binding Fragments

[0377] One embodiment also provides a single, tandem, DuoCAR, multi-targeted CAR (with or without one or more strengthening elements) for a patient-specific autologous anti-tumor lymphocyte group disclosed herein, which is specifically bound to one or more antigens disclosed herein, expressing single, tandem, DuoCAR, multi-targeted CAR (with or without one or more strengthening elements) T cells, antibodies or their antigen binding domains or parts. "T cells expressing single, tandem, DuoCAR, multi-targeted CAR (with or without one or more strengthening elements)" or "single, tandem, DuoCAR, multi-targeted CAR (with or without one or more strengthening elements) T cells" used herein means expressing single, tandem, DuoCAR, multi-targeted CAR (with or without one or more strengthening elements) T cells, and having antigen specificity determined by, for example, single, tandem, DuoCAR, multi-targeted CAR (with or without one or more strengthening elements) antibody-derived targeting domains.

[0378] As used herein, "antigen binding domain" may include antibodies and antigen binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antigen binding fragments thereof, as long as they exhibit the desired antigen binding activity. Some non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for an antigen.

[0379] "Monoclonal antibody" is an antibody obtained from a group of substantially homogeneous antibodies, that is, except for naturally occurring mutations that may be present in small amounts, the individual antibodies comprising the group are identical. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. The modifier "monoclonal" represents the characteristics of an antibody such as obtained from a substantially homogeneous antibody group, and is not interpreted as requiring the production of antibodies by any particular method. In some instances, a monoclonal antibody is an antibody produced by a monoclonal clone of a B lymphocyte or a cell or its progeny transfected with nucleic acid encoding a single antibody (or its antigen-binding fragment) of an antibody light chain and a heavy chain variable region. In some instances, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have a conservative amino acid substitution that has substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, referring to Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition. Cold Spring Harbor Publications, New York (2013).

[0380] Generally speaking, immunoglobulins have heavy chains (H) and light chains (L) interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes and numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (K). There are five major heavy chain species (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE.

[0381] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6.sup.th ed., WH Freeman and Co., p. 91 (2007)). In several embodiments, the heavy and light chain variable regions combine to specifically bind to an antigen. In other embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Reference to "VH" or "VH" refers to the variable region of an antibody heavy chain, including an antigen-binding fragment, such as a variable region of an Fv, ScFv, dsFv or Fab. Reference to "VL" or "VL" refers to the variable domain of an antibody light chain, including the variable domain of an Fv, ScFv, dsFv or Fab.

[0382] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework regions of the component light and heavy chains) is used to position and align the CDRs in three-dimensional space.

[0383] The CDR is primarily responsible for binding to the epitope of the antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2 and CDR3 (from N-terminus to C-terminus), and are also usually identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is a CDR3 of the variable domain of the heavy chain of an antibody in which it is present, and a VL CDR1 is a CDR1 of the variable domain of the light chain of an antibody in which it is present. Light chain CDRs are sometimes referred to as LCDR1, LCDR2 and LCDR3. Heavy chain CDRs are sometimes referred to as LCDR1, LCDR2 and LCDR3.

[0384] "Antigen binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize a cognate antigen, as well as various combinations of such portions. Some non-limiting examples of antigen binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments produced by modifying intact antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0385] Single-chain antibodies (ScFv) are genetically engineered molecules comprising the VH and VL domains of one or more antibodies connected by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242: 423426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85: 58795883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry, ID Rugs, 13: 543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is generally not critical for ScFv. Therefore, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0386] In dsFv, the heavy and light variable chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90: 64446448, 1993; Poljak et al., Structure, 2: 11211123, 1994).

[0387] Antibodies also include genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous conjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition., WH Freeman & Co., New York, 1997.

[0388] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening a combinatorial library consisting of a variable heavy chain and a variable light chain as described in Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference. These and other methods for preparing, for example, chimeric, humanized, CDR-grafted, single-chain and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14: 243-246 (1993); Ward et al., Nature 341: 544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0389] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and vice versa, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0390] "Humanized" antibodies or antigen-binding fragments include human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) antibodies or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is referred to as a "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as an "acceptor." In one embodiment, in a humanized immunoglobulin, all CDRs are from a donor immunoglobulin. The constant region does not need to be present, but if present, it may be substantially identical to a human immunoglobulin constant region, e.g., at least about 85% to 90%, e.g., about 95% or higher identity. Therefore, except for possible CDRs, all parts of a humanized antibody or antigen-binding fragment are substantially identical to corresponding parts of a natural human antibody sequence.

[0391] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies (which are generally from different species). In some instances, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0392] "Fully human antibodies" or "human antibodies" are antibodies that contain sequences from (or derived from) the human genome, but do not contain sequences from other species. In some embodiments, human antibodies contain CDRs, framework regions, and Fc regions (if present) that are from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for producing antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st edition. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0393] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0394] Methods for testing the ability of an antibody to bind to any functional portion of a single, tandem, DuoCAR, multi-targeting CAR (with or without one or more booster elements) are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., see below, U.S. Patent Application Publication No. 2002 / 0197266 A1 and U.S. Patent No. 7,338,929).

[0395] In addition, single, tandem, DuoCAR, multi-targeting CAR (with or without one or more boosting elements), T cells expressing single, tandem, DuoCAR, multi-targeting CAR (with or without one or more boosting elements), antibodies or antigen-binding portions thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles).

[0396] C. Conjugates

[0397] Single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements) for patient-specific autologous anti-tumor lymphocyte populations disclosed herein that are specific for one or more antigens disclosed herein, T cells expressing single, tandem, DuoCAR, multi-targeted CAR (with or without one or more reinforcing elements), or monoclonal antibodies or antigen-binding fragments thereof can be conjugated to reagents such as effector molecules or detectable markers using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which there are effector molecules or detectable markers covalently attached to antibodies or antigen-binding fragments, and the antibodies or antigen-binding fragments specifically bind to one or more antigens disclosed herein. Those skilled in the art will appreciate that a variety of effector molecules and detectable markers can be used, including (but not limited to) chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g. 125 I. 32 P. 14 C. 3 H and 35 S) and other labels, target moieties and ligands, etc.

[0398] The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to cause the death of a particular target cell (e.g., a tumor cell).

[0399] The methods used to attach effector molecules or detectable markers to antibodies or antigen-binding fragments vary depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acids (COOH), free amines (-NH 2 ) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the binding of effector molecules or detectable markers. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment of any of many known linker molecules (e.g., those available from Pierce Chemical Company, Rockford, IL). The linker can be any molecule used to connect the antibody or antigen binding fragment to the effector molecule or detectable marker. The linker can form a covalent bond with both the antibody or antigen binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be connected to the constituent amino acids (e.g., connected to cysteine ​​through a disulfide bond) or to the alpha carbon amino and carboxyl groups of the terminal amino acids through its side groups.

[0400] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill in the art and include those listed in: U.S. Pat. Nos. 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635, 483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414,

[0401] and U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0402] In some embodiments, the joint is cleavable under intracellular conditions, so that the cutting of the joint in the intracellular environment releases the effector molecule or detectable marker from the antibody or Fab. In other embodiments, the joint is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the joint can be cut by a cutting agent present in the intracellular environment (for example, in a lysosome or endosome or caveolea). The joint can be, for example, a peptide joint, which is cut by an intracellular peptidase or protease (including but not limited to a lysosomal protease or an endosomal protease). In some embodiments, the peptide joint is at least two amino acids long or at least three amino acids long. However, the joint can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, 1 to 15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptide linker (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker) that is cleavable by the thiol-dependent protease cathepsin B can be used. Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0403] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661). Such linkers are relatively stable under neutral pH conditions (e.g., conditions in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0404] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987); Phillips et al., ...

Claims

1. An isolated nucleic acid molecule encoding a boosted single, tandem, multi-targeted or DuoCAR chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) comprising at least one extracellular antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, the at least one extracellular antigen binding domain comprising a ROR1, MSLN, FOLR1 and / or CD276 / B7-H3 antigen binding domain operably linked to one or more booster elements, wherein the boosted single, tandem, multi-targeted or DuoCAR CAR is encoded by a nucleotide sequence comprising: SEQ ID NO:151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,226,228,230,232,234,236,238,240,242,244,245,247,249,251,253, or 255, or any combination thereof.

2. The isolated nucleic acid molecule of claim 1, wherein the enhanced single, tandem, multi-targeted or DuoCAR CAR further comprises one or more enhancer elements, the enhancer elements comprising: i) an arming element to overcome immunosuppression in the tumor microenvironment (TME); ii) a cytokine stimulating element to promote cytokine stimulation of autonomous T cells; iii) a digestive enzyme element to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and enable CAR T tumor penetration; iv) a neutrophil activation protein (NAP); or v) an on-switch element or an off-switch element to control the expression of the CAR; or any combination thereof, and wherein the enhanced single, tandem, multi-targeted or DuoCAR CAR exhibits one or more properties in a patient-specific manner.

3. The isolated nucleic acid molecule of claim 1, wherein the enhanced single, tandem, multi-targeted or DuoCAR CAR further comprises one or more enhancer elements, wherein the enhancer elements comprise armed elements comprising: TGFβRIIdn, truncated PD-1, PD-1dn, synthetic PD-1 activating receptor, truncated CTLA-4, truncated Tim-3, truncated TIGIT or any combination thereof, so as to overcome immunosuppression in the tumor microenvironment (TME) in a patient-specific manner.

4. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multi-targeted or DuoCAR CAR further comprises one or more booster elements, wherein the booster elements comprise a cytokine stimulation element comprising: membrane-bound IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21TNFα, IFNγ, or any combination thereof, for promoting cytokine stimulation of autonomous T cells in a patient-specific manner.

5. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multi-targeted or DuoCAR CAR further comprises one or more booster elements comprising a digestive enzyme element comprising: heparinase / HPSE, MMP-1, MMP-2, MMP-9, MMP-12, MMP-13 and hyaluronidase 1, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4, PH-20 and HYALP1, or any combination thereof, for overcoming the physical barrier of the tumor stroma / extracellular matrix (ECM) in a patient-specific manner and enabling CAR T tumor penetration.

6. The isolated nucleic acid molecule of claim 1, wherein the enhanced single, tandem, multi-targeting, HPSE, MMP-2, MMP-9 and PH-20 or DuoCAR CAR further comprises one or more enhancer elements, the enhancer elements comprising an on-switch element or an off-switch element comprising: a truncated EGF receptor, a truncated TGFβ receptor (TGFβRIIdn), a truncated CD19, a truncated CD20, a CD20 mimetic epitope, a truncated CD34, a truncated LNGF receptor, a chimeric co-stimulatory receptor (CCR), or any combination thereof, for controlling the expression of the CAR in a patient-specific manner.

7. The isolated nucleic acid molecule of claim 1, wherein the enhanced single, tandem, multi-targeted or DuoCAR CAR exhibits one or more properties in a patient-specific manner, wherein the properties further comprise: i) multi-targeting to overcome antigen escape; ii) high surface expression on transduced T cells; or iii) high cell lysis and in vivo expansion and persistence of transduced T cells to promote in vivo expansion and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, regression, elimination, remission of cancer or autoimmune disease, alloimmune disease or autoinvasive disease, or prevention or improvement of recurrence of cancer or autoimmune disease, alloimmune disease or autoinvasive disease, or any combination thereof.

8. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one ROR1 and / or MSLN antigen binding domain, the at least one intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

9. The isolated nucleic acid molecule of claim 8, wherein the encoded linker or spacer domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19 or CD28 and is connected to the transmembrane domain.

10. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular ROR1 and / or MSLN antigen binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

11. The isolated nucleic acid molecule of claim 10, wherein the leading nucleotide sequence comprises a nucleotide sequence comprising: SEQ ID NO:13 encoding the leading amino acid sequence of SEQ ID NO:14, or SEQ ID NO:39 encoding the leading amino acid sequence of SEQ ID NO:40, or SEQ ID NO:41 encoding the leading amino acid sequence of SEQ ID NO:42, or SEQ ID NO:43 encoding the leading amino acid sequence of SEQ ID NO:

44.

12. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta or zeta chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154 and TNFRSF19, or any combination thereof.

13. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular antigen binding domain comprises a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73, 75 or 77, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

14. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

15. The isolated nucleic acid molecule of claim 14, wherein the encoded at least one intracellular signaling domain is arranged on the C-terminal side relative to the CD3zeta intracellular domain.

16. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, a chimeric co-stimulatory receptor (CCR), or any combination thereof.

17. The isolated nucleic acid molecule of claim 16, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137), PD-1, GITR, and CTLA-4, or any combination thereof.

18. An enhanced single, tandem, multi-targeted or DuoCAR chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.

19. The boosted single, tandem, multi-targeting or DuoCAR CAR of claim 18, comprising at least one extracellular antigen binding domain, wherein the at least one extracellular antigen binding domain comprises a ROR1, MSLN, FOLR1 and / or CD276 antigen binding domain, wherein the boosted single, tandem, multi-targeting or DuoCAR comprises the following amino acid sequence: SEQ ID NO:152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256, or any combination thereof, at least one transmembrane domain and at least one intracellular signaling domain.

20. The boosted single, tandem, multi-targeted or DuoCAR CAR of claim 19, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta or zeta chain of the T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and TNFRSF19, or any combination thereof.

21. The boosted single, tandem, multi-targeted or DuoCAR CAR of claim 20, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

28.

22. The enhanced single, tandem, multi-targeting or DuoCAR CAR of claim 19, comprising at least one extracellular antigen binding domain, the at least one extracellular antigen binding domain comprising ROR1, MSLN, FOLR1 and / or CD276 antigen binding domains, the at least one intracellular signaling domain, or both, connected to the transmembrane domain via a linker or spacer domain; wherein the enhanced single, tandem, multi-targeting or DuoCAR comprises the following amino acid sequence: SEQ ID NO:152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,98,225,227,229,231,233,235,237,239,241,243,246,248,250,252,254,or 256, or any combination thereof, at least one transmembrane domain and at least one intracellular signaling domain.

23. The enhanced single, tandem, multi-targeted or DuoCAR CAR of claim 22, wherein the linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, IgG1, IgG2, IgG3, IgG4 or CD28 and is connected to the transmembrane domain.

24. The boosted single, tandem, multi-targeted or DuoCAR CAR of claim 19, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

25. The enhanced single, tandem, multi-targeted or DuoCAR CAR of claim 24, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain containing a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137), PD-1, GITR and CTLA-4, or a combination thereof.

26. A vector comprising the nucleic acid molecule of claim 1.

27. The vector of claim 26, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

28. The vector of claim 26, further comprising a promoter.

29. The vector of claim 28, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

30. A cell comprising the vector of claim 26.

31. The cell of claim 30, wherein the cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a T regulatory cell (Treg), an induced T regulatory (iTreg) cell, a B cell, a dendritic cell (DC), a γδ T cell, a macrophage, a stem cell, or an induced pluripotent stem (iPS) cell.

32. The cell of claim 30, wherein the T cell is a CD8 + T cells or CD4 + T cells.

33. The cell of claim 30, wherein the cell is a human cell.

34. A pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population, wherein the T cell comprises a nucleic acid sequence encoding a boosted single, tandem, multi-targeted or DuoCAR chimeric antigen receptor (CAR), wherein the boosted single, tandem, multi-targeted or DuoCAR CAR comprises at least one extracellular antigen binding domain, wherein the at least one extracellular antigen binding domain comprises a ROR1, MSLN, FOLR1 and / or CD276 antigen binding domain, which comprises the following amino acid sequence: SEQ ID NO:152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,225,227,229,231,233,235,237,239,241,243,246,248,250,252,254, or 256, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cell is a T cell of a human suffering from cancer, an autoimmune disease, an alloimmune disease, or an autoinvasive disease, or any combination thereof.

35. The pharmaceutical composition of claim 34, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta or zeta chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof.

36. The pharmaceutical composition of claim 34, wherein the T cells are T cells from a human suffering from a hematological cancer.

37. The pharmaceutical composition of claim 36, wherein the hematological cancer is leukemia or lymphoma.

38. The pharmaceutical composition of claim 37, wherein the leukemia is acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic T-cell leukemia (T-ALL), or acute lymphocytic B-cell leukemia (B-ALL).

39. The pharmaceutical composition of claim 37, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma.

40. The pharmaceutical composition of claim 36, wherein the hematological cancer is multiple myeloma.

41. The pharmaceutical composition of claim 36, wherein the autoimmune disease, alloimmune disease, or autoinvasive disease is rheumatoid arthritis, lupus, celiac disease, Sjögren's syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, poststreptococcal autoimmune disease, anti-neuronal antibody-mediated neuropsychiatric disorders, immune-mediated extrapyramidal movement disorders, Sydenham chorea, alloimmune hemolytic disease, pulmonary fibrosis, systemic scleroderma, and fibrotic diseases.

42. The pharmaceutical composition of claim 34, wherein the human cancer comprises adult epithelial cancers including oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinomas), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

43. A method for preparing cells, comprising transducing T cells with the vector of claim 26.

44. A method for producing an RNA-engineered cell population comprising introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule of claim 1.

45. A method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of the cell of claim 30.

46. ​​A method for treating or preventing cancer in a mammal, comprising administering to the mammal the boosted single, tandem, multi-targeted or DuoCAR CAR of claim 19 in an amount effective to treat or prevent cancer, an autoimmune disease, an alloimmune disease, an autoinvasive disease, or any combination thereof in the mammal.

47. A method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression, the method comprising administering to a subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a boosted single, tandem, multi-targeted or DuoCAR chimeric antigen receptor (CAR), wherein the boosted single, tandem, multi-targeted or DuoCAR CAR comprises at least one extracellular antigen binding domain, the at least one extracellular antigen binding domain comprising a ROR1, MSLN, FOLR1 and / or CD276 antigen binding domain comprising the following amino acid sequence: SEQ ID NO:152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,225,227,229,231,233,235,237,239,241,243,246,248,250,252,254, or 256, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cell is a T cell of a subject suffering from cancer.

48. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a boosted single, tandem, multi-targeted or DuoCAR chimeric antigen receptor (CAR), wherein the boosted single, tandem, multi-targeted or DuoCAR CAR comprises at least one extracellular antigen binding domain, the at least one extracellular antigen binding domain comprises a ROR1, MSLN, FOLR1 and / or CD276 antigen binding domain comprising the following amino acid sequence: SEQ ID NO:152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,225,227,229,231,233,235,237,239,241,243,246,248,250,252,254,or 256, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cell is a T cell of a subject having cancer.

49. The method of claim 47 or 48, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of the T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof.

50. A method for producing cells expressing enhanced single, tandem, multi-targeted or DuoCAR chimeric antigen receptors, the method comprising introducing the isolated nucleic acid of claim 1 into the cells.

51. A method for producing cells expressing enhanced single, tandem, multi-targeted or DuoCAR chimeric antigen receptors according to claim 50, wherein the cells are T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, dendritic cells (DCs), γδT cells, macrophages, stem cells or induced pluripotent stem (iPS) cells, or a cell population comprising T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, dendritic cells (DCs), γδT cells, macrophages, stem cells or induced pluripotent stem (iPS) cells.

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