Compositions and methods for treating cancer with DuoCAR

Abstract

In the field of cancer, particularly, compositions comprising at least two vectors encoding functional chimeric antigen receptors and methods for their use in patient-specific immunotherapy are provided. [Solution] An immunotherapeutic composition is provided, comprising one or more isolated nucleic acid molecules encoding at least one multicistronic vector, wherein each multicistronic vector encodes at least one functional CAR comprising a specific amino acid sequence, and at least one binding domain in at least one of the multicistronic vectors is non-identical, whereby combination of multicistronic vectors results in expression of two or more non-identical functional CAR molecules, each functional CAR molecule encoding at least one binding domain covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. patent application Ser. No. 16 / 692,957, filed November 22, 2019, which claims priority to PCT application Ser. No. PCT / US19 / 51734, filed September 18, 2019, which in turn claims priority to U.S. patent application Ser. No. 16 / 078,269, filed August 21, 2018, which is a continuation-in-part of U.S. patent application Ser. No. 16 / 078,269, filed September 18, 2018. No. 134,735, which in turn claims the benefit of priority to PCT Application No. PCT / US17 / 49923, filed September 1, 2017, which in turn claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 382,791, filed September 2, 2016, the entire contents of each of which are incorporated herein by reference.

[0002] Field of the Disclosure This application relates to the field of cancer, and in particular to compositions comprising at least two vectors encoding functional chimeric antigen receptors, and methods of use thereof in patient-specific immunotherapy.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 19, 2020, is named SequenceListing.txt and is 366 kilobytes in size.

[0004] Background of the Invention 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, making it the second leading cause of death after cardiovascular disease, accounting for approximately one-quarter of all deaths. Solid tumors are responsible for most of these deaths. While significant advances have been made in the medical treatment of certain cancers, the 5-year overall survival rate for all cancers has only improved by about 10% over the past 20 years. Cancers, or malignant tumors, rapidly metastasize and grow uncontrolled, making treatment extremely difficult. One of the challenges in modern cancer treatment is the time that elapses between cancer biopsy and diagnosis and effective treatment of the patient. During this time, the patient's tumor may grow unhindered, resulting in the disease progressing further before treatment can be applied. This negatively impacts cancer progression and outcomes.

[0005] Chimeric antigen receptors (DuoCARs) are hybrid molecules containing three essential units: (1) an extracellular antigen-binding motif, (2) a linking / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly active CD2-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is generally based on the single-chain fragment variable (scFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2 199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable work remains to be done in defining the most active T cell populations for transduction with CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0006] The linking motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed to be an extended, flexible linker. Structural motifs, such as those derived from the constant domain of IgG, can be used to extend the scFv binding domain away from the T cell plasma membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motif used in CARs has always included the CD3-ζ chain, because this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was also used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009; 183(9):5563-74). With the advancement of new technologies, activation of T cells with beads coupled to anti-CD3 and anti-CD28 antibodies, the presence of the canonical "signal 2" derived from CD28, encoded by the CAR itself, is no longer necessary. Using bead activation, third-generation vectors were found to be no superior to second-generation vectors in in vitro assays, and no clear benefit over second-generation vectors was observed 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 in line with 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 modes (Porter DL et al. This is supported by the clinical success of CD19-specific CARs in 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 sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). The culture conditions under which the CAR T-cell population is cultured are equally important.

[0007] A current challenge in broader and more effective application of CAR therapy for cancer relates to the lack of compelling targets. While creating binders to cell surface antigens is now readily achievable, discovering cell surface antigens specific for tumors while sparing normal tissues remains a formidable challenge. One potential way to confer higher target cell specificity to CAR-expressing T cells is to use a combinatorial CAR approach. In one system, the CD3-ζ and CD28 signaling units are split between two different CAR constructs expressed in the same cell; in another system, two DuoCARs are expressed in the same T cell, but one has a lower affinity, thus requiring the alternative CAR to be bound first for full activity of the second CAR (Lanitis E et al. Cancer Immunol Res. 2013;1(1)). (Issue): 43-53; Kloss CC et al. Nat Biotechnol. 2013; 31(1): 71-5). A second challenge for generating a single scFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma in which an effector cell population simultaneously targets multiple antigens (HER2, IL-13Ra, EphA2) in hopes of avoiding the growth of target antigen-negative populations (Hegde M et al. Mol Ther. 2013; 21(11): 2087-101).

[0008] T cell-based immunotherapy is an emerging field in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells via drug-induced dimerization of an inducible caspase-9 construct with AP1903 demonstrates one means by which a powerful switch can be pharmacologically triggered to control T cell populations (Di Stasi A et al., N Engl J Med. 2011;365(18):1673-83). Creation of an effector T cell population immune to the negative regulatory effects of transforming growth factor-β by expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al., J Immunother. 2008;31(5):500-5).

[0009] Thus, although CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors, the major obstacles to the clinical application of CAR-based technologies to date have been the limited in vivo expansion of CAR+ T cells, the rapid loss of cells after infusion, disappointing clinical activity, recurrence of the underlying medical disease or condition, and the excessive time that elapses between the diagnosis and timely treatment of cancer using such CAR+ T cells.

[0010] Thus, there is an urgent and long-felt need in the art to discover compositions and methods for the treatment of cancer using CAR-based therapies that can exhibit the intended cancer-specific therapeutic properties without the drawbacks mentioned above.

[0011] The present invention addresses these needs by providing compositions comprising at least two vectors encoding functional chimeric antigen receptors, and methods for their use in patient-specific immunotherapy that can be used to treat cancer and other diseases and / or conditions.

[0012] In particular, the invention disclosed and described herein provides an immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least two vectors, each vector encoding a functional DuoCAR, whereby the combination of vectors results in the expression of two or more non-identical binding domains, each vector-encoded binding domain(s) covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and this immunotherapeutic composition is used to transduce autologous lymphocytes to generate an active, patient-specific anti-tumor lymphocyte cell population that can be infused directly back into the patient to promote in vivo expansion, and the persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0013] Summary of the Invention Provided herein are novel adoptive immunotherapy compositions comprising lymphocytes transduced with two or more vectors, and methods for their use in patient-specific combination immunotherapy that can be used to treat cancer and other diseases and conditions.

[0014] Thus, in one aspect, the present specification provides a lentiviral vector expressing Duo chimeric antigen receptor (DuoCAR), and a nucleic acid molecule encoding the lentiviral vector expressing DuoCAR. Also provided are methods of using the disclosed lentiviral vector expressing DuoCAR, host cells, and nucleic acid molecules, for example, to treat cancer in a subject.

[0015] In one aspect, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCARs), each vector encoding a functional CAR, and wherein at least one binding domain(s) in one of the vectors is / are non-identical, whereby the combination of the vectors results in expression of two or more non-identical binding domains, and wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0016] In one embodiment, an immunotherapeutic composition is provided that includes one or more isolated nucleic acid molecules encoding at least three vectors (TRioCARs), each vector encoding a functional CAR, whereby the combination of the vectors results in expression of two or more non-identical binding domains, and the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0017] In one embodiment, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least four vectors (QuatroCARs), each vector encoding a functional CAR, whereby the combination of the vectors results in expression of two or more non-identical binding domains, wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0018] In yet another embodiment, an immunotherapeutic composition is provided that includes one or more isolated nucleic acid molecules encoding at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 vectors (e.g., "nCARs"), each vector encoding a functional CAR, whereby the combination of the vectors results in expression of two or more non-identical binding domains, wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and wherein each unique number of nCARs is defined when assembled into a CAR product that constitutes a unique CAR composition referred to herein as an "nCAR" (e.g., DuoCAR, TrioCAR, QuatroCAR, PentaCAR, HexaCAR, HeptaCAR, OctaCAR, NonaCAR, and DecaCAR, etc.).

[0019] In one embodiment, an immunotherapeutic composition is provided that includes: (a) at least two vectors each comprising a nucleic acid sequence that is functional in a cell; (b) each vector encodes a functional CAR molecule; (c) each CAR is composed of at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain in one of the vectors is not identical; and (e) the at least one binding domain, the single transmembrane domain, the at least one linker domain, and the at least one intracellular signaling motif are covalently linked in each of said vectors, and the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0020] In another embodiment, an immunotherapeutic composition is provided that comprises: (a) at least two vectors each comprising a nucleic acid sequence functional in a cell; (b) each vector encodes a functional CAR molecule; (c) each CAR comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain(s) in each vector are not identical; (e) the combination of at least one signaling motif is not identical between each of the vectors; and (f) the at least one binding domain, the single transmembrane domain, and the at least one intracellular signaling motif are covalently linked in each of said vectors, and a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0021] In one embodiment, an immunotherapeutic composition is provided in which each vector encodes more than one functional CAR.

[0022] In another embodiment, an immunotherapeutic composition is provided in which the combination of one or more signaling motifs is identical in one or more vectors.

[0023] In another embodiment, an immunotherapeutic composition is provided in which one or more multimeric domains are identical in one or more vectors.

[0024] In another embodiment, an immunotherapeutic composition is provided wherein the lymphocyte population(s) comprises a mixture of autologous T cells or peripheral blood-derived lymphocytes.

[0025] In another embodiment, an immunotherapy composition is provided, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one single-chain variable fragment of an antibody that binds to an antigen.

[0026] In another embodiment, an immunotherapy composition is provided, wherein at least one extracellular antigen-binding domain of the CAR comprises at least one heavy chain variable region of an antibody that binds to an antigen.

[0027] In another embodiment, an immunotherapeutic composition is provided, wherein at least one extracellular antigen-binding domain of the CAR, at least one intracellular signaling domain of the CAR, or both, is connected to the transmembrane domain by a linker or spacer domain.

[0028] In another embodiment, an immunotherapeutic composition is provided in which the extracellular antigen-binding domain of the CAR is preceded by a leader peptide.

[0029] In another embodiment, an immunotherapeutic composition is provided in which the extracellular antigen-binding domain of the CAR targets an antigen comprising CD19, CD20, CD22, ROR1, TSLPR, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1, MAGE-A3, PRAME peptide in combination with MHC, or any combination thereof.

[0030] In another embodiment, the extracellular antigen-binding domain of the CAR is selected from the group consisting of an anti-CD19 scFV antigen-binding domain, an anti-CD20 scFV antigen-binding domain, an anti-CD22 scFV antigen-binding domain, an anti-ROR1 scFV antigen-binding domain, an anti-TSLPR scFV antigen-binding domain, an anti-mesothelin 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-BCMA (CD269) scFV antigen-binding domain, an anti-GPC2 ... 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 (including single chain TCR constructs) antigen-binding domain, an anti-MAGE-A3 TCR, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0031] In another embodiment, an immunotherapeutic composition is provided in which the linker or spacer domain of the CAR is derived from the extracellular domain of CD8 and is linked to a transmembrane domain.

[0032] In another embodiment, an immunotherapeutic composition is provided wherein the CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, Fc epsilon R, or any combination thereof.

[0033] In another embodiment, an immunotherapeutic composition is provided wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0034] In another embodiment, an immunotherapeutic composition is provided wherein at least one intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

[0035] In another embodiment, an immunotherapeutic composition is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

[0036] In another embodiment, an immunotherapeutic composition is provided wherein at least one costimulatory 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.

[0037] In another embodiment, an immunotherapeutic composition is provided in which a single vector is used to encode all of the chimeric antigen receptors (e.g., lentivirus, adenovirus, SV40, herpes vector, POX vector, RNA, plasmid, cosmid, or any viral or non-viral vector) in combination with a CRISPR system for integration.

[0038] In another embodiment, an immunotherapeutic composition is provided in which each vector is an RNA vector or a DNA vector, alone or in combination with a transfection reagent or a method for delivering RNA or DNA to cells, a non-limiting example of which is electroporation.

[0039] In another embodiment, an immunotherapeutic composition is provided in which at least one vector expresses a nucleic acid molecule that regulates expression of a nucleic acid in a cell.

[0040] In another embodiment, an immunotherapeutic composition is provided in which the nucleic acid molecule inhibits or deletes expression of an endogenous gene.

[0041] In certain embodiments, immunotherapeutic compositions are provided in which an active, patient-specific, autologous anti-tumor lymphocyte cell population is generated within 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 21 days, or 1 month of lymphocyte collection or tumor biopsy, and the active, patient-specific, autologous anti-tumor lymphocyte cell population can be infused back into a patient afflicted with cancer and promoted in vivo proliferation, wherein persistence of the patient-specific anti-tumor lymphocyte cell population results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0042] In one aspect, provided herein is an isolated nucleic acid molecule encoding the chimeric antigen receptor described above.

[0043] In one aspect of the DuoCAR used in the patient-specific autologous lymphocyte population(s) of the immunotherapy composition of the present invention, the DuoCAR is modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcomes such as progression-free survival in cancer patients, or for monitoring the progress of such treatment. In one embodiment of the DuoCAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s), the nucleic acid molecule encoding the disclosed DuoCAR can be contained in a vector, such as a viral vector or a 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 lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0044] In certain embodiments of DuoCARs used in patient-specific autologous anti-tumor lymphocyte cell population(s), two or more lentiviral vectors are pseudotyped with different viral glycoproteins (GPs), including, for example, but not limited to, GPs derived from amphotropic murine leukemia virus (MLV-A), baboon endogenous virus (BaEV), GP164, gibbon ape leukemia virus (GALV), RD114, feline endogenous retrovirus, and GPs derived from non-retroviruses (vesicular stomatitis virus (VSV), measles virus, fowl plague virus (FPV), Ebola virus (EboV), lymphocytic choriomeningitis virus (LCMV)), as well as chimeric variants thereof, including, for example, but not limited to, chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GP fused to the cytoplasmic tail (termed TR) of MLV-A GP.

[0045] In certain embodiments of DuoCARs used in patient-specific autologous anti-tumor lymphocyte cell population(s), the vector further comprises a promoter that is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0046] In yet another embodiment of DuoCAR for use in patient-specific autologous anti-tumor lymphocyte cell population(s), the vector expressing the CAR can be further modified to include one or more operable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. The suicide switch can include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR can be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0047] In another embodiment of DuoCAR for use in the patient-specific autologous anti-tumor lymphocyte cell population(s), host cells containing nucleic acid molecule(s) encoding DuoCAR are also provided. In some embodiments, the host cells are T cells, e.g., primary T cells obtained from a subject. In one embodiment, the host cells are CD8 + T cells. In one embodiment, the host cells are CD4+ T cells. In one embodiment, the host cells are selected CD4+ and CD8+ lymphocytes purified directly from the patient's product without regard to balance. In another embodiment, the number of CD4+ and CD8+ T cells in the product is specific. In another embodiment, specific subsets of T cells are utilized, identified by phenotypic markers, including naive T cells (Tn), effector memory T cells (Tem), central memory T cells (Tcm), regulatory T cells (TReg), induced regulatory T cells (iTreg), suppressor T cells (Ts), stem cell memory T cells (Tscm), natural killer (NK) cells, and lymphokine-activated killer (LAK) cells.

[0048] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of an immunotherapeutic composition comprising a population of patient-specific autologous anti-tumor lymphocyte cell population(s) of a human with cancer, wherein the cells of the population comprise cells comprising nucleic acid molecules encoding at least two vectors, each vector encoding a functional CAR, whereby the combination of the vectors results in expression of two or more non-identical binding domains, and wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0049] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of an immunotherapeutic composition comprising a population of patient-specific autologous anti-tumor lymphocyte cell population(s) of a human with cancer, wherein the cells of the population (a) comprise cells comprising nucleic acid molecules encoding two or more vectors; (b) each vector encodes a functional CAR; (c) each CAR is composed of at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif; (d) the at least one binding domain in one of the vectors is non-identical; and (e) the at least one binding domain, single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each of said vectors, and a combination of vectors is used to genetically modify one or more lymphocyte populations.

[0050] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of an immunotherapeutic composition comprising a population of patient-specific autologous anti-tumor lymphocyte cell population(s) of a human with cancer, wherein the cells of the population (a) comprise cells comprising nucleic acid molecules encoding two or more vectors; (b) each vector encodes a functional CAR; (c) each CAR comprises at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif; (d) the at least one binding domain(s) in each vector are not identical; (e) the combination of the at least one signaling motif is not identical between each of the vectors; and (f) the at least one binding domain, single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each of said vectors, and a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0051] In one embodiment, the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancer and solid tumor, or any combination thereof. In another embodiment, the cancer is a hematological cancer, for example, a leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (AML), or a combination thereof. 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.

[0052] In yet another embodiment, the cancer comprises oral cavity and pharyngeal 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 bronchi), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicles, 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.

[0053] 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 (DuoCARs), thereby generating a patient-specific autologous anti-tumor lymphocyte cell population capable of promoting in vivo expansion, wherein persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0054] In another aspect, pharmaceutical compositions are provided comprising an autologous T cell population that has been transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs) to generate a patient-specific autologous anti-tumor lymphocyte cell population capable of promoting in vivo expansion, wherein persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0055] In another aspect, a method of generating lymphoid cells comprising an active, patient-specific, autologous anti-tumor DuoCAR is provided, the method comprising transducing lymphoid cells with two or more vectors or nucleic acid molecules encoding two or more chimeric antigen receptors (DuoCARs) that specifically bind to an antigen, thereby generating lymphoid cells comprising the active, patient-specific, autologous anti-tumor DuoCAR.

[0056] In yet another aspect, a method is provided for generating a population of RNA-engineered lymphocyte cells, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding two or more chimeric antigen receptors (DuoCARs) into a cell population of a subject, thereby generating a patient-specific autologous anti-tumor lymphocyte cell population capable of promoting in vivo proliferation, wherein persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0057] In another aspect, provided is a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of autologous lymphocyte cells transduced with one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs), thereby generating a patient-specific autologous anti-tumor lymphocyte cell population capable of promoting in vivo proliferation, wherein persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0058] In another aspect, provided is a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of an autologous lymphocyte cell population transduced with two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs) to generate a patient-specific autologous anti-tumor lymphocyte cell population that can be infused directly back into the patient to promote in vivo proliferation, wherein persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, or remission of the cancer, or prevention or amelioration of cancer recurrence, or any combination thereof, in a patient-specific manner.

[0059] In one embodiment, a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen is provided, comprising administering to a subject a pharmaceutical composition comprising at least two vectors, each vector encoding a functional CAR, whereby the combination of the vectors results in expression of two or more non-identical binding domains, wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and a pharmaceutically acceptable excipient, wherein the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0060] In another embodiment, a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen is provided, comprising administering to a subject a pharmaceutical composition that (a) comprises a nucleic acid molecule encoding two or more vectors; (b) each vector encodes a functional CAR; (c) each CAR is composed of at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain in one of the vectors is not identical; and (e) the at least one binding domain, single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of said vectors, wherein the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0061] In yet another embodiment, a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen is provided, comprising administering to a subject a pharmaceutical composition wherein: (a) the nucleic acid molecule encodes two or more vectors; (b) each vector encodes a functional CAR; (c) each CAR comprises at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain(s) in each vector are not identical; (e) the combination of the at least one signaling motif is not identical between each of the vectors; and (f) the at least one binding domain, single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of said vectors, and a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0062] In certain embodiments, the genetically modified lymphocytes are autologous T-cell lymphocytes, and the autologous or allogeneic T-cell lymphocytes are infused directly back into the patient to prevent or ameliorate the recurrence of malignant tumor disease.

[0063] In certain other embodiments, the genetically modified lymphocytes are autologous T-cell lymphocytes, which are directly infused back into the patient to promote in vivo expansion, and persistence of the patient-specific anti-tumor T-cell lymphocytes results in tumor stabilization, reduction, elimination, or cancer remission, or prevention or amelioration of cancer recurrence, or any combination thereof, in a patient-specific manner.

[0064] In yet another embodiment, the T cells are preselected by expression of surface markers associated with specific activation or memory.

[0065] In yet another embodiment, the T cells are derived from a hematopoietic stem cell donor and the procedure is performed in the context of a hematopoietic stem cell transplant.

[0066] In certain embodiments, methods are provided in which lymphoid cells are preselected by expression of surface markers associated with specific activation or memory.

[0067] In certain embodiments, methods are provided herein wherein the lymphoid cells are T cells and are derived from a hematopoietic stem cell donor, and the procedure is performed in the context of a hematopoietic stem cell transplant.

[0068] In yet another aspect, a method is provided for generating a persistent population of genetically engineered patient-specific autologous anti-tumor lymphocyte cell population(s) in a human diagnosed with cancer. In one embodiment, the method comprises administering one or more patient-specific autologous anti-tumor lymphocyte cell population(s) described herein to a human patient in need thereof, wherein the persistent population of the patient-specific autologous anti-tumor lymphocyte cell population(s), or a population of lymphocyte cell progeny, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0069] In one embodiment, the progeny lymphoid cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0070] In all of the aspects and embodiments of the methods described herein, any of the above-mentioned cancers, diseases, disorders or conditions associated with elevated expression of tumor antigens can be treated or prevented or ameliorated using patient-specific autologous anti-tumor lymphocyte cell population(s) comprising one or more of the DuoCAR immunotherapeutic compositions disclosed herein.

[0071] In yet another aspect, a kit is provided for making a DuoCAR immunotherapy composition comprising the patient-specific autologous anti-tumor lymphocyte cell population(s), or for preventing, treating, or ameliorating any cancer, disease, disorder, or condition associated with elevated expression of a tumor antigen in the subject, 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.

[0072] Although the compositions and methods of the present invention are exemplified with reference to the production and utilization of DuoCARs, it is contemplated herein that the compositions and methods are specifically intended to include the production and utilization of TrioCARs and QuatroCARs.

[0073] In yet another aspect, the immunotherapeutic composition comprises one or more isolated nucleic acids encoding at least one vector, wherein said vector comprises a nucleic acid sequence that results in at least one messenger RNA encoding DuoCAR (i.e., a multicistronic nucleic acid, or a nucleic acid that results in more than one transcript), conferring the ability to bind to two or more non-identical antigen targets, thereby resulting in multiple antigen specificities present in a single cell expressing said vector.

[0074] In yet another embodiment, the immunotherapeutic composition comprises one or more isolated nucleic acids encoding the at least two vectors described above, each vector comprising a soluble binder (tagged sc The vectors further encode a functional tag or anti-tag binding moiety (AT-CAR) that reconstitutes a functional chimeric antigen receptor upon co-incubation or co-administration of two or more vectors (such as an Fv, or scFv linked to an anti-tag binder), thereby providing the ability for the combination of the two vectors to bind to two or more non-identical antigen-binding domains, resulting in multiple antigen specificities present in cells expressing these two vectors.

[0075] In yet another embodiment, the immunotherapeutic composition comprises one or more isolated nucleic acids encoding at least two vectors as described above, each vector encoding a functional tag or anti-tag binding moiety (AT-CAR) that reconstitutes a functional chimeric antigen receptor upon co-incubation or co-administration of a soluble binder (such as a tagged scFv or an scFv linked to an anti-tag binder), and each vector expresses a unique tag (or anti-tag) that can bind to a soluble protein or a structurally engineered protein that provides multiple antigen specificities, or each vector expresses a unique tag (or anti-tag) that binds to a unique soluble binding domain that provides specific linkage of an AT-CAR that encodes an intracellular signaling motif to the antigen-binding domain of the tagged (or anti-tagged) binder.

[0076] In a non-limiting embodiment for the production of DuoCAR vectors, each of the compositions and methods disclosed in the above-referenced embodiments and aspects allows two vectors to be produced separately and then added to T cells, either sequentially or simultaneously. In another non-limiting embodiment, the plasmid DNA of two or more vectors can be mixed before or during transfection of producer cells or incorporated into the production of the cellular genome to produce a mixture of viral vectors containing multiple DuoCAR vector particles, which can then be used to transduce and genetically modify a patient's T cells.

[0077] For each of the various aspects and embodiments of DuoCAR, TrioCAR, and QuatroCAR specifically contemplated herein, the nucleotide sequence encoding the functional CAR comprises the nucleotide sequence of SEQ ID NO: 3, 9, 21, 25, 29, 31, 35, 39, 43, 47, 49, 51, 53, 55, 59, 61, 109, 111, 113, or 115, or any combination thereof.

[0078] For each of the various aspects and embodiments of DuoCAR, TrioCAR, and QuatroCAR specifically contemplated herein, each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO: 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, 62, 110, 112, 114, or 116, or any combination thereof.

[0079] It is understood that the above-mentioned patient-specific autologous anti-tumor lymphocyte cell population(s), two or more lentiviral vectors expressing chimeric antigen receptors (DuoCAR), host cells, and methods are useful beyond the specific aspects and embodiments described in detail herein. The above-mentioned features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

[0080] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0081] [Figure 1]Figure 1 shows four products (Examples 1 to 4) that can be produced as separate commercial entities. These DuoCAR sets can be created to target human B-cell malignancies expressing the three leukemia-associated antigens CD19, CD20, and CD22. In Product 1, two genetic vectors are used to co-transduce activated T-cell populations. The first vector encodes two antigen-binding domains (CD19, CD20) linked to a single intracellular domain (z, CD3 zeta chain) connected by the CD8 transmembrane region (8). The second vector encodes a CD22-binding domain and two signaling domains (BB, derived from CD137 / 4-1BB; and z). The second product in Example 2 features a first vector with CD19- and CD20-binding domains linked to CD28 and z signaling domains. The second vector encoded the CD22 binding domain and the BB and z signaling domains, essentially repeating the signaling package (three different signaling domains) of the third generation CAR vector. In the third product of Example 3, the first vector encoded the CD20 and CD22 binding domains linked to the BB and z signaling domains, and the second vector encoded the CD19 binding domain linked to the CD28 and z signaling domains. In the fourth product of Example 4, the first vector encoded the CD20 and CD22 binding domains and the BB and z signaling domains. The second vector encoded the CD19 binding domain and the z signaling domain. [Figure 2]

[0023] Figure 1 shows all potential single components that can be combined into DuoCAR for therapeutic products targeting B-cell malignancies. Nomenclature is the same as in Figure 1. [Figure 3]

[0023] Figure 1 shows a generalized schematic diagram of DuoCAR, which can be applied to multiple therapeutic needs, including inflammatory or autoimmune diseases and infectious diseases. In the diagram, a-CDX, a-CDY, and a-CDZ refer to antigen-binding domains specific for three different target antigens, CDX, CDY, and CDZ, respectively. All intracellular aspects of the CAR contain a CD8 linker and a transmembrane domain linked to either a CD3-zeta, CD28, or 4-1BB signaling domain (as in Figure 1). The specific combination of any of these two vectors into a single vector (e.g., example A plus F, where antigens X, Y, and Z are targeted while providing intracellular signaling via CD3-zeta and 4-1BB) is defined according to the specific therapeutic need. [Figure 4] Figure 4 shows a generalized schematic diagram of a set of DuoCARs in which two antigens are targeted by each vector. Vectors identical to those in Figure 3 retain their specific letter designations (A in Figure 3 and Figure 4 is the same). A new fourth antigen-binding domain is indicated by a-CDW. One product that targets four antigens is the A+T DuoCAR set. In this example, the extracellular antigens CDX, CDY, CDZ, and CDW are targeted, providing both CD3-zeta and CD28 intracellular signals. [Figure 5]Figures 5A, 5B, 5C, and 5D show current CARs in the literature compared to the DuoCAR of the present invention (Figures 5E, 5F, and 5G). A CAR expression vector can be created that directs the expression of a single binding domain (pairs of black, unfilled, or striped spheres, each with distinct specificities) connected to a linker and transmembrane domain (single open box). In the figure, the thick gray line indicates the plasma cell membrane. In this figure, the pair of black spheres represents anti-CD19-scFv, the pair of unfilled spheres represents anti-CD20-csFv, and the pair of striped spheres represents anti-CD22-scFv, all linked by joining multimers (1, 2, 3, 4, 5, or 6 repeats) of amino acid sequences, e.g., GGGGS. Intracellular lymphocyte signaling domains from 4-1BB (CD137), CD28, and CD3-zeta chain can be combined as shown. (Figure 5A) In a single CAR, a single binding domain is combined with a transmembrane and two signaling domains to create a second-generation CAR. (Figure 5B) In a split CAR, two different binders are expressed with a single signaling domain, which must be combined to allow effective T cell signaling upon recognition of two different antigens. (Figure 5C) In a tandem CAR, two binding domains are linked to a single signaling domain. In this case, binding of either domain induces full T cell activation. (Figure 5D) In ​​a multiple CAR from one vector, two fully functional CARs are expressed from a single vector, each capable of binding to a unique antigen. (Figure 5E) In contrast, a DuoCAR is composed of two vectors and expresses at least three binding domains with multiple possible signaling domain combinations. The essential features that distinguish a DuoCAR are the expression of two or more transcripts, the multiplicity of binding domains (at least one multitargeting), and the fully functional signaling characteristics of at least one of the two expressed cell surface proteins.(Figure 5F) In the DuoCAR monospecific soluble binder format, the CAR portion encoded by the vector expresses a tag or anti-tag motif that also encodes transmembrane and intracellular signaling motifs (CAR-based vectors are not identical in terms of intracellular motifs). The base vector binds to a soluble protein containing both an scFv domain that interacts with the antigen and a tag or anti-tag motif that mediates binding to the CAR-based protein itself. Once the soluble protein binds to the CAR-based protein, the same structural features that mediate the antitumor activity mediated by DuoCAR [as in (E)] are reconstituted. (Figure 5G) In the DuoCAR bispecific soluble binder format, the bispecific "tag"-"anti-tag" interaction is unique so that only one soluble binder can bind to only one base vector. In this example, the black diamond in the base vector and the angular binder in the soluble dual-scFv protein may indicate a "biotin"-"anti-biotin" interaction, and the black crescent in the second CAR-based vector may interact with the black oval in the monospecific scFv structure and indicate a "FITC"-"anti-FITC" interaction. [Figure 6] This graph shows the cell surface expression levels of CAR constructs in primary human T cells transduced with CAR expression vectors that differ between second-generation (two costimulatory domains) and third-generation (three costimulatory domains) formats. T cells were transduced to express the following CARs: no CAR (mock), second-generation CAR (CAR-A-28z), third-generation CAR (CAR-A-28BBz), and alternative second-generation CAR (CAR-A-BBz). The level of CAR surface expression was detected by flow cytometry and is reported as mean fluorescence intensity (MF) on the y-axis. The MFI of both second-generation CARs was very bright, even though all constructs expressed the exact same CAR-binding domain. [Figure 7]Figure 1 shows DuoCAR cell surface expression on human T cells. Human T cells were activated with CD3-CD28 nanomatrix (TRansAct, Miltenyi Biotec) in the presence of IL-2, transduced with two vectors (one encoding a tandem CD20-CD19 CAR and the other encoding a single CD22 CAR, thus a 2+1 Duo-Set format), and then analyzed for expression of CD19-, CD20-, or CD22-scFv domains by flow cytometry using recombinant CD19, CD20, or CD22 for staining. Paired columns show double staining: CD20 and CD19 scFv in the left column, and CD22 and CD19 scFv in the right column. The first row shows untransduced (UTD) T cells and therefore shows no binding. The second row shows T cells transduced with LV encoding the CD20_CD29 CAR vector, which has a CD8 transmembrane domain and intracellular CD28 and CD3-zeta signaling domains (20-19-28z). Double staining is seen for CD20 and CD19 binding (left panel), but only CD19 binding is seen in the right panel. The third row shows T cells transduced with a CD22 CAR vector, which has a CD8 transmembrane domain and intracellular 4-1BB and CD3-zeta signaling domains (22-BBz). Double staining is not seen for CD19 or CD20 (left panel), and only a single population of cells capable of binding CD22 is seen (right panel). In the fourth row, T cells are transduced with a DuoSet consisting of both vectors from rows two and three. Only DuoSet expresses all three CAR-encoded binding domains (42% of cells express CD20_19 (left panel) and 38% express CD22 and CD19 binding domains (right panel)). Because the CD22 and CD19 scFvs are located on two separate transmembrane proteins that comprise DuoSet, the 38% represents the true DuoSet-expressing population in this example. [Figure 8]

[0023] Figure 7 is a graph showing the antitumor cytolytic activity of DuoCAR-expressing T cells. As described in Figure 7, human T cells transduced with single CAR components (20_19-28z or 22-BBz) or DuoCAR (20_19-28z + 22-BBz) were used in cytotoxic T cell assays at four different effector-to-target ratios (20:1, 10:1, 5:1, 2.5:1, as indicated). The leukemia cell lines used as CAR-T targets were Raji (expressing all three target antigens), REH (expressing all three target antigens), K562 (control, no target expression), K562-CD19 (expressing CD19), K562-CD20 (expressing CD20), and K562-CD22 (expressing CD22). Only DuoCAR-transduced cells (20-19-28z+22-BBz, 2+1 DuoSet) showed high cytolytic activity against both leukemia cell lines (Raji and REH) and all three single-expressing K562 target cell lines (K562-CD19, K562-CD20, K562-CD22). [Figure 9]

[0033] Figure 7 shows DuoCAR cell surface expression in primary human T cells achieved by two different methods of LV preparation. Using the same methods and data analysis as in Figure 7, cells were then transduced with DuoCARs specific for CD19, CD20, and CD22 (a 2+1 DuoSet, where one CAR is a tandem CD20 and CD19 binder and the second CAR is a CD22 binder). The data in the first column show flow cytometry analysis for CD19 and CD20 binder expression, while the second column shows flow cytometry analysis for CD22 and CD19 binders present as CARs in DuoCAR-expressing cells for four different populations, corresponding to untransduced, single CD22-CAR transduction, double transduction with CD22 and CD20_19 CARs, and single transduction with tandem CD20_19 CARs, in the lower left, upper left, upper right, and lower right quadrants, respectively. Both the two LV transduction method (co-transduction) and the single LV transduction method (co-transfection) gave similar DuoCAR staining patterns, with over 30% of the T cell population specific for CD19, CD20, and CD22 by expressing both CAR cell surface proteins. [Figure 10] Figure 10 shows a schematic diagram of the DuoCAR2 cistronic construct. The DuoCAR construct is expressed from a single bicistronic open reading frame containing the sequences of two CAR chains separated by a 2A peptide. One CAR is composed of a CD22 scFv, a 4-1BB costimulatory domain, and a CD3 zeta activation domain linked in-frame to a CD8 hinge and transmembrane domain (Figure 10A). The other CAR is composed of a tandem CD20 CD19 scFv-based targeting domain, followed by a CD8 hinge and transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta activation domain (Figure 10B). [Figure 11]

[0023] Figure 1 shows cell surface expression of Set 1 bicistronic DuoCARs and controls in primary human T cells transduced with DuoCAR expression vectors, as measured by flow cytometry. T cells were transduced to express the following CARs: no CAR (UTD), construct number 2228 (2019 tandem CAR), construct numbers 2200, 2209, 2218, 2225, and 2227 (CD22 CAR variants), and construct numbers 2515, 2520, and 25212 (cistronic CARs containing one CAR chain targeted to CD22 and another tandem CAR chain targeted to CD20 and CD19 tumor antigens). In the duplicate plots shown, CAR22 expression is shown on the Y-axis, and CAR19 expression, representing the tandem 2019 CAR chain, is shown on the X-axis. The percentage of positive cells is indicated in each quadrant. Data are representative of three transduction experiments in T cells from separate healthy donors. [Figure 12] Figure 1 shows the cytokine response of bicistronic DuoCAR Set 1 co-incubated with Raji tumor cells. T cells were transduced to express the following CARs: no CAR (UTD), construct number 2228 (-2019 tandem CAR), construct number 2200 (-CD22 CAR), and construct numbers 2515, 2520, and 2521. DuoCAR T cells and controls were incubated overnight with triple-positive Raji cells, then supernatants were collected and analyzed by ELISA for IFNg, TNFa, and IL-2. N=3, + / -SD. One experiment representative of three independent experiments on T cells from separate donors is shown. [Figure 13]Figure 1 shows cell surface expression of Set 2 bicistronic DuoCARs and controls in primary human T cells transduced with DuoCAR expression vectors, as measured by flow cytometry. T cells were transduced to express the following CARs: no CAR (UTD), construct number 1497 (-2019 tandem CAR), construct number 2200 (-CD22 CAR), and bicistronic CAR construct numbers D0043, D0044, D0046, and D0047, which contain one CAR chain targeted to CD22 and another tandem CAR chain targeted to CD22 and the CD19 tumor antigen. In the duplicate plots shown, CAR22 expression is shown on the Y-axis, and CAR19 expression, representing the tandem 2019 CAR chain, is shown on the X-axis. The percentage of positive cells is shown in each quadrant. Data are representative of three transduction experiments in T cells from three separate healthy donors. [Figure 14] Figure 1 shows the antitumor cytolytic activity of bicistronic DuoCAR-expressing T cells from Set 2. Human T cells transduced with single CAR constructs (LTG1497, 20_19-28z, or LTG2200, 22-BBz) or DuoCARs (construct numbers D0043, D0044, D0046, and D0047, encoding 20_19-28z + 22-BBz) were used in cytotoxic T cell assays at four different effector-to-target ratios (10:1, 5:1, and 2.5:1, as indicated; the "D" in the construct name and the zeros between the numbers have been omitted for simplicity). The leukemia cell lines used as CAR-T targets were Raji (expressing all three target antigens), Reh (expressing all three target antigens), and 392T (lacking all three target antigens). DuoCAR lysed the triple-positive cell line in an E:T-dependent manner, whereas no lysis occurred in the target-negative 293T cell line. [Figure 15]Figure 1 shows the antitumor cytolytic activity of bicistronic DuoCAR Set 2-expressing T cells. Human T cells transduced with single CAR constructs (LTG1497, 20_19-28z, or LTG2200, 22-BBz) or DuoCARs (construct numbers D0043, D0044, D0046, and D0047, encoding 20_19-28z+22-BBz) were used in cytotoxic T cell assays at four different effector-to-target ratios (10:1, 5:1, and 2.5:1, as indicated; the "D" in the construct name and the zero between the numbers have been omitted for simplicity). The single-positive tumor cell lines used as CAR-T targets were K19 (expressing CD19), K20 (expressing CD20), and K22 (expressing CD22). Three single-positive tumor cell lines were developed in the background of the parental K562 erythroleukemia line, which naturally lacks CD19, CD20, or CD22 expression, by stable transduction of the desired single antigen (CD19, CD20, or CD22) and the firefly luciferase gene. DuoCARs lysed the single-positive cell lines in an E:T-dependent manner, and lysis above background levels was not mediated by CAR controls with mismatched antigen-targeting domains (CAR 22, LTG 2200 vs. K19 and K20; tandem CAR 2019, LTG 1479 vs. K22). [Figure 16]Figure 1 shows the cytokine response of bicistronic DuoCAR version 2 co-incubated with Raji tumor cells or in the absence of tumor (CAR alone). T cells were transduced to express the following CARs: no CAR (UTD), construct number 2273 (-2019 tandem single-chain CAR), construct number 2200 (CD22 single-chain CAR), and construct numbers D44 and D47 (referring to the D0044 and D0047 CAR constructs, respectively; the "D" in the construct name and the zero between the numerals have been omitted for simplicity). DuoCAR and T cells and controls were incubated overnight with triple-positive Raji cells, and then supernatants were collected and analyzed by ELISA for IFNg, TNFa, and IL-2. N=3, + / -SD. One experiment representative of three single experiments on T cells from separate donors is shown. [Figure 17]

[0023] Figure 1 shows a schematic diagram of two CAR chains that can be combined for co-expression in the same cell or population of cells by co-transfection or co-transduction to generate DuoCAR. One CAR chain is composed of a CD22 scFv, a 4-1BB costimulatory domain, and a CD3 zeta activation domain linked in-frame to a CD8 hinge and transmembrane domain. The other CAR chain is composed of a tandem CD20 CD19 scFv-based targeting domain, followed by a CD8 hinge and transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta activation domain. [Figure 18]Figure 1 shows cell surface expression of DuoCAR and controls in primary human T cells transduced with DuoCAR expression vector preparations generated by cotransfection of two transfer plasmids producing LVs, or individually transduced single-vector controls (top panel), as measured by flow cytometry. T cells were transduced to express the following CARs: construct numbers 2273, 2228 (-2019 tandem CAR), D1, D2, D3, CD22 CAR, and DuoCAR (construct numbers D1+2273, D2+2273, D3+2273). In the scatter plot shown, CAR22 expression is shown on the Y-axis, and CAR19 expression, representing the tandem 2019 CAR chain, is shown on the X-axis. The percentage of positive cells is shown in each quadrant. Representative data for three experiments using T cells from three donors. [Figure 19A]Graph showing the antitumor cytolytic activity of DuoCAR cells or single-chain CAR control. DuoCAR T cells were generated by cotransfection of two transfer plasmids producing lentiviral vectors. T cells were transduced with the resulting DuoCAR vector or single-chain CAR control to express the following CARs: construct number 2273 (-2019 single-chain tandem CAR); construct numbers D1, D2, and D3 (-CD22 single-chain CAR); and DuoCARs generated by combining two single CAR chains in the same CAR T product (construct numbers D1+2273, D2+2273, and D3+2273, where the "D" in the numerical designation is omitted for simplicity). The resulting CAR T cells were analyzed in a cytotoxic T cell assay at two different effector-to-target ratios (10:1, 5:1, as indicated) against native leukemia lines, CD19+CD20+CD22+ (Raji, Reh) or the CD19, CD20, and CD22 triple-negative control line 293T (Figure 19A). The native target lines Raji and Reh were lysed by single-chain CAR constructs with construct numbers D1+2273, D2+2273, and D3+2273 (the "D" in the numerical designation is omitted for simplicity) from all DuoCAR groups, as well as by the single-chain CAR control. In contrast, the DuoCAR and single-CAR control were not cytolytic against the CD19, CD20, and CD22 triple-negative line 293T, demonstrating the target specificity of the CAR constructs. Because DuoCAR simultaneously targets three target antigens, to further address the issue of target specificity, DuoCAR was tested against transgenic single-positive tumor lines generated in the background of K562 erythroleukemia cells, which naturally lack CD19, CD20, or CD22 expression. The single-positive tumor cell lines used as CAR-T targets were K19 (expressing CD19), K20 (expressing CD20), and K22 (expressing CD22) (Figure 19B). DuoCAR lysed the single-positive cell lines in an E:T-dependent manner, demonstrating that all targeting domains of DuoCAR are functional and specific for their cognate target molecules (Figure 19B).Furthermore, CAR single-chain controls with mismatched antigen-targeting domains (CAR 22, LTG 2200 vs. K19 and K20; tandem CAR 2019, LTG 1479 vs. K22) did not have specific lytic activity (Figure 19B). [Figure 19B]Graph showing the antitumor cytolytic activity of DuoCAR cells or single-chain CAR control. DuoCAR T cells were generated by cotransfection of two transfer plasmids producing lentiviral vectors. T cells were transduced with the resulting DuoCAR vector or single-chain CAR control to express the following CARs: construct number 2273 (-2019 single-chain tandem CAR); construct numbers D1, D2, and D3 (-CD22 single-chain CAR); and DuoCARs generated by combining two single CAR chains in the same CAR T product (construct numbers D1+2273, D2+2273, and D3+2273, where the "D" in the numerical designation is omitted for simplicity). The resulting CAR T cells were analyzed in a cytotoxic T cell assay at two different effector-to-target ratios (10:1, 5:1, as indicated) against native leukemia lines, CD19+CD20+CD22+ (Raji, Reh) or the CD19, CD20, and CD22 triple-negative control line 293T (Figure 19A). The native target lines Raji and Reh were lysed by single-chain CAR constructs with construct numbers D1+2273, D2+2273, and D3+2273 (the "D" in the numerical designation is omitted for simplicity) from all DuoCAR groups, as well as by the single-chain CAR control. In contrast, the DuoCAR and single-CAR control were not cytolytic against the CD19, CD20, and CD22 triple-negative line 293T, demonstrating the target specificity of the CAR constructs. Because DuoCAR simultaneously targets three target antigens, to further address the issue of target specificity, DuoCAR was tested against transgenic single-positive tumor lines generated in the background of K562 erythroleukemia cells, which naturally lack CD19, CD20, or CD22 expression. The single-positive tumor cell lines used as CAR-T targets were K19 (expressing CD19), K20 (expressing CD20), and K22 (expressing CD22) (Figure 19B). DuoCAR lysed the single-positive cell lines in an E:T-dependent manner, demonstrating that all targeting domains of DuoCAR are functional and specific for their cognate target molecules (Figure 19B).Furthermore, CAR single-chain controls with mismatched antigen-targeting domains (CAR 22, LTG 2200 vs. K19 and K20; tandem CAR 2019, LTG 1479 vs. K22) did not have specific lytic activity (Figure 19B). [Figure 20] Figure 2 shows the cytokine release activity of DuoCAR cells or single-chain CAR control in response to Raji13G11, a CD19+CD20+CD22+ clone. DuoCAR T cells were generated by cotransfection of two transfer plasmids producing lentiviral vectors. T cells were transduced with the resulting DuoCAR vector to express the following CARs: construct number 2273 (-2019 tandem CAR); construct numbers D1, D2, and D3 (-CD22 CAR); and three DuoCARs (D1+2273, D2+2273, and D3+2273; Figure 20, "D" in group labels omitted for simplicity). The resulting CAR T cells were mixed overnight with the triple-positive Raji tumor line at an E:T ratio of 10, and the culture supernatants were analyzed for IFNg, TNFa, and IL-2. All DuoCAR constructs produced high levels of the three cytokines in response to Raji cells. The DuoCAR-alone control, consisting of CAR T cells incubated in the absence of Raji target, produced no appreciable cytokines in response to Raji13G11 cells, demonstrating that the cytokine response was target-specific (Figure 20). [Figure 21]Figure 1 shows the construction of DuoCARs simultaneously targeting CD19, CD20, and CD22, as well as tandem and single-CAR controls. Each DuoCAR consists of a tandem CD20 and CD19 dual-targeting CAR co-expressed with a first- or second-generation single-targeting CD22 CAR. The two CAR constructs are co-expressed in a bicistronic format and linked by a ribosomal skip site 2A sequence to ensure stoichiometric expression of the two CAR chains. Due to the nature of this bicistronic expression cassette, both CAR chains are co-expressed in each transduced T cell. (Figure 21A) Triple-targeting anti-CD20 and anti-CD19 anti-CD22 DuoCAR D93 is composed of a 20-19 tandem ScFv, hinge and transmembrane domain, ICOS costimulatory domain, and CD3z activation domain, followed by a 2A sequence. Next, the single-targeting CD22 CAR is composed of a CD22 scFv, hinge and transmembrane domain, and CD3z activation domain. DuoCAR D94 is constructed as D93, except for the replacement of the ICOS costimulatory domain with an OX40 domain. DuoCAR construct D95 is constructed as D94, except for the addition of an ICOS costimulatory domain to the CD22 CAR chain. DuoCAR construct D96 is constructed as construct D95, except for the replacement of the OX40 costimulatory domain in the D95 construct with a CD27 costimulatory domain. All constructs contain a CD8-derived hinge and transmembrane domain. The tandem construct 1497 and the single CAR constructs D89, D92, 1538, 1497 represent functional controls. (Figure 21B) Schematic representation of DuoCAR T cells, tandem CARs, second-generation single CARs (without costimulatory domains), and first-generation single CARs (without costimulatory domains), in which one tandem CAR chain and one single CAR chain are co-expressed in the same T cell. [Figure 22]Surface expression of Duo-CAR T constructs D93, D94, D95, and D96, and tandem CAR 1497 (comparison) on human primary T cells. CAR T expression was determined by flow cytometry. T cells were activated with Miltenyi Biotec TransAct™ CD3 CD28 reagent in the presence of IL-2 and transduced with LV at an MOI of 80, as described in Materials and Methods. On day 8 of culture, viable transduced T cells (7-AAD negative) were assayed for CAR surface expression using one of three staining methods: CD19 Fc followed by anti-Fc-AF647, CD20 biotin reagent followed by streptavidin PE, or CD22-his reagent followed by anti-his-PE staining. Figure 22A shows one representative transduction experiment out of four. The upper panel shows the expression of CD20-targeting scFv relative to that of CD19-targeting scFv, and the lower panel shows the expression of CD22-targeting scFv relative to that of CD19-targeting scFv. The LV used in transduction is indicated at the top of each column. The percentage of the CAR T-positive population is shown in each quadrant of the histogram. Figure 22B shows the average DuoCAR expression percentage ± SEM for four transduction experiments performed on T cells from different donors. CAR-transduced T cells are defined as CAR19+CAR22+ cells, demonstrating the simultaneous detection of the two DuoCAR chains co-expressed in each cell. [Figure 23]Figure 23 shows graphs demonstrating in vitro CAR T cytotoxicity. Luciferase-based cytotoxicity assays were performed using stably luciferase-transduced Raji 13G11 CD19+CD20+CD22+, REH CD19+CD20lowCD22+, or CD19-CD22- cell lines (293T or K562). Specific lysis of target cells by DuoCARs D1-D4, tandem CAR 1497, or single CARs D89 and D92 is shown for Figure 23A) Raji cells, Figure 23B) Reh cells, Figure 23C) K562 cells, or Figure 23D) 293T cells. Negative control UTD untransduced T cells were included. CAR T cells and target tumor cells were co-incubated overnight at the indicated effector-to-target (E:T) ratio (x-axis). Error bars represent the mean value from three technical replicates. One experiment representative of three independent experiments on T cells from three donors is shown. Bars indicate mean + / - SD values ​​from three independent experiments performed with CAR T cells from three separate donors. [Figure 24] CAR T cytokine release in response to leukemia cell lines. Production of the cytokines IL-2, IFNγ, and TNFα by the CAR-Ts listed on the x-axis after overnight co-culture with the Raji leukemia line at an E:T ratio of 10:1 was measured using ELISA. Bars represent the mean + SD of triplicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. [Figure 25] 25A-25B are graphs showing the in vivo antitumor activity of DuoCAR. NSG mice bearing Raji tumors were treated with DuoCAR T cells D93, D94, D95, and D96, or tandem CAR 1497, or single CARs D89 or D92. CAR T cells were injected iv 7 days after tumor inoculation at a dose of either 5 million CAR T cells per mouse (FIG. 25A) or 2 million CAR T cells per mouse (FIG. 25B). Tumor burden was assessed by bioluminescence on the indicated days. N=6 mice per group, mean radiance±SEM is shown. [Figure 26]Figures 26A-26D show in vitro CAR T cytotoxicity against CD22 CD19-targeted CAR T cells (Figures 26A-26D), and the parental A431 negative control line, or Raji leukemia line clones engineered to lack expression of either CD19, CD20, or CD22, indicative of antigen escape clones, and A431 tumor line clones transduced to overexpress only one target antigen to confirm the specificity of the parental Raji line (Figures 26E-H). All target lines stably expressed firefly luciferase. Bars represent the mean ± SEM values ​​of triplicate determinations from one experiment, representative of three independent experiments performed with CAR T cells from three separate donors. [Figure 27]

[0023] Figure 1 shows the in vivo antitumor activity of DuoCAR in a model of tumor antigen escape. NSG mice were inoculated with an equal mixture of Raji CD19neg, Raji CD20neg, Raji CD22neg, and the parental Raji clone. Raji tumors were treated with DuoCAR T cells D93, D94, D95, or D96, which can target CD19, CD20, or CD22 antigens, or single CAR controls: CAR22 D92, CAR19 1538, or CAR20 1495. T cells were injected iv at a dose of 5 million CAR T cells per mouse 7 days after tumor inoculation, and tumor burden was assessed by bioluminescence on the indicated days. N=6 mice per group; mean radiance ± SEM is shown.

[0082] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes single or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including one antigen" without excluding other elements. The phrase "and / or" means "and" or "or." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for convenience unless otherwise specified. While many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided.

[0083] The term "about," when referring to a measurable value, e.g., amount, temporal duration, etc., is meant to encompass a variation of + / -20%, + / -10%, or more preferably + / -5%, or + / -1%, or even more preferably + / -0.1% from the specified value, where such variation is appropriate for practicing the disclosed methods.

[0084] Unless otherwise specified, technical terms herein are used according to conventional usage. Definitions of common terms in molecular biology are found in Benjamin Lewin, Genes VII, 1999, published by Oxford University Press; Bla (eds.), The Encyclopedia of Molecular Biology, 1994, published by Blackwell Science Ltd.; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 1995, published by VCH Publishers, Inc.; and other similar references.

[0085] The present invention relates to compositions and methods for treating diseases and / or conditions, and cancers, including but not limited to hematological malignancies and solid tumors. The present invention relates to patient-specific, tumor-specific strategies of adoptive cell transfer of T cells transduced with two or more vectors to express one or more DuoCARs.

[0086] The present invention more particularly relates to lentiviral vectors expressing chimeric antigen receptors (DuoCAR), as well as to host cells (e.g., lymphocytes, T cells) transduced with the lentiviral vectors expressing the CAR, nucleic acid molecules encoding the lentiviral vectors and chimeric antigen receptors, and methods of using same provided, e.g., to treat cancer in a subject.

[0087] Surprisingly and unexpectedly, it has now been discovered by the inventors that immunotherapeutic compositions comprising patient-specific autologous anti-tumor lymphocyte cell populations are highly effective as anti-tumor immunotherapeutics when the autologous lymphocyte cell populations are transduced with two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs). The use of at least two or more lentiviral vectors expressing single or multiple CARs in The persistence of patient-specific anti-tumor T cells appears to promote in vivo proliferation, resulting in tumor stabilization, reduction, elimination, or cancer remission, or prevention or amelioration of cancer recurrence, or any combination thereof, in a patient-specific manner.

[0088] Such active patient-specific anti-tumor T cell populations described herein can be directly infused back into the patient to promote in vivo expansion, and persistence of the patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, cancer remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner, including efficient expansion and rapid contraction of the therapeutic cell population.

[0089] Thus, in its broadest aspect, the novelty of this adoptive immunotherapy lies in the use of a combination of CAR-expressing vectors. The distinguishing feature is that, contrary to the traditional use of a single vector expressing one or more chimeric antigen receptors, the DuoCAR approach allows for the expression of multiple antigen specificities and in The goal of this approach is to provide both optimal signaling for anti-tumor T cell activity in vivo. Creating a system in which three or more antigens are efficiently targeted is far superior to single or tandem approaches that allow tumor cancer cells to generate escape variants that lead to tumor metastasis and / or tumor recurrence. The use of two or more vectors encoding single or multiple chimeric antigen receptors (DuoCARs) in which the specific combination of at least one binding domain(s) in each vector is not identical, coupled with the requirement that the combination(s) of at least one signaling motif is not identical between each vector, serves to ensure that one or more genetically modified lymphocyte populations transduced with such duo lentiviral vector-derived CARs generate patient-specific autologous anti-tumor lymphocyte cell populations capable of promoting in vivo proliferation, and the persistence of patient-specific anti-tumor lymphocyte cells results in tumor or cancer stabilization, reduction, elimination, or remission, and / or prevention or amelioration of tumor or cancer recurrence, or any combination thereof, in a patient-specific manner.

[0090] In one aspect, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCARs), each vector encoding a functional CAR, and wherein at least one binding domain(s) in one of the vectors is / are non-identical, such that the combination of the vectors results in expression of two or more non-identical binding domains, and wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0091] In another aspect, an immunotherapeutic composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCARs), each vector encoding a functional CAR, whereby the combination of vectors results in expression of two or more non-identical binding domains, wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, with the proviso that said immunotherapeutic composition specifically excludes a single CAR, a split CAR, a tandem CAR, or multiple CARs depicted in Figure 5(A), (B), (C), or (D), respectively.

[0092] The immunotherapeutic efficacy and prevention or amelioration of tumor or cancer recurrence achieved with the DuoCAR lentiviral vector-modified T cells of the present invention are significantly greater and synergistic than those achieved with either conventional CAR design alone. It is this unique combination of biological therapeutic benefit correlated with increased in vivo proliferation and persistence of patient-specific anti-tumor lymphocytes that results in tumor or cancer stabilization, reduction, elimination, or remission compared to conventional CAR-based T cell immunotherapy.

[0093] CAR expression vectors can be created that direct the expression of a single binding domain (black, solid, or striped sphere, Figure 5, each with a distinct specificity) connected to a linker and transmembrane domain (single solid box). Figure 5 below shows a comparison of a conventional CAR to the DuoCAR of the present invention. In Figure 5, the thick gray line indicates the plasma cell membrane. Intracellular lymphocyte signaling domains from 4-1BB (CD137), CD28, and the CD3-zeta chain can be combined as shown. All examples and uses of CD3 signaling domains in this document include modifications of the CD3-zeta chain by selective mutagenesis of tyrosine residues within one, two, or three immunoreceptor tyrosine-based activation motifs (ITAMs), or other such mutational alterations that render the ITAM motif no longer a target for phosphorylation. In a single CAR (Figure 5A), a single binding domain is combined with a transmembrane and two signaling domains. In split CARs (Figure 5B), two different binders are expressed with a single signaling domain that must be combined to effectively signal. In tandem CARs (Figure 5C), two binding domains are linked to a single signaling domain. In multiple CARs from one vector (Figure 5D), two fully functional CARs are expressed from a single vector. Duo-CARs of the present invention (e.g., Figure 5E) encode at least two vectors, each vector encoding a functional CAR, whereby the combination of the vectors results in the expression of two or more non-identical binding domains, each of which encodes a binding domain(s) covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs. The essential features that distinguish DuoCARs of the present invention are the use of two or more vectors, the multiplicity of binding domains, and the fully functional signaling characteristics (associated with in vivo T cell proliferation) of at least one of the two expressed cell surface proteins.

[0094] In another embodiment, the DuoCAR is a therapeutic T cell population-mediated tumor immunity. They are used to enhance the immune response. The immune response can be enhanced in at least three ways.

[0095] First, by providing T cells with additional signals for proliferation and survival in the body, the DuoCAR of the present invention allows for the persistence of a therapeutic T cell population by stimulating the T cell population upon encountering an autoantigen (e.g., CD19), the loss of which can be tolerated by the patient, while still serving to provide a stimulatory signal for the therapeutic cell population that is not present in the tumor tissue itself. It is well known / established that third-generation DuoCARs (intracellularly expressing three costimulatory domains linked to a single extracellular Ig-like binder) are not as well expressed in therapeutic T cells as DuoCARs expressing two intracellular costimulatory domains. For example, in Figure 6 below, the expression levels of CAR constructs in primary human T cells differ between second-generation (two costimulatory domains) and third-generation (three costimulatory domains) constructs. T cells were transduced to express the following CARs: no CAR (mock), second-generation CAR (CAR-A-28z), third-generation CAR (CAR-A-28BBz), and alternative second-generation CAR (CAR-A-BBz). The level of CAR surface expression was detected by flow cytometry and is reported as mean fluorescence intensity (MF) on the y-axis. The MFI of both second-generation CARs was very bright, even though all constructs expressed the exact same CAR-binding domain.

[0096] By providing a third T cell activating sequence on a separate vector CAR construct, the inventors are able to regain the benefits of expressing three costimulatory domains without the penalty of reduced expression of the CAR on the T cell surface.

[0097] In a second aspect, the DuoCAR of the present invention may target cell types other than tumors that mediate immunosuppressive effects. For example, if CD19-expressing B cells are present in tumor lesions and also inhibit anti-tumor immunity, such as by producing IL-4 or other mediators, then the second benefit to using a DuoCAR-expressing tumor-specific T cell population, an immunosuppressive cell population, is also eliminated.

[0098] For example, if immunosuppressive B cells are present within solid tumor lesions, they can be eliminated by the use of B cell-specific DuoCARs (such as CD19-specific DuoCARs). If immunosuppressive fibroblast-like cells are present, they can be eliminated by stroma-specific DuoCARs (e.g., by targeting fibroblast activation protein-alpha (FAP)). If malformed vasculature contributes to the lack of an effective immune response, DuoCARs specific to these types of blood or lymphatic vessels (such as anti-VEGFR) may also improve treatment outcomes.

[0099] In a third aspect, the DuoCAR of the present invention targets immunosuppressive populations distal to the tumor, i.e., present in another compartment in the body. For example, DuoCARs can be used to target myeloid-derived suppressor cells (MDSCs), which can be present either in the tumor lesion itself, or in regional lymph nodes or bone marrow. It is well established that tumor-draining lymph nodes can be either loci of immune activation or immunosuppression. This depends on the overall inflammatory tone of the lymph node and the differentiation of distant dendritic cells before migration to the lymph node. If tumor-draining lymph nodes are populated with myeloid-derived suppressor cells (MDSCs) or misdifferentiated antigen-presenting cells, such as dendritic cells, DuoCARs targeting these cell types, even though they are distal to the tumor itself, may also improve therapeutic outcomes. Beyond the immunotherapeutic application of cancer-specific DuoCARs, a second application of DuoCARs would be the prevention or treatment of autoimmune and / or inflammatory diseases. The difference from oncology-based applications is that the cellular substrate is regulatory T cells (TReg), or induced regulatory T cells (iTReg), or other cells cultured under conditions that promote a Th-2-like immune response. In therapeutic applications as diverse as graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT), allergic airway, intestinal, or other mucosal inflammation, or skin allergies, the presence of CAR-modified lymphocytes producing immunoinhibitory cytokines such as transforming growth factor-beta (TFG-beta) serves to exert a broad range of tolerogenic signals that ameliorate autoimmune or inflammatory-driven diseases. This approach includes peripheral or central nervous system (CNS) neurological inflammatory conditions such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, Parkinson's disease, and CTE (chronic traumatic encephalopathy due to repeated concussions or microconcussions). This approach also includes progressive scarring diseases such as COPD (chronic obstructive pulmonary disease).

[0100] In the treatment of inflammatory diseases, lymphocytes specific for tissue antigens, markers on the surface of inflamed cells, or misfolded proteins (such as tau protein or beta-amyloid) can be targeted by generating DuoCAR expression vectors specific for these targets. Single antibody-based therapies for Alzheimer's disease are already in clinical development (i.e., solanezumab by Eli Lilly and Company and aducanumab by Biogen, Inc.). In Alzheimer's disease, antibodies against monomeric or aggregated beta-amyloid can be used in the CAR format instead of binders against cell surface proteins. Binders against tau protein or tau peptides bound to MHC molecules can also be used as binding motifs for CARs. Receptors that mediate lymphocyte homing to specific peripheral tissues can also be included in the CAR format to provide local specificity for the CAR-expressing Treg population. Adhesion receptor domains known to drive lymphocyte inflammation to specific tissues and cytokine sequences, or cytokine or chemokine receptors, or binders can be used as part of the CAR domain. Adhesion molecules such as CD44 and integrin alpha-4 are known to target lymphocytes to the CNS, so including domains derived from adhesion molecules known to mediate the CNS migration behavior of lymphocyte populations can also be used to target CAR-expressing lymphocytes to diseased areas. The same is true for the intestine (i.e., binders for MAdCAm-1, CCR9 expression, or anti-CCL25), lung (i.e., P-selectin or mesothelin), skin (i.e., binders for E-selectin), or other mucosal surfaces.

[0101] To use this approach, patients with inflammatory conditions or whose diseases, such as Alzheimer's disease, can be treated by alleviating inflammatory pathology are admitted to the clinic, and peripheral blood is collected. Tregs can be directly selected using immunomagnetic beads (Regulatory T Cell Isolation Kit, Miltenyi Biotec) or induced by culture in an appropriate cytokine environment. These Tregs or iTregs are then transduced with DuoCAR vectors and, if necessary, expanded in vitro (Treg Expansion Kit, Miltenyi Biotec). The DuoCAR binding domain is derived from antibodies or receptors that mediate tissue-specific homing and disease-related binders, such as anti-beta amyloid. The engineered immune effector cells thus generated target appropriate sites and produce cytokines consistent with their Th2 or Treg differentiation pattern. It is also known that CAR-T cells can be engineered to secrete specific gene payloads upon CAR receptor activation. In addition to the DuoCAR payload expressed from the vector, additional therapeutic proteins or peptides can be expressed or secreted by the engineered T cells, such as a) A-beta DP (amyloid beta-degrading protease), b) matrix proteases (such as MMP-9 and MMP9 inhibitors in COPD), c) peptides or soluble antibody-like binders that interfere with plaque formation, and d) cytokines (such as TGF-beta, IL-4, IL-10, etc.).

[0102] miRNAs can also be expressed in cells that regulate T cell function. Examples of miRNAs include miRNAs These include miR-92a, miR-21, miR-155, miR-146a, miR-3162, miR-1202, miR-1246, and miR-4281, miR-142, and miR-17-92. Also, shRNAs targeting miRNAs can be developed. Examples include shRNAs targeting miR-28, miR-150, and miR-107, which normally bind to PD1 and increase its expression.

[0103] Beyond oncology-based applications, as well as inflammatory and autoimmune disease-based applications, a third application of DuoCAR technology is the generation of therapeutic lymphocyte populations specific for viral, bacterial, or fungal antigens. Thus, in relation to the oncology applications described for B-cell malignancies, targeting infectious diseases allows DuoCAR products to mediate immunoprotective or immunotherapeutic activity against infectious agents or the diseased tissues in which they reside, based on recognition of microbial antigens. Unlike T-cell receptor (TCR)-based approaches, where the T-cell receptor itself mediates recognition of the pathogen for which the peptide is encoded, the DuoCAR approach utilizes binding proteins expressed in a CAR vector format that confer antibody-like recognition (i.e., no antigen processing) to the transduced T-cell population. Activation of the therapeutic T-cell population results in immune activation loci that enable the elimination of infected cells and, if the microbial antigen is not cell-associated, the release of soluble mediator-like interferon-gamma, which enables an effective immune response to be mounted against the infectious agent.

[0104] For example, HIV is known to be highly variable and can be further classified into specific clades or families, allowing the generation of antibodies against clade-specific viral envelope proteins (env, gp120). Using the DuoCAR approach, three or more clade-specific antibody-like binders can be included in a CAR construct, resulting in broad anti-HIV immune activity. In addition to viral proteins, bacterial proteins can also be targeted. A current medical challenge is the treatment of antibiotic-resistant bacteria, which frequently arise in healthcare settings. These include VRE (vancomycin-resistant enterococci), MRSA (methicillin-resistant Staphylococcus aureus), and KPC (Klebsiella pneumoniae carbapenemase-producing Gram-negative bacteria, also known as CRKP). Klebsiella cell surface antigens include O antigens (9 variants) and K antigens (approximately 80 variants). The spectrum of O antigens, like many K antigens, can be easily covered with a small DuoCAR library. For use in oncology, CAR constructs featuring antibodies binding to different K or O serotypes have been created, and these CAR vectors are used to transduce isolated and activated Th1-like effector cell populations. In fungal diseases, research by L. Cooper et al. (Kumasesan, PR, 2014, PNAS USA, Vol. 111:10660) demonstrated that dectin-1, a fungal binding protein normally expressed in human cells, can be reconstituted as a CAR and used to control fungal growth in vitro. The human disease aspergillosis occurs in severely immunosuppressed individuals and is caused by the fungus A. fumigatus. Several groups have produced monoclonal antibodies specific to antigenic components on the cell surface of Aspergillus, thus opening the door to adoptive immunotherapy with DuoCARs that target three or more Aspergillus antigens on the fungal surface. Thus, in all of these infectious disease applications, the ability to create immunoglobulin-like binders to microbial antigens allows multiple antigens to be targeted by CAR-expressing effector lymphocyte populations.

[0105] Below follows a detailed description of DuoCARs that may be used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein, including a description of their extracellular, transmembrane, and intracellular domains, along with further description of DuoCARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, methods of treatment, compositions, and kits using the disclosed DuoCARs. While the compositions and methods of the present invention are exemplified with reference to the generation and utilization of DuoCARs, the compositions and methods of the present invention are not limited to the compositions and methods of the present invention. It is contemplated herein that the methods are specifically intended to include making and utilizing TrioCAR and QuatroCAR.

[0106] A. Chimeric Antigen Receptor (as present in DuoCAR) A DuoCAR disclosed herein comprises at least two vectors, each vector encoding a functional CAR, whereby the combination of the vectors results in the expression of two or more non-identical binding domains, wherein the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0107] CARs are artificially constructed hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. Features of DuoCARs include their ability to redirect T cell specificity and reactivity toward selected targets by utilizing the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition endows DuoCAR-expressing T cells with the ability to recognize antigens independently of antigen processing, thus bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, DuoCARs advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs).

[0108] As disclosed herein, the intracellular T cell signaling domain of a DuoCAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient lymphocyte response to an antigen. In some instances, the activation domain can be attenuated by mutation of specific sites of phosphorylation, i.e., the ITAM motif in the CD3 zeta chain, thus carefully regulating the degree of signal transduction mediated by that domain.

[0109] 1. Extracellular domain In one embodiment, the CAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein comprises a target-specific binding element, otherwise referred to as an antigen-binding domain or moiety. The selection of the domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a specific disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0110] In one embodiment, CARs can be engineered to target tumor antigens of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on tumor cells. Tumor antigens are proteins produced by tumor cells that elicit immune responses, particularly T cell-mediated immune responses. The choice of antigen-binding domain depends on the specific type of cancer being treated. Tumor antigens are well known in the art, and include, for example, glioma-associated antigens, 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 hs Examples of tumor antigens include p70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I receptor, IGF-II receptor, IGF-I receptor and mesothelin. The tumor antigens disclosed herein are included as examples only. This list is not intended to be exhaustive, and further examples will be readily apparent to those skilled in the art.

[0111] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several 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 the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, CD22, and CD37, are other candidate target antigens in B-cell lymphomas. 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.

[0112] The type of tumor antigen can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not exist on other cells in the body. TAAs are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. The expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or it can be an antigen that is normally present at very low levels on normal cells but is expressed at a significantly higher level on tumor cells.

[0113] Non-limiting examples of TSAs or TAAs include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage 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 resulting from 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, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-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-related protein, TA These include AL6, TAG72, TLP and TPS.

[0114] In preferred embodiments, the antigen binding domain portion of the CAR targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, etc. In yet another embodiment, provided herein is a DuoCAR comprising a tag or anti-tag binding domain.

[0115] Depending on the desired antigen to be targeted, the CAR can be engineered to contain an appropriate antigen-binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody specific for CD19 or its scFv subfragment can be used as the antigen-binding domain incorporated into the CAR.

[0116] In one exemplary embodiment, the antigen-binding domain portion of the CAR targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 scFV, wherein the nucleic acid sequence of the anti-CD19 scFV comprises the sequence set forth 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 the CAR comprises the amino acid sequence set forth in SEQ ID NO: 28. In a second exemplary embodiment, the antigen-binding domain of the CAR targets CD20. Preferably, the antigen-binding domain in the CAR is an anti-CD20 ScFv, wherein the nucleic acid sequence of the anti-CD20 ScFv comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the anti-CD20 ScFv portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 2. In a third exemplary embodiment, the antigen-binding domain of the CAR targets CD22. Preferably, the antigen-binding domain in the CAR is an anti-CD22 scFV, wherein the nucleic acid sequence of the anti-CD22 scFV comprises the sequence set forth in SEQ ID NO: 7. In another embodiment, the anti-CD22 scFV portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0117] In one embodiment of the present invention, a CAR is provided that is capable of binding to a non-TSA or non-TAA, including, for example, but not limited to, an antigen derived from Retroviridae (e.g., human immunodeficiency viruses, e.g., 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 types 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes viruses), Poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0118] In another aspect of the present invention, a CAR is provided that is capable of binding to antigens derived from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella, particularly infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, bacterial strains of Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus In one embodiment, CARs are provided that are capable of binding to antigens derived from Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof.

[0119] 2. Transmembrane domain In the DuoCARs used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein, the CAR comprises one or more transmembrane domains fused to the extracellular domain of the CAR.

[0120] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to the transmembrane domain.

[0121] 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.

[0122] In some cases, the transmembrane domain may be selected or by amino acid substitution to avoid binding of such domain to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0123] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present invention can be derived from (i.e., at least comprise) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, Fc epsilon R, or any combination thereof. Alternatively, the transmembrane domain can be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet or triple alanine motif provides a particularly suitable linker.

[0124] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 12.

[0125] In some cases, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 13. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 14.

[0126] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary DuoCAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein of the present invention include, for example, but not by way of limitation, a) the ability to induce leukemia and / or leukemia-associated ... These may include: a) improved lateral movement within the plasma membrane, allowing for more efficient signaling; b) superior location within plasma membrane microdomains such as lipid rafts and a greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane due to preferential movement away from inhibitory or down-regulatory interactions, e.g., less proximity to or fewer interactions with phosphatases such as CD45; and d) superior assembly into the T cell receptor signaling complex (i.e., immune synapse), or any combination thereof.

[0127] In one embodiment of the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein, non-limiting exemplary transmembrane domains for use in the DuoCAR disclosed herein include the TNFRSF16 and TNFRSF19 transmembrane domains that can be used to drive the TNFRSF transmembrane domain, and / or linker or spacer domains disclosed in applicant's co-pending provisional patent application Ser. No. 62 / 239,509, filed Oct. 9, 2015, entitled CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE, and in particular, other TNFRSF members listed within the tumor necrosis factor receptor superfamily listed in Table I, assigned Lentigen Technology, Inc., substance number LEN_015PRO.

[0128] 3. Spacer domain In the DuoCAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein, a spacer domain can be placed between the extracellular domain and the TNFRSF transmembrane domain, or between the intracellular domain and the TNFRSF transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to link the TNFRSF transmembrane domain with the extracellular domain and / or the TNFRSF transmembrane domain with the intracellular domain. A spacer domain contains up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0129] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in 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, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521 Nos. 5,284, 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, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0130] The spacer domain preferably has a sequence that promotes the binding of CAR to the antigen and enhances signal transduction into the cell. Examples of amino acids that are predicted 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 that constitute the spacer domain.

[0131] The spacer domain may be a whole or partial sequence of amino acids 137-206 (SEQ ID NO: 15) including the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). Alternatively, a portion of the constant region of an antibody heavy or light chain (CH1 or CL region, e.g., a peptide having the amino acid sequence set forth in SEQ ID NO: 16) may be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0132] Furthermore, a signal peptide sequence may be linked to the N-terminus of a CAR. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and have a length of 15 to 30 amino acids. Many of the protein molecules referred to above as intracellular domains have signal peptide sequences, and these signal peptides may be used as signal peptides for a CAR. 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.

[0133] 4. Intracellular domain The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0134] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capability.

[0135] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-dependently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0136] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0137] The CARs disclosed herein include primary cytoplasmic signaling sequences, which are particularly useful in Examples of ITAMs include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI RefSeq: NP_000724.1). The amino acids 402 to 495 of CD79a (NCBI RefSeq:NP_055022.2), 707 to 847 of CD79a (NCBI RefSeq:NP_001762.2), 166 to 226 of CD79a (NCBI RefSeq:NP_001774.1), 182 to 229 of CD79b (NCBI RefSeq:NP_000617.1), and CD66d (NCBI RefSeq:NP_000617.1) were identified. The present invention also includes peptides having a sequence of amino acids 177 to 252 of NCBI RefSeq:NP_001806.2, as well as variants thereof having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information in NCBI RefSeq ID or GenBank described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta. In another embodiment, one, two, or three of the ITAM motifs in CD3 zeta are attenuated by mutation or substitution of tyrosine residues with other amino acids.

[0138] In a preferred embodiment, the intracellular domain of the CAR can be designed to include a CD3-zeta signaling domain, either alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Specific, non-limiting examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI The present disclosure primarily exemplifies 4-1BB as a costimulatory signaling element, but other costimulatory elements are within the scope of the present disclosure.

[0139] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be randomly or specifically The amino acids may be linked to one another in a predetermined order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0140] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0141] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 17 and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 19.

[0142] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, 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-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0143] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 18 and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0144] 5. Further description of DuoCAR The functional portion of the DuoCAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein is also expressly included within the scope of the present invention. The term "functional portion," when used in reference to a CAR, refers to any one or more parts or fragments of the DuoCAR disclosed herein, which part or fragment retains the biological activity of the CAR (parent CAR) of which it is a part. A functional portion includes, for example, a portion of the CAR that retains the ability to recognize target cells or detect, treat, or prevent disease to a similar, the same, or greater extent than the parent CAR. With respect to the parent CAR, the functional portion may, for example, constitute about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0145] The functional portion can comprise additional amino acids at the amino or carboxy end of the portion, or at both ends, which additional amino acids are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional portion, such as, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc. More preferably, the additional amino acids enhance the biological activity of the functional portion compared to the biological activity of the parent CAR.

[0146] The functional variant of DuoCAR disclosed herein is included within the scope of this disclosure.The term "functional variant" as used herein refers to a CAR, polypeptide or protein that has substantial or significant sequence identity or similarity with the parent CAR, and this functional variant retains the biological activity of the CAR that it is a variant of.Functional variants can, for example, have a similar, the same or higher level of activity as the parent CAR. The term "functional variant" encompasses variants of the CARs described herein (parent CARs) that retain, to some extent, the ability to recognize target cells. With respect to the parent CAR, a functional variant can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.

[0147] A functional variant can, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR.

[0148] The amino acid substitutions in DuoCAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include those in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituting another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituting another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain substituting another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0149] A CAR can consist essentially of the specified amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not significantly alter the biological activity of the functional variant.

[0150] DuoCARs (including functional portions and functional variants) can be of any length, i.e., comprise any number of amino acids, provided that the DuoCAR (or functional portion or functional variant thereof) retains their biological activity, e.g., the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent a disease in a mammal, etc. For example, a CAR can be about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more amino acids in length.

[0151] DuoCARs (including functional portions and functional variants of the invention) can contain synthetic amino acids in place of 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-carboxyphenylalanine, 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'-methylaminopropanol, N ... These include α-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0152] DuoCARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts and / or optionally dimerized or polymerized, or conjugated.

[0153] DuoCAR (including its functional part and functional variant) can be obtained by methods known in the art.DuoCAR can be produced by any suitable method for producing polypeptide or protein.Suitable methods for de novo synthesis of polypeptide and protein 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, Reid, R. (ed.), Marcel Dekker, Inc., 2000; Epitope Mapping, Westwood et al. (ed.), Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Methods for making chimeric antigen receptors, T cells containing such receptors, and such uses (e.g., for the treatment of cancer) are known in the art and are further described herein (e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 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., 2013, pp. 1-9, each of which is incorporated herein by reference in its entirety). Oncol., 6:47, 2013; Tumaini et al., Cytotherapy, 15:1406-1417, 2013; Haso et al. (2013) Blood, 121:1165-1174; PCT Publication Nos. WO 2012 / 079000, WO 2013 / 12672; and U.S. Patent Application Publication No. 2012 / 0213783).For example, a nucleic acid molecule encoding a disclosed chimeric antigen binding receptor can be included in an expression vector (such as a lentiviral vector) that is used to transduce host cells, such as T cells, to make a disclosed CAR. In some embodiments, a method of using a chimeric antigen receptor includes isolating T cells from a subject, transducing the T cells with an expression vector (such as a lentiviral vector) that encodes the chimeric antigen receptor, and administering the CAR-expressing T cells to the subject for treatment, e.g., for treatment of a tumor in the subject.

[0154] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, an antibody or antigen-binding domain or portion thereof that specifically binds to one or more of the antigens disclosed herein, for use in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein. As used herein, "T cell expressing a CAR" or "CAR" refers to a T cell expressing a CAR. "T cell" means a T cell that expresses a CAR, e.g., has antigen specificity determined by the antibody-derived targeting domain of the CAR.

[0155] As used herein, an "antigen-binding domain" can include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as variants and fragments thereof that retain binding affinity to antigens.

[0156] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. In some examples, a monoclonal antibody is an antibody produced by a single clone of B lymphocytes, or an antibody produced by a cell transfected with nucleic acid encoding the antibody light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or its progeny. In some examples, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0157] Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules.

[0158] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy and light chains combine to specifically bind to an antigen. In further 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). References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including antigen-binding fragments such as Fv, scFv, dsFv, or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0159] The variable regions of the light and heavy chains 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, USDapartment of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, is arranged in three-dimensional space. It functions to position and align the CDRs.

[0160] CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be determined by the methods described in 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 scheme"). 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 typically referred to as CDR1, CDR2, and CDR3 (N- to C-terminus) and are also typically identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

[0161] "Antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize their cognate antigen, as well as various combinations of such portions. 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 engineering whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd Edition, Springer Press, 2010).

[0162] Single-chain antibodies (scFvs) are genetically engineered molecules containing the VH and VL domains of one or more antibodies (or antibodies) linked by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in scFvs is typically not critical for scFvs. Thus, scFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0163] In dsFv, the heavy and light variable chains are 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 use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain and create two antigen-binding sites (e.g., Holliger et al., Proc. Natl. Acad. Sci. 90:6444-6448, 1993; Poljak et al., Structure and Biology 1999, 10:1444-1448, 1993). , Vol. 2:1121-1123, 1994).

[0164] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0165] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of generating, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 14:243-246 (1993)). 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd Edition (Oxford University Press 1995); each of which is incorporated herein by reference.

[0166] 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; conversely, 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.

[0167] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly for the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0168] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0169] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from the human genome (or derived from the human genome) but does not contain sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region derived from the human genome (or derived from the human genome). Human antibodies can be identified and isolated using technology to create sequences based on sequences derived from the human genome, for example, by phage display, or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manual). uel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004 Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0170] An antibody can have one or more binding sites. If more than one binding site is present, these binding sites may be identical to one another or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0171] Methods for testing antibodies for the ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929).

[0172] The CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof, can also comprise a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., a gold particle).

[0173] C. Conjugate The DuoCAR, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments thereof specific for one or more of the antigens disclosed herein used in the patient-specific autologous anti-tumor lymphocyte cell population(s) disclosed herein can be conjugated to agents such as effector molecules or detectable markers using several means known to those skilled in the art. Both covalent and non-covalent binding means can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. Those skilled in the art will recognize that the conjugation of DuoCAR, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using several means known to those skilled in the art. Both covalent and non-covalent binding means can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. Those skilled in the art will recognize that the conjugation of DuoCAR, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments thereof specifically bind to one or more of the antigens disclosed herein can be conjugated to chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents, etc. 125 I, 32 P, 14 C. 3 H and 35 It will be understood that a variety of effector molecules and detectable markers may be used, including, but not limited to, S, as well as other labels, targeting moieties, ligands, and the like.

[0174] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to bring about the death of a particular target cell (e.g., a tumor cell).

[0175] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on an antibody to result in attachment of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization can be achieved using any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker may comprise any bond. The linker may be any molecule used to attach an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker is capable of forming covalent bonds to 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-chain or branched-chain 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 linkers may be attached to the constituent amino acids via their side groups (e.g., via disulfide linkages to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0176] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in U.S. Patent Nos. 7,964,566,7, 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, 5,532, 5,521, 5,533, 5,534, 5,535 ... Nos. 5,284, 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, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0177] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is non-cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolea). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids in length. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, incorporated herein by reference. In specific embodiments, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0178] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 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, such as in blood, but are unstable at pHs below 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Pat. No. 5,622,929).

[0179] In other embodiments, the linker is cleavable under reducing conditions (eg, 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-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford University Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0180] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0181] In still other embodiments, the linker is non-cleavable and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, the entire contents of which are incorporated herein by reference).

[0182] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating a conjugate containing the desired linker with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of free effector molecule or detectable marker present in the plasma. Various exemplary linkers that can be used in the conjugate are described in WO2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, and the like. No. 2005 / 0238649 and U.S. Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0183] In some embodiments, conjugates of a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecines, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0184] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthetically prepared maytansinol and maytansinol analogs according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; and 4,311,140. Nos. 3,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for making same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0185] Additional toxins can be used with CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Exemplary toxins include Pseudomonas exotoxin (PE), ricinus toxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, and botulinum toxins A-F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of these toxins (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401).

[0186] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin does not possess a mechanism for specific entry into cells and therefore requires an antibody that recognizes cell surface proteins that are internalized in order to be efficiently taken up by cells. Conjugation to the antibody or antigen-binding fragment is required.

[0187] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0188] Castor toxin is the lectin RCA60 from Ricinus communis (castor bean). For examples of castor toxins, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) is a lectin derived from the plant Ricinus communis (castor bean), according to its molecular weight of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 The toxin exists in two forms, termed the A chain and the B chain (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds the toxin to cell surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

[0189] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345(2):247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the enediyne antitumor antibiotic family, generating double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0190] Abrin includes toxic lectins from Abrus precatorius. The toxicants, abrins a, b, c, and d, have molecular weights of approximately 63 kD and 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0191] The CARs, CAR-expressing T cells, monoclonal antibodies specific for one or more of the antigens disclosed herein, antigen-binding fragments thereof used in the patient-specific autologous anti-tumor lymphocyte cell population(s) can also be conjugated to a detectable marker; for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic testing, and laparoscopic testing). Detection Specific, non-limiting examples of possible markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When a CAR, CAR-expressing T cells, antibody, or antigen-binding portion thereof is conjugated with a detectable enzyme, it can be detected by adding an additional reagent that the enzyme uses to produce a discernible reaction product. For example, in the presence of the drug horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CAR, CAR-expressing T cells, antibody, or antigen-binding portion thereof can also be conjugated with biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with an enzyme or fluorescent label.

[0192] CAR, CAR-expressing T cells, antibodies, or their antigen-binding portions can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag).

[0193] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can also be conjugated with radiolabeled amino acids.Radiolabels can be used for both diagnostic and therapeutic purposes.For example, radiolabels can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, emission spectroscopy, or other diagnostic techniques.In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, for example, for treating neuroblastoma.Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0194] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0195] D. Nucleotides, Expression, Vectors and Host Cells Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the DuoCARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein. The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0196] In one embodiment, an isolated nucleic acid molecule is provided that encodes a chimeric antigen receptor (CuoCAR) comprising, from N-terminus to C-terminus, at least one extracellular antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0197] In one embodiment of a CAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to an antigen.

[0198] In another embodiment of a CAR for use in the patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to an antigen.

[0199] In yet another embodiment of a CAR for use in the patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular antigen-binding domain comprises at least one antigen binding antigen of the lipocalin system (anticalin) that binds to the antigen.

[0200] In one embodiment of a CAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule is provided in which an encoded extracellular antigen-binding domain is connected to a transmembrane domain by a linker domain.

[0201] In another embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, in which the encoded extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0202] In yet another embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular antigen-binding domain 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, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0203] In certain embodiments of DuoCARs used in the patient-specific autologous anti-tumor lymphocyte cell population(s), the encoded extracellular antigen-binding domain is an anti-CD19 scFV antigen-binding domain, an anti-CD20 scFV antigen-binding domain, an anti-CD22 scFV antigen-binding domain, an anti-ROR1 scFV antigen-binding domain, an anti-TSLPR scFV antigen-binding domain, an anti-mesothelin scFV antigen-binding domain, an anti-CD33 / IL3Ra 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-BCMA (CD269) 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, anti-glycolipid F77 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 Provided is an isolated nucleic acid molecule encoding a CAR comprising a TCR scFV antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0204] In one embodiment of a DuoCAR used in a patient-specific autologous anti-tumor lymphocyte cell population(s), the DuoCAR provided herein further comprises a linker domain.

[0205] In one embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular antigen-binding domain, an intracellular signaling domain, or both, is connected to a transmembrane domain by a linker domain.

[0206] In one embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane domain.

[0207] In yet another embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the nucleic acid sequence encoding the transmembrane domain comprises a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0208] In one embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded transmembrane domain comprises an amino acid sequence that includes no more than 10 but at least one modification, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0209] In another embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0210] In yet another embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0211] In one embodiment of a CAR disclosed herein, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is positioned C-terminal to the CD3 zeta intracellular domain.

[0212] In another embodiment of a DuoCAR for use in a patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0213] DuoCAR used in patient-specific autologous anti-tumor lymphocyte cell population(s) In a further embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one costimulatory 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), or a combination thereof.

[0214] In one embodiment of a DuoCAR for use in the patient-specific autologous anti-tumor lymphocyte cell population(s), an isolated nucleic acid molecule encoding a CAR is provided that further comprises a leader or signal peptide sequence.

[0215] In some embodiments, nucleotide sequence can be codon modified.Without being bound by any particular theory, it is believed that codon optimization of nucleotide sequence can increase the translation efficiency of mRNA transcript.The codon optimization of nucleotide sequence can include replacing native codon with another codon that encodes the same amino acid but can be translated by tRNA that is more readily available in cells, thus increasing translation efficiency.The optimization of nucleotide sequence can also reduce the secondary mRNA structure that interferes with translation, thus increasing translation efficiency.

[0216] In embodiments of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the DuoCARs described herein (including functional portions and functional variants thereof).

[0217] "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that can be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and can contain natural, non-natural, or modified nucleotides, and can contain natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0218] Recombinant nucleic acids can have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques such as those described in Sambrook et al., supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosin, e), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil Examples of nucleic acids include, but are not limited to, uracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0219] The nucleic acid can comprise any isolated or purified nucleotide sequence encoding DuoCAR or any of its functional portions or variants. Alternatively, the nucleotide sequence can comprise a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0220] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0221] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably stronger than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that happen to have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length complements of 14-17 or more bases, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at temperatures of about 50-70°C. Such highly stringent conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any of the DuoCARs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0222] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein.

[0223] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For purposes of this specification, the term "recombinant expression vector" refers to a vector in which the construct is capable of expressing mRNA, protein, polypeptide, or other polypeptides. A vector refers to a genetically engineered oligonucleotide or polynucleotide construct that enables expression of an mRNA, protein, polypeptide, or peptide by a host cell when the vector contains a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. A vector as a whole does not exist in nature.

[0224] However, portions of the vector may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or derived from partially natural sources, and may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

[0225] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0226] Bacteriophage vectors, such as λυTΙO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149, can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, for example, but not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, etc. Non-clinical forms of lentiviral vectors are also available and known to those skilled in the art.

[0227] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:97 (1981)).

[0228] Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:101-102 (1980)), and direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)). :742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high velocity microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0229] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0230] Recombinant expression vectors, taking into account whether the vector is DNA- or RNA-based, can optionally include regulatory sequences, e.g., transcriptional and translational initiation and termination codons, specific to the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced. Recombinant expression vectors can include restriction sites to facilitate cloning.

[0231] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0232] The recombinant expression vector may comprise a native or non-native promoter operably linked to the nucleotide sequence encoding the CAR (including its functional portion and functional variant), or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR. The selection of a promoter, for example, strong, weak, inducible, tissue-specific, and developmentally specific, is within the skill of those skilled in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0233] Recombinant expression vectors can be designed for transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0234] Additionally, recombinant expression vectors can be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell that expresses the suicide gene. A suicide gene can be a gene that confers sensitivity to an agent, such as a drug, on the cell in which it is expressed, or a gene that causes the cell to die when contacted or exposed to the agent. Suicide genes are known in the art (e.g., Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK) K), Humana Press, 2004), which include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase (daminase), purine nucleoside phosphorylase, and nitroreductase.

[0235] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protist. The host cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. The host cell can be of any cell type, originate from any type of tissue, and be at any stage of development, but the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell can be a T cell.

[0236] For purposes herein, T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. When obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from a human. T cells can be any type of T cell, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc., and can be at any stage of development. T cells can be CD8+ T cells or CD4+ T cells.

[0237] In one embodiment, the DuoCAR described herein can be used in suitable non-T cells, such as cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0238] Also provided by one embodiment is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any recombinant expression vector, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, wherein the population primarily comprises host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0239] DuoCAR (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof) and antibodies (including antigen-binding portions thereof) are disclosed and described herein. and / or purified. For example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a host cell preparation is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, greater than about 60%, about 70%, or about 80%, or may be about 100%.

[0240] E. Treatment Method It is contemplated that the DuoCAR used in the patient-specific autologous anti-tumor lymphocyte cell population(s) can be used in a method for treating or preventing disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal a DuoCAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal. Additional methods for using the above-mentioned DuoCAR are disclosed above.

[0241] One embodiment further comprises lymphodepleting the mammal prior to administering the DuoCAR disclosed herein. Examples of lymphodepletion can include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0242] For the purpose of the method of administering host cells or a group of cells, the cells can be allogeneic or autologous to the mammal.Preferably, the cells are autologous to the mammal.As used herein, allogeneic refers to any material derived from a different animal of the same species as the individual into which the material is introduced.Two or more individuals are said to be allogeneic to each other if the genes are not identical at one or more loci.In some embodiments, allogeneic materials from individuals of the same species can be genetically sufficiently different to interact antigenically.As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0243] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, e.g., mice and hamsters, and logomorph mammals, e.g., rabbits. The mammal may be from the carnivora order, including felines (cats) and canines (dogs). The mammal may be from the artiodactyla order, including bovines (cows) and swines (pigs), or from the perissodactyla order, including equines (horses). The mammal may be from the primate order, ceboids or simoids (monkeys), or anthropoids (humans and apes). Preferably, the mammal is a human.

[0244] For these methods, the cancer may be selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, melanoma, leukemia ... The cancer may be any cancer, including any of the following: melanoma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0245] The terms "treat" and "prevent," and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art would recognize as having a potential beneficial or therapeutic effect. In this regard, the method may provide any amount or level of treatment or prevention of cancer in a mammal.

[0246] Furthermore, the treatment or prevention provided by this method can include treatment or prevention of one or more conditions or symptoms of the disease, such as cancer, being treated or prevented. Also, for purposes herein, "prevention" can include delaying the onset of the disease, or its symptoms or conditions.

[0247] Another embodiment provides a method for detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising one or more cells from the mammal with a DuoCAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0248] The sample can be obtained by any suitable method, for example, biopsy or autopsy.Biopsy is the removal of tissue and / or cells from an individual.Such removal can be the collection of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells.This experiment can include experiments to determine whether the individual has a certain condition or disease state and / or whether they are suffering from a certain condition or disease state.The condition or disease can be, for example, cancer.

[0249] For embodiments of methods for detecting the presence of a proliferation disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. When the sample contains whole cells, these cells can be any cells of a mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0250] The contacting step can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting step is in vitro.

[0251] Also, detection of complexes can be carried out by many methods known in the art.For example, the DuoCAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or antibody or its antigen-binding portion can be labeled with detectable label, such as the radioisotope disclosed above, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particle (e.g., gold particle), etc.

[0252] Methods for testing CARs for their ability to recognize target cells and antigen specificity are known in the art. For example, Clay et al., J. Immunol. 163:507-513 (1999) teaches a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin 2 (IL-2)). Furthermore, CAR function can be evaluated by measuring cytotoxicity, as described in Zhao et al., J. Immunol. 174:4415-4423 (2005).

[0253] Another embodiment provides the use of the DuoCAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody or antigen-binding portion thereof, and / or pharmaceutical composition of the invention to treat or prevent a proliferative disorder, such as cancer, in a mammal. The cancer can be any of the cancers described herein.

[0254] Any administration method, including local and systemic administration, can be used for the disclosed therapeutic agents. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific administration mode and dosing regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). When more than one agent or composition is administered, one or more administration routes can be used; for example, the chemotherapeutic agent can be administered orally, and the antibody or antigen-binding fragment or conjugate or composition can be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposome. In some embodiments, local administration of the disclosed compounds can be used, for example, by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment can be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops or intravitreally to the eye.

[0255] The disclosed therapeutic agents can be formulated in unit dosage forms suitable for individual administration of precise dosage amounts. Furthermore, the disclosed therapeutic agents can be administered in a single dose or in a multiple-dose schedule. A multiple-dose schedule is one in which the main course of treatment may involve more than one discrete dose, e.g., 1 to 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound(s) over a period of several days to several months or even years. Thus, the dosing regime will also be determined, at least in part, based on the specific needs of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0256] Typical dosages of antibodies or conjugates can range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0257] In particular examples, the subject is administered a therapeutic composition comprising one or more of the conjugate, antibody, composition, DuoCAR, CAR T cell, or additional agent in a multiple daily dosing schedule, e.g., at least 2 consecutive days, 10 consecutive days, etc., for a period of, e.g., weeks, months, or years. In one example, the subject is administered the conjugate, antibody, composition, or additional agent for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0258] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy, and / or chemotherapy in combination with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells (e.g., sequentially, substantially simultaneously, or simultaneously). Such agents and treatment methods and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for additional agents can be used according to manufacturer's instructions or can be as empirically determined by one skilled in the art. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) MC Perry (ed.), Williams & Wilkins, Baltimore, Md.

[0259] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor.Non-limiting examples of additional therapeutic agents that can be used in the combination therapy include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein.The methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by skilled clinicians.

[0260] Additional chemotherapeutic agents for combination immunotherapy include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids amides, such as podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; tumor-affinity photosensitizing dyes such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other drugs, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such drugs are known to those skilled in the art and can be determined by a skilled clinician.

[0261] In certain embodiments of the invention, the methods described herein or the T cells are administered at a therapeutic level. Cells activated and expanded using other methods known in the art or expanded at a low level are administered to patients in combination with (e.g., before, concurrently with, or after) any number of relevant treatment modalities, including, but not limited to, treatment with agents such as the antiviral therapy cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efalizumab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or p70S6 kinase (rapamycin), which is important for growth factor-induced signal transduction (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, concurrently with, or after) bone marrow transplantation, T-cell depletion therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATHT. In another embodiment, the cell compositions of the present invention are administered after B-cell depletion therapy, such as an agent reactive with CD20, e.g., Rituxan. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0262] The dosages of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Adjustments to dosages for human administration can be made according to art-accepted practice. Dosages for CAMPATH, for example, generally range from 1 to about 100 mg for an adult patient, usually administered daily for a period of 1 to 30 days. A preferred daily dose is 1 to 10 mg per day, although higher doses of up to 40 mg per day may be used in some cases.

[0263] Combination therapy can provide synergistic effects and can be proven to be synergistic, that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that can be obtained from using these compounds separately.Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen.When delivered alternately, synergistic effects can be achieved when these compounds are administered or delivered sequentially, for example, by different injections in separate syringes.Generally, during alternation, each active ingredient in an effective dosage is administered continuously, that is, sequentially, whereas in combination therapy, two or more active ingredients in an effective dosage are administered together.

[0264] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on each cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is effective.

[0265] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biologic compositions (hereinafter "compositions") for use in gene therapy, immunotherapy, adoptive immunotherapy, and / or cell therapy, comprising one or more of the disclosed CARs, or T cells expressing a CAR, an antibody, an antigen-binding fragment, a conjugate, a CAR, or T cells expressing a CAR that specifically binds to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier). These compositions can be prepared in unit dosage form for administration to a subject. The amount and timing of administration to achieve a desired outcome are at the discretion of the treating clinician. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed DuoCAR, or T cells expressing a CAR, an antibody, an antigen-binding fragment, a conjugate, is formulated for parenteral administration, such as intravenous administration. The compositions comprising the DuoCARs disclosed herein, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are used for, for example, treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some cases, these compositions are useful for the treatment or detection of cancer. The compositions comprising the DuoCARs disclosed herein, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are also used, for example, to detect pathological angiogenesis.

[0266] Compositions for administration can include a solution of CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. The compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant drugs, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are known or will become apparent to those skilled in the art.

[0267] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those of skill in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0268] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, can be provided in lyophilized form and rehydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. A solution of DuoCAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates, is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the field for administering antibody or antigen-binding fragment and conjugate drugs; for example, antibody drugs have been commercially available in the United States since the approval of RITUXAN® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates, can be administered by slow infusion rather than intravenous infusion or intravenous bolus. In one example, a higher load For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) can be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg for 4 to 8 weeks infused over a period of 30 minutes if the previous dose was well tolerated.

[0269] Controlled-release parenteral formulations can be prepared as implants, oily injections, or granular systems. For a broad review of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules, are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so only nanoparticles can be administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc., New York, NY, pp. 315-339 (1992).

[0270] Polymers can be used for ion-controlled release of the DuoCAR or CAR-expressing T cells, antibodies or antigen-binding fragments, or conjugate compositions disclosed herein. Various degradable and non-degradable polymer matrices used for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but still mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0271] G.Kit In one embodiment, a kit is also provided that uses the DuoCAR disclosed herein.For example, a kit for treating tumors in a subject or a kit for producing CAR T cells that express one or more of the DuoCARs disclosed herein.The kit typically includes the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or CAR-expressing T cells disclosed herein.More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, DuoCAR or CAR-expressing T cells can be included in the kit.

[0272] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, DuoCARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0273] The label or package insert typically further includes instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, DuoCAR, or CAR-expressing T cells, for example, in a method for treating or preventing tumors or a method for generating CAR T cells. The package insert typically includes instructions customarily included in the commercial packaging of a therapeutic product, including information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. The instructional material can be written in electronic form (e.g., a computer diskette or compact disc) or visual (e.g., a video file). The kit can also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit can further include a means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit can further include buffers and other reagents routinely used for the implementation of a particular method. Such kits and their appropriate contents are well known to those skilled in the art. [Example]

[0274] Example The present invention is further illustrated by the following attached drawings and examples of DuoCAR shown in the disclosure on pages 17-27, including the above, which examples should not be construed as imposing limitations on its scope in any way. On the contrary, reliance must be placed on various other embodiments, modifications, and equivalents thereof, which will readily be understood to occur to those skilled in the art after reading the description herein without departing from the spirit of the invention and / or the scope of the appended claims.

[0275] While various details have been described in conjunction with the exemplary implementations outlined above, various changes, modifications, variations, improvements and / or substantial equivalents, whether known or presently anticipated or foreseeable, may become apparent upon review of the foregoing disclosure.

[0276] Each application and patent cited herein, and each document or reference cited therein (including each issued patent in litigation, the "Application Citations"), and any other document or reference corresponding to and / or claiming priority to any of such applications and patents. Each PCT and foreign application or patent cited herein, and each document cited or referenced in each application cited document, is hereby expressly incorporated herein by reference and may be used in practicing the invention. More generally, documents or references are cited either in the text, in a reference list before the claims, or in the text itself, and each such document or reference ("herein-cited references"), and each document or reference cited in each herein-cited reference (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

[0277] The foregoing description of some specific embodiments provides sufficient information to enable others, by applying knowledge of the present invention, to easily modify or adapt the specific embodiments for various applications without departing from the general concept; therefore, such adaptations and modifications should, and are intended to, be understood as being within the meaning and range of equivalents of the disclosed embodiments. It is understood that the words or terms used herein are for purposes of description and not of limitation. In the drawings and description, exemplary embodiments are disclosed, and although specific terminology may be employed, unless otherwise noted, they are used in a generic and descriptive sense only, and not for purposes of limitation, and the claims are therefore not so limited. Furthermore, those skilled in the art will recognize that certain steps of methods discussed herein can be sequenced in a different order or steps can be combined. Accordingly, it is intended that the appended claims not be limited to the precise embodiments disclosed herein. Those skilled in the art will be able to recognize and ascertain, using no more than routine experimentation, many equivalents to the embodiments of the invention described herein. Such equivalents are encompassed by the following claims.

[0278] Description of the Examples Five examples are provided, whereby the expression of three functional binding domains on the surface of LV-transduced human T cell populations and the combination of different costimulatory intracellular domains demonstrates the feasibility of DuoSet technology (Example 1), and the functional activity of this population against three different leukemia antigens demonstrates its efficacy (Example 2). A comparison of the expression and function of DuoCARs generated by cotransfection, aka transduction, with a single LV product encoding both DuoCAR chains (generated by cotransfection of a packaging system with plasmids encoding the two CARs) is described in Example 3. In Example 4, DuoCARs transduced with LVs generated by the cotransfection method and a bicistronic DuoCAR, in which the two DuoCAR chains are encoded by a single construct separated by a ribosomal skip site, are compared for transduction efficiency and function.

[0279] An example of a monospecific CAR on which this technology is based and which can be included as a DuoSet component in a DuoCAR is the single CD20-targeting vector LTG1495, with the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. A second example is the CD22-specific monospecific CAR LTG2200, with the nucleotide sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10. A key molecular aspect in creating a DuoCAR is the inclusion of non-redundant matching sequences and the evaluation of these sequences in transduced T cells to prevent unexpected recombination or intracellular association. This can occur in both the vector's producer cell line or the target cell population. For this reason, we included variant CAR structures known to be compatible with the DuoCAR context. These are the CD19-specific CARs described in the nucleotide sequence of SEQ ID NO: 29 and the amino acid sequence of SEQ ID NO: 30, respectively. LTG1494. This sequence contains a sc linker called the Whitlow linker (amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 107); see Whitlow M. et al., 1993, Protein Eng. 6:989-995). It includes well-described linkers joining the heavy and light chains of FV4. In some cases, the Whitlow linker is, for example, (GGGGS) in the CD19 CAR format, as in LTG1538, which has the nucleotide sequence of SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 32, respectively. nThe linker was replaced with a linker. In another example, a CAR with an alternative transmembrane domain was created. The anti-CD19 CAR LTG1562, with the nucleotide sequence of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22, respectively, utilizes the CD4 (as opposed to CD8) transmembrane domain. Similarly, the anti-CD19 CAR LTG1563, with the nucleotide sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50, respectively, has an alternative transmembrane domain derived from TNFRSF19. DuoCARs can also be targeted to solid tumors, such as those expressing the mesothelin tumor antigen. For example, an scFV binder of the nucleotide sequence of SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 38, respectively, disclosed in Applicant's co-pending provisional patent application Ser. No. 62 / 444,201, filed Jan. 9, 2017, entitled "Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy," and assigned substance number LEN_017 to Lentigen Technology, Inc., was created for mesothelin, which can be incorporated into a functional CAR of the nucleotide sequence of SEQ ID NO: 39 and the amino acid sequence of SEQ ID NO: 40, respectively, and thereby incorporated into DuoCAR therapy. In addition to scFv sequences, single-chain antigen binders (as opposed to scFvs) can be incorporated into DuoCAR applications. For example, the CD33-specific heavy chain-only binder of the nucleotide sequence of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42, respectively, disclosed in Applicant's co-pending provisional patent application No. 62 / 476,438, filed March 24, 2017, entitled "Compositions and Methods for Treating Cancer with Anti-CD33 Immunotherapy," and assigned substance number LEN_018 to Lentigen Technology, Inc., can be incorporated into the functional CAR LTG1906 of the nucleotide sequence of SEQ ID NO: 43 and the amino acid sequence of SEQ ID NO: 44, respectively, which targets CD33-expressing malignancies. One example of a DuoCAR therapeutic application is the treatment of leukemias that express CD19, CD20, and TSLPR antigens.In this case, LTG1496 or LTG1497 (SEQ ID NOs: 35, 26, respectively) can be combined with the TSLPR-specific CAR (LTG1789) of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 48, respectively, which was created from the TSLPR-specific scFV domain of the nucleotide sequence of SEQ ID NO: 45 and the amino acid sequence of SEQ ID NO: 46.

[0280] Another example of a DuoCAR therapeutic application is the treatment of cancers that express the CD38 antigen. For example, the CD38-specific binder disclosed in the applicant's co-pending provisional patent application No. 62 / 773,940, entitled "Compositions and Methods for Treating Cancer with Anti-CD38 Immunotherapy," filed November 30, 2018; and assigned Lentigen Technology, Inc. substance number LEN_026, can be incorporated into one or more functional CARs that target CD38-expressing malignant tumors, as disclosed in the applicant's co-pending provisional patent application No. 62 / 773,940, the entire contents of which are incorporated herein by reference.

[0281] Another example of DuoCAR therapeutic application is the treatment of cancers that express CD123 antigen. For example, the CD123-specific binder disclosed in applicant's co-pending U.S. patent application Ser. No. 16 / 578,063, entitled "Compositions and Methods for Treating Cancer by Anti-CD123 Immunotherapy," filed on September 20, 2019; and assigned to Lentigen Technology, Inc., substance number LEN_024; and claiming the benefit of priority from provisional patent application Ser. No. 62 / 734,106, filed on September 20, 2018, can be incorporated into one or more functional CARs that target CD123-expressing malignant tumors, as disclosed in applicant's co-pending U.S. patent application Ser. No. 16 / 578,063, the entire contents of which are incorporated herein by reference.

[0282] Another example of a DuoCAR therapeutic application is the treatment of cancers that express the BCMA antigen. For example, the BCMA-specific binder disclosed in applicant's co-pending provisional patent application No. 62 / 854,574, filed May 30, 2019, entitled "Fully Human BCMA CAR T Cells for the Treatment of Multiple Myeloma and Other BCMA-Positive Malignancies" and assigned substance number MBG_13 to Lentigen Technology, Inc., can be incorporated into one or more functional CARs that target BCMA-expressing malignancies, as disclosed in applicant's co-pending provisional patent application No. 62 / 854,574, the entire contents of which are incorporated herein by reference.

[0283] An example of a tandem CAR (comprising two scFv domains set forth in the nucleotide sequence of SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 24) on which this technology is based includes the CD20_CD19 CAR LTG1497, which has the nucleotide sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 26. In some cases, reversing the order of the two binders may provide better DuoCAR expression in target cells. Thus, both LTG1497, in which the CD19 scFv is more proximal, as set forth in the nucleotide sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 26, and LTG1496, in which the CD19 scFv is more distal to the membrane, as set forth in the nucleotide sequence of SEQ ID NO: 33 and the amino acid sequence of SEQ ID NO: 34, can be used as one member of a DuoSet containing a DuoCAR.

[0284] Methods utilized in Examples 1 and 2: Cell lines (PBMC and target) All cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA) unless otherwise noted. The Burkitt lymphoma cell line Raji, the acute lymphoblastic leukemia cell line REH, and the chronic myeloid leukemia cell line K562 were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney cell line 293T was propagated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FBS.

[0285] The wild-type tumor line was stably transduced with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones to generate single-cell clones of the luciferase-expressing cell line. The mouse-adapted Raji-luc line was transduced into NSG mice (NOD.Cg-Prkd). scid Il2rg tm1Wjl Raji clones stably expressing firefly luciferase were generated by transplanting Raji clones into mice (J.I. / SzJ, The Jackson Laboratory, Sacramento, CA), isolating transplanted Raji-Luc tumor cells from mouse spleens by either positive selection (CD19 microbeads, human, Miltenyi Biotec, Bergisch Gladbach, Germany) or negative selection (mouse cell depletion kit, Miltenyi Biotec), expanding them in culture, and recloning to facilitate selection of clones with high expression of firefly luciferase at the Oklahoma Blood Institute (OBI, Oklahoma). Whole blood was collected from healthy volunteers with written donor consent at the University of California, San Diego, California. Processed buffy coats were purchased from OBI. CD4+ and CD8+ human T cells were purified from the buffy coats by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0286] Creation of chimeric antigen receptors (CARs) - Expression vectors containing DuoCAR The CAR antigen-binding domain ScFv sequences were derived from the murine hybridoma FMC-63 (FMC-63: AA1-267, GenBank ID: HM852952.1) for CD19 and Leu-16 for CD20 [1] for the entire VL and VH sequences. The CD22 scFv binding was created from publicly available sequences. Tandem CAR19_20 or CAR20_19 was generated by linking each antibody scFv in frame to the CD8 hinge and transmembrane domain (AA123-191, Ref Sequence ID NP_001759.3), 4-1BB (CD137, AA214-255, UniProt Sequence ID Q07011) transactivation domain, and CD3 zeta signaling domain (CD247, AA52-163, Ref Sequence ID: NP_000725.1). The scFv regions of 19A and 20A were linked in sequence by a flexible interchain linker (GGGGS)5 (SEQ ID NO: 108), followed by the CD8, 4-1BB, and CD3 zeta domains. A leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit was included in all constructs, as described in [2]. CAR construct sequences were codon-optimized (DNA2.0, Newark, CA) and cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD) under the control of the human EF-1α promoter. Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK 293T cells, as previously described in [3]. The harvested pelleted lentiviral supernatants were stored at -80°C.

[0287] Primary T cell transduction: Selected CD4+ and CD8+ human primary T cells from normal donors were cultured at 0.3–2 × 10 in TexMACS medium (serum-free) supplemented with 40 IU / ml IL-2. 6Cells were cultured at a density of 1000 cells / ml, activated with CD3 / CD28 MACS® GMP TransAct reagent (Miltenyi Biotec), and transduced overnight with a lentiviral vector encoding a CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 3, with the medium changed on day 4. On day 5, the cultures were transferred to TexMACS medium supplemented with 200 IU / ml IL-2 and propagated until harvest on days 10–13.

[0288] Immune effector assay: To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were mixed with CAR T cells at various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison, WI) was added to each well, and the resulting luminescence was analyzed in an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells with 1% Tween-20 (minimum CPS) were used to determine the assay range. Percent specific lysis was calculated as (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)).

[0289] Flow cytometry analysis: All cell staining reagents for flow cytometry were from Miltenyi Biotec unless otherwise noted. One million CAR T-transduced cells were harvested from culture, washed twice in cold staining buffer (AutoMACS solution with 0.5% bovine serum albumin), and pelleted at 350 x g for 5 minutes at 4 °C. CAR surface expression in transduced T cells was first stained with protein L-biotin conjugate (stock 1 mg / ml, 1:1000 dilution, GenScript, Piscataway, NJ) for 30 minutes at 4 °C, followed by two washes and 30 minutes at 4 °C with streptavidin-PE conjugate (stock: 1.0 ml, 1:20 dilution). CAR T-positive cells were detected by staining with 100 μl of 7AAD (Jackson ImmunoResearch Laboratories, West Grove, PA) at 100 μL dilution. Non-transduced and transduced cells stained with streptavidin-PE alone served as negative controls. The CD4 to CD8 ratio of the CAR T-positive population was determined using an anti-CD4 antibody, which was added during the second incubation step. Dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Specific DuoSet CAR T staining was performed on human T cells activated with DuoSet vector-transduced CD3-CD28 nanomatrix (TRansAct, Miltenyi Biotec) in the presence of IL-2 and analyzed for expression of CD19-, CD20-, or CD22-scFv domains by flow cytometry using recombinant CD19, CD20, or CD22 for staining against antibodies.

[0290] Anti-CD19 scFv activity was detected with CD19-Fc (R&D Biosystems) used at 1 μg / sample and stained with goat anti-human Fc-gamma-R-PE (Jackson ImmuoResearch Laboratories, Inc.) at 0.75 μg / sample. Anti-CD20 scFv activity was detected with CD20-biotin (Miltenyi Biotech) at 0.11 μg / sample and streptavidin pAPC (Miltenyi Biotec) at 0.2 μg / sample. Anti-CD22 scFc activity was detected with CD22-His (Thermo Fisher) at 0.1 μg / sample and anti-His FITC (Miltenyi Biotec). Flow cytometry analysis was performed on a MACSQuant® 10 Analyzer (Miltenyi Biotec). Target tumor lines and luciferase-positive subclones were characterized using CD19-FITC, CD20 VioBlue, and CD22-APC antibodies. Dead cells were excluded from the analysis by 7AAD staining (BD Biosciences, San Jose, CA).

[0291] Example 1 Expression of DuoCAR (2+1 DuoSet) in primary human T cells As a proof of principle, a DuoSet consisting of two CAR-T vectors was created. One member of the set expressed a tandem CD20_CD19 binding domain linked to a CD8 transmembrane and CD28 and CD3-zeta signaling domains (LTG2228), SEQ ID NO: 51 and SEQ ID NO: 52. The second member of the DuoSet was a CAR construct with a CD8 transmembrane and a single CD22 binder linked to a 4-1BB and CD3-zeta signaling domain (LTG2200), SEQ ID NO: 9 and SEQ ID NO: 10. In Figure 7, paired columns show double staining, with CD20 and CD19 scFv in the left column and CD22 and CD19 scFv in the right column. The first row shows untransduced (UTD) T cells and therefore shows no binding. The second row shows T cells transduced with LV encoding the CD20_CD19 CAR vector, which has a CD8 transmembrane domain and intracellular CD28 and CD3-zeta signaling domains (20-19-28z). Double staining is seen for CD20 and CD19 binding (left panel), but only CD19 binding is seen in the right panel. The third row shows T cells transduced with a CD22 CAR vector, which has a CD8 transmembrane domain and intracellular 4-1BB and CD3-zeta signaling domains (22-BBz). Double staining is not seen for CD19 or CD20 (left panel), and only a single population of cells capable of binding CD22 is seen (right panel). In the fourth row, T cells are transduced with a DuoSet consisting of both vectors from rows two and three. Only DuoSet expresses all three CAR-encoded binding domains (42% of cells express CD20_19 (left panel) and 38% express CD22 and CD19 binding domains (right panel)). Since v is on each of the two distinct transmembrane proteins that comprise DuoSet, 38% represents the population that expresses true DuoSet in this example.

[0292] Example 2 Anti-leukemic activity of human T cell preparations expressing DuoCAR generated by co-transduction Anti-leukemia activity of human T cell preparations expressing DuoCARs that simultaneously target three leukemia antigens (see Figure 7 for DuoCAR expression characteristics). A DuoSet consisting of a CD20_19 tandem CAR and a CD22-specific single CAR (prepared as in Example 1) was used as an effector T cell population in cytotoxic T cell assays using leukemia cell lines and model cell lines as targets. Human T cells transduced with single CAR components (20_19-28z or 22-BBz) or DuoCAR (20_19-28z + 22-BBz) were used in cytotoxic T cell assays at four different effector-to-target ratios (20:1, 10:1, 5:1, 2.5:1, as indicated) (see Figure 8 for DuoCAR expression characteristics). The leukemia cell lines used as CAR-T targets were Raji (expressing all three target antigens), REH (expressing all three target antigens), K562 (control, no target expression), K562-CD19 (expressing CD19), K562-CD20 (expressing CD20), and K562-CD22 (expressing CD22). Only DuoCAR-transduced cells (20-19-28z+22-BBz) showed high cytolytic activity against both the leukemia cell lines (Raji and REH) and all three single-expressing K562 target cell lines (K562-CD19, K562-CD20, K562-CD22). This demonstrates that Duo technology can simultaneously and uniquely target three leukemia antigens in the same effector T cell population, thus demonstrating superior anti-neoplastic activity by allowing for targeting of more than one or two target antigens at a time, thus reducing the likelihood of malignancies generating escape mutants (cell clones that lack or downregulate one or two antigens, which escape immune elimination). The end result is a high cure rate for patients who ultimately relapse due to escape and by-product of antigen-loss variants.

[0293] Example 3 Anti-leukemic activity of human T cell preparations expressing DuoCAR generated by co-transfection The DuoCAR technology described in this application generates a population of therapeutic lymphocytes, in this example, human T cells, that express more than two antigen specificities from more than one transmembrane protein encoded by a genetic vector. In this example, this is achieved by two different means. Figure 9 includes three rows of data labeled "untransduced," "co-transduced," and "co-transfection." Figure 9 includes two columns of data generated as in Figure 7, where the first column was analyzed by flow cytometry for the expression of CD20 and CD19 specific binding, and the second column was analyzed by flow cytometry for the expression of CD22 and CD19 binding activity. In the first row of data, untransduced human T cells are shown. No binding activity was observed for the CD19, CD20, or CD22 recombinant protein indicators of CAR-derived binding activity, demonstrating the absence of DuoCAR expression. In the second row, DuoCARs were generated using "co-transduction." In this dataset, two LVs were used to simultaneously transduce activated T cells. As in Figure 7, one CAR in the DuoSet containing DuoCARs was a tandem CD20 and CD19 binder linked to CD28 signaling and CD3-zeta signaling motifs, and the other CAR was a CD22 binder linked to 4-1BB and CD3-zeta signaling motifs. The upper right quadrant in column 1 shows the single CD20 and CD19 scFv activity. A very specific pattern of staining is shown. This is due to both binders on the same surface glycoprotein; therefore, they are co-expressed with equal intensity, resulting in the very specific linear pattern seen. In the second column of co-transduction data, a more traditional pattern is seen when the two glycoproteins are not expressed in a uniform pattern on each cell. Thus, four distinct population patterns are seen. In the lower left quadrant, cells expressing neither binder are seen. In the upper left, cells expressing only the CD22 CAR are seen. In the lower right quadrant, cells expressing only the CD20_CD19 tandem CAR are seen. Finally, in the upper right quadrant, cells expressing both members of the CAR DuoSet, including the DuoCAR, are seen.

[0294] In the bottom row, cell populations expressing DuoCAR are generated using different methods. Unlike the co-transduction method, in which two independently created LV preparations are used for T cell transduction, "co-transfection" refers to a method in which two backbone plasmids (encoding two CARs, including DuoCAR) are simultaneously transfected into the 293T packaging cell line for LV production. The helper plasmids containing this third-generation LV system are identical in both methods. The advantage of the co-transfection method is that a single preparation of LV containing vectors encoding both CARs is created. As can be seen from the data, the co-transfection method results in nearly identical patterns of expression of the CD20-CD19 CAR and CD22 CAR compared to the co-transduction method in the second row. The staining patterns for both glycoproteins induced by LVs generated by co-transfection in the upper right quadrant of the second column of data (CD22 for CD22-CAR, CD19 co-staining for CD20_CD19 CAR) demonstrate that both methods effectively generate DuoCARs.

[0295] References: 1)Wu, AM et al., Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein engineering, 2001, Vol. 14(12): 1025-1033. 2)Haso, W. et al. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood, 2013, volume 121 (issue 7): pages 1165-1174. 3) Kuroda, H. et al., Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection. Journal of virological methods, 2009, Volume 157 (No. 2): pp. 113-121.

[0296] Example 4 Comparison of DuoCAR generated by cotransfection method with bicistronic DuoCAR constructs Methods used in Example 4: Cell lines (PBMCs and targets): All cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA) unless otherwise noted. The Burkitt lymphoma cell line, Raji, the acute lymphoblastic leukemia cell line, REH, and the chronic myeloid leukemia cell line, K562, were cultured in 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Gr The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (Plant Island, NY). 293T, a human embryonic kidney cell line, was propagated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FBS.

[0297] The wild-type tumor line was stably transduced with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones to generate single-cell clones of the luciferase-expressing cell line. The mouse-adapted Raji-luc line was transduced into NSG mice (NOD.Cg-Prkd). cscid Il2rg tm1Wjl Raji-Luc tumor cells were generated by transplanting Raji clones stably expressing firefly luciferase into mice (SzJ, The Jackson Laboratory, Sacramento, CA). The transplanted Raji-Luc tumor cells were isolated from mouse spleens by either positive selection (CD19 microbeads, human, Miltenyi Biotec, Bergisch Gladbach, Germany) or negative selection (mouse cell depletion kit, Miltenyi Biotec), expanded in culture, and recloned to facilitate the selection of clones with high expression of firefly luciferase. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI, Oklahoma City, OK) with written donor consent. Processed buffy coats were purchased from OBI. CD4+ and CD8+ human T cells were purified from the buffy coats by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0298] Creation of Chimeric Antigen Receptor (CAR) - Expression Vector Containing DuoCAR: The CAR antigen-binding domain, ScFv, sequences were derived from the murine hybridoma FMC-63 (FMC-63: AA1-267, GenBank ID: HM852952.1) for CD19 and Leu-16 for CD20 [1], with the entire VL and VH sequences. Several anti-CD22 scFv binding sequences were used. Tandem CAR19_20 or CAR20_19 were generated by linking each antibody scFv in frame to the CD8 hinge and transmembrane domain (AA123-191, Ref Sequence ID NP_001759.3), 4-1BB (CD137, AA214-255, UniProt Sequence ID Q07011) transactivation domain, and CD3 zeta signaling domain (CD247, AA52-163, Ref Sequence ID: NP_000725.1). The scFv regions of 19A and 20A were linked in sequence by a flexible interchain linker (GGGGS)5 (SEQ ID NO: 108), followed by the CD8, 4-1BB, and CD3 zeta domains. A leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit was included in all constructs, as described in [2]. In the bicistronic CAR design, the two CAR chains were encoded within the same expression cassette, separated by a ribosomal skip site 2A. The CAR construct sequence was codon-optimized (DNA2.0, Newark, CA) and cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD) under the control of the human EF-1α promoter of MSCV. Supernatants containing lentiviral vectors (LV) were generated by transient transfection of HEK 293T cells, as previously described in [3]. For cotransfection experiments, equal amounts of the two transfer plasmids encoding each DuoCAR chain were mixed and applied, along with a helper plasmid, to the HEK293T packaging cell line during the transfection step. The resulting viral vector preparation was used to transduce primary human T cells. The collected pelleted lentiviral supernatant was stored at -80°C.

[0299] Primary T cell transduction: Selected CD4+ and CD8+ human primary T cells from normal donors were transduced at 0.3–2 × 10 in TexMACS medium (serum-free) supplemented with 40 IU / ml IL-2. 6 Cells were cultured at a density of 1000 cells / ml, activated with CD3 / CD28 MACS® GMP TransAct reagent (Miltenyi Biotec), and transduced overnight with a lentiviral vector encoding a CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 3, with the medium changed on day 4. On day 5, the cultures were transferred to TexMACS medium supplemented with 200 IU / ml IL-2 and propagated until harvest on days 10–13.

[0300] Immune effector assay: To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were mixed with CAR T cells at various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison, WI) was added to each well, and the resulting luminescence was analyzed in an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells with 1% Tween-20 (minimum CPS) were used to determine the assay range. The percent specific lysis was calculated as (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)).

[0301] Flow cytometry analysis: All cell staining reagents for flow cytometry were from Miltenyi Biotec unless otherwise noted. One million CAR T-transduced cells were harvested from culture, washed twice in cold staining buffer (AutoMACS solution with 0.5% bovine serum albumin), and pelleted at 350 × g for 5 minutes at 4 ° C. CAR surface expression on transduced T cells was detected by first staining with protein L-biotin conjugate (stock 1 mg / ml, 1:1000 dilution, GenScript, Piscataway, NJ) for 30 minutes at 4 ° C, followed by two washes and staining with streptavidin-PE conjugate (stock: 1.0 ml, 1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes at 4 ° C. As negative controls, untransduced and transduced cells stained with streptavidin-PE alone were used. The CD4 to CD8 ratio of the CAR T-positive population was determined using an anti-CD4 antibody, which was added during the second incubation step. Dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Specific DuoSet CAR T staining was performed on human T cells activated with DuoSet vector-transduced CD3-CD28 nanomatrix (TRansAct, Miltenyi Biotec) in the presence of IL-2 and analyzed for expression of CD19-, CD20-, or CD22-scFv domains by flow cytometry using recombinant CD19, CD20, or CD22 antibodies for staining.

[0302] Anti-CD19 scFv activity was detected with CD19-Fc (R&D Biosystems) used at 1 μg / sample and stained with goat anti-human Fc-gamma-R-PE (Jackson ImmuoResearch Laboratories, Inc.) at 0.75 μg / sample. Anti-CD20 scFv activity was detected with CD20-biotin (Miltenyi Biotech) at 0.1 μg / sample and streptavidin APC (Miltenyi Biotec) at 0.2 μg / sample. Anti-CD22 scFc activity was detected with CD22-His (Thermo Fisher) at 0.1 μg / sample and anti-His FITC (Miltenyi Biotec). Flow cytometry analysis was performed using a MACSQuant® 10 Analyzer (Miltenyi The target tumor lines and luciferase-positive subclones were analyzed by the National Institute of Biotec. Characterization was performed using CD19-FITC, CD20 VioBlue, and CD22-APC antibodies. Dead cells were excluded from the analysis by 7AAD staining (BD Biosciences, San Jose, CA).

[0303] Generation of bicistronic DuoCARs using 2A ribosomal skip sequences In addition to the co-transduction and co-transfection approaches described in Examples 2 and 3 above, DuoCAR simultaneously targets three hematological tumor antigens, CD19, CD20, and CD22, and features different costimulatory domains. Simultaneous expression of two CAR chains from a single mRNA transcript can be facilitated by the use of the self-cleaving element 2A. The 2A element mediates ribosomal skipping during translation of the mRNA transcript into protein, thus enabling the production of two different CAR protein chains in equimolar ratios. In this example, one CAR chain is composed of a CD22 scFv, a 4-1BB costimulatory domain, and a CD3 zeta activation domain linked in-frame to a CD8 hinge and transmembrane domain. The second CAR chain is composed of a tandem CD20 CD19 scFv-based targeting domain, followed by a CD8 hinge and transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta activation domain. The two designs differ in the order of the CAR chains; for example, in one design, the CD22 CAR comes first, followed by the 2A element and the tandem 2019 CAR, and vice versa (Figure 10).

[0304] First, a set of four bicistronic DuoCAR designs simultaneously targeting CD19, CD20, and CD22 antigens under the control of the EF1a promoter was constructed as described above (Set 1, Table 1 below).

[0305] [Table 1]

[0306] To facilitate optimal expression of the CD22-targeting CAR moiety in the DuoCAR format, the CD22-targeting CAR chain was incorporated into one of the CD22-reactive scFv sequences, 16P8 or 16P17. CD22 scFv M971 was used as a comparative control, and untransduced cells (UTD) served as a CAR-negative control. Co-expression of the CD20-CD19-targeting CAR chain and the CD22-targeting CAR chain was facilitated by a 2A ribosomal skip sequence, as described above. The individually encoded CAR chains were then transduced into the CD22-targeting CAR chains. , were included as expression controls. Human primary T cells from healthy donors were transduced with lentiviral vectors encoding each DuoCAR or a single CAR control. At the end of T cell culture expansion, CAR expression was assessed by flow cytometry. CAR20 in DuoCAR groups (LTG2515, LTG2520, LTG2521) represents co-expression of tandem CD20-CD19 CAR chains and CD22-CAR chains in the same cells. + CAR22 +The percentage of double-positive cells was relatively low, ranging from 28% (LTG2515, LTG2520) to 9% (LTG2521) (Figure 11). In contrast, expression of individual CAR controls was significantly higher, at approximately 72% for the CD22-targeting construct (LTG2200) and approximately 38% for the tandem CD20-CD19-targeting CAR (LTG 2228, Figure 11). The functionality of DuoCARs was then tested in a cytokine release assay. Control DuoCAR effector cells were mixed overnight with Raji target cells at an effector-to-target ratio (E:T) of 10. At the end of the incubation period, cell culture supernatants were collected and assayed for secreted T cell cytokines IFN-gamma, TNF-alpha, and IL-2 (Figure 12). Effectors incubated under similar conditions in the absence of tumor target cells were used as an additional control for spontaneous cytokine release. Co-incubation of Raji tumor cells with CAR effectors resulted in strong upregulation of IFN-gamma, IL-2, and TNF-alpha for all constructs. Notably, CARs did not spontaneously produce cytokines. However, as seen in Figure 11, cytokine secretion levels tended to be lower for all DuoCAR constructs compared to the positive control CAR22 LGT2200, and tandem 2019 CAR LGT2228, possibly due to relatively moderate expression of DuoCAR.

[0307] The moderate DuoCAR expression and cytokine response compared to the single CAR control (Figures 11 and 12) suggested that the large payload size may have a detrimental effect on the efficiency of DuoCAR expression in this configuration. To improve the efficiency of DuoCAR transduction, selected DuoCAR sequences were codon-reoptimized as necessary, and the expression cassette was recloned into a new expression backbone under the control of an MSCV internal promoter for improved bicistronic expression (Set 2, Table 1).

[0308] Lentiviral vectors were generated for each new DuoCAR construct, and CAR T cells were transduced and expanded as described in Materials and Methods. DuoCAR expression was determined by flow cytometry. The percentage of CD19+CD22+ T cells indicates cells co-expressing the two chains of DuoCAR (Figure 13). Here, high transduction efficiency was achieved for DuoCAR constructs D0044 (MSCV_20-19-28z-2A-16p8-BBz) and D0047 (MSCV_16p8-BBz-2A-20-19-28z), both of which contain the anti-CD22 scFv 16P8 (Figure 13, 51% and 45%, respectively). Unexpectedly, the control m971 CD22 scFv-containing DuoCAR, D0043, was well expressed in the distal orientation (MSCV_20-19-28z-2A-m971-BBz, 46% positive), but showed no expression in the reverse orientation (D0046, MSCV_m971-BBz-2A-20-19-28z). Thus, the choice of scFv sequence included in the DuoCAR design, as well as codon optimization of the sequence and choice of expression backbone, are all critical for optimal DuoCAR expression.

[0309] The cytotoxic function of DuoCAR Set 2-transduced T cells was assessed in overnight killing assays against a panel of tumor lines with varying expression of the tumor antigens CD19, CD20, and CD22. All lines were stably transduced to express firefly luciferase, and killing assays were performed as described in Materials and Methods. Initially, DuoCAR was transduced against CD19+CD20+CD22+ and Raji, a non-Hodgkin's lymphoma, or acute lymphoblastoid tumors. CAR 20-19-28z and CD22 CAR were mixed with Reh cells, a human leukemia leukemia, or the CD19-CD20-CD22- human fetal kidney 293T cell line (Figure 15). We included the DuoCARs D0044 and D0047, which bicistronically encode the 16p8-BBz CAR, as well as the single CAR 22 control LTG2200 and the tandem CAR control 20-19 LTG1497, as well as an untransformed T cell control (UTD) (Figure 14). For simplicity, constructs D0043, D0044, D0046, and D0047 are referred to as D43, D44, D46, and D47, respectively, in the figure legend (Figure 14). Effector and target cells were incubated overnight in triplicate at a ratio of 2.5, 5, or 10. Plates were then harvested and developed with SteadyGlo reagent, and luciferase activity of surviving tumor cells was determined by luminometry. Overall, CAR cytolytic function correlated with DuoCAR expression (Figure 13). DuoCARs D0047 and D0044 potently lysed the CD19, CD20, and CD22 triple-positive tumor lines Raji and Reh, as did the positive control DuoCAR D0043, whereas the suboptimally expressed construct D0046 had relatively poor lytic function (Figure 14). Lysis of the CD19-CD20-CD22 triple-negative line was not induced by any of the CAR constructs, highlighting the specificity of CAR-mediated lysis for the cognate antigen.

[0310] To further delineate the specificity of the DuoCAR constructs, we generated transgenic K562 lines expressing either the CD19, CD20, or CD22 antigens, designated K19, K20, and K22, respectively (Figure 15). In co-incubation assays with single-positive tumor lines, DuoCARs D44 and D47, featuring CAR chains targeting CD19, CD20, and CD22, potently lysed each target line in an effector-to-target ratio-dependent manner, and were similar in function to the control DuoCAR D0043 (the numerical designations of the constructs in the figure legends have been shortened from D0043, D0044, D0046, and D0047 to D43, D44, D46, and D47, respectively—Figure 15). Control T cells expressing the tandem 2019 CAR (1497) lysed the tumor lines K19 and K20, but had only a slight background lysis effect in K22 (less than 10% lysis at the highest E:T ratio of 10). The single CD22 control CAR potently lysed K22 tumor cells, but had no function in K20 cells and showed only background lysis in K19 cells (10% lysis at the highest E:T ratio of 10:1). Therefore, this experimental system allows for testing of CAR reactivity against each tumor antigen with high accuracy. In summary, both DuoCARs D0044 and D0047 demonstrated that their respective tumor-targeting domains were functional in this single-antigen expression test system (Figure 15).

[0311] To characterize the cytokine release response of DuoCAR constructs, DuoCAR T cell preparations D0044 and D0047 (figure legend: D44 and D47, respectively) and CD19+CD20+CD22+ were mixed with Raji tumor cells at an E:T ratio of 10 overnight, and culture supernatants were analyzed for the T cell cytokines IFNg, TNFa, and IL-2 by ELISA (Figure 16). The single CAR22 construct LTG2200 and the tandem 2019 CAR construct LTG2273 were included for comparison, and untransduced T cells (UTD) were used as a negative control. In parallel, CAR T cells from each group were incubated in the absence of tumor cells but under similar conditions to test for spontaneous cytokine release (Figure 16). We found that while the constructs did not result in spontaneous cytokine release, both DuoCARs D44 and D47 exhibited robust induction of IL-2, IFNg, and TNFa after co-incubation with the Raji target, highlighting the potency of these DuoCAR constructs. Notably, despite simultaneous co-expression of the two chains in the same cells, no evidence of sustained signaling was detected, as evidenced by the complete absence of spontaneous cytokine release. (Figure 16).

[0312] Having successfully developed a bicistronic DuoCAR consisting of two CAR chains targeting three distinct tumor antigens, CD19, CD20, and CD22, and harboring costimulatory domains with distinct preferential functions, the question arose: could similar constructs be generated using other approaches? Successful bicistronic expression of separate CAR chains within the same ORF requires multiple optimization and refinement steps and is unique to each new set of sequences. In contrast, combining two CAR sequences during lentiviral vector production or CAR T transduction may offer a more universal approach and a rapid method for creating CAR combinations expressed in the same cell or T cell population while using a single lentiviral preparation for T cell transduction. In this example, as in the DuoCAR approach, one CAR chain is composed of a CD22 scFv, a 4-1BB costimulatory domain, and a CD3 zeta activation domain linked in-frame to a CD8 hinge and transmembrane domain. The second CAR chain is composed of a tandem CD20 CD19 scFv-based targeting domain, followed by a CD8 hinge and transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta activation domain (Figure 17). In the co-transfection approach, two transfer plasmids, each encoding one CAR chain, are mixed together according to standard protocols (see Materials and Methods) and combined with a helper plasmid during the vector production step. The resulting lentiviral preparation thus encodes a mixture of the two CAR chains. Using this approach, we generated a set of lentiviral preparations simultaneously encoding two CAR chains (Table 2 below).

[0313] [Table 2]

[0314] Transfer plasmids for CAR22 were constructed utilizing scFvs 16P17, 16P8, and 16P13 CAR22-4-1BB-CD3 zeta (D1, D2, and D3, respectively) under the control of the MSCV promoter, and tandem CAR2019-28-CD3 zeta (LTG 2273) under the control of MSCV. Lentiviral vectors encoding each CAR chain alone were produced in parallel. High titers (10 for all DuoCAR cotransfection preparations) were obtained. 10 TU / ml, not shown) were routinely achieved, highlighting the efficiency of this technique.

[0315] To optimize DuoCAR function, a series of CAR22 constructs consisting of scFvs 16P17, 16P8, and 16P13 were designed under the control of the MSCV promoter (constructs D1, D2, and D3, respectively), and tandem CAR 2019 (LTG2273), also driven by the MSCV promoter, was used for DuoCAR cotransfection combinations (Table 2 and Figure 18). LVs were prepared by cotransfection of LTG2273 with one of the CD22 CAR plasmids, resulting in high infectious titers (not shown). Each LV was used at a multiplicity of infection (MOI) of 20 to transduce healthy donor T cells, and CAR expression was determined by flow cytometry (Figure 18). All control groups transduced with LVs encoding single CAR controls yielded high CAR expression (45% for D1, 82% for D2, 82% for D3, and 87% for 2273 (not shown)). Surprisingly and unexpectedly, in the cotransfection combinations, groups D2+73 and D3+73 yielded efficient and nearly identical coexpression of the two CAR chains (51%), while the D1+73 combination did not coexpress (2.8% CAR+), Figure 18. To determine whether these DuoCARs have lytic function, CAR T cells from each group were tested on a panel of tumor lines (Figure 19; in the labels for groups D1+2273, D2+2273, and D3+2273, the "D" has been omitted for simplicity). All DuoCAR preparations efficiently lysed the triple-positive tumor lines Raji and Reh, but not the triple-negative line 293T, confirming the specificity of the DuoCARs (Figure 19A). In addition, all DuoCARs demonstrated lytic function above background against the single-antigen tumor lines K19, K20, and K22, whereas single control CARs with mismatched targeting domains did not demonstrate specific lysis: D1 to D3 for K19; D1 to D3 for K20; and 2273 for K22 (Figure 19B). The ability of DuoCARs to induce cytokines upon co-incubation with specific tumor targets was then assayed. (Figure 20; in the labels of groups D1+2273, D2+2273, D3+2273, the "D" has been omitted for simplicity.) DuoCAR T cells, single CAR control, and untransduced T cells (UTD) were mixed with triple CD19+CD20+CD22+ Raji tumor cells and incubated overnight. In parallel, tumor-free CAR T cells were incubated under similar conditions to rule out spontaneous cytokine release.At the end of the incubation period, the culture supernatants were assayed for the cytokines IFNg, TNFa, and IL-2 by ELISA (Figure 20). All CAR groups produced high IFNg levels upon co-incubation with Raji. While some single CD22 CAR controls had moderate spontaneous IFNg release (D2, D3), DuoCARs did not spontaneously produce IFNg, suggesting a potentially high safety margin for DuoCARs. IL-2 and TNFa expression was also highly induced by co-incubation with Raji in all CAR groups, except for CAR 2272 (Figure 20).

[0316] In summary, this paper describes the generation of functional and highly specific DuoCAR by co-transfection of individual CAR chains during LV preparation and application of the resulting LV preparation in T cell transduction.Furthermore, using a transgenic cell line that expresses only a single target antigen (K19, K20, K22), we demonstrated that each CAR targeting domain is functional and can induce DuoCAR function against target-expressing tumor cells.Surprisingly and unexpectedly, only a few combinations can demonstrate both robust CAR expression and potent cytotoxicity function, and therefore, DuoCAR design is not trivial.

[0317] Example 5 The bicistronic DuoCAR potently eradicates lymphoma tumors. Materials and methods used in Example 5: (a) Cell line The Burkitt lymphoma cell line, Raji, and the chronic myeloid leukemia line, K562, were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). The REH leukemia line was purchased from DSMZ (Leibniz Institute DSMZ, Braunschwieg, Germany). Cells were cultured in a medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, U.K.). The cells were cultured in RPMI-1640 medium supplemented with ATP (T) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), cloning, and selecting luciferase-positive clones. The Raji 13G11 clone was generated by passage of luciferase-transduced Raji cells in mice and selected for its proliferation ability. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with written donor consent. Processed buffy coats were purchased from OBI (Oklahoma City, OK). CD4- and CD8-positive human T cells were purified from the buffy coat by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0318] (b) Creation of chimeric antigen receptor (CAR)-expression vector The DuoCAR construct was designed as a bicistronic sequence incorporating one tandem CD19- and CD20-targeting CAR and one single CD22-targeting CAR. Bicistronic expression of the two CAR constructs from the same mRNA template was facilitated by ribosomal skip element 2A. The CAR antigen-binding signal and tandem domains were derived from a human anti-CD22 single-chain variable fragment (ScFv) or a previously described tandem 20-19-targeting scFv (Schneider, D. et al., (2017). Journal for immunotherapy of cancer, Vol. 5(1), p. 42). The CAR T coding sequence was generated by linking the binder sequence in frame to the CD8a binding and transmembrane domains (aa 123-191, Ref. Sequence ID NP_001759.3). The C-terminal segment of the CAR construct contained the CD3 zeta signaling domain (CD247, aa 52-163, Ref Sequence ID: NP_000725.1). Some designs also contained costimulatory domains derived from human 4-1BB, ICOS, OX40, or CD27 proteins. CAR construct sequences were cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK 293T cells. The supernatants containing lentiviral vectors were centrifuged to pellet the vectors and stored at -80°C.

[0319] (c) Primary T cell purification and transduction CD4 according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany) + and CD8 + Human primary T cells from healthy volunteers were purified from whole blood or buffy coats (purchased from private donors with written donor consent) using immunomagnetic bead selection of cells. T cells were cultured at 0.3–2 × 10 in TexMACS medium supplemented with 200 IU / ml IL-2.6 Cells were cultured at a density of 1000 cells / ml, activated with CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), and transduced overnight with a lentiviral vector encoding a CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 2, with the medium changed on day 3. Cultures were propagated in TexMACS medium supplemented with 200 IU / ml IL-2 until harvest on days 8-10.

[0320] (d) Immune effector assays (CTL and cytokines) To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison, WI) was added to each well, and the resulting luminescence was quantified as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells plus 1% Tween-20 (minimum CPS) were used to determine the assay range. Percent specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants from 10:1 E:T co-cultures were removed and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0321] (e) Flow cytometry analysis For cell staining, 500,000 CAR T-transduced cells were harvested from culture and washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with CD19-Fc and CD20-biotin or CD19-Fc and CD22-His peptides followed by a secondary peptide-specific fluorescent conjugate (Jackson ImmunoResearch, West Grove, PA). Per the supplier's protocol, an anti-CD4 antibody conjugated to VioBlue fluorophore (Miltenyi Biotec) was used where indicated. Non-transduced cells served as a negative control. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Flow cytometry analysis was performed on a MACSQuant® 10 Analyzer (Miltenyi Biotec) and data plots were generated using FlowJo software (Ashland, OR).

[0322] Generation of trivalent DuoCARs targeting CD19, CD20, and CD22 A trivalent DuoCAR was constructed by linking a tandem 2019-targeting CAR chain to a 22-targeting CAR chain via a P2A ribosomal skip element. Four different DuoCAR constructs were designed based on the best combinations previously identified in optimization co-transduction experiments. The optimization studies included testing each CAR moiety containing 41BB, CD28, OX40, ICOS, or CD27 costimulatory domains or no costimulatory domains (i.e., a CD20 / 19 tandem CAR and a CD22 single CAR), alone or in combination. Specific parameters tested were CAR expression levels and in vitro antitumor activity. The structures of the DuoCAR constructs designated D93, D94, D95, and D96 are shown in Figure 21A. The DuoCAR construct D93 consisted of tandem scFv binder domains targeting the B cell antigens CD19 and CD20, a CD8-derived hinge and transmembrane domain, followed by an ICOS costimulatory domain and a CD3ζ activation domain. This CAR construct sequence was linked to a first-generation CAR targeting CD22 via a P2A ribosomal skip element, thus creating a bicistronic triple-targeting DuoCAR (Figure 21). The use of the 2A ribosomal skip element ensures the following CAR attributes: (i) production of a uniform cellular product, and (ii) stoichiometric expression of the two CAR moieties within individual cells; the combination achieves optimal antitumor function. The DuoCAR construct D94 was identical to D93, except that the ICOS costimulatory domain was replaced with an OX40 domain. Construct D95 consisted of a CD20 and CD19 tandem-targeting OX40z CAR chain identical to that of D94, followed by a second-generation CD22 CAR chain with an ICOS costimulatory domain and a CD3ζ activation domain. DuoCAR construct D96 contained a CD20 and CD19 tandem-targeting CAR chain with a CD27 costimulatory domain, followed by a CD22-targeting single CAR chain with an ICOS costimulatory domain and each CD3ζ activation domain (Figure 21A). The DuoCAR construct was encoded in a lentiviral vector and transduced into human primary T cells. DuoCAR was robustly expressed in T cells, ranging from 30% to 70% of CAR T+ cells across three different constructs and donors (Figures 22A and 22B).

[0323] In addition, several control CAR constructs were constructed, including single-targeting CARs and tandem-targeting CARs (Figure 21A). The single-targeting CARs contained either an anti-CD22 scFv, an anti-CD19 scFv, or an anti-CD20 scFv, followed by a CD8 hinge and transmembrane domain either directly linked to the CD3z activation domain (D92, CAR22, first generation) or also incorporating a costimulatory domain (D89, 1538, 1495, second generation) (Figure 21A). The tandem control CAR targeting CD20 and CD19, designated 1497 CAR, consisted of a tandem CD20-CD19 scFv linked in-frame to the CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3z activation domain. Tandem control constructs D88, D90, and D91 were designed as tandem CAR 1497, except for the replacement of the 4-1BB costimulatory domain with an ICOS, OX40, or CD27 domain, respectively (not shown). These constructs demonstrate antitumor activity against unmodified tumors expressing all three antigens, CD19, CD20, and CD22. However, in contrast to DuoCAR constructs D93, D94, D95, and D96, the tandem constructs are unable to prevent antigen escape by tumor cells double-negative for CD19 and CD20. The positioning and composition of CAR chains within T cells are shown schematically in Figure 21B for DuoCAR, tandem CAR, and first- and second-generation single CAR T cells. All single and tandem CAR constructs achieved robust expression in human primary T cells via lentiviral transduction.

[0324] DuoCAR potently and specifically lyses tumor targets in vitro To evaluate the functionality of the constructed DuoCAR, the constructed DuoCAR was mixed with luciferase-expressing target tumor cells for an overnight killing assay. The antigen-positive NHL line Raji (CD19+CD20+CD22+) and B-ALL line Reh (CD19+CD20lowCD22+) were used to test the ability of DuoCAR to lyse tumor cells in an antigen-specific manner. Negative control lines, myeloid leukemia K562 and human embryonic kidney cell line 293T, both of which are CD19-CD20-CD22-, were also included (Figure 23).

[0325] The tandem CAR 1497 and the single CAR controls D89 and D92, second and first generation CD22-targeting CARs, respectively, were included for comparison.

[0326] CAR T cells and target cells were mixed at effector to target (E:T) ratios of 10:1, 5:1, or 2.5:1, and at the end of the incubation period, specific lysis was calculated for each condition as described in Materials and Methods.

[0327] All single and tandem CARs lysed the target-positive tumor lines Raji and Reh in an E:T-dependent manner (Figures 23A, 23B). DuoCAR constructs potently lysed target cell lines expressing the target antigens CD19, CD20, and CD22 at all E:T ratios (Figures 23A, 23B). The tandem control CAR 1497, targeting the CD19 and CD20 antigens, resulted in relatively moderate tumor lysis at the tested E:T ratios compared to DuoCARs D93, D94, D95, and D96 in Raji cells (Figure 23A). In comparison, the lytic potency of 1497 was similar to that of DuoCARs D93, D94, D95, and D96 in Reh cells (Figure 23B). The single-targeting CAR22 constructs D89 and D92 tended to be the most potent tumor cell killers against the CD22 antigen-positive target lines Raji and Reh. In contrast, except for the single-targeting CAR22 construct D92 in K562 cells, which produced 27% nonspecific lysis at the highest E:T ratio of 10 (Figure 23C), neither the DuoCAR constructs nor the control lysed the target-negative tumor lines K562 and 293T (Figures 23C, 23D). Thus, DuoCAR performed as well as or better than the 1497 tandem construct in lysing antigen-positive target lines and did not produce background lysis in antigen-negative lines, demonstrating antigen dependence. Notably, despite the nonspecific lytic activity of a single CAR 22, D92, in K562 cells, incorporating the D92 sequence into D1 and D2 of the DuoCAR construct does not result in nonspecific target lysis by DuoCAR. Thus, the DuoCAR design appears to attenuate the undesirable spontaneous lytic activity seen in the first-generation CAR D92 (Figure 23C).

[0328] DuoCAR cytokine response To characterize cytokine production of DuoCARs in response to target cells, supernatants were collected from DuoCAR cocultures with CD19+CD20+CD22+ Raji target cells after overnight incubation. The concentrations of T cell proinflammatory and homeostatic cytokines IL-2, IFNγ, and TNFα in the culture supernatants were determined by ELISA (Figure 24, blue bars). Untransduced T cells (UTD) from the same donor and batch were included as CAR negative controls. Additionally, each CAR T cell group was incubated without Raji targets to control for possible spontaneous cytokine release by CAR T cells in the absence of activating target cells (Figure 24, light gray bars). DuoCAR, as well as single and tandem control CARs, strongly induced the production of IL-2, IFNγ, and TNFα in response to target Raji cells compared to UTD, but the DuoCAR lines or CAR controls were not prone to spontaneous release of these soluble factors in the absence of target cells (Figure 24).

[0329] DuoCAR efficiently lyses CD19+CD20+CD22 Raji tumors in vivo After establishing the cytotoxicity and cytokine release functionality of DuoCAR against antigen-positive target cells in vitro, DuoCAR function was then demonstrated in vivo. NSG (NOD.Cg-Prkdc) cells stably transduced with firefly luciferase were transduced with Raji cells. scid Il2rg tm1WjlWe utilized (SzJ) mouse xenografts. DuoCARs D93, D94, D95, and D96 were included, as well as the tandem control CAR 1497 and single controls D89 and D92 (Figure 25A). Tumor-bearing mice were administered either 5 million CAR T cells, or 2 million CAT T cells, respectively, or a dose-matched UTD control on study day 7, and tumor rejection was measured periodically by bioluminescence imaging until study day 28 (Figures 25A and 25B). In a high CAR T dose regimen of 5 million cells per mouse, DuoCARs potently suppressed Raji tumor progression from day 14 onward, whereas tumors in the tumor-alone group (TA) and the untransduced T cell group (UTD) progressed unabated. Compared with the TA and UTD negative controls, tumor suppression mediated by DuoCARs and single and tandem CAR controls was statistically significant. DuoCAR D93 and single CARs D89 and D92 tended to produce the highest tumor shrinkage over the study period, while DuoCAR D95 and tandem CAR 1497 tended to be the least potent CAR constructs. However, the differences between individual CAR constructs at this dosage level were not statistically significant (Figure 25A). To better identify minor differences between DuoCAR constructs and test whether they continued to function at lower dose regimens, Raji-bearing mice were transfected with each CAR T cell. 2 million cells were administered (Figure 25B). Despite the low CAR T dose, all CAR constructs significantly controlled Raji tumor burden compared to the TA and UTD controls at this level. While the DuoCAR constructs or control CARs were not significantly better than the other CARs, DuoCARs D93 and D94 tended to maintain good tumor control, and DuoCAR D96 tended to be less potent than the other CARs (Figure 25B). Of note, tumor shrinkage in 1497 of the tandem CAR group appeared slower compared to DuoCARs D93-D96, suggesting a possible advantage of the DuoCAR construct in this setting (Figure 25B). Additionally, single CAR D92 tended to reduce tumor burden faster than the other CAR constructs, consistent with its high potency, but lower specificity was also observed for this construct in in vitro cytotoxicity experiments against antigen-positive and antigen-negative tumor target lines (Figure 23).

[0330] The trispecific DuoCAR requires only a single antigen for antitumor function and potently kills antigen-depleted target cells in models of tumor antigen loss of either CD19, CD20, or CD22. By demonstrating that DuoCARs mediated potent rejection of CD19+CD20+CD22+ wild-type Raji xenografts in vivo, even at a low-dose regimen of 2 million CAR T+ cells / mouse, the sufficiency of each single antigen to trigger DuoCAR activation was validated in vitro. The CAR constructs included in this experiment are shown schematically in Figure 21A. Experimental groups included DuoCARs D93, D94, D95, and D96, tandem CAR control 1497, and single CAR controls D92, 1538, and 1495 targeting CD22, CD19, and CD20 antigens, respectively (Figure 26).

[0331] DuoCARs are predicted to function as logically gated (OR) constructs, i.e., the presence of any one or more of the three targeted antigens is sufficient to trigger CAR activation and antitumor function. We then confirmed that each of the three reactivities was intact in DuoCARs D93, D93, D95, and D96. To this end, the A431 squamous cell carcinoma line, which naturally lacks B cell surface molecules, was engineered to stably express either CD19, CD20, or CD22. To facilitate quantification of tumor lysis, each target A431 line also stably expressed firefly luciferase. DuoCAR T cells and control CARs were tested in in vitro cytotoxicity assays against each of the A431 clones expressing only one antigen, and the parental A431 line was included as a target-negative control (Figures 26A-26D).

[0332] DuoCARs D93, D93, D95, and D96 efficiently lysed tumors expressing only a single antigen: CD19 (Figure 26A), CD20 (Figure 26B), or CD22 (Figure 26C), respectively, but not the parental line A431, which lacks expression of these target molecules (Figure 26D). Tumor lysis by DuoCARs of target cells with cognate antigen expression depended on the effector-to-target ratio, demonstrating the precise specificity of the DuoCAR constructs. As expected, single CARs failed to lyse target clones when those clones lacked expression of the targeted antigen. Line A19 was lysed by single CARs 19 and 1538, but not by single CARs targeting CD22-D92 or targeting CD20-1495 (Figure 26A). Similarly, the target line A20 was lysed by CAR20 1495, but not by the single CARs CD22-D92 or CD19 CAR-1538 (Figure 26B). Furthermore, only the CD22-targeted single CARs D92 and D89, but not the CD19- and CD20-targeted CARs 1538 and 1495, respectively, lysed the A22 target line (Figure 26C). Consistent with the lack of expression of CD19, CD20, or CD22 in this tumor line, the constructs did not lyse the parental line A431 (Figure 26D). Thus, DuoCARs are capable of lysing the isolated form of DuoCARs independently of the other two antigens. In this study, the isolates were reactive with one of the target antigens CD19, CD20, or CD22. Furthermore, the presence of each single antigen, CD19, CD20, or CD22, in the isolate was sufficient to trigger DuoCAR function.

[0333] Next, in Raji clones with ablated expression of either CD19, CD20, or CD22, a model of tumor antigen escape, DuoCARs were able to lyse target cells despite the absence of any one of the three targeting molecules (Figures 26E-26G), and the magnitude of lysis was comparable to DuoCAR lysis of the parental Raji line, in which expression of all three antigens was intact (Figure 26H). In contrast, single-targeting CARs were only lytic to clones in which their toxic targets were present. Specifically, single-CARs 20 and 22, D92, and 1495, but not single-CARs 19 and 1538, lysed Raji 19KO (Figure 26E), and single-CARs 19 and CAR22 lysed CARs 19 and CAR22. CAR 19 lysed the Raji 20KO, but not CAR 20 (Figure 26F), and single CARs 19 and 20 lysed the Raji 22KO line, but not CAR22 (Figure 26G). In comparison, single CAR controls D92, 1538, or 1495 did not show any effect on lysis of the parental Raji clone expressing all three target antigens (Figure 26H). These results demonstrate the superiority of DuoCAR in targeting tumor cells lacking expression of one of the targeted antigens.

[0334] Tumor antigen escape, where expression of one or more of the targeted antigens is reduced or completely lost, and tumor heterogeneity, while the inability of a single agent / CAR to globally target a tumor cell population due to heterogeneous expression of the targeted antigen, remain major obstacles for CAR T immunotherapy. To demonstrate the ability of DuoCARs to combat tumors that have lost expression of some of their target antigens, we generated a heterogeneous xenograft tumor model (Figure 27). NSG mice were transfected with luciferase-positive, antigen-deficient Raji clones: Raji 19KO, Raji An equal mixture of 20KO, Raji 22KO, and parental Raji clones was implanted. Seven days after tumor implantation, mice were treated with 5 million CAR T+DuoCAR cells or single-CAR controls targeting CD19, CD20, or CD22. Tumor burden was measured by bioluminescence. Remarkably, starting from day 14 of the study, DuoCARs D93, D94, D95, or D96 completely rejected the xenogeneic Raji tumors. In contrast, tumors continued to grow in mice receiving single-targeting CARs CAR19-1538, CAR20-1495, or CAR22-D92 (Figure 27).

[0335] In summary, four novel DuoCAR designs, D93, D94, D95, and D96, are described herein that enable the generation of highly functional triple-targeted CAR T cells. DuoCAR T cells are highly specific and reactive to CD19, CD20, and CD22 antigens in vitro and in vivo, demonstrating highly potent function and complete tumor rejection in a disseminated in vivo xenograft Raji tumor model with differential expression of CD19, CD20, and CD22, whereas single-targeted CARs failed to prevent tumor progression in this model of tumor antigen escape. Thus, DuoCAR T cells represent a novel solution to confront antigenically heterogeneous tumor populations and mitigate tumor antigen escape, thus offering an opportunity to improve clinical outcomes in CAR T-treated patient populations.

[0336] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office Receiving Office in a PDF file titled "Sequence Listing." The Sequence Listing is incorporated by reference.

[0337] Sequences of the present disclosure The nucleic acid and amino acid sequences listed below are defined in 37 C.F.R. 1.822. The sequences are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids, as in: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 SEQ ID NO: 1 is the nucleotide sequence of the CD20-reactive scFv binding domain (LTG1495). GAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAACAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAA CCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGGTCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTGACCGTCAGCT CCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGACATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATGACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCGTCACCCAAGCCT TGGATCTACGCTACATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTCAGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGACGCCGCGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACTAAGCTGGAGATCAAA

[0338] SEQ ID NO: 2 is the amino acid sequence of the CD20-reactive scFv binding domain (LTG1495). EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVS SGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIK

[0339] SEQ ID NO: 3 is the nucleotide sequence of CAR LTG1495 (LP-1495-CD8 TM-41BB-CD3 zeta).

[0340] SEQ ID NO: 4 is the amino acid sequence of CAR LTG1495 (LP-1495-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCAR SNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAED AATYYCQQWSFNPPTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0341] SEQ ID NO:5 is the nucleotide sequence of the leader / signal peptide sequence. ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG

[0342] SEQ ID NO:6 is the amino acid sequence of the leader / signal peptide sequence. MLLLVTSLLLCELPHPAFLLIP

[0343] SEQ ID NO: 7 is the nucleotide sequence of the CD22-reactive scFv binding domain (LTG2200). CAGGTACAGCTCCAGCAGAGTGGCCCAGGGCTCGTGAAGCCAAGCCAGACGCTGTCCCTGACTTGTGCAATTTCAGGGGATTCAGTTTCATCAAATAGCGCGGCGTGGAATTGGATTCGACAATCTCCTTCCCGAGGGTTGGAATGGCTTGGACGAACATATTACAGATCCAAATGGTATAACGACTATGCGGTATCAGTAAAGTCAAGAATAACCATTAACCCCGACACAAGCAAGAACCAATTCTCTTTGCAGCTTAACTCTGTCACGCCAGAAGACACGGCAGTCTATTATTGCGCTCGCGAGGTAACGGGTGACCTGGAAGACGCTTTTGACATTTGGGGGCAGGGTACGATGGTGACAGTCAGTTCAGGGGGCGGTGGGAGTGGGGGAGGGGGTAGCGGGGGGGGAGGGTCAGACATTCAGATGACCCAGTCCCCTTCATCCTTGTCTGCCTCCGTCGGTGACAGGGTGACAATAACATGCAGAGCAAGCCAAACAATCTGGAGCTATCTCAACTGGTACCAGCAGCGACCAGGAAAAGCGCCAAACCTGCTGATTTACGCTGCTTCCTCCCTCCAATCAGGCGTGCCTAGTAGATTTAGCGGTAGGGGCTCCGGCACCGATTTTACGCTCACTATAAGCTCTCTTCAAGCAGAAGATTTTGCGACTTATTACTGCCAGCAGTCCTATAGTATACCTCAGACTTTCGGACAGGGTACCAAGTTGGAGATTAAGGCGGCCGCA

[0344] SEQ ID NO: 8 is the amino acid sequence of the CD22-reactive scFv binding domain (LTG2200). QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAA

[0345] SEQ ID NO: 9 is the nucleotide sequence of CAR LTG2200 (LP-2200-CD8 TM-41BB-CD3 zeta). CAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG

[0346] SEQ ID NO: 10 is the amino acid sequence of CAR LTG2200 (LP-2200-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR EVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATY YCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0347] SEQ ID NO: 11 is the nucleotide sequence of the DNA CD8 transmembrane domain. ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0348] SEQ ID NO: 12 is the amino acid sequence of the CD8 transmembrane domain. IWAPLAGTCGVLLLSLVITLYC

[0349] SEQ ID NO: 13 is the nucleotide sequence of the DNA CD8 hinge domain. ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCCAGTGCACACGAGGGGGCTGGACTTTGCCTGCGATATCTAC

[0350] SEQ ID NO: 14 is the amino acid sequence of the CD8 hinge domain. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY

[0351] SEQ ID NO: 15 is the amino acid sequence of amino acid numbers 137 to 206 of the hinge and transmembrane region of CD8 alpha (NCBI RefSeq: NP-001759.3). TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0352] SEQ ID NO: 16 is the amino acid sequence of the human IgG CL sequence. GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0353] SEQ ID NO: 17 is the nucleotide sequence of the DNA signaling domain of 4-1BB. AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0354] SEQ ID NO: 18 is the amino acid sequence of the signaling domain of 4-1BB. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0355] SEQ ID NO: 19 is the nucleotide sequence of the DNA signaling domain of CD3-zeta. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0356] SEQ ID NO: 20 is the amino acid sequence of CD3 zeta. RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0357] SEQ ID NO: 21 is the nucleotide sequence of CAR LTG1562 (LP-CD19 binder-CD8 linker-CD4tm-4-1BB-CD3-zeta).

[0358] SEQ ID NO: 22 is the amino acid sequence of CAR LTG1562 (LP-CD19 binder-CD8 link-CD4tm-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSAAAPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFVQPMALIVLGGVAGLLLFIGLGIFFCVRCRPRRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0359] SEQ ID NO: 23 is the nucleotide sequence of the CD20_19 reactive scFv binding domain (LTG1497 bispecific binder). GAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAACAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGGTCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTGACCGTCAGCTCCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGACATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATGACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCGTCACCCAAGCCTTGGATCTACGCTACATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTCAGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGACGCCGCGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACTAAGCTGGAGATCAAAGGAGGCGGCGGCAGCGGCGGGGGAGGGTCCGGAGGGGGTGGTTCTGGTGGAGGAGGATCGGGAGGCGGTGGCAGCGACATTCAGATGACTCAGACCACCTCCTCCCTGTCCGCCTCCCTGGGCGACCGCGTGACCATCTCATGCCGCGCCAGCCAGGACATCTCGAAGTACCTCAACTGGTACCAGCAGAAGCCCGACGGAACCGTGAAGCTCCTGATCTACCACACCTCC CGGCTGCACAGCGGAGTGCCGTCTAGATTCTCGGGTTCGGGGTCGGGAACTGACTACTCCCTTACTATTTCCAACCTGGAGCAGGAGGATATTGCCACCTACTTCTGCCAACAAGGAAACACCCTGCCGTACACTTTTGGCGGGGGA ACCAAGCTGGAAATCACTGGCAGCACATCCGGTTCCGGGAAGCCCGGCTCCGGAGAGGGCAGCACCAAGGGGGAAGTCAAGCTGCAGGAATCAGGACCTGGCCTGGTGGCCCCGAGCCAGTCACTGTCCGTGACTTGTACTGTGTCC GGAGTGTCGCTCCCGGATTACGGAGTGTCCTGGATCAGGCAGCCACCTCGGAAAGGATTGGAATGGCTCGGAGTCATCTGGGGTTCCGAAACCACCTATTACAACTCGGCACTGAAATCCAGGCTCACCATTATCAAGGATAACTCC AAGTCACAAGTGTTCCTGAAGATGAATAGCCTGCAGACTGACGACACGGCGATCTACTATTGCGCCAAGCACTACTACTACGGCGGATCCTACGCTATGGACTACTGGGGCCAGGGGACCAGCGTGACCGTGTCATCCGCGGCCGCA

[0360] SEQ ID NO: 24 is the amino acid sequence of the CD20_19-reactive scFv binding domain (LTG1497 bispecific binder). EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGGSGG GGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKGGGGSGGGGSGGGS GGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAA

[0361] SEQ ID NO: 25 is the nucleotide sequence of CAR LTG1497 (LP-LTG1497-CD8 TM-41BB-CD3 zeta) or (LP-CD20 VH-(GGGGS)3-CD20 VL-(GGGGS)5-CD19VL-Whitlow linker-CD19 VH-CD8 hinge+TM-41BB-CD3 zeta). TCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG

[0362] SEQ ID NO: 26 is the amino acid sequence of CAR LTG1497 (LP-LTG1497-CD8 TM-41BB-CD3 zeta) or (LP-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19VL-Whitlow linker-CD19 VH-CD8 hinge+TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0363] SEQ ID NO: 27 is the nucleotide sequence of the ScFV against CD19. GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCT...

Claims

1. 1. An immunotherapy composition comprising one or more isolated nucleic acid molecules encoding at least one multicistronic vector, wherein each multicistronic vector encodes at least one functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116, and wherein at least one binding domain in at least one of the multicistronic vectors is non-identical, whereby combination of multicistronic vectors results in expression of two or more non-identical functional CAR molecules, each functional CAR molecule encoding at least one binding domain covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

2. (a) at least one multicistronic vector, each multicistronic vector comprising a nucleic acid sequence that is functional in the cell; (b) each multicistronic vector encodes a functional CAR molecule comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116; (c) each functional CAR molecule comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain in one of the functional CAR molecules is not identical; (e) the at least one binding domain, the single transmembrane domain, the at least one linker domain, and the at least one intracellular signaling motif are covalently linked in each of the functional CAR molecules, and the combination of functional CAR molecules is used to genetically modify one or more lymphocyte populations. Immunotherapy composition.

3. (a) at least one multicistronic vector, each multicistronic vector comprising a nucleic acid sequence that is functional in the cell; (b) each multicistronic vector encodes one or more functional CARs comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116; (c) each functional CAR molecule comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain(s) in each CAR is not identical to at least one binding domain in another functional CAR molecule; (e) the at least one signaling motif is not identical among each of the multicistronic co-expressed functional CAR molecules; (f) the at least one binding domain, the single transmembrane domain, and the at least one intracellular signaling motif are covalently linked in each of the multicistronic vectors, and one or more multicistronic vectors are used to genetically modify one or more lymphocyte populations. Immunotherapy composition.

4. The immunotherapeutic composition of claims 1-3, wherein each multicistronic vector encodes more than one functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116.

5. The lymphocyte population(s) may comprise a mixture of autologous T cells or peripheral blood-derived lymphocytes. The immunotherapeutic composition of claim 2 or 3.

6. 4. The immunotherapy composition of claim 2 or 3, wherein at least one extracellular antigen-binding domain of the functional CAR molecule comprises at least one single-chain variable fragment of an antibody that binds to an antigen.

7. 4. The immunotherapy composition of claim 2 or 3, wherein at least one extracellular antigen-binding domain of the functional CAR molecule comprises at least one heavy chain variable region of an antibody that binds to an antigen.

8. 4. The immunotherapy composition of claim 2 or 3, wherein at least one extracellular antigen-binding domain of the functional CAR molecule, the at least one intracellular signaling domain of the CAR, or both, is connected to the transmembrane domain by a linker or spacer domain.

9. 4. The immunotherapy composition of claim 2 or 3, wherein the extracellular antigen-binding domain of the functional CAR molecule is preceded by a leader peptide.

10. 4. The immunotherapy composition of claim 2 or 3, wherein the extracellular antigen-binding domain of the CAR targets an antigen comprising CD19, CD20, CD22, ROR1, TSLPR, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

11. the extracellular antigen-binding domain of the CAR is selected from the group consisting of an anti-CD19 scFv antigen-binding domain, an anti-CD20 scFv antigen-binding domain, an anti-CD22 scFv antigen-binding domain, an anti-ROR1 scFv antigen-binding domain, an anti-TSLPR scFv antigen-binding domain, an anti-mesothelin 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-BCMA (CD269) 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, and an anti-PMSA 4. The immunotherapeutic composition of claim 2 or 3, comprising 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, an anti-MAGE A3 TCR scFv antigen binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

12. 4. The immunotherapeutic composition of claim 2 or 3, wherein the linker or spacer domain of the functional CAR molecule is derived from the extracellular domain of CD8 and is linked to the transmembrane domain.

13. 2. The method of claim 1, wherein the functional CAR molecule further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, or any combination thereof.

4. The immunotherapeutic composition according to claim 3.

14. The immunotherapeutic composition of claim 2 or 3, wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

15. The immunotherapeutic composition of claim 2 or 3, wherein the at least one intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

16. 4. The immunotherapy composition of claim 2 or 3, wherein the at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

17. 17. The immunotherapeutic composition of claim 16, wherein the at least one costimulatory 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), or any combination thereof.

18. The immunotherapy composition of claims 1-3, wherein a single viral vector is used to encode all of the chimeric antigen receptors in combination with a CRISPR system for integration (e.g., a lentiviral, retroviral, adenoviral, SV40, herpes, POX, or cosmid vector).

19. The immunotherapeutic composition of claims 1 to 3, wherein each multicistronic vector is an RNA vector or a DNA vector.

20. The immunotherapeutic composition of claims 1 to 3, wherein at least one multicistronic vector expresses a nucleic acid molecule that regulates the expression of a nucleic acid in a cell.

21. 21. The immunotherapy composition of claim 20, wherein the nucleic acid molecule inhibits or deletes expression of an endogenous gene.

22. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human lymphocyte cells, wherein the cells of the population comprise cells comprising a nucleic acid molecule encoding at least one multicistronic vector, each multicistronic vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116, and wherein at least one binding domain(s) in one of the multicistronic vectors is / are non-identical, whereby combination of multicistronic vectors results in expression of two or more non-identical binding domains, and wherein the binding domain(s) encoded by each multicistronic vector is / are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

23. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human lymphocyte cells, wherein the cells of the population (a) comprise cells comprising a nucleic acid molecule encoding one or more multicistronic vectors; (b) each multicistronic vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116; (c) each functional CAR molecule is composed of at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif; (d) the at least one binding domain in one of the multicistronic vectors is not identical; and (e) the at least one A pharmaceutical composition, wherein a binding domain, a single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each said multicistronic vector, and the combination of multicistronic vectors is used to genetically modify one or more lymphocyte populations.

24. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human lymphocyte cells, wherein the cells of the population (a) comprise cells comprising a nucleic acid molecule encoding one or more multicistronic vectors; (b) each multicistronic vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116; (c) each functional CAR molecule comprises at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif; and (d) each vector (e) the at least one binding domain(s) in each of the multicistronic vectors are not identical; (e) the combination of the at least one signaling motif is not identical between each of the multicistronic vectors; and (f) the at least one binding domain, single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each of the multicistronic vectors, and one or more combinations of multicistronic vectors are used to genetically modify one or more lymphocyte populations.

25. 25. The pharmaceutical composition of claim 23 or 24, wherein the lymphocyte cells are T cells of a human with hematological cancer.

26. 25. The pharmaceutical composition of claim 23 or 24, wherein the hematological cancer is leukemia or lymphoma.

27. 25. The pharmaceutical composition of claim 23 or 24, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

28. 25. The pharmaceutical composition of claim 23 or 24, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma.

29. 25. The pharmaceutical composition of claim 23 or 24, wherein the hematological cancer is multiple myeloma.

30. 25. The pharmaceutical composition of claim 23 or 24, wherein the human cancer comprises oral and pharyngeal 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 cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including cancer of the breast, reproductive system (cervix, uterus, 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 system, or any combination thereof.

31. 1. A method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to a mammal at least one multicistronic vector, each vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116, wherein at least one binding domain(s) in one of the multicistronic vectors is / are not identical, thereby providing a multicistronic vector.

1. A method comprising administering to a subject a pharmaceutical composition comprising multicistronic vectors, the combination of which results in the expression of two or more non-identical binding domains, each vector-encoded binding domain(s) being covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and a pharmaceutically acceptable excipient, wherein the combination of multicistronic vectors is used to genetically modify one or more lymphocyte populations.

32. 1. A method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to a subject a pharmaceutical composition comprising: (a) a nucleic acid molecule encoding two or more vectors; (b) each vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116; (c) each functional CAR molecule is composed of at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif; (d) the at least one binding domain in one of the multicistronic vectors is not identical; and (e) the at least one binding domain, single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of the multicistronic vectors, wherein a combination of multicistronic vectors is used to genetically modify one or more lymphocyte populations.

33. 1. A method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising: (a) a nucleic acid molecule encoding one or more multicistronic vectors; (b) each multicistronic vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO: 54, 56, 60, 62, 110, 112, 114, or 116; (c) each functional CAR molecule comprising at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif; and (d) one or more of said at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif in each multicistronic vector. (e) at least one binding domain(s) in each of the multicistronic vectors are not identical; (e) the combination of the at least one signaling motif is not identical between each of the multicistronic vectors; and (f) the at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of the multicistronic vectors, and one or more combinations of multicistronic vectors are used to genetically modify one or more lymphocyte populations.

34. 34. The method of claims 31-33, wherein the genetically modified lymphocytes are autologous lymphocytes, and the autologous or allogeneic lymphocytes are directly infused back into the patient to prevent recurrence of malignant tumor disease.

35. 34. The method of claims 31-33, wherein the genetically modified lymphocytes are autologous T cells, which are directly infused back into the patient to promote in vivo expansion, and wherein persistence of patient-specific anti-tumor T cells results in tumor stabilization, reduction, elimination, remission, or elimination of cancer or cancer recurrence in a patient-specific manner.

36. The method of claims 31-33, wherein the T cells have been preselected by expression of surface markers associated with specific activation or memory.

37. The method of claims 31 to 33, wherein the T cells and dendritic cells are derived from a hematopoietic stem cell donor and the procedure is performed in the context of a hematopoietic stem cell transplant.

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