Compositions and methods for treating cancer using DUOCAR

Abstract

To provide use of a population of human T cells for the manufacture of a pharmaceutical composition for treating a subject with an autoimmune disease.SOLUTION: Provided is use of a population of human T cells for manufacturing a pharmaceutical composition, the population of human T cells being autologous to a subject, each cell of the population of human T cells comprising at least one multicistronic vector, each of the at least one multicistronic vector comprising a promoter operably linked to a multicistronic nucleic acid sequence encoding at least two functional CARs, at least one of the at least two functional CARs comprising a specific amino acid sequence, thereby treating the subject with an autoimmune disease.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 382,791, filed 2 September 2016, which is incorporated herein by reference in its entirety under Section 119(e) of the U.S. Patent Act.

[0002] Areas of disclosure This application relates to the field of cancer, and more particularly to a composition comprising at least two vectors encoding a functional chimeric antigen receptor, and to a method of using the same in patient-specific immunotherapy. [Background technology]

[0003] Background of the Invention Cancer is one of the most significant threats to human health. In the United States alone, nearly 1.3 million people are diagnosed with cancer each year, making it the second leading cause of death after cardiovascular disease and accounting for about a quarter of all deaths. Solid tumors account for the vast majority of these deaths. While there have been significant advances in the medical treatment of certain cancers, the five-year overall survival rate for all cancers has improved by only about 10% in the last 20 years. Cancer, or malignant tumors, metastasize and grow rapidly without control, making them extremely difficult to treat. One of the challenges in modern cancer treatment is the time that passes between a patient's biopsy and the diagnosis of cancer, as well as the need for effective treatment. During this time, the patient's tumor can grow unchecked, and as a result, the disease may progress further before treatment can be applied. This negatively impacts the prognosis and outcome of cancer.

[0004] Chimeric antigen receptors (DuoCARs) are hybrid molecules containing three essential units: (1) an extracellular antigen-binding motif, (2) a ligation / 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; Vol. 2 (No. 4): e23621). The antigen-binding motif of CARs is generally constructed 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 is engineered to bind to the IL-13 receptor expressed by tumors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012; Vol. 18 (No. 8): pp. 2199-209; Lehner M et al. PLoS One. 2012; Vol. 7 (No. 2): e31210). Considerable effort is still required to define the most active T cell population for transduction of CAR vectors, determine optimal culture and proliferation techniques, and define the molecular details of the CAR protein structure itself.

[0005] The linking motif of CARs can be designed to be a relatively stable structural domain, such as the constant domain of IgG, or an extended flexible linker. Using structural motifs such as those derived from the constant domain of IgG, the scFv-binding domain can be extended 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 the disialoganglioside GD2; Orentas et al., unpublished observation). To date, in CARs... The signaling motifs used always include the CD3-ζ chain because this core motif is a crucial signal for T cell activation. The first reported second-generation CARs featured a CD28 signaling domain and a CD28 transmembrane sequence. This motif was similarly used in third-generation CARs containing the CD137(4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; Vol. 183 (No. 9): pp. 5563-74). With advances in new technologies, T cell activation by beads linked to anti-CD3 and anti-CD28 antibodies, as well as the presence of a canonical "signal 2" derived from CD28, no longer need to be encoded by the CAR itself. Using bead activation, third-generation vectors were found not to be superior to second-generation vectors in in vitro assays, and no clear benefit over second-generation vectors was obtained in a mouse model 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; Vol. 121 (No. 7): pp. 1165-1174; Kochenderfer JN et al. Blood. 2012; Vol. 119 (No. 12): pp. 2709-2720). This is the second-generation CD28 / CD3-ζ (Lee DW et al., American Society of Hematology Annual Meeting, New Orleans, LA; December 7-10, 2013) and CD137 / CD3-ζ signaling pathway (Porter This is supported by the clinical success of CD19-specific CARs described by DL et al. (N Engl J Med. 2011; vol. 365 (no. 8): pp. 725-733). In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 can also provide important sustained signaling in CAR-transduced T cells (Yvon E et al.). Clin Cancer Res. 2009; Vol. 15 (No. 18): pp. 5852-5860. The culture conditions under which the CAR T cell population was cultured are equally important.

[0006] A current challenge in the broader and more effective application of CAR therapy for cancer concerns the lack of compelling targets. While creating binding factors to cell surface antigens is now readily achievable, discovering tumor-specific cell surface antigens while overlooking normal tissue remains a challenging task. One potential method for conferring 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 lower affinity and therefore requires the alternative CAR to bind first for the full activity of the second CAR (Lanitis E et al. Cancer Immunol Res. 2013; Vol. 1 (No. 1): pp. 43-53; Kloss CC et al. Nat Biotechnol. 2013; Vol. 31 (No. 1): pp. 71-75). A second challenge for generating single scFv-based CARs as immunotherapeutic agents is the heterogeneity of tumor cells. At least one group is developing a CAR strategy for glioblastoma in which effector cell populations simultaneously target multiple antigens (HER2, IL-13Ra, EphA2) in the hope of avoiding the growth of target antigen-negative populations (Hegde M et al. Mol Ther. 2013; Vol. 21 (No. 11): pp. 2087-101).

[0007] T cell-based immunotherapy is a new field in synthetic biology; multiple promoters and gene products are hypothesized to guide these highly potent cells into the tumor microenvironment, where T cells can evade negative regulatory signals and effectively kill tumor cells. It can mediate this. Elimination of undesirable T cells via drug-induced dimerization of the inducible caspase 9 construct with AP1903 demonstrates one way in which a potent switch capable of controlling T cell populations can be pharmacologically initiated (Di Stasi A et al. N Engl J Med. 2011; vol. 365 (no. 18): pp. 1673-1683). The creation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β by decoy receptor expression further demonstrates the extent to which effector T cells can be manipulated for optimal antitumor activity (Foster AE et al. J Immunother. 2008; vol. 31 (no. 5): pp. 500-555).

[0008] Therefore, while 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 this CAR-based technology to date are the limited in vivo proliferation of CAR+ T cells, rapid cell disappearance after injection, disappointing clinical activity, recurrence of the underlying disease or condition, and the excessively long time elapsed between diagnosis and timely treatment of cancer using such CAR+ T cells. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, there is an urgent and long-standing need in the field to discover compositions and methods for treating cancer using CAR-based therapies that can exhibit cancer-specific and intended therapeutic properties without the aforementioned drawbacks. [Means for solving the problem]

[0010] The present invention addresses these needs by providing a composition comprising at least two vectors encoding a functional chimeric antigen receptor, as well as a method of using it in patient-specific immunotherapy that may be used to treat cancer and other diseases and / or conditions.

[0011] Specifically, the present invention, as disclosed and described herein, provides an immunotherapy composition comprising one or more isolated nucleic acid molecules, each vector encoding at least two vectors encoding a functional DuoCAR, wherein the combination of vectors results in the expression of two or more non-identical binding domains, and each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and which can be used for transduction into autologous lymphocytes to generate an active patient-specific antitumor lymphocyte cell population that can be returned to the patient by direct injection to promote in vivo proliferation and persistence of patient-specific antitumor T cells resulting in tumor stabilization, reduction, elimination, cancer remission, or prevention or remission of cancer recurrence or a combination thereof.

[0012] Summary of the Invention Novel adoptive immunotherapy compositions comprising lymphocytes transduced from two or more vectors, as well as methods of using them in patient-specific combination immunotherapy that may be used to treat cancer and other diseases and conditions, are provided herein.

[0013] Accordingly, in one embodiment, a lentiviral vector expressing a Duo-chimeric antigen receptor (DuoCAR) and a nucleic acid molecule encoding a lentiviral vector expressing DuoCAR are provided herein. Methods of using the disclosed lentiviral vector, host cells, and nucleic acid molecule expressing DuoCAR are also provided, for example, to treat cancer in a subject.

[0014] In one embodiment, the immunotherapy composition comprises one or more vectors encoding at least two vectors. Provided in the form of isolated nucleic acid molecules (DuoCAR), each vector encodes a functional CAR, where at least one binding domain(s) in one of the vectors are non-identical, so that the combination of vectors results in the expression of two or more non-identical binding domains, where the binding domain(s) encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0015] In one embodiment, the immunotherapy composition is provided in the form of one or more isolated nucleic acid molecules encoding at least three vectors (TrioCAR), each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains, where each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0016] In one embodiment, the immunotherapy composition is provided in the form of one or more isolated nucleic acid molecules encoding at least four vectors (QuatroCAR), each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains, where each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0017] In yet another embodiment, the immunotherapy composition is provided in the form of comprising one or more isolated nucleic acid molecules encoding at least two, three, four, five, six, seven, eight, nine or ten vectors (e.g., "nCAR"), each vector encoding a functional CAR, whereby the combination of vectors results in the expression of two or more non-identical binding domains, wherein each binding domain(s) encoded by each vector is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and each unique member of the nCAR set, when incorporated into the CAR product, constitutes a unique CAR composition referred to herein as "n-SET" (e.g., Duo-SET, Trio-SET, Quatro-SET, Penta-SET, Hexa-SET, Hepta-SET, Octa-SET, Nona-SET and Deca-SET, etc.).

[0018] In one embodiment, the immunotherapy composition is provided in the form of comprising at least two vectors each containing a nucleic acid sequence that is functional in a cell; (b) each vector encodes a functional CAR; (c) each CAR is composed of at least one binding domain, a single transmembrane domain and at least one intracellular signaling motif; (d) at least one binding domain in one of the vectors is non-identical; and (e) at least one binding domain, a single transmembrane domain, at least one linker domain and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0019] In another embodiment, the immunotherapy composition is provided in a form comprising: (a) at least two vectors each containing a nucleic acid sequence that is functional in cells; (b) each vector encodes a functional CAR; (c) each CAR comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif; (d) at least one binding domain(s) in each vector is not the same; (e) at least one combination of signaling motifs is not the same between each vector; and (f) at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each of the vectors, and a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0020] In one embodiment, an immunotherapy composition is provided wherein each vector encodes more than one functional CAR.

[0021] In another embodiment, an immunotherapy composition is provided wherein at least one combination of signaling motifs is the same on one or more vectors.

[0022] In another embodiment, an immunotherapy composition is provided wherein at least one multi-binding domain is the same on one or more vectors.

[0023] In another embodiment, an immunotherapy composition is provided wherein the lymphocyte population(s) comprises a mixture of autologous T cells or lymphocytes derived from peripheral blood.

[0024] The 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 the antigen.

[0025] 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 the antigen.

[0026] In another embodiment, an immunotherapy composition is provided in which at least one extracellular antigen-binding domain of the CAR, at least one intracellular signaling domain of the CAR, or both, are connected to a transmembrane domain by a linker or spacer domain.

[0027] In another embodiment, an immunotherapy composition is provided in which a leader peptide precedes the extracellular antigen-binding domain of a CAR.

[0028] In another embodiment, an immunotherapy composition is provided in which the extracellular antigen-binding domain of CAR targets antigens including CD19, CD20, CD22, ROR1, TSLPR, mesoserine, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1, MAGE-A3, PRAME peptides combined with MHC, or any combination thereof.

[0029] In another embodiment, the extracellular antigen-binding domains of CAR include anti-CD19 scFV antigen-binding domains, anti-CD20 scFV antigen-binding domains, anti-CD22 scFV antigen-binding domains, anti-ROR1 scFV antigen-binding domains, anti-TSLPR scFV antigen-binding domains, anti-mesoserine scFV antigen-binding domains, anti-CD33 scFV antigen-binding domains, anti-CD38 scFV antigen-binding domains, anti-CD123 (IL3RA) scFV antigen-binding domains, anti-CD138 scFV antigen-binding domains, anti-BCMA (CD269) scFV antigen-binding domains, anti-GPC2 scFV antigen-binding domains, anti-GPC3 scFV antigen-binding domains, anti-FGFR4 scFV antigen-binding domains, anti-c-Met scFV antigen-binding domains, anti-PMSA scFV antigen-binding domains, anti-glycolipid F77 scFV antigen-binding domains, and anti-EGFRvIII domains. scFV antigen-binding domain, anti-GD-2 scFV antigen-binding domain, anti-NY-ESO-1 An immunotherapy composition is provided comprising a TCR (including a single-stranded TCR construct) antigen-binding domain, an anti-MAGE-A3 TCR, or its amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0030] In another embodiment, an immunotherapy composition is provided in which the linker or spacer domain of CAR is derived from the extracellular domain of CD8 and linked to the transmembrane domain.

[0031] In another embodiment, an immunotherapy composition is provided in which the CAR further comprises a transmembrane domain containing a protein selected from the group consisting of the alpha, beta, or zeta chain of a 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.

[0032] In another embodiment, an immunotherapy composition is provided in which at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0033] In another embodiment, an immunotherapy composition is provided in which at least one intracellular signaling domain is positioned C-terminally relative to the CD3 zeta intracellular domain.

[0034] In another embodiment, an immunotherapy composition is provided in which at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

[0035] In another embodiment, an immunotherapy composition is provided in which at least one co-stimulatory domain comprises the functional signaling domains 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.

[0036] In another embodiment, an immunotherapy composition is provided in which a single vector is used in combination with a CRISPR system for incorporation to encode all chimeric antigen receptors (e.g., retroviruses, adenoviruses, SV40, herpes vectors, POX vectors, RNA, plasmids, cosmids, or any viral or non-viral vectors).

[0037] In another embodiment, each vector is an RNA or DNA vector, and the immunotherapy composition is provided either alone or in combination with a transfection reagent or, in a non-limiting example, a method for delivering RNA or DNA to cells, which is electroporation.

[0038] In another embodiment, an immunotherapy composition is provided in which at least one vector expresses a nucleic acid molecule that modulates nucleic acid expression in a cell.

[0039] In another embodiment, an immunotherapy composition is provided in which a nucleic acid molecule inhibits or deletes the expression of an endogenous gene.

[0040] In a particular embodiment, an immunotherapy composition is provided in which an active patient-specific autologous antitumor lymphocyte population is generated within 1, 2, 3, 4, 5, 7, 10, 12, 14, 21 days, or 1 month of lymphocyte retrieval or tumor biopsy and can be directly injected back into a patient with cancer. This active patient-specific autologous antitumor lymphocyte population is capable of patient-specifically promoting in vivo proliferation and persistence of patient-specific antitumor lymphocytes, resulting in tumor stabilization, reduction, elimination, cancer remission, or prevention or remission of cancer recurrence, or a combination thereof.

[0041] In one embodiment, isolated nucleic acid molecules encoding the above-mentioned chimeric antigen receptor are provided herein.

[0042] In one embodiment of DuoCAR used in a patient-specific autologous lymphocyte population(s) of the immunotherapy composition of the present invention, 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 progression of such treatment. In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population(s), the nucleic acid molecule encoding the disclosed DuoCAR may be contained in a vector such as a virus or a non-viral vector. The vector may be a DNA vector, RNA vector, plasmid vector, cosmid vector, herpesvirus vector, measles virus vector, lentiviral vector, adenovirus vector, or retroviral vector or a combination thereof.

[0043] In certain embodiments of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, two or more lentiviral vectors are pseudotyped with different viral glycoproteins (GPs), including, for example, but not limited to, ambidextrous mouse leukemia virus [MLV-A], baboon endogenous virus (BaEV), GP164, gibbon leukemia virus [GALV], RD114, feline endogenous virus retrovirus-derived GPs, and vesicular stomatitis virus [VSV], measles virus, avian plague virus [FPV], Ebola virus [EboV], lymphocytic choriomeningitis virus [LCMV] nonretrovirus-derived GPs, as well as their chimeric variants, 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 (referred to as TR) of MLV-A GP.

[0044] In certain embodiments of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, the vector further comprises a promoter which is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0045] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, the vector expressing CAR is controlled by a suicide switch. The vector may be further modified to include one or more activatable elements to control T cell expression or to eliminate CAR-T cells. The suicide switch may include, for example, an apoptosis-inducible signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0046] In another embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population, host cells containing the nucleic acid molecule encoding DuoCAR are also provided. In some embodiments, the host cells are T cells, e.g., primary T cells obtained from the 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 product without regard to ratio. In another embodiment, the number of CD4+ and CD8+ T cells in the product is specific. In another embodiment, a specific subset of T cells is T naive cells (Tn), T effector memory cells (Tem), T central memory cells (Tcm), T regulatory cells (Treg), inducible T regulatory cells (iTreg), T suppressor cells (Ts), and T stem cell memory cells (Tscm). These cells are used as phenotypic markers, such as natural killer (NK) cells and lymphokine-activated killer (LAK) cells, as identified by these markers.

[0047] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of an immunotherapy composition comprising a population of patient(s) of patient(s) of cancer-specific autologous antitumor lymphocyte cells, wherein the cells of the population comprise cells containing nucleic acid molecules, each vector encoding at least two vectors encoding functional CARs, the combination of vectors resulting in the expression of two or more non-identical binding domains, where each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0048] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of an immunotherapy composition comprising a population of patient(s) of cancer-specific autologous antitumor lymphocyte cells, wherein the cells of the population (a) comprise cells containing nucleic acid molecules encoding two or more vectors; (b) each vector encoding a functional CAR; (c) each CAR comprising at least one binding domain, at least one transmembrane domain, at least one linker domain and at least one intracellular signaling motif; (d) at least one binding domain in one of the vectors is not identical; and (e) at least one binding domain, a single transmembrane domain, at least one linker domain and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0049] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of an immunotherapy composition comprising a population of patient(s) of a human patient(s) with cancer, wherein the cells of the population (a) comprise cells comprising nucleic acid molecules encoding two or more vectors; (b) each vector encoding a functional CAR; (c) each CAR comprising at least one binding domain, at least one transmembrane domain, at least one linker domain and at least one intracellular signaling motif; (d) at least one binding domain(s) in each vector are not identical; (e) at least one combination of signaling motifs is not identical between each vector; and (f) at least one binding domain, a single transmembrane domain, at least one linker domain and at least one intracellular signaling motif are covalently linked in each vector, and a combination of two or more vectors is used to genetically modify one or more lymphocyte populations.

[0050] In one embodiment, the cancer is a refractory cancer that is unresponsive to one or more chemotherapy agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, 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 cancers and solid tumors, or any combination thereof. In another embodiment, the cancer includes hematological cancers, e.g., leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma), or multiple myeloma, or any combination thereof.

[0051] In yet another embodiment, cancer can be 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 bronchi), bone and joint cancer, soft tissue cancer, skin cancer (black This includes cancers of the breast, reproductive system (cervical, uterine body, ovaries, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidneys and renal pelvis, ureters), eyes and orbits, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

[0052] In another embodiment, a pharmaceutical composition is provided that generates a patient-specific autologous antitumor lymphocyte population transduced using two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs), thereby enabling patient-specific promotion of in vivo proliferation and persistence of patient-specific antitumor T cells resulting in tumor stabilization, reduction, elimination, remission of cancer, prevention or remission of cancer recurrence, or a combination thereof.

[0053] In another embodiment, a pharmaceutical composition is provided comprising an autologous lymphocyte population transduced using one or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs) to generate a patient-specific autologous antitumor lymphocyte population capable of promoting patient-specific in vivo proliferation and persistence of patient-specific antitumor T cells that result in tumor stabilization, reduction, elimination, remission of cancer, prevention or remission of cancer recurrence, or a combination thereof.

[0054] In another embodiment, a method is provided for producing active patient-specific autologous antitumor DuoCAR-containing lymphocytes. This method involves transducing lymphocytes with two or more vectors or nucleic acid molecules encoding two or more chimeric antigen receptors (DuoCARs) that specifically bind to an antigen, thereby producing active patient-specific autologous antitumor DuoCAR-containing lymphocytes.

[0055] In yet another embodiment, a method is provided for generating a population of RNA-modified lymphocytes, comprising introducing in vitro transcribed RNA or synthetic RNA of nucleic acid molecules encoding two or more chimeric antigen receptors (DuoCARs) into a target cell population, thereby generating a patient-specific autologous antitumor lymphocyte population capable of patient-specifically promoting in vivo proliferation and persistence of patient-specific antitumor T cells that result in tumor stabilization, reduction, elimination, cancer remission, or prevention or remission of cancer recurrence, or a combination thereof.

[0056] In another embodiment, a method is provided for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, the method comprising the step of administering to a subject a pharmaceutical composition comprising an antitumor-effective amount of an autologous antitumor lymphocyte cell population transduced with one or more lentiviral vectors encoding one or more chimeric antigen receptors (DuoCARs), thereby generating a patient-specific autologous antitumor lymphocyte cell population capable of patient-specifically promoting in vivo proliferation and persistence of patient-specific antitumor T cells resulting in tumor stabilization, reduction, elimination, remission or prevention of cancer recurrence or remission of cancer recurrence or a combination thereof.

[0057] In another embodiment, a method for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising the step of administering to the subject a pharmaceutical composition comprising an antitumor-effective amount of an autologous lymphocyte population transduced using two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs) to generate a patient-specific autologous antitumor lymphocyte population that can be returned to the patient by direct injection to promote in vivo proliferation and persistence of patient-specific antitumor T cells resulting in patient-specific stabilization, reduction, elimination of a tumor, or remission of cancer or prevention or remission of cancer recurrence, or a combination thereof. The law is provided.

[0058] In one embodiment, a method is provided for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising the step of administering a pharmaceutical composition to a subject comprising at least two vectors, each encoding a functional CAR, and a pharmaceutically acceptable excipient, wherein the combination of vectors results in the expression of two or more non-identical binding domains, and each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and the combination of vectors is used to genetically modify one or more lymphocyte populations.

[0059] In another embodiment, a method is provided for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising the step of administering a pharmaceutical composition to a subject, wherein (a) a nucleic acid molecule comprising two or more vectors; (b) each vector encoding a functional CAR; (c) each CAR comprising at least one binding domain, at least one transmembrane domain and at least one intracellular signaling motif; (d) at least one binding domain in the vectors is not identical; and (e) at least one binding domain, a single transmembrane domain and at least one intracellular signaling motif are covalently linked in each of the vectors, and the combination of vectors is used to genetically modify one or more lymphocyte populations.

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

[0061] In certain embodiments, the genetically modified lymphocytes are autologous T-cell lymphocytes, which are then injected directly into the patient to prevent relapse or induce remission of a malignant disease.

[0062] In certain other embodiments, the genetically modified lymphocytes are autologous T-cell lymphocytes, which are returned to the patient by direct injection to promote patient-specific in vivo proliferation and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, reduction, elimination, or remission of cancer, or prevention or remission of cancer recurrence, or any combination thereof.

[0063] In yet another embodiment, T cells are pre-selected by expressing specific activation or memory-related surface markers.

[0064] In yet another embodiment, the T cells are derived from a hematopoietic stem cell donor, and this procedure is performed in the context of hematopoietic stem cell transplantation.

[0065] In certain embodiments, a method is provided herein in which lymphocyte cells are pre-selected by expressing a specific activation or memory-related surface marker.

[0066] In a particular embodiment, the lymphocytes are T cells derived from a hematopoietic stem cell donor, and a method is provided herein in which the procedure is performed in the context of hematopoietic stem cell transplantation.

[0067] In yet another embodiment, a method is provided for generating a persistent population of genetically engineered patient-specific autologous antitumor lymphocyte cells in a person diagnosed with cancer. In one embodiment, the method comprises the steps of administering one or more patient-specific autologous antitumor lymphocyte cell populations described herein to a human patient in need thereof, wherein the persistent population of patient-specific autologous antitumor lymphocyte cell populations, or population of lymphocyte cell offspring, persists in the human for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, or three years after administration.

[0068] In one embodiment, the progeny lymphocyte cells in humans include memory T cells. In another embodiment, the T cells are autologous T cells.

[0069] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens may be treated, prevented, or mitigated using a patient-specific autologous antitumor lymphocyte cell population (may include one or more Duo Car immunotherapy compositions disclosed herein).

[0070] In yet another embodiment, a kit is provided for preparing a DuoCar immunotherapy composition comprising the patient-specific autologous antitumor lymphocyte cell population(s) described above, or for preventing, treating or relieving any of the cancers, diseases, disorders or conditions associated with elevated expression of a tumor antigen in the subject described above, the kit comprising a container containing one or any combination thereof of the nucleic acid molecules, vectors, host cells or compositions disclosed above, and instructions for using the kit.

[0071] While the compositions and methods of the present invention are illustrated with reference to the production and use of DuoCAR, it is intended herein that the compositions and methods specifically include the production and use of TrioCAR and QuatroCAR.

[0072] In yet another embodiment, the immunotherapy composition comprises one or more isolated nucleic acids encoding at least one vector, the vector containing a nucleic acid sequence (i.e., a multiple cistronic nucleic acid or a nucleic acid resulting in more than one transcript) that produces at least one messenger RNA encoding DuoCAR, thereby giving rise to the ability to bind to two or more non-identical antigen targets, and thereby resulting in the presence of multiple antigen specificity in a single cell expressing the vector.

[0073] In yet another embodiment, the immunotherapy composition described above comprises one or more isolated nucleic acids encoding at least two vectors, each vector further encoding a functional tag or anti-tag binding component (AT-CAR) that reconstitutes a functional chimeric antigen receptor when incubated together or administered together with a soluble binder (such as a tagged scFv or an scFv linked to an anti-tag binder), thereby giving the combination of the two vectors the ability to bind to two or more non-identical antigen-binding domains, resulting in the presence of multi-antigen specificity in cells expressing these two vectors.

[0074] In yet another embodiment, the immunotherapy composition described above comprises one or more isolated nucleic acids encoding at least two vectors, each vector containing a functional tag that reconstitutes a functional chimeric antigen receptor when incubated together or administered together with a soluble binder (such as a tagged scFv or an scFv linked to an anti-tag binder). Alternatively, each vector may encode an anti-tag binding component (AT-CAR), where each vector expresses a unique tag (or anti-tag) that can bind to a soluble protein or protein-modified structure to provide multi-antigen specificity, or each vector may express a unique tag (or anti-tag) that binds to only one of the soluble binding domains, resulting in the specific linkage of the tagged (or anti-tagged) binder of the intracellular signaling motif encoded by the AT-CAR to the antigen-binding domain.

[0075] In non-limiting embodiments of the production of DuoCAR vectors, their respective compositions and methods, as disclosed in the embodiments and aspects referenced above, the two vectors may be prepared separately and then added sequentially or simultaneously to T cells. In another non-limiting embodiment, the plasmid DNA of two or more vectors may be combined before or during transfection of the producing cells, or incorporated into the producing cell genome, to produce a mixture of viral vectors containing multiple DuoCAR vector particles, which is then used for gene transfer and genetic modification of patient T cells.

[0076] The patient-specific autologous antitumor lymphocyte cell population(s), two or more lentiviral vectors expressing a chimeric antigen receptor (DuoCAR), host cells, and methods described above are understood to be useful beyond the specific embodiments and models detailed herein. The aforementioned features and advantages of this disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

[0077] The following detailed description of preferred embodiments of the present invention will be better understood in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, preferred drawings of the present invention are shown. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings. [Brief explanation of the drawing]

[0078] [Figure 1]This figure shows four (four) products (Examples 1 to 4) that may be produced as separate commercial entities. These DuoCAR sets may be created to target human B-cell malignancies expressing three leukemia-associated antigens, CD19, CD20, and CD22. In Product 1, two gene vectors are used to co-transduce an activated T-cell population. The first vector encodes two antigen-binding domains (CD19, CD20) linked to a single intracellular domain (z, CD3 zeta chain) ligated by a 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, Example 2, features the first vector containing CD19 and CD20-binding domains linked to CD28 and z signaling domains. The second vector encodes the CD22-binding domain as well as the BB and z-signaling domains, essentially replicating the signaling package of a third-generation CAR vector (three distinct signaling domains). In the third product, Example 3, the first vector encodes the CD20- and CD22-binding domains linked to the BB and z-signaling domains, and the second vector encodes the CD19-binding domain linked to the CD28 and z-signaling domains. In the fourth product, Example 4, the first vector encodes the CD20- and CD22-binding domains as well as the BB and z-signaling domains. The second vector encodes the CD19-binding domain and the z-signaling domain. [Figure 2] This shows all possible single components that can be combined into the DuoCAR set for therapeutic products targeting B-cell malignancies. The nomenclature is the same as that in Figure 1. [Figure 3]This figure shows a general schema for a set of DuoCARs that can be applied to multiple therapeutic needs, including inflammatory or autoimmune diseases and infectious diseases. In the figure, a-CDX, a-CDY, and a-CDZ refer to antigen-binding domains specific to three different target antigens, CDX, CDY, and CDZ, respectively. The intracellular aspect of the CAR includes a CD8 linker linked to either the CD3-zeta, CD28, or 4-1BB signaling domain, and a transmembrane domain (as shown in Figure 1). The specific combination of any of these two vectors into a single vector (e.g., A plus F, where antigens X, Y, and Z are targeted while intracellular signaling is provided via CD3-zeta and 4-1BB) is determined by the specific therapeutic need. [Figure 4] This figure shows a general schema for DuoCAR sets in which two antigens are targeted by each vector. Vectors identical to those in Figure 3 retain their specific letter designations (A is the same in Figures 3 and 4). A novel fourth antigen-binding domain is indicated by a-CDW. A single product that targets four antigens is the A+T DuoCAR set. In this case, the extracellular antigens CDX, CDY, CDZ, and CDW are targeted while both CD3-zeta and CD28 intracellular signals are provided. [Figure 5]This figure compares current CARs in the literature (A, B, C, D) with the DuoCARs (E, F, G) of the present invention. It is possible to create CAR expression vectors that induce the expression of single-binding domains (paired black, empty, or striped spheres, each with distinct specificities) linked to linkers and transmembrane domains (single empty squares). In the figure, the thick gray line represents the plasma cell membrane. In this figure, paired black spheres represent anti-CD19 scFv, paired empty spheres represent anti-CD20 scFv, and paired striped spheres represent anti-CD22 scFv, all linked by amino acid sequences, such as multimers of GGGGS (1, 2, 3, 4, 5, or 6 repeats). Intracellularly, lymphocyte signaling domains derived from 4-1BB (CD137), CD28, and CD3 zeta chains may be combined as shown. (A) In a single CAR, a single binding domain is combined with a transmembrane and two signaling domains to create a second-generation CAR. (B) In a split CAR, two different binders are expressed with a single signaling domain that must be combined to enable T cell signaling for the recognition of two separate antigens. (C) In a tandem CAR, two binding domains are linked to a single signaling domain. In this case, binding of either domain induces complete T cell activation. (D) In ​​a multiple CAR from a single vector, two fully functional CARs are expressed from a single vector, each capable of binding to only one antigen. (E) In contrast, a DuoCAR consists of two vectors and expresses at least three binding domains, including multiple possible combinations of signaling domains. Essential features that distinguish DuoCARs from others are the expression of two or more transcripts, the multiplicity of binding domains (at least one of which is multitargeted), and at least one fully functional signaling feature of the two expressed cell surface proteins. (F) In the DuoCAR single-specific 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; these are not identical with respect to the intracellular motifs).The base vector binds to a soluble protein containing an scFv domain that interacts with the antigen, and also containing a tag or anti-tag motif to mediate binding to the CAR base protein itself. When the soluble protein binds to the CAR base protein, the same structural features that mediate the antitumor activity mediated by DuoCAR are reconstructed [as shown in (E)]. (G) In DuoCAR, the bispecific soluble binder format, the bispecific "tag"-"anti-tag" interaction is unique, and only one of the soluble binders can bind to only one of the base vectors. In this case, the black diamond-shaped binder on the base vector and the square-shaped binder on the soluble bispecific scFv protein represent a "biotin"-"anti-biotin" interaction, and the black crescent-shaped binder on the second CAR base vector interacts with the black ellipse on the single-specific scFv structure, which can represent a "FITC"-"anti-FITC" interaction. [Figure 6] This figure shows the cell surface expression levels of CAR constructs on primary human T cells transduced using different CAR expression vectors in second-generation (two co-stimulatory domains) and third-generation (three co-stimulatory domains) formats. T cells were transduced to express the following CARs: no CAR (fake), second-generation CAR (CAR-A-28z), third-generation CAR (CAR-A-28BBz), and alternative second-generation CAR (CAR-A-BBz). CAR surface expression levels were detected by flow cytometry and reported as mean fluorescence intensity (MF) on the y-axis. Despite all constructs expressing the exact same CAR-binding domain, the MFI of both second-generation CARs was brighter. [Figure 7]This figure shows DuoCAR cell surface expression in human T cells. Human T cells were activated in the presence of IL-2 using CD3-CD28 nanomatrix (TransAct, Miltenyi Biotec), transduced using two vectors (one encoding a tandem CD20-CD19 CAR and the other a single CD22 CAR, thus forming a 2+1 Duo-Set format), and then analyzed for CD19-, CD20-, or CD22-scFv domain expression by flow cytometry using recombinant CD19, CD20, or CD22 for staining. Paired columns show double staining for CD20 and CD19 scFvs in the left column and CD22 and CD19 scFvs in the right column. Column 1 shows T cells that have not been transduced (UTD) and therefore do not show binding. Column 2 shows T cells transduced using a LV encoding a CD20_CD19 CAR vector containing CD8 transmembrane and intracellular CD28 and CD3-zeta signaling domains (20-19-28z). Double staining is seen for CD20 and CD19 binding (left panel), while only CD19 binding is seen in the right panel. Column 3 shows T cells transduced using a CD22 CAR vector containing CD8 transmembrane and intracellular 4-1BB and CD3-zeta signaling domains (22-BBz). Double staining is not seen for CD19 or CD20 (left panel), and it can be seen that only a single population of cells can bind to CD22 (right panel). In column 4, T cells are transduced using a DuoSet composed of both vectors from columns 2 and 3. 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 CD22 and CD19 scFv are located on two separate transmembrane proteins, including DuoSet, the 38% represents the true DuoSet expression population in this example. [Figure 8]This figure shows the antitumor cell lytic activity of DuoCAR-expressing T cells. As shown in Figure 7, human T cells transduced using a single CAR component (20_19-28z or 22-BBz) or DuoSet (20_19-28z + 22-BBz) were used in cytotoxic T cell assays at four different effector target ratios (20:1, 10:1, 5:1, and 2.5:1 as shown). 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 monoexpressing K562 target cell lines (K562-CD19, K562-CD20, K562-CD22). [Figure 9]This figure shows DuoCAR cell surface expression in primary human T cells achieved by two different methods of LV preparation. The same methods and data analysis were used as shown in Figure 7 to generate cells transduced using CD19, CD20, and CD22-specific DuoCARs (2+1 DuoSet, where one CAR is a tandem CD20 and CD19 binder and the second CAR consists of a CD22 binder). The first column of the data shows flow cytometry analysis of CD19 and CD20 binder expression, while the second column shows flow cytometry analysis of CD22 and CD19 binders present as CARs in DuoCAR-expressing cells for four different populations corresponding to non-transduction, single CD22-CAR transduction, dual transduction with CD22 and CD20_19 CARs, and single transduction with tandem CD20_CD19 CARs, respectively, in the lower left, upper left, upper right, and lower right quadrants. Both the two-LV transduction method (co-transduction) and the single-LV transduction method (co-transfection) resulted in expression of both CA cell surface proteins, and more than 30% of the T cell population showed similar DuoCAR staining patterns, being specific to CD19, CD20, and CD22. [Modes for carrying out the invention]

[0079] Detailed explanation definition As used herein, the singular forms “a,” “an,” and “the” refer to both the singular and plural forms unless the context clearly indicates otherwise. For example, the term “one antigen” may include one or more antigens and may be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” Therefore, “comprising one antigen” means “including one antigen” without excluding other elements. The phrase “and / or” means “and” or “or.” Any and all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides, should be understood to be approximate and provided for convenience unless otherwise noted. Many methods and materials similar or equivalent to those described herein may be used, but particularly suitable methods and materials are described below. In case of any conflict, this specification, including the explanation of terms, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting. To facilitate further consideration of the various embodiments, the following definitions of terms are provided.

[0080] The term "approximately" means, when referring to measurable values ​​such as quantity or duration of time, to include variations of ±20%, ±10%, more preferably ±5%, ±1%, or even more preferably ±0.1% from the specified value, such variations being appropriate for carrying out the disclosed method.

[0081] Unless otherwise specified, technical terms in this specification shall be used in accordance with their conventional usage. Definitions of general terms in molecular biology are found in Benjamin Lewin, Genes VII, 1999, published by Oxford University Press; Kendrew et al., The Encyclopedia of Molecular Biology, 1994, published by Blackwell Science Ltd.; and Robert, published by VCH Publishers, Inc. A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, 1995; and other similar references can be found.

[0082] 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 for adoptive cell transfer of transduced T cells using two or more vectors expressing one or more DuoCARs.

[0083] More specifically, the present invention relates to transduction using a lentiviral vector expressing a chimeric antigen receptor (DuoCAR) and a lentiviral vector expressing CARS. The present invention relates to host cells (e.g., lymphocytes, T cells), lentiviral vectors, and nucleic acid molecules encoding chimeric antigen receptors, as well as methods of using them, for example, to treat cancer in a subject.

[0084] Surprisingly and unexpectedly, the inventors discovered that immunotherapy compositions containing patient-specific autologous antitumor lymphocyte cell populations are even more effective as antitumor immunotherapy when the autologous lymphocyte cell population is transduced using two or more lentiviral vectors encoding single or multiple chimeric antigen receptors (DuoCARs). The use of at least two lentiviral vectors expressing single or multiple CARS appears to specifically promote in vivo proliferation and persistence of patient-specific antitumor T cells, resulting in tumor stabilization, reduction, elimination, or remission or prevention of cancer recurrence or a combination thereof.

[0085] Such active patient-specific antitumor T cell populations described herein may be returned to the patient by direct injection to promote in vivo proliferation and persistence of patient-specific antitumor T cells in a patient-specific manner, resulting in tumor stabilization, reduction, elimination, remission of cancer, prevention or remission of cancer recurrence, or a combination thereof. This also includes effective proliferation and rapid reduction of the therapeutic cell population.

[0086] Therefore, in its broadest form, the novelty of this adoptive immunotherapy lies in the use of a combination of CAR-expression vectors. A distinguishing feature is that, in contrast to the conventional use of single vectors expressing one or more chimeric antigen receptors, the Duo CAR approach confers both multi-antigen specificity and optimal signaling to antitumor T cell activity in vivo. Creating a system in which three or more antigens are efficiently targeted is 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 condition that at least one or more signaling motif combinations are not identical across the vectors, works to ensure that one or more genetically modified lymphocyte populations transduced using such dual lentiviral vector-derived CARs generate patient-specific autologous antitumor lymphocyte populations that can specifically promote in vivo proliferation and persistence of patient-specific antitumor lymphocytes resulting in stabilization, reduction, elimination or remission of tumors or cancers and / or prevention or remission of tumor or cancer recurrence, or any combination thereof.

[0087] In one embodiment, the immunotherapy composition is provided comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCAR), each vector encoding a functional CAR, wherein at least one binding domain(s) in one of the vectors are not identical, so that the combination of vectors results in the expression of two or more non-identical binding domains, where each vector-encoded binding domain(s) are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0088] In another embodiment, the immunotherapy composition is provided in the form of one or more isolated nucleic acid molecules encoding at least two vectors (DuoCAR), provided that single CARs, split CARs, tandem CARs, or multiple CARs shown in Figure 5(A), (B), (C), or (D), respectively, are specifically excluded, each vector encoding a functional CAR, thereby resulting in the expression of two or more non-identical binding domains, where each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0089] The efficacy of immunotherapy achieved using DuoCAR lentiviral vector-modified T cells of the present invention, as well as the prevention or remission of tumor or cancer recurrence, is significantly greater and synergistically greater than that achieved using a single conventional CAR design. This is a unique combination of biological therapeutic benefits associated with increased in vivo proliferation and maintenance of patient-specific antitumor lymphocytes, resulting in tumor or cancer stabilization, reduction, elimination, or remission, compared to conventional CAR-based T cell immunotherapy.

[0090] CAR expression vectors can be created that induce the expression of a single binding domain (black, empty, or striped spheres, each with distinct specificities, Figure 5) linked to a linker and a transmembrane domain (single empty square). Figure 5 below shows a comparison of the DuoCAR of the present invention with a conventional CAR. In Figure 5, the thick gray line represents the plasma cell membrane. Intracellularly, the lymphocyte signaling domains derived from the 4-1BB (CD137), CD28, and CD3 zeta chains may be combined as shown. All examples and uses of the CD3 signaling domain in this document involve modification of the CD3 zeta chain by selective mutagenesis of its tyrosine residues or by modification of one, two, or three of the immune receptor tyrosine-based activation motifs (ITAMs) by other mutations that prevent the ITAM motif from being targeted for phosphorylation. In a single CAR (Figure 5A), the single binding domain is combined with the transmembrane and two signaling domains. In a split CAR (Figure 5B), two different binders are expressed in a form that has a single signaling domain that must be combined to enable signaling. In a tandem CAR (Figure 5C), two binding domains are linked to a single signaling domain. In a multiplex CAR from one vector (Figure 5D), two fully functional CARs are expressed from a single vector. In the DuoCAR of the present invention (e.g., Figure 5E), each vector encodes at least two vectors that encode a functional CAR, thereby the combination of vectors results in the expression of two or more non-identical binding domains, and each vector-encoded binding domain(s) is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs. The essential features that distinguish the DuoCAR of the present invention from others are the use of two or more vectors, the multiplicity of binding domains, and the expression of at least one fully functional signaling feature (in) of the two cell surface proteins. This concerns T cell proliferation in vivo.

[0091] In another embodiment, DuoCAR is used to enhance an immune response against tumors mediated by a therapeutic T cell population. The immune response is enhanced in at least three ways.

[0092] Firstly, by providing additional signals to T cells for proliferation and survival throughout the body, the DuoCAR of the present invention enables the persistence of a therapeutic T cell population by stimulating the T cell population upon encountering an autoantigen (e.g., CD19) that may be tolerable by the patient when lost and still function to provide stimulating signals to the therapeutic cell population that is not present in the tumor tissue itself. It is well known and established that third-generation DuoCARs (expressing three costimulatory domains intracellularly linked to a single extracellular Ig-like binder) are not expressed similarly in therapeutic T cells compared to these DuoCARs expressing two intracellular costimulatory domains. For example, in Figure 6 below, the expression levels of CAR constructs on 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: CAR-free (false), second-generation CAR (CAR-A-28z), third-generation CAR (CAR-A-28BBz), and modified second-generation CAR (CAR-A-BBz). CAR surface expression levels were detected by flow cytometry and reported as mean fluorescence intensity (MF) y-axis. Even though all constructs expressed exactly the same CAR-binding domain... Regardless, the MFI on both second-generation CARs was even brighter.

[0093] By providing a third T cell activation sequence on separate vector CAR constructs, the inventors can restore the benefits of expressing three co-stimulatory domains without incurring the disadvantage of reducing CAR expression on the T cell surface.

[0094] In a second aspect, the DuoCAR of the present invention may target non-tumor cell types that mediate immunosuppressive effects. For example, if CD19-expressing B cells are present in a tumor lesion and also inhibit anti-tumor immunity through the production of IL-4 or other mediators, a second benefit of using a DuoCAR-expressing tumor-specific T cell population is that the immunosuppressive cell population is also eliminated.

[0095] For example, if immunosuppressive B cells are present within a solid tumor lesion, 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 stromal-specific DuoCARs (e.g., by targeting fibroblast-activating protein-alpha (FAP)). If malformed vascular structures are the cause of a lack of effective immune response, DuoCARs specific to these types of blood vessels or lymphatic vessels (such as anti-VEGFRs) can also improve treatment outcomes.

[0096] In a third aspect, the DuoCAR of the present invention targets immunosuppressive populations distal to the tumor, i.e., located in another compartment of the body. For example, it targets myeloid-derived suppressor cells (MDSCs) and uses DuoCARs that may be present in the tumor lesion itself, in regional lymph nodes, or in the bone marrow. It is well established that tumor inflow lymph nodes may be either immunoactivating or immunosuppressive loci. This depends on the overall inflammatory tone of the lymph nodes and distal dendritic cell differentiation prior to migration to the lymph nodes. If there are misdifferentiated antigen-presenting cells such as myeloid-derived suppressor cells (MDSCs) or dendritic cells in tumor inflow lymph nodes, DuoCARs targeting these cell types can improve therapeutic outcomes even distal to the tumor itself. Beyond cancer-specific DuoCAR immunotherapy applications, a second application of DuoCAR is the prevention or treatment of autoimmune and / or inflammatory diseases. The difference from oncology-based applications is that T regulatory cells (Tregs), or inducible T regulatory cells (iTregs), or other cells cultured under conditions that promote Th-2-like immune responses are cytoplasmic. For oncological applications, Th-1-like cells are cytoplasmic. In therapeutic applications, the presence of CAR-modified lymphocytes that produce immunosuppressive cytokines such as transforming growth factor-beta (TFG-beta) works to emit broad immunotolerogenic signals that restore autoimmune or pro-inflammatory diseases, as well as in diverse cases such as graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT), allergic airway, intestinal or other mucosal inflammation, or skin allergies. This approach includes inflammatory conditions of peripheral or central nervous system (CNS) nerves, such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, Parkinson's disease, and CTE (chronic traumatic brain injury due to frequent concussions or micro-concussions). This method also applies to progressive scarring diseases such as COPD (chronic obstructive pulmonary disease).

[0097] In the treatment of inflammatory diseases, tissue antigen-specific lymphocytes, distress markers on the surface of inflammatory cells, or misfolded proteins (such as tau protein or beta-amyloid) are created by generating DuoCAR expression vectors that are specific to 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 CAR format instead of as binders to cell surface proteins. Binders to MHC molecules-bound tau protein or tau peptides can also be used as binding motifs to CARs. Receptors that mediate lymphocyte homing to peripheral tissues may also be included in the CAR format to confer region specificity to CAR-expressing Treg populations. Adhesion receptor domains and cytokine sequences, or cytokine or chemokine receptors or binders, known to drive lymphocyte infiltration into specific tissues, 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, thereby mediating the CNS migratory behavior of lymphocyte populations. Including adhesion molecule-derived domains can also be used to target CAR-expressing lymphocytes to disease areas. The same applies to the intestinal tract (i.e., binders to MAdCAm-1, expression of CCR9 or anti-CCL25, etc.), the lungs (i.e., P-selectin or mesothelin), the skin (i.e., binders to E-selectin), or other mucosal surfaces.

[0098] To use this method, patients with inflammatory conditions or those whose disease can be treated by reducing the inflammatory state, such as Alzheimer's disease, 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 under appropriate cytokine conditions. These Tregs or iTregs are then transduced using a DuoCAR vector and expanded in vitro as needed (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. Thus, the generated, engineered immune effector cells are targeted to appropriate sites and produce cytokines that match their Th2 or Treg differentiation patterns. It is also known that CAR-T cells may be engineered to secrete specific gene payloads upon activation of the CAR receptor. In addition to the DuoCAR payload expressed from the vector, further therapeutic proteins or peptides may be expressed or secreted by engineered T cell populations 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 inhibit plaque formation, and d) cytokines (such as TGF-beta, IL-4, and IL-10).

[0099] MiRNAs may also be expressed intracellularly to regulate T cell function. Examples of miRNAs include miR-92a, miR-21, miR-155, miR-146a, miR-3162, miR-1202, miR-1246, and miR-4281, miR-142, and miR-17-92. MiRNAs may also be developed from shRNAs. Examples include shRNAs targeted to miR-28, miR-150, and miR-107, which typically bind to PD1 and increase its expression.

[0100] Beyond oncology-based and inflammatory and autoimmune disease-based applications, a third application of Duo CAR technology is the generation of therapeutic lymphocyte populations specific to viral, bacterial, or fungal antigens. Thus, in relation to the oncology applications described for B-cell malignancies, the targeting of infectious diseases allows DuoCAR products to mediate immunoprotective or immunotherapeutic activity against diseased tissues based on the recognition of infectious agents or microbial antigens. Unlike T-cell receptor (TCR)-based methods, where the T-cell receptor itself mediates the recognition of peptides encoded by pathogens, the Duo CAR method utilizes binding proteins expressed in a CAR vector format that give the transduced T-cell population antibody-like recognition (i.e., no antigen processing is required). Activation of the therapeutic T-cell population creates an immunoactivating locus that can release soluble mediators such as interferon-gamma, which can eliminate infected cells and, if the microbial antigen is not cell-bound, enable an effective immune response initiated against the infectious agent.

[0101] For example, HIV is known to be highly variable, but it can be classified into specific clades or families, and antibodies against clade-specific viral envelope proteins (env, gp120) can be produced. Using the DuoCAR method, three or more clade-specific antibody-like binders are included in the 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 bacterial strains that frequently occur in clinical settings. These include VRE (vancomycin-resistant enterococci), MRSA (methicillin-resistant Staphylococcus aureus), and KPC (Klebsiella pneumoniae carbapenemase-producing Gram-negative bacteria, similarly CRKP). Klebsiella cell surface antigens include the O antigen (9 variants) and the K antigen (approximately 80 variants). The O antigen spectrum can be easily covered using a small DuoCAR library, as can the numerous K antigens. For use, CAR constructs characterized by antibodies binding to different K or O serotypes are created, and as in oncological applications, these CAR vectors are used for transduction into Th1-like effector bacterial populations, isolated, and activated. In fungal diseases, the work of L. Cooper et al. (Kumasesan, PR, 2014, PNAS USA, vol. 111: p. 10660) demonstrated that Dectin 1, a fungus-binding protein normally expressed on human cells, can be reconstituted as a CAR and used to control in vitro fungal growth. The human disease aspergillosis occurs in severely immunosuppressed individuals and is caused by the fungus A. fumigatus. Several groups produce monoclonal antibodies specific to antigenic components on the surface of Aspergillus cells, thereby enabling adoptive immunotherapy using DuoCARs that target three or more Aspergillus antigens on the fungal surface. Therefore, in all of these infectious disease applications, the ability to create immunoglobulin-like binders against microbial antigens allows multiple antigens to be targeted by a population of CAR-expressing effector lymphocytes.

[0102] A detailed description of DuoCARs that may be used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein is provided below, along with further descriptions of DuoCARs, antibodies and their antigen-binding fragments, conjugates, nucleotides, expression, vectors and host cells, methods of treatment using the disclosed DuoCARs, compositions and kits, and their extracellular, transmembrane and intracellular domains. While the compositions and methods of the present invention are illustrated with reference to the production and use of DuoCARs, it is intended herein that the compositions and methods may specifically include the production and use of TrioCARs and QuatroCARs.

[0103] A. Chimeric antigen receptor (present in DuoCAR) The DuoCARs disclosed herein comprise at least two vectors, each encoding a functional CAR, thereby resulting in a vector combination that produces 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, at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain and at least one intracellular domain.

[0104] CARs are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain (e.g., a single-chain variable fragment (scFv)) of an antibody linked to a T cell signaling domain via a transmembrane domain. DuoCARs are characterized by their ability to redirect the specificity and responsiveness of T cells toward a selected target by leveraging the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition allows T cells expressing DuoCARs to recognize antigens independently of antigen processing. It provides the ability to recognize tumors, thereby bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, DuoCAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0105] As disclosed herein, the intracellular T cell signaling domain of DuoCAR may include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of the CAR that includes the intracellular domain of the T cell receptor, for example, the intracellular portion of the CD3 zeta protein, not to the extent of the T cell receptor. The costimulatory signaling domain refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand, required for an efficient lymphocyte response to an antigen. In some cases, the activating domain may be attenuated by mutations at specific sites of phosphorylation, namely the ITAM motif in the CD3 zeta chain, thereby carefully regulating the degree of signaling mediated by that domain.

[0106] 1. Extracellular domain In one embodiment, the CAR used in the patient-specific autologous antitumor lymphocyte cell population(s) disclosed herein includes a target-specific binding element, otherwise called an antigen-binding domain or portion. The choice of domain depends on the type and number of ligands that define the surface of the target cells. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for antigen-binding domains in a CAR include those associated with viruses, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0107] In one embodiment, a CAR may be engineered to target a desired tumor antigen by manipulating a desired antigen-binding domain that specifically binds to the antigen on tumor cells. The tumor antigen is a protein produced by tumor cells that elicits an immune response, particularly a T-cell-mediated immune response. The selection of the antigen-binding domain depends on the specific type of cancer being treated. Tumor antigens are well known in this field and include, for example, glioma-associated antigens, carcinoembryonic antigens (CEAs), beta-human chorionic gonadotropins, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF)-I receptor, IGF-II receptor, IGF-I receptor, and mesoserine. The tumor antigens disclosed herein are for illustrative purposes only. This list is not intended to be exclusive, and further examples will be readily apparent to those skilled in the art.

[0108] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with the malignant tumor. Malignant tumors express several proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins are used to target individual tumors. The tumor has its own unique, truly tumor-specific immunoglobulin antigens. B-cell differentiation antigens, e.g., CD19, CD20, CD22, and CD37, are other candidate target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0109] Tumor antigens can be either tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and are not present on other cells throughout the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions that do not induce a state of immunological tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unresponsive, or they may be antigens that are normally present at very low levels on normal cells but expressed at considerably high levels on tumor cells.

[0110] Non-limiting examples of TSA or TAA include: differentiation antigens, e.g., MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multiseries antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens arising from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., 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 This includes 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 / cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0111] In preferred embodiments, the antigen-binding domain portion of the CAR targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesoserine, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, and MAGE A3 TCR. In yet another embodiment, the DuoCAR is provided herein in a form comprising a tag or anti-Tag binding domain.

[0112] Depending on the desired antigen to be targeted, the CAR may be engineered to include an appropriate antigen-binding domain that is specific to the desired antigen target. For example, if CD19 is the desired antigen to be targeted, a CD19-specific antibody or its scFv fragment may be used as the antigen-binding domain incorporated into the CAR.

[0113] 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, where the nucleic acid sequence of the anti-CD19 scFV includes the sequence shown in SEQ ID NO: 27. In one embodiment, the anti-CD19 scFV includes 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 includes the amino acid sequence shown 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 anti-CD20 scFv, and the nucleic acid sequence of anti-CD20 scFv includes the sequence described in SEQ ID NO: 1. In another embodiment, the anti-CD20 scFV portion of the CAR includes the amino acid sequence described 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 anti-CD22 scFv, and the nucleic acid sequence of anti-CD22 scFv includes the sequence described in SEQ ID NO: 7. In another embodiment, the anti-CD22 scFV portion of the CAR includes the amino acid sequence described in SEQ ID NO: 8.

[0114] In one aspect of the present invention, a non-TSA or non-TAA CAR is provided that contains, for example, but is not limited to, a retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 and HIV-LP), picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus and echovirus), rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, polynuclear respiratory virus, influenza virus, hepatitis B virus, parvovirus, adenoviridae, herpesviridae [e.g., herpes simplex virus type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesviruses], poxviridae (e.g., smallpox virus, vaccinia virus and poxvirus), or hepatitis C virus antigens, or any combination thereof.

[0115] In another aspect of the present invention, CARs capable of binding to antigens derived from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella are provided. In particular, CARs capable of binding to antigens derived from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof, are provided.

[0116] 2. Transmembrane domain In a DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population(s) disclosed herein, the CAR comprises one or more transmembrane domains fused to the extracellular domain of the CAR.

[0117] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded linker domain originates from the extracellular domain of CD8 and is linked to the transmembrane domain.

[0118] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded linker domain originates from the extracellular domain of the transmembrane domain and is linked to the transmembrane domain.

[0119] In some cases, the transmembrane domain minimizes interaction with other members of the receptor complex, and the transmembrane domain of the same or different surface membrane protein of such domain. To avoid binding to [the target molecule], this can be achieved by selecting or by amino acid substitutions.

[0120] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. The transmembrane regions particularly used in the present invention may originate from the alpha, beta, or zeta chains of T cell receptors, 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 (i.e., may include at least one of their transmembrane regions). Alternatively, the transmembrane domain may 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 terminus of the synthetic transmembrane domain. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2-amino acid and 10-amino acid lengths, can form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine bi or triple alanine motifs provide particularly suitable linkers.

[0121] 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 contains the nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the CD8 transmembrane domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12. In another embodiment, the CD8 transmembrane domain contains the amino acid sequence of SEQ ID NO: 12.

[0122] In some cases, the transmembrane domain of the CAR includes a CD8 alpha-hinge domain. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, the CD8 hinge domain includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14. In yet another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 14.

[0123] While not intended to limit to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with exemplary DuoCARs used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein include, for example, but not limited to, a) improved lateral movement within the plasma membrane, enabling more efficient signaling; b) superior location within plasma membrane microdomains such as lipid rafts and a higher ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane, such as less proximity to or less interaction with phosphatases like CD45, due to preferential movement away from inhibitory or downregulatory interactions; and d) superior assembly to T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0124] In one embodiment of a patient-specific autologous antitumor lymphocyte cell population disclosed herein, non-limiting exemplary transmembrane domains used in the DuoCAR disclosed herein include TNFRSF16, and the TNFRSF19 transmembrane domain may be used to induce TNFRSF transmembrane domains and / or linker or spacer domains, in particular, other TNFRSF members listed within the tumor necrosis factor receptor superfamily listed in Table I therein, as disclosed in the applicant's concurrently pending provisional patent application 62 / 239,509, titled CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE, filed 9 October 2015, and assigned to Lentigen Technology, Inc., with case number LEN_015PRO.

[0125] 3. Spacer Domain In DuoCARs used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein, the spacer domain may be located between the extracellular domain and the TNFRSF transmembrane domain, or between the intracellular domain and the TNFRSF transmembrane domain. The spacer domain means any oligopeptide or polypeptide that functions to link the TNFRSF transmembrane domain to the extracellular domain and / or to link the TNFRSF transmembrane domain to the intracellular domain. The spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids.

[0126] In some embodiments, the linker may include a spacer element, if present, which 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 skilled in the art and include U.S. Patents 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, and 5,521 This includes U.S. Patent Nos. 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 U.S. Patent Application Publication Nos. 20110070248, each of which is incorporated herein by reference in its entirety.

[0127] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signaling into the cell. Examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in the potential glycosylation site, and these amino acids can be used as amino acids constituting the spacer domain.

[0128] As spacer domains, all or part 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) may be used. Additionally, a portion of the constant region of the antibody H chain or L chain (CH1 region or CL region, e.g., a peptide with the amino acid sequence shown in SEQ ID NO: 16) may be used as a spacer domain. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0129] Furthermore, in CARs, a signal peptide sequence may be ligated to the N-terminus. Signal peptide sequences are present at the N-terminus of many secreted and membrane proteins and have a length of 15 to 30 amino acids. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for CARs. 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.

[0130] 4. Intracellular domains The cytoplasmic domain or, otherwise, the 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 may be cytolytic activity or helper activity, including cytokine secretion. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. Insofar as a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain, as long as it transmits effector function signals. Thus, the term “intracellular signaling domain” means including any truncated portion of an intracellular signaling domain that is sufficient to transmit effector function signals.

[0131] Preferred examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that work together to initiate signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0132] It is known that the signals generated through the TCR alone are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is 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 co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0133] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either in a stimulative or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulative manner may contain signaling motifs known as immunoreceptor-activating tyrosine motifs or ITAMs.

[0134] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly used in CARs disclosed herein 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 ITAM include amino acid numbers 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acid numbers 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acid numbers 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acid numbers 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acid numbers 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acid numbers 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI Amino acid numbers 402-495 of RefSeq:NP_055022.2, amino acid numbers 707-847 of 0022 (NCBI RefSeq:NP_001762.2), amino acid numbers 166-226 of CD79a (NCBI RefSeq:NP_001774.1), amino acid numbers 182-229 of CD79b (NCBI RefSeq:NP_000617.1), and CD66d (NCBI This includes peptides having amino acid sequences 177-252 of RefSeq:NP_001806.2), as well as variants of these peptides having the same function. NCBI RefSeq IDs or GenBan IDs described herein The amino acid numbering based on the amino acid sequence information of k is assigned based on the full length of each protein precursor (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling sequence derived from CD3 zeta. In another embodiment, one, two, or three ITAM motifs in CD3 zeta are attenuated by mutations or substitutions of tyrosine residues with other amino acids.

[0135] In preferred embodiments, the intracellular domain of a CAR may be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of a CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such costimulatory molecules include ligands that specifically bind to 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 CD83. Specific non-limiting examples of such co-stimulatory molecules include amino acid numbers 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acid numbers 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acid numbers 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acid numbers 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acid numbers 196-210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI This includes peptides having the sequences of amino acid numbers 214-255 of RefSeq:NP_001552.2), amino acid numbers 241-277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acid numbers 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants of these peptides having the same functions. Therefore, while this disclosure primarily exemplifies 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of this disclosure.

[0136] The cytoplasmic signaling sequences within the cytoplasmic signaling region of a CAR can be linked to each other randomly or in a specified order. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2-amino acid and 10-amino acid lengths, can form the linkage. Glycine-serine pairs provide particularly suitable linkers.

[0137] In one embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain. In yet another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain as well as CD28 and 4-1BB signaling domains.

[0138] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 17, and the CD3-zeta signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 19.

[0139] In one embodiment, the intracellular domain in CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where 4-1BB The signaling domain of CD3-zeta includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, and the signaling domain of CD3-zeta includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0140] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes the amino acid sequence shown in SEQ ID NO: 18, and the CD3-zeta signaling domain includes the amino acid sequence shown in SEQ ID NO: 20.

[0141] 5. Further description of DuoCAR Functional portions of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein are also expressly included within the scope of the invention. The term “functional portion,” when used in relation to a CAR, means any part or fragment of one or more DuoCARs disclosed herein, which retain the biological activity of the CAR from which it is a part (parent CAR). A functional portion includes, for example, a portion of a CRA that retains the ability to recognize target cells or to detect, treat, or prevent disease to a similar, equal, or greater degree than that of the parent CAR. With respect to the parent CAR, a functional portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0142] The functional moiety may contain additional amino acids at its amino-terminus, carboxy-terminus, or both, which 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 moiety, such as recognizing target cells, detecting cancer, or treating or preventing cancer. More preferably, the additional amino acids enhance the biological activity of the functional moiety compared to that of the parent CAR.

[0143] Functional variants of DuoCAR disclosed herein are included within the scope of this disclosure. The term “functional variant” as used herein means a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to the parent CAR, wherein the functional variant retains the biological activity of the CAR from which it is a variant. Functional variants include, for example, variants of the CAR described herein (parent CAR) that retain the ability to recognize target cells to a similar, identical, or greater degree than the parent CAR. With respect to the parent CAR, functional variants may, for example, have amino acid sequence identity with respect to the parent CAR by at least about 30%, 50%, 75%, 80%, 90%, 98% or more.

[0144] A functional variant may, for example, include the amino acid sequence of a parent CAR having at least one conserved amino acid substitution. Alternatively, the functional variant may further include the amino acid sequence of a parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the parent CAR.

[0145] 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 having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution is an acidic / negatively charged polar amino acid that is replaced with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), or another amino acid with a nonpolar side chain (e.g., Ala, Gly, Va). These may include amino acids having nonpolar side chains that substitute for l, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc., basic / positively charged polar amino acids that substitute for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), uncharged amino acids having polar side chains that substitute for another uncharged amino acid with polar side chains (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having beta-branched side chains that substitute for another amino acid with beta-branched side chains (e.g., He, Thr, and Val), and amino acids having aromatic side chains that substitute for another amino acid with aromatic side chains (e.g., His, Phe, Trp, and Tyr).

[0146] CARs may essentially consist of one or more of the specified amino acid sequences described herein, and as a result, other components, such as other amino acids, do not significantly alter the biological activity of the functional variant.

[0147] DuoCAR (including functional parts and functional variants) can be of any length, i.e., contain any number of amino acids, provided that DuoCAR (or its functional part or functional variant) retains its biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells in mammals, or the ability to treat or prevent disease in mammals. For example, a CAR can be approximately 50 to 5000 amino acid long, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more amino acid long.

[0148] DuoCAR (including the functional parts and functional variants of the present invention) may contain synthetic amino acids instead 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-acetylaminomethylcysteine, 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, and 1,2,3,4-tetrahydroxy It contains diisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0149] DuoCAR (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinks, etc., or converted to acid addition salts and / or optionally dimerized, polymerized, or conjugated.

[0150] DuoCAR (including its functional parts and functional variants) can be obtained by methods known in the art. DuoCAR can be prepared by any suitable method for producing polypeptides or proteins. Suitable methods for the de novo synthesis of polypeptides and proteins are: 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. (eds.), Oxford References include University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Methods for generating chimeric antigen receptors, T cells containing such receptors, and their uses (e.g., for the treatment of cancer) are publicly known in the art and are further described herein (e.g., each of which is incorporated herein by reference in its entirety: Brentjens et al., 2010, Molecular Therapy, Vol. 18: No. 4, pp. 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pp. 1-9; Till et al., 2008, Blood, Vol. 112: pp. 2261-2271; Park et al., Trends Biotechnol., Vol. 29: pp. 550-557, 2011; Grupp et al., N Engl J Med., Vol. 368: pp. 1509-1518, 2013; Han et al., J. Hematol See Oncol., vol. 6: p. 47, 2013; Tumaini et al., Cytotherapy, vol. 15, pp. 1406-1417, 2013; Haso et al., (2013) Blood, vol. 121, pp. 1165-1174; PCT Publications WO2012 / 079000, WO2013 / 126726; and U.S. Patent Application Publication No. 2012 / 0213783). For example, the nucleic acid molecule encoding the disclosed chimeric antigen-binding receptor may be contained in an expression vector (e.g., a lentiviral vector) used to transduce host cells, such as T cells, to produce the disclosed CAR. In some embodiments, a method using a chimeric antigen receptor includes the steps of isolating T cells from a subject, transduction of an expression vector (e.g., a lentiviral vector) encoding the chimeric antigen receptor into the T cells, and administration of CAR-expressing T cells to the subject for treatment of the subject, e.g., treatment of a tumor.

[0151] B. Antibodies and antigen-binding fragments One embodiment further provides CARs, CAR-expressing T cells, antibodies that specifically bind to one or more of the antigens disclosed herein, or an antigen-binding domain or portion thereof, for use in patient-specific autologous antitumor lymphocyte cell populations disclosed herein. As used herein, “CAR-expressing T cells” or “CAR T cells” means T cells that express a CAR and have antigen specificity determined, for example, by the antibody-derived targeting domain of the CAR.

[0152] As used herein, “antigen-binding domain” may include an antibody and its antigen-binding fragments. The term “antibody” is used herein in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and their antigen-binding fragments, as 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 their variants and fragments that retain binding affinity to an antigen.

[0153] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may exist in trace amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production by any particular method. In some cases, monoclonal antibodies are antibodies produced by a single clone of B lymphocytes, or antibodies produced by cells transfected with nucleic acids encoding the light and heavy chain variable regions of a single antibody (or its antigen-binding fragment), or by their offspring. In some cases, monoclonal antibodies are isolated from the subject. Monoclonal antibodies may have conserved amino acid substitutions that substantially have no effect on antigen binding or other immunoglobulin functions. Exemplary methods of monoclonal antibody production This is publicly known; see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition, Cold Spring Harbor Publications, New York (2013).

[0154] Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0155] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, for example, Kindt et al., Kuby Immunology, 6th edition, WHFreeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy chain and light chain combine to specifically bind to the antigen. In further embodiments, only the variable region of the heavy chain is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of the light chain. This is definitive (see, for example, Hamers-Casterman et al., Nature, Vol. 363: pp. 446-448, 1993; Sheriff et al., Nat. Struct. Biol., Vol. 3: pp. 733-736, 1996). A reference to "VH" or "VH" refers to the variable region of the antibody heavy chain, including antigen-binding fragments, e.g., those of Fv, scFv, dsFv, or Fab. A reference to "VL" or "VL" refers to the variable domain of the antibody light chain, including those of Fv, scFv, dsFv, or Fab.

[0156] 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, for example, Kabat et al., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1991). The sequences of different light or heavy chain framework regions are relatively conserved within a species. The antibody framework region, which is the combined framework region of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0157] CDRs are primarily responsible for binding antigens to epitopes. The amino acid sequence boundaries of a given CDR are described in Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB Vol. 273, pp. 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor"). The CDRs can be easily determined using one of several well-known schemes, including the one described in “Variable Domains and Ig Superfamily V-Like Domains,” Dev. Comp. Immunol., Vol. 27: pp. 55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically called CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are also typically identified by the chain on which a particular CDR is located. Thus, VH CDR3 is the CDR3 derived from the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes called LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are LCDR1, L They are sometimes referred to as CDR2 and LCDR3.

[0158] An "antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize a congener 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; diabody; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of the entire antibody, or antigen-binding fragments synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, vols. 1-2, 2nd edition, Springer Press, 2010).

[0159] Single-chain antibodies (scFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies linked by a suitable polypeptide linker as a single-chain molecule (see, e.g., Bird et al., Science, vol. 242: pp. 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., vol. 85: pp. 5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, vol. 13: pp. 543-549, 2010). The intramolecular orientation of the VH and VL domains in scFv is typically not definitive for the scFv. Therefore, scFv can be used with both possible configurations (VH domain - linker domain - VL domain; VL domain - linker domain - VH domain).

[0160] In dsFv, the variable chains of the heavy and light chains are mutated to introduce disulfide bonds to stabilize chain association. Diabodies are also included, 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 pairing that domain with a complementary domain on another chain and creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., vol. 90: pp. 6444-6448, 1993; Poljak et al., Structure, vol. 2: pp. 1121-1123, 1994).

[0161] Antibodies also include genetically modified forms such as chimeric antibodies (e.g., humanized mouse 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 edition, WH Freeman & Co., New York, 1997.

[0162] Antibodies not found in nature can be constructed using solid-phase peptide synthesis, recombinantly produced, or obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science Vol. 246: pp. 1275-1281 (1989), incorporated herein by reference. These and other methods for producing chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today Vol. 14: pp. 243-246 (1993); Ward et al., Nature). Volume 341: pp. 544-546 (1989); Harlow and Lane, above, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabec k, Antibody Engineering, 2nd edition (Oxford University Press 1995); each of these is incorporated herein by reference).

[0163] A reference antibody and an antibody that "binds to the same epitope" refer to an antibody that blocks the binding of the reference antibody to the antigen by 50% or more in a competitive assay, and conversely, a reference antibody blocks the binding of its antibody to the antigen by 50% or more in a competitive assay. Antibody competitive assays are well known, and exemplary competitive assays are provided herein.

[0164] 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 called the “donor,” and the human antibody or antigen-binding fragment providing the framework is called the “acceptor.” In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. A constant region is not required to be present, but if present, it may be substantially identical to the human immunoglobulin constant region, e.g., at least about 85–90%, e.g., about 95% or more. Thus, all parts of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding parts of the natural human antibody sequence, except perhaps the CDRs.

[0165] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some cases, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody as well as CDRs and / or framework regions from another human antibody.

[0166] A “fully human antibody” or “human antibody” is an antibody that contains sequences derived from (or originating from) the human genome but does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions derived from (or originating from) the human genome. Human antibodies can be identified and isolated, for example, by phage display, using technologies for generating sequences based on sequences derived from the human genome, or by using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004 Print.; Lonberg, Nat. Biotech., vol. 23: pp. 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., vol. 20: pp. 450-459, 2008).

[0167] Antibodies may have one or more binding sites. If there are more than one binding sites, these sites may be identical 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.

[0168] Methods for testing antibodies for their ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assays, such as radioimmunoassays (RIA), ELISA, Western blotting, immunoprecipitation, and competitive inhibition assays (see, for example, Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266Al, and U.S. Patent No. 7,338,929).

[0169] Furthermore, CARs, CAR-expressing T cells, antibodies, or their antigen-binding portions are detectable. The labels may include, for example, radioactive isotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0170] C. Conjugate DuoCARs, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments specific to one or more of the antigens disclosed herein, used in patient-specific autologous antitumor lymphocyte cell populations disclosed herein, may be conjugated to agents such as effector molecules or detectable markers by means of several means known to those skilled in the art. Both covalent and non-covalent means may be used. The conjugate includes, but is 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 know that chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents, etc., may be used. 125 I, 32 P, 14 C, 3 H and 35 It is understood that various effector molecules and detectable markers, including (but not limited to) S, as well as other labels, target moieties, and ligands, may be used.

[0171] The selection of a specific effector molecule or detectable marker depends on the specific target molecule or cell and the desired biological effect. For example, an effector molecule could be a cytotoxic substance used to induce the death of a specific target cell (e.g., tumor cells).

[0172] The procedure for conjugating an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups; for example, carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or bind further reactive functional groups. Derivatization may involve the binding of one of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to conjugate the antibody or antigen-binding fragment to the effector molecule or detectable marker. The linker can form a covalent bond 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, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker may be attached to the constituent amino acids via their side groups (e.g., via disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acids.

[0173] In some embodiments, the linker may include a spacer element, if present, which 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 skilled in the art and include U.S. Patents 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, 5,521,284, and U.S. This includes U.S. Patent No. 5,504,191, U.S. Patent No. 5,410,024, U.S. Patent No. 5,138,036, U.S. Patent No. 5,076,973, U.S. Patent No. 4,986,988, U.S. Patent No. 4,978,744, U.S. Patent No. 4,879,278, U.S. Patent No. 4,816,444 and U.S. Patent No. 4,486,414, as well as those listed in U.S. Patent Application Publication No. 20110212088 and U.S. Patent Application Publication No. 20110070248, each of which is incorporated herein by reference in its entirety.

[0174] In some embodiments, the linker is cleavable under intracellular conditions, and as a result, cleavage of the linker causes the release of an effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is incleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by cleavage agents present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). The linker may be a peptide linker cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptide linker is at least 2 amino acid long or at least 3 amino acid long. However, the linker may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid long, for example, 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acid long. Proteases may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics, Vol. 83: pp. 67-123). For example, peptide linkers cleavable by cathepsin B, a thiol-dependent protease, may be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linkers). 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 intracellular proteases may be a valine-citrulline linker or a phenylalanine-lysine linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0175] In other embodiments, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) may be used (see, e.g., 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, Vol. 83: pp. 67-123; Neville et al., 1989, Biol. Chem., Vol. 264: pp. 14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH levels below 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether conjugated to the therapeutic agent via an acylhydrazone linkage) (see, for example, U.S. Patent No. 5,622,929).

[0176] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), and SPDB (N-succinimidyl). This includes imidyl-3-(2-pyridyldithio)butyrate and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene), which can be formed using SPDB and SMPT (e.g., Thorpe et al., 1987, Cancer Res. Vol. 47: pp. 5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody C onjugates in Radioimagery and Therapy of See also Cancer (CW Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res., Vol. 68, pp. 9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0177] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. Vol. 15: pp. 1387-1393), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. Vol. 3 (No. 10): pp. 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. Vol. 3 (No. 10): pp. 1305-1312).

[0178] In yet another embodiment, the linker is incapable of cleavage, and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication 2005 / 0238649, the full contents of which are incorporated herein by reference).

[0179] In some embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., plasma), approximately 20%, 15%, 10%, 5%, 3%, or 1% of the linker in the conjugate sample is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. Various exemplary linkers that may be used in a conjugate are described in WO2004-010957, U.S. Patent Application Publication 2006 / 0074008, U.S. Patent Application Publication 20050238649, and U.S. Patent Application Publication 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0180] In some embodiments, CAR conjugates, CAR-expressing T cells, antibodies or their antigen-binding moieties, and one or more small molecule toxins, such as calitiamycin, maytansinoid, drastatin, auristatin, trichothecin, and CC1065, as well as derivatives of these toxins having toxic activity.

[0181] Maytansine compounds suitable for use as the toxin portion of mitansinoids 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 Vol. 99: pp. 7968-7973), or synthesized using known methods to produce maytansinol and maytansinol analogs. Mitansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Synthetic maytansinol and its Derivatives and analogs include, for example, U.S. Patent 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; 4,313,946; U.S. National Patent No. 4,315,929; U.S. Patent No. 4,317,821; U.S. Patent No. 4,322,348; U.S. Patent No. 4,331,598; U.S. Patent No. 4,361,650; U.S. Patent No. 4,364,866; U.S. Patent No. 4,424,219; U.S. Patent No. 4,450,254; U.S. Patent No. 4,362,663; and U.S. Patent No. 4,371,533, each of which is incorporated herein by reference. Conjugates containing mytansinoids, methods for preparing them, and their therapeutic uses are disclosed, for example, U.S. Patent No. 5,208,020; U.S. Patent No. 5,416,064; U.S. Patent No. 6,441,163 and European Patent No. EP0 425 235 B1, each of which is expressly incorporated herein by reference.

[0182] Further toxins may be used in conjunction with CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties. Exemplary toxins include Pseudomonas exotoxin (PE), hematopoxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calitiamycin, 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). The toxins intended may also include variants of these toxins (see, for example, U.S. Patent Nos. 5,079,163 and 4,689,401).

[0183] Saporins are toxins derived from Saponaria officinalis that disrupt protein synthesis by inactivating the 60S portion of ribosome complexes (Stirpe et al., Bio / Technology, Vol. 10: pp. 405-412, 1992). However, these toxins lack a mechanism for specific entry into cells and therefore require conjugation to antibodies or antigen-binding fragments that recognize internalized cell surface proteins in order to be efficiently taken up by cells.

[0184] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A variant 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. Patents No. 5,792,458 and 5,208,021.

[0185] Castor bean toxin is lectin RCA60 derived from Ricinus communis (castor bean). For examples of castor bean toxin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis aglutinin (RCA) has molecular weights of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 It exists in two forms, known as (Nicholson and Blaustein, J. Biochim. Biophys. Acta vol. 266: p. 543, 1972). Chain A is responsible for inactivating protein synthesis and cell death. Chain B binds hematin to galactose residues on the cell surface and promotes the transport of chain A into the cytosol (Olsnes et al., Nature vol. 249: pp. 627-631, 1974 and US Patent No. 3,060,165).

[0186] Ribonucleases have also been conjugated into targeted molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. Vol. 17: pp. 265-267, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene Vol. 190: pp. 31-35, 1997; and in Goyal and Batra, Biochem. Vol. 345 Part 2: pp. 247-244, 2000. Kalithiamycin, first isolated from Micromonospora echinospora, is a member of the enediin antitumor antibiotic family, inducing double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. Vol. 42: pp. 1070-1087, 1989). This drug is the toxic portion of an immunotoxin currently undergoing clinical trials (see, for example, Gillespie et al., Ann. Oncol. Vol. 11: pp. 735-741, 2000).

[0187] Abrin contains toxic lectins derived from Abrus precatorius. The toxic elements 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. Chain A inhibits protein synthesis; chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. Vol. 52: p. 1095, 1988; and Olsnes, Methods Enzymol. Vol. 50: pp. 330-335, 1978).

[0188] CARs used in patient-specific autologous antitumor lymphocyte cell populations, CAR-expressing T cells, monoclonal antibodies specific to one or more of the antigens disclosed herein, and their antigen-binding fragments may also be conjugated with detectable markers; for example, detectable markers detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic testing, and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic ligatures, 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 such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanidrin phosphors. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties are conjugated with a detectable enzyme, they can be detected by adding further reagents used by the enzyme to produce a identifiable 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. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties may also be conjugated with biotin and can be detected through indirect measurement of avidin or streptavidin binding.It should be noted that avidin itself can be conjugated with an enzyme or a fluorescent label.

[0189] A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can be conjugated with any paramagnetic agent. Paramagnetic agents such as superparamagnetic iron oxide are also used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. An antibody or antigen-binding fragment can also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pairing sequence, binding site for a secondary antibody, metal-binding domain, epitope tag) recognized by a secondary reporter. A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can also be conjugated with a radiolabeled amino acid. The radiolabel can be used for both diagnostic and therapeutic purposes. For example, the radiolabel can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, luminescence spectroscopy or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for the treatment of tumors in a subject, e.g., for the treatment of neuroblastoma. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides:

[0190] A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can also be conjugated with a radiolabeled amino acid. The radiolabel can be used for both diagnostic and therapeutic purposes. For example, the radiolabel can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, luminescence spectroscopy or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for the treatment of tumors in a subject, e.g., for the treatment of 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

[0191] Means for detecting such detectable markers are well known to those skilled in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted illumination. Enzymatic labels are typically detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0192] D. Nucleotides, expression, vectors, and host cells A nucleic acid comprising a nucleotide sequence encoding any of the DuoCARs, antibodies or their antigen-binding portions (including their functional portions and functional variants) described herein is further provided by one embodiment of the present invention. The nucleic acid 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.

[0193] In one embodiment, an isolated nucleic acid molecule is provided that encodes a chimeric antigen receptor (DuoCAR) comprising at least one extracellular antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, from the N-terminus to the C-terminus.

[0194] In one embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, 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.

[0195] In another embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, 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 the antigen.

[0196] In yet another embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular antigen-binding domain comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to the antigen.

[0197] In one embodiment of a CAR used in a patient-specific autologous antitumor lymphocyte cell population, the encoded extracellular antigen-binding domain is transmembrane via a linker domain. Isolated nucleic acid molecules are provided, to which domains are attached.

[0198] Another embodiment of DuoCAR for use in patient-specific autologous antitumor lymphocyte cell populations is provided, which is an isolated nucleic acid molecule encoding a CAR, wherein the extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0199] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, in which the encoded extracellular antigen-binding domain targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesoserine, 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.

[0200] In certain embodiments of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, the encoded extracellular antigen-binding domains include: anti-CD19 scFV antigen-binding domain, anti-CD20 scFV antigen-binding domain, anti-CD22 scFV antigen-binding domain, anti-ROR1 scFV antigen-binding domain, anti-TSLPR scFV antigen-binding domain, anti-mesoserin scFV antigen-binding domain, anti-CD33 / IL3Ra scFV antigen-binding domain, anti-CD38 scFV antigen-binding domain, anti-CD123(IL3RA) scFV antigen-binding domain, anti-CD138 scFV antigen-binding domain, anti-BCMA(CD269) scFV antigen-binding domain, anti-GPC2 scFV antigen-binding domain, anti-GPC3 scFV antigen-binding domain, anti-FGFR4 scFV antigen-binding domain, anti-c-Met scFV antigen-binding domain, and anti-PMSA. Isolated nucleic acid molecules encoding CARs are provided, comprising an scFV antigen-binding domain, an anti-glycolipid F77 scFV antigen-binding domain, an anti-EGFRvIII scFV antigen-binding domain, an anti-GD-2 scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or their amino acid sequences having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0201] In one embodiment of DuoCAR used in a patient-specific autologous antitumor lymphocyte cell population (may include multiple), the DuoCAR provided herein further comprises a linker domain.

[0202] In one embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular antigen-binding domain, an intracellular signaling domain, or both are linked to a transmembrane domain by a linker domain.

[0203] In one embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and linked to the transmembrane domain.

[0204] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, 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.

[0205] DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations (multiple populations are possible) In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded transmembrane domain comprises an amino acid sequence having at least one but 10 or fewer modifications, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[0206] Another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations is provided, which provides an isolated nucleic acid molecule encoding a CAR, further comprising a transmembrane domain containing a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or combinations thereof.

[0207] In yet another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0208] One embodiment of the CAR disclosed herein provides an isolated nucleic acid molecule encoding a CAR, wherein the encoded intracellular signaling domain is located C-terminally relative to the CD3 zeta intracellular domain.

[0209] Another embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations provides an isolated nucleic acid molecule encoding a CAR, wherein at least one encoded intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0210] Further embodiments of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations are provided, which are isolated nucleic acid molecules encoding a CAR, wherein at least one of the encoded co-stimulatory domains comprises the functional signaling domains of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0211] In one embodiment of DuoCAR used in patient-specific autologous antitumor lymphocyte cell populations, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or a signal peptide sequence.

[0212] In some embodiments, nucleotide sequences may be codon-modified. While not bound by any particular theory, codon optimization of nucleotide sequences is thought to increase the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences may involve replacing native codons with other codons that encode the same amino acid but can be translated by tRNA that is more readily available in the cell, thereby increasing translation efficiency. Optimization of nucleotide sequences may also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0213] In embodiments of the present invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the present invention. In another embodiment of the present invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding any of the DuoCARs (including their functional portions and functional variants) described herein.

[0214] As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from natural sources (e.g., isolated and / or purified), may contain natural, unnatural or modified nucleotides, and may contain natural, unnatural or modified nucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, the nucleic acid may appropriately contain one or more insertions, deletions, inversions, and / or substitutions.

[0215] Recombinant nucleic acids may have sequences that do not exist in nature, or sequences that are created by artificial combinations of two or otherwise separated segments of a sequence. These artificial combinations are often achieved by chemical synthesis, or more generally, by artificial manipulation of isolated segments of nucleic acids by genetic engineering techniques, such as those described above by Sambrook et al. 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., and Ausubel et al., above. 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 the physical stability of the double helix formed during 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-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine This includes, but is not limited to, nin, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 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 companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0216] The nucleic acid may include any isolated or purified nucleotide sequence encoding either DuoCAR or a functional portion or functional variant thereof. Alternatively, the nucleotide sequence may include a nucleotide sequence that is degenerate with any of the sequences, or a combination of degenerate sequences.

[0217] One embodiment includes a nucleotide sequence that is complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes with any of the nucleotide sequences of the nucleic acids described herein under stringent conditions, and is isolated or purified We also provide the nucleic acids that have been produced.

[0218] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. “High stringency conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectably strong amount compared to nonspecific hybridization. High stringency conditions include conditions that allow for the identification of polynucleotides with precisely complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that coincidentally have several subregions (e.g., 3–10 bases) that match the nucleotide sequence. These complementary subregions are more readily fused than full-length complements of 14–17 or more bases, and high stringency hybridization makes them readily identifiable. Relatively high stringency conditions include low-salt and / or high-temperature conditions, such as those provided at a temperature of about 50–70°C with about 0.02–0.1 M NaCl or equivalent. Such highly stringent conditions tolerate little mismatch between the nucleotide sequence and the template or target strand, if any, and are particularly suitable for detecting the expression of any of the DuoCARs of the present invention. Generally, it is understood that the conditions can be made more stringent by the addition of gradually increasing amounts of formamide.

[0219] Also provided are nucleic acids having nucleotide sequences that are identical to any of the nucleic acids described herein by at least about 70%, for example, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0220] In one embodiment, nucleic acids 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 the purposes of this specification, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide or peptide by a host cell, provided that the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is brought into contact with the cell under conditions sufficient for the mRNA, protein, polypeptide or peptide to be expressed in the cell. Vectors do not exist in nature as a whole.

[0221] However, some vector components may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or partially derived from 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-natural nucleotide linkages, or both. Preferably, non-natural or modified nucleotides or nucleotide linkages do not interfere with the transcription or replication of the vector.

[0222] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector that can be used to transform or transfect any suitable host cell. Suitable vectors include plasmids and viruses, which are designed for reproduction and growth, or for expression, or both. Vectors 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).

[0223] Bacteriophage vectors, such as λυTI O, λυTI 1, λZapII (Stratagene), EMBL4, and λNM149, may also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors may be viral vectors, such as retroviral vectors or lentiviral vectors. Lentiviral vectors are vectors derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. Vol. 17 (No. 8): pp. 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinics include, but are not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX® vector system. Non-clinical lentiviral vectors are also available and known to those skilled in the art.

[0224] Several transfection techniques are generally known in the field (see, for example, Graham et al., Virology, Vol. 52: pp. 456-467 (1973); Sambrook et al., above; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, Vol. 13: p. 97 (1981)).

[0225] Transfection methods include calcium phosphate coprecipitation (e.g., Graham et al., see above), direct microinjection into cultured cells (e.g., Capecchi, Cell, Vol. 22: pp. 479-488 (1980)), electroporation (e.g., Shigekawa et al., BioTechniques, Vol. 6: pp. 742-751 (1988)), liposome-mediated gene transfer (e.g., Mannino et al., BioTechniques, Vol. 6: pp. 682-690 (1988)), lipid-mediated transduction (e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, Vol. 84: pp. 7413-7417 (1987)), and high-velocity micropropellants. This includes nucleic acid delivery using microprojectiles (see, for example, Klein et al., Nature, Vol. 327: pp. 70-73 (1987)).

[0226] In one embodiment, recombinant expression vectors may be prepared using standard recombinant DNA techniques, such as those described above by Sambrook et al. and Ausubel et al. Circular or linear expression vector constructs may be prepared to contain a functional replication system in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0227] Recombinant expression vectors, taking into account whether the vector is DNA-based or RNA-based, may, if necessary, include regulatory sequences specific to the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), such as transcription and translation start and termination codons. Recombinant expression vectors may also include restriction sites to facilitate cloning.

[0228] Recombinant expression vectors may contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes may include those for biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., or nutritional complementation in the host to provide protrophotrophy. Suitable marker genes for the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine. This includes tetracycline resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0229] Recombinant expression vectors may include native or non-native promoters operably ligated to nucleotide sequences encoding CARs (including their functional portions and functional variants), or to nucleotide sequences complementary to or hybridizing to CAR-encoding nucleotide sequences. The choice of promoter, e.g., strong, weak, inducible, tissue-specific, and developmentally specific, is within the scope of the art. Similarly, combining nucleotide sequences with promoters is also within the scope of the art. Promoter may be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the terminal repeat sequences of mouse stem cell viruses.

[0230] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[0231] Furthermore, recombinant expression vectors can be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to drugs or other agents to cells expressing it, or a gene that causes cells to die when they come into contact with or are exposed to a drug. Suicide genes are publicly known in the art (see, for example, 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), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase (daminase), purine nucleoside phosphorylase, and nitroreductase.

[0232] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term “host cell” means any type of cell that may contain the recombinant expression vectors of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protist. The host cell may be a cultured cell or a primary cell, i.e., it may be isolated directly from an organism such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a 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. If the purpose is to amplify or replicate the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5a cell. If the purpose is to produce recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell can be of any cell type, originate from any type of tissue, and be at any developmental stage, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0233] For the purposes of this specification, T cells may be any T cells, such as cultured T cells, such as primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupTl, etc., or T cells obtained from mammals. When obtained from mammals, T cells may be found in, but are not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can be obtained from numerous sources. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from humans. T cells can be any type of T cell, and can be from any developmental stage, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Thi and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. T cells can be CD8+ T cells or CD4+ T cells.

[0234] In one embodiment, the DuoCAR described herein may be used in appropriate non-T cells. Such cells are cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0235] A population of cells comprising at least one host cell described herein is also provided by one embodiment. The population of cells may be a heterogeneous population comprising at least one other cell, e.g., a host cell (e.g., a T cell), 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., in addition to a host cell comprising one of the recombinant expression vectors described herein. Alternatively, the population of cells may be a substantially homogeneous population, where the population mainly comprises host cells comprising (e.g., essentially consisting of) recombinant expression vectors. The population may also be a clonal population of cells, where all cells in the population are clones of a single host cell comprising a recombinant expression vector, and as a result all cells in the population contain 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 vectors described herein.

[0236] DuoCAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including its population), and antibodies (including its antigen-binding parts) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is a preparation in which the host cells are purer than those in their natural environment within the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the host cell preparation is purified so that the host cells exhibit at least about 50% of the total cell content of the preparation, for example, at least about 70%. For example, purity can be at least about 50%, and can be above about 60%, about 70%, or about 80%, or it can be about 100%.

[0237] E. Treatment Method DuoCARs used in patient-specific autologous antitumor lymphocyte cell populations are intended to be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in mammals, comprising the step of administering DuoCAR, nucleic acid, recombinant expression vector, host cells, cell populations, antibodies and / or their antigen-binding moieties, and / or pharmaceutical compositions to a mammal in an amount effective for treating or preventing cancer in the mammal. Further uses of the DuoCAR described above are disclosed above.

[0238] One embodiment further includes a step of lymphodepleting the mammal prior to the step of administering the DuoCAR disclosed herein. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepletion chemotherapy, myeloablative lymphodepletion chemotherapy, and total body irradiation.

[0239] For methods of administering host cells or populations of cells, the cells may be homogeneous or autologous cells to the mammal. Preferably, the cells are autologous to the mammal. As used herein, homogeneous means different animals of the same species as the individual into which the material is introduced. This refers to any material originating from the same individual. Two or more individuals are said to be of the same species if their genes are not identical at one or more loci. In some embodiments, congenital material from individuals of the same species may be genetically distinct enough to interact antigenically. As used herein, “autologous” means any material originating from the same individual that is later reintroduced into the individual.

[0240] Mammals referred to herein may be any mammal. As used herein, the term “mammal” means any mammal, including but not limited to rodents, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals may be of Carnivora origin, including felines (cats) and canines (dogs). Mammals may be of Artiodactyla origin, including bovines (cows) and swines (pigs), or Perissodactyla origin, including equines (horses). Mammals may be of Primates, New World Cebioids or Simoids (monkeys) or Anthropoids (humans and apes). Preferably, the mammal is human.

[0241] Regarding these methods, cancers include acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, and chronic myeloid cancer. It may be any cancer, including any of the following: cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, humoral tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and Burkitt lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, retinoplasm, and mesentery, 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.

[0242] The terms “treat” and “prevent,” and words derived therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that a person skilled in the art would recognize as having a potentially beneficial or therapeutic effect. In this regard, this method may provide treatment or prevention of cancer in mammals of any amount or level.

[0243] Furthermore, the treatment or prevention provided by this method may include the treatment or prevention of one or more conditions or symptoms of a disease, such as cancer, being treated or prevented. Also, for the purposes of this specification, “prevention” may include delaying the onset of the disease, or its symptoms or conditions.

[0244] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising the steps of (a) contacting a sample comprising one or more cells of mammalian origin with DuoCAR, nucleic acid, recombinant expression vector, host cells, a population of cells, an antibody and / or its antigen-binding moiety, or a pharmaceutical composition to form a complex, and (b) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0245] Samples can be obtained by any suitable method, such as biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may involve collecting tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells. These experiments may include experiments to determine whether an individual has and / or is suffering from a particular condition or disease. The condition or disease may be, for example, cancer.

[0246] With regard to embodiments of a method for detecting the presence of proliferative disorders in mammals, such as cancer, a sample containing mammalian cells may include whole cells, their lysates, or fractions of whole cell lysates, such as nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions. If the sample contains whole cells, these cells may be any cells of a mammal, such as blood cells or cells of any organ or tissue, including endothelial cells.

[0247] The contact step can occur in vitro or in vivo for mammals. Preferably, the contact step is in vitro.

[0248] Furthermore, the detection of the complex can be carried out by many methods known in the art. For example, the DuoCARs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or antibodies or their antigen-binding moieties described herein and disclosed herein can be labeled with detectable labels, such as the radioisotopes disclosed herein, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0249] Methods for testing CARs for their ability to recognize target cells and antigen specificity are well known in the field. For example, Clay et al., J.Immunol, Vol. 163: pp. 507-513 (1999) teach 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 by Zhao et al., J.Immunol, Vol. 174: pp. 4415-4423 (2005).

[0250] Another embodiment of the present invention provides the use of DuoCAR, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or their antigen-binding moieties, and / or pharmaceutical compositions to treat or prevent proliferative disorders such as cancer in mammals. Cancer may be any of the cancers described herein.

[0251] Any method of administration, including topical and systemic administration, may be used for the disclosed therapeutic agent. For example, topical, oral, intravascular, intramuscular, intraperitoneal, intranasal, intradermal, subarachnoid, and subcutaneous administration may be used. The specific mode of administration and drug 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 prophylactic). If more than one agent or composition is administered, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, and antibodies or antigen-binding fragments or conjugates or compositions may be administered intravenously. Methods of administration include injections in which CARs, CAR T cells, conjugates, antibodies, antigen-binding fragments or compositions are delivered 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 liposomes. In some embodiments, topical administration of the disclosed compound is, for example, For example, it may be used 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 having a tendency toward tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically as eye drops to the cornea or intravitreally to the eye.

[0252] The disclosed therapeutic agents can be formulated in unit dosage forms appropriate for individual administrations of precise dosages. Furthermore, the disclosed therapeutic agents can be administered in single doses or in a multi-dose schedule. A multi-dose schedule is one in which the main course of treatment may consist of more than one separate 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 over a period of several days to several months or even several years. Thus, the dosing regimen is also determined at least in part based on the specific requirements of the subject being treated and depends on the judgment of the practitioner administering it.

[0253] Typical dosages of antibodies or conjugates may range from approximately 0.01 to approximately 30 mg / kg, for example, from approximately 0.1 to approximately 10 mg / kg.

[0254] In certain cases, subjects are administered a therapeutic composition comprising one or more of the following in multiple daily dosing schedules, for example, at least two consecutive days, at least ten consecutive days, etc., over a period of several weeks, months, or years. In one example, subjects are administered the conjugate, antibody, composition, or further drug over a period of at least 30 days, for example, at least two months, at least four months, at least six months, at least twelve months, at least 24 months, or at least 36 months.

[0255] In some embodiments, the disclosed methods include providing surgical, radiotherapy, and / or chemotherapeutic agents to a target (e.g., sequentially, substantially simultaneously, or concurrently) in combination with disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells. Such agents and methods of treatment and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for further agents may be used according to the manufacturer's instructions or may be determined experimentally by those skilled in the art. Preparation and dosing schedules for such chemotherapeutic agents are also described in Chemotherapy Service, (1992), edited by MCPerry, Williams & Wilkins, Baltimore, Md.

[0256] In some embodiments, combination therapy may involve administering a therapeutically effective dose of an additional cancer inhibitor to the subject. Non-limiting examples of additional therapeutic agents that may be used in 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 doses) and treatments may be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent may be administered in combination with CARs, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. Methods and therapeutic doses of such agents are known to those skilled in the art and may be determined by a skilled clinician.

[0257] Further chemotherapeutic agents for combination immunotherapy include alkylating agents, e.g., nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), and platinum compounds (e.g., carboplatin, cisplatin). (Oxaliplatin and BBR3464), busulfan, dacarbazine, mechloretamine, procarbazine, temozolomide, thiotepa and uramustine; antimetabolites, e.g., folic acid (e.g., methotrexate, pemetrexed and larcitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil and gemcitabine ; Plant alkaloids, e.g., podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine and vinorelbine); cytotoxic / antitemonic antibiotics, e.g., members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and barurubicin), bleomycin, rifampicin, hydroxyu Rare and mitomycin; topoisomerase inhibitors, e.g., topotecan and irinotecan; monoclonal antibodies, e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab and trastuzumab; tumor affinity photosensitive dyes, e.g., aminolevulinic acid, methyl aminolevulinic acid, porfimer sodium and verteporfin; and other drugs, e.g., alitretinoin, altretamine, amsacrine, anagrelide, This list includes, but is not limited to, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin difutitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masopropyl alcohol, 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.

[0258] In certain embodiments of the present invention, cells activated and proliferated using the methods described herein, or other methods known in the art in which T cells are proliferated to therapeutic levels, are administered to a patient in conjunction with several appropriate treatment modalities, including, but not limited to, drug treatments for MS patients, such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment, or efalizumab treatment for psoriasis patients, or other treatments for PML patients (for example, before, simultaneously with, or after). 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, cytotoxicities such as fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66: pp. 807-815, 1991; Henderson et al., Immun 73: pp. 316-321, 1991; Bierer et al., Curr. Opin. Immun 5: pp. 763-773, 1993). In further embodiments, the cell composition of the present invention is administered to the patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, or T cell depletion therapy. In another embodiment, the cell composition of the present invention is administered after B cell depletion therapy with a CD20-reactive agent such as Rituxan. For example, in one embodiment, the subject may receive standard treatment consisting of high-dose chemotherapy followed by peripheral blood stem cell transplantation. In a particular embodiment, after transplantation, the subject The patient receives an injection of the proliferated immune cells of the present invention. In a further embodiment, the proliferated cells are administered before or after surgery.

[0259] The dosage of the above treatment administered to a patient varies depending on the condition being treated and the exact nature of the recipient. Dosage adjustments for human administration may be carried out in accordance with accepted practices in the field. For example, the dose of CAMPATH is generally in the range of 1 to about 100 mg for adult patients and is usually administered daily over a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day, but in some cases, a larger dose of up to 40 mg per day may be used.

[0260] Combination therapy can provide and prove to be synergistic; that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that could result from using those compounds separately. Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously with the unit dose formulations used in combination; (2) delivered alternately or in parallel as separate formulations; or (3) in part with other regimens. When delivered alternately, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation, effective doses of each active ingredient are administered sequentially, i.e., in order; however, in combination therapy, effective doses of two or more active ingredients are administered together.

[0261] 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 having a tumor after anti-cancer treatment. After sufficient time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on the respective cancer cells, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in the immune complex compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in the immune complex compared to a control taken before treatment indicates that the treatment is effective.

[0262] F. Pharmaceutical Compositions Provided herein are biopharmaceutical compositions or biologic compositions (hereinafter “compositions” herein) for use in gene therapy, immunotherapy, adoptive immunotherapy and / or cell therapy, comprising one or more of the disclosed DuoCARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates, DuoCARs, 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 forms for administration to a subject. The amount and timing of administration to achieve the desired outcome is at the discretion of the clinician performing the treatment. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed DuoCARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates are formulated for parenteral administration such as intravenous administration. Compositions comprising a CAR, or T cells expressing a CAR, conjugate, antibody or antigen-binding fragment disclosed herein are used for, e.g., treatment and detection of tumors, such as, without limitation, neuroblastoma. In some examples, these compositions are useful for the treatment or detection of cancer. Compositions comprising a CAR, or T cells expressing a CAR, conjugate, antibody or antigen-binding fragment disclosed herein are also used, for example, for the detection of pathological angiogenesis.

[0263] Compositions for administration may include a CAR dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier, or a solution of T cells, conjugates, antibodies or antigen-binding fragments that express the CAR. Various 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 adjusters and buffers, toxicity modifiers, adjuvant agents, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the CAR, or T cells expressing the CAR, antibody or antigen-binding fragment or conjugate in these formulations can vary widely and is selected mainly based on the volume of liquid, viscosity, body weight, etc., according to the particular mode of administration selected and the requirements of the subject. The actual methods for preparing such dosage forms for use in gene therapy, immunotherapy and / or cell therapy are known or will be apparent to those skilled in the art.

[0264] Typical compositions for intravenous administration contain from about 0.01 to about 30 mg / kg per day per subject of an antibody or antigen-binding fragment or conjugate (or a corresponding dose of a CAR, or a conjugate containing a T cell expressing the CAR, antibody or antigen-binding fragment). The actual methods for preparing administrable compositions are known or apparent to those skilled 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).

[0265] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be supplied in lyophilized form and rehydrated with sterile water before administration, or they may also be supplied in sterile solutions of known concentrations. The 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 administered in some cases at doses of 0.5–15 mg / kg body weight. Considerable experience in the administration of antibody or antigen-binding fragments and conjugate drugs is available in this field; 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 may be administered by slow infusion rather than by intravenous injection or bolus. In one example, a higher loading dose is administered along with a subsequent maintenance dose administered at a lower level. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or a conjugate containing the antibody or antigen-binding fragment at a corresponding dose) may be infused over a period of approximately 90 minutes, followed by a weekly maintenance dose of 2 mg / kg over 30 minutes for 4 to 8 weeks, provided that the previous dose was well tolerated.

[0266] Controlled-release parenteral formulations can be prepared as implants, oily injections, or granular systems. For a broad overview 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 generally called nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. For example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drugs See elivery, A. Kydonieus (ed.), Marcel Dekker, Inc., New York, NY, pp. 315-339 (1992).

[0267] The polymers may be used for the ion-controlled release of 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. Vol. 26: pp. 537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but 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. Vol. 9: pp. 425-434, 1992; and Pec et al., J.Parent. Sci. Tech. Vol. 44 (No. 2): pp. 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. Vol. 112: pp. 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 further systems for the 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).

[0268] G. Kitt In one embodiment, a kit using the DuoCARs disclosed herein is also provided. For example, a kit for treating a tumor in a subject or a kit for producing CAR T cells expressing one or more of the DuoCARs disclosed herein. The kit typically includes, as disclosed herein, a disclosed antibody, an antigen-binding fragment, a conjugate, a nucleic acid molecule, a DuoCAR, or T cells expressing a CAR. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, a CAR, or T cells expressing a CAR may be included in the kit.

[0269] The kit may include a container and labels or accompanying documents on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Typically, a container holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, or DuoCAR or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). Labels or accompanying documents indicate that the composition is used to treat a particular condition.

[0270] The label or accompanying leaflet is typically an instruction for the use of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, DuoCAR, or CAR-expressing T cells in methods for treating or preventing tumors, or in methods for producing CAR T cells. The kit may further include the following. The package insert typically includes instructions that are customarily included in the market packaging of the therapeutic product, which typically include information about the use of the therapeutic product, including indications, usage, dosage, administration, contraindications and / or warnings. The teaching materials may be written in electronic form (e.g., computer diskette or compact disk) or visual (e.g., video file). The kit may also include further components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may further include means for detecting a label (e.g., an enzyme substrate for enzyme labeling, a filter set for detecting a fluorescent label, a suitable secondary label such as a secondary antibody, etc.). The kit may further include buffers and other reagents that are customarily used for carrying out a particular method. Such kits and suitable contents are well known to those skilled in the art. [Examples]

[0271] The present invention is further illustrated by the DuoCAR examples shown in the following appendix and pages 17–27, including both ends, without any interpretation that such examples limit its scope. Conversely, it will be readily apparent that various other embodiments, modifications, and equivalents thereof can be conceived after reading this specification without departing from the spirit of the invention and / or the scope of the appendix claims, and these may come to mind for those skilled in the art.

[0272] While various details have been outlined above, along with the exemplary implementations mentioned earlier, various alternatives, modifications, variations, improvements, and / or substantial equivalents may become apparent by reviewing the aforementioned disclosures, whether publicly known or currently unexpected or potentially unexpected.

[0273] Each application and patent cited herein, and each document or reference cited in each application and patent (including each granted patent in litigation, “Application Reference Documents”), and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and each document cited or referenced in each Application Reference Document, are incorporated herein by express reference and can be used in practicing the present invention. More generally, documents or references are cited in the text, in the list of references preceding the claims, or in the text itself, and each of these documents or references (“In-Specified References”), and each document or reference cited in each In-Specified Reference (including any manufacturer’s specifications, instructions, etc.), are incorporated herein by express reference.

[0274] The aforementioned descriptions of some specific embodiments provide sufficient information to enable others to easily modify or adapt the invention to various applications, such as specific embodiments, without departing from the general concept, by applying the knowledge of the invention. Therefore, such adaptations and modifications should and are intended to be understood as being within the meaning and scope of equivalents of the disclosed embodiments. It is understood that the terms or predicates used herein are for descriptive purposes only, not limiting purposes. Exemplary embodiments are disclosed in the drawings and description, and specific terminology may be used, but unless otherwise noted, they are used only in a general and descriptive sense, not limiting purposes, and therefore the claims are not so limited. Furthermore, those skilled in the art will understand that certain steps of the methods discussed herein can be arranged in a different order or combined. Therefore, the appended claims are not intended to be limited to the detailed embodiments disclosed herein. Those skilled in the art can understand and grasp many equivalents of the embodiments of the invention described herein using commonplace experiments. Such equivalents are encompassed by the following claims.

[0275] Two examples are provided, in which the expression of three functional binding domains on the surface of an LV transduced human T cell population 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).

[0276] Based on this technology, examples of single-specific CARs that may be included as DuoSet components in DuoCAR include the single CD20-targeting vector LTG1495, with nucleotide sequence SEQ ID NO: 3 and amino acid sequence SEQ ID NO: 4. A second example is the CD22-specific single-specific CAR LTG2200, with nucleotide sequence SEQ ID NO: 9 and amino acid sequence SEQ ID NO: 10. A key molecular aspect in creating DuoCAR is the inclusion of non-duplication-compatible sequences and the evaluation of these sequences in transduced T cells to ensure that no harmful recombination or intracellular association occurs. This can occur in both the vector-producing cell line and the target cell population. For this reason, we include variant CAR structures that are known to be compatible in DuoCAR settings. These include the CD19-specific CAR LTG1494, described in nucleotide sequence SEQ ID NO: 29 and amino acid sequence SEQ ID NO: 30, respectively. This sequence contains a well-described linker that connects the heavy and light chains of scFv, known as the Whitlow linker (see amino acid sequence GTSTGSGKPGSGEGSTKG, Whitlow M. et al., 1993, Protein Eng. Vol. 6: pp. 989-995). In some cases, the Whitlow linker is present in the CD19 CAR format at sequence number 31 of the nucleotide sequence and sequence number 32 of the amino acid sequence, respectively (GGGGS), as seen in LTG1538, for example. nThe linker is substituted. In another example, the CAR was created in a form that has an alternative transmembrane domain. Anti-CD19 CAR LTG1562, with nucleotide sequence SEQ ID NO: 21 and amino acid sequence SEQ ID NO: 22, utilizes the CD4 (as the opposite of CD8) transmembrane domain. Similarly, anti-CD19 CAR LTG1563 has an alternative transmembrane domain derived from TNFRSF19, with nucleotide sequence SEQ ID NO: 49 and amino acid sequence SEQ ID NO: 50, respectively. DuoCARs may be targeted to solid tumors, such as those expressing mesothelin tumor antigen. For example, the scFV binder may be created for mesothelin as disclosed in the applicant's concurrently pending provisional patent application No. 62 / 444,201, titled Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy, filed January 9, 2017, and assigned to Lentigen Technology, Inc., in transaction number LEN_017, with nucleotide sequence SEQ ID NO. 37 and amino acid sequence SEQ ID NO. 38, respectively, and may be incorporated into a functional CAR, with nucleotide sequence SEQ ID NO. 39 and amino acid sequence SEQ ID NO. 40, respectively, thereby being incorporated into DuoCAR therapy. In addition to the scFv sequence, single-chain antigen binders (as the opposite of scFv) may be incorporated into DuoCAR applications. For example, a CD33-specific heavy-chain-only binder is disclosed in the applicant's concurrently pending provisional patent application No. 62 / 476,438, titled Compositions and Methods As disclosed in the application filed March 24, 2017, for Treating Cancer With Anti-CD33 Immunotherapy, and assigned to Lentigen Technology, Inc., under transaction number LEN_018, the functional CAR, LTG1906, may be incorporated into the nucleotide sequence sequence 43 and the amino acid sequence sequence 44, respectively, to target CD33-expressing malignancies. One example of a DuoCAR therapeutic application is the treatment of leukemia expressing CD19, CD20, and TSLPR antigens. In this case, LTG1496 or LTG 1497 (sequences 35 and 26, respectively) may be combined with a TSLPR-specific CAR (LTG1789), sequence number 47 and the amino acid sequence sequence 48, respectively, which was created from a TSLPR-specific scFV domain, sequence number 45 and the amino acid sequence sequence 46.

[0277] Examples of tandem CARs (containing two scFv domains, described as nucleotide sequence SEQ ID NO: 23 and amino acid sequence SEQ ID NO: 24) on which this technology is based include CD20_CD19 CAR LTG1497, nucleotide sequence SEQ ID NO: 25 and amino acid sequence SEQ ID NO: 26. In some cases, reversing the order of the two binders can provide even better DuoCAR expression in target cells. Therefore, LTG1497, where the CD19 scFV is more proximal and shown in nucleotide sequence SEQ ID NO: 25 and amino acid sequence SEQ ID NO: 26; and LTG1496, where the CD19 scFV is more distal to the membrane and shown in nucleotide sequence SEQ ID NO: 33 and amino acid sequence SEQ ID NO: 34; can both be used as one of the members of a DuoSet containing a DuoCAR.

[0278] Methods used in Examples 1 and 2: Cell lines (PBMCs and targets) Unless otherwise noted, all cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). Burkitt lymphoma cell line Raji, acute lymphoblastic leukemia cell line REH, and chronic myeloid leukemia cell line K562 were cultured in RPMI-1640 medium supplemented with 10% thermo-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). Human fetal kidney cell line 293T was grown in Dulbecco's modified Eagle medium supplemented with 10% thermo-inactivated FBS.

[0279] Single-cell clones of luciferase-expressing cell lines were generated from wild-type tumor cells stably transduced using a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones. The mouse-adapted Raji-luc strain was created by stably expressing firefly luciferase in Raji clones, using NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ), and the Raji-luc tumor cells were transplanted into Jackson Laboratory (Sacramento, CA), isolated from mouse spleens by positive selection (CD19 microBeads, human, Miltenyi Biotec, Bergisch Gladbach, Germany) or negative selection (mouse cell depletion kit, Miltenyi Biotec), grown in culture, and re-cloned to facilitate selection of clones with high firefly luciferase expression. Whole blood was collected from healthy individuals with donor consent forms at Oklahoma Blood Institute (OBI, Oklahoma City, OK). Processed buffy coat was purchased from OBI. CD4- and CD8-positive human T cells were purified from buffy coat via positive selection using a 1:1 mixture of CD4- and CD8-MicroBeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0280] Creation of chimeric antigen receptor (CAR) expression vectors including DuoSet The CAR antigen-binding domains, scFvs, and sequences were derived from the complete sequences of mouse fusion cells FMC-63 (FMC-63:AA 1-267, GenBank ID:HM852952.1) for CD19 and Leu-16[1], VL, and VH for CD20. CD22 scFv binding was generated from published sequences. Tandem CAR19_20 or CAR20_19 were used, with the scFv of each antibody in frame, along with the CD8 hinge and transmembrane domain (AA 123-191, reference sequence ID NP_001759.3) and 4-1BB (CD137, AA 214-255, UniProt sequence ID Q07). 011) It was generated by linking to the transactivation domain and the CD3 zeta signaling domain (CD247, AA 52 - 163, reference sequence ID: NP_000725.1.). The scFv regions of 19A and 20A were linked in the sequence by a flexible inter-chain linker (GGGGS)5, followed by the CD8, 4-1BB, and CD3 zeta domains. The leader sequence derived from the human granulocyte macrophage colony-stimulating factor receptor alpha subunit was included in all constructs as described in [2]. 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 regulation of the human EF-1α promoter. The lentiviral vector (LV) - containing supernatant was generated by transient transfection of HEK 293T cells as already described in [3]. The recovered precipitated lentiviral supernatant was stored at -80 °C.

[0281] Primary T cell transduction: CD4+ and CD8+ human primary T cells selected from normal donors were cultured at a density of 0.3 to 2x10 6 cells / ml in TexMACS medium (serum-free) supplemented with 40 IU / ml IL-2 and activated using CD3 / CD28 MACS® GMP TransAct reagent (Miltenyi Biotec). On the third day, they were transduced overnight with the lentiviral vector encoding the CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), and the medium was changed on the fourth day. On the fifth day, the cultures were transferred to TexMACS medium supplemented with 200 IU / ml IL-2 and grown until recovery on days 10 - 13.

[0282] Immune effector assay: To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced using firefly luciferase were combined with CAR T cells in various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was analyzed on an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). Wells containing only targets (maximum CPS) and wells containing only targets with 1% Tween-20 added (minimum CPS) were used to determine the assay range. The percentage of specific lysis was calculated as: (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)).

[0283] Flow cytometry analysis: Unless otherwise noted, all cell staining reagents for flow cytometry were from Miltenyi Biotec. One million CAR T transduced cells were harvested from the culture, washed twice in chilled staining buffer (AutoMACS solution containing 0.5% bovine serum albumin), and precipitated at 350xg for 5 minutes at 4°C. CAR surface expression on transduced T cells was initially detected by 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 for 30 minutes at 4°C (stock: 1.0 ml, 1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA). Untransduced cells and transduced cells stained with streptavidin-PE alone were used as negative controls. Anti-CD4 antibody was used to determine the CD4 to CD8 ratio in the CAR T-positive population and 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 ul staining buffer before quantitative analysis by flow cytometry. Specific DuoSet CAR T staining was performed. The D3-CD28 nanomatrix (TransAct, Miltenyi Biotec) was activated and the cells were transformed using a DuoSet vector in the presence of IL-2 on human T cells. Expression of the CD19-, CD20-, or CD22-scFv domains was analyzed by flow cytometry using recombinant CD19, CD20, or CD22 for antibody staining.

[0284] Anti-CD19 scFv activity was detected using CD19-Fc (R&D Biosystems) at 1 ug / sample and stained with goat anti-human Fc-gamma-R-PE (Jackson ImmuoResearch Laboratories, Inc.) at 0.75 ug / sample. Anti-CD20 scFv activity was detected using CD20-biotin (Miltenyi Biotech) at 0.1 ug / sample and streptavidin pAPC (Miltenyi Biotec) at 0.2 ug / sample. Anti-CD22 scFc activity was detected using CD22-His (Thermo Fisher) at 0.1 ug / 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 analysis by 7AAD staining (BD Biosciences, San Jose, CA).

[0285] Example 1 Expression of DuoCAR (2+1 DuoSet) on primary human T cells As proof of principle, a DuoSet consisting of two CAR-T vectors was created. One member of the set expressed a CD8 transmembrane and CD28 and CD3-zeta signaling domain (LTG2228), and a tandem CD20_CD19 binding domain linked to sequence numbers 51 and SEQ ID NO: 52. The second member of the DuoSet was a CAR construct containing a CD8 transmembrane and 4-1BB and CD3-zeta signaling domain (LTG2200), and a CD22 binder linked to SEQ ID NO: 9 and SEQ ID NO: 10. In Figure 7, paired columns show double staining for CD20 and CD19 scFvs, left column, and CD22 and CD19 scFvs, right column. Column 1 shows T cells that have not been transduced and therefore do not show binding (UTD). Column 2 shows T cells transduced using an LV encoding a CD20_CD19 CAR vector containing CD8 transmembrane and intracellular CD28 and D3 zeta signaling domains (20-19-28z). Double staining is seen for CD20 and CD19 binding (left panel), while only CD19 binding is seen in the right panel. Column 3 shows T cells transduced using a CD22 CAR vector containing CD8 transmembrane and intracellular 4-1BB and CD3-zeta signaling domains (22-BBz). Double staining is not seen for CD19 or CD20 (left panel), and it can be seen that only a single population of cells can bind to CD22 (right panel). In column 4, T cells are transduced using a DuoSet composed of both vectors from columns 2 and 3. 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 CD22 and CD19 scFv are located on two separate transmembrane proteins, including DuoSet, the 38% represents the true DuoSet expression population in this example.

[0286] Example 2 Antileukemia activity of human T cell preparations expressing DuoCAR The anti-leukemic activity of human T cell preparations expressing DuoCAR simultaneously targets three leukemic antigens (see Figure 7 for DuoCAR expression characteristics). CD20_1 DuoSets consisting of nine tandem CARs and a CD22-specific single CAR (as prepared in Example 1) were used as effector T cell populations in cytotoxic T cell assays using leukemia cell lines and target model cell lines. Transduced human T cells using a single CAR component (20_19-28z or 22-BBz) or DuoSet (20_19-28z + 22-BBz) were used in cytotoxic T cell assays at four different effector target ratios (20:1, 10:1, 5:1, and 2.5:1 as shown) (see Figure 8). 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 leukemia cell lines (Raji and REH) and all three monoexpressing K562 target cell lines (K562-CD19, K562-CD20, K562-CD22). This demonstrates that DuoCAR technology can uniquely target three leukemia antigens simultaneously within the same effector T cell population, thereby demonstrating superior antineoplastic activity due to the ability to simultaneously target more than one or two target antigens, thereby reducing the likelihood of malignancies generating escape variants (cell clones with one or two antigens lost or downregulated, which escape from immunoablation). The ultimate outcome is a higher cure rate for patients, attributed to the escape and proliferation of antigen-loss variants, which ultimately result in relapse.

[0287] Example 3 DuoCAR production method The DuoCAR technology described in this application generates a population of therapeutic lymphocytes, in this embodiment, human T cells, that express more than two antigen specificities from more than one transmembrane protein encoded by a gene vector. In this embodiment, this is achieved by two separate means. Figure 9 contains three data columns labeled “Untransduced,” “Cotransduced,” and “Cotransfection.” Figure 9 contains two columns of data generated similarly to those in Figure 7, where the first column is analyzed by flow cytometry for the expression of CD20- and CD19-specific binding, and the second column is analyzed by flow cytometry for the expression of CD22- and CD19-binding activity. The first column of data shows untransduced human T cells. Binding activity is not shown for CD19, CD20, or CD22 recombinant protein indicators of CAR-derived binding activity, demonstrating the absence of DuoCAR expression. In the second column, “Cotransduced” was used to generate DuoCAR. In this dataset, two LVs were used to simultaneously transduce activated T cells. As in Figure 7, one CAR in the DuoSet containing the DuoCAR was a tandem CD20 and CD19 binder linked to CD28 signaling and CD3 zeta signaling motifs; the other CAR was a CD22 binder linked to 4-1BB and CD3 zeta signaling motifs. The second quadrant (Q2) in column 1 shows a very specific pattern of unitary staining for CD20 and CD19-scFv activity. This is because both binders are on the same surface glycoprotein, thereby they are co-expressed at the same intensity, resulting in the very specific linear pattern seen. In the second column of the cotransduction data, a more traditional pattern is seen where the two glycoproteins are not expressed in a uniform pattern on each cell. Thus, patterns for four different populations are seen. In the lower left quadrant, cells are seen where neither binder is expressed. In the upper left quadrant, cells expressing only the CD22 CAR can be seen. In the lower right quadrant, cells expressing only the CD20_CD19 tandem CAR can be seen.Finally, in the upper right quadrant, we can see cells expressing both members of the CAR DuoSet, including DuoCAR.

[0288] In the lower column, cell populations expressing DuoCAR are generated in different ways. Unlike cotransduction, which uses two independently generated LV preparations during T cell transduction, "cotransfection" refers to a method in which two skeletal plasmids (encoding two CARs, including DuoCAR) are transfected into 293 T cells, simultaneously generating LV. Other plasmids, including this third-generation LV system, are identical in both methods. The advantage of the cotransfection method is that a single preparation of LV containing vectors encoding both CARs is generated. As can be seen from the data, almost identical patterns of CD20-CD19 CAR and CD22 CAR expression are observed compared to cotransduction in the second column. The staining patterns for both glycoproteins induced by LV generated by cotransfection (CD22 co-staining for CD22-CAR and CD19 co-staining for CD20-19 CAR) in the upper right quadrant of the data in the second column demonstrate that both methods efficiently generate DuoCAR.

[0289] References:

[0290] [Table 1]

[0291] References regarding sequence listings This application includes a sequence listing electronically filed with the United States Patent and Trademark Office as a PDF file titled “Sequence Listing”. That sequence listing is incorporated herein by reference.

[0292] The array of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37C.FR1.822. Only single strands of each nucleic acid sequence are shown, but it is understood that complementary strands are included by some reference to the shown strand. In the attached sequence listing: Sequence ID 1 is the nucleotide sequence of the CD20-reactive scFv-binding domain (LTG1495). GAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAACAGACCCCGGGACAAGGGCTCGAATG GATTGGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACT ACTACTGCGCACGGTCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTGACCGTCAGCTCCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGACATTGTG CTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATGACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCGTCACCCAAGCCTTGGATCTACGCTAC ATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTCAGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGACGCCGGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACTAAGCTGGAGATCAAA Sequence ID 2 is the amino acid sequence of the CD20-reactive scFv-binding domain (LTG1495). EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVS SGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIK Sequence ID 3 is the nucleotide sequence of CAR LTG1495 (LP-1495-CD8 TM-41BB-CD3 zeta). Sequence ID 4 is the amino acid sequence of CAR LTG1495 (LP-1495-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSN YYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYY CQQWSFNPPTFGGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 5 is the nucleotide sequence of the leader / signal peptide sequence. ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG Sequence ID 6 is the amino acid sequence of the leader / signal peptide sequence. MLLLVTSLLLCELPHPAFLLIP Sequence ID 7 is the nucleotide sequence of the CD22-reactive scFv-binding domain (LTG2200). CAGGTACAGCTCCAGCAGAGTGGCCCAGGGCTCGTGAAGCCAAGCCAGACGCTGTCCCTGACTTGTGCAATTTCAGGGGATTCAGTTTCATCAAATAGCGCGGCGTGGAATTGGATTCGACAATCTCCTTCCCGAGGGTTGGAATGGCTTGGACGAACATATTACAGATCCAAATGGTATAACGACTATGCGGTATCAGTAAAGTCAAGAATAACCATTAACCCCGACACAAGCAAGAACCAATTCTCTTTGCAGCTTAACTCTGTCACGCCAGAAGACACGGCAGTCTATTATTGCGCTCGCGAGGTAACGGGTGACCTGGAAGACGCTTTTGACATTTGGGGGCAGGGTACGATGGTGACAGTCAGTTCAGGGGGCGGTGGGAGTGGGGGAGGGGGTA GCGGGGGGGGAGGGTCAGACATTCAGATGACCCAGTCCCCTTCATCCTTGTCTGCCTCCGTCGGTGACAGGGTGACAATAACATGCAGAGCAAGCCAAACAATCTGGAGCTATCTCAACTGGTACCAGCAGCGACCAGGAAAAGCGCCAAACCTGCTGATTTACGCTGCTTCCTCCCTCCAATCAGGCGTGCCTAGTAGATTTAGCGGTAGGGGCTCCGGCACCGATTTTACGCTCACTATAAGCTCTCTTCAAGCAGAAGATTTTGCGACTTATTACTGCCAGCAGTCCTATAGTATACCTCAGACTTTCGGACAGGGTACCAAGTTGGAGATTAAGGCGGCCGCA SEQ ID NO: 8 is the amino acid sequence of the CD22-reactive scFv binding domain (LTG2200). QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAA Sequence ID 9 is the nucleotide sequence of CAR LTG2200 (LP-2200-CD8 TM-41BB-CD3 zeta). Sequence ID 10 is the amino acid sequence of CAR LTG2200 (LP-2200-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR EVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATY YCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 11 is the nucleotide sequence of the DNA CD8 transmembrane domain. ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC Sequence ID 12 is the amino acid sequence of the CD8 transmembrane domain. IWAPLAGTCGVLLLSLVITLYC Sequence ID 13 is the nucleotide sequence of the DNA CD8 hinge domain. ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCCAGTGCACACGAGGGGGCTGGACTTTGCCTGCGATATCTAC Sequence ID 14 is the amino acid sequence of the CD8 hinge domain. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY Sequence ID 15 is the amino acid sequence from amino acid numbers 137 to 206 of the hinge and transmembrane region of CD8 alpha (NCBI RefSeq: NP.sub.--001759.3). TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC Sequence ID 16 is the amino acid sequence of the human IgG CL sequence. GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Sequence ID 17 is the nucleotide sequence of the DNA signaling domain at 4-1BB. AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG AAGAAGAAGAAGGAGGATGTGAACTG Sequence ID 18 is the amino acid sequence of the signaling domain at 4-1BB. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Sequence ID 19 is the nucleotide sequence of the DNA signaling domain of CD3-zeta. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC Sequence ID 20 is the amino acid sequence of CD3 zeta. RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 21 is the nucleotide sequence of CAR LTG1562 (LP-CD19 binder-CD8 linker-CD4tm-4-1BB-CD3-zeta). CTGCATATGCAGGCGCTGCCGCCGCGC Sequence ID 22 is the amino acid sequence of CAR LTG1562 (LP-CD19 binder-CD8 link-CD4tm-4-1BB-CD3-zeta). MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFVQPMALIVLGGVAGLLLFIGLGIFFCVRCRPRRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 23 is the nucleotide sequence of the CD20_19-reactive scFv binding domain (LTG1497 bispecificity binder). Sequence ID 24 is the amino acid sequence of the CD20_19-reactive scFv binding domain (LTG1497 bispecific binder). EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGGSGG GGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKGGGGSGGGGSGGGS GGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAA Sequence ID 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-Wittrow linker-CD19 VH-CD8 hinge+TM-41BB-CD3 zeta). Sequence ID 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 MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 27 is the nucleotide sequence of scFV for CD19. GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA Sequence number 28 is the amino acid sequence of the scFV for CD19. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG SEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS Sequence ID 29 is the nucleotide sequence of CAR LTG 1494 (LP-CD19 binder-CD8 link-CD8™-41BB-CD3 zeta). ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACTGACATTCAGATGACTCAGACCACCTCTTCCTTGTCCGCGTCACTGGGAGACAGAGTGACCATCTCGTGTCGCGCAAGCCAGGATATCT Sequence ID 30 is the amino acid sequence of CAR LTG 1494 (LP-CD19 binder-CD8 link-CD8™-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPDTDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 31 is the nucleotide sequence of CAR LTG1538 (LP-CD19 binder-CD8 link-CD8™ signal (LTI remanipulated CD19 CAR)). Sequence ID 32 is the amino acid sequence of CAR LTG1538 (LP-CD19 binder-CD8 link-CD8™ signal (LTI remanipulated CD19 CAR)). MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 33 is the nucleotide sequence of the CD19_20 reactive scFv binding domain (LTG1496). Sequence ID 34 is the amino acid sequence of the CD19_20-reactive scFv-binding domain (LTG1496). DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKL QESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGGSGGGGSGGG GSGGGGSGGGGSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTT VTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKAAA Sequence ID 35 is CAR LTG1496 (LP-LTG1496-CD8 TM-41BB-CD3 Zeta) or (LP-CD19 VL-Whitlowlinker-CD19) This is the nucleotide sequence of VH(GGGGS)5CD20 VH (GGGGS)3-CD20 VL CD8 hinge + TM-41BB-CD3 zeta). ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCTGCCTTCCTGCTGATTCCCGACATTCAGATGACTCAGACCACCTCCTCCCTGTCCGCCTCCCTGGGCGACCGCGTGACCATCTCATGCCGCGCCAGCCAGGACATCTCGAAGTACCTCAACTGGTACCAGCAGAAGCCCGACGGAACCGTGAAGCTCCTGATCTACCACACCTCCCGGCTGCACAGCGGAGTGCCGTCTAGATTCTCGGGTTCGGGGTCGGGAACTGACTACTCCCTTACTATTTCCAACCTGGAGCAGGAGGATATTGCCACCTACTTCTGCCAACAAGGAAACACCCTGCCGTACACTTTTGGCGGGGGAACCAAGCTGGAAATCACTGGCAGCACATCCGGTTCCGGGAAGCCCGGCTCCGGAGAGGGCAGCACCAAGGGGGAAGTCAAGCTGCAGGAATCAGGACCTGGCCTGGTGGCCCCGAGCCAGTCACTGTCCGTGACTTGTACTGTGTCCGGAGTGTCGCTCCCGGATTACGGAGTGTCCTGGATCAGGCAGCCACCTCGGAAAGGATTGGAATGGCTCGGAGTCATCTGGGGTTCCGAAACCACCTATTACAACTCGGCACTGAAATCCAGGC Sequence ID 36 is CAR LTG1496 (LP-LTG1496-CD8 TM-41BB-CD3 Zeta) or (LP-CD19 VL-Whitlow Linker-CD19) This is the amino acid sequence of VH(GGGGS)5CD20 VH (GGGGS)3-CD20 VL CD8 hinge + TM-41BB-CD3 zeta. MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTS EDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 37 is the nucleotide sequence of the mesothelin-reactive scFv-binding domain (LTG1904). GAGGTCCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCG GACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATTCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATTTATCGTCAGTGGCTGGACCCTTTAACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGT GGCGGGTCTGGTGGAGGCGGTAGCGGCGGTGGCGGATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTAT GGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGCGGAGGCACCCAGCTGACCGTCCTCGGT Sequence ID 38 is the amino acid sequence of the mesothelin-reactive scFv-binding domain (LTG1904). EVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTALYYCAKDLSSVAGPFNYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTQLTVLG Sequence ID 39 is the nucleotide sequence of CAR LTG1904 (LP-LTG1904-CD8 TM-41BB-CD3 zeta). Sequence ID 40 is the amino acid sequence of CAR LTG1904 (LP-LTG1904-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLS SVAGPFNYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNS RDSSGNHLVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 41 is the nucleotide sequence of the CD33-reactive single-strand binding domain VH-4 (LTG1906). GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGCCAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTG AGAAATACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAAGAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA Sequence ID 42 is the amino acid sequence of the CD33-reactive single-strand binding domain VH-4 (LTG1906). EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKENVDWGQGTLVTVSS Sequence ID 43 is the nucleotide sequence of CAR LTG1906 (LP-VH4-CD8 TM-41BB-CD3 zeta). ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCT ATGGCATGAGCTGGGTCCGCCAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAG CCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAAGAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 44 is the amino acid sequence of CAR LTG1906 (LP-VH4-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKENVDWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 45 is the nucleotide sequence of the TSLPR-reactive scFv binding domain (LTG1789). ATGGCACTGCCCGTGACCGCCCTGCTTCTGCCGCTTGCACTTCTGCTGCACGCCGCTAGGCCCCAAGTCACCCTCAAAGAGTCAGGGCCAGGAATCCTCAAGCCCTCACAGACTCTGTCTCTTACTTGCTCATTCAGCGGATTCAGCCTTTCCACCTCTGGTATGGGCGTGGGGTGGATTAGGCAACCTAGCGGAAAGGGGCTTGAATGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCTCACTGAAGTCCCAGCTCACTATTTCCAAAGATACTTCCCGGAATCAGGTGTTCCTCAAGATTACCTCTGTCGACACCGCTGATACCGCCACTTACTATTGTTCACGCAGACCGAGAGGTACCATGGACGCAATGGACTACTGGGGACAGGGCACCAGCGTGACCGTGTCATCTGGCGGTGGAGGGTCAGGAGGTGGAGGTAGCGGAGGCGGTGGGTCCGACATTGTCATGACCCAGGCCGCCAGCAGCCTGAGCGCTTCACTGGGCGACAGGGTGACCATCAGCTGTCGCGCATCACAAGATATCTCTAAGTATCTTAATTGGTACCAGCAAAAGCCGGATGGAACCGTGAAGCTGCTGATCTACTACACCTCACGGCTGCATTCTGGAGTGCCTAGCCGCTTTAGCGGATCTGGGTCCGGTACTGACTACAGCCTCACCATTAGAAACCTTGAACAGGAGGACATCGCAACTTATTTCTGCCAACAGGTCTATACTCTGCCGTGGACCTTCGGCGGAGGTACCAAACTGGAGATTAAGTCCGG SEQ ID NO: 46 is the amino acid sequence of the TSLPR-reactive scFv binding domain (LTG1789). MALPVTALLLPLALLLHAARPQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCSRRPRGTMDAMDYW GQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAASSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQQVYTLPWTFGGGTKLEIKS Sequence ID 47 is the nucleotide sequence of CAR LTG1789 (LP-3G11-CD8 TM-41BB-CD3 zeta). AGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Sequence ID 48 is the amino acid sequence of CAR LTG1789 (LP-3G11-CD8 TM-41BB-CD3 zeta). MALPVTALLLPLALLLHAARPQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCSRRP RGTMDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAASSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFC QQVYTLPWTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 49 is the nucleotide sequence of CAR LTG1563 (LP-CD19-TNFRSF19TM-41BB-CD3 zeta). Sequence ID 50 is the amino acid sequence of CAR LTG1563 (LP-CD19-TNFRSF19TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSAAAPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQPRRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 51 is the amino acid sequence of CAR LTG2228 (LP-CD20_CD19-CD8TM-CD28-CD3 zeta). ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCTGCCTTCCTGCTGATTCCCGAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAACAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGGTCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTGACCGTCAGCTCCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGACATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATGACTTGTAGAGCGTCGTCCAG Sequence ID 52 is the amino acid sequence of CAR LTG2228 (LP-CD20_CD19-CD8TM-CD28-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. Use of a population of human T cells for manufacturing a pharmaceutical composition for treating a subject having an autoimmune disease, wherein the population of human T cells is autologous to the subject, each cell in the population of human T cells comprises at least one multiple cistron vector, each of the at least one multiple cistron vector comprises a promoter operably ligated to a multiple cistron nucleic acid sequence encoding at least two functional CARs, the at least two functional CARs comprising non-identical amino acid sequences independently selected from the group consisting of the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 52, thereby treating a subject having an autoimmune disease.

2. The use according to claim 1, wherein a population of human T cells is injected directly into the subject.

3. The use according to claim 1, wherein the autoimmune disease is multiple sclerosis.

4. The use according to claim 1, wherein the autoimmune disease is a graft-versus-host disease.

5. The use according to claim 1, wherein the autoimmune disease is a skin allergy.

6. The use according to claim 1, wherein the autoimmune disease is a CD19+ autoimmune disease.

7. The use according to claim 1, wherein the population of human T cells is T regulatory cells.

8. The use according to claim 1, wherein the population of human T cells is helper T2 cells (Th2 cells).

9. The use according to claim 1, wherein the population of human T cells is T cells that promote a Th-2-like immune response.

Images