Bispecific chimeric antigen receptors and therapeutic uses thereof

专利摘要:
The invention is directed to a bispecific chimeric antigen receptor, comprising: (a) at least two antigen-specific targeting regions; (b) an extracellular spacer domain; (c) a transmembrane domain; (d) at least one co-stimulatory domain; and (e) an intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, and wherein the bispecific chimeric antigen receptor is co-expressed with a therapeutic control. The invention also provides methods and uses of the bispecific chimeric antigen receptors.
公开号:AU2013221672A1
申请号:U2013221672
申请日:2013-02-13
公开日:2014-08-07
发明作者:Michael Jensen
申请人:Seattle Childrens Hospital；
IPC主号:C07K16-46

专利说明:
WO 2013/123061 PCT/US2013/025953 BISPECIFIC CHIMERIC ANTIGEN RECEPTORS AND THERAPEUTIC USES THEREOF FIELD OF INVENTION 5 The invention relates to chimeric antigen receptors and to genetically engineered cells using the same. BACKGROUND OF THE INVENTION 10 Current immunotherapies are designed to target single antigens on cancer cells. However, for example, cancer cells are unstable and some cells may no longer possess the target antigen. These cells, referred to as antigen loss escape variants, escape destruction by the therapy and may continue to grow and spread unchecked. Therefore there is a 15 need in the art for therapies which prevent or minimize therapeutic failures in cancer and other diseases. SUMMARY OF THE INVENTION 20 In an embodiment, the invention provides a bispecific chimeric antigen receptor, comprising (a) at least two antigen-specific targeting regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one co-stimulatory domain and (e) an intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, and 25 wherein the bispecific chimeric antigen receptor is co-expressed with a therapeutic control. In an embodiment, the invention further provides a combination of a bispecific chimeric antigen receptor and a therapeutic control, wherein the bispecific chimeric antigen 30 receptor comprises (a) at least two antigen-specific targeting regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one co-stimulatory domain and (e) an intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen. 1 WO 2013/123061 PCT/US2013/025953 In an embodiment, the invention further provides a bispecific chimeric antigen receptor, comprising (a) at least two antigen-specific targeting regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one co-stimulatory domain and (e) an intracellular signaling domain, wherein each antigen-specific targeting region comprises 5 an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, and wherein the bispecific chimeric antigen receptor is co-expressed with truncated epidermal growth factor receptor (EGFRt). In an embodiment, the invention further provides a bispecific chimeric antigen receptor, 10 comprising (a) at least two antigen-specific targeting regions, (b) a CD8ahinge extracellular spacer domain, (c) a CD8a transmembrane domain, (d) a 4-1BB co stimulatory domain and (vi) a CD3 zeta intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, wherein the bispecific chimeric antigen receptor 15 is co-expressed with EGFRt and wherein the bispecific chimeric antigen receptor and EGFRt are linked via a T2A linker. In an embodiment, also provided are pharmaceutical compositions comprising the above described bispecific chimeric antigen receptors, a combination of the bispecific chimeric 20 antigen receptors and therapeutic controls, polypeptides encoding the bispecific chimeric antigen receptors, vectors, viruses and genetically engineered cells comprising the bispecific chimeric antigen receptors, vectors, viruses and genetically engineered cells comprising a combination of the bispecific chimeric antigen receptors and therapeutic controls, or combinations thereof, and a pharmaceutically acceptable carrier. 25 BRIEF DESCRIPTION OF FIGURES Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than 30 restrictive. Figure 1 depicts a schematic representation of a chimeric antigen receptor of the invention, in accordance with an embodiment of the present invention. ASTR is an 2 WO 2013/123061 PCT/US2013/025953 antigen-specific targeting region, L is a linker, ESD is an extracellular spacer domain, TM is a transmembrane domain, CSD is a co-stimulatory domain, and ISD is an intracellular signaling domain, 5 Figure 2 depicts (a) the components of an anti-CD19xCD20 CAR, and (b) a complete cDNA packaged into an epHIV-7 lentivirus vector transfer plasmid, in accordance with an embodiment of the present invention. Figure 3 depicts, in accordance with an embodiment of the present invention, the nucleic 10 acid sequence of a bispecific CAR CD19scFv-Gly4Serllinker-CD20scFv-IgG4Hinge CD28tm-41BB-CD3zeta-T2A-EGFRt epHIV7. Figure 4 depicts, in accordance with an embodiment of the present invention, the nucleic acid and amino acid sequences of a bispecific CAR CD19scFv-Gly4Serl linker 15 CD20scFv-IgG4Hinge-CD28tm-41BB-CD3zeta-T2A-EGFRt epHIV7. Figure 5 depicts, in accordance with an embodiment of the present invention, a CD 1 9scFv-Gly4Serl linker-CD20scFv-IgG4hinge-CD28tm-CD28gg-CD3Zeta transgene construct. 20 Figure 6 depicts, in accordance with an embodiment of the present invention, development of a CyCR platform to support exogenous yc independent growth. (a) Schematic diagrams of wild type versus chimeric cytokine receptors. The IL-7Ra constitutive cytokine receptor (CyCR7) consists of the human IL-7 cytokine tethered to 25 the full length human IL-7Ra chain via a (G 4
S)
2 linker. The IL-2RP constitutive cytokine receptor (CyCR2) is identical to CyCR7 except that the IL-7Ra intracellular signaling domain is replaced with the human IL-2/IL-15RP cytoplasmic domain. (b) Diagram of the expression construct CyCR-T2A-CD 19t. 30 Figure 7 depicts, in accordance with an embodiment of the present invention, the nucleic acid and amino acid sequences of an embodiment of the invention, namely a backbone CAR comprising the hinge region of IgG4, the transmembrane domain of CD28, the costimulatory domain of 4-1BB and the cytoplasmic domain of CD3zeta. 3 WO 2013/123061 PCT/US2013/025953 Figure 8 depicts, in accordance with an embodiment of the present invention, the nucleic acid sequence of an embodiment of the invention, namely GMCSFRss-CD19scFv Gly4Serlinker-CD20scFv-huIgGHinge/CH2/CH3 -CD28tm/CD28cyto-4 1 BB-CD3zeta. GMCSFRss is the signal sequence from GMCSFR. 5 Figure 9 depicts, in accordance with an embodiment of the present invention, the nucleic acid and amino acid sequences of an embodiment of the invention, namely GMCSFRss CD19scFv-Gly4Serlinker-CD20scFv-huIgGHinge/CH2/CH3-CD28tm/CD28cyto-41BB CD3zeta. GMCSFRss is the signal sequence from GMCSFR. 10 Figure 10 depicts, in accordance with an embodiment of the present invention, the nucleic acid sequence of an embodiment of the invention, namely the GMCSFRss-CD19scFv Gly4Serlinker-CD20scFv-CD8aHinge-CD8atm-41BB-CD3zeta-T2A-EGFRt. GMCSFRss is the signal sequence from GMCSFR. 15 Figure 11 depicts, in accordance with an embodiment of the present invention, the nucleic acid and amino acid sequences of an embodiment of the invention, namely GMCSFRss CD19scFv-Gly4Serlinker-CD20scFv-CD8aHinge-CD8atm-41BB-CD3zeta-T2A EGFRt. GMCSFRss is the signal sequence from GMCSFR. 20 Figure 12 depicts, in accordance with an embodiment of the present invention, the nucleic acid sequence of an embodiment of an invention namely T2A-EGFRt. Figure 13 depicts, in accordance with an embodiment of the present invention, the nucleic 25 acid and amino acid sequences of an embodiment of the invention, namely T2A-EGFRt. 4 WO 2013/123061 PCT/US2013/025953 DETAILED DESCRIPTION OF THE INVENTION All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the 5 same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5 th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold 10 Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. 15 Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below. The invention described herein provides chimeric antigen receptors. Chimeric antigen receptors are engineered receptors which graft an immune specificity onto a genetically 20 engineered cell. By housing specificities to multiple antigens in a single chimeric antigen receptor (CAR), various benefits may be achieved, including, among others, a significant reduction in effort as compared to making multiple T-cell products per patient. Definitions 25 Components of the Chimeric Antigen Receptors "Antigen-specific targeting region" (ASTR) as used herein refers to the region of the CAR which targets specific antigens. The CARs of the invention comprise at least two 30 targeting regions which target at least two different antigens. In an embodiment, CARs comprise three or more targeting regions which target at least three or more different antigens. The targeting regions on the CAR are extracellular. In some embodiments, the antigen-specific targeting regions comprise an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof and each of the targeting regions target a 5 WO 2013/123061 PCT/US2013/025953 different antigen. The targeting regions may comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen. There are, however, numerous alternatives, such as linked cytokines (which leads to recognition of cells bearing the 5 cytokine receptor), affibodies, ligand binding domains from naturally occurring receptors, soluble protein/peptide ligand for a receptor (for example on a tumor cell), peptides, and vaccines to prompt an immune response, which may each be used in various embodiments of the invention. In fact, almost any molecule that binds a given antigen with high affinity can be used as an antigen-specific targeting region, as will be 10 appreciated by those of skill in the art. "Chimeric antigen receptor" or "CAR" or "CARs" as used herein refers to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naYve T cells, central memory T cells, effector memory T cells or combination thereof). CARs 15 are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. The CARs of the invention comprise at least two antigen-specific targeting regions, an extracellular domain, a transmembrane domain, one or more co stimulatory domains, and an intracellular signaling domain. The two or more antigen specific targeting regions target at least two different antigens and may be arranged in 20 tandem and separated by linker sequences. In an embodiment, the extracellular spacer domain is optional. In another embodiment, the CAR is a bispecific CAR. A bispecific CAR is specific to two different antigens. "Co-stimulatory domain" (CSD) as used herein refers to the portion of the CAR which 25 enhances the proliferation, survival and/or development of memory cells. The CARs of the invention may comprise one or more co-stimulatory domains. Each co-stimulatory domain comprises the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD1la/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, 30 CD40 or combinations thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. 6 WO 2013/123061 PCT/US2013/025953 "Extracellular spacer domain" (ESD) as used herein refers to the hydrophilic region which is between the antigen-specific targeting region and the transmembrane domain. In some embodiments, the CARs of the invention comprise an extracellular spacer domain. In other embodiments, the CARs of the invention do not comprise an extracellular spacer 5 domain. The extracellular spacer domains include but are not limited to Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences or combinations thereof. Examples of extracellular spacer domains include but are not limited to CD8a hinge, and artificial spacers made of polypeptides 10 which may be as small as, for example, Gly3 or CH 1 and CH3 domains of IgGs (such as human IgG4). In some embodiments, the extracellular spacer domain is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgGI, (vi) a hinge region of IgGI or (vi) a hinge and CH2 region of IgGI. Other extracellular 15 spacer domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. "Intracellular signaling domain" (ISD) or "cytoplasmic domain" as used herein refer to the portion of the CAR which transduces the effector function signal and directs the cell 20 to perform its specialized function. Examples of domains that transduce the effector function signal include but are not limited to the Q chain of the T-cell receptor complex or any of its homologs (e.g., q chain, FcRly and P chains, MB1 (Iga) chain, B29 (Ig) chain, etc.), human CD3 zeta chain, CD3 polypeptides (A, 6 and 8), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other 25 molecules involved in T-cell transduction, such as CD2, CD5 and CD28. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. "Linker" (L) or "linker domain" or "linker region" as used herein refer to an oligo- or 30 polypeptide region from about 1 to 100 amino acids in length, which links together any of the domains/regions of the CAR of the invention. Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that 7 WO 2013/123061 PCT/US2013/025953 two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-like linker, 5 CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. In other embodiments, the linker sequences may comprise Asp-Val/Ile-Glu-X-Asn-Pro-Gly(2A>-Pro(2B> motif, which results in cleavage between the 2A glycine and the 2B proline. Other linkers will be apparent to those of skill in the art and may be used in connection with alternate 10 embodiments of the invention. "Transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I 15 transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Others 20 "Antigen loss escape variants" as used herein refer to cells which exhibit reduced or loss of expression of the target antigen, which antigens are targeted by the CARs of the invention. 25 "B-cell associated diseases" as used herein include B-cell immunodeficiencies, autoimmune diseases and/or excessive/uncontrolled cell proliferation associated with B cells (including lymphomas and/or leukemias). Examples of such diseases, wherein bispecific CARs of the invention may be used for therapeutic approaches include but are not limited to systemic lupus erythematosus (SLE), diabetes, rheumatoid arthritis (RA), 30 reactive arthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X linked agammaglobulinaemis, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus, common variable immunodeficiency, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and/or IgG subclass deficiency, B 8 WO 2013/123061 PCT/US2013/025953 lineage lymphomas (Hodgkin's lymphoma and/or non-Hodgkin's lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinaemia and/or hyper IgM syndrome, as well as virally-mediated B-cell diseases such as EBV mediated lymphoproliferative disease, and chronic infections in which B-cells participate in the 5 pathophysiology. "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the 10 chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, 15 but are not limited to B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkins lymphomas), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to 20 androgen-dependent prostate cancer and androgen-independent prostate cancer. "Co-express" as used herein refers to simultaneous expression of two or more genes. Genes may be nucleic acids encoding, for example, a single protein or a chimeric protein as a single polypeptide chain. For example, the CARs of the invention may be co 25 expressed with a therapeutic control (for example truncated epidermal growth factor (EGFRt)), wherein the CAR is encoded by a first polynucleotide chain and the therapeutic control is encoded by a second polynucleotide chain. In an embodiment, the first and second polynucleotide chains are linked by a nucleic acid sequence that encodes a cleavable linker. The polynucleotides encoding the CAR and the therapeutic control 30 system may be linked by IRES sequences. Alternately, the CAR and the therapeutic control are encoded by two different polynucleotides that are not linked via a linker but are instead encoded by, for example, two different vectors. Further, the CARs of the invention may be co-expressed with a therapeutic control and CCR, a therapeutic control and DHFR (for example mutant DHFR) or a therapeutic control and CCR and DHFR (for 9 WO 2013/123061 PCT/US2013/025953 example mutant DHFR). The CAR, therapeutic control and CCR may be co-expressed and encoded by first, second and third polynucleotide sequences, respectively, wherein the first, second and third polynucleotide sequences are linked via IRES sequences or sequences encoding cleavable linkers. Alternately, these sequences are not linked via 5 linkers but instead are encoded via, for example, separate vectors. The CAR, therapeutic control and DHFR (for example mutant DHFR) may be co-expressed and encoded by first, second and fourth polynucleotide sequences, respectively, wherein the first, second and fourth polynucleotide sequences are linked via IRES sequences or via sequences encoding cleavable linkers. Alternately, these sequences are not linked via linkers but 10 instead encoded via, for example, separate vectors. The CAR, therapeutic control, CCR and DHFR (for example mutant DHFR) may be co-expressed and encoded by first, second, third and fourth polynucleotide sequences, respectively, wherein the first, second, third and fourth polynucleotide sequences are linked via IRES sequences or sequences encoding cleavable linkers. Alternately, these sequences are not linked via linkers but 15 instead are encoded via, for example, separate vectors. If the aforementioned sequences are encoded by separate vectors, these vectors may be simultaneously or sequentially transfected. "Conditions", "disease conditions," "diseases" and "disease state" as used herein include 20 physiological states in which diseased cells may be targeted with the CARs of the invention, expressing, for example, antibodies against specific antigens on the diseased cells. Examples of antigens which may be targeted include but are not limited to antigens expressed on B-cells (such as CD19 and CD20), antigens expressed on carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on 25 various immune cells, and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, and/or inflammatory diseases. "Disease targeted by genetically modified cells" as used herein encompasses the targeting of any cell involved in any manner in any disease by the genetically modified cells of the 30 invention, irrespective of whether the genetically modified cells target diseased cells or healthy cells to effectuate a therapeutically beneficial result. The genetically modified cells include but are not limited to genetically modified T-cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells or embryonic stem cells. The genetically modified cells express the CARs of the invention, which CARs may target any of the 10 WO 2013/123061 PCT/US2013/025953 antigens expressed on the surface of target cells. Examples of antigens which may be targeted include but are not limited to antigens expressed on B-cells; antigens expressed on carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, and blastomas; antigens expressed on various immune cells; and antigens expressed on cells associated 5 with various hematologic diseases, autoimmune diseases, and/or inflammatory diseases. Other antigens that may be targeted will be apparent to those of skill in the art and may be targeted by the CARs of the invention in connection with alternate embodiments thereof. "Effector function" refers to the specialized function of a differentiated cell. Effector 10 function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. "Genetically modified cells", "redirected cells", "genetically engineered cells" or "modified cells" as used herein refer to cells that express the CAR of the invention. 15 "Immune cell" as used herein refers to the cells of the mammalian immune system including but not limited to antigen presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, 20 phagocytes, plasma cells and T-cells. "Immune response" as used herein refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and/or overactive immunity. 25 "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats 30 and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term. 11 WO 2013/123061 PCT/US2013/025953 "Polynucleotide" as used herein includes but is not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and/or tRNA. 5 "Naked DNA" as used herein refers to DNA encoding a CAR cloned in a suitable expression vector in proper orientation for expression. Viral vectors which may be used include but are not limited SIN lentiviral vectors, retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, hybrid vectors and/or plasmid transposons (for 10 example sleeping beauty transposon system) or integrase based vector systems. Other vectors that may be used in connection with alternate embodiments of the invention will be apparent to those of skill in the art. "Single chain variable fragment", "single-chain antibody variable fragments" or "scFv" 15 antibodies as used herein refer to forms of antibodies comprising the variable regions of only the heavy and light chains, connected by a linker peptide. "Target cell" as used herein refers to cells which are involved in a disease and can be targeted by the genetically modified cells of the invention (including but not limited to 20 genetically modified T-cells, NK cells, hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells). Other target cells will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. The terms "T-cell" and "T-lymphocyte" are interchangeable and used synonymously 25 herein. Examples include but are not limited to naYve T cells, central memory T cells, effector memory T cells or combinations thereof. "Therapeutic agents" as used herein refers to agents that are used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of 30 developing, slow the progression of and/or cure, a disease. Diseases targeted by the therapeutic agents include but are not limited to carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, and/or inflammatory diseases. 12 WO 2013/123061 PCT/US2013/025953 "Therapeutic controls" as used herein refers to agents that regulate cell proliferation, facilitate cell selection (for example selecting cells which express the chimeric antigen receptors of the invention), facilitate cell tracking or a combination thereof. In one 5 embodiment, regulating cell proliferation comprises up-regulating cell proliferation to promote cell propagation. In another embodiment, regulating cell proliferation comprises down-regulating cell proliferation so as to reduce or inhibit cell propagation. In some embodiments, the agents that serve as therapeutic controls may promote enrichment of cells which express the bispecific chimeric antigen receptors which may result in a 10 therapeutic advantage. "Transduction" as used herein refers to the introduction of a foreign nucleic acid into a cell using a viral vector. 15 "Transfection" as used herein refers to the introduction of a foreign nucleic acid into a cell using recombinant DNA technology. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, such as a protein or enzyme coded by the introduced gene or sequence. 20 The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" 25 and is a "transformant" or a "clone." The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species "Treatment" and "treating," as used herein refer to both therapeutic treatment and 30 prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those 13 WO 2013/123061 PCT/US2013/025953 already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented. "Tumor," as used herein refers to all neoplastic cell growth and proliferation, whether 5 malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Vector", "cloning vector" and "expression vector" as used herein refer to the vehicle by which a polynucleotide sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of 10 the introduced sequence. Vectors include plasmids, phages, viruses, etc. Description of the Invention Chimeric Antigen Receptors 15 While not wishing to be limited by any one premise, it is believed that the chimeric antigen receptors (for example bispecific CARs) of the instant invention may overcome conventional therapeutic failures due to, for example, outgrowth of antigen loss escape variants that can arise in the course of various therapies when a single antigen is targeted. 20 Accordingly, the invention is directed to, among other things, nucleic acid sequences and amino acid sequences encoding CARs, vectors comprising CARs, viruses comprising CARs, genetically modified cells comprising the CARs (redirected cells) and methods of making and using them. In some embodiments, the CARs are bispecific CARs. In other embodiments, the CARs target and bind three or more different antigens. 25 In general embodiments, the present invention relates to CARs (for example bispecific CARs), nucleic acid sequences encoding the CARs (for example bispecific CARs), the vectors comprising the nucleic acids encoding the CARs (for example bispecific CARs), viruses comprising the nucleic acid sequences encoding the CARs (for example bispecific 30 CARs), host cells (such as genetically modified cells) expressing the CARs (for example bispecific CARs), combinations of CARs (for example bispecific CARs) and therapeutic controls and methods of making and using the CARs (for example bispecific CARs) as therapeutic agents. 14 WO 2013/123061 PCT/US2013/025953 The CARs of the invention target at least two different antigens. The CARs (such as bispecific CARs) are co-expressed with a therapeutic control; for instance, truncated epidermal growth factor receptor (EGFRt), chimeric cytokine receptors (CCR) and/or dihydroxyfolate receptor (DHFR) (e.g., mutant DHFR). The polynucleotides encoding 5 the CAR and the therapeutic control(s) may be linked via IRES sequences or via polynucleotide sequences encoding cleavable linkers. The CARs of the invention are constructed so that they may be expressed in cells, which in turn proliferate in response to the presence of at least one molecule that interacts with at least one antigen-specific targeting region, for instance, an antigen. 10 In some embodiments, therapeutic controls for use with the CARs of the invention comprise any one or more of truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, 15 linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39) or AP1903/Fas system. In an embodiment, the CARs of the invention are linked to EGFRt via a cleavable linker or IRES sequences. In another 20 embodiment, a bispecific CAR is linked to EGFRt via a cleavable linker or IRES sequences. The CARs described herein may be synthesized as single polypeptide chains and may comprise at least two antigen-specific targeting regions, an extracellular spacer domain, a 25 transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain. In this embodiment, the antigen-specific targeting regions are at the N terminus, arranged in tandem and are separated by a linker peptide. The antigen-specific targeting region is linked to an extracellular spacer domain which is linked to the transmembrane domain. The transmembrane domain is linked to the co-stimulatory 30 domain. The co-stimulatory domain is linked to the intracellular signaling domain which is at the C-terminus. If more than one co-stimulatory domain is used, the multiple co stimulatory domains may be arranged in tandem with the transmembrane domain at its N terminus and the intracellular signaling domain at its C-terminus. Polynucleotides encoding these polypeptides may further comprise an N-terminal signal sequence which 15 WO 2013/123061 PCT/US2013/025953 directs the CAR to the cell surface as a type I transmembrane protein. The antigen specific targeting region may be extracellular-facing and the intracellular signaling domain may be cytoplasmic. 5 Figure 1 shows a schematic of a chimeric antigen receptor of the invention. In an embodiment, an extracellular spacer domain in the CAR is optional. In such a CAR, the antigen-specific targeting regions are at the N-terminus, arranged in tandem, and separated by a linker peptide. The antigen-specific targeting region may be linked to 10 the transmembrane domain. The transmembrane domain may be linked to the co stimulatory domain. The co-stimulatory domain may be linked to the intracellular signaling domain, which is at the C-terminus. If more than one co-stimulatory domain is used, the multiple co-stimulatory domains may be arranged in tandem with the transmembrane domain at its N-terminus and the intracellular signaling domain at its C 15 terminus. Polynucleotides encoding these polypeptides may further comprise an N terminal signal sequence which directs the CAR to the cell surface as a type I transmembrane protein. The antigen-specific targeting region may be extracellular-facing and the intracellular signaling domain may be cytoplasmic. 20 Antigen-Specific Targeting Regions of Chimeric Antigen Receptors The CARs of the invention may target several (such as two or more, three or more) different antigens. In an embodiment, the CAR is a bispecific CAR and targets two different antigens. As described above, the antigen-specific targeting regions of the CAR 25 may be arranged in tandem and may be separated by linker peptides. The antigens targeted by the CAR may be antigens on single diseased cell (such as a cancerous B-cell) or antigens that are expressed on separate cells that each contribute to the disease. The antigens targeted by the CAR are antigens which are either directly or indirectly involved in the disease. 30 In a bispecific CAR, at least two different antigen-specific antibodies or fragments thereof or derivatives thereof may be cloned into the antigen-specific targeting region. The antibodies may be specific for any, but at least two, distinct antigens of choice. The 16 WO 2013/123061 PCT/US2013/025953 antibody specific to the antigen may be the Fab fragment of the antibody or the single chain variable fragment (scFv) of the antibody. For example, Figure 2 shows an embodiment of the invention depicting a CAR specific to 5 CD19 and CD20. Using methods well known to one skilled in the art, scFvs specific to multiple, but at least two different antigens, may be cloned upstream (i.e., to N-terminus) of the IgG 4 -CD28-zeta domains so long as the target-antigens are expressed on cells that are targetable by the genetically modified cells described below. Such techniques are explained fully in the literature. (Sambrook et al, "Molecular Cloning: A Laboratory 10 Manual" (1989), Current Protocols in Molecular Biology. Volumes I-III [Ausubel, R. M., ed. (1994)], Cell Biology: A Laboratory Handbook. Volumes I-III [J. E. Celis, ed. (1994))], Current Protocols in Immunology. Volumes I-III [Coligan, J. E., ed. (1994)], Oligonucleotide Synthesis. (M. J. Gait ed. 1984), Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)], Transcription And Translation [B. D. Hames & S. J. 15 Higgins, eds. (1984)], Animal Cell Culture [R. I. Freshney, ed. (1986)], Immobilized Cells And Enzymes [IRL Press, (1986)], Practical Guide To Molecular Cloning B. Perbal (1984), Current Prptocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology as well as monographs in journals such as Advances in Immunology). 20 In one embodiment, each antigen-specific targeting region comprises the full-length IgG heavy chain (specific for the target antigen) having the VH, CHI, hinge, and the CH2 and CH3 (Fc) Ig domains, if the VH domain alone is sufficient to confer antigen-specificity ("single-domain antibodies"). The full length IgG heavy chain may be linked to the co 25 stimulatory domain and the intracellular signaling domain via the appropriate transmesmbrane domain. If both, the VH and the VL domains, are necessary to generate a fully active antigen-specific targeting region, the VH-containing CAR and the full-length lambda light chain (IgL) are both introduced into the cells to generate an active antigen specific targeting region. In an embodiment, an extracelluar spacer domain may be 30 linked between the antigen-specific binding domain and the transmembrane domain. The cells include but are not limited to T-lymphocytes (T-cells), natural killer cells, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells capable of giving rise to therapeutically relevant progeny. 17 WO 2013/123061 PCT/US2013/025953 In another embodiment, each antigen-specific targeting region of the CAR comprises at least two single chain antibody variable fragments (scFv), each specific for a different target antigen. scFvs, in which the C-terminus of one variable domain (VH or VL) is tethered to the N-terminus of the other (VL or VH, respectively) via a polypeptide linked, 5 have been developed without significantly disrupting antigen binding or specificity of the binding. (Chaudhary et al., A recombinant single-chain immunotoxin composed of anti Tac variable regions and a truncated diphtheria toxin. 1990 Proc. Natl. Acad. Sci., 87:9491; Bedzyk et al. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody. 1990 J. Biol. Chem., 265:18615). The linker 10 connects the N-terminus of the VH with the C-terminus of VL or the C-terminus of VH with the N-terminus of VL. These scFvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. The scFvs, specific for at least two different antigens, are arranged in tandem and linked to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In an embodiment, an 15 extracelluar spacer domain may be linked between the antigen-specific binding region and the transmembrane domain. In another aspect, each scFv fragment may be fused to all or a portion of the constant domains of the heavy chain. The resulting antigen-specific targeting region, specific for 20 at least two different antigens, is joined to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In an embodiment, an extracelluar spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain. 25 In a further embodiment, each antigen-specific targeting region of the CAR comprises a divalent (or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs). In CARs comprising di-scFVs, two scFvs specific for each antigen are linked together by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. (Xiong, Cheng-Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). 30 "Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding". Protein Engineering Design and Selection 19 (8): 359-367; Kufer, Peter; Lutterbiise, Ralf; Baeuerle, Patrick A. (2004). "A revival of bispecific antibodies". Trends in Biotechnology 22 (5): 238-244). CARs comprising at least two antigen-specific targeting regions would express two scFvs 18 WO 2013/123061 PCT/US2013/025953 specific for each of the two antigens. The resulting antigen-specific targeting region, specific for at least two different antigens, is joined to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In an embodiment, an extracelluar spacer domain may be linked between the antigen-specific binding domain 5 and the transmembrane domain. In an additional embodiment, each antigen-specific targeting region of the CAR comprises a diabody. In a diabody, the scFvs are created with linker peptides that are too short for the two variable regions to fold together, driving the scFvs to dimerize. Still 10 shorter linkers (one or two amino acids) lead to the formation of trimers, the so-called triabodies or tribodies. Tetrabodies may also be used. To create the CARs of the present invention, two or more individual antigen-specific targeting regions are connected to each other, either covalently or noncovalently, on a 15 single protein molecule. An oligo- or polypeptide linker, an Fc hinge or membrane hinge region may be used to connect these domains to each other. The CARs of the present invention may comprise two or more of the different antigen-specific targeting regions connected together in different combinations. For example, two or more antigen-specific targeting regions containing immunoglobulin sequences (e.g. scFvs and/or single-domain 20 antibodies) may be linked to each other. Targets of Antigen-specific targeting regions of chimeric antigen receptors In some embodiments, the antigen-specific targeting region of the CAR (for example 25 bispecific CAR) targets antigens specific for cancer, inflammatory disease, neuronal disorders, diabetes, cardiovascular disease, infectious diseases or a combination thereof. Examples of antigens which may be targeted by the CARs (for example bispecific CARs) of the invention include but are not limited to antigens expressed on B-cells, antigens expressed on carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, 30 antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, and/or inflammatory diseases. The CARs of the invention, which are specific for at least two different target antigens, may be capable of redirecting the effector function of the expressing-cells to either of both of the target antigens. This feature of the construct may overcome the issue of 19 WO 2013/123061 PCT/US2013/025953 antigen loss escape variants when targeting, for example, genetically unstable B-cell lineage malignancies using single antigen-specificity. Antigens specific for cancer which may be targeted by the CARs (for example bispecific 5 CARs) of the invention include but are not limited to any one or more of 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, 10 EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, Li-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin a531, integrin av33, MORAb-009, MS4A1, MUCi, mucin CanAg, N glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic 15 carcinoma cells, RANKL, RON, RORi, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-P, TRAIL-Ri, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin. Other antigens specific for cancer will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Examples of CARs which target the above antigens 20 include but are not limited to bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co expressed with EGFRt and DHFR (for example mutant DHFR) or bispecific CARs co expressed with EGFRt and CDR and DHFR (for example mutant DHFR). 25 In some embodiments, the bispecific chimeric antigen receptors target and bind at least two different antigens. Examples of pairings of at least two antigens bound by the bispecific CARs of the invention include but are not limited to CD19 and CD20, CD19 and CD22, CD20 and LI-CAM, LI-CAM and GD2, EGFR and LI-CAM, EGFR and C MET, EGFR and HER2, C-MET and HER2 and EGFR and ROR1. Other pairings of 30 antigens specific for cancer will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In yet other embodiments, the bispecific chimeric antigen receptor targets CD19 and CD20. Examples of CARs which target the above antigens include but are not limited to bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, 20 WO 2013/123061 PCT/US2013/025953 bispecific CARs co-expressed with EGFRt and DHFR (for example mutant DHFR) or bispecific CARs co-expressed with EGFRt and CDR and DHFR (for example mutant DHFR). 5 Antigens specific for inflammatory diseases which may be targeted by the CARs of the invention include but are not limited to any one or more of AOC3 (VAP-1), CAM-3001, CCL1 1 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-i, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 10 receptor, integrin a4, integrin a407, Lama glama, LFA-1 (CD1 1a), MEDI-528, myostatin, OX-40, rhuMAb 37, scleroscin, SOST, TGF beta 1, TNF-a or VEGF-A. Other antigens specific for inflammatory diseases will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Examples of CARs which target the above antigens include but are not limited to bispecific CARs, bispecific 15 CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (for example mutant DHFR) or bispecific CARs co-expressed with EGFRt and CDR and DHFR (for example mutant DHFR). 20 Antigens specific for neuronal disorders which may be targeted by the CARs of the invention include but are not limited to any one or more of beta amyloid or MABT5102A. Other antigens specific for neuronal disorders will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Examples of CARs which target the above antigens include but are not limited to bispecific CARs, 25 bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (for example mutant DHFR) or bispecific CARs co-expressed with EGFRt and CDR and DHFR (for example mutant DHFR). 30 Antigens specific for diabetes which may be targeted by the CARs of the invention include but are not limited to any one or more of L-1 or CD3. Other antigens specific for diabetes or other metabolic disorders will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Examples of 21 WO 2013/123061 PCT/US2013/025953 CARs which target the above antigens include but are not limited to bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (for example mutant DHFR) or bispecific CARs co-expressed with EGFRt and CDR and DHFR (for example 5 mutant DHFR). Antigens specific for cardiovascular diseases which may be targeted by the CARs of the invention include but are not limited to any one or more of C5, cardiac myosin, CD41 (integrin alpha-Ib), fibrin II, beta chain, ITGB2 (CD18) and sphingosine- 1-phosphate. 10 Other antigens specific for cardiovascular diseases will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Examples of CARs which target the above antigens include but are not limited to bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR 15 (for example mutant DHFR) or bispecific CARs co-expressed with EGFRt and CDR and DHFR (for example mutant DHFR). Antigens specific for infectious diseases which may be targeted by the CARs of the invention include but are not limited to any one or more of anthrax toxin, CCR5, CD4, 20 clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus and TNF-a. Other antigens specific for infectious diseases will be apparent to those of skill in the art and may be used in connection with alternate 25 embodiments of the invention. Examples of CARs which target the above antigens include but are not limited to bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co expressed with EGFRt and DHFR (for example mutant DHFR) or bispecific CARs co expressed with EGFRt and CDR and DHFR (for example mutant DHFR). 30 Further examples of target antigens include but are not limited to surface proteins found on cancer cells in a specific or amplified fashion (e.g. the IL-14 receptor, CD19, CD20 and CD40 for B-cell lymphoma, the Lewis Y and CEA antigens for a variety of carcinomas, the Tag72 antigen for breast and colorectal cancer, EGF-R for lung cancer, 22 WO 2013/123061 PCT/US2013/025953 folate binding protein and the HER-2 protein which is often amplified in human breast and ovarian carcinomas), or viral proteins (e.g. gp120 and gp41 envelope proteins of HIV, envelope proteins from the Hepatitis B and C viruses, the glycoprotein B and other envelope glycoproteins of human cytomegalovirus, the envelope proteins from 5 oncoviruses such as Kaposi's sarcoma-associated Herpes virus). Other potential targets of the CARs of the invention include CD4, where the ligand is the HIV gp120 envelope glycoprotein, and other viral receptors, for example ICAM, which is the receptor for the human rhinovirus, and the related receptor molecule for poliovirus. 10 Additional targets of the CARs of the invention include antigens involved in B-cell associated diseases. Yet further targets of the CARs of the invention will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. 15 Co-stimulatory domains of chimeric antigen receptors The CARs of the invention may also comprise a co-stimulatory domain. This domain may enhance cell proliferation, cell survival and development of memory cells. The CARs of the invention may comprise one or more co-stimulatory domains. Each co 20 stimulatory domain comprises the co-stimulatory domain of any one or more of, for example, members of the TNFR super family, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40 or combinations thereof. Co-stimulatory domains from other proteins may also be used with the CARs of the invention. Additional co-stimulatory domains will be apparent to 25 those of skill in the art and may be used in connection with alternate embodiments of the invention. If a CAR comprises more than one co-stimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. Extracellular spacer domain of chimeric antigen receptor 30 The CARs of the invention may further comprise an extracellular spacer domain. In some embodiments, this domain facilitates proper protein folding. The extracellular spacer domain comprises a hydrophilic region which is attached to the antigen-specific targeting region and the transmembrane domain. Extracellular spacer domains may 23 WO 2013/123061 PCT/US2013/025953 include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions antibodies, artificial spacer sequences or combinations thereof. Examples of extracellular spacer domains include but are not limited to CD8a hinge, 5 artificial spacers made of polypeptides such as Gly3, or CHI, CH3 domains of IgG's (such as human IgG4). Specifically, the extracellular spacer domain may be (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgGI, (vi) a hinge region of IgGI or (vi) a hinge and CH2 of IgGI or a combination thereof. Additional 10 extracellular spacer domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Transmembrane domain of chimeric antigen receptors 15 The CARs of the invention may also comprise a transmembrane domain. The transmembrane domain may comprise the transmembrane sequence from any protein which has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR of the invention may also comprise an artificial hydrophobic sequence. The transmembrane domains of the 20 CARs of the invention may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. Intracellular signaling domain of chimeric antigen receptors 25 The CARs of the invention may also comprise an intracellular signaling domain. This domain may be cytoplasmic and may transduce the effector function signal and direct the cell to perform its specialized function. Examples of intracellular signaling domains include, but are not limited to, ; chain of the T-cell receptor or any of its homologs (e.g., 30 q chain, FcRly and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc.), CD3 polypeptides (A, 6 and 8), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain may be human CD3 zeta 24 WO 2013/123061 PCT/US2013/025953 chain, FeyRIII, FecRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. 5 Linkers in chimeric antigen receptors In some embodiments, two or more components of the CARs of the invention are separated by one or more linkers. For example, in CARs comprising at least two antigen 10 specific targeting regions, the first targeting region on the CAR may be separated from the second targeting region on the CAR via a linker. Additionally, the CAR may be linked to therapeutic controls via a linker. Linkers are oligo- or polypeptides region from about 1 to 100 amino acids in length, that link together any of the domains/regions of the CAR of the invention. In some embodiments, the linkers may be for example, 5-12 15 amino acids in length, 5-15 amino acids in length or 5 to 20 amino acids in length. Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers, for example those longer than 100 amino acids, may be used in connection with alternate embodiments of the invention, and may be selected to, for example, ensure that two 20 adjacent domains do not sterically interfere with one another. Examples of linkers which may be used in the instant invention include but are not limited to 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof. Therapeutic controls 25 Therapeutic controls regulate cell proliferation, facilitate cell selection (for example selecting cells which express the chimeric antigen receptors of the invention) or a combination thereof. In one embodiment, regulating cell proliferation comprises up regulating cell proliferation to promote cell propagation. In another embodiment, 30 regulating cell proliferation comprises down-regulating cell proliferation so as to reduce or inhibit cell propagation. In some embodiments, the agents that serve as therapeutic controls may promote enrichment of cells which express the bispecific chimeric antigen receptors which may result in a therapeutic advantage. In some embodiments, agents 25 WO 2013/123061 PCT/US2013/025953 which serve as therapeutic controls may biochemically interact with additional compositions so as to regulate the functioning of the therapeutic controls. For example, EGFRt (a therapeutic control) may biochemically interact with cetuximab so as to regulate the function of EGFRt in selection, tracking, cell ablation or a combination 5 thereof. Examples of therapeutic controls include but are not limited to any one or more of truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 10 8, human caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), a selection marker, and combinations 15 thereof. In some embodiments, the therapeutic controls are co-expressed with the bispecific chimeric antigen receptor. Examples of agents which regulate the functioning of the therapeutic controls include but are not limited to any one or more of Herceptin, methotrexate, cetuximab, thymidine 20 analogs (for example ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5 flurocytosine (5-FC), 5-(azaridin-1-yl)-2, 4-dinitrobenzamide (CB1954), 6-thioguanine, a synthetic dimerizing drug (for example AP1903), fludarabine phosphate, linamarin (lin), nucleoside analogs (for exmaple BVDU, difluorodeoxycytidine (dFdC), 1-P-D arabinofuranosylthymine (ara-T)), indole-3-acetic (IAA), 1-buthionine-S,R-sulfoximine 25 (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors or combinations thereof. These agents may be administered before, during or after the use of the therapeutic controls. As described above, the CARs of the invention may be synthesized as single polypeptide 30 chains. If the CAR is a bispecific CAR, the polynucleotide sequence encoding the CAR may be, for example, in the following configuration in the N-terminal to C-terminal direction: N-terminal signal sequence - antigen-specific targeting region 1 - linker antigen-specific targeting region 2 - extracellular spacer domain - transmembrane 26 WO 2013/123061 PCT/US2013/025953 domain - co-stimulatory domain - intracellular signaling domain. In an embodiment, such a CAR may comprise two or more co-stimulatory domains. Alternatively, the polynucleotide sequence encoding the CAR may be in the following 5 configuration in the N-terminal to C-terminal direction: N-terminal signal sequence antigen-specific targeting region 1 - linker - antigen-specific targeting region 2 transmembrane domain - co-stimulatory domain - intracellular signaling domain. In an embodiment, such a CAR may comprise two or more co-stimulatory domains. 10 If a CAR comprises more than two antigen-specific targeting regions, the polynucleotide sequence encoding the CAR may be in the following configuration in the N-terminal to C-terminal direction: N-terminal signal sequence - antigen-specific targeting region 1 linker - antigen-specific targeting region 2 - linker - (antigen-specific targeting region) transmembrane domain - co-stimulatory domain - intracellular signaling domain. Such a 15 CAR may further comprise an extracellular spacer domain. Each antigen-specific targeting region may be separated by a linker. In an embodiment, such a CAR may comprise two or more co-stimulatory domains. The invention provides a nucleic acid sequence of the backbone of an exemplary CAR of 20 the invention comprising an extracellular spacer domain, a transmembrane domain, a co stimulatory domain and an intracellular signaling domain. Specifically, an exemplary backbone for a may CAR comprise, in the N-terminus to C-terminus orientation, IgG4hinge-CD28tm-41BB-CD3zeta, wherein the extracellular spacer domain is the IgG4 hinge region, the transmembrane domain is the transmembrane region from CD28, the co 25 stimulatory domain is from 4-1BB and the intracellular signaling domain is from the CD3 zeta chain (Figure 7). At least two or more antigen-specific targeting regions may be inserted N-terminal to the IgG4 hinge. The invention provides nucleic acid sequences of an exemplary embodiment of the 30 invention where the CAR is specific to CD19 and CD20. In one embodiment, the sequence encoding a bispecific anti-CD 19xCD20 CAR is set forth in Figure 3, 8 or 10. In another embodiment, the sequence encoding a bispecific anti-CD19xCD20 CAR is set forth in Figure 4, 9 or 11. In this exemplary embodiment, the bispecific CAR comprises 27 WO 2013/123061 PCT/US2013/025953 scFvs specific for CD19 and CD20 with each scFv separated by a linker, joined to an extracellular spacer domain, which is joined to the co-stimulatory and intracellular signaling domains via a transmembrane domain. Although the exemplary CAR depicts a set of scFv sequences, any scFv specific for CD19 and CD20 may be used. In a particular 5 embodiment, the bispecific CAR specific for CD19 and CD20 is CD19scFv Gly4Serlinker-CD20scFv-IgG4-Hinge-CD28tm-41BB(cyto)-zeta(cyto) and is encoded by the sequences set forth in Figures 3 and 4. This bispecific CAR comprises single chain Fv fragments specific for CD19 and CD20 linked by a Gly4Ser linker, an IgG4 hinge extracellular spacer domain, a CD28 transmembrane domain, a 41BB costimulatory 10 domain and the cytoplasmic domain from CD3 zeta chain. In another embodiment, the bispecific CAR specific for CD19 and CD20 comprises CD19scFv-Gly4serlinker-CD20scFv-hulgG4-hingeCH2CH3 -CD28tm/cyto-4 1 BB-zeta (Figures 9-10). This bispecific CAR comprises single chain Fv fragments specific for 15 CD19 and CD20 linked by a Gly4Ser linker, a human IgG4 hinge, CH2 and CH3 extracellular spacer domain, a CD28 transmembrane domain, a 4-1BB costimulatory domain and the cytoplasmic domain from CD3 zeta chain. In a further embodiment, the bispecific CAR specific for CD19 and CD20 is CD19 20 Gly4serlinker-CD20scFv-CD8ahinge-CD8aTM-41BBcostim-zetacyto (Figures 11-12). This bispecific CAR comprises single chain Fv fragments specific for CD19 and CD20 linked by a Gly4Ser linker, a CD8alpha hinge extracellular spacer domain, a CD8alpha transmembrane domain, a 41BB costimulatory domain and the cytoplasmic domain from CD3 zeta chain. 25 Truncated epidermal growth factor receptor (EGFRt) Human epidermal growth factor receptor (huEGFR)(EGFR; ErbB-1, HERI in humans) is a receptor tyrosine kinase of the ErbB family of growth factor receptors that is not 30 expressed by cells of the hematopoietic and lymphopoietic systems. Ligand (EGF, TGF a) binding occurs within N-terminal extracellular domains I and II of EGFR resulting from transition of receptor tyrosine kinase inactive monomers to active homodimers. 28 WO 2013/123061 PCT/US2013/025953 Extracellular domain III of EGFR contains the binding sites of antibodies (for example cetuximab (Erbitux), an IgGI chimeric antibody). It is believed that human EGFR may be rendered incapable of binding ligands (EGF, TGF-a) by removal of domains I and II, and devoid of signaling activity by deletion of its cytoplasmic tail, while retaining an 5 intact antibody binding site (for example cetuximab binding site), for example in extracellular domain III, IV or a combination thereof (Wang et al., A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells Blood 118(5)1255-1263). 10 A truncated EGFRt polypeptide described herein has at least three uses for genetic engineering of cell-based therapies: ex vivo cell purification, in vivo cell tracking, and cell ablation. In an embodiment, EGFRt, for use as a therapeutic control with the CARs of the invention, binds any one or more of EGFR-specific siRNA, a small molecule that targets EGFR, an anti-EGFR-antibody or a combination thereof. In another embodiment, 15 EGFRt comprises the sequence set forth in Figures 12 or 13 or sequences that are about 70%, about 75%, about 80%, about 85%, about 90% or about 95% homologous to the sequences set forth in Figures 12 or 13. In an embodiment of the invention, huEGFRt may be co-expressed with the CARs of the 20 invention so as to purify cells expressing the CARs (for example ex vivo cell purification), track cells (for example in vitro or in vivo cell tracking) expressing the CARs or regulate cells (for example in vivo or in vitro or ex vivo) expressing the CARs by triggering cell ablation as required. In one embodiment, the CARs are bispecific CARs. 25 Chimeric cytokine receptor (CCR) Based on the limitations of using exogenous yc cytokines in adoptive immunotherapy, the invention provides T cells with an intrinsic yc cytokine signaling mechanism. The utility 30 of forced constitutive chimeric cytokine receptors IL-2/IL-15RO (CyCR2) and IL-7Ra (CyCR7) receptor signals were compared. As described below, the chimeric cytokine receptors have the ability to improve the survival, persistence, and in vivo engraftment of cytotoxic T cells (CTLs). 29 WO 2013/123061 PCT/US2013/025953 Accordingly, in an embodiment of the invention, the CARs of the invention may be co expressed with CCR. For example, a bispecific CAR may be co-expressed with EGFRt and CCR. Alternately, a bispecific CAR may be co-expressed with CCR. Examples of chimeric cytokine receptor include but are not limited to IL-7 cytokine-linker- IL7Ra, IL 5 7 cytokine-linker-extracellular domain of IL-7Ra-transmembrane domain of IL-7Ra cytoplasmic domain of IL-2R, IL-7 cytokine-linker-IL2R. A CCR comprising IL-7 cytokine-linker- IL7Ra comprises an N-terminal signal sequence joined to the N-terminus of the IL-7 cytokine which is linked via a linker to 10 extracellular, transmembrane and cytoplasmic domains of IL-7Ra (the alpha chain of the IL-7 receptor). A CCR comprising IL-7 cytokine-linker-extracellular domain of IL-7Ra-transmembrane domain of IL-7Ra-cytoplasmic domain of IL-2R comprises an N-terminal signal 15 sequence joined to the N-terminus of the IL-7 cytokine which is linked via a linker to the extracellular domain and transmembrane domain of IL-7Ra and to the cytoplasmic domain of IL-2R (the beta chain of the IL-2 receptor). A CCR comprising IL-7 cytokine-linker-IL2R comprises N-terminal signal sequence 20 joined to the N-terminus of the IL-7 cytokine which is linked via a linker to extracellular, transmembrane and cytoplasmic domains of IL-2R. Dihydroxyfolate Receptor (DHFR) 25 Genetic modification of T cells to co-express a therapeutic transgene and a drug resistant transgene that confers resistance to lymphotoxic drugs provides the opportunity to select for therapeutic cells both in vivo and ex vivo. A mutated human enzyme transgene, dihydrofolate reductase double mutant (DHFRFS; L22F, F3 1S), which confers resistance of engineered T cells to methotrexate (MTX), allowing selection of cells co-expressing a 30 CD19-specific chimeric antigen receptor (CD19CAR) that specifically targets B-lineage tumor cells. 30 WO 2013/123061 PCT/US2013/025953 In an embodiment, the CARs of the invention (for example bispecific CARs) may be co expressed with DHFR (for example mutant DHFR). In a further embodiment, the bispecific CAR may be co-expressed with EGFRt, CCR and DHFR (including mutant DHFR). Alternately, the bispecific CAR may be co-expressed with EGFRt and DHFR 5 (including mutant DHFR). Other selection markers that may be used with the CARs of the invention include but are not limited to methylated-DNA-protein-cysteine methyltransferase (MDMT), inosine monophosphate dehydrogenase II (IMDHP2) or a combination thereof. MDMT makes 10 cells resistant to chemotherapy and therefore may be used if synergy between chemotherapy and T cell therapy is desired. Vectors encoding the CARs of the invention are also provided herein. Vectors encoding CARs also encode EGFRt. In some embodiments, vectors encoding CARs and EGFRt 15 also encode CCR or DHFR (for example mutant DHFR). In other embodiments, vectors encoding CARs and EGFRt also encode CCD and DHFR (for example mutant DHFR). In some specific embodiments, the vectors may encode a bispecific CAR and EGFRt, a bispecific CAR and EGFRt and CCR, a bispecific CAR and EGFRt and DHFR (for example mutant DHFR) or a bispecific CAR and EGFRt and CCR and DHFR (for 20 example mutant DHFR). Vectors which may be used to express the CARs of the invention include but are not limited to lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, AAV vectors, adeno virus vectors, engineered hybrid viruses, naked DNA (including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggybak, and Integrases such as Phi3 1. 25 In an exemplary embodiment of the invention, the bisepcific CAR specific to CD19 and CD20 disclosed herein is expressed via a lentiviral vector as illustrated in Figure 5. Genetically engineered cells of the invention 30 The invention also provides genetically engineered cells which comprise and stably express the CAR of the invention. The CAR expressed by the genetically engineered cell may comprise at least two antigen-specific targeting regions, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular 31 WO 2013/123061 PCT/US2013/025953 signaling domain. The polynucleotide sequence encoding the CAR may also comprise an N-terminal signal sequence. In an embodiment, the CAR is a bispecific CAR. Each of the at least two antigen-specific targeting regions, extracellular spacer domain, transmembrane domain, one or more co-stimulatory domains and an intracellular 5 signaling domain are described above. The antigen-specific targeting domains may be capable of specifically binding, in an MHC unrestricted manner, an antigen which is not normally bound by a T-cell receptor in that manner. In an embodiment, the genetically engineered cells that express the CARs (for example 10 bispecific CARs) of the invention co-express EGFRt. In a further embodiment, the genetically engineered cells that express the CARs (for example bispecific CARs) co express EGFRt and CCR. In an additional embodiment, the genetically engineered cells that express the CARs (for example bispecific CARs) co-express EGFRt and DHFR (for example mutant DHFR). In another embodiment, the genetically engineered cells that 15 express the CARs (for example bispecific CARs) co-express EGFRt, CCR and DHFR (for example mutant DHFR). The genetically engineered cells express a CAR having at least two antigen-specific targeting regions which are specific for at least two different target antigens. In one 20 embodiment, the antigen-specific targeting regions comprise target-specific antibodies or functional equivalents or fragments or derivatives thereof. The antigen-specific antibody may be the Fab fragment of the antibody or the single chain variable fragment (scFv) of the antibody. 25 Genetically engineered cells which may comprise and express the CARs of the invention include, but are not limited to, T-lymphocytes (T-cells), naYve T cells (TN), memory T cells (for example, central memory T cells (TcM), effector memory cells (TEM)), natural killer cells, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells capable of giving rise to therapeutically relevant progeny. In an embodiment, the 30 genetically engineered cells are autologous cells. By way of example, individual T-cells of the invention may be CD4+/CD8-, CD4-/CD8+, CD4-/CD8- or CD4+/CD8+. The T cells may be a mixed population of CD4+/CD8- and CD4-/CD8+ cells or a population of a single clone. CD4+ T-cells of the invention may produce IL-2, IFNy, TNFa and other 32 WO 2013/123061 PCT/US2013/025953 T-cell effector cytokines when co-cultured in vitro with cells expressing the target antigens (for example CD20+ and/or CD19+ tumor cells). CD8' T-cells of the invention may lyse antigen-specific target cells when co-cultured in vitro with the target cells. In some embodiments, T cells may be any one or more of CD45RA7 CD62L naYve cells, 5 CD45RO* CD62L central memory cells, CD62L- effector memory cells or a combination thereof (Berger et al., Adoptive transfer of virus-specific and tumor-specific T cell immunity. Curr Opin Immunol 2009 21(2)224-232). Genetically modified cells may be produced by stably transfecting cells with DNA 10 encoding the CAR of the invention. DNA encoding the CAR of the invention (for example bispecific CAR) may also encode EGFRt, CCR and/or DHFR (for example mutant DHFR). In one embodiment, a first polynucleotide encodes the CAR (for example bispecific CAR) and is linked via IRES sequences or a polynucleotide that encodes a cleavable linker, to a second polynucleotide that encodes EGFRt. In another 15 embodiment, the first polynucleotide encodes the CAR (for example bispecific CAR) and is linked via IRES sequences or a polynucleotide that encodes a cleavable linker, to a second polynucleotide that encodes EGFRt and the first or second polynucleotides are linked to a third polynucleotide that encodes CCR or DHFR (for example mutant DHFR), also via IRES sequences or a polynucleotide that encodes a cleavable linker. In a further 20 embodiment, the first polynucleotide encodes the CAR (for example bispecific CAR) and is linked via IRES sequences or a polynucleotide that encodes a cleavable linker, to a second polynucleotide that encodes EGFRt and the first and second polynucleotides are linked to a third polynucleotide that encodes CCR and a fourth polynucleotide that encodes DHFR (for example mutant DHFR) via IRES sequences or a polynucleotide that 25 encodes a cleavable linker. Viral vectors are commonly used to carry heterologous genes into cells (e.g., T-cells). Examples of viral vectors which may be used to generate genetically modified cells include but are not limited to SIN lentiviral vectors, retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors and/or plasmid transposons (e.g., sleeping beauty transposon system). 30 Various methods produce stable transfectants which express the CARs of the invention. In one embodiment, a method of stably transfecting and re-directing cells is by electroporation using naked DNA. By using naked DNA, the time required to produce redirected cells may be significantly reduced. Additional methods to genetically engineer 33 WO 2013/123061 PCT/US2013/025953 cells using naked DNA encoding the CAR of the invention include but are not limited to chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or 5 particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). The transfected cells demonstrating presence of a single integrated un-rearranged vector and expression of the CAR may be expanded ex vivo. In one embodiment, the cells selected for ex vivo expansion are CD8' and demonstrates the capacity to specifically recognize and lyse antigen-specific target cells. 10 Viral transduction methods may also be used to generate redirected cells which express the CAR of the invention. Cell types that may be used to generate genetically modified cells expressing the bispecific CAR of the invention include but are not limited to T lymphocytes (T-cells), natural killer cells, hematopoietic stem cells and/or pluripotent 15 embryonic/induced stem cells capable of giving rise to therapeutically relevant progeny. Stimulation of the T-cells by an antigen under proper conditions results in proliferation (expansion) of the cells and/or production of IL-2. The cells comprising the CAR of the invention will expand in number in response to the binding of one or more antigens to the 20 antigen-specific targeting regions of the CAR. The invention also provides a method of making and expanding cells expressing a CAR. The method comprises transfecting or transducing the cells with the vector expressing the CAR and stimulating the cells with cells expressing the target antigens, recombinant target antigens, or an antibody to the receptor to cause the cells to proliferate, so as to make and expand T-cells. In an 25 embodiment, the cells may be any one or more of T-lymphocytes (T-cells), natural killer (NK) cells, hematopoietic stem cells (HSCs) or pluripotent embryonic/induced stem cells capable of giving rise to therapeutically relevant progeny. In an exemplary embodiment, the genetically engineered cells of the invention express a 30 bispecific CAR which is specific for CD19 and CD20 antigens. In a further embodiment, a genetically engineered T-cell expresses the bispecific CARs CD19scFv-Gly4ser-linker CD20scFv-hulgG4-hinge-CD28-41BB(cyto)-zeta(cyto) or CD19scFv-Gly4ser-linker CD20scFv-hulgG4-hingeCH2CH3-CD28tm/cyto-zeta or CD 1 9-Gly4serlinker CD20scFv-CD8alphahinge-CD8alphaTM-4 1 BBcostim-zetacyto. 34 WO 2013/123061 PCT/US2013/025953 In an exemplary embodiment, the invention provides a method of making and expanding T-cells expressing a CD19-specific and CD20-specific CAR. The method comprises using a lentivirus to transduce CD3xCD28 bead-stimulated purified central memory T 5 cells (such as T-cells from peripheral blood) with the vector expressing the CD19 and CD20 bispecific CAR, growing the T-cells in the presence of rhuIL-2 and/or IL-15 and restimulating the T-cells with CD19* and CD20' cells, recombinant CD19 and CD20, or an antibody to the receptor to cause the T-cells to proliferate, so as to make and expand CD19-specific and CD20-specific T-cells. 10 Therapeutic methods of the invention The CARs of the invention may be used to overcome therapeutic failures arising from antigen loss escape variants, to reduce resistance to existing therapies and/or to treat 15 diseases associated with the antigens targeted by the CARs. Accordingly, the invention also provides methods for treating a disease associated with the antigen targeted by the CAR of the invention in a subject in need thereof. The method comprises providing a composition comprising the CAR of the invention and 20 administering an effective amount of the composition so as to treat the disease associated with the antigen in the subject. The invention also provides methods for overcoming therapeutic failures arising from antigen loss escape variants in disease states (e.g., B-cell diseases) in subjects in need 25 thereof. The method comprises providing a composition comprising the CAR of the invention and administering an effective amount of the composition so as to treat the disease associated with the antigen in the subject. In some embodiments, the composition comprises a polynucleotide encoding the CAR, a 30 protein comprising the CAR or genetically modified cells comprising the CAR. In another embodiment, the genetically modified cells of the composition are T-lymphocytes (T-cells), naive T cells (TN), memory T cells (for example, central memory T cells (TcM), effector memory cells (TEM)), natural killer (NK) cells, hematopoietic stem cells (HSCs) or pluripotent embryonic/induced stem cells capable of giving rise to therapeutically 35 WO 2013/123061 PCT/US2013/025953 relevant progeny, which express the CAR of the invention. The compositions of the invention may be administered alone or in conjunction with existing therapies. If other therapies are used in conjunction, the compositions of the invention may be administered concurrently or sequentially with the other the existing therapies. 5 Pharmaceutical compositions In various embodiments, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective 10 amount of the CAR (for example, bispecific CAR) of the invention. The CAR of the invention in the composition may be any one or more of a polynucleotide encoding the CAR, a protein comprising the CAR or genetically modified cells comprising the CAR. The composition may further comprise polynucleotides encoding EGFRt, CCR and/or DHFR (for example mutant DHFR), proteins co-expressed with the CAR including 15 EGFRt, CCR and/or DHFR or genetically modified cells that express the CAR and co express EGFRt, CCR and/or DHFR. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, 20 or, in the case of an aerosol composition, gaseous. In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to 25 aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and/or injections. The pharmaceutical compositions according to the invention can also contain any 30 pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a 36 WO 2013/123061 PCT/US2013/025953 combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, 5 immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically 10 acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as 15 glyceryl monostearate or glyceryl distearate, alone or with a wax. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid 20 carrier is used, the preparation will be in the form of syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule. The pharmaceutical compositions according to the invention may be delivered in a 25 therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological 30 condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for 37 WO 2013/123061 PCT/US2013/025953 instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000). 5 EXAMPLES The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific 10 materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. 15 Example 1 Figure 1 is a schematic representation of the bispecific chimeric antigen receptor of the invention. In an exemplary embodiment of the invention, Figure 2 depicts the components of bispecific anti-CD19xanti-CD20 bispecific CAR. Figure 2 also depicts a 20 schematic of the complete cDNA packaged into epHIV-7 lentivirus vector transfer plasmid. Figures 3 and 4 show the nucleic and amino acid sequences of an exemplary bispecific CAR, namely GMCSFss-CD19scFv-Gly4Serllinker-CD20scFv-IgG4Hinge CD28tm-41BBzeta-T2A-EGFRt epHIV7. 25 Example 2 Figure 5 is a schematic showing the vector construct of an exemplary CAR of the invention, namely, the CD 1 9scFv-CD20scFv-IgG4-CD28tm-CD28costim-CD3zeta transgene construct. The CD 19scFv-CD20scFv-IgG4-CD28tmCD28costim-CD3zeta 30 transgene was assembled using the one-step isothermal DNA assembly method previously described by Gibson et. al. (Enzymatic assembly of DNA molecules upto several hindred kilobases. Nature Methods. 2009;6:343-345). The VL and VH domains of the CD19 scFv construct was sequenced from a CD 19CAR-CD28-Zeta transgene previously described. Schmitz N, Dreger P, Glass B, Sureda A. Allogeneic transplantation 38 WO 2013/123061 PCT/US2013/025953 in lymphoma: current status. Haematologica. 2007;92(11):1533-1548) through polymerase chain reaction (PCR). The VH and VL domains of the CD20 scFv were assembled by spliced-overlap polymerase chain reaction using a CD20R-CD28-Zeta transgene previously described (Michael Jensen et al., CD20 is a molecular target for 5 scFvFc:zeta receptor redirected T-cells: implications for cellular immunotherapy of CD20' malignancy. Biology of Blood and Marrow Transplant. 1998;4:75-83). The VH and the VL domains of CD19 scFv and CD20 scFv were linked with an 18-residue linker peptide as previously described. The IgG4-CD28tm-CD28costim domain was sequenced using the CD19R-CD28-CD3zeta transgene by PCR. The CD3zeta-T2A-EGFRt epHIV7 10 lentiviral destination vector was prepared by NheI and RsrII restriction digestion of the CD19R-CD28 portion from a CD19R-CD28-Zeta-T2A-EGFRt_ epHIV7 plasmid previously described (Seitaro Terakura et al., Generation of CD19-CAR modified CD8+ T-cells derived from virus-specific central memory T-cells. Blood. Oct. 26, 2011). The final CD 1 9scFv-CD20scFv-IgG4-CD28tm-CD28costim-CD3zeta construct was 15 assembled by the one-step isothermal Gibson DNA assembly method using the restriction digested Zeta-epHIV7 destination vector and the CD19scFv, CD20scFv, and IgG4 CD28tm-CD28costim- DNA fragments with primers for each containing a 30 bp overlap at the 5' terminus. 39 WO 2013/123061 PCT/US2013/025953 Table 1: Regulatory Elements Present in the bispecific CAR epHIV-7 Transfer Plasmid Regulatory Element Function U5 5' Unique sequence Psi Packaging signal RRE Rev-responsive element flap Contains polypurine track sequence and central termination sequence to facilitate nuclear import of pre-integration complex EFIp Promoter EF1-alpha Eukaryotic Promoter sequence driving expression of CD 1 9xCD20 CAR WPRE Woodchuck hepatitis virus derived regulatory element to enhance viral RNA transportation delU3 3' U3 with deletion to generate SIN vector R Repeat sequence within LTR U5 3' U5 sequence in LTR AmpR Ampicillin-resistance gene CoEl ori Replication origin of plasmid SV40 ori Replication origin of SV40 CMV promoter CMV promoter to generate viral genome RNA R Repeat sequence within LTR Example 3 5 HEK 293T-cells were transfected with anti-CD19xCD20CAR-T2A-EGFRt epHIV-7 transfer plasmid or with anti-CD20xCD19CAR-T2A-EGFRt epHIV-7 transfer plasmid. Transfected cells were stained with biotinylated anti-Fc antibodies and streptavidin PE (SA-PE) and then were subjected to flow cytometric analysis for detection of expression of the above two CARs. Both the anti-CD 19xCD20 CAR and the anti-CD20xCD19 CAR 10 were expressed on transfected HEK 293T cells. 40 WO 2013/123061 PCT/US2013/025953 The epHIV-7 transfer plasmid co-expressed EGFRt with the above two bispecific CARs. EGFRt co-expression was detected on the same transfected cells using a combination of biotinlylated anti-EGFR antibodies/SA-PE staining and flow cytometric analysis. 5 Example 4 Primary human peripheral blood derived T-cells were activated with OKT3 and then were lentivirally transduced with monospecific anti-CD 19 CAR, monospecific anti-CD20 CAR or bispecific anti-CD19xCD20CAR-T2A-EGFRt epHIV7 lentivirus vector. epHIV7 10 lentivirus vector also encoded EGFRt together with monospecific anti-CD19 CAR, monospecific anti-CD20 CAR or bispecific anti-CD19xCD20. Thus, cells expressing the CARs co-expressed EGFRt. Transfected cells were stained with biotinlylated anti-EGFR antibodies and SA-PE and then were subjected to flow cytometric analysis for detection of EGFRt expression and co-expression of monospecific or bispecific CARs. Of the cells 15 transfected with monospecific anti-CD19 CAR, 51% expressed EGFRt; of the cells transfected with monospecific anti-CD20 CAR, 38.5% expressed EGFRt; of the cells transfected with the bispecific anti-CD 19xCD20 CAR, 63.8% expressed EGFRt. T cell receptor (TCR) complex in transfected cells was also detected in the same 20 transfected cells using FITC-conjugated anti-TCRa and anti-TCR3 antibodies staining and flow cytometric analysis. Example 5 25 H9 cells were genetically modified to express CD 19, or CD20, or both CD19 and CD20. Cells were stained with anti-CD19 and anti-CD20 antibodies and then were subject to flow cytometric analysis to detect the expression of CD19 and CD20. Cytometric analysis confirmed the desired expression profile of CD19 *CD20-, CD19-CD20*, and CD19*CD20* H9 cells, namely, genetically engineered H9 cells expressed CD19, or 30 CD20, or both CD19 and CD20 thereby simulating cancer target cells, which contain antigen-negative antigen loss escape variants. As described later, these cell lines were subsequently used as target cells to stimulate CAR-expressing T-cell lines, which act as effector cells to kill target cells. 41 WO 2013/123061 PCT/US2013/025953 Also, endogenous levels of CD 19 and CD20 expression in SUP-B 15 and DHL-6 cell lines was analyzed using anti-CD19 APC and anti-CD20 PE staining and flow cytometric analysis. SUP-B 15 cell line expressed high level of CD19 with low level of CD20 (thus CD19CD20-), and DHL-16 cell line expressed high level of CD20 with low level of 5 CD19 (thus CD19-CD20'). Example 6 A 4-hour chromium release assay was used to measure the lysis of the target cells by the 10 effector cells. Effector cells are primary human T-cells lentivirally transduced to express monospecific anti-CD19 CAR, monospecific anti-CD20 CAR or bispecific anti CD19xCD20 CAR. The bispecific anti-CD19xCD20 CAR effector T-cells effectively lysed all CD19 *CD20-, CD19-CD20', and CD19 *CD20' target cells, which include CD19*CD20- H9 cells, CD19-CD20' H9 cells, CD19*CD20' H9 cells and SUP-B 15 cells. 15 At effector to target ratios of 1:1, 3:1, 10:1, and 30:1, about 25%, 45%, 50% and 60%, respectively, target cells were lysed. In contrast, monospecific CAR expressing T-cell lines fail to lyse antigen-negative antigen loss escape variants, which escaped from the monospecific CAR effector cells. 20 The anti-CD19 CAR effector T-cells failed to lyse CD19-CD20* targets and the anti CD20 CAR effector T-cells failed to lyse CD19*CD20- targets. Example 7 25 Bispecific CAR-expressing CD4 enriched T-cells were activated for cytokine secretion (Interferon gamma (IFN-g, IFN-y)) upon stimulation by CD 1 9CD20-, CD 1 9-CD20, and CD19*CD20* target cells, which include CD19*CD20- H9 cells, CD19-CD20* H9 cells, CD19*CD20* H9 cells and SUP-B 15 cells. IFN-y content was measured by cytokine bead array of culture supernatants of T-cells and target cells after 24-hours of co-culture. 30 Activated bispecific CAR-expressing CD4 enriched T-cells secreted at least 2500 pg/ml INF-g upon stimulation by every type of target cells. In contrast, monospecific CAR expressing T-cell lines were not activated for cytokine INF-g secretion upon stimulation by antigen-negative antigen loss escape variants, which escaped from the monospecific 42 WO 2013/123061 PCT/US2013/025953 CAR effector cells. CD19 CAR T-cells failed to secrete IGN-y upon co-culture with CD19-CD20' target cells and CD20 CAR T-cells failed to secrete IGN-y upon co-culture with CD19CD20- target cells. In-vitro Stimulation Assay - Stimulators (3xlO5): - TM-LCL - H9 parent - OKT3-TM-LCL - H9 CD19R - SUP-B15 - H9 CD20R - DHL-6 - H9 CD19/20R - Responders (x1 0
^
6 on SjR2D17: - CD4 enriched mock - CD8 enriched mock - CD4 enriched CD19R - CD8 enriched CD19R - CD4 enriched CD20R - CD8 enriched CD20R - CD4 enriched CD19/20R - CD8 enriched CD19/20R - Cells incubated for 24 hrs, and cell free supernatant will be harvested today for BioPlex assay 5 Example 8 The example below describes a CD19 specific chimeric antigen receptor linked to truncated epidermal growth factor receptor (EGFRt) via a T2A sequence. EGFRt may be linked to and co-expressed with other chimeric antigen receptors, for example, bispecific 10 chimeric antigen receptors. Applicants demonstrated the utility of such a truncated EGFR (huEGFRt) expressed by transduced T cells for immunomagnetic purification using biotinylated cetuximab, cell tracking by flow cytometry and immunohistochemistry, and in vivo cell ablation after 15 systemic cetuximab administration. In this exemplary embodiment, domain I and II of EGFRt have been deleted while domains III and IV have been retained. The CD19CAR-T2A-EGFRt-epHIV7 lentiviral construct contains: (1) the chimeric antigen receptor (CAR) sequence consisting of the VH and VL gene segments of the 20 CD19-specific FMC63 monoclonal antibody (mAb), an IgG4 hinge-CH2-CH3, the transmembrane, and cytoplasmic signaling domains of the co-stimulatory molecule 43 WO 2013/123061 PCT/US2013/025953 CD28, and the cytoplasmic domain of the CD3Q chain (Kowolik CK. et al., CD28 costimuation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 2006, 66(22):10995-11004); (2) the self-cleaving T2A sequence (Szymczak AL. et al., 5 Correction of multi-gene deficiency in vivo using a "self-cleaving" 2A peptide-based retroviral vector. Nat Biotechnol 2004; 22(5)589-594); and (3) the truncated EGFR sequence as indicated. Immunomagnetic enrichment of huEGFRt' human T cells after lentiviral transduction 10 The biotinylated cetuximab was used for either immunomagnetic selection or FACS sorting of huEGFRt' cells. Applicants used biotinylated cetuximab in conjunction with commercially available antibiotin microbeads for the immunomagnetic selection of human T cells transduced with a self-inactivating lentivirus that directs the co-expression 15 of CD19CAR and huEGFRt. PBMCs or purified central memory (CD45RO*CD62L TcM) or effector memory (CD45ROCD62L TEM) T-cell subsets were stimulated with anti-CD3/anti-CD28 beads and then transduced by lentiviral vector to generate a panel of primary human T-cell 20 lines, of which 2.6%-40% expressed huEGFRt and CAR. The unselected cells were labeled with biotinylated cetuximab and anti-biotin microbeads; and then were separated to consistently obtain a selected cell population, of which 90% express huEGFRt and CAR. 25 Unselected T cells and selected fraction were stained with biotinylated-cetuximab and either PE-conjugated streptavidin or PE-conjugated anti-biotin Ab, and then were subject to flow cytometric analysis. Selection of CD19CAREGFRtf cells was performed either 3 days after transduction of OKT3 blasts (enriched from 38% to 98%), or after 1 rapid expansion cycle of transduced effector memory CD62LCD45RO*-derived cells (enriched 30 from 20% to 96%), after 3 rapid expansion cycles of transduced CMVpp65-specific TCM-derived cells (enriched from 12% to 91%), or after 2 rapid expansion cycles of transduced CD8'TCM-derived cells (enriched from 3% to 97%). Selection of CD19CAREGFRt*IMPDH2dm* cells was performed after 1 rapid expansion cycle of transduced TCM-derived cells (enriched from 25 to 92%). 44 WO 2013/123061 PCT/US2013/025953 CD19CAR-T2A-EGFRt-IMPDH2dm constructs contained in lentiviral vectors include codon optimized sequence portions of the CD19-specific, CD28 co-stimulatory CAR (CD19CAR), followed by the self-cleavable T2A, and selection markers huEGFRt and 5 IMPDH2dm (a double mutant of the inosine monophosphate dehydrogenase 2 gene that allows for cell survival upon addition of mycophenolate 27), along with the Elongation Factor 1 promoter sequences (EF-1p), the GM-CSF receptor alpha chain signal sequences (GMCSFRss), and the 3 nucleotide stop codon. 10 Before immunomagnetic selection, a proliferative advantage of huEGFRt- cells over huEGFRt+ cells was observed in cultures of unselected transduced T cells subjected to OKT3-mediated expansion. However, after immunomagnetic selection, the level of huEGFRt expression and the frequency of expressing cells remained stable over 3 consecutive 14-day cycles of OKT3-based expansion 1 4 . The fold expansion of EGFRt+ 15 cells after immunomagnetic selection was significantly enhanced over that of huEGFRt+ cells in the unselected cultures. These data demonstrate that huEGFRt can serve as a cell surface marker unique to transduced human T cells and enable subsequent cetuximab-based immunomagnetic 20 purification of stable huEGFRt-expressing cell populations which also express CARs. Tracking of adoptively transferred huEGFRt' T cells using flow cytometry and immunohistochemistry 25 To test the utility of huEGFRt for tracking the engraftment of adoptively transferred T cells, Applicants harvested blood and bone marrow specimens from NOD/Scid IL-2RyC nun mice engrafted with CD19CAR+EGFRt+ human T cells. First, unfixed peripheral blood and bone marrow mononuclear cell samples were 30 subjected to flow cytometric analysis after being stained with biotinylated cetuximab and PE-conjugated streptavidin. Although the level of human CD45+ T-cell engraftment (20%-25%) was similar in animals administered either EGFRt-negative or -positive T cells, double staining for human CD45 and EGFR allowed for the resolution of huEGFRt+ (ie, transgeneexpressing) human T cells from their huEGFRt-negative counterparts. 45 WO 2013/123061 PCT/US2013/025953 Second, Applicants sought to determine whether standard paraffin embedded fixed tissue specimens were amenable to detection of huEGFRt' T-cell infiltrates using EGFR specific diagnostic kits. Applicants performed immunohistochemical analysis of paraffin 5 embedded femurs from engrafted mice and detected huEGFRtf cells in the bone marrow. These data support the utility of huEGFRt to serve as a tracking marker for quantifying the frequency and tissue distribution of adoptively transferred T cells. Cetuximab binding to huEGFRt sensitizes human T cells to ADCC 10 A valuable feature of a cell surface selection/tracking marker would be its capacity to serve as a target for in vivo cell ablation. Applicants evaluated the extent to which Cetuximab bound to huEGFRt on T cells activates ADCC of huEGFRt+ T cells in vitro, and whether Cetuximab administration could attenuate the engraftment of adoptively 15 transferred huEGFRt+ T cells in NOD/scid mice. 5 1 Cr-labeled huEGFRt+ T cells as the target cells and human GM-CSF activated fresh PBMCs as effectors were co-cultured. Then, the addition of Cetuximab specifically sensitized huEGFRt+ T cells to ADCC cytolysis by effectors. Lysis of huEGFRt+ T cells 20 was measured by 4-hour chromium release assay and results showed that Cetuximab addition significantly increased lysis from less than 5% to about 50%, 45%, 40% and 15% respectively at effector to target (effector:target) ratios 50:1, 25:1, 5:1 and 1:1. In contrast, the addition of the CD20-specific mAb Rituxan had no effect on triggering 25 ADCC of huEGFRt+ T cells in this assay. Applicants next derived huEGFRt+ CTLL-2 murine T cells that were additionally modified to secrete autocrine IL-2 and express the firefly luciferase biophotonic reporter, and adoptively transferred these ffLuc'huEGFRt+ CTLL-2 cells via intravenous injection 30 to NOD/scid mice, which subsequently received Cetuximab or Rituxan. The in vivo engraftment of transferred CTLL-2, as measured by in vivo biophotonic imaging, was significantly inhibited (97%, P< .05) in mice that received Erbitux (1 mg intraperitoneally daily). The Cetuximab-mediated elimination of the ffLuc'huEGFRt+ CTLL-2 cells 46 WO 2013/123061 PCT/US2013/025953 occurred between 4 and 6 days. These data support the use of Cetuximab administration as a therapeutic control for patients receiving huEGFRt T cells. Example 9 5 This example describes T cells with an intrinsic yc cytokine signaling mechanism, and shows that chimeric cytokine receptors (CCR) IL-2/IL-15RO (CyCR2) and IL-7Ra (CyCR7) have the ability to improve the survival, persistence, and in vivo engraftment of cytotoxic T cells (CTLs). Truncated CD19 antigen (CD19t) was linked to CyCR via a 10 T2A linker to show the expression of CyCR on the cell surface. The chimeric cytokine receptors described herein may be linked to the chimeric antigen receptors of the invention, such as bispecific CARs described herein. To develop a cell-intrinsic, ligand-independent yc cytokine platform, Applicants 15 engineered chimeric yc cytokine receptors (CyCR) comprised of the IL-7 cytokine tethered by ten amino acids to the extracellular domain of IL-7Ra. To engineer a CyCR that confers an IL-7R signal, IL-7 cytokine was tethered to the full length IL-7Ra chain (CyCR7). A CyCR that provides an IL-2/IL-15RO signal was engineered by tethering the IL-7 cytokine to the extracellular and transmembrane domain of IL-7Ra fused to the 20 cytoplasmic domain of IL-2/IL-15RO (CyCR2). These single chain chimeric receptors are expected to require endogenous yc chain for signaling. Constructs were then generated where the CyCR transgenes were followed by the self cleavable T2A sequence, and a cytoplasmically truncated CD19 antigen (CD 19t). CyCR 25 and CD19t are expressed as a single transcript and cleaved post-translationally at the C terminus of the T2A self-cleaving peptide to yield two separate type 1 membrane proteins CyCR(T2A) and CD19t. Based on expression of two proteins from a single transcript, the ratio of CyCR(T2A) to CD19t expression is 1:1, therefore, cell surface CD19t is an indication of CyCR cell surface expression. Lentiviral transduction and expression of 30 these constructs could then be measured by surface CD19t expression, such as that seen in both Jurkat and NK-92 cell lines. A third CyCR was also engineered, having IL-7 cytokine tethered to a truncated IL-7Ra (CyCR7t), which is missing amino acids 1-126 from the extracellular domain of the IL 47 WO 2013/123061 PCT/US2013/025953 7Ra . A molecular model of CyCR7t dimerization with the endogenous ye chain is necessary for signal transduction. The lack of amino acids 1-126 of the extracellular domain of IL-7Ra renders the CyCR7t nonfunctional. 5 Truncated CyCR7 expression does not functionally signal or support cytokine independent cell growth. Flow cytometric detected cell-surface CD19t on lenti transduced Jurkat (95% CD19tfCyCR7tf) and Teff cell lines (97% CD19tfCyCR7t). Western blot analysis of STAT5 phosphorylation within CyCR7t expressing Jurkat cell line did not detect obvious increase of phosphorylated STAT5 as compared to non 10 transduced control Jurkat cell line. Positive controls OKT3 stimulated PBMC cultured in 50U/ml IL-2 and 1Ong/ml IL-15 and K562 showed activation of increased phosphorylated STAT5. Accordingly, expansion and viability of CTLs transduced with CyCR7t cultured for 20 days were still dependent on cytokines. 15 To determine if functional CyCRs such as CyCR2 and CyCR7 could support the growth of CD8' human primary T cells in the absence of exogenous cytokine, we measured the expansion of CTLs expressing each CyCR. Human primary T cells expressing CyCR7t were unable to expand in the absence of exogenous cytokine . Both CyCR2 and CyCR7 were able to support the survival and proliferation of the CD8' T cells through 20 maintenance of viability, in a manner similar to that of parental cells cultured in 5U/ml and 0.5 U/ml IL-2, respectively. The increased total cell expansion measured for CyCR2 versus CyCR7' CTL correlates with increased expression (i.e., MFI of 26 for CyCR7 versus 52 for CyCR2) of Ki67, a nuclear antigen protein present in GI, S, G2, and M phase of the cell cycle. Higher Bcl-2, an key antiapoptotic protein induced in response to 25 IL-2 and IL-7 signaling, expression was observed for CyCR7 versus CyCR2 CTL, supporting the ability of CyCR7 to maintain the survival of the human primary T cells. Together this data suggests that, although both CyCRs support cytokine-independent T cell viability and expansion, CyCR2 provides a proliferative advantage while CyCR7 maintains survival for effector CD8' CTLs. 30 48 WO 2013/123061 PCT/US2013/025953 CyCR expressing CD8' T cells exhibit cytokine independent engraftment in vivo Studies by our lab and others indicate that human CTL engraftment in NOD/Scid IL 2RyC nun mice is dependent on exogenous administration of human IL-15 or IL-2. To 5 test the potential of CyCR expression in CTLs to overcome this dependence, parental effector T cells, CyCR7' CTLs, and CyCR2' CTLs were injected into the tail vein of immunodeficient NOD/Scid IL-2RyC nul mice in the absence of exogenous cytokine administration. Total engraftment was compared by harvesting at least four mice per group at day 8, 17, 24, and 48 and analyzing T cell levels in the blood and bone marrow. 10 In the blood, CyCR2' CTLs had impressive significant (P<0.007) exogenous cytokine independent engraftment compared to CyCR7' CTLs and the parental cells. In the bone marrow, both CyCR7' CTLs (P<0.03) and CyCR2' CTLs (P<0.0005) had significant exogenous cytokine independent engraftment compared to the parental cells. CyCR2' 15 CTLs had higher engraftment compared to CyCR7' CTLs. This indicates that both CyCR7' CTLs and CyCR2' CTLs are capable of supporting exogenous cytokine independent engraftment but the total percentage of cells was different. The blood supported higher percent engraftment of CyCR2' CTLs compared to bone marrow. The bone marrow supported the engraftment of CyCR7' CTLs over a longer period of time. 20 Importantly, the engraftment was not infinite as the cells were no longer present in the blood and bone marrow at day 48 in either group. Cell intrinsic yc cytokine signals can replace the need for exogenous cytokine administration for the support of adoptively transferred CTLs. Providing cell intrinsic 25 cytokine receptors can overcome the major limitation of adoptive immunotherapy; the long-term persistence of adoptively transferred CTL. This may eliminate the need for administration of exogenous cytokine, which may reduce toxicities and bystander effects on endogenous cell types. 30 Example 10 This example shows that CD19 chimeric antigen receptor linked to EGFRt and DHFR can be regulated by methotrexate. Using the methods described herein, the 49 WO 2013/123061 PCT/US2013/025953 dihydroxyfolate receptor described herein may be linked to the bispecific chimeric antigen receptors of the invention. Applicants developed a human selectable transgene using a variant of human 5 dihydrofolate reductase (hDHFR) that would enable selection of T cells with the less toxic, pharmaceutically available drug methotrexate (MTX). MTX exerts its anti proliferative effect through competitive inhibition of DHFR, a key enzyme essential for de novo synthesis of thymidylate nucleotides. 10 In the instant example, Applicants evaluated the potential of DHFRFS (hDHFR L22F/F3 IS variant) mediated in vitro selection of primary human T cells that co-express a CD19-specific chimeric antigen receptor (CD19CAR for targeting of CD19-expressing tumors). In this strategy, we hypothesized that exposure of a transduced mixed population of T cells to the lymphotoxic drug MTX should lead to elimination of 15 untransduced T cells and selective expansion of DHFRFS/CD19CAR T cells co expressing T cells, increasing the anti-tumor efficacy of the T cell population as a whole. Here Applicants show that DHFRFS-mediated selection of gene modified T cells enforced the CD19CAR therapeutic transgene expression, and allowed for the derivation of CAR-' stable integrants in the presence of clinically attainable concentrations of MTX (e.g., 0.1 20 tM MTX). To translate the hDHFRFS selection approach for potential therapeutic utility, Applicants designed a lentiviral vector co-expressing hDHFRFS in conjunction with a CD19-specific chimeric antigen receptor (CD19CAR) and a truncated human EGFR polypeptide as a 25 tracking marker (huEGFRt) each separated by a ribosomal skip T2A sequence. CTLL2 T cells were first transduced with this CD19CAR-huEGFRt-hDHFRFS lentiviral vector and evaluated for their resistance to MTX. Ten days after lenti-transduction, 7-8 % of the cells were positive for CD 1 9CAR and huEGFRt expression. 30 In the absence of MTX, the non-transduced and transduced CTLL2 cells expanded at an equivalent rate (21- and 27-fold respectively). After incubation with MTX (0-0.1 gM) for 8 days, a 7-fold expansion with 80% survival was observed with transduced cells, while 50 WO 2013/123061 PCT/US2013/025953 exposure of non-transduced CTLL2 cells to ;> 0.05 gM MTX resulted in strong inhibition of non-transduced CTLL2 cell expansion and viability. Evaluation of huEGFRt expression levels of transduced CTLL2 cells after 8 days in 5 culture with varying concentrations of MTX further revealed significant MTX-mediated enrichment of transgene-expressing huEGFRt' cells (49%, 93%, 98.5%, 99% at 0.01, 0.025, 0.05 and 0.1 ptM MTX respectively). To further characterize the maximum dose of MTX that could be tolerated by selected 10 CTLL2 cells, transduced CTLL2 cells that had been cultured in 0.1 gM MTX for 8 days were re-plated at a wider range of MTX concentrations (up to 0.75 gM). These transduced and pre-MTX selected cells were able to expand 90-100 fold at MTX concentrations up to 0.25 gM, which is equivalent to non-transduced control CTLL2 expansion in the absence of MTX. 15 Applicants transduced primary human T cells with the same CD19CAR-huEGFRt hDHFRFS lentiviral vector. Purified CD62LCD45RO* T cells were used as a starting population based on their potential for persistence after adoptive transfer. Ten days after transduction, these T cells were cultured in varying concentrations of MTX and assessed 20 for cell number and viability over time. After 10 days, transduced and non-transduced T cells expanded equally (80-fold) in the absence of MTX. Furthermore, even at 0.1 piM MTX, transduced T cells maintained a viability of 63%, while non-transduced primary human T cells exhibited strong inhibition of both viability and fold-expansion starting at concentrations as low as 0.025 [tM MTX. 25 Flow cytometric evaluation of transduced T cells after 10 days in culture with varying concentrations of MTX revealed significant MTX-mediated enrichment of transgene expressing cells (e.g., 0.025[tM MTX enriched about 54% CD19CAR* and 79% EGFRtf; 0.05ptM MTX enriched about 76% CD19CAR* and 89% EGFRtf) 30 Comparison of CD19CAR and EGFRt expression at day 6 vs. day 10 of culture revealed the steady progression of this MTX/DHFRFS-mediated selection over time (Day 0: 18% CD19CAR, 28% EGFRtf; Day 6: 48% CD19CAR, 71% EGFRtf; Day 10: 70% CD19CAR+, 88% EGFRt). 51 WO 2013/123061 PCT/US2013/025953 All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 5 invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5 th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in 10 the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. 15 For purposes of the present invention, the following terms are defined below. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall 20 within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s). 25 The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments 30 described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention. 52 WO 2013/123061 PCT/US2013/025953 While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader 5 aspects. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). 53

权利要求:
Claims (104)
[1] 1. A bispecific chimeric antigen receptor, comprising: a. at least two antigen-specific targeting regions; b. an extracellular spacer domain; c. a transmembrane domain; d. at least one co-stimulatory domain; and e. an intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, and wherein the bispecific chimeric antigen receptor is co-expressed with a therapeutic control.
[2] 2. The bispecific chimeric antigen receptor of claim 1, wherein the therapeutic control comprises any one or more of truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3 -acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), a selection marker, and combinations thereof.
[3] 3. The bispecific chimeric antigen receptor of claim 2, wherein the EGFRt binds any one or more of an EGFR-specific siRNA , a small molecule, an anti-EGFR antibody or a fragment thereof, or a combination thereof.
[4] 4. The bispecific chimeric antigen receptor of claim 2, wherein the selection marker comprises any one or more of dihydroxyfolate receptor (DHFR), mutant DHFR, methylated-DNA-protein-cysteine methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2) and combinations thereof. 54 WO 2013/123061 PCT/US2013/025953
[5] 5. The bispecific chimeric antigen receptor of claim 2, wherein the CCR comprises any one or more of (i) IL-7 cytokine-linker- IL7Ra, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Ra-transmembrane domain of IL-7Ra-cytoplasmic domain of IL-2R, (iii) IL-7 cytokine-linker-IL2RP, and (iv) combinations thereof.
[6] 6. The bispecific chimeric antigen receptor of claim 1, wherein the bispecific chimeric antigen receptor and the therapeutic control are linked via a cleavable linker.
[7] 7. The bispecific chimeric antigen receptor of claim 6, wherein the cleavable linker is a self cleaving cleavable linker.
[8] 8. The bispecific chimeric antigen receptor of claim 7, wherein the cleavable linker is any one or more of a 2A linker, 2A-like linker or a functional equivalent thereof.
[9] 9. The bispecific chimeric antigen receptor of claim 1, wherein the extracellular spacer domain comprises any one or more of an Fc fragment of an antibody or a functional equivalent, fragment or derivative thereof, a hinge region of an antibody or a functional equivalent, fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence and combinations thereof.
[10] 10. The bispecific chimeric antigen receptor of claim 9, wherein the extracellular spacer domain comprises any one or more of (i) a hinge, CH2 and CH3 region of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 region of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 region of IgG1, (vi) a hinge region of IgG1, (vi) a hinge and CH2 region of IgG 1, or (vii) combinations thereof.
[11] 11. The bispecific chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises any one or more of a transmembrane region of a Type I transmembrane protein, an artificial hydrophobic sequence, and combinations thereof.
[12] 12. The bispecific chimeric antigen receptor of claim 11, wherein the transmembrane domain comprises any one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, and combinations thereof. 55 WO 2013/123061 PCT/US2013/025953
[13] 13. The bispecific chimeric antigen receptor of claim 1, wherein the co-stimulatory domain comprises a signaling domain from any one or more of CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and combinations thereof.
[14] 14. The bispecific chimeric antigen receptor of claim 1, wherein the intracellular signaling domain comprises a signaling domain of one or more of a human CD3 zeta chain, FcyRIII, FcRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.
[15] 15. The bispecific chimeric antigen receptor of claim 1, wherein each of the at least two antigen-specific targeting domains target an antigen independently selected from the group consisting of antigens specific for cancer, an inflammatory disease, a neuronal disorder, diabetes, a cardiovascular disease, an infectious disease, an autoimmune disease, and combinations thereof.
[16] 16. The bispecific chimeric antigen receptor of claim 15, wherein the antigen specific for cancer comprises any one or more of 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin a531, integrin av33, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, RORI, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-P, TRAIL-Ri, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof.
[17] 17. The bispecific chimeric antigen receptor of claim 1, wherein the at least two antigen specific targeting regions bind (i) CD19 and CD20, (ii) CD20 and Li-CAM, (iii) Li 56 WO 2013/123061 PCT/US2013/025953 CAM and GD2, (iv) EGFR and LI-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and RORI.
[18] 18. The bispecific chimeric antigen receptor of claim 1, wherein the at least two antigen specific targeting regions bind CD19 and CD20.
[19] 19. The bispecific chimeric antigen receptor of claim 15, wherein the antigen specific for an inflammatory disease comprises any one or more of AOC3 (VAP-1), CAM-3001, CCL1 1 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-i, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin a407, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb 37, scleroscin, SOST, TGF beta 1, TNF-a, VEGF-A, and combinations thereof.
[20] 20. The bispecific chimeric antigen receptor of claim 15, wherein the antigen specific for a neuronal disorder comprises any one or more of beta amyloid, MABT5102A, and combinations thereof.
[21] 21. The bispecific chimeric antigen receptor of claim 15, wherein the antigen specific for diabetes comprises any one or more of L-1 3, CD3, and combinations thereof.
[22] 22. The bispecific chimeric antigen receptor of claim 15, wherein the antigen-specific for a cardiovascular disease comprises any one or more of C5, cardiac myosin, CD41 (integrin alpha-Ib), fibrin II, beta chain, ITGB2 (CD18), sphingosine- 1-phosphate, and combinations thereof.
[23] 23. The bispecific chimeric antigen receptor of claim 15, wherein the antigen specific for an infectious disease comprises any one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-a, and combinations thereof. 57 WO 2013/123061 PCT/US2013/025953
[24] 24. In combination, the bispecific chimeric antigen receptor of claim 1 and the therapeutic control.
[25] 25. The combination of claim 24, wherein the therapeutic control comprises any one or more of truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), a selection marker, and combinations thereof.
[26] 26. The combination of claim 25, wherein the EGFRt binds any one or more of an EGFR specific siRNA, a small molecule, an anti-EGFR antibody or a fragment thereof, or a combination thereof.
[27] 27. The combination of claim 25, wherein the selection marker comprises any one or more of dihydroxyfolate receptor (DHFR), mutant DHFR, methylated-DNA-protein-cysteine methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2) and combinations thereof.
[28] 28. The combination of claim 25, wherein the CCR comprises any one or more of (i) IL-7 cytokine-linker- IL7Ra, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Ra transmembrane domain of IL-7Ra-cytoplasmic domain of IL-2R, (iii) IL-7 cytokine linker-IL2RP, and (iv) combinations thereof.
[29] 29. The combination of claim 24, wherein the bispecific chimeric antigen receptor and the therapeutic control are linked via a cleavable linker.
[30] 30. The combination of claim 29, wherein the cleavable linker is a self-cleaving cleavable linker. 58 WO 2013/123061 PCT/US2013/025953
[31] 31. The combination of claim 29, wherein the cleavable linker is any one or more of a 2A linker, 2A-like linker or a functional equivalent thereof.
[32] 32. A polynucleotide encoding the bispecific chimeric antigen receptor of claim 1 or the combination of claim 24.
[33] 33. A polypeptide encoded by the polynucleotide of claim 32.
[34] 34. A vector comprising the polynucleotide of claim 32.
[35] 35. A virus comprising the polynucleotide of claim 32.
[36] 36. The virus of claim 35, wherein the virus is an RNA virus.
[37] 37. The virus of claim 35, wherein the virus is a retrovirus, an adenovirus, an adeno associated virus, a lentivirus, a pox virus or a herpes virus.
[38] 38. A genetically engineered cell, comprising the polynucleotide of claim 32, the chimeric antigen receptor of claim 1, or the combination of claim 24.
[39] 39. The genetically engineered cell of claim 38, wherein the cell is a T-lymphocyte (T-cell)
[40] 40. The genetically engineered cell of claim 39, wherein the cell is a naYve T cells, a central memory T cells, an effector memory T cell or a combination thereof.
[41] 41. The genetically engineered cell of claim 38, wherein the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.
[42] 42. A pharmaceutical composition, comprising: a. any one or more of the bispecific chimeric antigen receptor of claim 1, the combination of claim 24, the polypeptide of claim 32, the vector of claim 34, the virus of claim 35, the genetically engineered cell of claim 38, and combinations thereof; and b. a pharmaceutically acceptable carrier. 59 WO 2013/123061 PCT/US2013/025953
[43] 43. In combination, the pharmaceutical composition of claim 42 and a composition adapted to biochemically interact with the therapeutic control to inhibit proliferation of a cell expressing the therapeutic control.
[44] 44. The combination of claim 43, wherein the composition adapted to biochemically interact with the therapeutic control is any one or more of Herceptin, methotrexate, cetuximab, thymidine analogs (for example ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2, 4-dinitrobenzamide (CB1954), 6 thioguanine, a synthetic dimerizing drug (for example AP1903), fludarabine phosphate, linamarin (lin), nucleoside analogs (for exmaple BVDU, difluorodeoxycytidine (dFdC), 1-0-D-arabinofuranosylthymine (ara-T)), indole-3-acetic (IAA), 1-buthionine-S,R sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors or combinations thereof.
[45] 45. A method of producing a quantity of T-cells expressing a chimeric antigen receptor, comprising: (i) transfecting one or more T-cells with the vector of claim 34; and (ii) stimulating the one or more T-cells with cells expressing antigens targeted by the at least two antigen-specific targeting regions, recombinant antigens targeted by the at least two antigen-specific targeting regions, or an antibody to the chimeric antigen receptor, whereby the T-cells proliferate so as to produce the quantity of T-cells.
[46] 46. A method for treating a disease in a subject in need thereof, comprising: (i) providing the composition of claim 42; and (ii) administering a therapeutically effective amount of the composition to the subject so as to treat the disease, wherein the at least two antigen-specific targeting regions each target an antigen, and at least one such antigen is associated with the disease.
[47] 47. A bispecific chimeric antigen receptor, comprising: a. at least two antigen-specific targeting regions; b. an extracellular spacer domain; c. a transmembrane domain; 60 WO 2013/123061 PCT/US2013/025953 d. at least one co-stimulatory domain; and e. an intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, and wherein the bispecific chimeric antigen receptor is co-expressed with truncated epidermal growth factor receptor (EGFRt).
[48] 48. The bispecific chimeric antigen receptor of claim 47, wherein the bispecific chimeric antigen receptor is co-expressed with a therapeutic control, comprising any one or more of thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), a selection marker, and combinations thereof.
[49] 49. The bispecific chimeric antigen receptor of claim 47, wherein the EGFRt binds any one or more of an EGFR-specific siRNA, a small molecule, an anti-EGFR antibody or a fragment thereof, or a combination thereof.
[50] 50. The bispecific chimeric antigen receptor of claim 48, wherein the selection marker comprises any one or more of dihydroxyfolate receptor (DHFR), mutant DHFR, methylated-DNA-protein-cysteine methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2) and combinations thereof.
[51] 51. The bispecific chimeric antigen receptor of claim 48, wherein the CCR comprises any one or more of (i) IL-7 cytokine-linker- IL7Ra, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Ra-transmembrane domain of IL-7Ra-cytoplasmic domain of IL-2R, (iii) IL-7 cytokine-linker-IL2RP, and (iv) combinations thereof.
[52] 52. The bispecific chimeric antigen receptor of claim 47, wherein the bispecific chimeric antigen receptor and the therapeutic control are linked via a cleavable linker. 61 WO 2013/123061 PCT/US2013/025953
[53] 53. The bispecific chimeric antigen receptor of claim 52, wherein the cleavable linker is a self-cleaving cleavable linker.
[54] 54. The bispecific chimeric antigen receptor of claim 52, wherein the cleavable linker is any one or more of a 2A linker, 2A-like linker or a functional equivalent thereof.
[55] 55. The bispecific chimeric antigen receptor of claim 47, wherein the extracellular spacer domain comprises any one or more of an Fc fragment of an antibody or a functional equivalent, fragment or derivative thereof, a hinge region of an antibody or a functional equivalent, fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence and combinations thereof.
[56] 56. The bispecific chimeric antigen receptor of claim 55, wherein the extracellular spacer domain comprises any one or more of (i) a hinge, CH2 and CH3 region of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 region of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 region of IgG1, (vi) a hinge region of IgG1, (vi) a hinge and CH2 region of IgG 1, and (vii) combinations thereof.
[57] 57. The bispecific chimeric antigen receptor of claim 47, wherein the transmembrane domain comprises any one or more of a transmembrane region of a Type I transmembrane protein, an artificial hydrophobic sequence, and combinations thereof.
[58] 58. The bispecific chimeric antigen receptor of claim 57, wherein the transmembrane domain comprises any one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, and combinations thereof.
[59] 59. The bispecific chimeric antigen receptor of claim 47, wherein the co-stimulatory domain comprises a signaling domain from any one or more of CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 and combinations thereof.
[60] 60. The bispecific chimeric antigen receptor of claim 47, wherein the intracellular signaling domain comprises a signaling domain of one or more of a human CD3 zeta chain, 62 WO 2013/123061 PCT/US2013/025953 FeyRIII, FecRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.
[61] 61. The bispecific chimeric antigen receptor of claim 47, wherein each of the at least two antigen-specific targeting domains target an antigen independently selected from the group consisting of antigens specific for cancer, an inflammatory disease, a neuronal disorder, diabetes, a cardiovascular disease, an infectious disease, an autoimmune disease, and combinations thereof.
[62] 62. The bispecific chimeric antigen receptor of claim 61, wherein the antigen specific for cancer comprises any one or more of 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin a531, integrin av33, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, RORi, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-P, TRAIL-Ri, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof.
[63] 63. The bispecific chimeric antigen receptor of claim 47, wherein the at least two antigen specific targeting regions bind (i) CD19 and CD20, (ii) CD20 and Li-CAM, (iii) Li CAM and GD2, (iv) EGFR and LI-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and RORi.
[64] 64. The bispecific chimeric antigen receptor of claim 47, wherein the at least two antigen specific targeting regions bind CD19 and CD20. 63 WO 2013/123061 PCT/US2013/025953
[65] 65. The bispecific chimeric antigen receptor of claim 61, wherein the antigen specific for an inflammatory disease comprises any one or more of AOC3 (VAP-1), CAM-3001, CCL1 1 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-i, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin a407, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb 37, scleroscin, SOST, TGF beta 1, TNF-a, VEGF-A, and combinations thereof.
[66] 66. The bispecific chimeric antigen receptor of claim 61, wherein the antigen specific for a neuronal disorder comprises any one or more of beta amyloid, MABT5102A, and combinations thereof.
[67] 67. The bispecific chimeric antigen receptor of claim 61, wherein the antigen specific for diabetes comprises any one or more of L-1 3, CD3, and combinations thereof.
[68] 68. The bispecific chimeric antigen receptor of claim 61, wherein the antigen-specific for a cardiovascular disease comprises any one or more of C5, cardiac myosin, CD41 (integrin alpha-Ib), fibrin II, beta chain, ITGB2 (CD18), sphingosine- 1-phosphate, and combinations thereof.
[69] 69. The bispecific chimeric antigen receptor of claim 61, wherein the antigen specific for an infectious disease comprises any one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-a, and combinations thereof.
[70] 70. In combination, the bispecific chimeric antigen receptor of claim 47 and the EGFRt.
[71] 71. The combination of claim 70, wherein the EGFRt binds any one or more of an EGFR specific siRNA, a small molecule, an anti-EGFR antibody or a fragment thereof, or a combination thereof. 64 WO 2013/123061 PCT/US2013/025953
[72] 72. The combination of claim 70, wherein the bispecific chimeric antigen receptor and the EGFRt are linked via a cleavable linker.
[73] 73. The combination of claim 72, wherein the cleavable linker is a self-cleaving cleavable linker.
[74] 74. The combination of claim 72, wherein the cleavable linker is any one or more of a 2A linker, 2A-like linker or a functional equivalent thereof.
[75] 75. A polynucleotide encoding the bispecific chimeric antigen receptor of claim 47 or the combination of claim 70.
[76] 76. A polypeptide encoded by the polynucleotide of claim 75.
[77] 77. A vector comprising the polynucleotide of claim 75.
[78] 78. A virus comprising the polynucleotide of claim 75.
[79] 79. The virus of claim 78, wherein the virus is an RNA virus.
[80] 80. The virus of claim 78, wherein the virus is a retrovirus, an adenovirus, an adeno associated virus, a lentivirus, a pox virus or a herpes virus.
[81] 81. A genetically engineered cell, comprising the polynucleotide of claim 75, the chimeric antigen receptor of claim 47, or the combination of claim 70.
[82] 82. The genetically engineered cell of claim 81, wherein the cell is a T-lymphocyte (T-cell)
[83] 83. The genetically engineered cell of claim 82, wherein the cell is a naYve T cells, a central memory T cells, an effector memory T cell or a combination thereof.
[84] 84. The genetically engineered cell of claim 81, wherein the cell is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.
[85] 85. A pharmaceutical composition, comprising: a. any one or more of the bispecific chimeric antigen receptor of claim 47, the combination of claim 70, the polypeptide of claim 76, the vector of claim 77, the 65 WO 2013/123061 PCT/US2013/025953 virus of claim 78, the genetically engineered cell of claim 81, and combinations thereof; and b. a pharmaceutically acceptable carrier.
[86] 86. In combination, the pharmaceutical composition of claim 85 and a composition adapted to biochemically interact with the therapeutic control to inhibit proliferation of a cell expressing the EGFRt.
[87] 87. The combination of claim 86, wherein the composition adapted to biochemically interact with the therapeutic control is any one or more of Herceptin, methotrexate, cetuximab, thymidine analogs (for example ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2, 4-dinitrobenzamide (CB1954), 6 thioguanine, a synthetic dimerizing drug (for example AP1903), fludarabine phosphate, linamarin (lin), nucleoside analogs (for exmaple BVDU, difluorodeoxycytidine (dFdC), 1-0-D-arabinofuranosylthymine (ara-T)), indole-3-acetic (IAA), 1-buthionine-S,R sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors or combinations thereof.
[88] 88. A method of producing a quantity of T-cells expressing a chimeric antigen receptor, comprising: (i) transfecting one or more T-cells with the vector of claim 77; and (ii) stimulating the one or more T-cells with cells expressing antigens targeted by the at least two antigen-specific targeting regions, recombinant antigens targeted by the at least two antigen-specific targeting regions, or an antibody to the chimeric antigen receptor, whereby the T-cells proliferate so as to produce the quantity of T-cells.
[89] 89. A method for treating a disease in a subject in need thereof, comprising: (i) providing the composition of claim 85; and (ii) administering a therapeutically effective amount of the composition to the subject so as to treat the disease, wherein the at least two antigen-specific targeting regions each target an antigen, and at least one such antigen is associated with the disease. 66 WO 2013/123061 PCT/US2013/025953
[90] 90. A bispecific chimeric antigen receptor comprising the sequence set forth in Figures 4, 9 or 11.
[91] 91. A bispecific chimeric antigen receptor, comprising: a. at least two antigen-specific targeting regions; b. a CD8ahinge extracellular spacer domain; c. a CD8a transmembrane domain; d. a 4-1BB co-stimulatory domain; and e. a CD3 zeta intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen, wherein the bispecific chimeric antigen receptor is co-expressed with truncated epidermal growth factor receptor (EGFRt), and wherein the bispecific chimeric antigen receptor and EGFRt are linked via a T2A linker.
[92] 92. The bispecific chimeric antigen receptor of claim 91, wherein the bispecific chimeric antigen receptor is co-expressed with a therapeutic control, comprising any one or more of thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-depedent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), a selection marker, and combinations thereof.
[93] 93. The bispecific chimeric antigen receptor of claim 91, wherein the EGFRt binds any one or more of an EGFR-specific siRNA, a small molecule, an anti-EGFR antibody or a fragment thereof, or a combination thereof.
[94] 94. The bispecific chimeric antigen receptor of claim 92, wherein the selection marker comprises any one or more of dihydroxyfolate receptor (DHFR), mutant DHFR, 67 WO 2013/123061 PCT/US2013/025953 methylated-DNA-protein-cysteine methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2) and combinations thereof.
[95] 95. The bispecific chimeric antigen receptor of claim 92, wherein the CCR comprises any one or more of (i) IL-7 cytokine-linker- IL7Ra, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Ra-transmembrane domain of IL-7Ra-cytoplasmic domain of IL-2R, (iii) IL-7 cytokine-linker-IL2RP, and (iv) combinations thereof.
[96] 96. The bispecific chimeric antigen receptor of claim 91, wherein each of the at least two antigen-specific targeting domains target an antigen independently selected from the group consisting of antigens specific for cancer, an inflammatory disease, a neuronal disorder, diabetes, a cardiovascular disease, an infectious disease, an autoimmune disease, and combinations thereof.
[97] 97. The bispecific chimeric antigen receptor of claim 96, wherein the antigen specific for cancer comprises any one or more of 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin a531, integrin av33, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, RORi, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-P, TRAIL-Ri, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof.
[98] 98. The bispecific chimeric antigen receptor of claim 96, wherein the at least two antigen specific targeting regions bind (i) CD19 and CD20, (ii) CD20 and Li-CAM, (iii) Li CAM and GD2, (iv) EGFR and LI-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and RORi. 68 WO 2013/123061 PCT/US2013/025953 Via Electronic Submission Attorney Docket No. 067505-000001WOOO
[99] 99. The bispecific chimeric antigen receptor of claim 96, wherein the at least two antigen specific targeting regions bind CD19 and CD20.
[100] 100. The bispecific chimeric antigen receptor of claim 96, wherein the antigen specific for an inflammatory disease comprises any one or more of AOC3 (VAP-1), CAM-3001, CCL1 1 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-i, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin a407, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb 37, scleroscin, SOST, TGF beta 1, TNF-a, VEGF-A, and combinations thereof.
[101] 101. The bispecific chimeric antigen receptor of claim 96, wherein the antigen specific for a neuronal disorder comprises any one or more of beta amyloid, MABT5102A, and combinations thereof.
[102] 102. The bispecific chimeric antigen receptor of claim 96, wherein the antigen specific for diabetes comprises any one or more of L-1 3, CD3, and combinations thereof.
[103] 103. The bispecific chimeric antigen receptor of claim 96, wherein the antigen-specific for a cardiovascular disease comprises any one or more of C5, cardiac myosin, CD41 (integrin alpha-Ib), fibrin II, beta chain, ITGB2 (CD18), sphingosine- 1-phosphate, and combinations thereof.
[104] 104. The bispecific chimeric antigen receptor of claim 96, wherein the antigen specific for an infectious disease comprises any one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-a, and combinations thereof. 69

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法律状态:
2018-03-08| FGA| Letters patent sealed or granted (standard patent)|

优先权:

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申请号 | 申请日 | 专利标题

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US201261598216P| true| 2012-02-13|2012-02-13||

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US61/598,216||2012-02-13||

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