Compositions and Methods for Treating Cancer with DuoCARs

Abstract

Novel therapeutic immunotherapy compositions comprising at least two vectors, each vector encoding a functional CAR, whereby the combination of vectors results in the expression of two or more non-identical binding domains, wherein each vector encoded binding domain(s) are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs are provided herein as well as are methods of use of same in a patient-specific immunotherapy that can be used to treat cancers and other diseases and conditions.

What is claimed is: 
     
         1 . An immunotherapy composition comprising one or more isolated nucleic acid molecules encoding at least two vectors, each vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62, wherein at least one binding domain(s) in one of the vectors are non-identical, and whereby the combination of vectors results in the expression of two or more non-identical binding domains, wherein each vector encoded binding domain(s) are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs. 
     
     
         2 . An immunotherapy composition comprising:
 (a) at least two vectors, each comprising nucleic acid sequences that are functional in cells;   (b) wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62;   (c) wherein each CAR comprises of at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif;   (d) wherein the at least one binding domains in one of the vectors are non-identical; and   (e) wherein the at least one binding domain, a single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each said vector, wherein the combination of vectors are used to genetically modify one or more lymphocyte populations.   
     
     
         3 . An immunotherapy composition comprising:
 (a) at least two vectors, each comprising nucleic acid sequences that are functional in cells;   (b) wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62;   (c) wherein each CAR comprises at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif;   (d) wherein the at least one binding domain(s) in each vector are non-identical;   (e) wherein the at least one signaling motif combinations are non-identical between each of the vectors; and   (f) wherein the at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each said vector, wherein the combination of two or more vectors are used to genetically modify one or more lymphocyte populations.   
     
     
         4 . The immunotherapy composition of  claims 1 - 3 , wherein each vector encodes more than one functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62. 
     
     
         5 . The immunotherapy composition of  claim 2  or  3 , wherein the lymphocyte population(s) comprise autologous T-cells or a mixture of peripheral blood derived lymphocytes. 
     
     
         6 . The immunotherapy composition of  claim 2  or  3 , wherein the at least one extracellular antigen binding domain of the CAR comprises at least one single chain variable fragment of an antibody that binds to the antigen. 
     
     
         7 . The immunotherapy composition of  claim 2  or  3 , wherein the at least one extracellular antigen binding domain of the CAR comprises at least one heavy chain variable region of an antibody that binds to the antigen. 
     
     
         8 . The immunotherapy composition of  claim 2  or  3 , wherein the at least one extracellular antigen binding domain of the CAR, the at least one intracellular signaling domain of the CAR, or both are connected to the transmembrane domain by a linker or spacer domain. 
     
     
         9 . The immunotherapy composition of  claim 2  or  3 , wherein the extracellular antigen binding domain of the CAR is preceded by a leader peptide. 
     
     
         10 . The immunotherapy composition of  claim 2  or  3 , wherein the extracellular antigen binding domain of the CAR targets an antigen comprising CD19, CD20, CD22, ROR1, TSLPR, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof. 
     
     
         11 . The immunotherapy composition of  claim 2  or  3 , wherein the extracellular antigen binding domain of the CAR comprises an anti-CD19 scFV antigen binding domain, an anti-CD20 scFV antigen binding domain, an anti-CD22 scFV antigen binding domain, an anti-ROR1 scFV antigen binding domain, an anti-TSLPR scFV antigen binding domain, an anti-mesothelin scFV antigen binding domain, an anti-CD33 scFV antigen binding domain, an anti-CD38 scFV antigen binding domain, an anti-CD123 (IL3RA) scFV antigen binding domain, an anti-CD138 scFV antigen binding domain, an anti-BCMA (CD269) scFV antigen binding domain, an anti-GPC2 scFV antigen binding domain, an anti-GPC3 scFV antigen binding domain, an anti-FGFR4 scFV antigen binding domain, an anti-c-Met scFV antigen binding domain, an anti-PMSA scFV antigen binding domain, an anti-glycolipid F77 scFV antigen binding domain, an anti-EGFRvIII scFV antigen binding domain, an anti-GD-2 scFV antigen binding domain, an anti-NY-ESo-1 TCR scFV antigen binding domain, an anti-MAGE A3 TCR scFV antigen binding domain, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, or any combination thereof. 
     
     
         12 . The immunotherapy composition of  claim 2  or  3 , wherein the linker or spacer domain of the CAR is derived from the extracellular domain of CD8, and is linked to the transmembrane domain. 
     
     
         13 . The immunotherapy composition of  claim 2  or  3 , wherein the CAR further comprises a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, or any combination thereof. 
     
     
         14 . The immunotherapy composition of  claim 2  or  3 , wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain. 
     
     
         15 . The immunotherapy composition of  claim 2  or  3 , wherein the at least one intracellular signaling domain is arranged on a C-terminal side relative to the CD3 zeta intracellular domain. 
     
     
         16 . The immunotherapy composition of  claim 2  or  3 , wherein the at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof. 
     
     
         17 . The immunotherapy composition of  claim 16 , wherein the at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof. 
     
     
         18 . The immunotherapy composition of  claims 1 - 3 , wherein a single viral vector is used to encode all chimeric antigen receptors (e.g. adeno, SV40, herpes vector, POX vector, or cosmid vector), in combination with CRISPR system for integration. 
     
     
         19 . The immunotherapy composition of  claims 1 - 3 , wherein each vector is an RNA or DNA vector. 
     
     
         20 . The immunotherapy composition of  claims 1 - 3 , wherein at least one vector expresses a nucleic acid molecule that modulates the expression of a nucleic acid in the cell. 
     
     
         21 . The therapeutic composition of  claim 20 , wherein the nucleic acid molecule inhibits or deletes the expression of an endogenous gene. 
     
     
         22 . A pharmaceutical composition comprising an antitumor effective amount of a population of human lymphocyte cells, wherein the cells of the population include cells comprising nucleic acid molecules encoding at least two vectors, each vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62, wherein at least one binding domain(s) in one of the vectors are non-identical, and whereby the combination of vectors results in the expression of two or more non-identical binding domains, wherein each vector encoded binding domain(s) are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs. 
     
     
         23 . A pharmaceutical composition comprising an antitumor effective amount of a population of human lymphocyte cells, wherein the cells of the population include cells comprising (a) nucleic acid molecules encoding two or more vectors; (b) wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62; (c) wherein each CAR comprises of at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif; (d) wherein the at least one binding domains in one of the vectors are non-identical; and (e) wherein the at least one binding domain, a single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each said vector, wherein the combination of vectors are used to genetically modify one or more lymphocyte populations. 
     
     
         24 . A pharmaceutical composition comprising an antitumor effective amount of a population of human lymphocyte cells, wherein the cells of the population include cells comprising (a) nucleic acid molecules encoding two or more vectors; (b) wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62; (c) wherein each CAR comprises at least one binding domain, at least one transmembrane domain, at least one linker domain, and at least one intracellular signaling motif, (d) wherein the at least one binding domain(s) in each vector are non-identical; (e) wherein the at least one signaling motif combinations are non-identical between each of the vectors; and (f) wherein the at least one binding domain, a single transmembrane domain, at least one linker domain, and at least one intracellular signaling motif are covalently linked in each said vector, wherein the combination of two or more vectors are used to genetically modify one or more lymphocyte populations. 
     
     
         25 . The pharmaceutical composition of  claim 23  or  24 , wherein the lymphocyte cells are T cells of a human having a hematological cancer. 
     
     
         27 . The pharmaceutical composition of  claim 23  or  24 , wherein the hematological cancer is leukemia or lymphoma. 
     
     
         28 . The pharmaceutical composition of  claim 23  or  24 , wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML). 
     
     
         29 . The pharmaceutical composition of  claim 23  or  24 , wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma. 
     
     
         30 . The pharmaceutical composition of  claim 23  or  24 , wherein the hematological cancer is multiple myeloma. 
     
     
         31 . The pharmaceutical composition of  claim 23  or  24 , wherein the human cancer includes an adult carcinoma comprising coral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bones and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, the genital system (uterine cervix, uterine corpus, ovary, vulva, vagina, prostate, testis, penis, endometrium), the urinary system (urinary bladder, kidney and renal pelvis, ureter), the eye and orbit, the endocrine system (thyroid), and the brain and other nervous system, or any combination thereof. 
     
     
         32 . A method of treating a mammal having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising at least two vectors, each vector encoding a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62, wherein at least one binding domain(s) in one of the vectors are non-identical, and whereby the combination of vectors results in the expression of two or more non-identical binding domains, wherein each vector encoded binding domain(s) are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and a pharmaceutically acceptable excipient, wherein the combination of vectors are used to genetically modify one or more lymphocyte populations. 
     
     
         33 . A method of treating a mammal having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising (a) nucleic acid molecules encoding two or more vectors; (b) wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62; (c) wherein each CAR comprises of at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif; (d) wherein the at least one binding domains in one of the vectors are non-identical; and (e) wherein the at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each said vector, wherein the combination of vectors are used to genetically modify one or more lymphocyte populations. 
     
     
         34 . A method of treating a mammal having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising (a) nucleic acid molecules encoding two or more vectors; (b) wherein each vector encodes a functional CAR comprising the amino acid sequence of SEQ ID NO. 4, 10, 22, 26, 30, 32, 36, 40, 44, 48, 50, 52, 54, 56, 60, or 62; (c) wherein each CAR comprises at least one binding domain, at least one transmembrane domain, and at least one intracellular signaling motif; (d) wherein the at least one binding domain(s) in each vector are non-identical; (e) wherein the at least one signaling motif combinations are non-identical between each of the vectors; and (f) wherein the at least one binding domain, a single transmembrane domain, and at least one intracellular signaling motif are covalently linked in each said vector, wherein the combination of two or more vectors are used to genetically modify one or more lymphocyte populations. 
     
     
         35 . The method of  claims 32 - 34 , wherein the genetically modified lymphocytes are autologous lymphocytes, and wherein the autologous or allogeneic lymphocytes are infused directly back into the patient so as to prevent malignant disease relapse. 
     
     
         36 . The method of  claims 32 - 34 , wherein the genetically modified lymphocytes are autologous T-cells, and wherein the autologous T-cells are infused directly back into the patient to promote in vivo expansion, persistence of patient-specific anti-tumor T-cells resulting in tumor stabilization, reduction, elimination, remission, or elimination of cancer or relapse of cancer in a patient-specific manner. 
     
     
         37 . The method of  claims 32 - 34 , wherein the T cell has been preselected by virtue of expressing specific activation or memory-associated surface markers. 
     
     
         38 . The method of  claims 32 - 34 , wherein the T cell and dendritic cells are derived from a hematopoietic stem cell donor, and wherein the procedure is carried out in the context of hematopoietic stem cell transplantation.