Compositions and methods for treating cancer with anti-cd19/cd20 immunotherapy

Abstract

Chimeric antigen receptors containing CD19/CD20 or CD20/CD19 antigen binding domains are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the chimeric antigen receptors are also disclosed. Methods of treating or preventing cancer in a subject, and methods of making chimeric antigen receptor T cells are also disclosed.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule encoding a CD19/CD20 tandem chimeric antigen receptor (CAR) comprising at least one extracellular antigen binding domain comprising a CD19/CD20 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the CD19/CD20 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO. 1 or 3. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded at least one CD19/CD20 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD19/CD20. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded at least one CD19/CD20 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19/CD20. 
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded at least one CD19/CD20 antigen binding domain, the at least one intracellular signaling domain, or both are connected to the transmembrane domain by a linker or spacer domain. 
     
     
         5 . The isolated nucleic acid molecule of  claim 4 , wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 or CD28, and is linked to a transmembrane domain. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded extracellular CD19/CD20 antigen binding domain is preceded by a leader nucleotide sequence encoding a leader peptide. 
     
     
         7 . The isolated nucleic acid molecule of  claim 6 , wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 11 encoding the leader amino acid sequence of SEQ ID NO: 12. 
     
     
         8 . The isolated nucleic acid molecule of  claim 1 , wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof. 
     
     
         9 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence encoding the CD19/CD20 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO. 1 or 3, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof. 
     
     
         10 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain. 
     
     
         11 . The isolated nucleic acid molecule of  claim 10 , wherein the encoded at least one intracellular signaling domain is arranged on a C-terminal side relative to the CD3 zeta intracellular domain. 
     
     
         12 . The isolated nucleic acid molecule of  claim 1 , wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof. 
     
     
         13 . The isolated nucleic acid molecule of  claim 12 , wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof. 
     
     
         14 . A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of  claim 1 . 
     
     
         15 . The CAR of  claim 14 , comprising at least one extracellular antigen binding domain comprising a CD19/CD20 antigen binding domain comprising the amino acid sequence of SEQ ID NO. 6, 8, or 10, at least one transmembrane domain, and at least one intracellular signaling domain. 
     
     
         16 . The CAR of  claim 15 , wherein the CD19/CD20 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD19/CD20. 
     
     
         17 . The CAR of  claim 15 , wherein the CD19/CD20 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19/CD20. 
     
     
         18 . The CAR of  claim 15 , wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19 or any combination thereof. 
     
     
         19 . The CAR of  claim 18 , wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO: 28. 
     
     
         20 . The CAR of  claim 15 , wherein the at least one extracellular antigen binding domain comprising a CD19/CD20 antigen binding domain comprising the amino acid sequence of SEQ ID NO. 6, 8, or 10, and the at least one intracellular signaling domain, or both are connected to the transmembrane domain by a linker or spacer domain. 
     
     
         21 . The CAR of  claim 20 , wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28, and is linked to a transmembrane domain. 
     
     
         22 . The CAR of  claim 17 , wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain. 
     
     
         23 . The CAR of  claim 22 , wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof. 
     
     
         24 . A vector comprising a nucleic acid molecule of  claim 1 . 
     
     
         25 . The vector of  claim 24 , wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, adenoviral vector, or a retrovirus vector, or a combination thereof. 
     
     
         26 . The vector of  claim 24 , further comprising a promoter. 
     
     
         27 . The vector of  claim 26 , wherein the promoter is an inducible promoter, a constitutive promoter, a tissue specific promoter, a suicide promoter or any combination thereof. 
     
     
         28 . A cell comprising the vector of  claim 24 . 
     
     
         29 . The cell of  claim 28 , wherein the cell is a T cell. 
     
     
         30 . The cell of  claim 28 , wherein the T cell is a CD8+ T cell. 
     
     
         31 . The cell of  claim 28 , wherein the cell is a human cell. 
     
     
         32 . A method of making a cell comprising transducing a T cell with a vector of  claim 24 . 
     
     
         33 . A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid molecule of  claim 1 . 
     
     
         34 . A method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of a cell of  claim 28 . 
     
     
         35 . A method of treating or preventing cancer in a mammal, comprising administering to the mammal the CAR of  claim 15 , in an amount effective to treat or prevent cancer in the mammal. 
     
     
         36 . A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising a CD19/CD20 antigen binding domain comprising the amino acid sequence of SEQ ID NO. 2 or 4, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a human having a cancer. 
     
     
         37 . The pharmaceutical composition of  claim 36 , wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof. 
     
     
         38 . The pharmaceutical composition of  claim 36 , wherein the T cells are T cells of a human having a hematological cancer. 
     
     
         39 . The pharmaceutical composition of  claim 38 , wherein the hematological cancer is leukemia or lymphoma. 
     
     
         40 . The pharmaceutical composition of  claim 39 , wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML). 
     
     
         41 . The pharmaceutical composition of  claim 39 , wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma. 
     
     
         42 . The pharmaceutical composition of  claim 38 , wherein the hematological cancer is multiple myeloma. 
     
     
         43 . The pharmaceutical composition of  claim 36 , wherein the human cancer includes an adult carcinoma comprising oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, interhepatic 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. 
     
     
         44 . 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 an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising a CD19/CD20 antigen binding domain comprising the amino acid sequence of SEQ ID NO. 2 or 4, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cells are T cells of the subject having cancer. 
     
     
         45 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising a CD19/CD20 antigen binding domain comprising the amino acid sequence of SEQ ID NO. 2 or 4, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cells are T cells of the subject having cancer. 
     
     
         46 . The method of  claim 44  or  45 , wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof. 
     
     
         47 . A process for producing a chimeric antigen receptor-expressing cell, the process comprising introducing the isolated nucleic acid of  claim 1  into a cell. 
     
     
         48 . The process for producing a chimeric antigen receptor-expressing cell according to  claim 47 , wherein the cell is a T cell or a cell population containing a T cell.