Method and compositions for cellular immunotherapy

Abstract

The present invention provides nucleic acids, vectors, host cells, methods and compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively transferring CD8+ central memory T cells or combinations of central memory T cells with CD4+ T cells that are genetically modified to express a chimeric receptor. In embodiments the genetically modified host cell comprises a nucleic acid comprising a polynucleotide coding for a ligand binding domain, a polynucleotide comprising a customized spacer region, a polynucleotide comprising a transmembrane domain, and a polynucleotide comprising an intracellular signaling domain. It has been surprisingly found that the length of the spacer region can affects the ability of chimeric receptor modified T cells to recognize target cells in vitro and affects in vivo efficacy of the chimeric receptor modified T cells. Pharmaceutical formulations produced by the method, and methods of using the same, are also described.

That which is claimed is: 
     
         1 . A nucleic acid encoding a chimeric receptor, the nucleic acid comprising:
 (a) a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to an epitope in the Ig-like/Frizzled domain of a Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1);   (b) a polynucleotide encoding a transmembrane domain;   (c) a polynucleotide encoding a polypeptide spacer located between the antibody or antigen-binding fragment thereof and the transmembrane domain, wherein the polypeptide spacer is about 15 amino acids or less in length and comprises an amino acid sequence of X 1 PPX 2 P, wherein X 1  is a cysteine, glycine, or arginine and X 2  is a cysteine or a threonine (SEQ ID NO:1); and   (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the antibody or antigen-binding fragment thereof comprises:
 a variable light chain (VL) region having a least 90% sequence identity to the VL region of the anti-ROR1 antibody R12; and a variable heavy chain (VH) region having at least 90% identity to the VH region of the anti-ROR1 antibody R12;   a variable light chain (VL) region comprising a CDRL1, a CDRL2, and a CDRL3 of the anti-ROR1 antibody R12, and a variable heavy chain (VH) region comprising a CDRH1, a CDRH2, and a CDRH3 of the anti-ROR1 antibody R12; or   a variable light chain (VL) region of the anti-ROR1 antibody 2A2; and a variable heavy chain (VH) region of the anti-ROR1 antibody 2A2.   
     
     
         3 . The nucleic acid of  claim 1 , wherein the polypeptide spacer comprises a human IgG1, IgG2, or IgG4 hinge or a modified version thereof, the polypeptide spacer comprises the amino acid sequence set forth in SEQ ID NO:17, 18, 20, 21, 51, 52, or 53; or the polypeptide spacer consists of the amino acid sequence set forth in SEQ ID NO:21. 
     
     
         4 . The nucleic acid of  claim 1 , wherein the CD3ζ signaling domain comprises the amino acid sequence encoded by SEQ ID NO:7. 
     
     
         5 . The nucleic acid of  claim 1 , wherein the transmembrane domain comprises a transmembrane domain of a CD8 or of a CD28 comprising the amino acid sequence encoded by SEQ ID NO:5 or encoded by the nucleotide sequence atgttctgggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg or comprising the amino acid sequence MFWVLVVVGGVLACYSLLVTVAFIIFWV. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the costimulatory domain comprises:
 the signaling domain of a 4-1BB or a modified version thereof, optionally wherein the 4-1BB signaling domain or a modified version thereof comprises amino acids 214-255 of SEQ ID NO:15 or comprises the amino acid sequence encoded by SEQ ID NO:6; or   the signaling domain of a CD28 or a modified version thereof, optionally wherein the CD28 signaling domain or a modified version thereof comprises amino acids 180-220 of SEQ ID NO:14 or comprises a modified version thereof comprising an LL→GG substitution located at positions 186-187 of SEQ ID NO:14.   
     
     
         7 . The nucleic acid of  claim 1 , wherein:
 the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) comprising a variable light chain (VL) region of the anti-ROR1 antibody R12; and a variable heavy chain (VH) region of the anti-ROR1 antibody R12 or the sequence   
       
         
           
                 
               
                   QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATI 
                 
                     
                 
                   YPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYA 
                 
                     
                 
                   DDALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPA 
                 
                     
                 
                   KITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGS 
                 
                     
                 
                   SSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG; 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
         the polypeptide spacer comprises the amino acid sequence of SEQ ID NO:21; 
         the transmembrane domain comprises the amino acid sequence encoded by SEQ ID NO:5; and 
         the intracellular signaling domain comprises a 4-1BB signaling domain comprising the amino acid sequence encoded by SEQ ID NO:6 and a CD3ζ signaling domain comprising the amino acid sequence encoded by of SEQ ID NO:7; or 
         wherein the nucleic acid encodes a chimeric receptor encoded by a nucleic acid comprising the sequence of SEQ ID NO:41 or a sequence having at least 90% identity to the sequence of SEQ ID NO:41. 
       
     
     
         8 . A nucleic acid encoding a chimeric receptor, the nucleic acid comprising:
 (a) a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to an epitope in the Kringle domain of a Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1);   (b) a polynucleotide encoding a transmembrane domain;   (c) a polynucleotide encoding a polypeptide spacer located between the antibody or antigen-binding fragment thereof and the transmembrane domain, wherein the polypeptide spacer is an immunoglobulin hinge-CH2-CH3 region; and   (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.   
     
     
         9 . The nucleic acid of  claim 8 , wherein the antibody or antigen-binding fragment thereof comprises:
 a variable light chain (VL) region having a least 90% sequence identity to the VL region of the anti-ROR1 antibody R11; and a variable heavy chain (VH) region having at least 90% identity to the VH region of the anti-ROR1 antibody R11; or   a variable light chain (VL) region comprising a CDRL1, a CDRL2, and a CDRL3 of the anti-ROR1 antibody R11, and a variable heavy chain (VH) region comprising a CDRH1, a CDRH2, and a CDRH3 of the anti-ROR1 antibody R11.   
     
     
         10 . The nucleic acid of  claim 8 , wherein the polypeptide spacer comprises a human IgG4 hinge region or modified version thereof, a human IgG4 CH2 domain and a human IgG4 CH3 domain; or the polypeptide spacer comprises the amino acid sequence set forth in SEQ ID NO:50. 
     
     
         11 . The nucleic acid of  claim 8 , wherein the CD3ζ signaling domain comprises the amino acid sequence encoded by SEQ ID NO:7. 
     
     
         12 . The nucleic acid of  claim 8 , wherein the transmembrane domain comprises a transmembrane domain of a CD8 or of a CD28 comprising the amino acid sequence encoded by SEQ ID NO:5 or encoded by the nucleotide sequence atgttctgggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg or comprising the amino acid sequence MFWVLVVVGGVLACYSLLVTVAFIIFWV. 
     
     
         13 . The nucleic acid of  claim 8 , wherein the costimulatory domain comprises:
 the signaling domain of a 4-1BB or a modified version thereof, optionally wherein the 4-1BB signaling domain or a modified version thereof comprises amino acids 214-255 of SEQ ID NO:15 or comprises the amino acid sequence encoded by SEQ ID NO:6; or   the signaling domain of a CD28 or a modified version thereof, optionally wherein the CD28 signaling domain or a modified version thereof comprises amino acids 180-220 of SEQ ID NO:14 or comprises a modified version thereof comprising an LL→GG substitution located at positions 186-187 of SEQ ID NO:14.   
     
     
         14 . The nucleic acid of  claim 8 , wherein:
 the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) comprising a variable light chain (VL) region of the anti-ROR1 antibody R11; and a variable heavy chain (VH) region of the anti-ROR1 antibody R11 or the amino acid sequence   
       
         
           
                 
               
                   QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFIN 
                 
                     
                 
                   SGGSTWYASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGD 
                 
                     
                 
                   FNIWGPGTLVTISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTIN 
                 
                     
                 
                   CQASQSIDSNLAWFQQKPGQPPTLLIYRASNLASGVPSRFSGSRSGTEYTL 
                 
                     
                 
                   TISGVQREDAATYYCLGGVGNVSYRTSFGGGTEVVVK; 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
         the polypeptide spacer comprises the amino acid sequence of SEQ ID NO:50; 
         the transmembrane domain comprises the amino acid sequence encoded by SEQ ID NO:5; and 
         the intracellular signaling domain comprises a 4-1BB signaling domain comprising the amino acid sequence encoded by SEQ ID NO:6 and a CD3ζ signaling domain comprising the amino acid sequence encoded by of SEQ ID NO:7; or 
         wherein the nucleic acid encodes a chimeric receptor encoded by a nucleic acid comprising the sequence of SEQ ID NO:43 or a sequence having at least 90% identity to the sequence of SEQ ID NO:43. 
       
     
     
         15 . A nucleic acid encoding a chimeric receptor, the nucleic acid comprising:
 (a) a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to Receptor tyrosine-protein kinase erbB-2 (HER2);   (b) a polynucleotide encoding a transmembrane domain;   (c) a polynucleotide encoding a polypeptide spacer located between the antibody or antigen-binding fragment thereof and the transmembrane domain, wherein the polypeptide spacer is an immunoglobulin hinge-CH2-CH3 region; and   (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.   
     
     
         16 . The nucleic acid of  claim 15 , wherein the antibody or antigen-binding fragment thereof comprises:
 a variable light chain (VL) region having a least 90% sequence identity to the VL region of the anti-HER2 antibody Herceptin; and a variable heavy chain (VH) region having at least 90% identity to the VH region of the anti-HER2 antibody Herceptin; or   a variable light chain (VL) region comprising a CDRL1, a CDRL2, and a CDRL3 of the anti-HER2 antibody Herceptin, and a variable heavy chain (VH) region comprising a CDRH1, a CDRH2, and a CDRH3 of the anti-HER2 antibody Herceptin.   
     
     
         17 . The nucleic acid of  claim 15 , wherein the polypeptide spacer comprises a human IgG4 hinge region or modified version thereof, a human IgG4 CH2 domain and a human IgG4 CH3 domain; or the polypeptide spacer comprises the amino acid sequence set forth in SEQ ID NO:50. 
     
     
         18 . The nucleic acid of  claim 15 , wherein the CD3ζ signaling domain comprises the amino acid sequence encoded by SEQ ID NO:7. 
     
     
         19 . The nucleic acid of  claim 15 , wherein the transmembrane domain comprises a transmembrane domain of a CD8 or of a CD28 comprising the amino acid sequence encoded by SEQ ID NO:5 or encoded by the nucleotide sequence atgttctgggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg or comprising the amino acid sequence MFWVLVVVGGVLACYSLLVTVAFIIFWV. 
     
     
         20 . The nucleic acid of  claim 15 , wherein the costimulatory domain comprises:
 the signaling domain of a 4-1BB or a modified version thereof, optionally wherein the 4-1BB signaling domain or a modified version thereof comprises amino acids 214-255 of SEQ ID NO:15 or comprises the amino acid sequence encoded by SEQ ID NO:6; or   the signaling domain of a CD28 or a modified version thereof, optionally wherein the CD28 signaling domain or a modified version thereof comprises amino acids 180-220 of SEQ ID NO:14 or comprises a modified version thereof comprising an LL→GG substitution located at positions 186-187 of SEQ ID NO:14.   
     
     
         21 . The nucleic acid of  claim 15 , wherein:
 the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) comprising a variable light chain (VL) of the anti-HER2 antibody Herceptin and a variable heavy chain (VH) region of the anti-HER2 antibody Herceptin or the amino acid sequence encoded by   
       
         
           
                 
               
                   gcattcctcctgatcccagatatccagatgacccagtccccgagctccctg 
                 
                     
                 
                   tccgcctctgtgggcgatagggtcaccatcacctgccgtgccagtcaggat 
                 
                     
                 
                   gtgaatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaa 
                 
                     
                 
                   ctactgatttactcggcatccttcctctactctggagtccettctcgcttc 
                 
                     
                 
                   tctggttccagatctgggacggatttcactctgaccatcagcagtctgcag 
                 
                     
                 
                   ccggaagacttcgcaacttattactgtcagcaacattatactactcctccc 
                 
                     
                 
                   acgttcggacagggtaccaaggtggagatcaaaggcagtactagcggcggt 
                 
                     
                 
                   ggctccgggggcggatccggtgggggcggcagcagcgaggttcagctggtg 
                 
                     
                 
                   gagtctggcggtggcctggtgcagccagggggctcactccgtttgtectgt 
                 
                     
                 
                   gcagettctggcttcaacattaaagacacctatatacactgggtgcgtcag 
                 
                     
                 
                   gccccgggtaagggcctggaatgggttgcaaggatttatcctacgaatggt 
                 
                     
                 
                   tatactagatatgccgatagcgtcaagggccgtttcactataagcgcagac 
                 
                     
                 
                   acatccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggac 
                 
                     
                 
                   actgccgtctattattgttctagatggggaggggacggcttctatgctatg 
                 
                     
                 
                   gactactggggtcaaggaaccctggtcaccgtctcgagt; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         the polypeptide spacer comprises the amino acid sequence of SEQ ID NO:50; 
         the transmembrane domain comprises the amino acid sequence encoded by atgttctgggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg; and 
         the intracellular signaling domain comprises a 4-1BB signaling domain comprising the amino acid sequence encoded by SEQ ID NO:6 and a CD3ζ signaling domain comprising the amino acid sequence encoded by of SEQ ID NO:7; or 
         wherein the nucleic acid encodes a chimeric receptor encoded by a nucleic acid comprising the sequence of SEQ ID NO:56 or a sequence having at least 90% identity to the sequence of SEQ ID NO:56. 
       
     
     
         22 . A chimeric receptor polypeptide encoded by the nucleic acid of  claim 1 . 
     
     
         23 . An expression vector, comprising the nucleic acid of  claim 1 . 
     
     
         24 . A host cell comprising the nucleic acid of  claim 1 . 
     
     
         25 . A composition comprising the host cell of  claim 24  in a pharmaceutically acceptable excipient. 
     
     
         26 . A chimeric receptor polypeptide encoded by the nucleic acid of  claim 8 . 
     
     
         27 . An expression vector, comprising the nucleic acid of  claim 8 . 
     
     
         28 . A host cell comprising the nucleic acid of  claim 8 . 
     
     
         29 . A composition comprising the host cell of  claim 28  in a pharmaceutically acceptable excipient. 
     
     
         30 . A chimeric receptor polypeptide encoded by the nucleic acid of  claim 15 . 
     
     
         31 . An expression vector, comprising the nucleic acid of  claim 15 . 
     
     
         32 . A host cell comprising the nucleic acid of  claim 15 . 
     
     
         33 . A composition comprising the host cell of  claim 32  in a pharmaceutically acceptable excipient. 
     
     
         34 . An in vitro method for preparing a host cell, comprising:
 (1) introducing a nucleic acid encoding a chimeric receptor into cells of a cell population, wherein the nucleic acid comprises:   (a) a polynucleotide encoding a ligand binding domain that binds to an epitope in an extracellular region of a ligand, wherein the ligand is expressed on a tumor or is a viral molecule;   (b) a polynucleotide encoding a transmembrane domain;   (c) a polynucleotide encoding a polypeptide spacer located between the antibody or antigen-binding fragment thereof and the transmembrane domain, wherein the polypeptide spacer is about 10 to about 229 amino acids or less in length and comprises an amino acid sequence of X 1 PPX 2 P, wherein X 1  is a cysteine, glycine, or arginine and X 2  is a cysteine or a threonine (SEQ ID NO:1); and   (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain; and   (2) culturing the cells in the presence of at least one homeostatic cytokine.   
     
     
         35 . The method of  claim 34 , wherein the ligand is a CD19, a ROR1 or a HER2. 
     
     
         36 . The method of  claim 34 , wherein the cell population comprises lymphocytes. 
     
     
         37 . The method of  claim 36 , wherein the lymphocyte has a CD45RA−, CD45RO+, and CD62L+ phenotype. 
     
     
         38 . A method of treating a subject having a cancer or a viral infection expressing a ligand, the method comprising administering to a subject a host cell comprising a nucleic acid encoding a chimeric receptor into cells of a cell population, wherein the nucleic acid comprises:
 (a) a polynucleotide encoding a ligand binding domain that binds to an epitope in an extracellular region of a ligand, wherein the ligand is expressed on a tumor or is a viral molecule;   (b) a polynucleotide encoding a transmembrane domain;   (c) a polynucleotide encoding a polypeptide spacer located between the antibody or antigen-binding fragment thereof and the transmembrane domain, wherein the polypeptide spacer is about 10 to about 229 amino acids or less in length and comprises an amino acid sequence of X 1 PPX 2 P, wherein X 1  is a cysteine, glycine, or arginine and X 2  is a cysteine or a threonine (SEQ ID NO:1); and   (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.   
     
     
         39 . The method of  claim 38 , wherein the cancer is a solid tumor or a hematologic malignancy; and/or wherein the cancer is selected from the group consisting of a lymphoma, a leukemia and an epithelial cancer; and/or wherein the cancer is a tumor, which tumor is a B cell malignancy, a CLL or an MCL, or which tumor is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a renal cancer, a pancreatic cancer, a prostate cancer, and an ovarian cancer. 
     
     
         40 . The method of  claim 39 , wherein the ligand is a CD19, a ROR1 or a HER2. 
     
     
         41 . An in vitro method for identifying a chimeric receptor, comprising:
 (a) providing a library of nucleic acids encoding a chimeric receptor, wherein each of the plurality of nucleic acids encodes a chimeric receptor that differs in spacer length;   (b) introducing each of the plurality of the nucleic acids into a separate isolated T cell population and expanding each T cell population in vitro;   (c) administering each genetically modified T cell population into an animal model bearing a tumor and determining whether a genetically modified T cell population has anti-tumor efficacy; and   (d) selecting a nucleic acid encoding the chimeric receptor that provides for anti-tumor efficacy in vitro and/or in an animal model.   
     
     
         42 . The method of  claim 41 , further comprising introducing the selected nucleic acid encoding the chimeric receptor into a host cell.