Co-stimulatory domains for use in genetically-modified cells

Abstract

The present disclosure provides novel co-stimulatory domains useful in genetically-modified cells to promote cell proliferation and/or promote cytokine secretion after antigen recognition. For example, disclosed herein are genetically-modified cells comprising a chimeric antigen receptor or an inducible regulatory construct incorporating the co-stimulatory domains disclosed herein. Also disclosed herein are plasmids and viral vectors comprising a nucleic acid sequence encoding the co-stimulatory domains, and methods of administering compositions comprising the novel co-stimulatory domains to subjects in order to reduce the symptoms, progression, or occurrence of disease, such as cancer.

1 . A nucleic acid molecule comprising a nucleotide sequence encoding a co-stimulatory domain having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 8, 5, 6, or 7. 
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein said co-stimulatory domain comprises at least one TRAF-binding motif. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein said TRAF-binding motif is selected from the group consisting of SEQ ID NOs: 10, 9, and 11. 
     
     
         4 . The nucleic acid molecule of any one of  claims 1 - 3 , wherein said co-stimulatory domain comprises two TRAF-binding motifs separated by a spacer sequence. 
     
     
         5 . The nucleic acid molecule of  claim 4 , wherein said spacer is selected from the group consisting of SEQ ID NOs: 15 and 12-14. 
     
     
         6 . The nucleic acid molecule of any one of  claims 1 - 5 , wherein said co-stimulatory domain comprises two TRAF-binding motifs selected from the group consisting of SEQ ID NOs: 10, 9, and 11 separated by a spacer sequence, wherein said spacer sequence is selected from the group consisting of SEQ ID NOs: 15 and 12-14. 
     
     
         7 . The nucleic acid molecule of any one of  claims 1 - 6 , wherein said co-stimulatory domain comprises TRAF binding motifs of SEQ ID NOs: 9 and 11, separated by a spacer sequence. 
     
     
         8 . The nucleic acid molecule of  claim 7 , wherein said spacer sequence comprises SEQ ID NO: 12. 
     
     
         9 . The nucleic acid molecule of  claim 7  or  claim 8 , wherein said nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1, wherein said nucleotide sequence encodes a co-stimulatory domain. 
     
     
         10 . The nucleic acid molecule of any one of  claims 7 - 9 , wherein said nucleotide sequence encodes a co-stimulatory domain comprising an amino acid sequence set forth in SEQ ID NO: 5. 
     
     
         11 . The nucleic acid molecule of any one of  claims 1 - 6 , wherein said co-stimulatory domain comprises TRAF binding motifs of SEQ ID NOs: 10 and 11, separated by a spacer sequence. 
     
     
         12 . The nucleic acid molecule of  claim 11 , wherein said spacer sequence is selected from the group consisting of SEQ ID NOs: 15, 13, and 14. 
     
     
         13 . The nucleic acid molecule of  claim 11  or  claim 12 , wherein said nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 2, or 3, wherein said nucleotide sequence encodes a co-stimulatory domain. 
     
     
         14 . The nucleic acid molecule of any one of  claims 11 - 13 , wherein said nucleotide sequence encodes a co-stimulatory domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 8, 6, or 7. 
     
     
         15 . The nucleic acid molecule of any one of  claims 1 - 14 , wherein said nucleic acid molecule comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein said CAR comprises at least one co-stimulatory domain of any one of  claims 1 - 14 . 
     
     
         16 . The nucleic acid molecule of  claim 15 , wherein said CAR comprises at least one co-stimulatory domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 8, 5, 6, or 7. 
     
     
         17 . The nucleic acid molecule of  claim 15  or  claim 16 , wherein said CAR comprises at least two co-stimulatory domains, wherein at least one of said co-stimulatory domains is a co-stimulatory domain of any one of  claims 1 - 14 . 
     
     
         18 . The nucleic acid molecule of  claim 17 , wherein said CAR comprises at least two co-stimulatory domains of any one of  claims 1 - 14 . 
     
     
         19 . The nucleic acid molecule of any one of  claims 15 - 18 , wherein said CAR further comprises at least one intracellular signaling domain. 
     
     
         20 . The nucleic acid molecule of  claim 19 , wherein said intracellular signaling domain is a CD3 ζ domain. 
     
     
         21 . The nucleic acid molecule of any one of  claims 1 - 14 , wherein said nucleic acid molecule comprises a nucleotide sequence encoding an inducible regulatory construct, wherein said inducible regulatory construct comprises at least one co-stimulatory domain of any one of  claims 1 - 14 . 
     
     
         22 . The nucleic acid molecule of  claim 21 , wherein said inducible regulatory construct further comprises a binding domain which allows two of said inducible regulatory constructs to dimerize, wherein dimerization initiates a co-stimulatory signal to a cell. 
     
     
         23 . The nucleic acid molecule of  claim 22 , wherein said binding domain binds a small molecule or an antibody. 
     
     
         24 . The nucleic acid molecule of  claim 22  or  claim 23 , wherein said binding domain comprises an analogue of FKBP12. 
     
     
         25 . The nucleic acid molecule of any one of  claims 1 - 24 , wherein said nucleic acid molecule is an mRNA, a recombinant DNA construct, or a viral genome of a viral vector. 
     
     
         26 . A recombinant DNA construct comprising said nucleic acid molecule of any one of  claims 1 - 24 . 
     
     
         27 . The recombinant DNA construct of  claim 26 , wherein said recombinant DNA construct encodes a viral vector, wherein said viral vector comprises said nucleic acid molecule of any one of  claims 1 - 24 . 
     
     
         28 . The recombinant DNA construct of  claim 27 , wherein said viral vector is a recombinant adeno-associated viral (AAV) vector. 
     
     
         29 . A viral vector comprising said nucleic acid molecule of any one of  claims 1 - 24 . 
     
     
         30 . The viral vector of  claim 29 , wherein said viral vector is a recombinant AAV vector. 
     
     
         31 . A genetically-modified cell comprising said nucleic acid molecule of any one of  claims 1 - 24 . 
     
     
         32 . The genetically-modified cell of  claim 31 , wherein said genetically-modified cell comprises an expression cassette comprising said nucleic acid molecule of any one of  claims 1 - 24 . 
     
     
         33 . The genetically-modified cell of  claim 32 , wherein said nucleic acid molecule or said expression cassette is present within the genome of said genetically-modified cell. 
     
     
         34 . The genetically-modified cell of  claim 32 , wherein said nucleic acid molecule or said expression cassette is not integrated into the genome of said genetically-modified cell. 
     
     
         35 . The genetically-modified cell of  claim 34 , wherein said nucleic acid molecule or said expression cassette is present in said genetically-modified cell in a recombinant DNA construct, in an mRNA, or in a viral genome, which are not integrated into the genome of said genetically-modified cell. 
     
     
         36 . The genetically-modified cell of any one of  claims 31 - 35 , wherein said genetically-modified cell comprises an expression cassette comprising a nucleotide sequence encoding a CAR that comprises said co-stimulatory domain of any one of  claims 1 - 24 . 
     
     
         37 . The genetically-modified cell of any one of  claims 31 - 35 , wherein said genetically-modified cell comprises:
 (i) a CAR expression cassette comprising a nucleotide sequence encoding a CAR that does not comprise a co-stimulatory domain of any one of  claims 1 - 24 ; and   (ii) a regulatory expression cassette encoding an inducible regulatory construct of any one of  claims 21 - 24 .   
     
     
         38 . The genetically-modified cell of any one of  claims 31 - 35 , wherein said genetically-modified cell comprises an expression cassette comprising:
 (i) a nucleotide sequence encoding a CAR that does not comprise a co-stimulatory domain of any one of  claims 1 - 24 ; and   (ii) a nucleotide sequence encoding an inducible regulatory construct of any one of  claims 21 - 24 .   
     
     
         39 . The genetically-modified cell of any one of  claims 31 - 35 , wherein said genetically-modified cell comprises:
 (i) a CAR that does not comprise said co-stimulatory domain of any one of  claims 1 - 24 ; and   (ii) an inducible regulatory domain of any one of  claims 21 - 24 .   
     
     
         40 . The genetically-modified cell of any one of  claims 31 - 39 , wherein said genetically-modified cell is a genetically-modified eukaryotic cell. 
     
     
         41 . The genetically-modified cell of any one of  claims 31 - 40 , wherein said genetically-modified cell is a genetically-modified human T cell. 
     
     
         42 . The genetically-modified cell of any one of  claims 31 - 41 , wherein said genetically-modified cell exhibits increased proliferation and/or cytokine secretion compared to a control cell that does not comprise a co-stimulatory domain having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 8, 5, 6, or 7. 
     
     
         43 . A method for producing a genetically-modified cell comprising at least one co-stimulatory domain of any one of  claims 1 - 24 , said method comprising introducing into a cell at least one nucleic acid molecule of any one of  claims 1 - 24 . 
     
     
         44 . The method of  claim 43 , wherein said nucleic acid molecule is said nucleic acid molecule of any one of  claims 15 - 20  which encodes a CAR. 
     
     
         45 . The method of  claim 43 , wherein said nucleic acid molecule is said nucleic acid molecule of any one of  claims 21 - 24  which encodes an inducible regulatory construct. 
     
     
         46 . The method of any one of  claims 43 - 45 , wherein said method further comprises introducing into said cell:
 (i) a second nucleic acid molecule encoding an engineered nuclease, wherein said engineered nuclease is expressed in said cell, or;   (ii) an engineered nuclease protein;   wherein said engineered nuclease recognizes and cleaves a recognition sequence in the genome of said cell to produce a cleavage site, and wherein said nucleic acid molecule encoding said at least one co-stimulatory domain is inserted into the genome of said cell at said cleavage site.   
     
     
         47 . The method of  claim 46 , wherein said engineered nuclease is an engineered meganuclease, a recombinant zinc-finger nuclease (ZFN), a recombinant transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease. 
     
     
         48 . The method of  claim 46  or  claim 47 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         49 . The method of any one of  claims 46 - 48 , wherein said nucleic acid molecule of any one of  claims 1 - 24  further comprises sequences homologous to sequences flanking said cleavage site, such that said nucleic acid molecule is inserted into the genome of said cell at said cleavage site by homologous recombination. 
     
     
         50 . The method of any one of  claims 46 - 48 , wherein said nucleic acid molecule of any one of  claims 1 - 24  lacks substantial homology to said cleavage site, such that said nucleic acid molecule is inserted into the genome of said cell by non-homologous end joining. 
     
     
         51 . The method of any one of  claims 43 - 50 , wherein said genetically-modified cell is a genetically-modified eukaryotic cell. 
     
     
         52 . The method of any one of  claims 43 - 51 , wherein said genetically-modified cell is a genetically-modified human T cell. 
     
     
         53 . The method of any one of  claims 43 - 52 , wherein said nucleic acid molecule of any one of  claims 1 - 24  is introduced into said cell using said mRNA of  claim 25 . 
     
     
         54 . The method of any one of  claims 43 - 52 , wherein said nucleic acid molecule of any one of  claims 1 - 24  is introduced into said cell using said recombinant DNA construct of any one of  claims 26 - 28 . 
     
     
         55 . The method of any one of  claims 43 - 52 , wherein said nucleic acid molecule of any one of  claims 1 - 24  is introduced into said cell using said viral vector of  claim 29  or  claim 30 . 
     
     
         56 . The method of any one of  claims 43 - 55 , wherein said genetically-modified cell exhibits increased activation, proliferation, and/or cytokine secretion following introduction of the nucleic acid molecule of any one of  claims 1 - 24  when compared to a control cell that does not comprise a co-stimulatory domain. 
     
     
         57 . A pharmaceutical composition comprising a pharmaceutically-acceptable carrier and said genetically-modified cell of any one of  claims 31 - 42 . 
     
     
         58 . A method of immunotherapy for treating cancer in a subject in need thereof, said method comprising administering to said subject said genetically-modified cell of any one of  claims 36 - 42 . 
     
     
         59 . The method of  claim 58 , wherein said method comprises administering said pharmaceutical composition of  claim 57 , wherein said pharmaceutical composition comprises said genetically-modified cell of any one of  claims 36 - 42 . 
     
     
         60 . The method of  claim 58  or  claim 59 , wherein said cancer is a carcinoma, lymphoma, sarcoma, blastoma, or leukemia. 
     
     
         61 . The method of any one of  claims 58 - 60 , wherein said cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. 
     
     
         62 . The method of  claim 61 , wherein said cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.