New patent application published for: ENGINEERED HUMAN T CELLS COMPRISING A SWITCHABLE CHIMERIC ANTIGEN CELL SURFACE RECEPTOR AND METHODS FOR GENERATING THEM

ENGINEERED HUMAN T CELLS COMPRISING A SWITCHABLE CHIMERIC ANTIGEN CELL SURFACE RECEPTOR AND METHODS FOR GENERATING THEM

Claims

1. An engineered human T cell, comprising: i. a genetic modification in a T cell receptor alpha chain gene that reduces or eliminates surface expression of endogenous T cell receptor alpha chain relative to an unmodified T cell; ii. a genetic modification in an HLA-A gene that reduces or eliminates surface expression of endogenous HLA-A relative to an unmodified T cell, iii. a genetic modification in a CIITA gene that reduces or eliminates surface expression of endogenous HLA class II relative to an unmodified T cell, and iv. a reversed universal chimeric antigen cell surface receptor that comprises: a tag, an extracellular hinge and a transmembrane domain, and a signal transduction domain.
2. The engineered human T cell according to claim 1, wherein the tag is a peptide epitope tag.
3. The engineered human T cell according to claim 2, wherein the peptide epitope tag is a myc-tag, a His-tag, a short linear peptide sequence from yeast transcription factor GCN4, a leucine zipper sequence or a short linear peptide sequence from a human nuclear protein.
4. The engineered human T cell according to claim 1, wherein the extracellular hinge and the transmembrane domain are selected from hinges and transmembrane domains of a human CD28 molecule, a CD8a chain, or an NK cell receptor; parts of an IgG1 or IgG4 constant region, mutants thereof and combinations thereof.
5. The engineered human T cell according to claim 1, wherein the signal transduction domain is selected from cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10, CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD3 chains, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activating Fc receptors, mutants thereof and combinations thereof.
6. The engineered human T cell according to claim 1, wherein the T cell is homozygous for HLA-B and homozygous for HLA-C genotypes.
7. A pharmaceutical composition comprising the engineered human T cell according claim 1 and a pharmaceutically acceptable excipient.
8. (canceled)
9. A kit comprising: a) an engineered human T cell, comprising: i. a genetic modification in a T cell receptor alpha chain gene that reduces or eliminates surface expression of endogenous T cell receptor alpha chain relative to an unmodified T cell, ii. a genetic modification in an HLA-A gene that reduces or eliminates surface expression of endogenous HLA-A relative to an unmodified T cell, iii. a genetic modification in a CIITA gene that reduces or eliminates surface expression of endogenous HLA class II relative to an unmodified T cell, and iv. a reversed universal chimeric antigen cell surface receptor that comprises: a tag, an extracellular hinge and a transmembrane domain, and a signal transduction domain, and b) a targeting module comprising a tag-binding domain and at least one target cell-binding domain, a nucleic acid encoding the targeting module, a vector comprising the nucleic acid encoding the targeting module, or a cell comprising the nucleic acid encoding the targeting module, wherein the tag-binding domain of the targeting module binds to the tag of the reversed universal chimeric antigen cell surface receptor.
10. The kit according to claim 9, wherein the at least one target cell-binding domain is an antibody or antigen-binding fragment thereof, a protein, a peptide or a low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366, CD371, IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1, CXCR4, c-Met, mesothelin, ErbB1, ErbB2, ErbB3, ErbB4, tumor necrosis factor receptor claudin, ephrin, EphA1-10, EphA5, EphB1, EphB2, EphB3, EphB4, EphB5, EphB6, fucosyl transferase, PSCA, PSMA, CEA, fetal acetylcholine receptor, vascular endothelia growth factor EpCAM, AFP, intercellular adhesion molecule, C-type lectin, integrin, mucin, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, NKG2D receptor, epithelia glycoprotein, diasialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer-testis antigen, fibroblast activation protein, carbonic anhydrase, carbohydrate antigen, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycan.
11. The kit according to claim 9, wherein the kit further comprises at least one further targeting module, a nucleic acid encoding the at least one further targeting module, a vector comprising the nucleic acid encoding the at least one further targeting module or a cell comprising the nucleic acid encoding the at least one further targeting module, wherein the at least one further targeting module comprises at least one target cell-binding domain and a tag-binding domain, wherein the at least one target cell-binding domain is an antibody or antigen-binding fragment thereof, a protein, a peptide or a low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6 CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD133, CD135, CD150 CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366, CD371, IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1, CXCR4, c-Met, mesothelin, ErbB1, ErbB2, ErbB3, ErbB4, tumor necrosis factor receptor, claudin, ephrin, EphA1-10, EphA5, EphB1, EphB2, EphB3, EphB4, EphB5, EphB6, fucosyl transferase, PSCA, PSMA, CEA, fetal acetylcholine receptor, vascular endothelia growth factor, EpCAM, AFP, intercellular adhesion molecule, C-type lectin, integrin, mucin, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, NKG2D receptor, epithelia glycoprotein, diasialoganglioside, glypican, G protein-coupled receptor, human papillomavirus protein, cancer-testis antigen, fibroblast activation protein, carbonic anhydrase, carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycan.
12-13. (canceled)
14. A kit comprising: a) a CRISPR system comprising a nucleic acid that targets a region of an endogenous T cell receptor alpha chain (TRAC) gene, a nucleic acid that targets a region of an endogenous HLA-A gene, and/or a nucleic acid that targets a region of an endogenous CIITA gene, b) a nucleic acid encoding a reversed universal chimeric antigen cell surface receptor that comprises: a tag, an extracellular hinge and a transmembrane domain, and a signal transduction domain,  or a vector comprising the nucleic acid encoding a reversed universal chimeric antigen cell surface receptor, and c) a targeting module comprising a tag-binding domain and at least one target cell-binding domain, a nucleic acid encoding the targeting module, a vector comprising the nucleic acid encoding the targeting module, or a cell comprising the nucleic acid encoding the targeting module, wherein the tag-binding domain of the targeting module binds to the tag of the reversed universal chimeric antigen cell surface receptor.
15. The kit according to claim 14, wherein the CRISPR system further comprises an mRNA that encodes CRISPR-associated protein 9 and a nucleic acid that targets a region of an endogenous TRAC, HLA-A, or CIITA gene.
16. The kit according to claim 15, wherein the nucleic acid that targets a region of an endogenous TRAC gene comprises a nucleotide sequence according to SEQ ID NO. 1, the nucleic that targets a region of an endogenous HLA-A gene comprises a nucleotide sequence according to SEQ ID NO. 2 and/or the nucleic acid that targets a region of an endogenous CIITA gene comprises a nucleotide sequence according to SEQ ID NO. 3.
17. A method for generating an engineered human T cell expressing a reversed universal chimeric antigen cell surface receptor, the method comprising: (1) providing a human T cell, (2) introducing into the human T cell a CRISPR system comprising: a. a nucleic acid that targets a region of an endogenous T cell receptor alpha chain (TRAC) gene, b. a nucleic acid that targets a region of an endogenous HLA-A gene, and c. a nucleic acid that targets a region of an endogenous CIITA gene, and (3) introducing into the human T cell a nucleic acid encoding a reversed universal chimeric antigen cell surface receptor that comprises: a tag, an extracellular hinge and a transmembrane domain, and a signal transduction domain, or a vector comprising the nucleic acid encoding the reversed universal chimeric antigen cell surface receptor.
18. The method according to claim 17, wherein the human T cell is homozygous for HLA-B and homozygous for HLA-C genotypes.
19. The method according to claim 18, wherein the method further comprises matching the HLA-B and HLA-C alleles of the engineered human T cell to the HLA-B and HLA-C alleles of a patient.
20. The method according to claim 17, wherein the CRISPR system further comprises an endonuclease or an mRNA encoding an endonuclease.
21. The method according to claim 20, wherein the endonuclease is CRISPR-associated enzyme 9.
22. The according to claim 17, wherein the nucleic acid that targets a region of an endogenous TRAC gene comprises a nucleotide sequence according to SEQ ID NO. 1, the nucleic acid that targets a region of an endogenous HLA-A gene comprises a nucleotide sequence according to SEQ ID NO. 2, and/or the nucleic acid that targets a region of an endogenous CIITA gene comprises a nucleotide sequence according to SEQ ID NO. 3.
23. The method according to claim 20, wherein the CRISPR system is packaged in a lipid nanoparticle.
24. The method according to claim 23, wherein the lipid nanoparticle comprises a cationic and/or ionizable lipid and a helper lipid.
25. The method according to claim 23, wherein the lipid nanoparticle comprises an ionizable lipid, a helper lipid and PEG-DMG.
26. The method according to claim 17, wherein introducing into the human T cell of the nucleic acid encoding the reversed universal chimeric antigen cell surface receptor is a retroviral, lentiviral, or adenoviral vector particle.
27. A method of treating a cancer, an infectious disease or an autoimmune disease in a mammal, comprising: a) administering to the mammal an effective amount of a targeting module comprising a tag-binding domain and at least one target cell-binding domain, a nucleic acid encoding the targeting module, or a pharmaceutical composition comprising the targeting module, and b) administering to the mammal an effective amount of an engineered human T cell according to claim 1 or a pharmaceutical composition comprising the engineered human T cell, wherein the tag-binding domain of the targeting module binds to the tag of the reversed universal chimeric antigen cell surface receptor, and wherein the targeting module, the nucleic acid encoding the targeting module, or the pharmaceutical composition comprising the targeting module is administered to the mammal prior to, concurrent with or after the administration of the engineered human T cell or the pharmaceutical composition comprising the engineered human T cell.
28. The kit according to claim 15, wherein the CRISPR system is packaged in a lipid nanoparticle.