Genetically engineered t cells with regnase-1 and/or TGFBRII disruption have improved functionality and persistence
A population of genetically engineered T cells, comprising a disrupted Reg1 gene and/or a disrupted TGFBRII gene. Such genetically engineered T cells may comprise further genetic modifications, for example, a disrupted CD70 gene. The population of genetically engineered T cells exhibit one or more of (a) improved cell growth activity; (b) enhanced persistence; and (c) reduced T cell exhaustion, (d) enhanced cytotoxicity activity, (e) resistant to inhibitory effects induced by TGF-b, and (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby, as compared to non-engineered T cell counterparts.
1. A method for treating cancer, the method comprising administering to a subject in need thereof a population of genetically engineered T cells, wherein the genetically engineered T cells comprise:
(i) a disrupted Regnase-1 (Reg1) gene;
(ii) a disrupted Transforming Growth Factor Beta Receptor II (TGFBRII) gene; and
(iii) a nucleic acid encoding a chimeric antigen receptor (CAR) that binds a tumor antigen.
2. The method of claim 1 , wherein the disrupted Reg1 gene is genetically edited in exon 2 and/or exon 4; and/or wherein the disrupted TGFBRII gene is genetically edited in exon 4 or exon 5.
3. The method of claim 1 , wherein the disrupted Reg1 gene, the disrupted TGFBRII gene, or both are genetically edited by a CRISPR/Cas-mediated gene editing system.
4. The method of claim 3 ,
wherein the CRISPR/Cas-mediated gene editing comprises a guide RNA (gRNA) targeting a site in the Reg1 gene, wherein the target site comprises a nucleotide selected from the group consisting of SEQ ID NO: SEQ ID NO: 320, 322, 323, and 327; and/or
wherein the CRISPR/Cas-mediated gene editing system comprises a guide RNA (gRNA) targeting a site in the TGFBRII gene that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 275, 305, 311, and 317.
5. The method of claim 4 ,
wherein the gRNA targeting the Reg1 gene comprises the nucleotide sequence selected from the group consisting of SEQ ID NO: 22, 30, 34, and 50; and/or
wherein the gRNA targeting the TGFBRII gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 270, 300, 306, and 312.
6. The method of claim 1 , wherein the genetically engineered T cells further comprise:
(iv) a disrupted T cell receptor alpha chain constant region (TRAC) gene,
(v) a disrupted beta-2-microglobulin (β2M) gene,
(vi) a disrupted CD70 gene, or
(vii) a combination of any of (iv)-(vi).
7. The method of claim 6 , wherein the genetically engineered T cells comprise a disrupted T cell receptor alpha chain constant region (TRAC) gene and a disrupted beta-2-microglobulin (β2M) gene.
8. The method of claim 7 , wherein the nucleic acid encoding the CAR is inserted in the disrupted TRAC gene.
9. The method of claim 7 , the CAR binds CD19 and comprises an extracellular antigen binding domain specific to CD19, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv) that comprises the amino acid sequence of SEQ ID NO: 120.
10. The method of claim 9 , wherein the CAR that binds CD19 comprises the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO:353.
11. The method of claim 9 , wherein the subject is a human patient having a CD19+ cancer.
12. The method of claim 11 , wherein the CD19+ cancer is a hematologic cancer.
13. The method of claim 7 , wherein the genetically engineered T cells further comprise a disrupted CD70 gene.
14. The method of claim 13 , wherein the CAR is specific to CD70 and comprises an extracellular antigen binding domain that binds CD70, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv) that comprises the amino acid sequence of SEQ ID NO: 140 or 142.
15. The method of claim 14 , wherein the CAR specific to CD70 comprises the amino acid sequence of SEQ ID NO:138 or SEQ ID NO:354.
16. The method of claim 14 , wherein the subject is a human patient having a CD70+ cancer.
17. The method of claim 16 , wherein the CD70+ cancer is a hematopoietic cancer or a solid cancer.
18. The method of claim 1 , wherein the tumor antigen is CD19, BCMA, CD70, CD33, or PTK7.
19. The method of claim 1 , wherein the genetically engineered T cells are prepared from immune cells of one or more human donors.
20. The method of claim 1 , wherein the genetically engineered T cells are allogeneic to the subject.