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 population of genetically engineered T cells, comprising T cells that comprise:
(i) a knockout Regnase-1 (Reg1) gene; and
(ii) a knockout Transforming Growth Factor Beta Receptor II (TGFBRII) gene.
2. The population of genetically engineered T cells of claim 1 , wherein the T cells are further engineered to express a chimeric antigen receptor (CAR).
3. The population of genetically engineered T cells of claim 1 , wherein the knockout Reg1 gene is genetically edited in exon 2 and/or exon 4.
4. The population of genetically engineered T cells of claim 1 , wherein the knockout TGFBRII gene is genetically edited in exon 4 or exon 5.
5. The population of genetically engineered T cells of claim 1 , wherein the knockout Reg1 gene, the knockout TGFBRII gene, or both are genetically edited by a CRJSPR/Cas-mediated gene editing system.
6. The population of genetically engineered T cells of claim 5 , wherein the CRISPR/Cas-mediated gene editing comprises a guide RNA (gRNA) targeting a site in the Reg1 gene, and wherein the target site comprises the nucleotide selected from the group consisting of SEQ ID NO: SEQ ID NO: 320, 322, 323, and 327.
7. The population of genetically engineered T cells of claim 6 , 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.
8. The population of genetically engineered T cells of claim 1 , wherein the CRISPR/Cas-mediated gene editing system comprises a guide RNA (gRNA) targeting a site in the TGFBRII gene, and wherein the targeting site comprises the nucleotide sequence selected from the group consisting of SECS ID NOs: 275, 305, 311, and 317.
9. The population of genetically engineered T cells of claim 8 , wherein the gRNA targeting the TGFBRII gene comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 270, 300, 306, and 312.
10. The population of genetically engineered T cells of claim 1 , wherein the genetically engineered T cells further comprise:
(iii) a knockout T cell receptor alpha chain constant region (TRAC) gene,
(iv) a knockout beta-2-microglobulin (β2M) gene,
(v) a knockout CD70 gene, or
(vi) a combination of any of (iii)-(v).
11. The population of genetically engineered T cells of claim 10 , wherein the genetically engineered T cells comprise a knockout T cell receptor alpha chain constant region (TRAC) gene and a knockout beta-2-microglobulin (β2M) gene.
12. The population of genetically engineered T cells of claim 11 , wherein the genetically engineered T cells further comprise a knockout CD70 gene.
13. The population of genetically engineered T cells of claim 12 , wherein the knockout TRAC gene, the knockout β2M gene, and/or the knockout CD70 gene is genetically edited by one or more CRISPR/Cas-mediated gene editing systems.
14. The population of genetically engineered T cells of claim 2 , wherein the genetically engineered T cells comprise a nucleic acid encoding the CAR, and wherein the nucleic acid is inserted in the genome of the T cells.
15. The population of genetically engineered T cells of claim 14 , wherein the nucleic acid encoding the CAR is inserted in the a knockout TRAC gene, and wherein the nucleic acid encoding the CAR replaces a deleted fragment comprising SEQ ID NO: 69 in the TRAC gene.
16. The population of genetically engineered T cells of claim 2 , wherein the CAR comprises an extracellular antigen binding domain specific to a tumor antigen, a co-stimulatory signaling domain of 4-1BB or CD28, and a cytoplasmic signaling domain of CD3ζ.
17. The population of genetically engineered T cells of claim 16 , wherein the tumor antigen is CD19, CD70, CD33, or PTK7.
18. The population of genetically engineered T cells of claim 17 , wherein the CAR hinds CD19 (anti-CD19 CAR), wherein the extracellular antigen binding domain in the anti-CD19 CAR is a single chain variable fragment (scFv) that binds CD19 (anti-CD19 scFv), and wherein the anti-CD19 scFv comprises (i) a heavy chain variable region (V H ) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 124; and (ii) a light chain variable region (V L ) that comprises the same light chain CDRs as those in SEQ ID NO: 125.
19. The population of genetically engineered T cells of claim 18 , wherein the V H comprises the amino acid sequence of SEQ ID NO: 124 and the V L comprises the amino acid sequence of ID NO: 125.
20. The population of genetically engineered T cells of claim 19 , wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 120.
21. The population of genetically engineered T cells of claim 20 , wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO:353.
22. The population of genetically engineered T cells of claim 21 , wherein the genetically engineered T cells comprise a knockout T cell receptor alpha chain constant region (TRAC) gene and a knockout beta-2-microglobulin (β2M) gene, and wherein the nucleic acid encoding the anti-CD19 CAR is inserted in the knockout TRAC gene.
23. The population of genetically engineered T cells of claim 17 , wherein the CAR binds CD70 (anti-CD70 CAR), wherein the extracellular antigen binding domain in the anti-CD70 CAR is a single chain variable fragment (scFv) that binds CD70 (anti-CD70 scFv), and wherein the anti-CD70 scFv comprises (i) a heavy chain variable region (V H ) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 143; and (ii) a light chain variable region (V L ) that comprises the same light chain CDRs as those in SEQ ID NO: 144.
24. The population of genetically engineered T cells of claim 23 , wherein the V H comprises the amino acid sequence of SEQ ID NO: 143 and the V L comprises the amino acid sequence of SEQ ID NO: 144.
25. The population of genetically engineered T cells of claim 24 , wherein the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 140 or 142.
26. The population of genetically engineered T cells of claim 25 , wherein the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO:354.
27. The population of genetically engineered T cells of claim 26 , wherein the genetically engineered T cells comprise a knockout T cell receptor alpha chain constant region (TRAC) gene, a knockout beta-2-microglobulin (β2M) gene and a knockout CD70 gene, and wherein the nucleic acid encoding the anti-CD70 CAR is inserted in the knockout TRAC gene.
28. The population of genetically engineered T cells of claim 1 , wherein the genetically engineered T cells are isolated from primary T cells of one or more human donors.
29. A pharmaceutical composition comprising the population of genetically engineered T cells of claim 1 and a pharmaceutically acceptable carrier.