T cell modifying compounds and uses thereof

• US 9,597,357 B2
• Filed: 10/10/2013
• Issued: 03/21/2017
• Est. Priority Date: 10/10/2012
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Gregory, Philip D., Holmes, Michael C.
• Primary Examiner(s): OUSPENSKI, ILIA I
• Application Number:
  US14/051,244
• Publication Number:
  US 20140120622A1
• Time in Patent Office: 1,258 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 35/26 Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464406 Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
• CPC Class Codes: A61K 39/464409 Vascular endothelial growth factor receptors [VEGFR]
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/64 comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Methods and compositions for modifying T-cells in which PD1 and/or CTLA-4 is repressed and/or inactivated using fusion proteins such as artificial transcription factors and nucleases.

Claims

1. A genetically modified T-cell that expresses a chimeric antigen receptor (CAR), wherein an exogenous sequence encoding a CAR is integrated into the genome of the T-cell using one or more zinc finger nucleases, and further wherein expression of at least one endogenous immunological checkpoint gene is repressed by genetically modifying the immunological checkpoint gene in the T-cell, wherein the immunological checkpoint gene is a programmed death receptor PDCD1 gene or a CTLA-4 gene, wherein the genetic modification is to a sequence as shown in any SEQ ID NO:

21-33 and 116-120.

2. The T-cell of claim 1 wherein the immunological checkpoint gene is a CTLA-4 gene.

3. The T-cell of claim 1, wherein the immunological checkpoint gene is PDCD1.

4. The T-cell of claim 1, wherein the T-cell is selected from the group consisting of a CD4+ cell, a CD8+ cell and a tumor infiltrating cell (TIL).

5. The T-cell of claim 1, wherein the exogenous sequence encoding the CAR is integrated into the T-cell genome at a safe harbor locus.

6. The T-cell of claim 1, wherein the exogenous sequence encoding the CAR is randomly integrated into the T-cell genome.

7. The T-cell of claim 1, wherein the CAR comprises a signaling domain of a T-cell receptor (TCR).

8. The T-cell of claim 7, wherein the CAR comprises a scFv specificity domain.

9. The T-cell of claim 1, further comprising at least one additional transgene.

10. The T-cell of claim 9, wherein the at least one additional transgene encodes a tumor-associated antigen (TAA)-specific T-cell receptor (TCR).

11. The T-cell of claim 1, wherein the T-cell is stimulated.

12. The T-cell of claim 11, wherein the T-cell is stimulated with anti-CD28/CD3 beads.

13. A method of making the T-cell of claim 5, the method comprising:

cleaving the safe harbor gene in the T-cell using a pair of zinc finger nucleases (ZFNs) such that the exogenous sequence encoding the CAR is integrated into the safe harbor gene.

14. The method of claim 13, where the safe harbor gene is selected from the group consisting of AAVS1, CCR5, HPRT and Rosa.

15. The method of claim 13, wherein the exogenous sequence encoding the CAR is carried by a plasmid vector or a viral vector.

16. The method of claim 13, wherein the ZFNs are introduced into the cell as mRNA.

17. The method of claim 13, wherein the T-cell is selected from the group consisting of a CD4+ cell, a CD8+ cell and a tumor infiltrating cell (TIL).

18. The method of claim 13, wherein the CAR comprises a signaling domain of a T-cell receptor (TCR).

19. The method of claim 13, wherein the CAR comprises a scFv specificity domain.

20. The method of claim 13, further comprising stimulating the T-cell.

21. The method of claim 13, wherein the T-cell is stimulated with anti-CD28/CD3 beads.

22. The method of claim 13, further comprising integrating at least additional transgene into the T-cell genome.

23. The method of claim 22, wherein the at least one additional transgene encodes a TAA-specific T-cell receptor (TCR).