Genetically engineered T cells with PTPN2 knockout have improved functionality and anti-tumor activity

Patent metadata

Field	Value
Publication number	US-12553030-B2
Application number	US-202217689071-A
Country	US
Kind code	B2
Filing date	Mar 8, 2022
Priority date	Mar 9, 2021
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

Abstract

A population of genetically engineered T cells, comprising a disrupted protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene and optionally a disrupted TRAC gene, a disrupted β2M gene, and/or a disrupted CD70 gene. Also provided herein are methods for making such genetically engineered T cells and therapeutic uses thereof.

First claim

What is claimed is:

1 . A population of genetically engineered T cells, comprising:
a nucleic acid encoding a chimeric antigen receptor (CAR); a disrupted protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene; a disrupted T cell receptor alpha chain constant region (TRAC) gene; and a disrupted beta-2-microglobulin (β2M) gene, wherein the cells exhibit enhanced survival in the presence of natural killer (NK) cells.

2 . The population of genetically engineered T cells of claim 1 , wherein the disrupted PTPN2 gene is genetically edited in exon 1, exon 2, and/or exon 3.

3 . The population of genetically engineered T cells of claim 1 , wherein the disrupted PTPN2 gene is genetically edited by CRISPR/Cas-mediated gene editing.

4 . The population of genetically engineered T cells of claim 3 , wherein the CRISPR/Cas-mediated gene editing comprises a guide RNA (gRNA) targeting a site in the PTPN2 gene that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-13.

5 . The population of genetically engineered T cells of claim 4 , wherein the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 56, 57, 60, 61, 64-67, 70, and 71.

6 . The population of genetically engineered T cells of claim 5 , wherein the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26, 27, 30, 31, 34, 38, 39, 42, 43, 46, 47, 50, 51, 54, 55, 58, 59, 32, 63, 68, 69, 192, 193.

7 . The population of genetically engineered T cells of claim 1 , wherein the disrupted TRAC gene is genetically edited by a CRISPR/Cas-mediated gene editing system.

8 . The population of genetically engineered T cells of claim 7 , wherein the CRISPR/Cas-mediated gene editing system comprises a gRNA comprising the nucleotide sequence of SEQ ID NO: 76 or 77.

9 . The population of genetically engineered T cells of claim 8 , wherein the disrupted TRAC gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 93 to 100.

10 . The population of genetically engineered T cells of claim 1 , wherein the disrupted T cell TRAC gene has a deleted fragment comprising SEQ ID NO: 87.

11 . The population of genetically engineered T cells of claim 1 , wherein the nucleic acid is inserted in the genome of the T cells.

12 . The population of genetically engineered T cells of claim 11 , wherein the disrupted TRAC gene comprises the nucleic acid encoding the CAR.

13 . The population of genetically engineered T cells of claim 12 , wherein the nucleic acid encoding the CAR replaces the deleted fragment in the disrupted TRAC gene.

14 . The population of genetically engineered T cells of claim 1 , wherein the disrupted β2M gene is genetically edited by CRISPR/Cas-mediated gene editing.

15 . The population of genetically engineered T cells of claim 14 , wherein the CRISPR/Cas-mediated gene editing comprises a gRNA comprising the nucleotide sequence of SEQ ID NO: 80 or 81.

16 . The population of genetically engineered T cells of claim 15 , wherein the disrupted β2M gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101 to 106.

17 . The population of genetically engineered T cells of claim 1 , wherein the T cells further comprise a disrupted CD70 gene.

18 . The population of genetically engineered T cells of claim 17 , wherein the disrupted CD70 gene is genetically edited by CRISPR/Cas-mediated gene editing.

19 . The population of genetically engineered T cells of claim 18 , wherein the CRISPR/Cas-mediated gene editing comprises a gRNA comprising the nucleotide sequence of SEQ ID NO: 72 or 73.

20 . The population of genetically engineered T cells of claim 19 , wherein the disrupted CD70 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 107 to 112.

21 . The population of genetically engineered T cells of claim 1 , 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 CD35.

22 . The population of genetically engineered T cells of claim 21 , wherein the tumor antigen is CD19, BCMA, or CD70.

23 . The population of genetically engineered T cells of claim 22 , wherein the extracellular antigen binding domain is a single chain variable fragment (scFv) that binds CD19, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 137.

24 . The population of genetically engineered T cells of claim 23 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 135.

25 . The population of genetically engineered T cells of claim 22 , wherein the extracellular antigen binding domain is a single chain variable fragment (scFv) that binds CD70, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 157 or 159.

26 . The population of genetically engineered T cells of claim 25 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 155.

27 . The population of genetically engineered T cells of claim 22 , wherein the extracellular antigen binding domain is a single chain variable fragment (scFv) that binds BCMA, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 165.

28 . The population of genetically engineered T cells of claim 27 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 163.

29 . The population of genetically engineered T cells of claim 1 , wherein the genetically engineered T cells are derived from primary T cells of one or more human donors.

30 . The population of genetically engineered T cells of claim 1 , wherein the population of genetically engineered T cells expressing the CAR has enhanced CAR potency and/or increased CAR copies as compared to non-engineered T cell counterparts.

31 . A method for preparing the population of genetically engineered T cells of claim 1 , the method comprising:
(a) providing a plurality of cells, which are T cells or precursor cells thereof;
genetically editing the PTPN2, TRAC and β2M genes; and
(b) producing the population of genetically engineered T cells having disrupted PTPN2, TRAC and β2M genes.

Assignees

• Crispr Therapeutics Ag

Inventors

• Tetteh Paul

Classifications

• A61K40/50

  characterised by the use of allogeneic cells · CPC title

• A61K40/4232

  Tumor necrosis factors [TNF] or CD70 · CPC title

• A61K40/4215

  Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30 · CPC title

• A61K40/4211

  CD19 or B4 · CPC title

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title