Dna constructs for improved t cell immunotherapy

Patent metadata

Field	Value
Publication number	US-2025382340-A1
Application number	US-202318837837-A
Country	US
Kind code	A1
Filing date	Feb 14, 2023
Priority date	Feb 14, 2022
Publication date	Dec 18, 2025
Grant date	—

Abstract

Provided herein are methods and compositions for modifying the genome of human T cells. Further, the compositions and methods described herein can be used to generate human T cells with altered specificity and functionality, while limiting the side effects associated with T cell therapies. Provided herein is a human T cell that heterologously expresses one or more polypeptides. In some embodiment, the one or more polypeptides, for example, two or more polypeptides, are encoded by a nucleic acid construct inserted into the TCR locus of the cell.

First claim

1 . A human T cell that heterologously expresses:
a polypeptide comprising a TFAP4 protein, and a polypeptide comprising a BATF protein; a polypeptide comprising a BATF3 protein, and a polypeptide comprising a TFAP4 protein; a polypeptide comprising a FOXJ2 protein, and a polypeptide comprising a RARA protein; a polypeptide comprising an ID3 protein, and a polypeptide comprising TFAP4 protein; a polypeptide comprising a BATF protein, and a polypeptide comprising IRF2 protein; a polypeptide comprising a NANOG protein, and a polypeptide comprising TFAP4 protein; a polypeptide comprising a MAFF protein, and a polypeptide comprising SATB1 protein; a polypeptide comprising a BATF protein, and a polypeptide comprising IRF1 protein; a polypeptide comprising a TFAP4 protein and a polypeptide comprising HOPX protein; a polypeptide comprising a BATF protein, and a polypeptide comprising HES2 protein; a polypeptide comprising an ATF2 protein, and a polypeptide comprising BATF protein; a polypeptide comprising a BATF3 protein, and a polypeptide comprising SMAD4 protein; a polypeptide comprising a FOXP3 protein, and a polypeptide comprising RELA protein; a polypeptide comprising an ID3 protein, and a polypeptide comprising BATF3 protein; a polypeptide comprising an ID2 protein, and a polypeptide comprising a TP73 protein; a polypeptide comprising a TIGIT protein, and a polypeptide comprising an ICOS protein; a polypeptide comprising a LTBR protein, and a polypeptide comprising a 4-1BB protein; a polypeptide comprising a DR5 protein, and a polypeptide comprising a ICOS protein; a polypeptide comprising a SMAD3 protein, and a polypeptide comprising a BATF3 protein; a polypeptide comprising a HES2 protein; a polypeptide comprising a FOXJ2 protein; a polypeptide comprising a CCR4 protein; a polypeptide comprising a human TFAP4 protein, and a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain) via a transmembrane domain; a polypeptide comprising a human SOX5 protein, and a truncated TIGIT protein comprising the human TIGIT extracellular domain or a portion thereof, the TIGIT transmembrane domain, and about 1-10 (e.g., 7) amino acids of the human TIGIT intracellular domain; a polypeptide comprising a human MYC protein, and a polypeptide comprising a human TGFbR2 extracellular domain or a portion thereof linked to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g. 7) amino acids of the TGFbR2 intracellular domain) via a transmembrane domain; a polypeptide comprising a BATF3 protein and a polypeptide comprising 4-1BB protein; a polypeptide comprising a TFAP4 protein, and a polypeptide comprising a human LTBR extracellular domain or a portion thereof linked to the intracellular domain of human IL-4R (and optionally 1-10 (e.g. 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; a polypeptide comprising a ATF1 protein, and a polypeptide comprising a human IL4RA extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB; a polypeptide comprising a BATF protein, and a polypeptide comprising a human 2B4 extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising a RORC protein, and a polypeptide comprising a VISTA extracellular domain or a portion thereof linked to the intracellular domain of human CD28, and optionally about 1-15 (e.g., 12) amino acids of the human CD28 extracellular domain; a polypeptide comprising a BATF protein and a polypeptide comprising CXCR 1 protein; a polypeptide comprising a MAFF protein and a polypeptide comprising IL2RA protein; a polypeptide comprising a ATF1 protein, and a polypeptide comprising a human TNFRSF12 extracellular domain or a portion thereof linked to the intracellular domain of human OX40 (and optionally 1-10 (e.g. 7) amino acids of the TNFRSF12 intracellular domain) via a transmembrane domain; a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain) via a transmembrane domain; a polypeptide comprising a human BATF2 extracellular domain or a portion thereof and a truncated PD-1 comprising the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain and about 1-12 (e.g., 10) amino acids of the human PD-1 intracellular domain; a polypeptide comprising a human TCF7 protein and a polypeptide comprising a human LAT1 protein; a polypeptide comprising a human 4-1BB protein; a polypeptide comprising a human STAT5 protein and a human IL-2RA protein; a polypeptide comprising a human FOXJ2 protein and a polypeptide comprising a LTBR extracellular domain or a portion thereof linked to the intracellular domain of human CD28 (and optionally 1-10 (e.g. 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; a polypeptide comprising a human FOXJ2 protein and a polypeptide comprising a LTBR extracellular domain or a portion thereof linked to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g. 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; a polypeptide comprising a human HOPX protein and a polypeptide comprising a IL4RA extracellular domain or a portion thereof linked to the intracellular domain of human 41-BB via a transmembrane domain; a polypeptide comprising a ID2 protein, and a polypeptide comprising a BLTA extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising a human SMAD1 protein, and a polypeptide comprising a MCT4 protein; a polypeptide comprising a human SMAD1 protein and a polypeptide comprising a LTBR extracellular domain or a portion thereof linked to the intracellular domain of human IL-4R (and optionally 1-10 (e.g. 7) amino acids of the LTBR intracellular domain) via a transmembrane domain, a polypeptide comprising about 1-12 amino acids (e.g., 7 amino acids) of the DR5 intracellular domain and a polypeptide comprising the intracellular domain of human ICOS via a transmembrane domain; a polypeptide comprising a TIGIT extracellular domain or a portion thereof linked to the intracellular domain of human ICOS (and optionally about 1-15 (e.g., 12) amino acids of the human ICOS extracellular domain) via a transmembrane domain; a polypeptide comprising a LTBR extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 7) amino acids of the human LTBR intracellular domain) via a transmembrane domain, wherein the one or more polypeptides are encoded by a heterologous nucleic acid construct inserted into a target genomic locus of the cell, optionally wherein the target genomic locus is the T-cell receptor (TCR) locus of the cell, optionally wherein the heterologous nucleic acid construct is non-virally inserted.

2 . The human T cell of claim 1 , wherein the T cell heterologously expresses one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 30-SEQ ID NO: 58, SEQ ID NO: 69-85, SEQ ID NO:160, SEQ ID NO:161 and SEQ ID NO:162.

3 . The human T cell of claim 1 , wherein the target insertion site is in exon 1 of a TCR-alpha subunit constant gene (TRAC) or in exon 1 of a TCR-beta subunit constant gene (TRBC).

4 . (cancele

Assignees

• Univ California
• The J David Gladstone Inst A Testamentary Trust Established Under The Will Of J David Glads

Inventors

• Blaeschke Franziska
• Roth Theodore Lee
• Marson Alexander
• Chen yan yi
• Apathy Ryan

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N2510/00

  Genetically modified cells · CPC title

• C12N5/0636

  T lymphocytes · CPC title

• C07K14/7051

  T-cell receptor (TcR)-CD3 complex · CPC title

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title