Cells expressing a chimeric receptor from a modified invariant cd3 immunoglobulin superfamily chain locus and related polynucleotides and methods

Patent metadata

Field	Value
Publication number	US-2023398148-A1
Application number	US-202118035277-A
Country	US
Kind code	A1
Filing date	Nov 3, 2021
Priority date	Nov 4, 2020
Publication date	Dec 14, 2023
Grant date	—

Abstract

Provided herein are engineered T cells, expressing a chimeric receptor comprising an antigen-binding domain fused to an endogenous invariant CD3 chain of the immunoglobulin superfamily (invariant CD3-IgSF). In some embodiments, the engineered T cells contain a modified invariant CD3-IgSF chain locus that encodes the chimeric receptor. Also provided are cell compositions containing the engineered T cells, nucleic acids for engineering cells, and methods, kits and articles of manufacture for producing the engineered cells, such as by targeting a transgene encoding a portion of a chimeric receptor for integration into an invariant CD3-IgSF chain genomic locus. In some embodiments, the engineered cells, e.g. T cells, can be used in connection with cell therapy, including in connection with cancer immunotherapy comprising adoptive transfer of the engineered cells.

First claim

1 . An engineered T cell, comprising a modified invariant CD3-immunoglobulin superfamily (invariant CD3-IgSF) chain locus comprising a nucleic acid sequence encoding a mini chimeric antigen receptor (miniCAR), wherein the miniCAR is a fusion protein comprising a heterologous antigen-binding domain and an endogenous invariant CD3 chain of the invariant CD3-IgSF chain locus, wherein:
the nucleic acid sequence comprises an in-frame fusion of (i) a transgene comprising a sequence encoding the antigen-binding domain and (ii) an open reading frame of the endogenous invariant CD3-IgSF chain locus encoding the invariant CD3-IgSF chain.

2 . An engineered T cell expressing a mini chimeric antigen receptor (miniCAR), wherein the miniCAR is a fusion protein comprising a heterologous antigen-binding domain and an endogenous invariant CD3 chain of the immunoglobulin superfamily (invariant CD3-IgSF chain).

3 . An engineered T cell comprising a transgene encoding an antigen-binding domain inserted in-frame with an open reading frame of a locus encoding an endogenous invariant CD3 chain of the immunoglobulin superfamily (invariant CD3-IgSF chain), wherein the engineered T cell expresses a miniCAR fusion protein comprising a heterologous antigen-binding domain and the endogenous invariant CD3-IgSF chain.

4 . The engineered T cell of claim 2 or 3 , wherein the miniCAR is expressed from a modified invariant CD3-immunoglobulin superfamily (invariant CD3-IgSF) chain locus comprising a nucleic acid sequence encoding the miniCAR, wherein:
the nucleic acid sequence comprises an in-frame fusion of (i) a transgene comprising a sequence encoding the antigen-binding domain and (ii) an open reading frame of the endogenous invariant CD3-IgSF chain locus encoding the invariant CD3-IgSF chain.

5 . The engineered T cell of claim 1 or 4 , wherein the modified invariant CD3-IgSF chain locus is a modified CD3 epsilon (CD3E) locus encoding a CD3e chain, a modified CD3 delta (CD3D) locus encoding a CD3d chain, or a modified CD3 gamma (CD3G) locus encoding a CD3g chain.

6 . The engineered T cell of any of claim 1 , 4 , or 5 , wherein the modified invariant CD3-IgSF chain locus is a modified CD3E locus encoding a CD3e chain.

7 . The engineered T cell of any of claims 1 - 6 , wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

8 . The engineered T cell of any of claims 1 - 7 , wherein the antigen-binding domain comprises a Fab fragment, a Fab 2 fragment, a single domain antibody, or a single chain variable fragment (scFv).

9 . The engineered T cell of any one of claim 1 , or 5 - 8 , wherein the modified invariant CD3-IgSF chain locus comprises, in order from 5′ to 3′, a sequence of nucleotides encoding the heterologous antigen-binding domain and the endogenous invariant CD3-IgSF chain.

10 . The engineered T cell of any of claims 1 - 9 , wherein the antigen-binding domain and the invariant CD3-IgSF chain are directly linked.

11 . The engineered T cell of any of claims 1 - 9 , wherein the antigen-binding domain and the invariant CD3-IgSF chain are linked indirectly via a linker.

12 . The engineered T cell of any of claim 1 , or 3 - 11 , wherein the transgene further comprises a nucleic acid sequence encoding a linker.

13 . The engineered T cell of claim 12 , wherein the linker is positioned 3′ to the antigen-binding domain.

14 . An engineered T cell, comprising a modified CD3E locus comprising a nucleic acid sequence encoding a miniCAR, the miniCAR comprising a heterologous antigen-binding domain and an endogenous CD3e chain, wherein:
the nucleic acid sequence comprises an in-frame fusion of (i) a transgene comprising a sequence encoding the antigen-binding domain, wherein the antigen-binding domain is an scFv, and a sequence encoding a linker, and (ii) an open reading frame of an endogenous CD3E locus encoding the CD3e chain.

15 . The engineered T cell of any one of claims 12 - 14 , wherein the transgene sequence comprises, in order from 5′ to 3′, a sequence of nucleotides encoding the antigen-binding domain and a sequence of nucleotides encoding the linker.

16 . The engineered T cell of claim 15 , wherein the modified invariant CD3-IgSF chain locus comprises, in order from 5′ to 3′, a sequence of nucleotides encoding the antigen-binding domain, the linker, and the invariant CD3-IgSF chain.

17 . The engineered T cell of any of claims 12 - 16 , wherein the linker is a polypeptide linker.

18 . The engineered T cell of any of claims 12 - 17 , wherein the linker is a polypeptide that is 3 to 18 amino acids in length.

19 . The engineered T cell of any of claims 12 - 18 , wherein the linker comprises GS, GGS, GGGGS (SEQ ID NO:122), GGGGGS (SEQ ID NO:128) and combinations thereof.

20 . The engineered T cell of any of claims 12 - 18 , wherein the linker comprises (GGS)n, wherein n is 1 to 10, (GGGGS)n (SEQ ID NO: 121), wherein n is 1 to 10, or (GGGGGS)n (SEQ ID NO:129), wherein n is 1 to 4.

21 . The engineered T cell of any of claim 1 , or 3 - 20 , wherein the transgene further comprises a nucleic acid sequence encoding one or more multicistronic element.

22 . The engineered T cell of claim 21 , wherein the P2A element comprises the sequence set forth in SEQ ID NO: 3.

23 . The engineered T cell of claim 21 or claim 22 , wherein at least one of the one or more multicistronic elements is positioned 5′ to the antigen-binding domain.

24 . The engineered T cell of any of claims 21 - 23 , wherein the transgene sequence comprises, in order from 5′ to 3′, a sequence of nucleotides encoding the multicistronic element, optionally the P2A element; the antigen-binding domain; and the linker.

25 . The engineered T cell of any of claim 1 , or 3 - 24 , wherein the transgene further comprises a nucleic acid sequence encoding an affinity tag.

26 . The engineered T cell of claim 25 , wherein the affinity tag is a streptavidin binding peptide.

27 . The engineered T cell of any of claims 21 - 26 , wherein the modified invariant CD3-IgSF chain locus comprises, in order from 5′ to 3′, a sequence of nucleotides encoding the multicistronic element; the antigen-binding domain; the linker; and the invariant CD3-IgSF chain.

28 . The engineered T cells of any of claim 1 , 3 - 13 , or 15 - 27 , wherein the open reading frame of the endogenous invariant CD3-IgSF chain locus encodes a full length mature invariant CD3-IgSF chain.

29 . The engineered T cell of any of claim 1 , 4 - 13 , or 15 - 28 , wherein the modified invariant CD3-IgSF chain locus comprises the promoter and/or regulatory or control element of the endogenous locus operably linked to control expression the nucleic acid sequence encoding the miniCAR.

30 . The engineered T cell of any of claim 1 , 4 - 13 , or 15 - 28 , wherein the modified invariant CD3-IgSF chain locus comprises one or more heterologous regulatory or control elements operably linked to control expression of the miniCAR or a portion thereof.

31 . The engineered T cell of any of claims 1 - 30 , wherein the antigen-binding domain binds to a target antigen that is associated with, specific to, and/or expressed on a cell or tissue of a disease, disorder or condition.

32 . The engineered T cell of claim 31 , wherein the target antigen i

Assignees

• Juno Therapeutics Inc

Inventors

• Poltorak Mateusz Pawel
• Germeroth Lothar
• Stemberger Christian

Classifications

• A61K40/11 Primary

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

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title

• A61K40/4211

  CD19 or B4 · CPC title

• A61K40/421

  Immunoglobulin superfamily · CPC title

• A61K2239/48

  Blood cells, e.g. leukemia or lymphoma · CPC title