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

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).

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.

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.

A polynucleotide, comprising:

• (a) a nucleic acid sequence encoding an antigen-binding domain;
• and
• (b) one or more homology arms linked to the nucleic acid sequence, wherein the one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of an invariant CD3 chain of the immunoglobulin superfamily (invariant CD3-IgSF chain) locus of a T cell, wherein the invariant CD3-IgSFchain locus encodes an invariant CD3-IgSF chain.

A polynucleotide, comprising:

• (a) a nucleic acid sequence encoding an antigen-binding domain;
• and
• (b) one or more homology arms linked to the nucleic acid sequence encoding the transgene, wherein the one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of a CD3E locus encoding a CD3e chain.

The method of claim 91 or 92, wherein the incubation is carried out subsequent to the introducing of the one or more agents and the introducing of the polynucleotide or vector, and wherein the incubation is for up to 21 days, optionally up to or about 7 days.

A T cell comprising a TCR/CD3 complex comprising a mini chimeric antigen receptor (CAR), 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) of the TCR/CD3 complex.