Genetically engineered mice expressing components of the human cellular immune system

Abstract

Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) that have been genetically engineered to express a humanized or human T cell receptor (TCR) comprising a variable domain encoded by (a) at least one human TCR variable region gamma gene segment and a (human) TCR gamma constant region gene sequence, and / or (b) or at least one human TCR variable region delta gene segment and a (human) TCR delta constant region gene sequence. Also provided are embryos, tissues, and cells expressing these. Methods for producing genetically engineered animals that express humanized or human gamma and / or delta TCRs are also provided. Methods for using genetically engineered animals to mount substantially humanized T cell immune responses for the development of human therapeutics are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 63 / 376,706, filed September 22, 2022, and U.S. Provisional Patent Application No. 63 / 383,213, filed November 10, 2022, each of which is incorporated herein by reference.

[0002] Sequence Listing An official copy of the Sequence Listing was submitted electronically via EFS-Web and filed concurrently with the specification under the file name 11224.xml, created on September 21, 2023, and 41 kilobytes in size. The Sequence Listing contained in this XML format document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses: (i) (a) a human (humanized) TCR δ polypeptide or a human (humanized) hybrid TCR polypeptide comprising a human hybrid TCR α / δ variable domain and a human TCR δ constant domain, and / or (b) a human (humanized) gamma delta T cell receptor (TCR) comprising a human (humanized) TCR gamma polypeptide; and (ii) (a) an endogenous or human (humanized) TCRα polypeptide, and (b) an endogenous or human (humanized) TCR β polypeptide, and optionally, (iii) one or more human (humanized) T cell co-receptors (e.g., CD4 and / or CD8 (e.g., CD8α and / or CD8β)), and / or (iv) one or more human (humanized) T cell co-receptors (e.g., one or more human (humanized) major histocompatibility complex (MHC II) complexes (e.g., MHC IIα and / or MHC IIβ) and / or MHC I (e.g., human (humanized) MHC Iα polypeptides alone or optionally complexed with human (humanized) β2 microglobulin). The present disclosure also relates to embryos, tissues, cells and / or nucleic acids isolated from the non-human animals, methods of making the non-human animals, and methods of using the non-human animals, for example, as non-human animal models for studying gamma and / or delta T cell responses for the development of human therapeutics. [Background technology]

[0004] In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (eg, immunoglobulins) and T lymphocytes (eg, T cell receptors, also called TCRs).

[0005] Not all antigens induce T cell activation due to tolerance mechanisms. However, in some diseases (e.g., cancer, autoimmune diseases), peptides derived from self-proteins are targeted by cellular components of the immune system, leading to the destruction of cells presenting such peptides. Significant progress has been made in recognizing clinically significant antigens (e.g., antigens associated with various types of cancer) and / or TCR sequences that bind to clinically significant antigens. However, there remains a need for in vivo and in vitro systems that mimic aspects of the human immune system to improve the identification and selection of clinically significant peptides that will induce appropriate responses in human T cells and / or TCRs capable of binding clinically significant antigens (e.g., for adoptive immunotherapy of cancer, T cell vaccination for autoimmunity, etc.). Thus, there is a need for biological systems (e.g., genetically modified non-human animals and cells) that can exhibit components of the human immune system, particularly components of the T cell immune response. Summary of the Invention [Means for solving the problem]

[0006] Described herein are non-human animals (e.g., rodents (e.g., rats or mice)) with humanized TRD loci (encoding TCR-δ) and / or TRG loci (encoding TCR-γ polypeptides), which may result in potential therapeutics that utilize human γ / δ TCRs and / or T cells.

[0007] In some embodiments, the humanized TCRγ mice described herein: (I) Germline and CD3 containing unrearranged TCR γ variable region sequences comprising unrearranged human TCR Vγ segments and unrearranged human TCR Jγ segments - Contains T cells, the unrearranged TCR gamma variable region sequence is optionally operably linked to a human TCR gamma constant region gene sequence at the endogenous TCR gamma locus (e.g., a human TCR gamma constant region gene sequence at the endogenous TCR gamma locus), and a nucleotide sequence comprising an endogenous TCR gamma constant region gene sequence (e.g., an endogenous Trgc1 constant region gene sequence, an endogenous Trgc2 constant region gene sequence, an endogenous Trgc3 constant region gene sequence, and / or an endogenous Trgc4 constant region gene sequence) is replaced with a human TCR gamma constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a TRGC2 constant region sequence); the unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vγ / Jγ variable region gene operably linked to a human TCR gamma constant region gene sequence; a rearranged human TCR Vγ / Jγ variable region gene operably linked to a human TCR gamma constant region gene sequence, which together encode a human TCR gamma polypeptide; (II) CD3 expressing on its surface a TCR comprising a human TCRγ polypeptide. + T cells, including

[0008] In some humanized TCRγ mouse embodiments, germline and CD3 -The T cell further comprises an unrearranged T cell receptor (TCR) delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged human TCR D delta segment, and an unrearranged human TCR J delta segment, wherein the unrearranged TCR delta variable region sequence is optionally operably linked to a human TCR delta constant region gene sequence at an endogenous TCR delta locus, wherein the unrearranged human TCR V delta segment, the unrearranged human TCR D delta segment, and the unrearranged human TCR J delta segment can be rearranged (or rearranged) in the T cell to form a rearranged human TCR V delta / D delta / J delta variable region gene operably linked to the human TCR delta constant region gene sequence, wherein the rearranged human TCR V delta / D delta / J delta variable region gene operably linked to the human TCR delta constant region gene sequence together encodes a human TCR delta polypeptide, and the mouse is a CD3 mouse that expresses on its surface a functional TCR comprising both a human TCR gamma polypeptide and said human TCR delta polypeptide. + In some embodiments, germ cells and CD3 - The T cell further comprises an unrearranged human TCR Vα segment upstream of the unrearranged TCRδ variable region sequence and the human TCRδ constant region gene sequence, wherein the unrearranged human TCR Vα segment, unrearranged human TCR Dδ, and unrearranged human TCR Jδ segment can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to the human TCRδ constant region gene sequence, wherein the rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence together encodes a human hybrid TCR comprising a human hybrid TCR α / δ variable domain and a human TCRδ constant domain, and the mouse expresses a functional TCR on its surface comprising a human hybrid TCR polypeptide comprising a human hybrid α / δ variable domain and a human TCRδ constant domain. + Contains T cells.

[0009] The humanized TCRδ mice described herein are (I) From 5' to CD3 an unrearranged human TCR Vα segment, and an unrearranged TCR delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged human TCR D delta segment, and an unrearranged human TCR J delta segment; an unrearranged TCR delta variable region sequence operably linked to a human TCR delta constant region gene sequence, optionally at an endogenous TCR delta locus; the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ, and the unrearranged human TCR Jδ segment can be rearranged (or rearranged) in a T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to a human TCRδ constant region gene sequence; a rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to a human TCRδ constant region gene sequence, which together encodes a human hybrid TCR polypeptide comprising a human hybrid TCR α / δ variable domain and a human TCRδ constant domain; (II) CD3 expressing on its surface a functional TCR comprising a human hybrid TCR comprising a human hybrid α / δ variable domain and a human TCR δ constant domain. + Contains T cells.

[0010] In some humanized TCR δ mouse embodiments, germline and CD3 -the T cell comprises a replacement of an endogenous TCR Vα segment with an unrearranged human TCR Vα segment and a replacement of an endogenous TCR Jα segment with an unrearranged human TCR Jα segment, wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are operably linked to each other with a TCRα constant region gene sequence, e.g., a mouse TCRα constant region gene sequence, wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment can rearrange (or rearrange) in the T cell to form a rearranged TCR Vα / Jα variable region gene operably linked to the TCRα constant region gene sequence, wherein the rearranged human TCR Vα / Jα variable region gene operably linked to the TCRα constant region gene sequence together encodes a TCRα polypeptide comprising a human TCRα variable domain operably linked to a TCRα constant domain (e.g., a human or mouse TCRα constant domain), and the mouse is a CD3 + In some humanized TCR δ mouse embodiments, germline and CD3 T cells are -The T cell comprises a replacement of all endogenous TCR Vα segments with a complete repertoire of unrearranged human TCR Vα segments, and a replacement of all endogenous TCR Jα segments with a complete repertoire of unrearranged human TCR Jα segments, wherein the complete repertoire of unrearranged human TCR Vα segments and the complete repertoire of unrearranged human TCR Jα segments are operably linked to each other at the endogenous TCRα locus with a murine TCRα constant region gene sequence, and the complete repertoire of unrearranged human TCR Vα segments and the complete repertoire of unrearranged human TCR Jα segments can be rearranged (or reconstituted) in the T cell to form a rearranged human TCR Vα / Jα variable region gene operably linked to the murine TCRα constant region gene sequence, and a rearranged TCR operably linked to the murine TCRα constant region gene sequence. The Vα / Jα variable region genes together encode a chimeric TCRα polypeptide comprising a human TCRα variable domain operably linked to a mouse TCRα constant domain, and the mouse expresses a functional TCR comprising the chimeric TCRα polypeptide on its surface. + Contains T cells.

[0011] In some humanized gamma and / or delta TCR mouse embodiments, germline and CCD3 - T cells are (A) Replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; or (B) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacement of an endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; or (C) (i) replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; and (ii) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

[0012] In some embodiments, (A) The unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; or (B) The unrearranged human TCR Vδ segments comprise a complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise a complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise a complete repertoire of unrearranged human TCR Jδ segments; (C) (i) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; and (ii) the unrearranged human TCR Vδ segments comprise the full repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise the full repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise the full repertoire of unrearranged human TCR Jδ segments.

[0013] In some humanized γ and / or δ TCR mouse embodiments, (I) Germ cells and CD3 - Somatic T cells are (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence; (II) The mice express a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + Contains T cells.

[0014] In some humanized γ and / or δ TCR mouse embodiments, germline and CD3 - The T cell further comprises an unrearranged TCR β variable region sequence comprising at least one unrearranged human TCR variable region Vβ segment, at least one unrearranged human TCR variable region Dβ segment, and at least one unrearranged TCR variable region Jβ segment, wherein the unrearranged TCR β variable region sequence is operably linked to a TCR β constant region gene sequence, such as a mouse TCR β constant region gene sequence, optionally at an endogenous TCR β locus, and the unrearranged human TCR Vβ segment, unrearranged human TCR Dβ segment, and unrearranged human TCR Jβ segment can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vβ / Dβ / Jβ variable region gene operably linked to the TCR β constant region gene sequence, and the rearranged human TCR Vβ / Dβ / Jβ variable region gene operably linked to the TCR β constant region gene sequence together comprise a human TCR β variable domain and a TCR β constant domain (e.g., a mouse or human TCR The mice are coated with a TCRβ polypeptide containing a TCRβ constant domain (a TCRβ constant domain), and the mice express a functional TCR containing the TCRβ polypeptide on their surface. + In some embodiments, the unrearranged TCR β variable region sequence comprises a mouse TCRB non-coding sequence.

[0015] In some humanized γ and / or δ TCR mouse embodiments, (I) Germ cells and CD3 - T cells are (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence; (C) At the endogenous TCRα locus Replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) At the endogenous TCRβ locus replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; Replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; and comprising the replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (II) The mouse expresses on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells and CD3 expressing a human or humanized TCR comprising a human or humanized TCR alpha polypeptide and a human or humanized TCR beta polypeptide on their surface. + Further included are T cells.

[0016] In some humanized γ and / or δ TCR mouse embodiments, (I) Germ cells and CD3 - T cells are (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence; (C) At the endogenous TCRα locus Replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) At the endogenous TCRβ locus replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; replacement of all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments; comprising the replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 co-receptor comprising the D1, D2, and D3 domains of a human CD4 polypeptide operably linked to the D4, transmembrane domain, and cytoplasmic domain of a mouse CD4 polypeptide; (F) a second nucleotide sequence encoding a chimeric human / mouse CD8 α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8 β polypeptide, the chimeric human / mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8α polypeptide, and the chimeric human / mouse CD8β polypeptide comprises a second and third nucleotide sequence comprising an IgV-like domain of a human CD8β polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8β polypeptide; (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC IIα polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC IIβ polypeptide, the chimeric human / mouse MHC IIα polypeptide comprises the α1 and α2 domains of a human HLA class IIα polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIα polypeptide, and the chimeric human / mouse MHC IIβ polypeptide comprises the β1 and β2 domains of a human HLA class IIβ polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIβ polypeptide, a first nucleic acid sequence and a second nucleic acid sequence; (H) a third nucleic acid sequence encoding a chimeric human / mouse MHC I polypeptide comprising the α1 domain, α2 domain, and α3 domain of a human HLA class I polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC class I polypeptide; (I) a polynucleotide encoding a human or humanized β2 microglobulin polypeptide, the polynucleotide comprising the nucleotide sequence set forth in exon 1 of the mouse β2 microglobulin gene operably linked to the nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene; (II) The mouse expresses on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells and CD3s expressing on their surface a functional human or humanized TCR comprising a human or humanized TCR alpha polypeptide and a human or humanized TCR beta polypeptide. + and optionally further comprising T cells. The mouse (e) chimeric human / mouse CD4 coreceptor; (f) a chimeric CD8 co-receptor comprising a chimeric human / mouse CD8α polypeptide and a chimeric human / mouse CD8β polypeptide; (h) a chimeric MHC II complex comprising a chimeric human / mouse MHC IIα polypeptide and a chimeric human / mouse MHC IIβ polypeptide, wherein the chimeric MHC II complex is capable of binding a chimeric human / mouse CD4 co-receptor; (i) a chimeric human / mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding to a chimeric CD8 co-receptor; and (j) expressing a human or humanized β2 microglobulin polypeptide;

[0017] In some humanized gamma and / or delta TCR mouse embodiments, germline and CCD3 -The T cells each comprise a human CTCF binding element upstream of the TCR gamma locus, the TCR alpha locus, or a first human CTCF binding element upstream of the TCR gamma locus and a second human CTCF binding element upstream of the TCR alpha locus. In some embodiments, the germ cells and somatic cells each comprise a human CTCF binding element upstream of the TCR alpha locus.

[0018] In some humanized gamma and / or delta TCR mouse embodiments, the mouse comprises gamma / delta T cells in its thymus, its spleen, its skin, and / or its intestinal mucosa, and the gamma / delta T cells may comprise human TCRgamma and human TCRdelta polypeptides. In some gamma and / or delta TCR mouse embodiments described herein, the mouse optionally further comprises human or humanized TCRalpha and beta MHC I, MHC IIalpha, and CD4beta, CD8, and / or beta2M loci, and the mouse may comprise a population of CD45+CD3+ T cells among intraepithelial lymphocytes (IELs) isolated from the spleen, thymus, mesenteric lymph node (MLN), skin, intestinal mucosa, and / or large or small intestine, and a proportion of the CD45+CD3+ T cell population expresses the human gamma / delta TCR. In some embodiments, the percentage of spleen, thymus, MLN, skin, intestinal mucosa, and / or IEL CD45+CD3+ T cells expressing the human γ / δ TCR in the humanized γ and / or δ TCR mouse embodiments described herein is comparable to the percentage of CD45+CD3+ T cells expressing the murine γ / δ TCR in wild-type mice (e.g., not significantly different, any differences are not statistically significant, within 10 percentage points of each other, etc.). In some embodiments, the percentage of spleen, thymus, MLN, and / or IEL CD45+CD3+ T cells expressing the human γ / δ TCR in the γ and / or δ TCR mouse embodiments described herein is greater (e.g., 1.5- to 3-fold greater) than the percentage of CD45+CD3+ T cells expressing the murine γ / δ TCR in wild-type mice.

[0019] In some humanized gamma and / or delta TCR embodiments, the human TCR gamma polypeptide is derived from a human TRGV2 gene segment, a human TRGV3 gene segment, a human TRGV4 gene segment, a human TRGV5 gene segment, a human TRGV8 gene segment, a human TRGV9 gene segment, a human TRGV10 gene segment, or a human TRGV11 gene segment. In some embodiments, the human TCR gamma polypeptide is derived from a human TRGJ1 gene segment, a human TRGJP gene segment, a human TRGJP1 gene segment, a human TCRGJ2 gene segment, or a human TRGJP2 gene segment. In some embodiments, the human TCR delta polypeptide is derived from a human TRDV1 gene segment, a human TRAV17 gene segment, a human TRAV19 gene segment, a human TRAV21 gene segment, a human TRAV21 gene segment, a human TRAV26-2 gene segment, a human TRAV29 / TRDV5 gene segment, a human TRAV31 gene segment, a human TRAV38-2 / TRDV8 gene segment, a human TRAV39 gene segment, a human TRAV40 gene segment, a human TRAV41 gene segment, a human TRDV2 gene segment, or a human TRDV3 gene segment. In some embodiments, the human TCR delta polypeptide is derived from a human TRDJ1 gene segment, a human TRDJ2 gene segment, a human TRDJ3 gene segment, or a human TRDJ4 gene segment.

[0020] Also described herein is a mouse embryonic stem (ES) cell or germ cell comprising an unrearranged TCR gamma variable region sequence comprising an unrearranged human TCR V gamma segment and an unrearranged human TCR J gamma segment, wherein the unrearranged TCR gamma variable region sequence is operably linked to a human TCR gamma constant region gene sequence, optionally at an endogenous TCR gamma locus (e.g., a human TCR gamma constant region gene sequence at an endogenous TCR gamma locus), and wherein a nucleotide sequence comprising the endogenous TCR gamma constant region gene sequence (e.g., an endogenous Trgc1 constant region gene sequence, an endogenous Trgc2 constant region gene sequence, an endogenous Trgc3 constant region gene sequence, and / or an endogenous Trgc4 constant region gene sequence) has been replaced with a nucleotide sequence comprising a human TCR gamma constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence). In some embodiments, the ES cell or germ cell further comprises an unrearranged T cell receptor (TCR) delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged human TCR D delta segment, and an unrearranged human TCR J delta segment, wherein the unrearranged TCR delta variable region sequence is optionally operably linked to a human TCR delta constant region gene sequence at the endogenous TCR delta locus. In some embodiments, the ES cell or germ cell further comprises an unrearranged human TCR V alpha segment upstream of the unrearranged TCR delta variable region sequence and the human TCR delta constant region gene sequence.

[0021] Also, in the 5' to 3' direction, an unrearranged human TCR Vα segment, and an unrearranged TCR delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged TCR D delta, and an unrearranged human TCR J delta segment; Mouse ES or germline cells are described in which unrearranged TCR delta variable region sequences are optionally operably linked to human TCR delta constant region gene sequences at the endogenous TCR delta locus.

[0022] In some embodiments, the ES cells or germ cells comprise a replacement of endogenous TCR Vα segments with unrearranged human TCR Vα segments, and a replacement of endogenous TCR Jα segments with unrearranged human TCR Jα segments, wherein the unrearranged human TCR Vα segments and the unrearranged human TCR Jα segments are operably linked to a TCR α constant region gene sequence (e.g., a mouse TCR α constant region gene sequence (optionally at the endogenous TCR α locus) or a human TCR α constant region gene sequence). In some embodiments, the mouse ES cells or germ cells comprise a replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and a replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments, wherein the full repertoire of unrearranged human TCR Vα segments and the full repertoire of unrearranged human TCR Jα segments are operably linked to a mouse TCR α constant region gene sequence at the endogenous TCR α locus.

[0023] In some embodiments, the mouse ES cells or germ cells are (A) Replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with unrearranged human TCRγ constant region gene sequences; or (B) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacement of an endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or (C)(i) replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; and (ii) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

[0024] In some ES or germ cell embodiments, (A) The unrearranged TCR Vγ segments comprise the full repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the full repertoire of unrearranged human TCR Jγ segments, or (B) The unrearranged human TCR Vδ segments comprise the full repertoire of unrearranged human TCR Vδ segments; the unrearranged human TCR Dδ segments comprise the full repertoire of unrearranged human TCR Dδ segments; the unrearranged human TCR Jδ segments comprise the full repertoire of unrearranged human TCR Jδ segments; or (C)(i) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; and (ii) The unrearranged human TCR Vδ segments comprise the complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise the complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise the complete repertoire of unrearranged human TCR Jδ segments.

[0025] In some embodiments, (I) ES cells or germ cells are (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all endogenous TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and It involves replacing the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence. In some embodiments, the endogenous TCR gamma locus described herein comprises a replacement of an endogenous genomic sequence comprising all endogenous TCR Vγ segments, all endogenous TCR Jγ segments, and all endogenous TCR gamma constant region gene sequences with a human genomic sequence comprising a complete repertoire of unrearranged human TCR Vγ segments, a complete repertoire of unrearranged human TCR Jγ segments, and a complete repertoire of human TCR gamma constant region gene sequences (e.g., hTCRGC1 and hTCRGC2).

[0026] In some embodiments, the ES cell or germ cell further comprises an unrearranged TCR β variable region sequence comprising at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment, wherein the unrearranged TCR β variable region sequence is optionally operably linked to a TCR β constant region gene sequence (e.g., a murine TCR β constant region gene sequence) at the endogenous TCR β locus. In some embodiments, the unrearranged TCR β variable region sequence comprises a murine TCR β non-coding sequence.

[0027] In some embodiments, the ES cells or germ cells described are (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence, (C) At the endogenous TCRα locus Replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) At the endogenous TCR β locus replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; Replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; and It involves replacing all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments.

[0028] In some embodiments, the ES cells or germ cells described herein are (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence; (C) At the endogenous TCRα locus Replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) At the endogenous TCRβ locus replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; replacement of all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments; comprising the replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (a) a first nucleotide sequence encoding a chimeric human / mouse CD4 co-receptor comprising the D1, D2, and D3 domains of a human CD4 polypeptide operably linked to the D4, transmembrane domain, and cytoplasmic domain of a mouse CD4 polypeptide; (F) a second nucleotide sequence encoding a chimeric human / mouse CD8 α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8 β polypeptide, the chimeric human / mouse CD8α polypeptide comprises second and third nucleotide sequences, wherein the chimeric human / mouse CD8 polypeptide comprises an IgV-like domain of a human CD8 polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8α polypeptide, and the chimeric human / mouse CD8 polypeptide comprises an IgV-like domain of a human CD8 polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8β polypeptide; (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC IIα polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC IIβ polypeptide, the chimeric human / mouse MHC IIα polypeptide comprises the α1 and α2 domains of a human HLA class IIα polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIα polypeptide, and the chimeric human / mouse MHC IIβ polypeptide comprises the β1 and β2 domains of a human HLA class IIβ polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIβ polypeptide, a first nucleic acid sequence and a second nucleic acid sequence; (H) a third nucleic acid sequence encoding a chimeric human / mouse MHC I polypeptide comprising the α1 domain, α2 domain, and α3 domain of a human HLA class I polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC class I polypeptide; (I) A polynucleotide encoding a human or humanized β2 microglobulin polypeptide, comprising the nucleotide sequence shown in exon 1 of the mouse β2 microglobulin gene operably linked to the nucleotide sequences shown in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.

[0029] In some ES cell or germ cell embodiments, the ES cell or germ cell further comprises a human CTCF binding element upstream of the TCRγ locus, upstream of the TCRα locus, or a first human CTCF binding element upstream of the TCRγ locus and a second human CTCF binding element upstream of the TCRα locus. In some embodiments, the mouse ES cell or germ cell further comprises a human CTCF binding element upstream of the TCRα locus. In some embodiments, the mouse ES cell or germ cell comprises the human nucleotide sequence set forth in chr7:38383439-38230960 (GRCh38 coordinates) and / or the human nucleotide sequence set forth in Chr14:22421820-22464666 (GRCh38 coordinates).

[0030] Also described herein are targeting vectors, for example, targeting vectors that include a 5' mouse homology arm and a 3' mouse homology arm, as follows, from 5' to 3': (a) 5' mouse homology arm; (b) the human nucleotide sequence set forth in chr7:38383439-38230960 (GRCh38 coordinates); and (c) 3' mouse homologous arm.

[0031] Also described is a targeting vector comprising (i) a selection cassette and (ii) a human nucleotide sequence set forth in Chr14:22421820-22464666 (GRCh38 coordinates).

[0032] In some methods described herein, the method includes immunizing a genetically modified mouse described herein with a target antigen and allowing the mouse to mount an immune response to the target antigen; and obtaining therefrom a nucleic acid sequence encoding a human TCR variable domain that binds to the target antigen, e.g., a nucleic acid sequence encoding a human TCRγ polypeptide (or domain thereof) of a TCR that binds to the target antigen, a nucleic acid sequence encoding a human TCRδ polypeptide (or variable domain thereof) of a TCR that binds to the target antigen, or a nucleic acid sequence encoding a human TCRα / δ polypeptide (or variable domain thereof) that binds to the target antigen, wherein the human TCRα / δ polypeptide is encoded by a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to a human TCRδ constant region gene sequence, a nucleic acid sequence encoding a human TCRα polypeptide (or variable domain thereof) of a TCR that binds to the target antigen, a nucleic acid sequence encoding a human TCRβ polypeptide (or variable domain) of a TCR that binds to the target antigen, or any combination thereof.

[0033] Also described herein are methods for producing a human therapeutic (or human TCR protein), comprising immunizing a genetically modified mouse described herein with an antigen of interest; obtaining T cells from the mouse that are reactive to the antigen of interest; and obtaining from the T cells a TCR (e.g., γδ TCR, α / δγ TCR, αβ TCR) that binds to the antigen of interest, and / or a nucleic acid sequence encoding the TCR or encoding a variable domain of the TCR, wherein the TCR comprises a human TCR variable domain, and optionally, using the human TCR variable domain in the human therapeutic. In some embodiments, the human therapeutic (or human TCR protein) is a soluble T cell receptor. In some embodiments, the human therapeutic (or human TCR protein) is a single-chain TCR. In some embodiments, the human therapeutic (or human TCR protein) is an scTv. In some embodiments, the soluble T cell receptor is fused to a moiety that can kill infected or cancer cells (e.g., a cytotoxic molecule (e.g., a chemotherapeutic agent), a toxin, a radionuclide, a prodrug, or an antibody), an immunomodulatory molecule (e.g., a cytokine or chemokine), and / or an immunoinhibitory molecule (e.g., a molecule that inhibits T cells from killing other cells that carry the antigen recognized by the T cell).

[0034] Also described herein are host cells comprising a nucleic acid molecule produced according to any of the methods described herein.

[0035] 1. A method for producing a genetically modified mouse or mouse ES cell, comprising: (a) an unrearranged TCR gamma variable region sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment, the unrearranged TCR gamma variable region sequence is operably linked to a human TCR gamma constant region gene sequence, and optionally, at the endogenous TCR gamma locus, a nucleotide sequence comprising an endogenous TCR gamma constant region gene sequence (e.g., an endogenous TCR gamma constant region gene sequence (e.g., an endogenous Trgc1 constant region gene sequence, an endogenous Trgc2 constant region gene sequence, an endogenous Trgc3 constant region gene sequence, and / or an endogenous Trgc4 constant region gene sequence) is replaced with a nucleotide sequence comprising a human TCR gamma constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence); the unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vγ / Jγ variable region gene operably linked to a human TCR gamma constant region gene sequence; modifying the genome of a mouse or mouse ES cell containing germ cells and CD3- somatic cells containing rearranged human TCR Vγ / Jγ variable region genes operably linked to a human TCR γ constant region gene sequence that together encode a human TCR γ polypeptide; and / or (b) an unrearranged T cell receptor (TCR) delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged human TCR D delta segment, and an unrearranged human TCR J delta segment; an unrearranged TCR delta variable region sequence operably linked to a human TCR delta constant region gene sequence; an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment can be rearranged (or rearranged) in a T cell to form a rearranged human TCR Vδ / Dδ / Jδ variable region gene operably linked to a human TCRδ constant region gene sequence; Provided herein are methods that include modifying the genome of a mouse or mouse ES cell, wherein a rearranged human TCR Vδ / Dδ / Jδ variable region gene operably linked to a human TCRδ constant region gene sequence comprises an unrearranged T cell receptor (TCR) δ variable region sequence, which together encode a human TCRδ polypeptide.

[0036] In some embodiments, the modifying comprises: (a) replacing an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes with a heterologous sequence comprising unrearranged human TCR Vγ segments and unrearranged human TCR Jγ segments operably linked to a human TCRγ constant region gene sequence (e.g., replacing all endogenous TCR Vγ segments, all endogenous TCR Jγ segments, and all endogenous TCRγ constant region gene sequences with a human genomic sequence comprising a complete repertoire of unrearranged human TCR Vγ segments, a complete repertoire of unrearranged human TCR Jγ segments, and a complete repertoire of human TCRγ constant region gene sequences); and / or (b) Replacing an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and an endogenous TCR Cδ gene with a heterologous sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, an unrearranged human TCR Jδ segment and a human TCRδ constant region gene sequence.

[0037] In some method embodiments, (a) the endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes comprises a complete repertoire of endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes; and / or (b) The endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ genes includes all endogenous TCR Vδ, Dδ, Jδ gene segments and TCR Cδ genes located between the TCR Vα and TCR Jα gene segments.

[0038] In some method embodiments, (a) the heterologous sequence comprising unrearranged human TCR Vγ segments and unrearranged human TCR Jγ segments operably linked to human TCR gamma constant region gene sequences includes the complete repertoire of unrearranged TCR Vγ and unrearranged human TCR Jγ segments and all human TCR gamma constant region gene sequences (e.g., hTCRGC1 and hTCRGC2); and / or (b) The heterologous sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, an unrearranged human TCR Jδ segment, and a human TCRδ constant region gene sequence comprises a complete repertoire of unrearranged TCR Vδ, unrearranged Dδ, unrearranged human TCR Jδ segment, and human TCR Cδ gene sequences located between a human TCR Vα gene segment and a human TCR Jα gene segment on chromosome 14 of the human genome.

[0039] In some method embodiments, the modification comprises homologous recombination in the ES cells such that the heterologous sequences comprising unrearranged human TCR Vγ segments and unrearranged human TCR Jγ segments are operably linked to a human TCR γ constant region gene sequence, and the heterologous sequences comprising unrearranged human TCR Vδ segments, unrearranged human TCR Dδ segments, unrearranged human TCR Jδ segments, and human TCR δ constant region gene sequences have been added, in any order, to the genome of the one or more ES cells. Some methods further comprise generating a mouse from the one or more ES cells.

[0040] In some embodiments, the modifying comprises: (a) obtaining a first mouse comprising a homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with a heterologous sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment operably linked to a human TCR γ constant region gene sequence; (b) obtaining a second mouse comprising a homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and an endogenous TCR Cδ gene with a heterologous sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, an unrearranged human TCR Jδ segment operably linked to a human TCRδ constant region gene sequence; (c) mating the first mouse and the second mouse to obtain a genetically modified mouse; Genetically modified mice (i) replacement of endogenous genomic sequences comprising endogenous TCR Vγ and Jγ gene segments, and endogenous TCR Cγ genes, with heterologous sequences comprising unrearranged human TCR Vγ and unrearranged human TCR Jγ segments operably linked to a human TCRγ constant region gene sequence; and (ii) comprising a replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, and Jδ gene segments, and an endogenous TCR Cδ gene, with a heterologous sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment operably linked to a human TCRδ constant region gene sequence; The genetically modified mice express human TCRγ and human TCRδ polypeptides. [Brief explanation of the drawings]

[0041] [Figure 1]Figure 1 illustrates antigen recognition by various T cell receptor (TCR) embodiments. The left panel shows a non-limiting α / β T cell embodiment interacting with a non-limiting antigen-presenting cell embodiment, where the chimeric α / β TCR recognizes antigen (gray circle) presented in the context of a chimeric major histocompatibility complex (MHC) I polypeptide, (i) comprising the extracellular domain of a human leukocyte antigen (HLA) class I polypeptide, and (ii) associated with human β2 microglobulin (β2M). A non-limiting α / β T cell embodiment interacts with a non-limiting professional antigen-presenting cell embodiment, where the chimeric α / β TCR recognizes antigen (gray circle) presented in the context of a chimeric MHC II, (i) comprising the extracellular domain of an HLA class II complex. The right panel shows a non-limiting γ / δ T cell embodiment, where antigen is recognized in an MHC-independent manner. MHC I and MHC II domains and components are shown, along with their respective co-receptors, CD8 and CD4. Mouse regions are solid lines and human regions are unfilled / dotted lines. In various embodiments presented in the figures, the illustrated associations can be made in genetically modified animals (e.g., mice) described herein. [Figure 2]Figure 2 shows (not to scale) the strategy for inserting a human TCRδ locus into a humanized TCRα allele (MAID1771) of the mouse TCRα locus, where the TCRδ locus is added downstream of a human TCR Vα segment and upstream of a human TCR Jα segment. Also shown is the strategy for inserting a human CTCF binding element upstream of the humanized TCRα locus. Mouse coding sequences are indicated by closed symbols, and human coding sequences are indicated by open symbols unless otherwise indicated. Mouse intergenic or non-coding sequences are indicated by solid lines between coding sequences, and human intergenic or non-coding sequences are indicated by unfilled double lines between coding sequences unless otherwise specified. MAID refers to the modified allele ID number. H = human, TRAV = TCR Vα segment, TRAJ = TCR Jα segment (hTRAJ = human TRAJ), TRAC = TCR Cα domain, TCRD = TCRδ, BHR = bacterial homologous recombination, EP = electroporation, CTCF = CCCTC-binding factor, Loxp-Ub-Hyg = floxed hygromycin resistance gene under the control of the ubiquitin promoter. [Figure 3A] FIG. 3A shows (not to scale) the deletion of all mouse TCRγ V and J segments and TCRγ C genes from the mouse TCRγ locus to form the MAID20143 allele. [Figure 3B]Figure 3B shows the strategy for inserting human TCRγV and J segments and the TCRγC gene into the TCRγ locus of a mouse lacking all mouse TCRγV and J segments and the TCRγC gene to form a human TCRγ allele (MAID20200). Mouse coding sequences are indicated by closed symbols, and human coding sequences are indicated by open symbols unless otherwise indicated. Mouse intergenic or noncoding sequences are indicated by solid lines between coding sequences, and human intergenic or noncoding sequences are indicated by unfilled double lines between coding sequences unless otherwise specified. Although not shown in Figure 3B, MAID20200 contains all 15 hTRGV segments, all 5 hTRGJ segments, and both hTRGC genes in the germline configuration. MAID refers to the modified allele ID number. H = human, TRGV = TCR Vγ segment, TRGJ = TCR Jγ segment, TRGC = TCR Cγ gene, TCRG = TCRγ, BHR = bacterial homologous recombination, EP = electroporation, CTCF = CCCTC-binding factor, Loxp-Hyg-Loxp = floxed hygromycin resistance gene under the control of the ubiquitin promoter. [Figure 4]Figure 4 shows (not to scale) the homozygous MAID6981 allele after removal of the selection cassette from MAID6980 and the homozygous MAID20201 allele after removal of the selection cassette from MAID20200. Although not shown in Figure 4, MAID20201 contains all 15 hTRGV segments, all 5 hTRGJ segments, and both hTRGC genes in germline configuration. Mouse coding sequences are indicated by closed symbols, and human coding sequences are indicated by open symbols unless otherwise indicated. Mouse intergenic or non-coding sequences are indicated by solid lines between coding sequences, and human intergenic or non-coding sequences are indicated by unfilled double lines between coding sequences unless otherwise specified. MAID refers to the modified allele ID number. H = human, TRAV = TCR Vα segment, TRAJ = TCR Jα segment (hTRAJ = human TRAJ), TRAC = TCR Cα domain, TCRD = TCRδ locus, enh = enhancer, CTCF = CCCTC binding factor, Loxp = lox recombination site after removal of the floxed drug cassette with Cre recombinase. [Figure 5A] Figure 5A shows representative FACS dot plots of mouse splenocytes from three different wild-type (WT) mice homozygous for the humanized TCR α / δ and γ and wild-type TCR β loci (6981 OH 20201HO), as well as three mice, stained with anti-human γ / δ (Y-axis) and anti-mouse γ / δ (X-axis) antibodies. [Figure 5B] Figure 5B shows representative FACS plots of mouse thymocytes from three different wild-type ("WT") and three mice homozygous for the humanized TCR α / δ and γ and wild-type TCR β loci (6981 HO 20201HO) stained with anti-human γ / δ (Y-axis) and anti-mouse γ / δ (X-axis) antibodies. [Figure 6A]Figure 6A shows the percentage of TCRd mRNAs (Y-axis) utilizing the indicated human TCRα V and TCRδ V segments in thymic or splenic T cells of homozygous mice for humanized TCRα / δ and γ and wild-type TCRβ loci, as determined by NGS (6981 HO 20201HO). [Figure 6B] Figure 6B shows the percentage of TCRd mRNAs (Y-axis) utilizing the indicated TCRδJ segments present in thymic or splenic T cells of homozygous mice relative to the humanized TCRα / δ and γ and wild-type TCRβ loci, as determined by NGS (6981 HO 20201HO). [Figure 7A] Figure 7A shows the percentage of TCRg mRNAs utilizing the indicated human TCRγ V segments (Y-axis) present in thymic or splenic T cells of homozygous mice for humanized TCRα / δ and γ and wild-type TCR β loci (6981 HO 20201HO hTCRα). [Figure 7B] Figure 7B shows the percentage of TCRg mRNAs (Y-axis) utilizing the indicated human TCRγ J segments present in thymic or splenic T cells of homozygous mice for humanized TCRα / δ and γ and wild-type TCR β loci (6981HO 20201HO hTCRα). [Figure 8A] Figure 8A shows the incremental strategy for humanizing the mouse TCR alpha locus (not to scale), in which TCR alpha variable region gene segments are successively added upstream of the initial humanization of the deleted mouse locus (MAID1540). Mouse coding sequences are shown in solid form, and human coding sequences are shown in open form unless otherwise indicated. Although not shown in this figure, each non-coding sequence between each human TRAV and each human TRAJ is human. The remaining intergenic or non-coding sequences are mouse. MAID refers to the modified allele ID number. TRAV = TCR V alpha segment, TRAJ = TCR J alpha segment (hTRAJ = human TRAJ), TRAC = TCR C alpha domain, TCRD = TCR delta. [Figure 8B]Figure 8B shows a stepwise strategy for humanizing the mouse TCRβ locus (not to scale), in which TCRβ variable region gene segments are sequentially added to a deleted mouse TCRβ variable locus. Mouse coding sequences are shown as filled shapes, and human coding sequences are shown as empty shapes unless otherwise indicated. Although not depicted in this figure, the non-coding sequences between each human TRBV, between human D1 and the nearest human J, between human D2 and each human J, and between each J are human. The remaining intergenic or non-coding sequences are mouse. MAID refers to the modified allele ID number. TRBV or TCRBV = TCR Vβ segment. [Figure 8C] Figure 8C shows schematic diagrams (not to scale) of (a) the TRBDJ1 cluster, in which the D1 and J1 gene segments are human and the non-coding sequences between them, including RSS and other intergenic sequences, are mouse; (b) the mouse TRB C1 constant region gene; (c) the TRBDJ2 cluster, in which the D2 and J2 gene segments are human and the non-coding sequences between them, including RSS and other intergenic sequences, are mouse; and (d) the mouse TRB C2 constant region gene. Mouse coding sequences are shown as filled shapes, and human coding sequences are shown as empty shapes. As depicted in this figure, the non-coding sequences between each human TRBD segment and each human TRBJ segment, and between each TRBJ segment, are mouse. LoxP sequences are shown as arrows labeled accordingly. [Figure 9A] Figure 9A shows a schematic diagram (not to scale) of the chimeric CD4 locus. Human coding exon sequences are indicated by striped shapes, mouse coding exons are indicated by filled shapes, and non-coding exons are indicated by empty shapes. The immunoglobulin-like domain (Ig), transmembrane (TM), cytoplasmic (CYT), and signal peptide (signal) coding exons, as well as the 3' untranslated region (UTR), are shown. The floxed (loxP) neomycin phosphotransferase (Pgk-neo) cassette is depicted with an arrowhead labeled accordingly. [Figure 9B]Figure 9B shows a schematic diagram (not to scale) of the chimeric CD8a and CD8b loci. Human coding exon sequences are indicated by striped shapes, mouse coding exons are indicated by filled shapes, and non-coding exons are indicated by empty shapes. The immunoglobulin-like domain (IgV), transmembrane (TM), cytoplasmic (CYT), and signal peptide (signal) coding exons, as well as the 3' untranslated region (UTR), are indicated. The floxed (loxP) hygromycin (Hyg) and neomycin phosphotransferase (Pgk-neo) cassettes are indicated by correspondingly labeled arrows. [Figure 10A] Figures 10A-10C provide schematic diagrams (not to scale) of exemplary chimeric MHC I and MHC II loci, e.g., a chimeric HLA-A2 / H-2K locus (Figure 10A), a chimeric HLA-DR2 / H-2E locus (Figure 10B), and a humanized β2M locus (Figure 10C). Unless otherwise indicated, human sequences are depicted as empty shapes, while mouse sequences are depicted as filled shapes. Striped shapes represent exon 1 of H-2E, which is derived from a mouse strain different from the endogenous locus (see Figure 11B). The floxed neomycin phosphotransferase cassette is depicted with an arrowhead labeled accordingly. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 11A]Figures 11A-11C show a strategy for generating a humanized MHC locus containing humanized MHC I and MHC II genes. In the specific embodiment depicted in Figure 11A, the resulting mouse MHC locus contains chimeric HLA-A2 / H-2K and HLA-DR2 / H-2E sequences (H2-K+ / 1666 MHC-II+ / 6112) and lacks H2-D (H2-D+ / deleted) and H-2A sequences (the genetic engineering scheme also results in the deletion of H-2A). The large targeting vector (LTVEC) or Cre recombinase construct introduced into the ES cells at each stage of humanization is depicted to the right of the arrow. The MAID or four-digit number refers to the altered allele ID number. [Figure 11B] Figure 11B is a schematic diagram (not to scale) of an exemplary HLA-DR2 / H-2E large targeting vector. Unless otherwise indicated, human sequences are depicted as empty shapes and mouse sequences are depicted as filled shapes. Striped shapes represent exon 1 of H-2E derived from a mouse strain different from the endogenous locus. Floxed hygromycin cassettes are depicted as arrows labeled accordingly. [Figure 11C] Figure 11C is a schematic diagram (not to scale) of an exemplary genotype of a chimeric human / mouse MHC locus (** represents the H-2L gene, which is absent in all mouse strains, e.g., absent in the C57BL / 6 or 129 mouse strains), in which the endogenous mouse H-2K and H-2E loci are replaced by the chimeric human / mouse HLA-A2 / H-2K and HLA-DR2 / H-2E loci, respectively (striped shapes), the H-2A and H-2D loci are deleted (empty shapes outlined by dotted lines), and the remaining loci are endogenous mouse genes (solid shapes outlined by solid lines). [Figure 12A]Figures 12A-12D show the presence of γδ T cells in the spleen, thymus, intestinal tissue, and skin of TCRα / δ, TCRβ, TCRγ, MHC I, MHC IIα and β, CD4, CD8α and β, and β2M loci (VelociT αβγδ mice) or wild-type (WT) mice. Figure 12A shows representative FACS dot plots of mouse CD45+CD3+ splenic T cells from two different VelociT αβγδ mice and two different WT mice stained with anti-human γ / δ (Y-axis) and anti-mouse γ / δ (X-axis) antibodies. [Figure 12B] Figure 12B shows representative FACS dot plots of mouse CD45+CD3+ thymic T cells from two different VelociT αβγδ mice and two different WT mice stained with anti-human γ / δ (Y-axis) and anti-mouse γ / δ (X-axis) antibodies. [Figure 12C] Figure 12C shows representative FACS dot plots of CD45+CD3+ T cells from mesenteric lymph nodes (MLN, first column) and intraepithelial lymphocytes (IEL) isolated from the colon (second column) and small intestine (third column) from two different VelociT αβγδ mice and two different WT mice stained with anti-human γ / δ (Y-axis) and anti-mouse γ / δ (X-axis). [Figure 12D] Figure 12D shows representative FACS contour plots of CD45+CD3+ skin epidermal T cells from two different VelociT αβγδ mice and two different WT mice stained with anti-human γ / δ (Y-axis) and anti-mouse γ / δ (X-axis) antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0042] Described herein are non-human animals (e.g., rodents (e.g., rats or mice)) with humanized TRG (encoding TCR-γ polypeptides) and / or TRD (encoding TCR-δ polypeptides) loci, which may provide potential therapeutics utilizing human γ / δ T cells. As shown herein for such genetically engineered mice, mice with human or humanized TRD (encoding TCR-δ polypeptides) and / or TRG (encoding TCR-γ polypeptides) loci contain human or humanized γ / δ T cells in the thymus and spleen at levels comparable to mice with intact mouse components (Figures 5A and 5B). These mice also utilize a diverse repertoire of human TCRγ and / or human TCRδ gene segments, when present (Figures 6A, 6B, 7A, and 7B), demonstrating proper recombination, expression, and thymic selection of human or humanized γ / δ TCRs. Also described herein are tissues and cells expressing human or humanized γ / δ TCRs, animals expressing human or humanized γ / δ TCRs, methods of using the tissues and cells, methods of producing animals, and embryonic stem (ES) cells and germ cells that can be used to produce such animals. Additionally, described herein are non-human animals (e.g., mice) that contain human or humanized TRD and TRG loci, along with human or humanized TRA and TRAB loci and human or humanized loci for other components of T cell immunity, including TCR co-receptors (CD4 and CD8) and MHC loci. Thus, such mice achieve full humanization of both the α / β and γ / δ T cell lineages, in contrast to the published "VelociT" mouse (Moore, M. et al. Sci Immunol 6 (2021); see above), in which α / β T cells but not γ / δ T cells were humanized.VelociT αβγδ mice homozygous for one or more unrearranged human or humanized TCRγ, TCRδ, TCRα, and / or TCRβ loci, one or more human or humanized coreceptor loci, and / or one or more MHC loci contain human or humanized γ / CD45+CD3+ T cells in the thymus, spleen, skin, mesenteric lymph nodes (MLN), and intraepithelial lymphocytes (IELs) isolated from the colon or small intestine at levels comparable to or exceeding those of mice with fully mouse components (Figures 12A-12D). Human or humanized α / β and γ / δ T cell lineages, tissues and cells expressing human or humanized T cell coreceptors and MHC, methods of using and generating such humanized animals, and embryonic stem (ES) and germ cells that can be used to generate such animals are also described.

[0043] The terms "chain" and "polypeptide" encompass consecutive amino acids that are covalently linked and have a particular amino acid sequence, and such terms may be used interchangeably herein. Generally, a T cell receptor comprises two TCR chains / polypeptides (e.g., a TCRγ polypeptide associated with a TCRδ polypeptide, a TCRγ polypeptide associated with a hybrid TCRα / δ polypeptide comprising a hybrid TCRα / δ variable domain and a TCRδ constant domain, or a TCRα polypeptide associated with a TCRβ polypeptide).

[0044] Those skilled in the art will understand that in addition to the nucleic acids whose nucleic acid residues encode TCR variable region gene segments (e.g., TCR V, D, and J gene segments) and / or any of the humanized T-cell co-receptor polypeptides, humanized MHC polypeptides, and β2 microglobulin described herein, due to the degeneracy of the genetic code, other nucleic acids can encode the polypeptides of the invention. Thus, in addition to genetically modified non-human animals comprising unrearranged human TCR gamma variable region gene segments in their genome, also provided are non-human animals whose genome comprises such gene segments, including human TCR gamma constant region gene sequences, unrearranged human TCR delta variable region gene segments, and / or human TCR delta constant region gene sequences (and optionally unrearranged human TCR alpha variable region gene segments, unrearranged human TCR beta variable region gene segments, nucleotide sequences encoding humanized T-cell co-receptor polypeptides, e.g., CD4 or CD8 polypeptides, and / or nucleic acid sequences encoding humanized MHC polypeptides, constant region gene sequences, humanized T-cell co-receptor polypeptides (e.g., CD4 or CD8 polypeptides), and optionally nucleic acid sequences encoding humanized MHC polypeptides that can associate with humanized T-cell co-receptor polypeptides that differ from those described herein due to degeneracy in the genetic code or that differ by encoding conservative amino acid substitutions.

[0045] Also described herein are genetically modified non-human animals whose genomes comprise (e.g., at an endogenous locus) nucleotide sequences encoding non-variable amino acid sequences or polypeptides (e.g., TCR framework regions, TCR constant domains, CD4 or CD8 polypeptides, MHC polypeptides, etc.), wherein the non-variable amino acid sequences or polypeptides comprise conservative amino acid substitutions of the amino acid sequences described herein.

[0046] Conservative amino acid substitutions include those in which an amino acid residue is replaced with another amino acid residue having a side chain, R group, with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying a nucleotide sequence to introduce nucleotide changes that encode the conservative substitution. Generally, conservative amino acid substitutions do not substantially alter the desired functional property of a protein, such as the ability of CD4 or CD8 to bind to MHC II or MHC I, respectively. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Conservative amino acid substitutions include valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine. In some embodiments, conservative amino acid substitutions can be the substitution of any naturally occurring residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Sequence Database, Science 256:1443-45, which is incorporated herein by reference. In some embodiments, the substitution is a moderately conservative substitution, and the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0047] Sequence identity can be determined by a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments described herein, identity is determined using ClustalW v.1.83 (slow) alignment with an open gap penalty of 10.0 and an extended gap penalty of 0.1, and using the Gonnet similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared for sequence identity depends on the particular sequence. In various embodiments, identity is determined by comparing the sequence of the mature protein from its N-terminus to its C-terminus. In various embodiments, when comparing a chimeric human / non-human sequence to a human sequence, the human portion (but not the non-human portion) of the chimeric human / non-human sequence is used in the comparison to determine the level of identity between the human sequence and the human portion of the human / non-human chimeric sequence (e.g., comparing the human ectodomain of a chimeric human / mouse protein to the human ectodomain of a human protein).

[0048] The term "homology" or "homologous" with respect to a sequence, e.g., a nucleotide sequence or an amino acid sequence, refers to two sequences that, upon optimal alignment and comparison, are identical, e.g., at least about 75% of the nucleotides or amino acids, e.g., at least about 80% of the nucleotides or amino acids, e.g., at least about 90-95% of the nucleotides or amino acids, e.g., greater than 97% of the nucleotides or amino acids. Those skilled in the art will understand that for optimal gene targeting, the targeting construct should contain arms homologous to the endogenous DNA sequence (i.e., "homologous arms"). Thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

[0049] The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. As such, a nucleic acid sequence encoding a protein can be operably linked to regulatory sequences (e.g., promoters, enhancers, silencer sequences, etc.) to maintain proper transcriptional regulation. Also, various portions of a chimeric or humanized protein of the invention can be operably linked to maintain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless otherwise stated, the various domains of a chimeric or humanized protein of the invention are operably linked to each other.

[0050] The term "replacement," in reference to gene replacement, refers to the placement of exogenous genetic material at an endogenous locus, thereby replacing all or part of the endogenous gene with an orthologous or homologous nucleic acid sequence. As shown in the Examples below, in one embodiment, endogenous gene segments or constant region gene sequences at the TCR gamma locus have been replaced with (orthologous) human TCR gamma gene segments or human TCR gamma constant region gene sequences, respectively, and / or endogenous gene segments or constant region gene sequences at the TCR delta locus have been replaced with (orthologous) human TCR delta gene segments or human TCR delta constant region gene sequences, respectively.

[0051] As used herein, "functional," e.g., in reference to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with a native protein. For example, in some embodiments of the invention, replacement of a non-human animal TCR gene with a human TCR gene at an endogenous non-human animal TCR locus results in a locus that is incapable of expressing functional endogenous TCR polypeptides, but is capable of expressing functional human TCR polypeptides. As a non-limiting example, replacement of an endogenous non-human TCRγ gene and / or an endogenous non-human TCRδ gene with a human TCRγ gene and / or a human TCRδ gene, respectively, in a non-human animal results in the non-human animal having a functional TCR (e.g., capable of binding antigen, transmitting an activation signal, and / or expressing CD3) comprising a human TCRγ polypeptide and / or a human TCRδ polypeptide, respectively. + This can result in non-human animals expressing TCRs that are capable of initiating T cell-mediated immune responses. Non-human animals, tissues, cells and macromolecules

[0052] The Vertebrate adaptive immune system has two T cell lineages that utilize diverse antigen receptors resulting from somatic recombination of DNA segments encoding their antigen recognition or variable domains. T cells bind to epitopes on small antigenic determinants on the surface of antigen-presenting cells associated with major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complexes. T cells bind to these epitopes via T cell receptor (TCR) complexes on the surface of T cells, which primarily recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. Non-limiting, exemplary interactions of α / β TCRs with MHC class I molecules (presenting antigens to CD8+ T cells) and MHC class II molecules (presenting antigens to CD4+ T cells), or γ / δ TCRs with antigens, according to embodiments described herein, are shown in Figure 1 (closed symbols represent non-human sequences, striped symbols represent human sequences).

[0053] T cell receptors are heterodimeric structures composed of two types of chains: α (alpha) and β (beta), or (gamma) and (delta) chains. Alpha / beta (α / β) T cells are the most abundant T cell lineage. The α chain is encoded by a nucleic acid sequence within the α locus (human or mouse chromosome 14) and also encompasses the entire δ locus, which encodes the δ chain; the β chain is encoded by a nucleic acid sequence within the β locus (mouse chromosome 6 or human chromosome 7); and the γ chain is encoded by a nucleic acid sequence within the γ locus (mouse chromosome 13 or human chromosome 7). The majority of T cells have α / β TCRs, while the majority of T cells have γ / δ TCRs.

[0054] The T cell receptor gamma and delta polypeptides (and similarly the alpha and beta polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides that make up the TCR contains an extracellular domain consisting of a variable domain and a constant domain, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable domain of the TCR determines its antigen specificity and contains three complementarity-determining regions (CDRs).

[0055] The three-dimensional structure of the antigen-recognition site of a T-cell receptor is similar to that of an antibody and mainly comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) flanked by framework regions. The periphery of the antigen-binding site includes the CDR1 and CDR2 loops, while the center of the antigen-binding site of a TCR is formed by CDR3.

[0056] The structural diversity of T cell receptors is primarily due to the combinatorial and junctional diversity generated during rearrangement. T cell receptor variable loci in germline DNA (e.g., DNA found in all germ cells) contain a number of unrearranged TCR variable (V) segments, TCR diversity (D) segments (for the TCRβ and TCRδ loci), and TCR joining (J) segments. During rearrangement, one of each of the multiple V(D)J segments joins together during recombination to form a rearranged V / (D) / J variable region gene sequence operably linked to a TCR constant region gene sequence, such that the variable region gene sequence and the TCR constant region gene sequence together encode a TCR polypeptide. Unrearranged TCRs The V, D, and J gene segments are flanked by 12-mer or 23-mer spacer-length recombination signal sequences (RSSs) that direct recombination according to the well-established "12 / 23 rule" of recombination. The D genes of the TCRβ and TCRδ loci are flanked by 12 and 23 RSSs, and their recombination may be controlled by mechanisms in addition to the 12 / 23 rule. See, for example, Olaru A., et al. (2005) J. Immunol. 174(10):6220-6226, the entire contents of which are incorporated herein by reference. Any TCR segment operably linked to an RSS has not undergone recombination and can therefore be considered an unrearranged segment. Thus, each unrearranged TCR V, D, or J segment is operably linked (e.g., flanked and contiguous on one or both sides) to a recombination signal sequence (RSS), which may be a 12-mer RSS or a 23-mer RSS.Thus, according to the 12 / 23 rule, unrearranged TCR Vα segments can rearrange with unrearranged TCR Jα segments to form TCR Vα / Jα gene sequences encoding TCR α variable domains; unrearranged TCR Vβ segments can rearrange with unrearranged TCR Dβ segments and unrearranged TCR Jβ segments to form TCR Vβ / Dβ / Jβ gene sequences encoding TCR β variable domains; unrearranged TCR Vγ segments can rearrange with unrearranged TCR Jγ segments to form TCR Vγ / Jγ gene sequences encoding TCR γ variable domains; unrearranged TCR Vδ segments can rearrange with unrearranged TCR Dδ segments and unrearranged TCR Jδ segments to form TCR Vδ / DδJδ gene sequences encoding TCR δ variable domains; and in some cases, unrearranged TCR Vα segments can rearrange with unrearranged TCR Dδ segments and unrearranged TCR Jδ segments to form TCR Vα / DδJδ gene sequences encoding hybrid TCR α / δ variable domains. Upon reconstitution, T cells mature and enter the peripheral blood, and therefore do not express T cell maturation markers, such as CD3. - T cells may also comprise "germline" or unrearranged T cell receptor variable region sequences.

[0057] The TCRα locus (chromosome 14 in mice and humans) contains a cluster of Vα gene segments, each preceded by an exon encoding a leader sequence (L). A cluster of Jα gene segments is located a considerable distance from the Vα gene segment. The Jα gene segment is followed by a single α constant (C) region gene sequence, which contains separate exons for the constant and hinge domains, and a single exon encoding the transmembrane and cytoplasmic regions. The TCRβ locus (chromosome 6 in mice and chromosome 7 in humans) has a different organization, with the cluster of Vβ gene segments located apart into two separate clusters (e.g., the TCRBDJ1 and TCRBDJ2 clusters), each containing a single D gene segment along with six or seven J gene segments and a single Cβ gene. Each TCR Cβ gene has separate exons encoding the constant domain, hinge, transmembrane region, and cytoplasmic region. The TCRα locus is interrupted between the V and J gene segments by another T cell receptor locus, the TCRδ locus.

[0058] The genomic structure of the TCRγ and TCRδ loci is particularly different from that of the TCRα or TCRβ loci. The TCRδ locus is located within the TCRα locus. Three Dδ gene segments, three Jδ gene segments, and a single δC gene are located between a cluster of Vα gene segments and a cluster of Jα gene segments, and the Vδ gene segments are interspersed between the Vα gene segments, with the Vδ3 gene segment between the cluster of TCR Cδ and Jα segments. See Janeway's Immunobiology, Chapter 4, 5th Ed., Murphy et al. eds., Garland Science, 2001. The TCRγ locus consists of four different constant (C) region gene sequences (three functional) in mice and two gamma constant region gene sequences (TCRGC1 and TCRGC2) in humans, with each Cγ region gene sequence containing its own cluster of Jγ gene segments. See, for example, Figure 3B.

[0059] Specific T cell receptor (TCR) repertoires are generated during T cell development through a complex developmental program in the thymus. The diversity of this repertoire is maintained by the expression of distinct TCR α / β or γ / δ chains and their variable regions, which are encoded by several variable (V), joining (J), and diversity (D) gene segments.

[0060] It is generally understood that TCR gene segments rearrange during T cell development to form complete variable domain exons: TCR alpha variable (Vα) and joining (Jα) gene segments undergo rearrangement such that the resulting TCR alpha chain is encoded by a specific combination of V-J segments (Vα / Jα sequences) operably linked to a TCR alpha constant (Cα) region gene sequence; TCR beta variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments rearrange such that the resulting TCR beta chain is encoded by a specific combination of V-DJ segments (Vβ / Dβ / Jβ sequences) operably linked to a TCR beta constant (Cβ) region gene sequence; TCR gamma variable (Vγ) and joining (Jγ) gene segments rearrange such that the resulting TCR gamma chain is encoded by a specific combination of V-J segments (Vγ / Jγ sequences) operably linked to a TCR gamma (Cγ) gene; and TCR delta variable (Vδ) gene segments rearrange such that the resulting TCR gamma chain is encoded by a specific combination of V-J segments (Vγ / Jγ sequences) operably linked to a TCR gamma (Cγ) gene sequence. Diversity (Dδ) and joining (Jδ) gene segments are rearranged so that the resulting TCR δ chain is encoded by a specific combination of VDJ segments (Vδ / Dδ / Jδ sequences), and sometimes includes a V-DJ (Vδ / Dδ / Dδ / Jδ sequence) operably linked to a TCR δ (Cδ) gene. See Hata et al. (188) Science 240:1541-1544; Hata et al. (1989) J. Exp. Med. 169:41-57. The use of two D segments greatly increases the variability of the δ chain, primarily because extra N-region nucleotides can be added at the junction between the two D gene segments and at the VD and DJ junctions.

[0061] The diversity of TCRs can be mainly attributed to the combinatorial and junctional diversity generated during the process of gene rearrangement. Most of the variation in TCR chains is found in the junction region, which is encoded by V, D, and J gene segments and modified by P and N nucleotides. This region encodes the CDR3 loop of the TCR chain, which forms the center of the antigen-binding site. Therefore, the center of the TCR chain is highly variable, while the periphery is relatively less variable.

[0062] Upon interaction with the thymic stroma, thymocytes are induced to undergo several developmental stages and are characterized by the expression of various cell surface markers. A summary of the characteristic cell surface markers at various developmental stages in the thymus is presented in Table 1. Rearrangement at the TCRβ variable locus begins at the DN2 stage and ends during the DN4 stage, while rearrangement of the TCR variable locus occurs at the DP stage. After rearrangement of the TCRβ locus is complete, cells express TCR chains along with the alternative α chain, pTα, at the cell surface. See Janeway's Immunobiology, Chapter 7, 7th Ed., Murphy et al. eds., Garland Science, 2008.

[0063] [Table 1]

[0064] Naive CD4+ and CD8+ T cells exit the thymus and enter peripheral lymphoid organs (e.g., the spleen), where they are exposed to antigen, activated, clonally expand, and differentiate into large numbers of effector T cells (Teff), such as cytotoxic T cells, TREG cells, TH17 cells, TH1 cells, TH2 cells, etc. Following infection, large numbers of T cells persist as memory T cells and are classified as either central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708-12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401:659-60. Sallusto and co-authors proposed that after initial infection, Tem cells represent a readily available pool of antigen-primed memory T cells in peripheral tissues with effector function, while Tcm cells represent antigen-primed memory T cells in peripheral lymphoid organs that can become new effector T cells upon secondary challenge. While all memory T cells express the CD45RO isoform of CD45 (naive T cells express the CD45RA isoform), Tcm are characterized by the expression of L-selectin (also known as CD62L) and CCR7+, which are important for binding to and signaling within peripheral lymphoid organs and lymph nodes. Id. Thus, all T cells found in peripheral lymphoid organs (e.g., naive T cells, Tcm cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express a number of different cell surface markers, such as CD44. For a review of various cell surface markers on T cells, see Janeway's Immunobiology, Chapter 10, supra.

[0065] Early studies led scientists to believe that α / β and γ / δ T cells arise sequentially, e.g., T cells arise from developing α / β thymocytes when γ / δ TCR rearrangement is not functional. See Allison, J., et al., Immunol Today (1987) 8:293-296; Pardoll, D., et al., Nature (1987) 326:79-81; Boismenu R Curr. Biol. (1995) 5:829-831, each of which is incorporated herein by reference in its entirety. However, studies expressing integrated rearranged TCRγ and / or TCRδ sequences in the germ cells of genetically engineered mice (KN6 mice with Vγ4Jγ1Cγ1 and Vδ5DJδ1Cδ rearrangements) contradicted the sequential rearrangement model, as such expression did not prevent normal development of α / β T cells. It was also shown that knockout of the TCR Cα gene did not affect the generation of gamma / delta thymocytes or the development of gamma / delta T cells, but instead resulted in an increased number of gamma / delta thymocytes that may have originated from atypical developmental pathways. Knockout of TCR Cβ in mice also did not show a developmental block in gamma / delta T cell development, and it was therefore concluded that double-positive cells do not appear to be intermediate in the development of TCRγ / delta T cells. Kreslavasky, T., et al. (2010) Curr Opin Immunol. 22:185-192; Mombaerts P., et al. (1992) 360:225-231. Extensive studies using various knockout and TCR transgenic mice have suggested several different models for the conditions required for specific T cell lineage commitment and maturation, as reviewed in Hahn, AM, and Winkler, TH, (2020) J. Leuk. Biol. 107:2019, incorporated herein by reference in its entirety. The stochastic model proposes that fate decision occurs stochastically prior to TCR expression. Using Vγ4-Jγ1Cγ1-modified mice, it was shown that IL-7Rα-high cells exhibit biased differentiation toward a gamma / delta fate.A signal strength model, based on studies with Vγ6Jγ1Cγ1- and Vδ1Dδ1Jδ2Cδ-engineered mice, proposes that the strength of TCR signals is a critical determinant of lineage fate decisions and that γ / δ TCR signaling may be a fundamental parameter for thymic differentiation of γ / δ T cell subsets. Hayes,SM,et al.(2002)Immunity,16:827-38;Hayes,SM,et al.(2005)Immunity,22:583-93;Haks et al.(2005)Immunity,22:595-606;Jensen,KD,et al.(2008)Immunity 29:90-100;Dent A., et al. (1990) Nature 406:524; Sim, G., et al. (1995) J. Immunol. 154:5827-31.

[0066] While the TCR variable domain primarily functions in antigen recognition, the extracellular portion of the constant domain, as well as the transmembrane and cytoplasmic domains of the TCR, also play important roles. A complete TCR receptor complex requires more than just the α and β polypeptides, or the γ and δ polypeptides; additional molecules include CD3γ, CD3δ, and CD3ε, as well as the ζ chain homodimer (ζζ). Upon TCRβ rearrangement, if the cell expresses TCR / pTα, this pre-TCR complex is present on the cell surface together with CD3. TCRαε (or pTα) on the cell surface has two basic residues in its transmembrane domain, one of which recruits the CD3γ heterodimer and the other recruits ζζ through its respective acidic residues. TCRβ has an additional basic residue in its transmembrane domain that is thought to recruit the CD3ε heterodimer. See, for example, Kuhns et al. (2006) Deconstructing the Form and Function of the TCR / CD3 Complex, Immunity 24:133-39; Wucherpfennig et al. (2009) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2:a005140. The assembled complex, containing the TCRβ heterodimer, CD3γε, CD3δε, and ζζ, is expressed on the surface of T cells. Polar residues in the transmembrane domain have been suggested to function as quality control for exiting the endoplasmic reticulum, and it has been demonstrated that in the absence of the CD3 subunit, TCR chains are retained in the ER and targeted for degradation.See, e.g., Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Function, Annu. Rev. Immunol. 23:101-25.

[0067] The CD3 and ζ chains of the assembled complex provide the building blocks for TCR signaling as a TCRαβ heterodimer (or TCRγ / δ heterodimer), which lack signaling activity by themselves. Each CD3 chain has one immune-receptor tyrosine-based activation motif (ITAM), while the ζ chain contains three tandem ITAMs. ITAMs contain tyrosine residues that can be phosphorylated by associated kinases. Thus, the assembled TCR-CD3 complex contains 10 ITAM motifs. See, e.g., Love and Hayes (2010) ITAM-Mediated Signaling by the T-Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. 2:e002485. Following TCR engagement, ITAM motifs are phosphorylated by the Src family tyrosine kinases Lck and Fyn, thereby initiating a signaling cascade that results in Ras activation, calcium mobilization, actin cytoskeletal rearrangements, and activation of transcription factors, all of which ultimately lead to T cell differentiation, proliferation, and effector functions. See also Janeway's Immunobiology, supra, both of which are incorporated herein by reference.

[0068] Additionally, the TCR β transmembrane and cytoplasmic domains are thought to play a role in mitochondrial targeting and apoptosis induction; indeed, naturally occurring N-terminally truncated TCR β molecules exist in thymocytes. Shani et al. (2009) Incomplete T-cell receptor—peptides target the mitochondrion and induce apoptosis, Blood 113:3530-41. Thus, several important functions are provided by the TCR constant region (which, in various embodiments, includes the extracellular domain and portions of the transmembrane and cytoplasmic domains), and in various embodiments, the structure of this region should be taken into consideration when designing a humanized TCR or a genetically modified non-human animal that expresses it.

[0069] The signal strength model is related to the observation that γ / δ TCRs have different signaling potentials and CD3 complex compositions. Hayes, S. M., and Love, P. E., (2002) Immunity, supra. γ / δ T cell development also begins at the immature DN stage; however, γ / δ T lymphocytes do not undergo a developmental stage defined by the expression of the pre-T cell receptor (pre-TCR) or CD4 / CD8 coreceptors. Instead, in-frame rearrangement of the TCRγ and TCRδ genes in immature thymocytes leads to the surface expression of the mature γ / δ TCR, which transduces signals that regulate the differentiation of immature DN cells into the γ / δ T cell lineage and are required for their differentiation into mature γ / δ T cells. The structure of the γ / δ TCR has been reported to be similar to that of the α / β TCR, except that the γ / δ TCR may contain an FcεRIγ (FcRγ) chain. See Qian, D., et al. (1993) Proc. Natl. Acad. Sci. USA 90:11875-879; Park, SW, et al. (1995) Eur. J. Immunol. 25:2107-2110, each of which is incorporated herein by reference in its entirety. Deletion of CD3γ, CD3ε, or CD3ζ blocks the development of γ / δ T cells, whereas loss of CD3δ or FcRγ has no effect on the generation of mature γ / δ TCR+ cells. Hayes et al. (2002) Immunity demonstrates that in the absence of CD3δ, signaling by γ / δ TCRs is superior to that of α / β TCRs, as measured by their ability to induce calcium mobilization, MAP kinase activation, and cell proliferation.

[0070] Gamma / delta (γ / δ) T cells remain poorly characterized. T cells with γ / δ TCRs are a distinct lineage of T cells that appear to be able to recognize antigens directly, similar to antibodies, without the need for presentation by MHC molecules or processing of the antigen, and also to recognize a wide range of antigens (e.g., phosphoproteins, lipids, glycolipids) independently of MHC. Although rare in peripheral blood, gamma / delta T cells are detectable in the thymus, spleen, and lymph nodes and are highly abundant in select tissue sites, such as intestinal tissue (e.g., colon, small intestine) and skin, where they are under continuous surveillance for microbial and other environmental antigens (J.C. Ribot, N. Lopes, B. Silva-Santos, γ / δ T cells in tissue physiology and surveillance. Nat Rev Immunol 21, 221–232 (2021), incorporated herein by reference in its entirety). Compared to α / β T cells, gamma / δ cells have a more restricted TCR repertoire and acquire specific cytokine production and tissue-homing properties early in development. These features, along with the lack of MHC restriction and recognition of antigens associated with "danger" signals, characterize this T lymphoid subset, which has been linked to innate immunity (B. Silva-Santos, S. Mensurado, S.B.Coffelt, γ / δ T cells: pleiotropic immune effectors with therapeutic potential in cancer. Nature Reviews Cancer, 1-13 (2019), incorporated herein by reference in its entirety).

[0071] Described herein are genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that can be used as animal models of human or humanized cellular responses involving gamma / delta T cells, to study such responses, and potentially develop human therapeutics. Generally, genetically engineered non-human animals as described herein include animals that contain unrearranged human or humanized (e.g., human gamma and / or delta; and / or human alpha and / or beta) T cell variable loci, e.g., capable of rearrangement (or rearrangement) in T cells to form nucleic acid sequences encoding the variable domains of a human T cell receptor, rearranged human variable domains, and human or non-human (e.g., mouse or rat) constant domains. Also described herein are non-human animals (e.g., rodents, e.g., rats, mice) capable of generating a diverse repertoire of human T cell receptor variable region sequences. Accordingly, the present invention provides non-human animals that express fully human variable domains (e.g., human TCR gamma variable domains and / or human TCR delta variable domains) and / or fully human TCRs (e.g., human TCR gamma chains and / or human TCR delta chains) and bind to epitopes of the antigen of interest in response to an antigen of interest. In some embodiments, provided herein are non-human animals that generate a diverse T cell receptor repertoire (gamma / delta T cells and alpha / beta T cells) capable of reacting with a variety of antigens, including, but not limited to, antigens not presented by APCs (recognized by gamma / delta T cells) and antigens presented by APCs (recognized by alpha / beta T cells).

[0072] In one embodiment, the invention provides genetically modified non-human animals (e.g., rodents, e.g., rats, mice) comprising unrearranged human TCR variable region segments (V(D)J segments) in their genomes, where the unrearranged human TCR variable region segments replace endogenous non-human TCR variable region segments at endogenous non-human (e.g., rodent) TCR variable loci (e.g., TCRγ, and / or TCRδ loci, and / or TCRα and / or TCRβ loci). In one embodiment, the unrearranged human TCR variable loci replace endogenous non-human TCR variable loci.

[0073] In another embodiment, the present invention provides genetically modified non-human animals (e.g., rodents, e.g., rats, mice) comprising in their genomes unrearranged human TCR variable region segments (V(D)J segments), which are operably linked to human or non-human TCR constant region gene sequences, resulting in a human or humanized TCR locus, respectively, wherein the human or humanized TCR locus is at a genomic site other than the endogenous non-human TCR locus. Thus, in one embodiment, also provided are non-human animals (e.g., rodents, e.g., mice, rats) comprising a transgene comprising an unrearranged human TCR variable region segment operably linked to a human or non-human TCR constant region gene sequence.

[0074] In some embodiments, the genetically modified non-human animals of the present invention have in their genome (a) human TCR variable region segments operably linked to (b) human or retained non-human (e.g., rodent, e.g., mouse, rat) TCR constant region gene sequences encoding a TCR constant domain. As described above, the constant domain of the TCR participates in the signaling cascade initiated during antigen-stimulated T cell activation, resulting in the TCR constant region interacting with various anchors and signaling proteins within the T cell. Thus, in one aspect, the genetically modified non-human animals of the present invention express human or humanized T cell receptors that retain the ability to recruit various endogenous non-human anchors or signaling molecules, such as CD3 molecules (e.g., CD3γ, CD3δ, CD3ε), zeta chain, Lck, Fyn, ZAP-70, etc. A non-limiting list of molecules recruited to the TCR complex is described in Janeway's Immunobiology, supra. In some embodiments, the non-human animals, or cells of the non-human animals described herein, comprise a human T cell (e.g., a human TCRγ polypeptide and / or a human δ polypeptide) described herein in association with a non-human animal (e.g., a rodent, e.g., a rat or mouse) TCR anchor or signaling molecule, e.g., a CD3 molecule (e.g., CD3γ, CD3δ, CD3ε), zeta chain, Lck, Fyn, ZAP-70, etc., and / or a non-human animal FcεRI γ (FcRγ) chain.

[0075] Thus, in various embodiments, the invention generally provides genetically modified non-human animals, wherein the non-human animals are configured with a humanized TCR variable locus in an unrearranged state within the genome, e.g., the unrearranged human TCR variable gene region is configured with human TCR variable segments that can recombine (or recombine) within, e.g., a T cell (e.g., of a mouse) to form a human rearranged TCR variable gene sequence. As used herein, "TCR locus" refers to a location on genomic DNA that contains the coding region of a TCR, including the entire coding region of the TCR, including unrearranged V(D)J sequences, enhancer sequences, constant region gene sequences, and sequences upstream or downstream thereof (e.g., UTRs, regulatory regions), or intervening DNA sequences (e.g., introns). As used herein, TCR variable locus, TCR variable region, or TCR variable locus refers to a portion of genomic DNA that contains a TCR variable region segment (V(D)J region), but does not include the TCR constant sequences, and, in various embodiments, enhancer sequences. Other sequences may be included in the TCR variable locus for purposes of genetic manipulation (e.g., selection cassettes, restriction sites, etc.), and these are encompassed herein. In various embodiments, the non-human animal comprises a contiguous portion of a human genomic TCR variable locus, including a V segment, a D segment, and a J segment, or a D segment and a J segment, or a V segment and a J segment, arranged in an unrearranged human genomic variable locus, including, for example, a promoter sequence, leader sequence, intergenic sequence, regulatory sequence, etc., arranged in a human genomic TCR variable locus. A contiguous human sequence with respect to a TCRG, TCRD, TCRA, and / or TCRB sequence may also generally refer to the complete human sequence, e.g., when the TCR coding sequence (e.g., TCR gene segment) and the TCR non-coding sequence (e.g., non-coding DNA flanking and separating the TCR gene segments, such as non-coding recombination signal sequences (RSS) and other non-coding intergenic sequences) are human, and preferably, the TCR gene segments and TCR non-coding sequences are in the same order as found in a human germline.

[0076] In various embodiments of a human or humanized TCR gamma locus and / or a human and / or humanized TCR delta locus, and optionally a human or humanized TCR alpha and / or TCR beta locus, the humanized locus may comprise human coding sequences (e.g., TCR gene segments) and non-human, e.g., murine, TCR non-coding sequences. In some embodiments, a human TCR gene segment replaces an orthologous non-human (e.g., murine) TCR gene segment such that the human TCR gene segment is flanked by the same non-human (e.g., murine) TCR non-coding sequences as are flanked by the replaced orthologous non-human (e.g., murine) TCR gene segment, e.g., such that the human TCR gene segments and non-human (e.g., murine) TCR non-coding sequences are in the same order as found in the non-human (e.g., murine) germline, but due to replacement of the orthologous (e.g., non-human) TCR gene segment. See, e.g., Figure 8C.

[0077] In other aspects, the various segments are arranged as in an unrearranged, non-human genomic TCR variable locus. In various embodiments of the humanized TCR α, β, δ and / or γ loci, the humanized loci are arranged by juxtaposition of two or more human genomic segments that do not appear to be juxtaposed in the human genome, such as a fragment of a V segment of a human variable locus located near a constant region of the human genome and a fragment of a V segment of a human variable locus located in the human genome at the upstream end of the human variable locus. Human or humanized gamma and / or delta TCR responses

[0078] In some embodiments, a non-human animal is provided that comprises unrearranged human TCR variable region segments in its genome, wherein the unrearranged human TCR gamma variable region segments are operably linked to human or non-human TCR constant region gene sequences, respectively, to provide a humanized TCR locus. In one embodiment, the human or humanized TCR gamma locus is at a site in the genome other than the endogenous non-human TCR gamma locus. In another embodiment, the unrearranged human TCR gamma variable region segment replaces the endogenous non-human TCR gamma variable region segment, and the human TCR gamma constant region gene sequence is such that an endogenous non-human TCR gamma constant region gene sequence (e.g., a nucleotide sequence comprising an endogenous TCR gamma constant region gene sequence (e.g., an endogenous Trgc1 constant region gene sequence, an endogenous Trgc2 constant region gene sequence, an endogenous Trgc3 constant region gene sequence, and / or an endogenous Trgc4 constant region gene sequence) is replaced with a nucleotide sequence comprising a human TCR gamma constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence). In one embodiment, the unrearranged human TCR gamma variable locus replaces the endogenous non-human TCR variable locus.

[0079] In some embodiments, an unrearranged TCR gamma variable locus comprising human variable region segments (e.g., human Vγ and Jγ segments) is positioned in a non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, an endogenous TCR variable locus is replaced by an unrearranged TCR variable locus comprising human variable region segments. In one aspect, the endogenous non-human Vγ and Jγ segments are incapable of rearranging to form a rearranged Vγ / Jγ sequence. Thus, in one aspect, the human Vγ and Jγ segments in the unrearranged TCR gamma variable locus are capable of rearranging to form a rearranged human Vγ / Jγ sequence.

[0080] In some embodiments, the non-human animals of the invention comprise an unrearranged humanized TCRγ locus, e.g., a TCRγ locus comprising at least one functional unrearranged human Vγ segment and at least one functional unrearranged human Jγ segment (e.g., a complete repertoire of functional unrearranged human Vγ segments and a complete repertoire of functional unrearranged human Jγ variable region segments). In some embodiments, the non-human animals of the invention comprise an unrearranged human TCRγ locus, e.g., at least one functional unrearranged human Vγ segment and at least one functional unrearranged human Jγ segment (e.g., a complete repertoire of functional unrearranged human Vγ segments and a complete repertoire of functional unrearranged human Jγ segments, at least one functional human TCR Cγ gene, e.g., a complete repertoire of functional human TCR Cγ genes), optionally wherein the functional unrearranged human Vγ segment, functional unrearranged human Jγ segment, and functional human TCR Cγ gene are in the same order as found in a germline human TCRγ locus. The number and positions of the various TCRγ segments can be determined from the IMGT database. The human TCRγ locus is located on human chromosome 7, while the mouse TCRγ locus is located on mouse chromosome 13.

[0081] The mouse TCR gamma variable locus is approximately 200 kilobases long and contains seven TRGV gene segments, four TRGJ gene segments, and four TRGC genes (three functional) belonging to five subgroups. The human TCR gamma variable locus is approximately 160 kilobases long and contains 12-15 TRGVs (TRGV1, TRGV2, TRGV3, TRGV3P, TRGV4, TRGV5, TRGV5P, TRGV6, TRGV7, TRGV8, TRGV9, TRGV10, TRGV11, TRGVA, and TRGVB) belonging to six subgroups, which includes three TRGJs (TRGJP1, TRGJP, and TRGJ1) for the first portion and two TRGJs (TRGJP2 and TRGJ2), TRGC1, and TRGC2 genes for the second portion. Although not shown in Figure 3B, MAID20200 contains all 15 hTRG V segments, all 5 hTRG J segments, and both hTRGC genes in germline configuration. Unless otherwise stated, the number of human V(D)J segments referred to throughout this specification refers to the total number of V(D)J segments. In one embodiment of the present invention, a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) comprises at least one human Vγ and at least one human Jγ segment. In one embodiment, the non-human animal comprises a humanized TCRγ locus comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more human Vγ segments. In some embodiments, the human or humanized TCRγ locus comprises at least one functional human Vγ gene segment. In some embodiments, the human or humanized TCRγ locus comprises two functional human Vγ gene segments. In some embodiments, the human or humanized TCR gamma locus comprises three of the functional human V gamma gene segments. In some embodiments, the human or humanized TCR gamma locus comprises four of the functional human V gamma gene segments. In some embodiments, the human or humanized TCR gamma locus comprises five of the functional human V gamma gene segments.In some embodiments, the human or humanized TCR gamma locus comprises six of functional human V gamma gene segments. In some embodiments, the functional human V gamma gene segments are selected from the group consisting of human TRGV2, human TRGV3, human TRGV4, human TRGV5, human TRGV8, human TRGV9, and variants thereof. Thus, in some embodiments, the humanized TCR gamma locus in a non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% human V gamma, and in some embodiments, about 2%, about 3%, about 15%, about 65%, about 90%, or 100% human V gamma. In one embodiment, the non-human animal comprises a humanized TCRγ locus comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more human Vγ segments. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus comprising a human TRGJP1 gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus comprising a human TRGJP gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus comprising a human TRGJ1 gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus comprising a human TRGJP2 gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus comprising a human TRGJ2 gene segment. In some embodiments, the non-human animal comprises an endogenous TCRγ locus, a human TRGC1 gene, operably linked to a human TRGV segment and a TRJV segment. In some embodiments, the non-human animal comprises an endogenous TCR gamma locus, a human TRGC2 gene, operably linked to human TRGV and TRJV segments.

[0082] In one embodiment, the non-human animal comprises a human TCRγ locus comprising a DNA fragment comprising contiguous human sequences of human Vγ1 (Vγ segments are also referred to as "TRGV" or "TCRGV") and human Cγ2 ("TCRGC2"; Cγ is also referred to as "TRGC" or "TCRGC"), and comprises a human Jγ gene segment (Jγ segments are also referred to as "TRGJ" or "TCRGJ") and a human Cγ1 ("TCGRC1") gene between the human Vγ gene segment and the human Cγ2 gene. TCRG non-coding sequence refers to the non-coding recombination signal sequence (RSS) and the contiguous non-coding sequence found between any two consecutive unrearranged TRGV segments, between any unrearranged TRGV segment and unrearranged TRGJ segment, and between any two consecutive unrearranged TRGJ segments.

[0083] In some embodiments, the humanized TCRγ mice described herein: (I) Germline and CD3 containing unrearranged TCR γ variable region sequences comprising unrearranged human TCR Vγ segments and unrearranged human TCR Jγ segments - Contains T cells, the unrearranged TCR gamma variable region sequence is operably linked to a human TCR gamma constant region gene sequence (e.g., a human TCR gamma constant region gene sequence at the endogenous TCR gamma locus), optionally at the endogenous TCR gamma locus, and a nucleotide sequence comprising the endogenous TCR gamma constant region gene sequence (e.g., an endogenous Trgc1 constant region gene sequence), an endogenous Trgc2 constant region gene sequence, an endogenous Trgc3 constant region gene sequence, and / or an endogenous Trgc4 constant region gene sequence) is replaced with a nucleotide sequence comprising a human TCR gamma constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence); the unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vγ / Jγ variable region gene operably linked to a human TCR gamma constant region gene sequence; germline and CD3- somatic cells comprising a rearranged human TCR Vγ / Jγ variable region gene operably linked to a human TCR gamma constant region gene sequence, which together encode a human TCR gamma polypeptide; (II) CD3 expressing on its surface a functional TCR comprising a human TCRγ polypeptide. + Contains T cells.

[0084] In some embodiments, the human TCR gamma polypeptide is derived from a human TRGV2 gene segment, a human TRGV3 gene segment, a human TRGV4 gene segment, a human TRGV5 gene segment, a human TRGV8 gene segment, a human TRGV9 gene segment, a human TRGV10 gene segment, or a human TRGV11 gene segment. In some embodiments, the human TCR gamma polypeptide is derived from a human TRGJ1 gene segment, a human TRGJP gene segment, a human TRGJP1 gene segment, a human TCRGJ2 gene segment, or a human TRGJP2 gene segment.

[0085] In some humanized TCRγ mouse embodiments, germline and CD3 -the T cell further comprises an unrearranged T cell receptor (TCR) delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged human TCR D delta segment, and an unrearranged human TCR J delta segment, wherein the unrearranged TCR delta variable region sequence is operably linked to a human TCR delta constant region gene sequence, optionally at an endogenous TCR delta locus, and the unrearranged human TCR V delta segment, the unrearranged human TCR D delta segment, and the unrearranged human TCR J delta segment can be rearranged (or rearranged) in the T cell to form a rearranged human TCR V delta / D delta / J delta variable region gene operably linked to the human TCR delta constant region gene sequence, wherein the rearranged human TCR V delta / D delta / J delta variable region gene operably linked to the human TCR delta constant region gene sequence together encodes a human TCR delta polypeptide, and the mouse expresses a functional TCR comprising both a human TCR gamma polypeptide and a human TCR delta polypeptide on its surface. + Further included are T cells.

[0086] In some embodiments, the human TCR delta polypeptide is derived from a human TRDV1 gene segment, a human TRAV17 gene segment, a human TRAV19 gene segment, a human TRAV21 gene segment, a human TRAV21 gene segment, a human TRAV26-2 gene segment, a human TRAV29 / TRDV5 gene segment, a human TRAV31 gene segment, a human TRAV38-2 / TRDV8 gene segment, a human TRAV39 gene segment, a human TRAV40 gene segment, a human TRAV41 gene segment, a human TRDV2 gene segment, or a human TRDV3 gene segment. In some embodiments, the human TCR delta polypeptide is derived from a human TRDJ1 gene segment, a human TRDJ2 gene segment, a human TRDJ3 gene segment, or a human TRDJ4 gene segment.

[0087] In some embodiments, a non-human animal is provided that comprises in its genome an unrearranged human TCRδ variable region segment (with or without a human or humanized TCRγ locus as described herein), wherein the unrearranged human TCRδ variable region segment is operably linked to a human or non-human TCRδ constant region gene sequence, resulting in a human or humanized TCRδ locus. In one embodiment, the humanized TCRδ locus is at a site in the genome other than the endogenous non-human TCRδ locus. In another embodiment, the unrearranged human TCRδ variable region segment replaces the endogenous non-human TCRδ variable region segment while replacing the endogenous non-human TCRδ constant region gene sequence. In one embodiment, the unrearranged human TCRδ variable locus replaces the endogenous non-human TCRδ variable locus.

[0088] In some embodiments, an unrearranged TCRδ variable locus comprising a human variable region segment (e.g., a human Vδ segment, a Dδ segment, and a Jδ segment) is positioned in a non-human genome such that the human variable region segment replaces the corresponding non-human variable region segment. In one embodiment, an endogenous TCRδ variable locus is replaced by an unrearranged TCRδ variable locus comprising a human variable region segment. In one aspect, the endogenous non-human Vδ segment, a Dδ segment, and a Jδ segment are incapable of rearranging to form a rearranged Vδ / Dδ / Jδ sequence. Thus, in one aspect, the human Vδ segment, a Dδ segment, and a Jδ segment in the unrearranged TCRδ variable locus are capable of rearranging to form a rearranged human Vδ / Dδ / Jδ sequence.

[0089] In some embodiments, the non-human animals of the invention comprise an unrearranged humanized TCRδ locus, e.g., a TCRδ locus that comprises at least one functional unrearranged human Vδ segment, at least one functional unrearranged human Dδ segment, and at least one functional unrearranged human Jδ segment (e.g., a complete repertoire of functional unrearranged human Vδ segments, a complete repertoire of functional unrearranged human Dδ segments, and a complete repertoire of functional unrearranged human Jδ segments). In some embodiments, the non-human animals of the invention comprise an unrearranged human TCRδ locus, e.g., a TCRδ locus comprising at least one functional unrearranged human Vδ segment, at least one functional unrearranged human Dδ segment, and at least one functional unrearranged human Jδ segment (e.g., a complete repertoire of functional unrearranged human Vδ segments, a complete repertoire of functional unrearranged human Dδ segments, and a complete repertoire of functional unrearranged human Jδ segments), operably linked to a functional human TCR Cδ gene, optionally wherein the functional unrearranged human Vδ segment, functional unrearranged human Dδ segment, functional unrearranged human Jδ segment, and functional human TCR Cδ gene are in the same order as found in the germline human TCRδ locus. The number and location of the various TCR segments can be determined from the IMGT database. In both mice and humans, TCRδ gene segments are located within the TCRα locus on chromosome 14 (Figure 2). The TCRδ J and D segments are located between the Vα and Jα segments, while the TCRδ V segments are scattered throughout the TCRα locus, mostly located between the various Vα segments. Due to the genomic arrangement of the TCRδ gene segments within the TCRα locus, successful rearrangements at the TCRα locus can result in the deletion or inactivation of TCRδ gene segments.

[0090] The mouse TCR delta variable locus is approximately 275 kilobases and contains six TRDVs (16, including 10 upstream TRAV / DVs), two TRDDs, two RDJs, and one TRDC. One TRDV is in the reverse orientation 3' of TRDC. The human TCR delta cluster, located between the TRAV and TRAJ gene segments, contains one TRDV gene segment (TRDV2), three TRDD gene segments (TRDD1, TRDD2, TRDD3), four TRDJ gene segments (TRDJ1, TRDJ4, TRDJ2, and TRDJ3), one TRDC gene, and one TRDV in the reverse orientation downstream of the TRDC gene (TRDV3). This cluster spans 60 kilobases. One TRDV gene segment (TRDV1) is located between the TRAV gene segments, and five gene segments within the TRAV gene segment, designated TRAV / DV gene segments (TRAV14 / DV4, TRAV23 / DV6, TRAV29 / DV5, TRAV36 / DV7, and TRAV38-2 / DV8), can be used to synthesize the TCRδ chain or the TCRα chain. In one embodiment of the present invention, a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) comprises at least one human Vδ, one Dδ, and at least one human Jδ segment. In one embodiment, the non-human animal comprises a human TCRδ locus comprising one human Vδ segment. In one embodiment, the non-human animal comprises a human TCRδ locus comprising two human Vδ segments. In one embodiment, the non-human animal comprises a human TCRδ locus comprising three human Vδ segments. In one embodiment, the non-human animal comprises a human TCRδ locus comprising one human TRAV / DV segment. In one embodiment, the non-human animal comprises a human TCRδ locus comprising two human TRAV / DV segments. In one embodiment, the non-human animal comprises a human TCRδ locus comprising three human TRAV / DV segments. In one embodiment, the non-human animal comprises a human TCRδ locus comprising four human TRAV / DV segments. In one embodiment, the non-human animal comprises a human TCRδ locus comprising five human TRAV / DV segments.In some embodiments, the non-human animal comprises a human TCRδ locus comprising one human TRDJ gene segment. In some embodiments, the non-human animal comprises a human TCRδ locus comprising two human TRDJ gene segments. In some embodiments, the non-human animal comprises a human TCRδ locus comprising three human TRDJ gene segments. In some embodiments, the non-human animal comprises a human TCRδ locus comprising four human TRDJ gene segments. In some embodiments, the non-human animal comprises a human TCRδ locus comprising a human TRDC gene.

[0091] In one embodiment, the non-human animal comprises a humanized TCRδ locus comprising a DNA fragment comprising a contiguous human sequence comprising human Vδ1-Vδ3 gene segments (Vδ segments are also referred to as "TRDV" or "TCRDV"), including Dδ1, Dδ2, and Dδ3 (Dδ segments are also referred to as "TRDD" or "TCRDD"), and the Cδ gene (Cδ is also referred to as "TRDC" or "TCRDC"). In some embodiments, the DNA fragment also comprises a human Jα segment. TCRD non-coding sequence refers to the contiguous non-coding sequence found between the non-coding recombination signal sequence (RSS) and any two consecutive unrearranged TRDV segments, between any unrearranged TRDV segment and an unrearranged TRDD segment, between any two consecutive unrearranged TRDD segments, and between any TRDD segment and the TRCD gene. In various embodiments, the DNA fragment comprising the contiguous human sequence of a human TCR variable region segment also comprises restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with the proper function (e.g., rearrangement, splicing, etc.) of the various genes at the TCR delta locus.

[0092] In some embodiments, the mice described herein are capable of expressing germ cells and CD3 - Contains T cells, (A) Replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; or (B) comprising a replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or (C)(i) replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; and (ii) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

[0093] In some embodiments, (A) The unrearranged TCR Vγ segments comprise the full repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the full repertoire of unrearranged human TCR Jγ segments, or (B) The unrearranged human TCR Vδ segments comprise the full repertoire of unrearranged human TCR Vδ segments; the unrearranged human TCR Dδ segments comprise the full repertoire of unrearranged human TCR Dδ segments; the unrearranged human TCR Jδ segments comprise the full repertoire of unrearranged human TCR Jδ segments; or (C)(i) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; and (ii) The unrearranged human TCR Vδ segments comprise the complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise the complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise the complete repertoire of unrearranged human TCR Jδ segments.

[0094] In some embodiments, the mouse described herein: (I) (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and Germline and human TCR gamma constant region gene sequences, including replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences. (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Jδ segments, and A germline and CD3 T cell line comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence. - somatic T cells, and (II) CD3 expressing a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide on its surface. + Contains T cells.

[0095] In some embodiments, non-human animals described herein with humanized TRD and / or TRG loci possess γ / δ T cells in the spleen and thymus, which comprise a small fraction of the total T cells observed in WT mice (Figures 5A-5B) and in the skin and intestinal mucosa (Figures 12A-12D). Furthermore, TCR repertoire analysis of thymic and splenic mRNA revealed successful and diverse V(D)J gene usage, indicating proper recombination, expression, and thymic selection of humanized γ / δ TCRs (Figures 6A, 6B, 7A, and 7B). Recombination of a diverse repertoire of V(D)J gene segments was also observed in γ / δ T cells in the skin and intestinal mucosa (data not shown).

[0096] Because the TRD locus is located within the TRA locus, in some embodiments, germline and CD3 -The T cell further comprises an unrearranged human TCR Vα segment upstream of the unrearranged TCRδ variable region sequence and the human TCRδ constant region gene sequence, and the unrearranged human TCR Vα segment, unrearranged human TCR Dδ, and unrearranged human TCR Jδ segment can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to the human TCRδ constant region gene sequence, and the rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence together encodes a human hybrid TCR polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, and the mouse expresses a CD3 + Further included are T cells.

[0097] In some embodiments, the humanized TCRγ and / or humanized TCRδ non-human animals described herein may also have a human or humanized TCRα locus. Mice with a human or humanized α locus are described in U.S. Patent No. 9,113,616, which is incorporated herein by reference. See also Figure 8A. Fully humanized T cell responses

[0098] Described herein are non-human animals that contain human or humanized TRD and TRG loci, as well as human or humanized TRA and / or TRB loci, and human or humanized loci for other components involved in α / β TCR rearrangement and / or activation, such as TCR co-receptor (CD4 and CD8) and MHC loci. Human or humanized α and / or β TCR loci

[0099] In some embodiments, a non-human animal is provided that comprises an unrearranged human TCR variable region segment in its genome, the unrearranged human TCR variable region segment operably linked to a human or non-human TCR constant region gene sequence, resulting in a human or humanized TCR locus. In one embodiment, the humanized TCR alpha locus is at a site in the genome other than the endogenous non-human TCR locus. In another embodiment, the unrearranged human TCR alpha variable region segment replaces the endogenous non-human TCR alpha variable region segment, while the human TCR alpha constant region gene sequence replaces the endogenous non-human TCR alpha constant region gene sequence. In one embodiment, the unrearranged human TCR alpha variable locus replaces the endogenous non-human TCR alpha variable locus.

[0100] In one embodiment, an unrearranged TCR α variable locus comprising human variable region segments (e.g., human Vα and Jα segments) is positioned in a non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, an endogenous TCR α variable locus is replaced by an unrearranged TCR α variable locus comprising human variable region segments. In one aspect, the endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα / Jα sequence. Thus, in one aspect, the human Vα and Jα segments in the unrearranged TCR α variable locus are capable of rearranging to form a rearranged human Vα / Jα sequence.

[0101] In some embodiments, the non-human animals of the invention comprise an unrearranged humanized TCR α locus, e.g., a TCR α locus comprising at least one functional unrearranged human Vα segment and at least one functional unrearranged human Jα segment (e.g., a complete repertoire of functional unrearranged human Vα segments and a complete repertoire of functional unrearranged human Jα variable region segments). In some embodiments, the non-human animals of the invention comprise an unrearranged human TCR α locus, e.g., at least one functional unrearranged human Vα segment and at least one functional unrearranged human Jα segment (e.g., a complete repertoire of functional unrearranged human Vα segments and a complete repertoire of functional unrearranged human Jα segments), a functional TCR C α gene (e.g., an endogenous TCR C α gene), optionally wherein the functional unrearranged human Vα segment(s), functional unrearranged human Jα segment(s), and functional TCR C α gene are in the same order as found in a germline human TCR α locus. The number and positions of the various TCR α segments can be determined from the IMGT database.

[0102] The mouse TCR α variable locus is approximately 1.5 megabases and contains a total of 110 Vα segments and 60 Jα segments. The human TCR α variable locus is approximately 1 megabase and contains a total of 54 Vα segments and 61 Jα segments, with 45 Vα and 50 Jα segments considered functional. Unless otherwise stated, the number of human V(D)J segments referred to throughout this specification refers to the total number of V(D)J segments. In one embodiment of the present invention, a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) contains at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal contains a humanized TCR locus containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Vα segments. In some embodiments, the humanized TCR alpha locus comprises 2, 8, 23, 35, 48, or 54 human Vα segments. Thus, in some embodiments, the humanized TCR alpha locus in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% human Vα, and in some embodiments, about 2%, about 3%, about 15%, about 65%, about 90%, or 100% human Vα.

[0103] In one embodiment, a non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human Vα40 to Vα41 (Vα segments are also referred to as "TRAV" or "TCRAV") and a DNA fragment comprising the contiguous human sequence of 61 human Jα segments (Jα segments are also referred to as "TRAJ" or "TCRAJ"). TCRA non-coding sequence refers to the contiguous non-coding sequence, including non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, found between any two contiguous unrearranged TRAV segments, between any unrearranged TRAV segment and an unrearranged TRAJ segment, and between any two contiguous unrearranged TRAJ segments. In one embodiment, a non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV35 to TRAV41 and a DNA fragment comprising the contiguous human sequence of 61 human TRAJα. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV22 to TRAV41 and a DNA fragment comprising the contiguous human sequence of 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV13-2 to TRAV41 and a DNA fragment comprising the contiguous human sequence of 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV6 to TRAV41 and 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV1-1 to TRAV41 and 61 human TRAJ. In various embodiments, the DNA fragment comprising the contiguous human sequence of a human TCR α variable region segment also comprises restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with the proper function (eg, rearrangement, splicing, etc.) of the various genes in the TCR alpha locus.

[0104] In one embodiment, the humanized TCR alpha locus comprises 61 human Jα segments, or 100% human Jα segments. In a specific embodiment, the humanized TCR alpha locus comprises 8 human Vα segments and 61 human Jα segments, and in another specific embodiment, the humanized TCR alpha locus comprises 23 human Vα segments and 61 human Jα segments. In another embodiment, the humanized TCR alpha locus comprises human Vα and Jα segments, i.e., all human variable region gene segments encoded by the locus, or the complete repertoire of 54 human Vα segments and 61 human Jα segments. In various embodiments, the non-human animal does not comprise any endogenous non-human Vα or Jα segments at the TCR alpha locus.

[0105] In some embodiments, the mouse described herein: (I) From 5' to CD3 an unrearranged human TCR Vα segment, and an unrearranged TCR delta variable region sequence comprising an unrearranged human TCR V delta segment, an unrearranged TCR D delta, and an unrearranged human TCR J delta segment; the unrearranged TCR delta variable region sequence is optionally operably linked to a human TCR delta constant region gene sequence at an endogenous TCR delta locus; the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ, and the unrearranged human TCR Jδ segment can be rearranged (or rearranged) in a T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to a human TCRδ constant region gene sequence; and a germline and CD3 -T cells, and (II) CD3 expressing on its surface a functional TCR comprising a human hybrid TCR α / δ variable domain operably linked to a human TCR δ constant domain. + Contains T cells.

[0106] In some embodiments, the germ cells and CD3 - The T cell may comprise a replacement of an endogenous TCR Vα segment with an unrearranged human TCR Vα segment, and a replacement of an endogenous TCR Jα segment with an unrearranged human TCR Jα segment, wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are operably linked to each other with a TCR α constant region gene sequence, e.g., a mouse TCR α constant region gene sequence, and the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment can rearrange (or rearrange) in the T cell to form a rearranged TCR Vα / Jα variable region gene operably linked to the TCR α constant region gene sequence, and the rearranged human TCR Vα / Jα variable region gene operably linked to the TCR α constant region gene sequence together encodes a TCR α polypeptide comprising a human TCR α variable domain, and the mouse is a CD3 + In some embodiments, the target cell population comprises germline and CD3 T cells. -a T cell comprising a replacement of all endogenous TCR Vα segments with a complete repertoire of unrearranged human TCR Vα segments, and a replacement of all endogenous TCR Jα segments with a complete repertoire of unrearranged human TCR Jα segments, wherein the complete repertoire of unrearranged human TCR Vα segments and the complete repertoire of unrearranged human TCR Jα segments are operably linked to each other and to a TCR α constant region gene sequence at the endogenous TCR α locus, and wherein the complete repertoire of unrearranged human TCR Vα segments and the complete repertoire of unrearranged human TCR Jα segments can be rearranged (or reconstituted) in the T cell to form a rearranged human TCR Vα / Jα variable region gene operably linked to the endogenous TCR α constant region gene sequence ... The Vα / Jα variable region genes together encode a chimeric TCRα polypeptide comprising a human TCRα variable domain operably linked to a mouse TCRα constant domain, and the mouse expresses on its surface a functional TCR comprising the chimeric TCRα polypeptide. + Further included are T cells.

[0107] The humanized TRA / D and TRG loci can be introduced into mice carrying a humanized TRB locus and other components of T cell immunity, such as TCR co-receptors (CD4 and CD8) and MHC, thus achieving complete humanization in both T cell lineages.

[0108] As a non-limiting example, a humanized TRD and / or TRG locus may be introduced into a mouse containing a human or humanized TRB locus (the TRB locus, which comprises the humanized TCRBDJ1 and TCRBDJ2 clusters, consists of mouse TCRB non-coding sequences and human coding sequences). These are the mice described in U.S. Patent No. 9,113,616 and Moore, M. et al. Sci Immunol 6 (2021); doi:10.1126 / sciimmunol.abj4026, each of which is incorporated herein by reference in its entirety. See Figures 8B and 8C.

[0109] In some embodiments, a non-human animal is provided that comprises an unrearranged human TCR β variable region segment in its genome, the unrearranged human TCR β variable region segment operably linked to a non-human TCR β constant region gene sequence, resulting in a humanized TCR β locus. In one embodiment, the humanized TCR β locus is at a site in the genome other than the endogenous non-human TCR β locus. In another embodiment, the unrearranged human TCR β variable region segment replaces the endogenous non-human TCR β variable region segment while retaining the endogenous non-human TCR β constant region gene sequence. In one embodiment, the unrearranged human TCR β variable locus replaces the endogenous non-human TCR β variable locus.

[0110] In some embodiments, an unrearranged TCR β variable locus comprising human variable region segments (e.g., human Vβ segments, Dβ segments, and J segment β) is positioned in a non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, an endogenous TCR β variable locus is replaced by an unrearranged TCR β variable locus comprising human variable region segments. The human TCR α variable locus is approximately 1 megabase and contains a total of 54 Vα segments and 61 Jα segments, with 45 Vα and 50 Jα segments believed to be functional. Thus, in one aspect, the human Vβ segments, Dβ segments, and J segment β in an unrearranged TCR β variable locus are capable of rearranging (or recombining), e.g., in a T cell, to form a rearranged human Vβ / DβJβ sequence.

[0111] The mouse TCR beta variable locus is approximately 0.6 megabases and contains a beta, a total of 33 V segments, 2 D beta segments, and 14 J beta segments. The human TCR beta variable locus is approximately 0.6 megabases and contains a total of 67 V beta segments, 2 D beta segments, and 14 J beta segments. In one embodiment, the genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) contains at least one human V beta segment, at least one human D beta segment, and at least one human J alpha segment.

[0112] In one embodiment, the non-human animal comprises a humanized TCR beta locus comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or up to 67 human V beta segments. In some embodiments, the humanized TCR beta locus comprises 8, 14, 40, 66, or 67 human V beta segments. Thus, in some embodiments, a humanized TCR β locus in a non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of human Vβs; in some embodiments, it may comprise about 20%, about 60%, about 15%, about 98%, or 100% of human Vβs.

[0113] In some embodiments, the endogenous TCR β variable locus, e.g., the endogenous TCR β mouse variable locus, is Replacement of one or all of the adjacent endogenous T-cell variable region Vβ gene segments, e.g., one or all of the adjacent endogenous T-cell variable region Vβ gene segments between the first 5' trypsinogen cluster and the second 3' trypsinogen cluster, with one or all of the unrearranged human T-cell variable region gene segments of TRBV1 to TRBV29-1; and / or This includes replacement of one or more non-contiguous endogenous Vβ gene segments (e.g., endogenous mouse TCRBV31 gene segments) with human TCRBV gene segments (e.g., replacement of mouse TCRBV31 gene segments with orthologous human TCRBV30 gene segments).

[0114] In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising the contiguous human sequence of human Vβ18 through Vβ29-1 (Vβ segments are also referred to as "TRBV" or "TCRBV"). In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising the contiguous human sequence of human TRBV18 through TRBV29-1, a separate DNA fragment comprising the contiguous human TCRBDJ1 sequence comprising human Dβ1-Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 segment), and a separate DNA fragment comprising the contiguous human TCRBDJ2 sequence comprising human Dβ2-Jβ2 (i.e., the human Dβ2-Jβ2-1-Jβ2-7 segment). An unrearranged TCRBDJ1 sequence, which may also be referred to as an unrearranged TCRBJD1 cluster, includes an unrearranged TCRBD1 segment, one to all of the unrearranged TCRBJ1 segments (e.g., the Jβ1-1, Jβ1-2, Jβ1-3, Jβ1-4, Jβ1-5, and Jβ1-6 segments), and the TCRBDJ1 non-coding sequences between an unrearranged TCRBD1 segment and an unrearranged TCRBJ1 segment and between any two consecutive unrearranged TCRBJ1 gene segments. A TCR non-coding sequence refers to the flanking non-coding sequences, including non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, found between any two consecutive unrearranged TRBV segments, and may include TCRBDJ1 non-coding sequences, for example, the flanking non-coding sequences, including non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, found between an unrearranged TRBD1 segment and a TRBJ1 segment, and between any two consecutive unrearranged TRBJ1 segments, or TCRBDJ2 non-coding sequences, for example, the flanking non-coding sequences, including non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, found between an unrearranged TRBD2 segment and a TRBJ2 segment, and between any two consecutive unrearranged TRBJ2 segments.The rearranged TCRBDJ1 sequence may be operably linked to multiple unrearranged TRBV segments and a TCRBC1 constant region sequence (which may also be referred to as a TRBC1 region sequence). The unrearranged TCRBDJ2 sequence, which may also be referred to as an unrearranged TCRBJD2 cluster, includes an unrearranged TCRBD2 segment, one to all of the unrearranged TCRBJ2 segments (e.g., Jβ2-1, Jβ2-2, Jβ2-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments), and the TCRBDJ2 non-coding sequences between the unrearranged TCRBD2 segment and the unrearranged TCRBJ2 segment and between any two consecutive unrearranged TCRBJ2 gene segments. The unrearranged TCRBDJ2 sequence may be operably linked to multiple unrearranged TRBV segments and a TCRBC2 constant region gene sequence (which may also be referred to as a TRBC2 region sequence).In one embodiment, the non-human animal comprises (i) a TCRBDJ1 cluster, in which at least all or at least one of the Dβ1-Jβ1 segments (i.e., the Dβ1, Jβ1-1, Jβ1-2, Jβ1-3, Jβ1-4, Jβ1-5, and Jβ1-6 segments) are human, and the non-coding sequences (including RSSs and other intergenic sequences) between the Dβ1-Jβ1 segments are non-human, e.g., mouse, optionally flanked by the same mouse TCR non-coding sequences as the Dβ1 and Jβ1-1 through JβJ1-6 segments are typically flanked by mouse TCR non-coding sequences, and / or (ii) a TCRBDJ2 cluster. The cluster includes a humanized TCR β locus comprising either or both of the Dβ2-Jβ2 segments (i.e., the Dβ2, Jβ2-1, Jβ2-2, Jβ2-3, Jβ2-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments) in which at least one or all of the Dβ2-Jβ2 segments are human and the non-coding sequences (including RSSs and other intergenic sequences) between the Dβ2-Jβ2 segments are non-human, e.g., mouse, and optionally the Dβ2 and JβJ2-1 to Jβ2-7 gene segments are flanked by the same mouse TCR non-coding sequences normally flanked by the mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments. In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising contiguous human sequences of human TRBV6-5 through TRBV29-1, a separate DNA fragment comprising contiguous human sequences of human Dβ1-Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 segment), and a separate DNA fragment comprising contiguous human sequences of human Dβ2-Jβ2 (i.e., the human Dβ2-Jβ2-1-Jβ2-7 segment). In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising contiguous human sequences of human TRBV1 through TRBV29-1, a separate DNA fragment comprising contiguous human sequences of human Dβ1-Jβ1, and a separate DNA fragment comprising contiguous human sequences of human Dβ2-Jβ2.In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising contiguous human sequences of human TRBV1 through TRBV29-1, a separate DNA fragment comprising contiguous human sequences of human Dβ1-Jβ1, a separate DNA fragment comprising contiguous human sequences of human Dβ2-Jβ2, and a separate DNA fragment comprising a sequence of human TRBV30. In various embodiments, the DNA fragments comprising the contiguous human sequences of human TCR β variable region segments also comprise restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with the proper function (e.g., rearrangement, splicing, etc.) of various genes at the TCR β locus.

[0115] In one embodiment, the humanized TCR beta locus comprises 14 human J beta segments, or 100% human J beta segments, and two human D beta segments, or 100% human D beta segments. In another embodiment, the humanized TCR beta locus comprises at least one human V beta segment, e.g., 14 human V beta segments, and all mouse D beta and J beta segments. In a specific embodiment, the humanized TCR beta locus comprises 14 human V beta segments, two human D beta segments, and 14 human J beta segments. In another specific embodiment, the humanized TCR beta locus comprises the complete repertoire of human V beta segments, D beta segments, and J beta segments, i.e., all human variable beta region gene segments encoded by the beta locus or 67 human V beta segments, two human D beta segments, and 14 human J beta segments. In one embodiment, the non-human animal comprises one (e.g., 5') non-human V beta segment in the humanized TCR beta locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ segments, Dβ segments, or J segment β at the TCR β locus.

[0116] In one embodiment, the humanized TCR beta locus comprises 13 human J beta segments, or 100% functional human J beta segments, and 2 human D beta segments, or 100% functional human J beta segments. In another embodiment, the humanized TCR beta locus comprises at least one human V beta segment, e.g., 14 human V beta segments, and all functional mouse D beta and J beta segments. In a specific embodiment, the humanized TCR beta locus comprises 14 human V beta segments, 2 human D beta segments, and 13 functional human J beta segments. In another specific embodiment, the humanized TCR beta locus comprises the complete repertoire of human V beta segments, D beta segments, and J beta segments, i.e., all human variable beta region gene segments encoded by the beta locus or 67 human V beta segments, 2 human D beta segments, and 13 functional human J beta segments. In one embodiment, the non-human animal comprises one (e.g., 5') non-human V beta segment in the humanized TCR beta locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ segments, Dβ segments, or J segment β at the TCR β locus.

[0117] In one aspect, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR beta variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 10% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 15% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ1 cluster and at least 15% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 20% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ1 cluster and at least 20% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster.In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 30% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ1 cluster and at least 30% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a T CRB non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 40% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a T CRB non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 50% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR beta variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., CD4+ and / or CD8+ T cells, wherein at least 60% of the TCRs expressed by the population of splenocytes are derived from gene segments from the TCRBDJ2 cluster.In some embodiments, the non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR beta variable locus comprising a TCR non-coding sequence comprises a population of spleen cells, e.g., CD4+ and / or CD8+ T cells, wherein at least 70% of the TCRs expressed by the population of spleen cells are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., a population of CD4+ and / or CD8+ T cells, that express TCRs derived from gene segments from the TCRBDJ1 cluster and TCRs derived from gene segments from the TCRBDJ2 cluster in a ratio of 1:3, 3:7, 1:2, 2:3, or 1:1. In some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprising a humanized TCR β variable locus comprising a TCR non-coding sequence comprises a population of splenocytes, e.g., a population of CD4+ and / or CD8+ T cells, that express TCRs derived from gene segments from the TCRBDJ2 cluster and TCRs derived from gene segments from the TCRBDJ1 cluster in a ratio of 1:3, 3:7, 1:2, or 2:3.

[0118] In some embodiments, the germ cells and CD3 -The T cell comprises an unrearranged TCR β variable region sequence comprising at least one unrearranged human TCR variable region Vβ segment, at least one unrearranged human TCR variable region Dβ segment, and at least one unrearranged TCR variable region Jβ segment, wherein the unrearranged TCR β variable region sequence is operably linked to a TCR β constant region gene sequence, such as a mouse TCR β constant region gene sequence, optionally at an endogenous TCR β locus, and the unrearranged human TCR Vβ segment, unrearranged human TCR Dβ segment, and unrearranged human TCR Jβ segment can be rearranged (or rearranged) in the T cell to form a rearranged human TCR Vβ / Dβ / Jβ variable region gene operably linked to the TCR β constant region gene sequence, and the rearranged human TCR Vβ / Dβ / Jβ variable region gene operably linked to the TCR β constant region gene sequence together coats a TCR β polypeptide comprising a human TCR β variable domain, and the mouse produces CD3 T cells expressing a TCR comprising the TCR β polypeptide on its surface. + In some embodiments, the unrearranged TCR β variable region sequence comprises a mouse TCRB non-coding sequence.

[0119] In some mouse embodiments described herein, (I) Germ cells and CD3 - T cells (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence; (C) At the endogenous TCRα locus Replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) At the endogenous TCRβ locus replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; Replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; and comprising the replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Jδ segments; (II) The mouse expresses on its surface a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells and CD3 expressing on their surface a functional TCR comprising a human or humanized α polypeptide and a human or humanized TCR β polypeptide. + Further included are T cells.

[0120] In some embodiments, germ cells and CD3 -The T cells each comprise a human CTCF binding element. In some embodiments, the germ cells and somatic cells each comprise a human CTCF binding element upstream of the TCR alpha locus. In some embodiments, the germ cells and somatic cells each comprise a human CTCF binding element upstream of the TCR gamma locus.

[0121] In some embodiments, the germ cells and somatic cells each comprise a human CTCF binding element upstream of the TCR alpha locus and a human CTCF binding element upstream of the TCR gamma locus.

[0122] In one aspect, the non-human animal expresses a humanized T cell receptor with a non-human constant region on the surface of the T cell, and the receptor is capable of interacting with a non-human molecule, e.g., an anchoring molecule or a signaling molecule expressed in the T cell (e.g., a CD3 molecule, a zeta chain, or another protein anchored to the TCR through a CD3 molecule or a zeta chain). Thus, in one aspect, a cell complex is provided, comprising: (a) a non-human T cell expressing (i) a TCR comprising a humanized TCR α chain described herein and a humanized TCR β chain described herein and (ii) a chimeric co-receptor described herein; and (b) a non-human antigen-presenting cell comprising an antigen bound to a chimeric MHC I and / or a chimeric MHC II described herein. In one embodiment, the human TCR γ chain and / or TCR δ chain are complexed with a non-human, e.g., endogenous, zeta (ζ) chain homodimer and a non-human, e.g., endogenous, CD3 heterodimer. In one embodiment, the cell complex is an in vivo cell complex.

[0123] In various embodiments, the non-human animals (e.g., rodents, e.g., mice or rats) described herein produce T cells that are capable of undergoing thymic development, progressing from DN1 to DN2 to DN3 to DN4 to DP, and to CD4 or CD8 SP T cells. Such T cells of the non-human animals of the invention typically express cell surface molecules (e.g., CD25, CD44, Kit, CD3, pTα, etc.) produced by T cells during specific stages of thymic development.

[0124] In various embodiments, the non-human animals described herein produce T cells that are capable of undergoing T cell differentiation in the periphery. In additional embodiments, the non-human animals described herein produce CD3 T cells in the periphery, e.g., in the spleen, skin, and intestinal mucosa. + Contains T cells.

[0125] DN1 and DN2 cells that do not receive sufficient signals (e.g., Notch signals) can develop into B cells, myeloid cells (e.g., dendritic cells), mast cells, and NK cells. See Yashiro-Ohtani et al. (2010) Notch regulation of early thymocyte development, Seminars in Immunology 22:261-69. In some embodiments, the non-human animals described herein develop B cells, myeloid cells (e.g., dendritic cells), mast cells, and NK cells. In some embodiments, the non-human animals described herein develop a dendritic cell population in the thymus.

[0126] The predominant type of T cell receptor expressed on the surface of T cells is TCR / β, with a minority of cells expressing TCR / . In some embodiments, T cells of non-human animals comprising humanized TCR gamma and / or delta loci exhibit utilization of the TCR alpha / beta and TCR gamma / delta loci similar to wild-type animals (e.g., T cells of non-human animals described herein express TCR alpha / beta and TCR delta / gamma proteins in proportions comparable to those expressed by wild-type animals). Thus, in some embodiments, non-human animals comprising humanized TCR alpha / beta and human TCR gamma / delta loci exhibit utilization of all loci. Humanized T cell co-receptors

[0127] Although antigen recognition by gamma / delta T cells may not require interaction of T cell coreceptors (e.g., CD4 and CD8) with MHC, the non-human animals described herein may comprise human or humanized TRA and TRB loci in addition to the human or humanized TRD (and TRG) loci, and the non-human animals described herein may also comprise human or humanized CD4 loci and / or human or humanized CD8 (e.g., CD8α and CD8β) loci. See, e.g., U.S. Patent Nos. 9,848,587 and 10,820,581, each of which is incorporated herein by reference.

[0128] Disclosed herein are non-human animals that express at least one human or humanized T cell co-receptor, e.g., CD4, CD8, and / or CD8. Accordingly, the non-human animals described herein comprise at least one first, second, and / or third nucleotide sequence, each of which encodes a different human or chimeric human / non-human T cell co-receptor polypeptide selected from a human or humanized CD4 polypeptide, a human or humanized CD8α polypeptide, and a human or humanized CD8β polypeptide. The use of the designations first, second, and third herein should not be construed as limiting the non-human animals disclosed herein as requiring the presence of all three nucleotide sequences or any of the co-receptor nucleotide sequences in any order. Thus, the non-human animals disclosed herein can comprise a nucleic acid sequence or sequences encoding a human or humanized CD4 and / or a human or humanized CD8 (e.g., human or humanized CD8α and / or CD8β) polypeptide.

[0129] In one embodiment, the non-human animals described herein comprise a first nucleotide sequence encoding a human or humanized CD4 polypeptide. In another embodiment, the non-human animals described herein comprise a first nucleotide sequence encoding a human or humanized CD8α polypeptide and a second nucleotide sequence encoding a human or humanized CD8β polypeptide. In another embodiment, the non-human animals described herein comprise first and second nucleotide sequences encoding human or humanized CD8α and CD8β polypeptides, and further comprise a third nucleotide sequence encoding a human or humanized CD4 polypeptide.

[0130] In various embodiments, the present invention generally provides genetically modified non-human animals that include in their genome, e.g., at the endogenous CD4 locus, a nucleotide sequence encoding a human or humanized CD4 polypeptide, such that the animal expresses a human or humanized CD4 polypeptide.

[0131] The human CD4 gene is located on chromosome 12 and is thought to contain 10 exons. The CD4 gene encodes a protein with an amino-terminal hydrophobic signal sequence encoded by exons 2 and 3 of the gene. The protein contains four extracellular immunoglobulin-like domains, Ig1-Ig4, also commonly referred to as the D1-D4 domains. Maddon et al. (1987) Structure and expression of the human and mouse T4 genes, Proc. Natl. Acad. Sci. USA 84:9155-59. The D1 domain is thought to be encoded by exon 3 (sequence downstream of the signal peptide) and exon 4, while D2, D3, and D4 are encoded by separate exons—exons 5, 6, and 7, respectively (Figure 9A: The D1, D2, D3, and D4 domains are encoded by sequences designated Ig1, Ig2, Ig3, and Ig4, respectively). Littman (1987) The Structure of the CD4 and CD8 Genes, Ann. Rev. Immunol. 5:561-84; Hanna et al. (1994) Specific Expression of the Human CD4 Gene in Mature CD4+CD8- and Immature CD4+CD8+ T Cells and in Macrophages of Transgenic Mice, Mol. Cell. Biol. 14(2):1084-94; Maddon et al., supra. In areas of high protein concentration, such as areas of contact between T cells and antigen-presenting cells, the molecules tend to homodimerize through interactions between opposing D4 domains.Zamoyska (1998) CD4 and CD8: modulators of T cell receptor recognition of antigen and of immune responses? Curr. Opin. Immunol. 10:82-87; Wu et al. (1997) Dimeric association and segmental variability in the structure of human CD4,Nature 387:527; Moldovan et al. (2002) CD4 Dimers Constitute the Functional Component Required for T Cell Activation, J. Immunol. 169:6261-68.

[0132] The D1 domain of CD4 resembles an immunoglobulin variable (V) domain and, together with part of the D2 domain, is thought to bind to (associate with) MHC II, for example, at the MHC II coreceptor binding site. Huang et al. (1997) Analysis of the contact sites on the CD4 molecule with Class II MHC molecule, J. Immunol. 158:216-25. MHC II then interacts with the T cell coreceptor CD4 in the hydrophobic cleft at the junction between the MHC II α2 and β2 domains. Wang and Reinherz (2002) Structural Basis of T Cell Recognition of Peptides Bound to MHC Molecules, Molecular Immunology, 38:1039-49.

[0133] Domains D3 and D4 of the CD4 coreceptor are thought to interact with the TCR-CD3 complex, because substitutions in these two domains abrogated the ability of CD4 to bind to the TCR. Vignali et al. (1996) The Two Membrane Proximal Domains of CD4 Interact with the T Cell Receptor, J. Exp. Med. 183:2097-2107. The CD4 molecule exists as a dimer, and residues in the D4 domain of the molecule are thought to be involved in CD4 dimerization. Moldovan et al. (2002) CD4 Dimers Constitute the Functional Components Required for T Cell Activation, J. Immunol. 169:6261-68.

[0134] Exon 8 of the CD4 gene encodes the transmembrane domain, while the remainder of the gene encodes the cytoplasmic domain. The CD4 cytoplasmic domain has many different functions. For example, the cytoplasmic domain of CD4 recruits the tyrosine kinase Lck. Lck is a Src family kinase that associates with the CD4 and CD8 cytoplasmic domains. Simultaneous binding of co-receptors and TCRs to the same MHC leads to increased tyrosine phosphorylation of CD3 and chains of the TCR complex, which in turn leads to the recruitment of other factors that play a role in T cell activation. By designing and testing the expression of a hybrid protein containing the CD8 extracellular domain and the CD4 cytoplasmic tail in transgenic mice, Itano and co-authors propose that the CD4 cytoplasmic tail also promotes the differentiation of CD4+CD8+ T cells into the CD4+ lineage. Itano et al. (1996) The Cytoplasmic Domain of CD4 Promotes the Development of CD4 Lineage T Cells, J. Exp. Med. 183:731-41. Expression of the hybrid protein led to the development of MHC I-specific CD4 lineage T cells. Ibid.

[0135] The CD4 coreceptor appears to be the primary receptor for the HIV virus, and CD4+ T cell depletion is an indicator of disease progression. The cytoplasmic tail of CD4 appears to be essential for delivering the apoptotic signal to CD4+ T cells during HIV-induced apoptosis. Specifically, interaction of CD4 and Lck has been shown to enhance HIV-induced apoptosis in these cells. Corbeil et al. (1996) HIV-induced Apoptosis Requires the CD4 Receptor Cytoplasmic Tail and Is Accelerated by Interaction of CD4 with p56lck, J. Exp. Med. 183:39-48.

[0136] T cells develop in the thymus and progress from immature CD4- / CD8- (double-negative, or DN) thymocytes to CD4+ / CD8+ (double-positive, or DP) thymocytes, ultimately undergoing positive selection to become either CD4+ or CD8+ (single-positive, or SP) T cells. DP thymocytes signaling through an MHC I-restricted TCR differentiate into CD8+ T cells, whereas DP thymocytes signaling through an MHC II-restricted TCR differentiate into CD4+ T cells. The cues received by DP cells that lead to their differentiation into either CD4+ or CD8+ T cells have been the subject of much research. Various models for CD4 / CD8 lineage selection have been proposed and are reviewed in Singer et al. (2008) Lineage fate and intense debate: myths, models, and mechanisms of CD4- versus CD8- lineage choice, Nat. Rev. Immunol. 8:788-801.

[0137] Inactivation of specific T cell coreceptors as a result of positive selection is the product of transcriptional regulation. For CD4, an enhancer located 13 kb upstream of CD4 exon 1 has been shown to upregulate CD4 expression in CD4+ and CD8+ T cells. Killeen et al. (1993) Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4, EMBO J. 12:1547-53. A cis-acting transcriptional silencer located within the first intron of the mouse CD4 gene functions to silence CD4 expression in cells other than CD4+ T cells. Siu et al. (1994) A transcriptional silencer controls the developmental expression of the CD4 gene, EMBO J. 13:3570-3579.

[0138] Because key transcriptional regulators (e.g., promoters, enhancers, silencers) controlling CD4 lineage selection were missing in several previously developed transgenic mouse lines expressing human CD4, these mice failed to recapitulate normal T cell lineage development and produced immune cells other than CD4+ T cells that express CD4. See, e.g., Law et al. (1994) Human CD4 Restores Normal T Cell Development and Function in Mice Deficient in CD4, J. Exp. Med. 179:1233-42 (CD4 expression in CD8+ T cells and B cells); Fugger et al. (1994) Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region β-chain T-cell repertoire and mediates an HLA-D-restricted immune response, Proc. Natl. Acad. Sci. USA, 91:6151-55 (CD4 expressed on all CD3+ thymocytes and B cells). Thus, in one embodiment, it may be advantageous to generate genetically modified animals in which the animals retain endogenous mouse promoters and / or other regulatory elements to produce T cells capable of undergoing T cell development and lineage selection.

[0139] Thus, in various embodiments, the invention provides a genetically modified non-human animal comprising, e.g., at its endogenous T cell co-receptor locus (e.g., CD4 locus), a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide. In one embodiment, the human portion of the chimeric polypeptide comprises all or substantially all of the extracellular portion (or portion thereof, e.g., one or more extracellular domains, e.g., at least two contiguous extracellular domains) of a human T cell co-receptor. In one embodiment, the non-human portion of the chimeric polypeptide comprises the transmembrane and cytoplasmic domains of a non-human T cell co-receptor. In one embodiment, the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. Thus, in one aspect, the invention provides a genetically modified non-human animal comprising, at its endogenous CD4 locus, a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide, wherein the human portion of the chimeric polypeptide comprises all or substantially all of the extracellular portion of human CD4 and the non-human portion comprises at least the transmembrane and cytoplasmic domains of non-human CD4, and the animal expresses a functional chimeric CD4 polypeptide. In one embodiment, the non-human animal expresses only a humanized CD4 polypeptide, ie, a chimeric human / non-human CD4 polypeptide, and does not express a functional non-human CD4 protein from its endogenous CD4 locus.

[0140] In one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of a human CD4 polypeptide. In another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises an MHC II binding domain of a human CD4 polypeptide (e.g., a substantial portion of the human D1 and D2 domains). In one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the D1, D2, and D3 domains of a human CD4 polypeptide. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the immunoglobulin-like domains of CD4, e.g., the domains designated D1, D2, D3, and D4. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises in its human portion all or substantially all of the human CD4 sequences involved in interaction with MHC II and / or the extracellular portion of the T-cell receptor. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of human CD4 involved in interactions with MHC II and / or the variable domain of the T-cell receptor. Thus, in one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises all or substantially all of the coding sequence for domains D1-D2 of human CD4 (e.g., a portion of exon 3 and exons 4-5 of the human CD4 gene), and in another embodiment, it comprises all or substantially all of the coding sequence for domains D1-D3 of human CD4 (e.g., a portion of exon 3 and exons 4-6 of human CD4). Thus, in one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD4 comprises nucleotide sequences encoding all or substantially all of the D1-D3 domains of human CD4. In another embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises the coding sequence for domains D1-D4 of human CD4. In another embodiment, the nucleotide sequence can include a nucleotide sequence encoding the mouse CD4 signal peptide, for example, the region encoded by part of exons 2-3 of the mouse gene.In another embodiment, the nucleotide sequence can include a nucleotide sequence encoding a human CD4 signal peptide. In one embodiment, the chimeric human / non-human CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, with the human portion of the chimeric polypeptide spanning approximately amino acids 27 to 319 of SEQ ID NO: 1 (set forth separately in SEQ ID NO: 79).

[0141] In one embodiment, the non-human animal expresses a chimeric human / non-human CD4 polypeptide sequence. In one embodiment, the human portion of the chimeric CD4 sequence contains one or more conservative or non-conservative modifications.

[0142] In one aspect, a non-human animal is provided that expresses a human CD4 sequence, wherein the human CD4 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence. In a specific embodiment, the human CD4 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence described in U.S. Patent No. 10,820,581. In one embodiment, the human CD4 sequence comprises one or more conservative substitutions. In one embodiment, the human CD4 sequence comprises one or more non-conservative substitutions.

[0143] In some embodiments, a portion, e.g., a human portion of a chimeric CD4, can include substantially all of the sequences set forth herein (e.g., substantially all of the protein domains set forth herein). Substantially all sequences generally include 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids believed to represent a particular portion of the protein (e.g., a particular functional domain, etc.). Those skilled in the art will understand that the boundaries of functional regions may vary slightly depending on the alignment and domain prediction methods used.

[0144] In one aspect, the nonhuman portion of the chimeric human / non-human CD4 polypeptide comprises at least the transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Due to the important functions performed by the CD4 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequence in the genetically engineered animal ensures preservation of proper intracellular signaling and other functions of the coreceptor. In one embodiment, the non-human animal is a mouse, and the non-human CD4 polypeptide is a mouse CD4 polypeptide. Specific mouse CD4 sequences are described in U.S. Pat. No. 10,820,581, cited in the Examples, but any suitable sequence derived therefrom, e.g., containing conservative / non-conservative amino acid substitutions, is encompassed herein. In one embodiment, the non-human portion of the chimeric CD4 coreceptor comprises any sequence of endogenous CD4 that has not been humanized.

[0145] The non-human animals described herein can comprise, at their endogenous loci, nucleotide sequences encoding chimeric human / non-human CD4 polypeptides. In one aspect, this results in the replacement of a portion of the endogenous CD4 gene with a nucleotide sequence encoding a portion of a human CD4 polypeptide. In one embodiment, such a replacement is, for example, a replacement of an endogenous nucleotide sequence encoding all or substantially all of the extracellular domain of non-human CD4 with a human nucleotide sequence encoding at least all or substantially all of the first immunoglobulin-like domain (i.e., D1) of non-human CD4 (e.g., a sequence encoding all or substantially all of domains D1-D2 of non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D3 of non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D4 of non-human CD4). In one embodiment, the replacement results in a chimeric protein comprising human CD4 sequences involved in interaction with the extracellular portion of MHC II and / or the T-cell receptor. In yet another embodiment, the substitution results in a chimeric protein comprising a human CD4 sequence involved in interaction with MHC II and / or the variable domain of a T cell receptor. In one embodiment, the substitution does not include substitution of the CD4 sequence encoding at least the transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Thus, in one aspect, the non-human animal expresses a chimeric human / non-human CD4 polypeptide from an endogenous non-human CD4 locus. In yet another embodiment, the substitution results in a protein comprising the polypeptide sequence set forth in SEQ ID NO: 1.

[0146] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD4 locus (e.g., a chimeric human / rodent CD4 locus, e.g., a chimeric human / mouse CD4 locus) described herein is provided. In one aspect, the chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD4 sequence is placed in an endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus such that it retains the CD4 enhancer element located upstream of the first CD4 exon. In one embodiment, the substitution in the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus comprises, for example, a portion of exon 3 encoding D1 and exons 4-6 encoding D2-D3 of the CD4 polypeptide with the remaining D1. Thus, in one aspect, the chimeric CD4 locus retains the cis-acting silencer located in intron 1 of the non-human (e.g., mouse) CD4 gene. Thus, in one embodiment, the chimeric locus retains the endogenous non-human (e.g., rodent, e.g., mouse) CD4 promoter and regulatory elements. In another embodiment, the chimeric locus may include human promoter and regulatory elements to the extent that they allow for proper CD4 expression, CD4+ T cell development, CD4 lineage selection, and coreceptor function. Thus, in some aspects, animals of the invention comprise genetic modifications that do not alter proper lineage selection and T cell development. In one aspect, animals of the invention (e.g., rodents, e.g., mice) do not express chimeric CD4 polypeptides on immune cells other than those that normally express CD4. In one aspect, the animals do not express CD4 on B cells or mature CD8+ T cells. In one embodiment, the replacement results in retention of elements that allow for proper spatial and temporal regulation of CD4 expression.

[0147] In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a functional chimeric CD4 protein from a chimeric CD4 locus described herein displays the chimeric protein on the cell surface, e.g., on the T cell surface. In one embodiment, the non-human animal expresses the chimeric CD4 protein on the cell surface in a cellular distribution that is the same as that observed in humans. In one aspect, the CD4 protein of the invention is capable of interacting with an MHC II protein expressed on the surface of a second cell, e.g., an antigen-presenting cell (APC).

[0148] In various embodiments, the present invention generally provides genetically modified non-human animals that comprise, in their genome, e.g., an endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8 polypeptide; thus, the animals express human or humanized CD8 polypeptides. In various embodiments, the present invention provides non-human animals that comprise, in their genome, e.g., an endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8α polypeptide, and / or a nucleotide sequence encoding a human or humanized CD8β polypeptide. Thus, the genetically modified non-human animals of the present invention express human or humanized CD8α and / or human or humanized CD8β polypeptides.

[0149] The human CD8 protein is typically expressed on the cell surface as a heterodimer of two polypeptides, CD8α and CD8β, although disulfide-linked homodimers and homomultimers have also been detected (e.g., in NK cells and intestinal γδ T cells that express CD8αα). The genes encoding human CD8α and CD8β are located close to each other on chromosome 2. Nakayama et al. (1992) Recent Duplication of the Two Human CD8 β-chain Genes, J. Immunol. 148:1919-27. The CD8α protein contains a leader peptide, an immunoglobulin V-like region, a hinge region, a transmembrane domain, and a cytoplasmic tail. Norment et al. (1989) Alternatively spliced ​​mRNA encodes a secreted form of human CD8α. Characterization of the human CD8α gene, J. Immunol. 142:3312-19. The exons / introns of the CD8α gene are depicted diagrammatically in Figure 9B.

[0150] The human CD8β gene is located upstream of the CD8α gene on chromosome 2. Multiple isoforms generated by alternative splicing of the CD8β gene have been reported, and one isoform lacks the transmembrane domain and is predicted to produce a secreted protein. Norment et al. (1988) A second subunit of CD8 is expressed in human T cells, EMBO J. 7:3433-39. The exons and introns of the CD8β gene are depicted diagrammatically in Figure 9B.

[0151] The membrane-bound CD8β protein contains an N-terminal signal sequence followed by an immunoglobulin V-like domain, a short extracellular hinge region, a transmembrane domain, and a cytoplasmic tail. See Littman (1987) The structure of the CD4 and CD8 genes, Ann Rev. Immunol. 5:561-84. The hinge region is a site of extensive glycosylation, which is thought to maintain its conformation and protect the protein from cleavage by proteases. Leahy (1995) A structural view of CD4 and CD8, FASEB J. 9:17-25.

[0152] The CD8 protein is typically expressed on cytotoxic T cells and interacts with MHC I molecules. The interaction is mediated through CD8 binding to the α3 domain of MHC I. Although MHC class I binding to CD8 is approximately 100-fold weaker than TCR binding to MHC class I, CD8 binding enhances the affinity of TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366.

[0153] Binding of CD8 to MHC class I molecules is species-specific, and the mouse CD8 homolog, Lyt-2, has been shown to bind H-2Dd molecules in the α3 domain but not to HLA-A molecules. See Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class I α3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168:325-341. The differential binding was likely due to CDR-like determinants (CDR1-like and CDR2-like) on CD8 that were not conserved between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174:371-379; Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice Carrying a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. 173:1007-1015; and, Gao et al. (1997) Crystal structure of the complex between human CD8α and HLA-A2, Nature 387:630-634. CD8 has been reported to bind to HLA-A2 in a conserved region of the α3 domain (positions 223-229). A single substitution in HLA-A (V245A) reduced CD8 binding to HLA-A, but with a concomitant large reduction in T cell-mediated lysis.Salter et al. (1989), Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8, Nature 338:345–348. In general, polymorphism in the α3 domain of HLA-A molecules also affected binding to CD8. Ibid. In mice, an amino acid substitution at residue 227 in H-2Dd affected the binding of mouse Lyt-2 to H-2Dd, and cells transfected with the mutant H-2Dd were not lysed by CD8+ T cells. Potter et al. (1989), Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73–75. Thus, expression of human or humanized CD8 may be useful for testing T cell responses to antigens presented by human or humanized MHC I.

[0154] Like CD4, the cytoplasmic domain of CD8 interacts with the tyrosine kinase Lck, which in turn leads to T cell activation. Lck appears to interact with the cytoplasmic domain of CD8α, and this interaction appears to be regulated by the presence of the cytoplasmic domain of CD8β, because mutation or deletion of the cytoplasmic domain of CD8β resulted in reduced CD8α-associated Lck activity. Irie et al. (1998) The cytoplasmic domain of CD8β Regulates Lck Kinase Activation and CD8 T cell Development, J. Immunol. 161:183-91. Decreased Lck activity has been associated with impaired T cell development. Id.

[0155] Expression of CD8 on appropriate cells, such as cytotoxic T cells, is tightly controlled by diverse enhancer elements located throughout the CD8 locus. For example, at least four regions of DNAse I hypersensitivity, regions frequently associated with regulatory factor binding, have been identified in the CD8 locus. Hosert et al. (1997) A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice, J. Immunol. 158:4270-81. Since the discovery of these DNAse I hypersensitive regions in the CD8 locus, at least five enhancer elements have been identified and found to be spread throughout the CD8 locus. These elements control expression of CD8α and / or β in T cells of various lineages, including DP, CD8 SP T cells, or cells expressing the γδ TCR. See, e.g., Kioussis et al. (2002) Chromatin and CD4, CD8A, and CD8B gene expression during thymic differentiation, Nature Rev. 2:909-919 and Online Erratum; Ellmeier et al. (1998) Multiple Development Stage-Specific Enhancers Regulate CD8 Expression in Developing Thymocytes and in Thymus-Independent T cells, Immunity 9:485-96.

[0156] Thus, similar to the benefits derived from retention of the endogenous CD4 promoter and regulatory elements for genetically modified animals of human or humanized CD4, in some embodiments, there may be benefits in developing genetically modified non-human animals that retain the endogenous mouse promoter and regulatory elements capable of controlling expression of human or humanized CD8. As described herein, there may be particular benefits in generating genetically modified animals that include replacement of endogenous non-human sequences encoding CD8α and / or β proteins with endogenous non-human sequences encoding human or humanized CD8α and / or proteins.

[0157] In various embodiments, the invention provides genetically modified non-human animals that comprise in their genome, e.g., in their endogenous CD8 locus, at least one nucleotide sequence encoding a chimeric human / non-human CD8 polypeptide (e.g., CD8α and / or β polypeptide), wherein the human portion of the polypeptide comprises all or substantially all of the extracellular portion (or a portion thereof, e.g., the extracellular domain) of the human CD8 polypeptide (e.g., CD8α and / or β), and the non-human portion comprises at least the transmembrane and cytoplasmic domains of the non-human CD8 (e.g., CD8α and / or β), and the animal expresses the chimeric CD8 polypeptide (e.g., CD8α and / or β polypeptide). Thus, in one embodiment, the invention provides a genetically modified non-human animal comprising, at its endogenous non-human CD8 locus, a first nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide, wherein the first nucleotide sequence comprises a sequence encoding all or substantially all of the extracellular portion of the human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of the non-human CD8α polypeptide, and the second nucleotide sequence comprises a sequence encoding all or substantially all of the extracellular portion of the human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of the non-human CD8β polypeptide, and the animal expresses a functional chimeric human / non-human CD8 protein. In one aspect, the non-human animal expresses only the humanized CD8 polypeptides (e.g., chimeric human / non-human CD8α and / or β polypeptides) and does not express the corresponding functional non-human CD8 polypeptide from the endogenous CD8 locus.

[0158] In one embodiment, a chimeric human / non-human CD8α polypeptide comprises, in its human portion, all or substantially all of the extracellular portion of a human CD8α polypeptide. In one embodiment, the human portion of a chimeric CD8α polypeptide comprises at least the MHC I-binding domain of a human CD8α polypeptide. In one embodiment, the human portion of a chimeric CD8α polypeptide comprises at least all or substantially all of the sequence of an immunoglobulin V-like domain of a human CD8α. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric CD8α polypeptide comprises at least exons encoding the extracellular portion of a human CD8α polypeptide. In one embodiment, the nucleotide sequence comprises at least exons encoding an Ig V-like domain. In one embodiment, the extracellular portion of a human CD8α polypeptide is a region encompassing a portion of the polypeptide that is not the transmembrane or cytoplasmic domain. In one embodiment, the nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide comprises a sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8α signal peptide. Alternatively, the nucleotide sequence may comprise a sequence encoding a human CD8α signal sequence. In one embodiment, the chimeric human / non-human CD8α polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3, with the human portion of the chimeric polypeptide set forth in amino acids 28-179 of SEQ ID NO: 3 (represented separately in SEQ ID NO: 4).

[0159] Similarly, in one embodiment, a chimeric human / non-human CD8 β polypeptide comprises, in its human portion, all or substantially all of the extracellular portion of a human CD8 β polypeptide. In one embodiment, the human portion of a chimeric CD8 β polypeptide comprises all or substantially all of the sequence of an immunoglobulin V-like domain of human CD8 β. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric CD8 β polypeptide comprises at least an exon encoding the extracellular portion of a human CD8 β polypeptide. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric human / non-human CD8 β polypeptide comprises at least an exon encoding an IgG V-like domain of human CD8 β. In one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD8 β polypeptide comprises a sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8 β signal peptide. Alternatively, the nucleotide sequence may comprise a sequence encoding a human CD8 β signal sequence. In one embodiment, the chimeric human / non-human CD8β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5, with the human portion of the chimeric polypeptide set forth in amino acids 15-165 of SEQ ID NO: 5 (separately represented in SEQ ID NO: 6).

[0160] In one embodiment, the non-human animal expresses a chimeric human / non-human CD8α and / or CD8β polypeptide. In some embodiments, the human portion of the chimeric human / non-human CD8α and / or β polypeptide comprises one or more conservative or non-conservative modifications.

[0161] In one aspect, a non-human animal is provided that expresses a human CD8α and / or β polypeptide sequence, wherein the human CD8α and / or β polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD8α and / or β polypeptide sequence, respectively. In a specific embodiment, the human CD8α and / or β polypeptide sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the respective human CD8α and / or β polypeptide sequence described in U.S. Patent No. 9,848,587. In one embodiment, the human CD8α and / or β polypeptide sequence comprises one or more conservative substitutions. In one embodiment, the human CD8α and / or β polypeptide sequence comprises one or more non-conservative substitutions.

[0162] In some embodiments, a portion, e.g., a human portion of a chimeric CD8, can include substantially all of the sequences set forth herein (e.g., substantially all of the protein domains set forth herein). Substantially all sequences generally include 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids that are believed to represent a particular portion of a protein (e.g., a particular functional domain, etc.). Those skilled in the art will understand that the boundaries of functional regions may vary slightly depending on the alignment and domain prediction methods used.

[0163] In one aspect, the nonhuman portion of the chimeric human / non-human CD8α and / or β polypeptide comprises at least the transmembrane and / or cytoplasmic domains of the non-human CD8α and / or β polypeptide, respectively. Due to the important functions performed by the CD8 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequences in the genetically engineered animal ensures preservation of proper intracellular signaling and other coreceptor functions. In one embodiment, the non-human animal is a mouse, and the non-human CD8α and / or β polypeptides are mouse CD8α and / or β polypeptides, respectively. Particular mouse CD8α and β sequences are described in U.S. Patent No. 9,848,587, cited in the Examples, although any suitable sequences derived therefrom, such as sequences containing conservative / non-conservative amino acid substitutions, are encompassed herein. In one embodiment, the non-human animal (e.g., a rodent, e.g., a mouse) retains any endogenous sequences that have not been humanized.

[0164] The non-human animals described herein can comprise, at their endogenous loci, nucleotide sequences encoding chimeric human / non-human CD8α and / or β polypeptides. In one aspect, this results in a replacement of a portion of the endogenous CD8α gene with a nucleotide sequence encoding a portion of a human CD8α polypeptide, and / or a replacement of a portion of the endogenous CD8β gene with a nucleotide sequence encoding a portion of a human CD8β polypeptide. In one embodiment, such a replacement is a replacement of endogenous nucleotide sequences encoding all or substantially all of the extracellular portion of non-human CD8α and / or β with a human nucleotide sequence encoding the same. In one embodiment, such a replacement is a replacement of sequences encoding at least all or substantially all of the immunoglobulin V-like domains of non-human CD8α and / or β with a human nucleotide sequence encoding the same. In one embodiment, the replacement does not include replacement of CD8α and / or β sequences encoding the transmembrane and cytoplasmic domains of non-human CD8α and / or β polypeptides. In this manner, the non-human animal expresses chimeric human / non-human CD8 α and / or β polypeptides from the endogenous non-human CD8 locus. In yet another embodiment, the substitution results in a CD8 α and / or β protein comprising the polypeptide sequence set forth in SEQ ID NOs: 3 and / or 5, respectively.

[0165] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD8 locus (e.g., a chimeric rodent CD8 locus, e.g., a chimeric mouse CD8 locus) is provided. In one aspect, the chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD8α and / or β sequence is placed at the respective endogenous non-human (e.g., rodent, e.g., mouse) CD8α and / or β locus such that it retains the endogenous CD8α and / or β promoter and regulatory elements. In another embodiment, the chimeric locus can include human CD8α and / or β promoter and regulatory elements to the extent that it allows proper CD8 and / or β expression (proper spatial and temporal protein expression), CD8+ T cell development, CD8 lineage selection, and coreceptor function. Thus, in one aspect, the animals of the invention comprise genetic modifications that do not alter proper lineage selection and T cell development. In one aspect, an animal (e.g., a rodent, e.g., a mouse) of the invention does not express the chimeric CD8 protein on immune cells other than those that normally express CD8, e.g., the animal does not express CD8 on B cells or mature CD4+ T cells. In one embodiment, the replacement results in retention of elements that allow for proper spatial and temporal regulation of CD8α and / or β expression.

[0166] In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a functional chimeric CD8 protein (e.g., CD8αβ or CD8αα) from a chimeric CD8 locus described herein displays the chimeric protein on the cell surface. In one embodiment, the non-human animal expresses the chimeric CD8 protein on the cell surface in a cellular distribution that is the same as that observed in humans. In one aspect, the CD8 protein of the invention is capable of interacting with an MHC I protein expressed on the surface of a second cell.

[0167] Human or humanized MHC molecules Although recognition of antigen by gamma / delta T cells may not require interaction between T cell co-receptors (e.g., CD4 and CD8) and MHC, the non-human animals described herein may further comprise one or more human or humanized MHC loci, as the non-human animals described herein may comprise human or humanized TRA, TRB, CD4 and / or CD8 (e.g., CD8α and CD8β) loci in addition to the human or humanized TRD (and TRG) loci.

[0168] In various embodiments, provided herein are at least one humanized T cell coreceptor, at least one humanized MHC associated with the humanized T cell coreceptor, and a human or humanized α / β TCR, which, upon recognition and binding to a peptide presented by the humanized MHC, engages the humanized coreceptor to transmit an activation signal to cells expressing the humanized TCR and chimeric T cell coreceptor polypeptides. Accordingly, the non-human animals disclosed herein comprise at least a first, second, and / or third nucleic acid sequence, each encoding a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC II α polypeptide, a human or humanized MHC II β polypeptide, and a human or humanized MHC I α polypeptide, and the non-human animals optionally also comprise human or humanized β2-microglobulin. The use of the designations first, second, and third herein should not be construed as limiting the non-human animals disclosed herein as requiring the presence of all three nucleic acid sequences or any of the human or humanized MHC polypeptides, in any particular order.

[0169] Thus, in some embodiments, the non-human animals described herein can comprise, for example, first and second nucleotide sequences encoding a human or chimeric CD8α polypeptide and a human or chimeric CD8β polypeptide operably linked to a non-human TCRα constant gene sequence, an unrearranged T cell receptor (TCR) variable locus comprising at least one human Vα segment and at least one human Jα segment, and / or an unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to a non-human TCRβ constant region gene sequence, and optionally, first and second nucleic acid sequences encoding, for example, a human or humanized MHC Iα polypeptide and a human or humanized β2-microglobulin polypeptide. Thus, in another embodiment, the non-human animals described herein may comprise, for example, a first nucleotide sequence encoding a chimeric CD4 polypeptide; an unrearranged T cell receptor (TCR) alpha variable locus comprising at least one human Valpha segment and at least one human Jalpha segment operably linked to a non-human TCR alpha constant region gene sequence; an unrearranged TCR beta variable locus comprising at least one human Vbeta segment, at least one human Dbeta segment, and at least one human Jbeta segment operably linked to a non-human TCR beta constant region gene sequence; and first and second nucleic acid sequences encoding, for example, a human or humanized MHC II alpha polypeptide and a human or humanized MHC II beta polypeptide.In some embodiments, the non-human animals described herein comprise first, second, and third nucleotide sequences encoding, for example, a chimeric CD4 polypeptide, a chimeric CD8α polypeptide, and a chimeric CD8β polypeptide, an unrearranged T cell receptor (TCR) variable locus comprising at least one human Vα segment and at least one human Jα segment operably linked to a non-human TCR α constant region gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to a TCR β constant region gene sequence, and optionally first, second, third, and fourth nucleic acid sequences encoding a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, a human or humanized MHC Iα polypeptide, and a human or humanized β2 microglobulin.

[0170] In various embodiments, provided herein are genetically modified non-human animals, e.g., rodents (e.g., mice or rats), comprising in their genomes nucleic acid sequences encoding human or humanized MHC I polypeptides and / or nucleic acid sequences encoding human or humanized MHC II proteins. The MHC I nucleic acid sequences can encode partially human and partially non-human MHC I polypeptides, e.g., chimeric human / non-human MHC I polypeptides, and the MHC II nucleic acid sequences can encode partially human and partially non-human MHC II proteins, e.g., chimeric human / non-human MHC II proteins (e.g., comprising chimeric human / non-human MHC II and polypeptides). In some aspects, the animal does not express endogenous MHC I and / or MHC II polypeptides, e.g., functional endogenous MHC I and / or MHC II polypeptides, on its cell surface. In some embodiments, the only MHC I and / or MHC II molecules expressed on the cell surface of the animal are chimeric MHC I and / or MHC II molecules.

[0171] Genetically modified non-human animals comprising nucleic acid sequences in their genomes, e.g., at endogenous loci, encoding human / non-human MHC I polypeptides, are disclosed in U.S. Patent Nos. 9,615,550 and 9,591,835, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals comprising nucleic acid sequences in their genomes, e.g., at endogenous loci, encoding humanized, e.g., chimeric, human / non-human MHC II polypeptides, are disclosed in U.S. Patent Nos. 8,847,005 and 9,043,966, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals comprising in their genomes, e.g., at endogenous loci, nucleic acid sequences encoding chimeric human / non-human MHC I polypeptides, and comprising in their genomes, e.g., at endogenous loci, nucleic acid sequences encoding humanized, e.g., chimeric human / non-human MHC II polypeptides, are disclosed in U.S. Patent Publication No. 20140245467, which is incorporated herein by reference in its entirety.

[0172] In various embodiments, disclosed herein is a genetically modified non-human animal that comprises in its genome, e.g., at one or more endogenous MHC loci: a first nucleic acid sequence encoding a chimeric human / non-human MHC I polypeptide, wherein the human portion of the chimeric MHC I polypeptide comprises an extracellular portion (or portion thereof, e.g., one or more extracellular domains) of a human MHC I polypeptide; a second nucleic acid sequence encoding a chimeric human / non-human MHC II α polypeptide, wherein the human portion of the chimeric MHC II α polypeptide comprises an extracellular portion (or portion thereof, e.g., one or more extracellular domains) of a human MHC II α polypeptide; and / or a third nucleic acid sequence encoding a chimeric human / non-human MHC II β polypeptide, wherein the human portion of the chimeric MHC II β polypeptide comprises an extracellular portion (or portion thereof, e.g., one or more extracellular domains) of a human chimeric MHC II β polypeptide, wherein the non-human animal is capable of expressing functional chimeric human / non-human MHC I and MHC I polypeptides from the endogenous non-human loci. The non-human animal expresses a non-human MHC II protein. In one embodiment, the first, second, and / or third nucleic acid sequence is located at an endogenous non-human MHC I, MHC IIα, and MHC IIβ locus, respectively. In one embodiment, the non-human animal is a mouse, and the first, second, and / or third nucleic acid sequence is located at an endogenous mouse MHC locus on mouse chromosome 17. In one embodiment, the first nucleic acid sequence is located at an endogenous non-human MHC I locus. In one embodiment, the second nucleic acid sequence is located at an endogenous non-human MHC IIα locus. In one embodiment, the third nucleic acid sequence is located at an endogenous non-human MHC IIβ locus.

[0173] In one embodiment, the non-human animal expresses only chimeric human / non-human MHC I polypeptides, MHC IIα polypeptides, and / or MHCIIβ polypeptides, and does not express endogenous non-human MHC polypeptides (e.g., functional endogenous MHC I polypeptides, IIα polypeptides, and / or IIβ polypeptides) from endogenous non-human MHC loci. In one embodiment, the animal described herein expresses a functional chimeric MHC I and a functional chimeric MHC II on the surface of its cells, such as antigen-presenting cells. In one embodiment, the MHC I and MHC II expressed by the animal on the cell surface are only chimeric MHC I and chimeric MHC II, and the animal does not express any endogenous MHC I and MHC II on the cell surface.

[0174] In one embodiment, the chimeric human / non-human MHC I polypeptide comprises, in its human portion, for example, the peptide-binding cleft of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises the extracellular portion of human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises the extracellular domain of the alpha chain of human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises the alpha 1 and alpha 2 domains of human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises the alpha 1, alpha 2, and alpha 3 domains of human MHC I.

[0175] In one aspect, the human portion of the chimeric MHC IIα polypeptide and / or the human portion of the chimeric MHC IIβ polypeptide comprises the peptide-binding domain of a human MHC IIα polypeptide and / or a human MHC IIβ polypeptide, respectively. In one aspect, the human portion of the chimeric MHC IIα and / or β polypeptide comprises the extracellular portion of a human MHC IIα and / or β polypeptide, respectively. In one embodiment, the human portion of the chimeric MHC IIα polypeptide comprises the α1 domain of a human MHC IIα polypeptide, and in another embodiment, the human portion of the chimeric MHC IIα polypeptide comprises the α1 domain and the α2 domain of a human MHC IIα polypeptide. In a further embodiment, the human portion of the chimeric MHC IIβ polypeptide comprises the β1 domain of a human MHC II polypeptide, and in another embodiment, the human portion of the chimeric MHC IIβ polypeptide comprises the β1 domain and the β2 domain of a human MHC IIβ polypeptide.

[0176] In some embodiments, human or humanized MHC I polypeptides can be derived from functional human HLA molecules encoded by any of the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci. Human or humanized MHC II polypeptides can be derived from functional human HLA molecules encoded by any of the HLA-DP, -DQ, and -DR loci. A list of commonly used HLA antigens and alleles is provided in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), which is incorporated herein by reference. Shankarkumar et al. also provide a brief description of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) "The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1 / 3 / 4 / 5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens," Tissue Antigens 73:95-170, and a recent update by Marsh et al. (2010) "Nomenclature for factors of the HLA system," 2010, Tissue Antigens 75:291-455, both of which are incorporated herein by reference. In some embodiments, the MHC I or MHC II polypeptide can be derived from any functional human HLA-A, B, C, DR, or DQ molecule. Thus, human or humanized MHC I and / or II polypeptides can be derived from any functional human HLA molecule described therein, hi some embodiments, all MHC I and MHC II polypeptides expressed on the cell surface include a portion derived from a human HLA molecule.

[0177] Of particular interest are human HLA molecules, specific polymorphic HLA alleles, which are known to be associated with numerous human diseases, such as human autoimmune diseases. Indeed, certain polymorphisms in HLA loci have been found to correlate with the development of rheumatoid arthritis, type I diabetes, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune disorders. For example, Wong and Wen (2004) What can the HLA transgenic mouse tell us about autoimmune diabetes?, Diabetologia 47:1476-87; Taneja and David (1998) HLA Transgenic Mice as Humanized Mouse Models of Disease and Immunity, J.Clin. Invest. 101:921-26; Bakker et al. (2006), A high-resolution See HLA and SNP haplotype map for disease association studies in the extended human MHC, Nature Genetics 38:1166-72 and Supplementary Information; and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases, Proc. Natl. Acad. Sci. USA 106:18680-85. Thus, human or humanized MHC I and / or II polypeptides can be derived from human HLA molecules known to be associated with particular diseases, eg, autoimmune diseases.

[0178] In one particular aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a particular embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., and HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., an HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is commonly used in North American populations. While this example cites U.S. Patent Nos. 9,615,550 and 10,154,658 to describe this particular HLA sequence, any suitable HLA-A sequence is encompassed herein, including, for example, polymorphic variants of HLA-A2 expressed in human populations, sequences with one or more conservative or non-conservative amino acid modifications, and nucleic acid sequences that differ from the sequences described herein due to the degeneracy of the genetic code.

[0179] In another specific embodiment, the human portion of the chimeric MHC I polypeptide is derived from a human MHC I selected from HLA-B and HLA-C. In one embodiment, it is derived from HLA-B, e.g., HLA-B27. In another embodiment, it is derived from HLA-A3, -B7, -Cw6, etc.

[0180] In one specific embodiment, the human portions of the humanized MHC II α and β polypeptides described herein are derived from human HLA-DR, e.g., HLA-DR2. Typically, the HLA-DR α chain is monomorphic; e.g., the α chain of the HLA-DR complex is encoded by an HLA-DRA gene (e.g., the HLA-DR *01 gene). On the other hand, the HLA-DR β chain is polymorphic. Thus, HLA-DR2 comprises an α chain encoded by an HLA-DRA gene and a β chain encoded by an HLA-DR1β*1501 gene. While the present examples cite U.S. Patent Nos. 8,847,005 and 9,043,996 to describe these specific HLA sequences, any suitable HLA-DR sequence is encompassed herein, including, for example, polymorphic variants expressed in the human population, sequences with one or more conservative or non-conservative amino acid modifications, and nucleic acid sequences that differ from the sequences described herein due to the degeneracy of the genetic code.

[0181] The human portion of the chimeric MHC II α and / or β polypeptide can be encoded by the nucleic acid sequence of an HLA allele known to be associated with a common human disease. Such HLA alleles include, but are not limited to, HLA-DRB1*0401, -DRB1*0301, -DQA1*0501, -DQB1*0201, DRB1*1501, -DRB1*1502, -DQB1*0602, -DQA1*0102, -DQA1*0201, -DQB1*0202, -DQA1*0501, and combinations thereof. For a summary of HLA allele / disease associations, see Bakker et al. (2006), supra, which is incorporated herein by reference in its entirety.

[0182] In one embodiment, the non-human portion of the chimeric human / non-human MHC I, MHC IIα, and / or MHC IIβ polypeptide comprises the transmembrane and / or cytoplasmic domain of an endogenous non-human (e.g., rodent, e.g., mouse, rat, etc.) MHC I, MHC IIα, and / or MHC IIβ polypeptide, respectively. Thus, the non-human portion of the chimeric human / non-human MHC I polypeptide may comprise the transmembrane and / or cytoplasmic domain of an endogenous non-human MHC I polypeptide. The non-human portion of the chimeric MHC IIα polypeptide may comprise the transmembrane and / or cytoplasmic domain of an endogenous non-human MHC IIα polypeptide. The non-human portion of the chimeric human / non-human MHC IIβ polypeptide may comprise the transmembrane and / or cytoplasmic domain of an endogenous non-human MHC IIβ polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human portion of the chimeric MHC I polypeptide is derived from a mouse H-2K protein. In one aspect, the animal is a mouse, and the non-human portion of the chimeric MHC IIα and β polypeptides is derived from a mouse H-2E protein. Thus, the non-human portion of the chimeric MHC II polypeptide may comprise a transmembrane domain and a cytoplasmic domain derived from a mouse H-2K, and the non-human portion of the chimeric MHC IIα and β polypeptide may comprise a transmembrane domain and a cytoplasmic domain derived from a mouse H-2E protein. While U.S. Patent Nos. 9,615,550 and 10,154,658, cited in the Examples, contemplate specific H-2K and H-2E sequences, any suitable sequence, e.g., polymorphic variants, conservative / non-conservative amino acid substitutions, etc., is encompassed herein. In one aspect, the non-human animal is a mouse, and the mouse does not express a functional endogenous MHC polypeptide from its H-2D locus. In some embodiments, the mouse is engineered to lack all or part of the endogenous H-2D locus. In other embodiments, the mouse does not express any functional endogenous mouse MHC I and MHC II on the cell surface.

[0183] A chimeric human / non-human polypeptide may comprise a human or non-human leader (signal) sequence. In one embodiment, a chimeric MHC I polypeptide comprises the non-human leader sequence of an endogenous MHC I polypeptide. In one embodiment, a chimeric MHC IIα polypeptide comprises the non-human leader sequence of an endogenous MHC IIα polypeptide. In one embodiment, a chimeric MHC IIβ polypeptide comprises the non-human leader sequence of an endogenous MHC IIβ polypeptide. In alternative embodiments, the chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC II polypeptide comprise the non-human leader sequence of an MHC I polypeptide, MHC IIα polypeptide, and / or MHC II polypeptide, respectively, from another non-human animal, e.g., another rodent or another mouse strain. Thus, a nucleic acid sequence encoding a chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide may be operably linked to a nucleic acid sequence encoding a non-human MHC I, MHC IIα, and / or MHC IIβ leader sequence, respectively. In yet another embodiment, the chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide comprises a human leader sequence for a human MHC I polypeptide, a human MHC IIα polypeptide, and / or a human MHC IIβ polypeptide, respectively (e.g., the leader sequence for human HLA-A2, human HLA-DRα, and / or human HLA-DR1*1501, respectively).

[0184] The human portion of a chimeric human / non-human MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide can comprise the complete or substantially complete extracellular domain of a human MHC I polypeptide, human MHC IIα polypeptide, and / or human MHC IIβ polypeptide, respectively. Thus, the human portion can comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g., 95% or more, of the amino acids encoding the extracellular domain of a human MHC I polypeptide, human MHC IIα polypeptide, and / or human MHC IIβ polypeptide (e.g., human HLA-A2, human HLA-DR, and / or human HLA-DR1*1501). In one example, the substantially complete extracellular domain of a human MHC I polypeptide, human MHC IIα polypeptide, and / or human MHC IIβ polypeptide lacks a human leader sequence. In another example, the chimeric human / non-human MHC I polypeptide, chimeric human / non-human MHC IIα polypeptide, and / or chimeric human / non-human MHC IIβ polypeptide comprises a human leader sequence.

[0185] Furthermore, the chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide can be operably linked to (e.g., expressed under the regulatory control of) an endogenous non-human promoter and regulatory elements, e.g., mouse MHC I regulatory elements, MHC IIα regulatory elements, and / or MHC IIβ regulatory elements, respectively. Such an arrangement facilitates appropriate expression of the chimeric MHC I polypeptide and / or MHC II polypeptide in the non-human animal, e.g., during an immune response in the non-human animal.

[0186] In further embodiments, the non-human animal of the invention, e.g., a rodent, e.g., a rat or mouse, comprises a nucleic acid sequence encoding human or humanized β2 microglobulin (e.g., at the endogenous β2 microglobulin locus). The β2 microglobulin or light chain (also abbreviated as "β2M") of the MHC class I complex is a small (12 kDa), non-glycosylated protein that functions primarily to stabilize the MHC Iα chain. The generation of human or humanized β2 microglobulin animals is described in detail in U.S. Patent Publication No. 9,615,550, which is incorporated herein by reference.

[0187] A nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide may contain nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may contain nucleic acid residues encoding the amino acid sequence set forth in amino acids 21-119 of the human β2 microglobulin protein (i.e., amino acid residues corresponding to mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may contain nucleic acid residues encoding the amino acid sequence set forth in amino acids 23-115 of the human β2 microglobulin protein, e.g., the amino acid sequence set forth in amino acids 23-119 of the human β2 microglobulin protein. The nucleic acid and amino acid sequences of human β2 microglobulin are described in Gussow et al., supra, and are incorporated herein by reference.

[0188] Thus, a human or humanized β2 microglobulin polypeptide can comprise the amino acid sequence set forth in amino acids 23 to 115 of a human β2 microglobulin polypeptide, such as the amino acid sequence set forth in amino acids 23 to 119 of a human β2 microglobulin polypeptide, such as the amino acid sequence set forth in amino acids 21 to 119 of a human β2 microglobulin polypeptide. Alternatively, human β2 microglobulin can comprise amino acids 1 to 119 of a human β2 microglobulin polypeptide.

[0189] In some embodiments, the nucleotide sequence encoding human or humanized β2 microglobulin comprises the nucleotide sequence represented in exons 2 through 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises the nucleotide sequence represented in exons 2, 3, and 4 of the human β2 microglobulin gene. In this embodiment, the nucleotide sequences represented in exons 2, 3, and 4 are operably linked to allow normal transcription and translation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exons 2 through 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to approximately 267 bp after exons 2 through 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises approximately 2.8 kb of the human β2 microglobulin gene.

[0190] Thus, a human or humanized β2 microglobulin polypeptide can be encoded by a nucleotide sequence comprising the nucleotide sequence set forth in exons 2 through 4 of human β2 microglobulin, e.g., the nucleotide sequence corresponding to exons 2 through 4 of the human β2 microglobulin gene. Alternatively, the polypeptide can be encoded by a nucleotide sequence comprising the nucleotide sequence set forth in exons 2, 3, and 4 of the human β2 microglobulin gene. In a specific embodiment, a human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to approximately 267 bp following exons 2 through 4 of the human β2 microglobulin gene. In another specific embodiment, a human or humanized polypeptide is encoded by a nucleotide sequence comprising approximately 2.8 kb of the human β2 microglobulin gene. Because exon 4 of the β2 microglobulin gene contains the 5' untranslated region, a human or humanized polypeptide can be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.

[0191] While specific nucleic acid and amino acid sequences for generating genetically engineered animals are described herein, it will be understood by those skilled in the art that sequences with one or more conservative or non-conservative amino acid substitutions, or sequences that differ from those described herein due to the degeneracy of the genetic code, are also provided.

[0192] Thus, non-human animals are provided that express a human β2 microglobulin sequence, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence. In certain embodiments, the β2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence described herein. In one embodiment, the human β2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence comprises one or more non-conservative substitutions.

[0193] Also provided are non-human animals in which the nucleotide sequence encoding the human or humanized β2 microglobulin protein also comprises the nucleotide sequence set forth in exon 1 of the non-human β2 microglobulin gene. Thus, in certain embodiments, the non-human animal comprises in its genome a nucleotide sequence encoding human or humanized β2 microglobulin, the nucleotide sequence comprising exon 1 of the non-human β2 microglobulin and exons 2, 3, and 4 of the human β2 microglobulin gene. Thus, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of the non-human β2 microglobulin gene and exons 2, 3, and 4 of the human β2 microglobulin gene (e.g., exons 2 and 3 of the human β2 microglobulin gene).

[0194] In one embodiment, the non-human animal (e.g., a rodent, e.g., a mouse) of the invention further comprises a nucleic acid sequence encoding a human or humanized MHC I protein in addition to a nucleotide sequence encoding the chimeric CD8 protein, wherein the chimeric CD8 protein expressed on the surface of a T cell of the animal is capable of associating, binding, and / or interacting with a human or humanized MHC I expressed on the surface of a second cell, e.g., an antigen-presenting cell. In one embodiment, the MHC I protein comprises the extracellular domain of a human MHC I polypeptide. In one embodiment, the animal further comprises a human or humanized β2 microglobulin polypeptide. Exemplary genetically modified animals expressing human or humanized MHC I polypeptides and / or β2 microglobulin polypeptides are described in U.S. Patent Nos. 9,615,550 and 9,591,835, both of which are incorporated herein by reference in their entireties. Thus, in one embodiment, an animal comprising a chimeric CD8 protein described herein may further comprise a humanized MHC I complex, wherein the humanized MHC I complex comprises: (1) a humanized MHC I polypeptide, e.g., the humanized MHC I polypeptide comprises a human MHC I extracellular domain and the transmembrane and cytoplasmic domains of an endogenous (e.g., murine) MHC I, e.g., the humanized MHC I comprises the α1, α2, and α3 domains of a human MHC I polypeptide; and (2) a human or humanized β2 microglobulin polypeptide (e.g., the animal comprises in its genome the nucleotide sequence set forth in exons 2, 3, and 4 of human β2 microglobulin). In one aspect, both the humanized MHC I polypeptide and the human or humanized β2 microglobulin polypeptide are encoded by nucleotide sequences located in the endogenous MHC I and β2 microglobulin loci, respectively; and in one aspect, the animal does not express functional endogenous MHC I and β2 microglobulin polypeptides. Thus, the MHC I expressed by the animal can be a chimeric human / non-human, eg, human / rodent (eg, human / mouse) MHC I polypeptide.The human portion of the chimeric MHC I polypeptide may be derived from a human HLA class I protein selected from the group consisting of HLA-A, HLA-B, and HLA-C, e.g., HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecule present in the human population. In embodiments where the animal is a mouse, the non-human (i.e., mouse) portion of the chimeric MHC I polypeptide may be derived from a mouse MHC I protein selected from H-2D, H-2K, and H-2L.

[0195] In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) of the invention further comprises a nucleotide sequence encoding a human or humanized MHC II protein, and the chimeric CD4 protein expressed on the surface of the animal's T cells is capable of interacting with a human or humanized MHC II expressed on the surface of a second cell, e.g., an antigen-presenting cell. In one embodiment, the MHC II protein comprises the extracellular domain of a human MHC IIα polypeptide and the extracellular domain of a human MHC IIβ polypeptide. Exemplary genetically modified animals expressing human or humanized MHC II polypeptides are described in U.S. Patent No. 8,847,005, issued September 30, 2014, and U.S. Patent No. 9,043,996, each of which is incorporated by reference in its entirety. Thus, in one embodiment, an animal comprising a chimeric CD4 protein described herein further comprises a humanized MHC II protein, the humanized MHC II protein being (1) a humanized MHC IIα polypeptide comprising a human MHC IIα ectodomain and an endogenous, e.g., mouse, MHC II transmembrane and cytoplasmic domain, the human MHC IIα ectodomain comprising the α1 and α2 domains of human MHC IIα, and (2) a humanized MHC IIβ polypeptide comprising a human MHC IIβ ectodomain and an endogenous, e.g., mouse, MHC II transmembrane and cytoplasmic domain, the human MHC IIβ ectodomain comprising the β1 and β2 domains of human MHC IIβ. In one aspect, both the humanized MHC IIα and β polypeptides are encoded by nucleic acid sequences located in endogenous MHC II and loci, respectively; in one aspect, the animal does not express functional endogenous MHC IIα and β polypeptides. Thus, the MHC II expressed by the animal can be a chimeric human / non-human, e.g., human / rodent (e.g., human / mouse) MHC II protein. The human portion of the chimeric MHC II protein can be derived from a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g., HLA-DR4, HLA-DR2, HLA-DQ2.5, HLA-DQ8, or any other HLA class II molecule present in the human population.In embodiments in which the animal is a mouse, the non-human (ie, mouse) portion of the chimeric MHC II polypeptide may be derived from a mouse MHC II protein selected from H-2E and H-2A.

[0196] Various other embodiments of genetically modified non-human animals, e.g., rodents, e.g., rats or mice, will be apparent to those skilled in the art from the present disclosure and from the disclosures of U.S. Pat. Nos. 8,847,005, 9,043,996, 9,591,835, 9,615,550, and 10,154,658, which are incorporated herein by reference.

[0197] In various embodiments, the genetically modified non-human animals described herein generate cells, e.g., APCs, with human or humanized MHC I and II on the cell surface, resulting in human-like presentation of peptides as epitopes for T cells. Because substantially all of the components of the complex are human or humanized, the genetically modified non-human animals of the present invention can be used to test the function of the human immune system in humanized animals, identify antigens and antigen epitopes (e.g., T cell epitopes, e.g., unique human cancer epitopes) that elicit an immune response, for example, for use in vaccine development, evaluation of vaccine candidates and other vaccine strategies; to study human autoimmunity, human infectious diseases, and to devise better treatment strategies based on human MHC expression.

[0198] In some embodiments, the mouse described herein: (I) (A) At the endogenous TCRγ locus replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; Replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments, and comprising the replacement of all TCR gamma constant region gene sequences with a complete repertoire of human TCR gamma constant region gene sequences; (B) At the endogenous TCRδ locus replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments, and comprising a replacement of the endogenous TCR delta constant region gene sequence with a human TCR delta constant region gene sequence; (C) At the endogenous TCRα locus Replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) At the endogenous TCRβ locus replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; replacement of all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments; comprising the replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 co-receptor comprising the D1, D2, and D3 domains of a human CD4 polypeptide operably linked to the D4, transmembrane domain, and cytoplasmic domain of a mouse CD4 polypeptide; (F) a second nucleotide sequence encoding a chimeric human / mouse CD8 α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8 β polypeptide, the chimeric human / mouse CD8α polypeptide comprises second and third nucleotide sequences, wherein the chimeric human / mouse CD8 polypeptide comprises an IgV-like domain of a human CD8 polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8α polypeptide, and the chimeric human / mouse CD8 polypeptide comprises an IgV-like domain of a human CD8 polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8β polypeptide; (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC IIα polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC IIβ polypeptide, the chimeric human / mouse MHC IIα polypeptide comprises the α1 and α2 domains of a human HLA class IIα polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIα polypeptide, and the chimeric human / mouse MHC IIβ polypeptide comprises the β1 and β2 domains of a human HLA class IIβ polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIβ polypeptide, a first nucleic acid sequence and a second nucleic acid sequence; (H) a third nucleic acid sequence encoding a chimeric human / mouse MHC I polypeptide comprising the α1 domain, α2 domain, and α3 domain of a human HLA class I polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC class I polypeptide; (I) a germline and CD3 polynucleotide encoding a human or humanized β2 microglobulin polypeptide, the polynucleotide comprising the nucleotide sequence shown in exon 1 of the mouse β2 microglobulin gene operably linked to the nucleotide sequence shown in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene; - Contains T cells, (II) a CD3 + T cells and CD3s expressing on their surface a functional human or humanized TCRβ comprising a human or humanized TCRα polypeptide and a human or humanized TCRβ polypeptide. + and optionally, the mouse further comprises: (e) chimeric human / mouse CD4 coreceptor; (f) a chimeric CD8 co-receptor comprising a chimeric human / mouse CD8α polypeptide and a chimeric human / mouse CD8β polypeptide; (h) a chimeric MHC II complex comprising a chimeric human / mouse MHC IIα polypeptide and a chimeric human / mouse MHC IIβ polypeptide, wherein the chimeric MHC II complex is capable of binding a chimeric human / mouse CD4 co-receptor; (i) a chimeric human / mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding to a chimeric CD8 co-receptor; and (j) expressing a human or humanized β2 microglobulin polypeptide;

[0199] In some embodiments, the non-human animals described herein comprise two copies of one or more modified loci described herein. In some embodiments, the non-human animals described herein comprise two copies of an unrearranged human or humanized TCRγ locus, two copies of an unrearranged human or humanized TCRδ locus, two copies of an unrearranged human or humanized TCRα locus, two copies of an unrearranged human or humanized TCRβ locus, two copies of a human or humanized CD4 locus, two copies of a human or humanized CD8α locus, two copies of a human or humanized CD8β locus, two copies of a human or humanized MHC I locus, and / or two copies of a human or humanized MHC IIα and / or MHC IIβ locus. Thus, the non-human animals are homozygous for one or more unrearranged human or humanized TCRγ, TCRδ, TCRα, and / or TCRβ loci, one or more human or humanized co-receptor loci, and / or one or more MHC loci. In some embodiments of the invention, the non-human animal comprises one copy of an unrearranged human or humanized TCR gamma locus, one copy of an unrearranged human or humanized TCR delta variable locus, one copy of an unrearranged human or humanized TCR alpha variable locus, one copy of an unrearranged human or humanized TCR beta variable locus, one copy of a human or humanized CD4 locus, one copy of a human or humanized CD8 alpha locus, one copy of a human or humanized CD8 beta locus, one copy of a human or humanized MHC I locus, and / or one copy of a human or humanized MHC II alpha and / or MHC II beta locus. Thus, the non-human animal may be heterozygous for an unrearranged human or humanized TCR gamma, TCR delta, TCR alpha, and / or TCR beta locus, one or more human or humanized co-receptor loci, and / or one or more human or humanized MHC loci.In some embodiments, the non-human animal (e.g., a mouse) is heterozygous or homozygous for an unrearranged human TCR gamma locus, e.g., the non-human animal comprises an unrearranged human TCR Vγ segment and an unrearranged human Jγ segment operably linked to a human TCR Cγ gene. In some embodiments, the non-human animal (e.g., a mouse) is heterozygous or homozygous for an unrearranged human TCR delta locus, e.g., the non-human animal comprises an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human Jδ segment operably linked to a human TCR Cδ gene.

[0200] In some embodiments, the non-human animals described herein that are heterozygous or homozygous for human TRD and TRG loci, and humanized TRA, TRB, MHC I, MHC II, CD4, CD8, and β2-microglobulin loci, comprise a population of CD45+CD3+ T cells in their spleen, thymus, mesenteric lymph nodes (MLN), skin, intestinal mucosa CD45, and / or intraepithelial lymphocytes (IEL) isolated from their colon or small intestine, wherein a percentage of the population of CD45+CD3+ T cells express a human γ / δ TCR. In some embodiments, the percentage of spleen, thymus, MLN, and / or IEL CD45+CD3+ T cells expressing human γ / δ TCR in the γ and / or δ TCR mouse embodiments described herein is comparable to the percentage of CD45+CD3+ T cells expressing murine γ / δ TCR in wild-type mice (e.g., not significantly different, any differences are not statistically significant, within 10 percentage points of each other, etc.). In some embodiments, the percentage of spleen, thymus, MLN, and / or IEL CD45+CD3+ T cells expressing human γ / δ TCR in the γ and / or δ TCR mouse embodiments described herein is greater (e.g., 1.5- to 3-fold greater) than the percentage of CD45+CD3+ T cells expressing murine γ / δ TCR in wild-type mice.

[0201] The genetically modified non-human animals of the present invention can be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets and rhesus monkeys). For non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are used to generate non-human animals containing this genetic modification. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent cells) and using nuclear transfer to transfer the modified genome into suitable cells, such as oocytes, and fertilizing the modified cells (e.g., modified oocytes) in non-human animals under appropriate conditions to form embryos.

[0202] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is, for example, a small mammal of the superfamily Rodentia or Murine superfamily. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the Murine superfamily. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, maned mice), Nesomyidae (climbing mice, rock mice, with-tailed rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In certain embodiments, the genetically modified rodent is selected from pure-breed mice or rats (Muridae), gerbils, spiny mice, and maned mice. In one embodiment, the genetically modified mouse is from a member of the Murine family. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0203] In certain embodiments, the non-human animal is a mouse of a C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836; see also Auerbach et al (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In one embodiment, the genetically modified mouse is a mixture of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another embodiment, the rodent is a mixture of the aforementioned 129 strains, or a mixture of the aforementioned BL / 6 strains. In a specific embodiment, the mixed 129 strain is the 129S6 (129 / SvEvTac) strain. In another embodiment, the mouse is a BALB strain, such as a BALB / c strain. In yet another embodiment, the mouse is a mixture of a BALB strain and another of the aforementioned strains. The non-human animals provided herein can be mice derived from any combination of the aforementioned strains.

[0204] In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0205] In some aspects of the invention, non-human animals comprising unrearranged human or humanized TCRγ, TCRδ, TCRα, and / or TCRβ loci (each comprising unrearranged human TCRγ, TCRδ, TCRα, and / or TCRβ variable V, (D), and segments) retain endogenous non-human TCRγ, TCRδ, TCRα, and / or TCRβ gene segments or loci. In one embodiment, the retained endogenous non-human TCRγ, TCRδ, TCRα, and / or TCRβ segments are functional gene segments, e.g., rearranged (or rearranged) in T cells and found at the same locus as human TCRγ, TCRδ, TCRα, and / or TCRβ segments, respectively, according to the 12 / 23 rule of recombination. See Olaru A., supra. In one embodiment, the endogenous non-human TCRγ, TCRδ, TCRα, and / or TCRβ are non-functional gene segments or loci. In one embodiment, the non-functional locus is an inactivated locus, e.g., an inverted locus (e.g., the coding nucleic acid sequence of the variable locus is in an inverted orientation relative to the constant region sequence, such that successful rearrangement using variable region segments from the inverted locus is not possible). In one embodiment, the humanized TCR gamma, TCR delta, TCR alpha, and / or TCR beta segment or variable locus is located between an endogenous non-human TCR gamma, TCR delta, TCR alpha, and / or TCR beta variable locus and an endogenous non-human TCR gamma, TCR delta, TCR alpha, and / or TCR beta constant locus, respectively. In one embodiment, the humanized TCR gamma, TCR delta, TCR alpha, and / or TCR beta locus and / or variable locus is located between an endogenous non-human TCR gamma, TCR delta, TCR alpha, and / or TCR beta variable locus and a human TCR gamma, TCR delta, TCR alpha, and / or TCR beta constant region locus, respectively.

[0206] In various embodiments of the invention, unrearranged human or humanized TCR variable loci (e.g., TCRα TCRβ, TCRγ, and / or TCRδ variable loci) are present in the germ cells of a non-human animal (e.g., a rodent, e.g., a mouse or rat), e.g., the non-human animal is comprised of germ cells (sperm and oocytes) having loci modified as described herein. In various embodiments, replacement of TCR V(D)J segments with unrearranged human TCR V(D)J segments (e.g., Vα and Jα; Vβ and Dβ and Jβ; Vδ and Dδ and Jδ; Vγ and Jγ segments) is at an endogenous non-human TCR variable locus (or loci), and the unrearranged human V and J, and / or V and D and J segments are operably linked to non-human TCR constant region gene sequences.

[0207] In one embodiment, a non-human animal (e.g., a rodent, e.g., a mouse or rat) comprising a human or humanized TCR gamma and / or TCR delta locus described herein, and optionally comprising a TCR alpha and / or TCR locus, expresses a humanized T cell receptor on the surface of the T cell comprising a human variable region and a non-human (e.g., rodent, e.g., a mouse or rat) constant domain. In some embodiments, the non-human animal is capable of expressing a diverse repertoire of humanized T cell receptors that recognize a variety of presented antigens.

[0208] In addition to genetically engineered non-human animals, non-human embryonic stem (ES) cell lines or germ cell lines, and embryos (e.g., rodent, e.g., mouse or rat embryos) comprising and / or derived from ES cells are also provided. The ES cells, germ cells, and / or embryos described herein comprise genetically modified loci described herein, e.g., human or humanized TCRG and / or TCRD loci, and optionally human or humanized TCRA, TCRB, CD4, CD8α, CD8β, MHC I and / or MHC IIα and / or MHC IIβ loci.

[0209] Also provided herein are tissues derived from non-human animals (e.g., rodents, e.g., mice or rats) that contain cells that express human TCRγ and / or TCRδ proteins from human or humanized TCRG and / or TCRD loci, respectively.

[0210] In some embodiments, a method for producing human TCRγ and / or human TCRδ proteins is provided, comprising expressing the human TCRγ and / or human TCRδ proteins in a single cell from a nucleotide construct described herein. In one embodiment, the nucleotide construct is a viral vector, and in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells (e.g., PERC.6™ cells) that express the viral nucleic acid sequence.

[0211] In one aspect, a cell expressing a human TCR gamma and / or human TCR delta protein is provided. In one embodiment, the cell comprises an expression vector comprising a human TCR gamma and / or human TCR delta protein described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells (e.g., PERC.6™ cells) that express a viral nucleic acid sequence.

[0212] Also provided are human TCR gamma and / or human TCR delta proteins produced by the non-human animals described herein. Thus, the human TCR proteins comprise human complementarity-determining regions (i.e., human CDRs 1, 2, and 3) in their variable domains and human constant regions. Also provided are nucleic acids encoding the human TCR variable domains produced by the non-human animals described herein.

[0213] Also provided are non-human cells isolated from the non-human animals described herein. In one embodiment, the cells are ES cells. In one embodiment, the cells are T cells, e.g., gamma / delta T cells. Also provided are non-human cells that express TCR proteins, including human TCR gamma and / or human TCR delta proteins.

[0214] Also provided is a non-human cell comprising a chromosome or fragment thereof of a non-human animal described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal described herein. In one embodiment, the non-human cell comprises a chromosome or fragment thereof as a result of nuclear transfer.

[0215] In one aspect, hybridomas or quadromas are provided and are derived from cells of a non-human animal described herein. In one embodiment, the non-human animal is a mouse or a rat. Genetically modified non-human animals are produced that mount substantially humanized T cell immune responses.

[0216] Also provided are methods for producing the genetically engineered non-human animals (e.g., genetically engineered rodents, e.g., mice or rats) described herein. Generally, the methods involve inserting into the non-human animal an unrearranged T cell receptor (TCR) gamma variable locus comprising at least one human Vγ segment and at least one human Jγ segment operably linked to a human or non-human TCR gamma constant region gene sequence, and / or an unrearranged TCR delta variable locus comprising at least one human Vδ segment, at least one human Dδ segment, and at least one human Jδ segment operably linked to a human or non-human TCR delta constant region gene sequence.In some embodiments, the method optionally includes: (a) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment operably linked to a non-human TCR α constant region gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to a non-human TCR β constant region gene sequence; (b) inserting into the genome of the non-human animal a first nucleotide sequence encoding a human T cell co-receptor polypeptide, a second nucleotide sequence encoding a second chimeric human / non-human T cell co-receptor polypeptide, and / or a third chimeric human / non-human T cell co-receptor polypeptide. The method may further include any one of, or a combination of, (a) introducing a third nucleotide sequence encoding a chimeric T cell co-receptor polypeptide, wherein the non-human portion of each chimeric T cell co-receptor polypeptide comprises at least the transmembrane and cytoplasmic domains of a non-human T cell co-receptor and the human portion of each chimeric polypeptide comprises the extracellular portion (or a portion thereof) of a human T cell co-receptor; (b) inserting into the genome a first nucleic acid sequence encoding the first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding the second chimeric human / non-human MHC polypeptide, and / or a third nucleic acid sequence encoding a third chimeric human / non-human MHC polypeptide; or (c) adding to the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide.In some embodiments, the method comprises: (a) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment operably linked to a non-human TCR α constant region gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to a non-human TCR β constant region gene sequence; and (b) inserting into the genome of the non-human animal a first nucleotide sequence encoding a human T cell co-receptor polypeptide, a second nucleotide sequence encoding a second chimeric human / non-human T cell co-receptor polypeptide, and / or a third chimeric human / non-human T cell co-receptor polypeptide. The method may further include any one or combination of: (a) introducing a third nucleotide sequence encoding a chimeric T cell co-receptor polypeptide, wherein the non-human portion of each chimeric T cell co-receptor polypeptide comprises at least the transmembrane and cytoplasmic domains of the non-human T cell co-receptor and the human portion of each chimeric polypeptide comprises the extracellular portion (or a portion thereof) of the human T cell co-receptor; (b) inserting into the genome a first nucleic acid sequence encoding the first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding the second chimeric human / non-human MHC polypeptide, and / or a third nucleic acid sequence encoding the third chimeric human / non-human MHC polypeptide; and (c) adding to the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the introducing, inserting, and / or positioning step comprises substituting sequences that target the extracellular domain of a T cell co-receptor, the variable domain of a TCR, and optionally a TCR constant region gene sequence, the extracellular domain of an MHC polypeptide, or a portion of β2 microglobulin for the extracellular domain of a human T cell co-receptor, the human TCR variable domain, and optionally a human TCR constant domain, the human MHC extracellular domain, and / or a portion of human β2 microglobulin, respectively.

[0217] In other embodiments, the introduction, insertion, placement, and / or addition may involve breeding, e.g., mating, of animals of the same species. In other embodiments, the introduction, insertion, placement, and / or addition involves sequential homologous recombination in ES cells. In some embodiments, the ES cells are derived from a non-human animal that has been genetically modified to contain one or more, but not all, of the desired genetic modifications, and homologous recombination in such ES cells completes the genetic modification. In other embodiments, the introduction, insertion, placement, and / or addition may involve a combination of breeding and homologous recombination in ES cells, e.g., mating an animal with another (or more) animal of the same species, where some or all of the animal may be generated from the genetically modified ES cells via a single homologous recombination or sequential homologous recombination events, and where some ES cells may be isolated from a non-human animal containing one or more of the genetic modifications disclosed herein.

[0218] In some embodiments, the method utilizes a targeting construct generated using VELOCIMOUSE® technology, as described in the Examples, to introduce the construct into ES cells, and introduce the targeted ES cell clones into mouse embryos using VELOCIMOUSE® technology. The targeting construct may be composed of 5' and / or 3' homology arms that target the endogenous sequence to be replaced, an insertion sequence that replaces the endogenous sequence, and one or more selection cassettes. A selection cassette is a nucleotide sequence inserted into the targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. Many suitable selection cassettes are known in the art. Generally, selection cassettes allow for positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). Additionally, the selection cassette may be flanked by recombination sites, which allow for deletion of the selection cassette upon treatment with a recombinase enzyme. Commonly used recombination sites are loxP and Frt, which are recognized by the Cre and Flp enzymes, respectively, but others are known in the art. The selection cassette can be located anywhere in the construct outside of the coding region. In one embodiment, the selection cassette is located at the 5' end of the human DNA fragment. In another embodiment, the selection cassette is located at the 3' end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment. In another embodiment, the selection cassette is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located at the junction of the human and mouse DNA fragments.

[0219] In some embodiments, a method for producing a genetically modified non-human animal results in an animal whose genome comprises an unrearranged humanized TCR locus (e.g., a human or unrearranged humanized TCRγ and / or TCRδ locus, and optionally a human or humanized TCRα, TCRβ locus). In one embodiment, a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) is provided that expresses a T cell receptor comprising a human variable region and a human or endogenous TCR constant domain on the surface of the T cell, the method comprising inserting, e.g., replacing, an endogenous non-human TCRγ variable locus into a first non-human animal, e.g., with an unrearranged humanized TCRγ variable locus comprising at least one human Vγ segment and at least one human Jγ segment, wherein the humanized TCRγ variable locus is a human or endogenous TCRγ constant region gene sequence. and in a second non-human animal, an endogenous non-human TCRδ variable locus is inserted, e.g., substituted, into the non-human endogenous TCRδ variable locus, an unrearranged humanized TCRδ variable locus comprising at least one human Vδ segment, one human Dδ segment, and one human Jδ segment, and the first and second non-human animals are bred to obtain a non-human animal that expresses a T cell receptor comprising a human or humanized gamma / δ TCR, in which the humanized TCRδ variable locus is operably linked to a human or endogenous TCRδ constant region gene sequence. In some embodiments, the methods for producing genetically modified non-human animals result in an animal whose genome comprises an unrearranged humanized TCR locus (e.g., an unrearranged humanized TCRα, TCRβ locus).In one embodiment, a method is provided for producing a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a T cell receptor comprising a human variable region and a human constant domain on the surface of the T cell, the method comprising inserting, e.g., replacing, into a first non-human animal an endogenous non-human TCR gamma locus (e.g., the first non-human animal has an unrearranged human TCR gamma locus in which at least one human V gamma segment and at least one human J gamma segment are operably linked to a human TCR gamma constant region gene sequence) (e.g., the method comprises inserting, e.g., replacing, into a first non-human animal an endogenous TCR gamma constant region gene sequence (e.g., an endogenous Trgc1 constant region gene sequence, an endogenous Trgc2 constant region gene sequence, an endogenous Trgc3 constant region gene sequence, and / or an endogenous Trgc4 constant region gene sequence)). In some embodiments, the method further comprises replacing the endogenous non-human TCR delta locus with a human TCR delta variable locus comprising at least one human V delta segment, one human D delta segment, and one human J delta segment operably linked to the human TCR delta constant region gene sequence (e.g., the method further comprises replacing the endogenous TCR delta constant region gene sequence with the human TCR delta constant region gene sequence), and breeding the first and second non-human animals to obtain a non-human animal that expresses a T cell receptor comprising a human gamma / delta TCR. In some embodiments, the second non-human animal comprises a human or humanized TCR alpha locus before and / or in addition to the human TCR delta locus.

[0220] In some embodiments, the methods for producing genetically modified non-human animals result in animals whose genomes comprise unrearranged humanized TCR loci (e.g., unrearranged humanized TCRγ and / or TCRδ genes and unrearranged human or humanized TCRs and / or TCR loci by Bayi). In some embodiments, a method is provided for generating a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a T cell receptor comprising a human variable region and a human constant domain on the surface of the T cell, the method comprising: replacing in a first non-human animal an endogenous non-human TCRγ sequence comprising at least one endogenous Vγ segment (e.g., all endogenous Vγ segments), at least one endogenous Jγ segment (e.g., all endogenous Jγ segments), and at least one endogenous Cγ gene (e.g., all endogenous Cγ genes) with an unrearranged TCRγ sequence comprising at least one human Vγ segment (e.g., all human Vγ segments), at least one human Jγ segment (e.g., all human Jγ segments), and at least one human Cγ gene (e.g., all human Cγ genes). and inserting, e.g., substituting, in a second non-human animal at least one endogenous Vδ segment (e.g., all endogenous Vδ segments), at least one endogenous Dδ segment (e.g., all endogenous Dδ segments), at least one endogenous Jδ segment (e.g., all endogenous Jδ segments), and endogenous Cδ into at least one human Vδ segment (e.g., all human Vδ segments), at least one human Dδ segment (e.g., all human Dδ segments), at least one human Jδ segment (e.g., all human Jδ segments), and a human Cδ gene; and breeding the first and second non-human animals to obtain a non-human animal that expresses a T cell receptor comprising a human gamma / δ TCR. In some embodiments, the second non-human animal comprises a human or humanized TCR alpha locus before and / or in addition to the human TCR delta locus.

[0221] In some embodiments, the method comprises inserting, e.g., replacing, in a first non-human animal, an unrearranged, humanized TCR α variable locus comprising at least one human Vα segment and at least one human Jα segment into an endogenous non-human TCR α variable locus, wherein the unrearranged, humanized TCR α variable locus comprises at least one human Vβ segment, one human Dβ segment, and one human Jβ segment into an endogenous non-human TCR β variable locus, wherein the humanized TCR β variable locus is operably linked to an endogenous TCR β constant region gene sequence, in a second non-human animal, and mating the first and second non-human animals to obtain a non-human animal that expresses a T cell receptor comprising the human variable region and the non-human constant region gene sequence. In other embodiments, the invention also provides methods for producing a genetically modified non-human animal whose genome comprises a humanized, unrearranged TCR α locus or a non-human animal whose genome comprises an unrearranged, humanized TCR β locus.

[0222] In various embodiments, the replacement is made at the endogenous locus. In various embodiments, the method involves a stepwise humanization strategy in which constructs containing additional variable region segments are introduced into ES cells, ultimately generating mice containing a full repertoire of human variable region segments and fully human constant region gene sequences (see, e.g., Figures 2 and 3).

[0223] Some method embodiments described herein include (1) replacing endogenous non-human (e.g., mouse) J1 sequences with human TRBD1 and human TRBJ1-1 through TRBJ1-6 gene segments and non-human (e.g., mouse) tcrbdj1 non-coding sequences (including non-coding recombination signal sequences (RSSs) and non-intergenic sequences), where the human TRBD1 and human TRBJ1 1 through TRBJ1-6 gene segments are the same non-human (e.g., mouse) tcrbdj1 sequences that are normally flanked by non-human (e.g., mouse) Trbd1 and non-human (e.g., mouse) Trbj1 1 through Trbj1-6 gene segments. and / or (2) replacing an endogenous non-human (e.g., mouse) tcrbdj2 sequence with a nucleic acid sequence comprising human TRBD2 and TRBJ2-1 to TRBJ2-7 gene segments and mouse tcrbdj2 non-coding sequences, wherein the human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments flank the same mouse Trbdj2 non-coding sequences that are normally flanked by mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments. In some embodiments, such replacements result in operable linkages between the nucleic acid sequences comprising human TRBD1 and human TRBJ1-1 and the TRBJ1-6 gene segment, and between the non-human (e.g., mouse) tcrbdj1 non-coding sequence (including non-coding recombination signal sequences (RSS) and other non-intergenic sequences) and the non-human (e.g., mouse) tcrbc1 constant region gene sequence, and insert operable linkages of the tcrbc1 constant region gene sequence and / or the sequences comprising human TRBD2 and human TRBJ2-1 through TRBJ2-7 gene segments and the mouse TCRBDJ2 non-coding sequence (including non-coding recombination signal sequences (RSS) and other non-intergenic sequences), respectively, into the non-human (e.g., mouse) tcrbc2 constant region gene sequence (see Figure 8C).In such embodiments, the resulting mouse may comprise one of the full repertoire of human TCRB variable region segments operably linked to the TCRBDJ1 and TCRBDJ2 clusters, but may retain endogenous TCRA and / or TCRB non-coding sequences, e.g., non-coding DNA (e.g., non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences).

[0224] The present disclosure also provides methods for modifying TCR variable gene loci (e.g., TCRα, TCRβ, TCRδ, and / or TCRγ gene loci) in non-human animals to express human or humanized TCR proteins described herein. This invention provides methods for modifying TCR variable loci to express human or humanized TCR proteins on the surface of T cells, e.g., by inserting, e.g., substituting, an unrearranged humanized TCR variable locus for an endogenous non-human TCR variable locus in a non-human animal. In one embodiment, the TCR variable locus is a TCRγ variable locus, the unrearranged humanized TCR variable locus consists of at least one human Vγ segment and at least one human Jγ segment, optionally operably linked to a human Cγ gene. In one embodiment, the TCR variable locus is a TCRδ variable locus, the unrearranged humanized TCR variable locus comprises at least one human Vδ segment, at least one human Dδ segment, and at least one human Jδ segment. In various embodiments, the unrearranged humanized TCR variable loci are operably linked to corresponding endogenous non-human TCR constant regions.

[0225] Thus, a nucleotide construct for generating a genetically modified animal comprising a humanized TCR variable region gene is also provided. In one embodiment, the nucleotide construct comprises 5' and 3' homology arms, a DNA fragment comprising a human TCR variable region gene segment and a human TCR constant region gene sequence, and a selection cassette flanked by recombination sites.

[0226] In one embodiment, at least one homologous arm is a non-human homologous arm, which is homologous to a non-human TCR locus (e.g., a non-human TCRγ), hi one embodiment, one or more homologous arms are non-human homologous arms, which are homologous to a human TCR locus (e.g., a human TCRα locus) in the genome of the non-human animal.

[0227] Various exemplary embodiments of the humanized loci described herein are presented in the figures and described in the Examples.

[0228] Upon completion of gene targeting, ES cells or genetically modified non-human animals are screened to confirm successful integration of the exogenous nucleotide sequence or expression of the exogenous polypeptide of interest. Numerous techniques are known to those skilled in the art, including (but not limited to) Southern blotting, long-range PCR, quantitative PCR (e.g., real-time PCR using TAQMAN), fluorescent in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, and the like. In one example, non-human animals (e.g., mice) carrying the desired genetic modification can be identified using a modification of the allelic assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659. Other assays for identifying specific nucleotide or amino acid sequences in genetically modified animals are known to those skilled in the art.

[0229] In some embodiments, the animals are produced herein by breeding. For example, in some embodiments, the mice are (a) obtaining a first mouse comprising a homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with a heterologous sequence comprising said unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment operably linked to a human TCR γ constant region gene sequence; (b) obtaining a second mouse comprising a homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and an endogenous TCR Cδ gene with a heterologous sequence comprising the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ segment operably linked to the human TCRδ constant region gene sequence; (c) mating the first mouse and the second mouse to obtain a genetically modified mouse; Genetically modified mice (i) replacement of endogenous genomic sequences comprising endogenous TCR Vγ and Jγ gene segments, and endogenous TCR Cγ genes, with heterologous sequences comprising unrearranged human TCR Vγ segments and unrearranged human TCR Jγ segments operably linked to human TCR γ constant region gene sequences; and (ii) comprising a replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, and Jδ gene segments, and an endogenous TCR Cδ gene, with a heterologous sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment operably linked to a human TCRδ constant region gene sequence; The genetically modified mice express human TCRγ and human TCRδ polypeptides.

[0230] Use of genetically modified non-human animals to mount substantially humanized T cell immune responses The role of gamma / delta T cells in immune defense remains poorly understood. See, for example, Vermijlen, D. et al. (2017) Seminars in Cell and Developmental Biol. 84:75-86. However, several features outlined above offer promise for therapeutic applications. A major barrier to T cell therapy derived from alpha / beta T cells is MHC restriction of the target antigen, limiting the application of any genetically engineered alpha / beta TCR to patients with the appropriate MHC haplotype. Furthermore, many alpha / beta T cells are "alloreactive to foreign MHC," which limits their use as allogeneic cell therapy due to the risk of graft-versus-host disease (GVHD). gamma / delta T cells lack these caveats and may offer a rapid route to off-the-shelf allogeneic therapy that requires less extensive manipulation and engineering to mitigate GVHD risk. Furthermore, the antitumor and antibacterial activities of gamma / delta T cells resemble "innate immune-like" cells such as natural killer cells, potentially offering additional clinical benefits currently being evaluated in various clinical trials. See, for example, Ferry GM and Anderson J. (2002) Exp. Immunol 2:168-79; Park JH and Lee HK (2021) Experimental & Molecular Medicine 53:318-327.

[0231] The TRG / TRD humanized mice described herein offer several novel areas of research into gamma / delta T cell biology and potential therapies. The published mice effectively model infectious and autoimmune disease responses driven by alpha / beta T cells (Moore, M. et al. Sci Immunol 6 (2021); doi:10.1126 / sciimmunol.abj4026, incorporated herein by reference in its entirety), and further humanization of the gamma / delta lineage may facilitate modeling of skin and mucosal immune responses. This includes, but is not limited to, studies of the role of gamma / delta T cells and antigens in inflammatory skin diseases, their interactions with the gut microbiota, and barrier tissue repair and homeostasis. The TRG / TRD humanized mice also provide new tools for investigating and identifying gamma / delta T cell antigens and cognate TCRs, which remain poorly characterized compared to their alpha / beta counterparts. For example, identifying antigen / TCR interactions abundant on tumor cells could lead to γ / δ-based antitumor cell therapies that lack the MHC restriction that limits existing TCR therapies. Furthermore, because graft-versus-host disease is driven by alloreactive α / β T cell receptors, γ / δ T cells are of increasing interest in allogeneic hematopoietic stem cell transplantation. Clinical strategies to maximize the function of these lymphocytes have been developed and are under development, including in vivo activation of γ / δ T cells or subsets after transplantation with specific drugs or antibodies, ex vivo expansion and manipulation of patient- or donor-derived γ / δ T cells and their subsets, and adoptive transfer of ex vivo activated lymphocytes. Handgretinger, R. and Schilbach, K. (2018) Blood 131:1063-72, incorporated herein by reference. Thus, the TRG / TRD humanized mice described herein may be a useful tool in preclinical studies of novel approaches aimed at expanding γ / δ T cells, directing them to tumors, and / or expanding γ / δ T cells in vivo or ex vivo.

[0232] Also described herein are embodiments in which the humanized TRA / D and TRG loci are introduced into mice that have a humanized TRB locus, as well as other components of T cell immunity, including TCR co-receptors (CD4 and CD8) and MHC loci. Thus, these mice are fully humanized in both T cell lineages to support research into cellular immunity.

[0233] Genetically modified non-human animals, e.g., rodents, e.g., mice or rats, containing either or both humanized CD4 and MHC II, or humanized CD8 and MHC I (and β2-microglobulin), present peptides in a human manner to T cells (CD4+ or CD8+ T cells, respectively) because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can also be used to study the function of the human immune system in humanized animals, e.g., for use in vaccine development, identification of antigens and antigen epitopes (e.g., T cell epitopes, e.g., unique human cancer epitopes) that elicit an immune response, i.e., identification of T cells that bind with high affinity to antigens in the context of the human MHC I complex, e.g., for use in adaptive and "innate" T cell therapy, evaluation of vaccine candidates and other vaccine strategies, study of human autoimmunity, study of human infectious diseases, and devising better therapeutic strategies based on human TCR expression.

[0234] Thus, in various embodiments, the genetically engineered animals of the present invention are useful, inter alia, for assessing the ability of antigens to initiate an immune response in humans and for generating antigenic diversity and identifying specific antigens that can be used in human vaccine development.

[0235] In one aspect, a method is provided for determining whether a peptide elicits a cellular immune response in a human, comprising exposing a genetically modified non-human animal described herein to the peptide, thereby allowing the non-human animal to mount an immune response, and detecting cells in the non-human animal (e.g., CD8+ or CD4+ T cells, comprising human CD8 or CD4, respectively) that bind to the sequence of the peptide presented by the chimeric human / non-human MHC I or II molecule described herein.

[0236] In one aspect, a method for identifying a candidate agent that proliferates and / or activates γ / δ T cells is described, comprising administering the candidate agent to a non-human animal as described herein (or an in vitro composition comprising γ / δ T cells isolated from the non-human animal as described herein) and measuring the level of γ / δ T cell proliferation and / or activation, wherein an increased level of γ / δ T cell proliferation and / or activation identifies the candidate agent as an agent capable of proliferating and / or activating γ / δ T cells. In some embodiments, the candidate agent is a tumor-associated antigen. In some embodiments, the agent is an antibody.

[0237] In one aspect, a method for stimulating and / or activating gamma / delta T cells (e.g., of V52 gamma / delta T cells) comprises administering an aminobisphosphonate (e.g., zoledronate, pamidronate, lizudronate) to a non-human animal described herein. See Latha et al. (2014) Front. Immunol, Vol. 5 DOI=10.3389 / fimmu.2014.00571, which is incorporated herein by reference in its entirety. Aminobisphosphonates act as inhibitors of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, causing the accumulation of isopentenyl pyrophosphate (IPP), thereby activating gamma / delta T cells in vivo. See, e.g., Park et al. (2021) Vol. 8 doi.org / 10.3389 / fchem.2020.612728.

[0238] In one aspect, a method for identifying a human T cell epitope is provided, the method comprising exposing a non-human animal as described herein to an antigen comprising the putative T cell epitope, allowing the non-human animal to mount an immune response, the non-human animal isolating MHC class I or MHC class II restricted T cells that bind the epitope, and identifying the epitope bound by the T cells.

[0239] In one aspect, a method is provided for identifying an antigen that generates a T cell response in humans, the method comprising exposing a mouse to a putative antigen as described herein, allowing the mouse to generate an immune response, and identifying the antigen that is presented by an HLA class I or class II restricted molecule.

[0240] In one aspect, a method is provided for determining whether a putative antigen contains an epitope that generates an HLA class I or class II restricted immune response upon exposure to the human immune system, the method comprising exposing a mouse described herein to the putative antigen and measuring an antigen-specific HLA class I or HLA class II restricted immune response in the mouse.

[0241] The genetically engineered non-human animals described herein can also be useful for identifying T cell receptors, e.g., high avidity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. This method includes exposing a non-human animal described herein to an antigen, allowing the non-human animal to mount an immune response to the antigen, isolating from the non-human animal T cells having a T cell receptor that binds to the antigen (whether presented by human or humanized MHC I or MHC II), and determining the sequence of the T cell receptor. In some embodiments, this method includes exposing a non-human animal described herein to an antigen, allowing the non-human animal to mount an immune response to the antigen, and isolating from the non-human animal γδ T cells having a γδ T cell receptor that binds the antigen in an MHC-independent manner. Instead of antigen exposure, gamma / delta T cells, like natural killer (NK) cells, can respond to stress-induced self-ligands, such as major histocompatibility complex class I-related chains A and B (MICA / B) and UL16-binding protein (ULBP), via activation of the NKG2D receptor (NKG2D ligand) expressed on gamma / delta T cells and Vδ1 receptors. Furthermore, gamma / delta T cells also express pattern recognition receptors, such as Toll-like receptors, which enhance their antitumor activity. Gamma / delta T cells also express the natural cytotoxic receptors KNp30 and NKp44, and gamma / delta T cells have been shown to be able to kill lymphoid leukemia cell lines and leukemic blasts from patients with chronic myeloid leukemia via NKp30.18. Finally, using the activating receptor DNAM-1, γ / δ T cells efficiently target nectin-2 (CD112) and poliovirus receptor (CD155) positive acute myeloid leukemia and multiple myeloma cells.

[0242] Non-human animals expressing a diverse repertoire of functional human TCR V(D)J gene segments may be useful for the testing of human diseases. Thus, in one embodiment, the genetically engineered non-human animals described herein may express a TCR repertoire substantially similar to that expressed in humans, although, for example, the TCR repertoire of the non-human animals described herein may be derived from at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 99% of all functional human TCR alpha, TCR beta, TCR gamma, and / or TCR delta gene segments.

[0243] In addition to the ability to identify antigens and antigenic epitopes from human pathogens or neoplasms, the genetically modified animals of the present invention can be used to identify autoantigens associated with human autoimmune diseases, such as type I diabetes, multiple sclerosis, etc. The genetically modified animals of the present invention can also be used to study various aspects of human autoimmune diseases and can be utilized as autoimmune disease models.

[0244] In various embodiments, the genetically modified non-human animals of the present invention generate T cells with human or humanized TCR molecules on their surface, which, as a result, optionally recognize peptides in a human-like manner when presented by an MHC complex. The genetically modified non-human animals described herein may be used to study human T cell development and function and the process of immunological tolerance. They may also be used to test human vaccine candidates, generate TCRs with specific specificities for TCR gene therapy, generate TCR libraries against disease-associated antigens (e.g., tumor-associated antigens (TAAs)), and the like.

[0245] There has been growing interest in T cell therapy in the art because T cells (e.g., cytotoxic T cells) can be directed to attack and destroy cells that present a target antigen, such as a viral antigen, bacterial antigen, or tumor antigen. Initial trials in cancer T cell therapy aim to isolate tumor-infiltrating lymphocytes (TILs; a lymphocyte population in the tumor mass that likely contains T cells reactive to tumor antigens) from tumor cell masses, expand them in vitro using T cell growth factors, and return them to patients in a process called adoptive T cell transfer. See, for example, Restifo et al. (2012) Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews 12:269-81; Linnermann et al. (2011) T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success, J. Invest. Dermatol. 131:1806-16. However, the success of these therapies has so far been limited to melanoma and renal cell carcinoma, and TIL adoptive transfer has not been specifically directed against a defined tumor-associated antigen (TAA). Linnermann et al., supra.

[0246] Attempts have been made to initiate TCR gene therapy, in which T cells are selected or programmed to target target antigens, such as TAA.Current TCR gene therapy relies on the identification of the sequence of TCR that is directed to a specific antigen, such as tumor-associated antigens.For example, Rosenberg and colleagues have published several studies, in which they transduced the genes encoding the TCR α and β chains specific for the melanoma-associated antigen MART-1 epitope into peripheral blood lymphocytes derived from melanoma patients, and used the resulting proliferated lymphocytes for adoptive T cell therapy. Johnson et al. (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen, Blood 114:535-46; Morgan et al. (2006) Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes, Science 314:126-29. MART-1-specific TCRs have been isolated from patients who experienced tumor regression after TIL treatment. However, the identification of such TCRs, especially high-avidity TCRs (potentially therapeutically useful), is complicated by the fact that most tumor antigens are self-antigens, and TCRs targeting these antigens are often deleted or have suboptimal affinity, primarily due to immunological tolerance.

[0247] In various embodiments, the present invention solves this problem by providing genetically engineered non-human animals that contain unrearranged human TCR variable loci in their genomes. The non-human animals described herein are capable of generating T cells with a diverse repertoire of humanized T cell receptors. Thus, the non-human animals described herein can be a source of a diverse repertoire of humanized T cell receptors, e.g., high avidity humanized T cell receptors, for use in adoptive T cell transfer.

[0248] Thus, in one embodiment, the invention provides a method for generating a T cell receptor for a human antigen, comprising immunizing a non-human animal (e.g., a rodent, e.g., a mouse or rat) as described herein with the antigen of interest, allowing the animal to mount an immune response, isolating activated T cells with specificity for the antigen of interest from the animal, and determining the nucleic acid sequence of the T cell receptor expressed by the antigen-specific T cells.

[0249] In one embodiment, the invention provides a method for producing a human T cell receptor specific to an antigen of interest (e.g., a disease-associated antigen), the method comprising immunizing a non-human animal described herein with the antigen of interest, initiating an immune response in the animal, isolating T cells from the animal that are responsive to the antigen of interest, determining the nucleic acid sequence of a human TCR variable region expressed by the T cell, (a) cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of a human TCR constant region gene sequence such that the human TCR variable region is operably linked to the human TCR constant region gene sequence, or (b) cloning the human TCR variable region operably linked to a human TCR constant region sequence into a nucleotide construct, wherein the non-human animal comprises a complete human TCR locus, and expressing a human T cell receptor from the construct that is specific for the antigen of interest. In one embodiment, the steps of isolating T cells, determining the nucleic acid sequence of at least the human TCR variable region expressed by the T cell, cloning the TCR-encoding sequence into a nucleotide construct, and expressing the human T cell receptor are performed using standard techniques known to those of skill in the art.

[0250] In one embodiment, a nucleotide sequence encoding a T cell receptor specific for an antigen of interest is expressed in a cell, hi one embodiment, the TCR-expressing cell is selected from CHO, COS, 293, HeLa, PERC.6™ cells, etc.

[0251] The target antigen can be any antigen known to cause or be associated with a disease or condition, such as a tumor-associated antigen, or an antigen from a virus, bacterium, or other pathogen. Many tumor-associated antigens are known in the art. A selection of tumor-associated antigens is presented in the Cancer Immunity (A Journal of the Cancer Research Institute) Peptide Database (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the invention, the target antigen is a human antigen, such as a human tumor-associated antigen. In some embodiments, the antigen is a cell-type-specific intracellular antigen, and a T cell receptor is used to kill cells expressing the antigen.

[0252] In one embodiment, provided herein is a method for identifying T cells with specificity for an antigen of interest, e.g., a tumor-associated antigen, comprising immunizing a non-human animal as described herein with the antigen of interest, allowing the animal to mount an immune response, and isolating T cells with specificity for the antigen from the non-human animal.

[0253] The present invention provides new methods for adoptive T cell therapy. Thus, provided herein are methods of treating or ameliorating a disease or condition (e.g., cancer) in a subject (e.g., a mammalian subject, e.g., a human subject), comprising immunizing a non-human animal as described herein with an antigen associated with the disease or condition, allowing the animal to mount an immune response, isolating a population of antigen-specific T cells from the animal, and infusing the isolated antigen-specific T cells into the subject. In one embodiment, the invention provides a method of treating or ameliorating a disease or condition in a human subject, the method comprising: immunizing a non-human animal described herein with an antigen of interest (e.g., a disease- or condition-associated antigen, e.g., a tumor-associated antigen); allowing the animal to mount an immune response; isolating a population of antigen-specific T cells from the animal; determining the nucleic acid sequence of a T cell receptor expressed by the antigen-specific T cells (e.g., a first and / or second nucleic acid sequence encoding a human rearranged TCR delta variable region gene or a TCR gamma variable region gene, or a third and / or fourth nucleic acid sequence encoding a human rearranged TCR alpha and / or human rearranged TCR beta variable region gene); cloning the nucleic acid sequence of the T cell receptor, e.g., the first, second, third, and / or fourth nucleic acid sequence, into an expression vector (e.g., a retroviral vector); introducing the vector into T cells from the subject such that the T cells express the antigen-specific T cell receptor; and infusing the T cells into the subject. In one embodiment, the T cell receptor nucleic acid sequence need not be further humanized (e.g., because some animal embodiments herein comprise fully human TRD and TRG loci). In one embodiment, the T cell receptor nucleic acid sequence is further humanized prior to introduction into T cells derived from the subject, e.g., the sequence encoding the non-human constant region is modified to more closely resemble a human TCR constant region (e.g., the non-human constant region is replaced with a human constant region). In some embodiments, the disease or condition is cancer. In some embodiments, the antigen-specific T cell population is expanded prior to infusion into the subject. In some embodiments, the subject's immune cell population is immunodepleted prior to infusion of the antigen-specific T cells.In some embodiments, the antigen-specific TCR is a high avidity TCR, e.g., a high avidity TCR for a tumor-associated antigen. In some embodiments, the T cell is a cytotoxic T cell. In another embodiment, the disease or condition is caused by a virus or bacteria.

[0254] In another embodiment, the disease or condition is an autoimmune disease.Treg cells are a subpopulation of T cells that maintain tolerance to self-antigens and prevent pathological autoreactivity.Thus, also provided herein is a method for treating autoimmune diseases that depend on the generation of antigen-specific Treg cells in the non-human animal of the present invention described herein.

[0255] Also provided herein are methods of treating or ameliorating a disease or condition (e.g., cancer) in a subject, the methods comprising introducing cells afflicted by the disease or condition (e.g., cancer cells) from the subject into a non-human animal, allowing the animal to mount an immune response against the cells, isolating a population of T cells from the animal that are responsive to the cells, determining the nucleic acid sequence of a T cell receptor variable domain expressed by the T cells, cloning the sequence encoding the T cell receptor variable domain into a vector (e.g., in-frame and operably linked to a human TCR constant gene), introducing the vector into T cells derived from the subject, and infusing the subject's T cells, bearing the T cell receptor, into the subject.

[0256] Also provided herein is the use of a non-human animal described herein for producing a nucleic acid sequence encoding a human TCR variable domain (e.g., a TCR gamma and / or delta variable domain). In one embodiment, a method for producing a nucleic acid sequence encoding a human TCR variable domain is provided, comprising immunizing a non-human animal described herein with an antigen of interest, allowing the non-human animal to mount an immune response against the antigen of interest, and obtaining therefrom a nucleic acid sequence encoding a human TCR variable domain that binds to the antigen of interest. In one embodiment, the method further comprises generating a nucleic acid sequence encoding a human TCR variable domain, optionally operably linked to a non-human TCR constant domain, the method comprising isolating T cells from the non-human animal described herein and obtaining therefrom a nucleic acid sequence encoding a TCR variable domain, optionally operably linked to a non-human TCR constant domain, and cloning the nucleic acid sequence encoding the TCR variable domain (e.g., a first, second, third, or fourth nucleic acid sequence encoding a human rearranged TCR gamma variable region gene, a human rearranged TCR delta variable region gene, a TCR alpha variable region gene, or a TCR beta variable region gene, respectively) in frame with a promoter with an appropriate human constant region, unless one is already present due to the nature of the genetic engineering of the mouse (e.g., a human TCR gamma constant region gene sequence, a human TCR delta constant region gene sequence, a TCR alpha constant region gene sequence, or a TCR beta constant region gene sequence, respectively).

[0257] Thus, provided herein are rearranged TCR variable nucleic acid sequences, such as rearranged TCRγ, TCRδ, or TCRα / δ variable region nucleic acid sequences generated in, for example, a non-human animal described herein, and encoded by, for example, a human rearranged Vγ / Jγ gene sequence, a rearranged human VδDδJδ gene sequence, or a rearranged human VαDδJδ gene sequence, respectively. Also provided are TCR variable region amino acid sequences encoded by such rearranged TCR variable region nucleic acid sequences. Such rearranged TCR variable region nucleic acid sequences (TCRγ and / or TCRδ variable region nucleic acid sequences) obtained in a non-human animal described herein may be utilized in humans for a variety of uses described herein, for example, as human therapeutic agents.

[0258] No TCR αδ variable region nucleic acid sequences have been previously described. Accordingly, such TCR αδ variable region nucleic acid sequences may be particularly useful in identifying new classes of TCR variable domains and / or surveying such TCR αδ variable region nucleic acid sequences in human populations. Such TCR αδ variable region nucleic acid sequences and the variable domains encoded therefrom may represent a novel class of therapeutic agents, as hybrid TCR α / δ variable domains may bind to different classes of antigens entirely or may bind antigens in a manner distinct from that of the original TCR α, TCR β, TCR γ, or TCR δ variable domains.

[0259] Also provided herein is the use of a non-human animal described herein to generate a human therapeutic, comprising immunizing the non-human animal with an antigen of interest (e.g., a tumor-associated antigen), allowing the non-human animal to mount an immune response, obtaining T cells from the animal that are reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence encoding a humanized TCR protein or a human TCR variable domain that binds to the antigen of interest, and using the nucleic acid sequence encoding the humanized TCR protein or human TCR variable domain in the human therapeutic.

[0260] Thus, also provided is a method for mak...

Claims

1. (I) A germline and CD3 containing an unrearranged TCRγ variable region sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment. － A T cell, the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally at an endogenous TCRγ locus; the unrearranged human TCR Vγ segment and the unrearranged human TCR Jγ segment are rearranged in a T cell of the mouse to form a rearranged human TCR Vγ / Jγ variable region gene sequence operably linked to the human TCRγ constant region gene sequence; the rearranged human TCR Vγ / Jγ variable region gene sequence operably linked to the human TCRγ constant region gene sequence, together encoding a human TCRγ polypeptide, － Somatic cells and (II) a CD3 that expresses on its surface a functional TCR comprising the human TCRγ polypeptide. ＋ and a T cell.

2. germ cells and CD3 － the T cell further comprises an unrearranged T cell receptor (TCR) δ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment; the unrearranged TCRδ variable region sequence is optionally operably linked to a human TCRδ constant region gene sequence at an endogenous TCRδ locus; the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ segment are rearranged in a T cell of the mouse to form a rearranged human TCR Vδ / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence; the rearranged human TCR Vδ / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence together encodes a human TCRδ polypeptide; the mouse expresses on its surface a functional TCR comprising both the human TCRγ polypeptide and the human TCRδ polypeptide; ＋ The mouse of claim 1 , comprising T cells.

3. germ cells and CD3 － the somatic cell further comprises an unrearranged human TCR Vα segment upstream of the unrearranged TCRδ variable region sequence and the human TCRδ constant region gene sequence; the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ, and the unrearranged human TCR Jδ segment are rearranged in a T cell of the mouse to form a rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence; the rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence together encodes a human hybrid TCR polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain; the mouse expressing on its surface a functional TCR comprising the human hybrid TCR polypeptide comprising the human hybrid α / δ variable domain and the human TCR δ constant domain, ＋ The mouse of claim 2 , comprising T cells.

4. (I) in the 5' to 3' direction, an unrearranged human TCR Vα segment, and and a germline and CD3 unrearranged TCRδ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment. － A somatic cell, the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus; the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ, and the unrearranged human TCR Jδ segment are rearranged in a T cell of the mouse to form a rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence; The rearranged human TCR Vα / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence together encodes a human hybrid TCR polypeptide comprising a human hybrid TCR α / δ variable domain and a human TCRδ constant domain. － Somatic cells and (II) a CD3 expressing on its surface a functional TCR comprising the human hybrid TCR comprising the human hybrid α / δ variable domain and the human TCRδ constant domain. ＋ and a T cell.

5. germ cells and CD3 － the somatic cell comprises a replacement of an endogenous TCR Vα segment with said unrearranged human TCR Vα segment, and a replacement of an endogenous TCR Jα segment with an unrearranged human TCR Jα segment; the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are operably linked to one another with a TCRα constant region gene sequence, e.g., a murine TCRα constant region gene sequence; the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are rearranged in a T cell of the mouse to form a rearranged TCR Vα / Jα variable region gene sequence operably linked to the TCRα constant region gene sequence; the rearranged human TCR Vα / Jα variable region gene sequence operably linked to the TCRα constant region gene sequence together encodes a TCRα polypeptide comprising a human TCRα variable domain and a TCRα constant domain; the mouse expressing on its surface a functional TCR comprising the TCRα polypeptide, ＋ The mouse of claim 4 , comprising T cells.

6. germ cells and CD3 － the somatic cells comprise a replacement of all endogenous TCR Vα segments with a complete repertoire of unrearranged human TCR Vα segments, and a replacement of all endogenous TCR Jα segments with a complete repertoire of unrearranged human TCR Jα segments; a complete repertoire of unrearranged human TCR Vα segments and a complete repertoire of unrearranged human TCR Jα segments operably linked to a murine TCRα constant region gene sequence at an endogenous TCRα locus; a complete repertoire of unrearranged human TCR Vα segments and a complete repertoire of unrearranged human TCR Jα segments are rearranged in a T cell of the mouse to form a rearranged human TCR Vα / Jα variable region gene sequence operably linked to a mouse TCRα constant region gene sequence; the rearranged TCR Vα / Jα variable region gene sequence operably linked to the murine TCRα constant region gene sequence together encodes a chimeric TCRα polypeptide comprising a human TCRα variable domain operably linked to a murine TCRα constant domain; the mouse expressing on its surface a functional TCR comprising the chimeric TCRα polypeptide; ＋ The mouse of claim 4 or 5, comprising T cells.

7. germ cells and CD3 － Somatic cells, (A) replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; or (B) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or (C) (i) comprising a replacement of an endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, a replacement of an endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and a replacement of an endogenous TCRγ constant region gene sequence with a human TCRγ constant region gene sequence; (ii) the mouse of any one of claims 1 to 6, comprising a replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence.

8. (A) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; or (B) the unrearranged human TCR Vδ segments comprise a complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise a complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise a complete repertoire of unrearranged human TCR Jδ segments; or (C) (i) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; and (ii) the unrearranged human TCR Vδ segments comprise a complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise a complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise a complete repertoire of unrearranged human TCR Jδ segments.

9. (I) the germ cells and CD3 － T cells, (A) at the endogenous TCRγ locus: replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments; and Replacement of all TCRγ constant region gene sequences with the complete repertoire of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus, Replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Dδ segments; replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments; and comprising a replacement of the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence, (II) the mouse expresses on its surface a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide; ＋ The mouse of any one of claims 1 to 8, comprising T cells.

10. germ cells and CD3 － the T cell further comprises an unrearranged TCR β variable region sequence comprising at least one unrearranged human TCR variable region Vβ segment, at least one unrearranged human TCR variable region Dβ segment, and at least one unrearranged TCR variable region Jβ segment; the unrearranged TCRβ variable region sequence is operably linked to a TCRβ constant region gene sequence, such as a murine TCRβ constant region gene sequence, optionally at an endogenous TCRβ locus; the unrearranged human TCR Vβ segment, the unrearranged human TCR Dβ segment, and the unrearranged human TCR Jβ segment are rearranged in a T cell of the mouse to form a rearranged human TCR Vβ / Dβ / Jβ variable region gene sequence operably linked to the TCR β constant region gene sequence; the rearranged human TCR Vβ / Dβ / Jβ variable region gene sequence operably linked to the TCR β constant region gene sequence together encodes a TCR β polypeptide comprising a human TCR β variable domain and a TCR β constant domain; the mouse expressing on its surface a functional TCR comprising the TCRβ polypeptide, ＋ The mouse of any one of claims 1 to 9, comprising T cells.

11. The mouse of claim 10, wherein the unrearranged TCR β variable region sequence comprises a mouse TCRB non-coding sequence.

12. (I) the germ cells and CD3 － T cells, (A) at the endogenous TCRγ locus: replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments; and Replacement of all TCRγ constant region gene sequences with the complete repertoire of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus, Replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Dδ segments; replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments; and comprising a replacement of the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence, (C) at the endogenous TCRα locus: replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments; and comprising the replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) at the endogenous TCRβ locus: replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; Replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; and comprising the replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (II) the mouse expresses on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide; ＋ T cells and CD3s expressing on their surface a functional human or humanized TCR comprising a human or humanized TCR alpha polypeptide and a human or humanized TCR beta polypeptide. ＋ The mouse of any one of claims 1 to 11, further comprising T cells.

13. (I) the germ cells and CD3 － T cells, (A) at the endogenous TCRγ locus: replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments; and Replacement of all TCRγ constant region gene sequences with the complete repertoire of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus, Replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Dδ segments; replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments; and Replacement of the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; (C) at the endogenous TCRα locus: replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments; and replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; (D) at the endogenous TCRβ locus: replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 co-receptor comprising the D1, D2, and D3 domains of a human CD4 polypeptide operably linked to the D4, transmembrane, and cytoplasmic domains of a mouse CD4 polypeptide; (F) a second nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide, a second nucleotide sequence and a third nucleotide sequence, wherein the chimeric human / mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8α polypeptide, and the chimeric human / mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8β polypeptide; (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC IIα polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC IIβ polypeptide, a first nucleic acid sequence and a second nucleic acid sequence, wherein the chimeric human / mouse MHC IIα polypeptide comprises the α1 and α2 domains of a human HLA class IIα polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIα polypeptide, and the chimeric human / mouse MHC IIβ polypeptide comprises the β1 and β2 domains of a human HLA class IIβ polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIβ polypeptide; (H) a third nucleic acid sequence encoding a chimeric human / mouse MHC I polypeptide comprising the α1 domain, α2 domain, and α3 domain of a human HLA class I polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC class I polypeptide; and (I) a polynucleotide comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, the nucleotide sequence comprising the nucleotide sequence set forth in exon 1 of the mouse β2 microglobulin gene operably linked to the nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene; (II) the mouse expresses on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide; ＋ T cells and CD3s expressing on their surface a functional human or humanized TCR comprising a human or humanized TCR alpha polypeptide and a human or humanized TCR beta polypeptide. ＋ and optionally further comprising T cells. The mouse (e) the chimeric human / mouse CD4 co-receptor; (f) a chimeric CD8 co-receptor comprising said chimeric human / mouse CD8α polypeptide and said chimeric human / mouse CD8β polypeptide; (h) a chimeric MHC II complex comprising the chimeric human / mouse MHC IIα polypeptide and the chimeric human / mouse MHC IIβ polypeptide, wherein the chimeric MHC II complex is capable of binding the chimeric human / mouse CD4 co-receptor; (i) the chimeric human / mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding to the chimeric CD8 co-receptor; and (j) a mouse described in any one of claims 1 to 12, which expresses the human or humanized β2 microglobulin polypeptide.

14. germ cells and CD3 － The mouse of any one of claims 1 to 13, wherein each of the T cells comprises a human CTCF binding element upstream of the TCRγ locus.

15. The mouse of any one of claims 1 to 14, wherein the germ cells and somatic cells each contain a human CTCF binding element upstream of the TCRα locus.

16. The mouse (a) γ / δ T cells in the thymus, the spleen, the skin, and / or the intestinal mucosa, and / or 16. The mouse of any one of claims 1 to 15, comprising (b) a population of CD45+CD3+ T cells expressing human γδ TCR among intraepithelial lymphocytes in its thymus, spleen, mesenteric lymph nodes, skin, intestinal mucosa, and / or its colon and / or small intestine, wherein optionally the proportion of the population of CD45+CD3+ T cells expressing human γδ TCR among intraepithelial lymphocytes in its thymus, spleen, mesenteric lymph nodes, skin, intestinal mucosa, and / or its colon and / or small intestine is equal to or greater than the proportion of the population of CD45+CD3+ T cells expressing mouse γδ TCR among intraepithelial lymphocytes in the thymus, spleen, mesenteric lymph nodes, skin, intestinal mucosa, and / or its colon and / or small intestine of a wild-type mouse.

17. 17. The mouse of any one of claims 1 to 16, wherein the human TCRγ polypeptide is derived from a human TRGV2 gene segment, a human TRGV3 gene segment, a human TRGV4 gene segment, a human TRGV5 gene segment, a human TRGV8 gene segment, a human TRGV9 gene segment, a human TRGV10 gene segment, or a human TRGV11 gene segment.

18. The mouse of any one of claims 1 to 17, wherein the human TCRγ polypeptide is derived from a human TRGJ1 gene segment, a human TRGJP gene segment, a human TRGJP1 gene segment, a human TCRGJ2 gene segment, or a human TRGJP2 gene segment.

19. 19. The mouse of any one of claims 2 to 18, wherein the human TCRδ polypeptide is derived from a human TRDV1 gene segment, a human TRAV17 gene segment, a human TRAV19 gene segment, a human TRAV21 gene segment, a human TRAV21 gene segment, a human TRAV26-2 gene segment, a human TRAV29 / TRDV5 gene segment, a human TRAV31 gene segment, a human TRAV38-2 / TRDV8 gene segment, a human TRAV39 gene segment, a human TRAV40 gene segment, a human TRAV41 gene segment, a human TRDV2 gene segment, or a human TRDV3 gene segment.

20. The mouse of any one of claims 1 to 19, wherein the human TCRδ polypeptide is derived from a human TRDJ1 gene segment, a human TRDJ2 gene segment, a human TRDJ3 gene segment, or a human TRDJ4 gene segment.

21. A mouse embryonic stem (ES) cell or germ cell comprising an unrearranged TCRγ variable region sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment, A mouse embryonic stem (ES) cell or germ cell, wherein the unrearranged TCRγ variable region sequence is optionally operably linked to a human TCRγ constant region gene sequence at the endogenous TCRγ locus.

22. the ES cell or germ cell further comprises an unrearranged T cell receptor (TCR) δ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment; 22. The mouse ES cell or germ cell of claim 21, wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at the endogenous TCRδ locus.

23. 23. The mouse ES cell or germ cell of claim 22, wherein the ES cell or germ cell further comprises an unrearranged human TCR Vα segment upstream of the unrearranged TCRδ variable region sequence and the human TCRδ constant region gene sequence.

24. In the 5' to 3' direction, an unrearranged human TCR Vα segment, and an unrearranged TCRδ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged TCR Dδ, and an unrearranged human TCR Jδ segment; A mouse ES cell or germ cell, wherein the unrearranged TCRδ variable region sequence is optionally operably linked to a human TCRδ constant region gene sequence at the endogenous TCRδ locus.

25. the ES cell or germ cell comprises a replacement of an endogenous TCR Vα segment with the unrearranged human TCR Vα segment, and a replacement of an endogenous TCR Jα segment with an unrearranged human TCR Jα segment; 25. The mouse ES cell or germ cell of claim 24, wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are operably linked to each other and to a TCRα constant region gene sequence.

26. replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments, and replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; The mouse ES cell or germ cell of claim 24 or 25, wherein a complete repertoire of unrearranged human TCR Vα segments and a complete repertoire of unrearranged human TCR Jα segments are operably linked to a mouse TCRα constant region gene sequence at the endogenous TCRα locus.

27. The ES cells or germ cells are (A) replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; or (B) Replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; or (C) (i) replacement of endogenous TCR Vγ segments with unrearranged human TCR Vγ segments, replacement of endogenous TCR Jγ segments with unrearranged human TCR Jγ segments, and replacement of endogenous TCRγ constant region gene sequences with human TCRγ constant region gene sequences; and (ii) the mouse ES cell or germ cell of any one of claims 21 to 26, comprising a replacement of an endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, a replacement of an endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, a replacement of an endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and a replacement of an endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence.

28. (A) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; or (B) the unrearranged human TCR Vδ segments comprise a complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise a complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise a complete repertoire of unrearranged human TCR Jδ segments; or (C) (i) the unrearranged TCR Vγ segments comprise a complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise a complete repertoire of unrearranged human TCR Jγ segments; and 28. The mouse ES cell or germ cell of Claim 27, wherein (ii) the unrearranged human TCR Vδ segments comprise a complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segments comprise a complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segments comprise a complete repertoire of unrearranged human TCR Jδ segments.

29. (I) the ES cells or germ cells are (A) at the endogenous TCRγ locus: replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments; and Replacement of all TCRγ constant region gene sequences with the complete repertoire of human TCRγ constant region gene sequences; optionally, replacement of an endogenous genomic sequence comprising all endogenous TCR Vγ segments, all endogenous TCR Jγ segments, and all endogenous TCRγ constant region gene sequences with a human genomic sequence comprising a complete repertoire of unrearranged human TCR Vγ segments, a complete repertoire of unrearranged human TCR Jγ segments, and a complete repertoire of human TCRγ constant region gene sequences (e.g., hTCRGC1 and hTCRGC2); and (B) at the endogenous TCRδ locus, Replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Dδ segments; Replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Jδ segments; and 29. The mouse ES cell or germ cell of any one of claims 21 to 28, comprising a replacement of an endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence.

30. the ES cells or germ cells further comprise an unrearranged TCR β variable region sequence comprising at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment; 30. The mouse ES cell or germ cell of any one of claims 21-29, wherein the unrearranged TCR β variable region sequence is optionally operably linked to a TCR β constant region gene sequence (e.g., a mouse TCR β constant region gene sequence) at an endogenous TCR β locus.

31. The mouse ES cell or germ cell of claim 30, wherein the unrearranged TCR β variable region sequence comprises a mouse TCRB non-coding sequence.

32. The ES cells or the germ cells are (A) at the endogenous TCRγ locus: replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments; and Replacement of all TCRγ constant region gene sequences with the complete repertoire of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus, Replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Dδ segments; replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments; and Replacement of the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; (C) at the endogenous TCRα locus: replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments; and Replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; and (D) at the endogenous TCRβ locus: replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; Replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; and 32. The mouse ES cell or germ cell of any one of claims 21-31, comprising replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments.

33. The ES cells or germ cells are (A) at the endogenous TCRγ locus: replacement of all endogenous TCR Vγ segments with a full repertoire of unrearranged human TCR Vγ segments; replacement of all endogenous TCR Jγ segments with a full repertoire of unrearranged human TCR Jγ segments; and Replacement of all TCRγ constant region gene sequences with the complete repertoire of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus, Replacement of all endogenous TCR Vδ segments with a full repertoire of unrearranged human TCR Vδ segments; replacement of all endogenous TCR Dδ segments with a full repertoire of unrearranged human TCR Dδ segments; replacement of all endogenous TCR Jδ segments with a full repertoire of unrearranged human TCR Jδ segments; and Replacement of the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; and (C) at the endogenous TCRα locus: replacement of all endogenous TCR Vα segments with a full repertoire of unrearranged human TCR Vα segments; and Replacement of all endogenous TCR Jα segments with a full repertoire of unrearranged human TCR Jα segments; and (D) at the endogenous TCRβ locus: replacement of all endogenous TCR Vβ segments with a full repertoire of unrearranged human TCR Vβ segments; replacement of all endogenous TCR Dβ segments with a full repertoire of unrearranged human TCR Dβ segments; replacement of all endogenous TCR Jβ segments with a full repertoire of unrearranged human TCR Jβ segments; (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 co-receptor comprising the D1, D2, and D3 domains of a human CD4 polypeptide operably linked to the D4, transmembrane, and cytoplasmic domains of a mouse CD4 polypeptide; (F) a second nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide, a second nucleotide sequence and a third nucleotide sequence, wherein the chimeric human / mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8α polypeptide, and the chimeric human / mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse CD8β polypeptide; (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC IIα polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC IIβ polypeptide, a first nucleic acid sequence and a second nucleic acid sequence, wherein the chimeric human / mouse MHC IIα polypeptide comprises the α1 and α2 domains of a human HLA class IIα polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIα polypeptide, and the chimeric human / mouse MHC IIβ polypeptide comprises the β1 and β2 domains of a human HLA class IIβ polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC IIβ polypeptide; (H) a third nucleic acid sequence encoding a chimeric human / mouse MHC I polypeptide comprising the α1 domain, α2 domain, and α3 domain of a human HLA class I polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC class I polypeptide; and (I) A mouse ES cell or germ cell described in any one of claims 21 to 32, comprising a polynucleotide encoding a human or humanized β2 microglobulin polypeptide, the polynucleotide comprising a nucleotide sequence comprising the nucleotide sequence shown in exon 1 of the mouse β2 microglobulin gene operably linked to the nucleotide sequences shown in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.

34. The mouse ES cell or germ cell of any one of claims 21 to 33, further comprising a human CTCF binding element upstream of the TCRγ locus.

35. 35. The mouse ES cell or germ cell of any one of claims 21 to 34, further comprising a human CTCF binding element upstream of the TCRα locus.

36. 36. The mouse ES cell or germ cell of any one of claims 21 to 35, comprising the human nucleotide sequence set forth in chr7:38383439-38230960 (GRCh38 coordinates).

37. 36. The mouse ES cell or germ cell of any one of claims 22 to 35, comprising a human nucleotide sequence set forth in Chr14:22421820-22464666 (GRCh38 coordinates).

38. In the 5' to 3' direction, (a) 5′ mouse homology arm; (b) the human nucleotide sequence set forth in chr7:38383439-38230960 (GRCh38 coordinates); and (c) A targeting vector containing a 3' mouse homology arm.

39. (i) a selection cassette, and (ii) a targeting vector comprising the human nucleotide sequence set forth in Chr14:22421820-22464666 (GRCh38 coordinates).

40. 1. A method for generating a nucleic acid sequence encoding a human T cell receptor (TCR) gamma variable domain, comprising: Immunizing the genetically modified mouse according to any one of claims 1 to 20 with a target antigen; allowing the mouse to mount an immune response to the antigen of interest; and obtaining therefrom a nucleic acid sequence encoding a human TCRγ variable domain that binds to said antigen of interest.

41. 1. A method for generating a nucleic acid sequence encoding a human TCRγ polypeptide, comprising: Immunizing the genetically modified mouse according to any one of claims 1 to 20 with a target antigen; allowing the mouse to mount an immune response to the antigen of interest; and obtaining therefrom a nucleic acid sequence encoding a human TCRγ polypeptide of a TCR that binds to said target antigen.

42. 1. A method for generating a nucleic acid sequence encoding a human T cell receptor (TCR) δ variable domain, comprising: Immunizing the genetically modified mouse according to any one of claims 2 to 20 with a target antigen; allowing the mouse to mount an immune response to the antigen of interest; and obtaining therefrom a nucleic acid sequence encoding a human TCRδ variable domain that binds to said antigen of interest.

43. 1. A method for generating a nucleic acid sequence encoding a human TCRδ polypeptide, comprising: Immunizing the genetically modified mouse according to any one of claims 2 to 20 with a target antigen; allowing the rodent to mount an immune response to the target antigen; and obtaining therefrom a nucleic acid sequence encoding a human TCRδ polypeptide of a TCR that binds to the target antigen.

44. 1. A method for generating a nucleic acid sequence encoding a human hybrid T-cell receptor (TCR) α / δ variable domain, comprising: Immunizing the genetically modified mouse according to any one of claims 4 to 20 with a target antigen; allowing the mouse to mount an immune response to the antigen of interest; and obtaining therefrom a nucleic acid sequence encoding a human hybrid TCR α / δ variable domain that binds to said antigen of interest.

45. 1. A method for producing a human therapeutic comprising: Immunizing the genetically modified mouse according to any one of claims 1 to 20 with a target antigen; allowing the mouse to mount an immune response; obtaining T cells reactive to the target antigen from the mouse; obtaining from the T cell a T cell receptor that binds to the antigen of interest and / or a nucleic acid sequence(s) encoding the T cell receptor, wherein the T cell receptor comprises a human TCR variable domain.

46. 46. ​​The method of claim 45, wherein the human therapeutic is a soluble T cell receptor.

47. 47. The method of claim 45 or 46, wherein the human therapeutic is a single chain TCR.

48. 48. The method of any one of claims 45 to 47, wherein the human therapeutic is a scTv.

49. 47. The method of claim 46, wherein the soluble T cell receptor is fused to a moiety capable of killing infected or cancerous cells, such as a cytotoxic molecule (e.g., a chemotherapeutic agent), a toxin, a radionuclide, a prodrug, an antibody, etc.

50. 47. The method of claim 46, wherein the soluble T cell receptor is fused to an immunomodulatory molecule, e.g., a cytokine or chemokine.

51. 47. The method of claim 46, wherein the soluble T cell receptor is fused to an immune inhibitory molecule, e.g., a molecule that inhibits T cells from killing other cells that carry the antigen recognized by the T cell.

52. A host cell comprising a nucleic acid molecule according to any one of claims 40 to 44.

53. A method for producing a genetically modified mouse or mouse ES cell, comprising: (a) an unrearranged TCRγ variable region sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment, the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, and the unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments are rearranged in a T cell of the mouse to form a rearranged human TCR Vγ / Jγ variable region gene sequence operably linked to the human TCRγ constant region gene sequence; an unrearranged TCRγ variable region sequence, wherein the rearranged human TCR Vγ / Jγ variable region gene sequence is operably linked to a human TCRγ constant region gene sequence, and together they encode a human TCRγ polypeptide; and / or (b) an unrearranged T cell receptor (TCR) δ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment; the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence; the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ are rearranged in a T cell of the mouse to form a rearranged human TCR Vδ / Dδ / Jδ variable region gene sequence operably linked to the human TCRδ constant region gene sequence; wherein the rearranged human TCR Vδ / Dδ / Jδ variable region gene sequence is modified to include a T cell receptor (TCR) δ variable region sequence operably linked to the human TCRδ constant region gene sequence, the two sequences together encoding a human TCRδ polypeptide.

54. The modification is (a) replacing an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes with a heterologous sequence comprising said unrearranged human TCR Vγ segments and said unrearranged human TCR Jγ segments operably linked to said human TCRγ constant region gene sequence; and / or 54. The method of claim 53, comprising (b) replacing an endogenous genomic sequence comprising endogenous TCR V5, D5, J5 gene segments and an endogenous TCR C5 gene with a heterologous sequence comprising the unrearranged human TCR V5 segment, the unrearranged TCR D5 segment, the unrearranged human TCR J5 segment, and the human TCR5 constant region gene sequence.

55. (a) the endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes comprises a complete repertoire of endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes; and / or (b) the endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and an endogenous TCR Cδ gene comprises all endogenous TCR Vδ, Dδ, Jδ gene segments located between a TCR Vα gene segment and a TCR Jα gene segment.

56. (a) the heterologous sequence comprising the unrearranged human TCR Vγ segments and the unrearranged human TCR Jγ segments operably linked to the human TCRγ constant region gene sequence comprises a complete repertoire of unrearranged TCR Vγ and unrearranged human TCR Jγ segments and all human TCR Cγ genes; and / or (b) the heterologous sequence comprising the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, the unrearranged human TCR Jδ segment, and the human TCRδ constant region gene sequence comprises a complete repertoire of unrearranged human TCR Vδ, unrearranged human TCR Dδ, and unrearranged human TCR Jδ segments and a human TCR Cδ gene sequence located between a human TCR Vα gene segment and a human TCR Jα gene segment on chromosome 14 of the human genome.

57. 57. The method of any one of claims 53-56, wherein the modification comprises homologous recombination in the ES cell such that a heterologous sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment is operably linked to a human TCRγ constant region gene sequence, and wherein the heterologous sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, an unrearranged human TCR Jδ segment, and a human TCRδ constant region gene sequence is added, in any order, to the genome of one or more ES cells.

58. 58. The method of claim 57, further comprising generating a mouse from the one or more ES cells.

59. The modification is (a) obtaining a first mouse comprising a homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with the heterologous sequence comprising the unrearranged human TCR Vγ segment and the unrearranged human TCR Jγ segment operably linked to the human TCRγ constant region gene sequence; (b) obtaining a second mouse comprising a homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and an endogenous TCR Cδ gene with a heterologous sequence comprising the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ segment operably linked to the human TCRδ constant region gene sequence; (c) mating the first mouse and the second mouse to obtain a genetically modified mouse; The genetically modified mouse is (i) replacing an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments, and an endogenous TCR Cγ gene, with a heterologous sequence comprising said unrearranged human TCR Vγ segments and said unrearranged human TCR Jγ segments operably linked to said human TCRγ constant region gene sequence; and (ii) comprising a replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments, and an endogenous TCR Cδ gene with a heterologous sequence comprising the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ segment operably linked to a human TCRδ constant region gene sequence; 57. The method of any one of claims 53 to 56, wherein the genetically modified mouse expresses a human TCRγ polypeptide and a human TCRδ polypeptide.

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