Genetically modified mice expressing components of human cellular immune system

Abstract

Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats), the non-human animal is 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) 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 the receptors. Also provided are methods of making genetically engineered animals expressing the humanized or human gamma and / or delta TCRs. Also provided are methods of developing human therapeutics using the genetically engineered animals that produce a substantially humanized T cell immune response.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 376,706, filed on September 22, 2022, and U.S. Provisional Application Serial No. 63 / 383,213, filed on November 10, 2022, under 35 U.S.C. §119(e), each of which is hereby incorporated by reference.

[0003] Sequence Listing

[0004] An official copy of the sequence listing is an XML-formatted sequence listing with a file name of 11224.xml and a size of 41 kilobytes, created on September 21, 2023, submitted electronically via EFS-Web and submitted simultaneously with this specification. The sequence listing contained in this XML-formatted document is part of the specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to non-human animals (e.g., rodents such as mice or rats) that express:

[0006] (i) a humanized γδ T cell receptor (TCR) comprising:

[0007] (a) a human(ized) TCRδ polypeptide or a human(ized) hybrid TCR polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, and / or

[0008] (b) humanized TCRγ polypeptide, and

[0009] (ii) an αβTCR comprising:

[0010] (a) endogenous or human(ized) TCRα polypeptide, and

[0011] (b) endogenous or human(ized) TCRβ polypeptide, and optionally

[0012] (iii) one or more human(ized) T cell co-receptors (e.g., CD4 and / or CD8 (e.g., CD8α and / or CD8β)), and / or (iv) one or more human(ized) major histocompatibility complexes associated with the one or more human(ized) T cell co-receptors (e.g., MHC II complex (e.g., MHC IIα and / or MHC IIβ) and / or MHC I (which can be, for example, a human(ized) MHC Iα polypeptide alone or in complex with human(ized) β2 microglobulin)). The present disclosure also relates to embryos, tissues, cells and / or nucleic acids isolated from these non-human animals; methods of producing these non-human animals; and methods of using these non-human animals, for example as non-human animal models, to study γ and / or δ T cell responses to develop human therapeutic agents. Background Art

[0013] 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 known as TCRs).

[0014] Due to tolerance mechanism, not all antigens can stimulate T cell activation. However, in some diseases (for example, cancer, autoimmune diseases), peptides derived from self proteins become targets of cellular components of the immune system, which results in the destruction of cells presenting such peptides. Significant progress has been made in identifying clinically important antigens (for example, antigens associated with various types of cancer) and / or in conjunction with clinically important antigen TCR sequences. However, in order to improve the identification and selection of clinically important peptides that can stimulate suitable responses in human T cells and / or TCRs that can be combined with clinically important antigens (for example, adoptive immunotherapy for cancer, T cell vaccination for autoimmunity, etc.), it is still necessary to simulate the in vivo and in vitro systems of various aspects of the human immune system. Therefore, it is necessary to display human immune system components, particularly biological systems of T cell immune response components (for example, genetically modified non-human animals and cells). Summary of the invention

[0015] Described herein are non-human animals (e.g., rodents (e.g., rats or mice)) having humanized TRD gene loci (encoding TCR-δ polypeptides) and / or TRG gene loci (encoding TCR-γ polypeptides) that can produce potential therapeutic agents utilizing human γ / δ TCRs and / or T cells.

[0016] In some embodiments, a humanized TCRγ mouse as described herein comprises:

[0017] (I) Germ cells containing unrearranged TCRγ variable region sequences and CD3 -somatic cell, the unrearranged TCRγ variable region sequence comprises an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment,

[0018] wherein the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally at an endogenous TCRγ locus (e.g., a human TCRγ constant region gene sequence at an endogenous TCRγ locus, wherein a nucleotide sequence comprising an endogenous TCRγ 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 by a nucleotide sequence comprising a human TCRγ constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence),

[0019] wherein the unrearranged human TCR Vγ segment and the unrearranged human TCR Jγ segment are capable of rearranging (or rearranging) in a T cell to form a rearranged human TCR Vγ / Jγ variable region gene operably linked to the human TCRγ constant region gene sequence, and

[0020] wherein the rearranged human TCR Vγ / Jγ variable region gene operably linked to the human TCRγ constant region gene sequence together encodes a human TCRγ polypeptide, and

[0021] (II) CD3 expressing a TCR comprising the human TCRγ polypeptide on its surface + T cells.

[0022] In some humanized TCRγ mouse embodiments, germ cells and CD3 - The somatic cell also comprises an unrearranged T cell receptor (TCR) δ variable region sequence, the 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, wherein the unrearranged TCR δ variable region sequence is operably linked to a human TCR δ constant region gene sequence, optionally at an endogenous TCR δ locus, and wherein the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ segment are capable of rearranging (or rearranging) in the T cell to form a rearranged human TCR Vδ / Dδ / Jδ variable region gene operably linked to a human TCR δ constant region gene sequence, wherein the rearranged human TCR δ constant region gene sequence is operably linked to the human TCR The Vδ / Dδ / Jδ variable region genes together encode a human TCRδ polypeptide, wherein the mouse comprises a CD3 T cell expressing on its surface a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. +In some embodiments, germ cells and CD3 - The somatic cell further comprises an unrearranged human TCR Vα segment located upstream of an unrearranged TCRδ variable region sequence and a human TCRδ constant region gene sequence, wherein the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ and the unrearranged human TCR Jδ segment are capable of rearranging (or rearranging) in the T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to a 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 polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, and wherein the mouse comprises a CD3+ TCR expressing a functional TCR comprising the human hybrid TCR polypeptide on its surface. + T cell, the human hybrid TCR polypeptide comprises a human hybrid α / δ variable domain and a human TCRδ constant domain.

[0023] A humanized TCRδ mouse as described herein may comprise:

[0024] (I) Germ cells and CD3 - A somatic cell, said cell comprising from 5' to 3':

[0025] Unrearranged human TCR Vα segment and

[0026] an unrearranged TCR delta variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment,

[0027] wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus,

[0028] wherein the unrearranged human TCR Vα segment, the unrearranged TCR Dδ and the unrearranged human TCR Jδ segment are capable of rearranging (or rearrangement) in a T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to the human TCRδ constant region gene sequence,

[0029] 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 polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, and

[0030] (II) a CD3 cell expressing on its surface a functional TCR comprising the human hybrid TCR +T cell, the human hybrid TCR comprises the human hybrid α / δ variable domain and the human TCRδ constant domain.

[0031] In some humanized TCRδ mouse embodiments, germ cells and CD3 - The somatic 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, 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, such as a mouse TCRα constant region gene sequence, and wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are capable of rearranging (or rearranging) in a 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 operably linked to the TCRα constant region gene sequence The Vα / Jα variable region genes together encode 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 wherein the mouse comprises a CD3 T cell expressing on its surface a functional TCR comprising the TCRα polypeptide. + In some humanized TCRδ mouse embodiments, germ cells and CD3 - The somatic cell comprises replacement of all endogenous TCR Vα segments with a complete repertoire of unrearranged human TCR Vα segments and replacement of all endogenous TCR Jα segments with a complete repertoire of unrearranged human TCR Jα segments, wherein said complete repertoire of unrearranged human TCR Vα segments and said complete repertoire of unrearranged human TCR Jα segments are operably linked to each other and to a mouse TCR α constant region gene sequence at an 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 are capable of rearranging (or rearrangement) in a T cell to form a rearranged human TCR Vα / Jα variable region gene operably linked to said mouse 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 wherein the mouse comprises a CD3 T cell expressing on its surface a functional TCR comprising the chimeric TCRα polypeptide. + T cells.

[0032] In some humanized γ and / or δ TCR mouse embodiments, germ cells and CD3 -Somatic cells contain:

[0033] (A) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence; or

[0034] (B) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or

[0035] (C)(i) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence, and

[0036] (ii) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

[0037] In some embodiments,

[0038] (A) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments; or

[0039] (B) 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;

[0040] (C)(i) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments, and

[0041] (ii) the unrearranged human TCR Vδ segment comprises the complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segment comprises the complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segment comprises the complete repertoire of unrearranged human TCR Jδ segments.

[0042] In some humanized gamma and / or delta TCR mouse embodiments:

[0043] (I) the germ cells and the CD3 - T cells contain:

[0044] (A) at the endogenous TCRγ locus:

[0045] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0046] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0047] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0048] (B) at the endogenous TCRδ locus:

[0049] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0050] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0051] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0052] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0053] (II) the mouse comprises a CD3 T cell expressing on its surface a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide + T cells.

[0054] In some humanized gamma and / or delta TCR mouse embodiments: germ cells and CD3 -The somatic cell further comprises an unrearranged TCRβ variable region sequence, the unrearranged TCRβ 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 TCRβ variable region sequence is operably linked to a TCRβ constant region gene sequence, such as a mouse TCRβ constant region gene sequence, optionally at an endogenous TCRβ locus, wherein the unrearranged human TCR Vβ segment, the unrearranged human TCR Dβ segment and the unrearranged human TCR Jβ segment are capable of rearranging (or rearranging) in a T cell to form a rearranged human TCR Vβ / Dβ / Jβ variable region gene operably linked to the TCRβ constant region gene sequence, and wherein the rearranged human TCR operably linked to the TCRβ constant region gene sequence The Vβ / Dβ / Jβ variable region genes together encode a TCRβ polypeptide comprising a human TCRβ variable domain and a TCRβ constant domain (e.g., a mouse or human TCRβ constant domain); and wherein the mouse further comprises a CD3 T cell expressing on its surface a functional TCR comprising the TCRβ polypeptide. + In some embodiments, the unrearranged TCRβ variable region sequence comprises a mouse TCRB noncoding sequence.

[0055] In some humanized gamma and / or delta TCR mouse embodiments:

[0056] (I) the germ cells and the CD3-somatic cells comprise:

[0057] (A) at the endogenous TCRγ locus:

[0058] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0059] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0060] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0061] (B) at the endogenous TCRδ locus:

[0062] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0063] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0064] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0065] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0066] (C) at the endogenous TCRα locus:

[0067] Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and

[0068] Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments, and

[0069] (D) at the endogenous TCRβ locus:

[0070] Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments,

[0071] Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments, and

[0072] replacing all endogenous TCR Jβ segments with the complete repertoire of unrearranged human TCR Jβ segments; and

[0073] (II) the mouse further comprises a CD3 T cell expressing on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide + T cells, and CD3 T cells expressing on their surface a human or humanized TCR comprising a human or humanized TCR alpha polypeptide and a human or humanized TCR beta polypeptide + T cells.

[0074] In some humanized gamma and / or delta TCR mouse embodiments:

[0075] (I) the germ cells and the CD3 - Somatic cells contain:

[0076] (A) at the endogenous TCRγ locus:

[0077] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0078] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0079] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0080] (B) at the endogenous TCRδ locus:

[0081] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0082] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0083] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0084] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0085] (C) at the endogenous TCRα locus:

[0086] Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and

[0087] Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments;

[0088] (D) at the endogenous TCRβ locus:

[0089] Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments,

[0090] Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments,

[0091] Replace all endogenous TCR Jβ segments with the complete repertoire of unrearranged human TCR Jβ segments; and

[0092] (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 coreceptor 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; and

[0093] (F) a second nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide,

[0094] 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 wherein 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; and

[0095] (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC II alpha polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC II beta polypeptide,

[0096] wherein the chimeric human / mouse MHC II alpha polypeptide comprises an alpha 1 domain and an alpha 2 domain of a human HLA class II alpha polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II alpha polypeptide, and wherein the chimeric human / mouse MHC II beta polypeptide comprises a beta 1 domain and a beta 2 domain of a human HLA class II beta polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II beta polypeptide;

[0097] (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 the transmembrane domain and cytoplasmic domain of a mouse MHC class I polypeptide; and

[0098] (I) a polynucleotide sequence encoding a human or humanized β2 microglobulin polypeptide and 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,

[0099] (II) wherein the mouse further comprises a CD3 T cell expressing on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells, and CD3 T cells 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 + T cells, and optionally

[0100] wherein the mouse expresses:

[0101] (e) the chimeric human / mouse CD4 coreceptor,

[0102] (f) a chimeric CD8 coreceptor comprising said chimeric human / mouse CD8 alpha polypeptide and said chimeric human / mouse CD8 beta polypeptide,

[0103] (h) a chimeric MHC II complex comprising the chimeric human / mouse MHC II α polypeptide and the chimeric human / mouse MHC II β polypeptide, wherein the chimeric MHC II complex is capable of binding to the chimeric human / mouse CD4 coreceptor,

[0104] (i) the chimeric human / mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding to the chimeric CD8 coreceptor, and

[0105] (j) the human or humanized β2 microglobulin polypeptide.

[0106] In some humanized γ and / or δ TCR mouse embodiments, germ cells and CD3 - The somatic cells each comprise 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 germ cells and the somatic cells each comprise a human CTCF binding element upstream of the TCRα locus.

[0107] In some humanized γ and / or δTCR mouse embodiments, the mouse comprises γ / δT cells in its thymus, its spleen, its skin and / or its intestinal mucosa, wherein the γ / δT cells may comprise human TCRγ polypeptides and human TCRδ polypeptides. In some γ and / or δTCR mouse embodiments as described herein; optionally wherein the mouse also comprises human or humanized TCRα and β, MHC I, MHC IIα and β, CD4, CD8α and β, and / or β2M loci; the mouse may comprise a CD45+CD3+T cell population in its spleen, thymus, mesenteric lymph nodes (MLN), skin, intestinal mucosa and / or in intraepithelial lymphocytes (IEL) isolated from its colon and / or small intestine, wherein a certain percentage of the CD45+CD3+T cell population expresses human γ / δTCR. In some embodiments, the percentage of spleen, thymus, MLN, skin, intestinal mucosa and / or IEL CD45+CD3+T cells expressing human γ / δTCR in humanized γ and / or δTCR mouse embodiments as described herein is comparable to the percentage of CD45+CD3+T cells expressing mouse γ / δTCR in wild-type mice (e.g., no significant difference, any difference is statistically insignificant, within 10 percentage points of each other, etc.). In some embodiments, in γ and / or δTCR mouse embodiments as described herein, the percentage of spleen, thymus, MLN and / or IEL CD45+CD3+T cells expressing human γ / δTCR is greater than the percentage of CD45+CD3+T cells expressing mouse γ / δTCR in wild-type mice (e.g., 1.5-fold to 3-fold).

[0108] In some humanized γ and / or δTCR embodiments, 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. In some embodiments, 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. In some embodiments, 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 / DV8 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 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.

[0109] Also described herein is 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, wherein the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally at an endogenous TCRγ locus (e.g., a human TCRγ constant region gene sequence at an endogenous TCRγ locus, wherein a nucleotide sequence comprising an endogenous TCRγ 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 by a nucleotide sequence comprising a human TCRγ 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) δ variable region sequence, wherein the unrearranged T cell receptor (TCR) δ variable region sequence comprises an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment, wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus. In some embodiments, the ES cell or germ cell further comprises an unrearranged human TCR Vα segment located upstream of the unrearranged TCRδ variable region sequence and the human TCRδ constant region gene sequence.

[0110] Also described herein is a mouse ES cell or germ cell comprising, from 5' to 3':

[0111] Unrearranged human TCR Vα segment and

[0112] an unrearranged TCRδ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged TCR Dδ and an unrearranged human TCR Jδ segment,

[0113] wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus.

[0114] In some embodiments, ES cells or germ cells comprise replacing endogenous TCR Vα segments with unrearranged human TCR Vα segments and replacing 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 each other and to a TCR α constant region gene sequence (e.g., a mouse TCR α constant region gene sequence (optionally at an endogenous TCR α locus) or a human TCR α constant region gene sequence). In some embodiments, mouse ES cells or germ cells comprise replacing all endogenous TCR Vα segments with a complete library of unrearranged human TCR Vα segments and replacing all endogenous TCR Jα segments with a complete library of unrearranged human TCR Jα segments, wherein the complete library of unrearranged human TCR Vα segments and the complete library of unrearranged human TCR Jα segments are operably linked to each other and to a mouse TCR α constant region gene sequence at an endogenous TCR α locus.

[0115] In some embodiments, the mouse ES cell or germ cell comprises:

[0116] (A) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with an unrearranged human TCR γ constant region gene sequence; or

[0117] (B) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or

[0118] (C)(i) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence, and

[0119] (ii) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

[0120] In some ES cell or germ cell embodiments:

[0121] (A) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments; or

[0122] (B) 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; or

[0123] (C)(i) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments, and

[0124] (ii) the unrearranged human TCR Vδ segment comprises the complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segment comprises the complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segment comprises the complete repertoire of unrearranged human TCR Jδ segments.

[0125] In some embodiments:

[0126] (I) The ES cell or germ cell comprises:

[0127] (A) at the endogenous TCRγ locus:

[0128] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0129] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0130] Replace all endogenous TCRγ constant region gene sequences with a complete library of human TCRγ constant region gene sequences; and

[0131] (B) at the endogenous TCRδ locus:

[0132] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0133] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0134] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0135] The endogenous TCRδ constant region gene sequence was replaced with the human TCRδ constant region gene sequence.

[0136] In some embodiments, an endogenous TCRγ locus as described herein comprises replacing 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).

[0137] In some embodiments, the ES cell or germ cell further comprises an unrearranged TCRβ variable region sequence, the unrearranged TCRβ variable region sequence comprises 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 TCRβ variable region sequence is operably linked to a TCRβ constant region gene sequence (e.g., a mouse TCRβ constant region gene sequence), optionally at an endogenous TCRβ locus. In some embodiments, the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence.

[0138] In some embodiments, the ES cell or germ cell as described comprises:

[0139] (A) at the endogenous TCRγ locus:

[0140] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0141] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0142] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0143] (B) at the endogenous TCRδ locus:

[0144] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0145] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0146] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0147] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0148] (C) at the endogenous TCRα locus:

[0149] Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and

[0150] Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments, and

[0151] (D) at the endogenous TCRβ locus:

[0152] Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments,

[0153] Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments, and

[0154] All endogenous TCR Jβ segments were replaced with the complete repertoire of unrearranged human TCR Jβ segments.

[0155] In some embodiments, an ES cell or germ cell as described herein comprises:

[0156] (A) at the endogenous TCRγ locus:

[0157] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0158] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0159] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0160] (B) at the endogenous TCRδ locus:

[0161] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0162] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0163] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0164] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0165] (C) at the endogenous TCRα locus:

[0166] Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and

[0167] Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments, and

[0168] (D) at the endogenous TCRβ locus:

[0169] Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments,

[0170] Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments,

[0171] Replace all endogenous TCR Jβ segments with the full repertoire of unrearranged human TCR Jβ segments,

[0172] (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 coreceptor 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;

[0173] (F) a second nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide,

[0174] 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 wherein 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;

[0175] (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC II alpha polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC II beta polypeptide,

[0176] wherein the chimeric human / mouse MHC II alpha polypeptide comprises an alpha 1 domain and an alpha 2 domain of a human HLA class II alpha polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II alpha polypeptide, and wherein the chimeric human / mouse MHC II beta polypeptide comprises a beta 1 domain and a beta 2 domain of a human HLA class II beta polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II beta polypeptide;

[0177] (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 the transmembrane domain and cytoplasmic domain of a mouse MHC class I polypeptide; and

[0178] (I) A polynucleotide sequence encoding a human or humanized β2 microglobulin polypeptide and 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.

[0179] In some ES cell or germ cell embodiments, the ES cell or germ cell also includes a human CTCF binding element located upstream of the TCRγ locus, located upstream of the TCRα locus, or a first human CTCF binding element located upstream of the TCRγ locus and a second human CTCF binding element located upstream of the TCRα locus. In some embodiments, the mouse ES cell or germ cell also includes a human CTCF binding element located upstream of the TCRα locus. In some embodiments, the mouse ES cell or germ cell includes a human CTCF binding element located upstream of the TCRα locus. In some embodiments, the mouse ES cell or germ cell includes a human nucleotide sequence shown in chr7:38383439-38230960 (GRCh38 coordinates) and / or a human nucleotide sequence shown in Chr14:22421820-22464666 (GRCh38 coordinates).

[0180] Also described herein are targeting vectors, e.g., targeting vectors comprising a 5' mouse homology arm and a 3' mouse homology arm; from 5' to 3':

[0181] (a) 5' mouse homology arm,

[0182] (b) the human nucleotide sequence shown at chr7:38383439-38230960 (GRCh38 coordinates), and

[0183] (c) 3' mouse homology arm.

[0184] Also described is a targeting vector comprising (i) a selection cassette and (ii) a human nucleotide sequence shown in Chr14:22421820-22464666 (GRCh38 coordinates).

[0185] In some methods described herein, the method may include immunizing a genetically modified mouse as described herein with an antigen of interest, allowing the mouse to produce an immune response to the antigen of interest, and obtaining therefrom a nucleic acid sequence encoding a human TCR variable domain that binds to the antigen of interest, such as a nucleic acid sequence encoding a human TCRγ polypeptide (or its variable domain) of a TCR that binds to the antigen of interest, a nucleic acid sequence encoding a human TCRδ polypeptide (or its variable domain) of a TCR that binds to the antigen of interest, a nucleic acid sequence encoding a human TCRα / δ polypeptide (or its variable domain) of a TCR that binds to the antigen of interest (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 its variable domain) of a TCR that binds to the antigen of interest, a nucleic acid sequence encoding a human TCRβ polypeptide (or its variable domain) of a TCR that binds to the antigen of interest, and any combination thereof.

[0186] Also described herein is a method for preparing a human therapeutic agent (or human TCR protein), the method comprising: immunizing a genetically modified mouse as described herein with an antigen of interest, allowing the mouse to produce an immune response, obtaining T cells reactive to an antigen of interest from the mouse from the mouse, and obtaining a TCR (e.g., γδTCR, α / δγTCR, αβTCR) and / or a nucleic acid sequence encoding the variable domain of the TCR or encoding the TCR that binds to an antigen of interest from the T cell, wherein the TCR comprises a human TCR variable domain, and optionally employing the human TCR variable domain in a human therapeutic agent. In some embodiments, a human therapeutic agent (or human TCR protein) is a soluble T cell receptor. In some embodiments, a human therapeutic agent (or human TCR protein) is a single-chain TCR. In some embodiments, a human therapeutic agent (or human TCR protein) is a 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 immunosuppressive molecule (e.g., a molecule that inhibits T cells from killing other cells bearing an antigen recognized by the T cell).

[0187] Also described herein is a host cell comprising a nucleic acid molecule prepared according to any of the methods described herein.

[0188] Described herein is a method for preparing a genetically modified mouse or mouse ES cell, the method comprising modifying the genome of the mouse or mouse ES cell to comprise:

[0189] (a) an unrearranged TCRγ variable region sequence comprising an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment,

[0190] wherein the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally wherein the human TCRγ constant region gene sequence at the endogenous TCRγ locus, e.g., a nucleotide sequence comprising an endogenous TCRγ 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 by a nucleotide sequence comprising a human TCRγ constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence),

[0191] wherein the unrearranged human TCR Vγ segment and the unrearranged human TCR Jγ segment are capable of rearranging (or rearranging) in a T cell to form a rearranged human TCR Vγ / Jγ variable region gene operably linked to the human TCRγ constant region gene sequence, and

[0192] wherein the rearranged human TCR Vγ / Jγ variable region gene operably linked to the human TCRγ constant region gene sequence together encodes a human TCRγ polypeptide; and / or

[0193] (b) an unrearranged T cell receptor (TCR) delta variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment and an unrearranged human TCR Jδ segment,

[0194] wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence,

[0195] wherein the unrearranged human TCR Vδ segment, the unrearranged TCR Dδ segment and the unrearranged human TCR Jδ segment are capable of rearranging (or rearranging) in a T cell to form a rearranged human TCR Vδ / Dδ / Jδ variable region gene operably linked to the human TCR δ constant region gene sequence, and

[0196] The rearranged human TCR Vδ / Dδ / Jδ variable region gene operably linked to the human TCRδ constant region gene sequence together encodes a human TCRδ polypeptide.

[0197] In some embodiments, the modification comprises:

[0198] (a) replacing an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and endogenous TCR Cγ genes with a 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 (e.g., replacing 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) and / or

[0199] (b) replacing the endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ gene with a heterologous sequence comprising said unrearranged human TCR Vδ segment, said unrearranged human TCR Dδ segment, said unrearranged human TCR Jδ segment and said human TCRδ constant region gene sequence.

[0200] In some method embodiments:

[0201] (a) said 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

[0202] (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 and a TCR Cδ gene located between the TCR Vα and TCR Jα gene segments.

[0203] In some method embodiments:

[0204] (a) said heterologous sequence comprising said unrearranged human TCR Vγ segment and said unrearranged human TCR Jγ segment operably linked to said human TCRγ constant region gene sequence comprises a complete repertoire of unrearranged TCR Vγ and unrearranged human TCR Jγ segments and all human TCRγ constant region gene sequences (e.g., hTCRGC1 and hTCRGC2), and / or

[0205] (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 library of unrearranged TCR Vδ, unrearranged Dδ and unrearranged human TCR Jδ segments, and a human TCR Cδ gene sequence located between the human TCR Vα gene segment and the human TCR Jα gene segment on chromosome 14 of the human genome.

[0206] In some method embodiments, the modification includes homologous recombination in one or more ES cells, so that a 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; and a 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 is added to the genome of the one or more ES cells in any order. Some methods also include generating mice from the one or more ES cells.

[0207] In some method embodiments, the modifying comprises:

[0208] (a) obtaining a first mouse, said first mouse comprising homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with said heterologous sequence comprising said unrearranged human TCR Vγ segment and said unrearranged human TCR Jγ segment operably linked to said human TCRγ constant region gene sequence,

[0209] (b) obtaining a second mouse, said second mouse comprising homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ gene with said heterologous sequence comprising said unrearranged human TCR Vδ segment, said unrearranged human TCR Dδ segment, and said unrearranged human TCR Jδ segment operably linked to said human TCR δ constant region gene sequence, and

[0210] (c) breeding the first mouse and the second mouse to obtain a genetically modified mouse,

[0211] The genetically modified mouse comprises:

[0212] (i) replacing an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with said heterologous sequence comprising said unrearranged human TCR Vγ segment and said unrearranged human TCR Jγ segment operably linked to said human TCRγ constant region gene sequence, and

[0213] (ii) replacing an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ gene with said heterologous sequence comprising said unrearranged human TCR Vδ segment, said unrearranged human TCR Dδ segment, and said unrearranged human TCR Jδ segment operably linked to said human TCR δ constant region gene sequence, and

[0214] The genetically modified mouse expresses human TCRγ polypeptide and human TCRδ polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0215] Figure 1 Depict antigen recognition of different T cell receptor (TCR) embodiments.Left figure shows non-restrictive α / βT cell embodiment interacting with non-restrictive antigen presenting cell embodiment, wherein chimeric α / βTCR recognizes antigen (grey circle) presented in chimeric major histocompatibility complex (MHC) I polypeptide background, the polypeptide (i) comprises the extracellular domain of human leukocyte antigen (HLA) I class polypeptide, and (ii) associates with human β2 microglobulin (β2M).Middle figure shows non-restrictive α / βT cell embodiment interacting with non-restrictive professional antigen presenting cell embodiment, wherein chimeric α / βTCR recognizes antigen (grey circle) presented in chimeric MHC II, the chimeric MHC II (i) comprises the extracellular domain of HLA II class complex.Right figure shows non-restrictive γ / δT cell embodiment, it is independent of MHC recognition antigen.The domains and components of MHC I and MHC II are shown together with their respective auxiliary receptors CD8 and CD4. Mouse regions are solid, while human regions are unfilled / stippled.In various embodiments presented in the figures, the depicted associations can occur in a genetically modified animal (eg, a mouse) described herein.

[0216] Figure 2A strategy for inserting a human TCR δ locus into a humanized TCR α allele (MAID1771) of a mouse TCR α locus is depicted (not to scale), wherein the TCR δ locus is added to the downstream of the human TCR V α segment and the upstream of the human TCR J α segment. A strategy for inserting human CTCF binding elements upstream of the humanized TCR α locus is also shown. Unless otherwise specified, mouse coding sequences are indicated by closed symbols; human coding sequences are indicated by open symbols. Unless otherwise specified, mouse intergenic or non-coding sequences between coding sequences are indicated by solid lines; human intergenic or non-coding sequences between coding sequences are indicated by unfilled double lines. MAID refers to a 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 controlled by the ubiquitin promoter.

[0217] Figure 3A Depicted (not to scale) are deletions of all mouse TCRγV and J segments and the TCRγC gene from the mouse TCRγ locus to form the MAID20143 allele. Figure 3B Depicted is a strategy for inserting human TCRγV and J segments and human TCRγC genes into a mouse TCRγ locus lacking all mouse TCRγV and J segments and TCRγC genes to form a human TCRγ allele (MAID20200). Unless otherwise noted, mouse coding sequences are indicated by closed symbols; human coding sequences are indicated by open symbols. Unless otherwise noted, mouse intergenic or noncoding sequences between coding sequences are indicated by solid lines; human intergenic or noncoding sequences between coding sequences are indicated by unfilled double lines. Although in Figure 3B Not depicted in , but MAID20200 contains all 15 hTRGV segments, all 5 hTRGJ segments, and two hTRGC genes in germline configuration. MAID refers to 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 controlled by ubiquitin promoter.

[0218] Figure 4Depicted (not to scale) are the homozygous MAID6981 allele resulting from removal of the selection cassette from MAID6980 and the homozygous MAID20201 allele resulting from removal of the selection cassette from MAID20200. Figure 4 Not depicted in , but MAID20201 contains all 15 hTRGV segments, all 5 hTRGJ segments, and two hTRGC genes in germline configuration. Mouse coding sequences are indicated by closed symbols unless otherwise noted; human coding sequences are indicated by open symbols. Mouse intergenic or noncoding sequences between coding sequences are indicated by solid lines unless otherwise noted; human intergenic or noncoding sequences between coding sequences are indicated by unfilled double lines. MAID refers to modified allele ID number. H = human, TRAV = TCRVα 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 Cre recombinase removes the floxed drug cassette.

[0219] Figure 5A Depicted are representative FACS dot plots of mouse splenocytes from three different wild-type ("WT") mice and three mice homozygous for humanized TCR α / δ and γ and wild-type TCR β loci (6981HO 20201HO) stained with anti-human γ / δ (Y axis) and anti-mouse γ / δ (X axis) antibodies. Figure 5B Depicted are representative FACS dot plots of mouse thymocytes from three different wild-type ("WT") mice and three mice homozygous for humanized TCR α / δ and γ and wild-type TCR β loci (6981HO 20201HO) stained with anti-human γ / δ (Y axis) and anti-mouse γ / δ (X axis) antibodies.

[0220] Fig. 6A Depicted are the proportions of TCRd mRNA for the indicated human TCRαV and TCRδV segments in thymic or splenic T cells of mice homozygous for humanized TCRα / δ and γ and wild-type TCRβ loci (6981HO 20201HO), as determined by NGS. Figure 6B Depicted are the proportions of TCRd mRNA for the indicated TCRδ J segments present in thymic or splenic T cells of mice homozygous for humanized TCRα / δ and γ and the wild-type TCRβ locus (6981HO20201HO), as determined by NGS (Y axis).

[0221] Fig. 7ADepicted are the proportions of TCRg mRNA for the indicated human TCRγV segments present in thymic or splenic T cells of mice homozygous for humanized TCRα / δ and γ and wild-type TCRβ loci (6981HO 20201HO hTCRα) (Y axis). Figure 7B Depicted are the proportions of TCRg mRNA for the indicated human TCRγJ segments present in thymic or splenic T cells of mice homozygous for humanized TCRα / δ and γ and wild-type TCRβ loci (6981HO 20201HO hTCRα) (Y axis).

[0222] Fig. 8A Depicted (not to scale) a progressive strategy for the humanization of the mouse TCR α locus, in which TCR α variable region gene segments are sequentially added upstream of the initial humanization of the deleted mouse locus (MAID1540). Unless otherwise specified, mouse coding sequences are indicated with filled shapes; human coding sequences are indicated with open shapes. Although not depicted in this figure, each non-coding sequence between each person TRAV and each person TRAJ is human. The remaining intergenic or non-coding sequences are mouse. MAID refers to a modified allele ID number. TRAV = TCR Vα segment, TRAJ = TCR Jα segment (hTRAJ = human TRAJ), TRAC = TCR Cα domain, TCRD = TCRδ. Figure 8B Depicted (not to scale) a progressive strategy for the humanization of the mouse TCRβ locus, in which the TCRβ variable region gene segments are sequentially added to the missing mouse TCRβ variable loci. Unless otherwise indicated, mouse coding sequences are indicated by filled shapes; human coding sequences are indicated by blank shapes. Although not depicted in this figure, each non-coding sequence between each human TRBV, between human D1 and the nearest human J, between human D2 and each human J, and between each J is human. The remaining intergenic or non-coding sequences are mouse. MAID refers to a modified allele ID number. TRBV or TCRBV = TCRβV segment. Figure 8CThe following schematic diagrams (not to scale) are depicted: (a) TRBDJ1 cluster, wherein D1 and J1 gene segments are human and the non-coding sequence between them, including RSS and other intergenic sequences, is mouse, (b) mouse TRB C1 constant region gene sequence, (c) TRBDJ2 cluster, wherein D2 and J2 gene segments are human and the non-coding sequence between them, including RSS and other intergenic sequences, is mouse, and (d) mouse TRB C2 constant region gene sequence. Mouse coding sequences are indicated by filled shapes; human coding sequences are indicated by blank shapes. As shown in the figure, each non-coding sequence between each human TRBD segment and each human TRBJ segment and between each TRBJ segment is mouse. LoxP sequences are depicted as correspondingly labeled arrows.

[0223] Fig. 9A A schematic diagram (not to scale) of a chimeric CD4 locus is depicted. Human coding exon sequences are presented with striped shapes, mouse coding exon sequences are presented with filled shapes, and non-coding exon sequences are presented with hollow shapes. Immunoglobulin-like domains (Ig), transmembrane regions (TM), cytoplasmic regions (CYT) and signal peptide (signal) coding exons, as well as 3' untranslated regions (UTRs) are indicated. The floxed (loxP) neomycin phosphotransferase (Pgk-neo) box is depicted with correspondingly labeled arrows. Fig. 9B The schematic diagram (not to scale) of mosaic CD8a and CD8b loci is depicted. Human coding exon sequences are presented with striped shapes, mouse coding exon sequences are presented with filled shapes, and non-coding exon sequences are presented with hollow shapes. Immunoglobulin-like domains (IgV), transmembrane regions (TM), cytoplasmic regions (CYT) and signal peptide (signal) coding exons, and 3' untranslated regions (UTRs) are indicated. Floxed (loxP) hygromycin (Hyg) and neomycin phosphotransferase (Pgk-neo) boxes are depicted with correspondingly labeled arrows.

[0224] Figures 10A-10C provide schematic diagrams (not to scale) of exemplary chimeric MHC I and MHC II loci, such as chimeric HLA-A2 / H-2K loci (Figure 10A), chimeric HLA-DR2 / H-2E loci (Figure 10B), and humanized β2M loci (Figure 10C). Unless otherwise indicated, human sequences are depicted as hollow 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 (see Fig. 11B ). The floxed neomycin phosphotransferase cassette is depicted with correspondingly labeled arrows.

[0225] Figure 11A-11CA strategy for generating a humanized MHC locus comprising humanized MHC I and MHC II genes is depicted. Fig.11A In a specific embodiment depicted in FIG. 1 , the MHC locus of the generated mouse comprises chimeric HLA-A2 / H-2K and HLA-DR2 / H-2E sequences (H2-K + / 1666 MHC-II + / 6112 ) and lacks the H2-D sequence (H2-D + / 缺失 ) and H-2A sequences (the genetic engineering protocol also resulted in the deletion of H-2A). The large targeting vector (LTVEC) or Cre recombinase construct introduced into ES cells at each stage of humanization is depicted to the right of the arrow. MAID or 4-digit number refers to the modified allele ID number. Fig. 11B is a schematic diagram of an exemplary HLA-DR2 / H-2E large targeting vector (not to scale). Unless otherwise indicated, human sequences are depicted as open 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 is depicted as a correspondingly labeled arrow. Fig. 11C is a schematic diagram (not to scale) of an exemplary genotype of a chimeric human / mouse MHC locus (** represents an H-2L gene that is not present in all mouse strains, e.g., not present in the C57BL / 6 or 129 mouse strains), wherein 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 (open shapes framed by dashed lines), and the remaining loci are endogenous mouse genes (solid shapes framed by real lines)

[0226] Figures 12A-12D Depicted are the presence of γδ T cells in the spleen, thymus, intestinal tissue, and skin of mice homozygous for humanization of the TCRα / δ, TCRβ, TCRγ, MHC I, MHC IIα and β, CD4, CD8α and β, and β2M loci ("VelociTαβγδ mice") or wild-type (WT) mice. Fig. 12A Depicted are representative FACS dot plots of mouse CD45+CD3+ splenic T cells from two different VelociTαβγδ mice and two different WT mice stained with anti-human γ / δ (Y axis) and anti-mouse γ / δ (X axis) antibodies. Fig. 12B Depicted are representative FACS dot plots of mouse CD45+CD3+ thymic T cells from two different VelociTαβγδ mice and two different WT mice stained with anti-human γ / δ (Y axis) and anti-mouse γ / δ (X axis) antibodies. Fig. 12CDepicted are representative FACS dot plots of CD45+CD3+ T cells from mesenteric lymph nodes (MLN; first column) and intraepithelial lymphocytes (IELs) isolated from the colon (second column) and small intestine (third column) of two different VelociTαβγδ mice and two different WT mice stained with anti-human γ / δ (Y axis) and anti-mouse γ / δ (X axis) antibodies. Fig.12D Depicted are representative FACS contour plots of CD45+CD3+ skin epidermal T cells from two different VelociTαβγδ mice and two different WT mice stained with anti-human γ / δ (Y axis) and anti-mouse γ / δ (X axis) antibodies. DETAILED DESCRIPTION

[0227] Described herein are non-human animals (e.g., rodents (e.g., rats or mice)) having humanized TRG (encoding TCR-γ polypeptides) and / or TRD (encoding TCR-δ polypeptides) loci that can produce potential therapeutic agents that utilize human γ / δ T cells. As shown herein, for such genetically engineered mice, mice having human or humanized TRD (encoding TCR-δ polypeptides) and / or human 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 having full murine components ( Figure 5A and Figure 5B When present, these mice also use a diverse repertoire of human TCRγ gene segments and / or human TCRδ gene segments ( Fig. 6A , Figure 6B , Fig. 7A and Figure 7B), indicating the correct recombination, expression and thymic selection of human or humanized γ / δTCR. Tissues and cells expressing human or humanized γ / δTCR are also described herein; methods using animals, tissues and cells expressing human or humanized γ / δTCR; methods for preparing these animals; and embryonic stem (ES) cells and germ cells that can be used to produce such animals. In addition, human or humanized TRD and TRG loci are described herein, together with human or humanized TRA and TRAB loci and other components of T cell immunity, including non-human animals (e.g., mice) of TCR auxiliary receptors (CD4 and CD8) and MHC loci. Therefore, such mice will carry the complete humanization of α / β and γ / δT cell lineages, which is very different from the disclosed "VelociT" mice (Moore, M. et al. Sci Immunol 6 (2021); see above), which have α / β but no γ / δT cell humanization. VelociTαβγδ 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 / δCD45+CD3+ T cells in the thymus, spleen, skin, mesenteric lymph nodes (MLN), and in intraepithelial lymphocytes (IELs) isolated from the colon or small intestine at levels comparable to or greater than those of mice with full murine components ( Figures 12A-12D ). Also described are tissues and cells expressing human or humanized α / β and γ / δ T cell lineages, human or humanized T cell co-receptors, and MHC; methods of using such humanized animals, methods of making such animals; and embryonic stem (ES) cells and germ cells that can be used to generate such animals.

[0228] The terms "chain" and "polypeptide" encompass contiguous amino acids that are covalently linked and have a specific amino acid sequence; such terms can be used interchangeably herein. Typically, 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, a TCRα polypeptide or associated with a TCRβ polypeptide).

[0229] Those skilled in the art will understand that due to the degeneracy of the genetic code, in addition to the nucleic acid residues encoding the TCR variable region gene segments (e.g., TCR V, D and J gene segments) and / or optional humanized T cell co-receptor polypeptides, humanized MHC polypeptides and β2 microglobulin described herein, other nucleic acids may also encode the polypeptides of the present invention. Therefore, in addition to a genetically modified non-human animal whose genome comprises an unrearranged human TCRγ variable region gene segment, a human TCRγ constant region gene sequence, an unrearranged human TCRδ variable region gene segment and / or a human TCRδ constant region gene sequence (and optionally an unrearranged human TCRα variable region gene segment, an unrearranged human TCRβ variable region gene segment; a nucleotide sequence encoding a humanized T cell co-receptor polypeptide, such as a CD4 or CD8 polypeptide; and / or a nucleic acid sequence encoding a humanized MHC polypeptide); a non-human animal whose genome comprises such gene segments, constant region gene sequences and optional nucleotide sequences encoding humanized T cell co-receptor polypeptides (e.g., CD4 or CD8 polypeptides); and optionally a nucleic acid sequence encoding a humanized MHC polypeptide capable of associating with a humanized T cell co-receptor polypeptide is also provided, wherein the nucleic acid sequence differs from the nucleic acid sequence described herein due to the degeneracy of the genetic code, or differs and encodes conservative amino acid substitutions.

[0230] Also described herein are genetically modified non-human animals whose genome comprises (e.g., at an endogenous locus) a nucleotide sequence encoding a non-variable amino acid sequence or polypeptide (e.g., a TCR framework region, a TCR constant domain, a CD4 or CD8 polypeptide, an MHC polypeptide, etc.), wherein the non-variable amino acid sequence or polypeptide comprises a conservative amino acid substitution of an amino acid sequence described herein.

[0231] Conservative amino acid substitutions include replacement of one amino acid residue by 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 the nucleotide sequence to introduce nucleotide changes that will encode conservative substitutions. In general, conservative amino acid substitutions will not significantly change the functional properties of interest of the protein, for example, the ability of CD4 or CD8 to associate (e.g., bind) with MHC II or MHC I, respectively. Examples of amino acid groups with similar chemical properties in side chains 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 substitution groups include, for example, 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 any native residue in a protein replaced by alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, conservative substitutions are made, which conservative substitutions have positive values ​​in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45) (incorporated herein by reference). In some embodiments, substitutions are moderately conservative substitutions, wherein substitutions have non-negative values ​​in the PAM250 log-likelihood matrix.

[0232] 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, alignment is performed using ClustalW v.1.83 (slow) (with an open gap penalty of 10.0 and an extension gap penalty of 0.1) and using the Gonnet similarity matrix (MacVector TM10.0.2, MacVector Inc., 2008) to determine identity. With respect to sequence identity, the length of the sequence compared will depend on the specific 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 a chimeric human / non-human sequence is compared with a human sequence, the human portion of the chimeric human / non-human sequence (rather than the non-human portion) is used for comparison to determine the level of identity between the human sequence and the human portion of the chimeric human / non-human sequence (e.g., the human extracellular domain of a chimeric human / mouse protein is compared with the human extracellular domain of a human protein).

[0233] The term "homology" or "homologous" with respect to a sequence (e.g., a nucleotide or amino acid sequence) means that two sequences, when optimally aligned and compared, are identical in, for example, at least about 75% of the nucleotides or amino acids, such as at least about 80% of the nucleotides or amino acids, such as at least about 90%-95% of the nucleotides or amino acids, such as greater than 97% of the nucleotides or amino acids. One skilled in the art will appreciate that for optimal gene targeting, the targeting construct should contain arms that are homologous to the endogenous DNA sequence (i.e., "homology arms"); thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

[0234] The term "operably connected" refers to a juxtaposition of components described in this way that allow them to function in their intended manner. Therefore, the nucleic acid sequence encoding the protein can be operably connected to a regulatory sequence (e.g., a promoter, an enhancer, a static subsequence, etc.) to retain appropriate transcriptional regulation. In addition, the different parts of the chimeric or humanized protein of the present invention can be operably connected to maintain the correct folding, processing, targeting, expression and other functional properties of the protein in the cell. Unless otherwise indicated, the various domains of the chimeric or humanized protein of the present invention are operably connected to each other.

[0235] The term "replacement" about gene replacement refers to placing exogenous genetic material at the endogenous locus, thereby replacing all or part of the endogenous gene with an orthologous or homologous nucleic acid sequence. As demonstrated in the following examples, in one embodiment, the endogenous gene segment or constant region gene sequence of the TCRγ locus is replaced by (orthologous) human TCRγ gene segment or human TCRγ constant region gene sequence, and / or the endogenous gene segment or constant region gene sequence of the TCRδ locus is replaced by (orthologous) human TCRδ gene segment or human TCRδ constant region gene sequence, respectively.

[0236] As used herein, "functional", for example, with respect to a functional polypeptide, refers to a polypeptide that retains at least one biological activity that is generally associated with a native protein. For example, in some embodiments of the present invention, the endogenous non-human animal TCR locus of a non-human animal TCR gene is replaced with a human TCR gene to produce a locus that may not be able to express a functional endogenous TCR polypeptide, but may be able to express a functional human TCR polypeptide. As a non-limiting example, replacing 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 in a non-human animal, respectively, can produce a non-human animal that expresses a functional TCR (e.g., a TCR that can bind to an antigen, transmit an activation signal, and / or induce a CD3+T cell-mediated immune response) comprising a human TCRγ polypeptide and / or a human TCRδ polypeptide, respectively.

[0237] Non-human animals, tissues, cells and macromolecules

[0238] The adaptive immune system of vertebrates carries two T cell lineages that utilize diverse antigen receptors generated by somatic recombination of DNA segments encoding their antigen recognition domains or variable domains. T cells bind epitopes on small antigenic determinants on the surface of antigen presenting cells that are associated with major histocompatibility complexes (MHC; in mice) or human leukocyte antigens (HLA; in humans). T cells bind to these epitopes through T cell receptor (TCR) complexes on the surface of T cells, primarily recognizing peptide antigens presented by major histocompatibility complex (MHC) molecules. According to the embodiments described herein, non-limiting and 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 are shown in Figure 1 (Closed symbols represent non-human sequences; striped symbols represent human sequences).

[0239] The T cell receptor is a heterodimeric structure composed of two types of chains: an alpha (alpha) chain and a beta (beta) chain, or a gamma (gamma) chain and a delta (delta) chain. Alpha / beta (α / β) T cells are the most abundant T cell lineage. The alpha chain is encoded by a nucleic acid sequence located at the alpha locus (on chromosome 14 in humans or mice), which also encompasses the entire delta locus encoding the delta chain; the beta chain is encoded by a nucleic acid sequence located within the beta locus (on chromosome 6 in mice or chromosome 7 in humans); and the gamma chain is encoded by a nucleic acid sequence located within the gamma locus (on chromosome 13 in mice or chromosome 7 in humans). Most T cells have an alpha / beta TCR; while a minority of T cells carry a gamma / delta TCR.

[0240] 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, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant domain) that make up the variable and constant domains. The variable domains of the TCR determine its antigen specificity and contain three complementarity determining regions (CDRs).

[0241] The three-dimensional structure of the antigen recognition site of the T cell receptor looks similar to that of an antibody and mainly contains three complementary determining regions (CDR1, CDR2, CDR3), which are flanked by framework regions. The periphery of the antigen binding site contains the CDR1 and CDR2 loops, and the center of the antigen binding site of the TCR is formed by CDR3.

[0242] The structural diversity of T cell receptor is mainly due to the combination and connection diversity produced during rearrangement.The T cell receptor variable gene locus in germline DNA (for example, the DNA found in all germ cells) contains many TCR variable (V) segments, TCR diversity (D) segments (for TCR β and TCR δ seats) and TCR connection (J) segments of each of the unrearranged segments.During rearrangement, each segment in each multiple V (D) J segments is connected together during recombination to form the V / (D) / J variable region gene sequence of rearrangement, and the V / (D) / J variable region gene sequence of the rearrangement is operably connected with the TCR constant region gene sequence in such a way that the variable region gene sequence and the TCR constant region gene sequence encode TCR polypeptides together.The flank of the unrearranged TCR V, D and J gene segments is the recombination signal sequence (RSS) of 12-mer or 23-mer spacer length, and RSS guides the recombination according to the reorganization "12 / 23 rule" established. The D gene flanks in the TCRβ and TCRδ loci are 12RSS and 23RSS, and their recombination can be controlled by mechanisms other than the 12 / 23 rule. See, for example, Olaru A. et al. (2005) J.Immunol.174(10):6220-6226, which is incorporated herein by reference in its entirety. Any TCR segment operably connected to the RSS has not yet undergone recombination and can therefore be considered to be an "unrearranged" segment. Therefore, each "unrearranged" TCRV segment, D segment, or J segment is operably connected to a recombination signal sequence (RSS) (e.g., associated with the RSS, one or both sides are flanked by the RSS, adjacent to the RSS, etc.), and the RSS can be a 12mer RSS or a 23mer RSS. In this manner and according to the 12 / 23 rule: an unrearranged TCR Vα segment can rearrange with an unrearranged TCR Jα segment to form a TCR Vα / Jα gene sequence encoding a TCRα variable domain, an unrearranged TCR Vβ segment can rearrange with an unrearranged TCR Dβ segment and an unrearranged TCR Jβ to form a TCR Vβ / Dβ / Jβ gene sequence encoding a TCRβ variable domain, an unrearranged TCR Vγ segment can rearrange with an unrearranged TCR Jγ segment to form a TCR Vγ / Jγ gene sequence encoding a TCRγ variable domain, an unrearranged TCR Vδ segment can rearrange with an unrearranged TCR Dδ segment and an unrearranged TCR Jδ segment to form a TCR Vδ / DδJδ gene sequence encoding a TCRδ variable domain, and in some cases, an unrearranged TCR Vα segment can rearrange with an unrearranged TCR Dδ segment and an unrearranged TCR The Jδ segments rearrange to form the TCR Vα / DδJδ gene sequence encoding the hybrid TCRα / δ variable domain. After rearrangement, the T cells mature and enter the periphery.Therefore, somatic cells do not express markers of T cell maturation, such as CD3. - Somatic cells may also contain "germline" or unrearranged T cell receptor variable region sequences.

[0243] The TCRα locus (chromosome 14 of mice and humans) contains a cluster of Vα gene segments, each of which is preceded by an exon encoding a leader sequence (L). The Jα gene segment cluster is located at a considerable distance from the Vα gene segment. Behind the Jα gene segment is a single α constant (C) region gene sequence, which contains a single exon of a constant domain and a hinge domain and a single exon encoding a transmembrane region and a cytoplasmic region. The TCRβ locus (chromosome 6 of mice, chromosome 7 of humans) has a different organizational structure, with the Vβ gene segment cluster located away from two separate clusters (e.g., TCRBDJ1 cluster and TCRBDJ2 cluster) - each cluster contains a single D gene segment, together with 6 or 7 J gene segments and a single Cβ gene. Each TCR Cβ gene has a single exon encoding a constant region, a hinge region, a transmembrane region, and a cytoplasmic region. The TCRα locus is interrupted by another T cell receptor locus - the TCRδ locus between the V and J gene segments.

[0244] The genomic organization of the TCRγ locus and the TCRδ locus is significantly different from that of the TCRα locus and the TCRβ locus. 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 the Vα gene segment cluster and the Jα gene segment cluster; the Vδ gene segments are interspersed between the Vα gene segments, and the Vδ3 gene segments are between the TCR Cδ gene and the Jα segment cluster. See Janeway's Immunobiology, Chapter 4, 5th Edition, edited by Murphy et al., Garland Science, 2001. The TCRγ locus consists of four different constant (C) region gene sequences (3 functional gene sequences) in mice and two γ constant region gene sequences (TCRGC1 and TCRGC2) in humans, each Cγ region gene sequence containing its own Jγ gene segment cluster. See, for example, Figure 3B .

[0245] The repertoire of specific T cell receptors (TCRs) is generated during T cell development through a complex developmental program in the thymus. The diversity of this repertoire is maintained by the expression of different TCR α / β or γ / δ chains and the variable regions encoded by several variable (V), joining (J) and diversity (D) gene segments.

[0246] Generally, it is understood that the TCR gene segments are rearranged during T cell development to form complete variable domain exons. The TCRα variable (Vα) and joining (Jα) gene segments undergo rearrangement so that the resulting TCRα chain is encoded by a specific combination of VJ segments (Vα / Jα sequences) operably linked to the TCRα constant (Cα) region gene sequence; the TCRβ variable (Vβ), diversity (Dβ) and joining (Jβ) gene segments undergo rearrangement so that the resulting TCRβ chain is encoded by a specific combination of VDJ segments (Vβ / Dβ / Jβ sequences) operably linked to the TCRβ constant (Cβ) region gene sequence; the TCRγ variable (V The TCRγ chain is encoded by a specific combination of VJ segments (Vγ / Jγ sequences) operably linked to the TCRγ (Cγ) gene; the TCRδ variable (Vδ), diversity (Dδ) and joining (Jδ) gene segments undergo rearrangement so that the resulting TCRδ chain is encoded by a specific combination of VDJ segments (Vδ / Dδ / Jδ sequences) operably linked to the TCRδ (Cδ) gene, and sometimes by VDDJ (Vδ / Dδ / Dδ / Jδ sequences) operably linked to the TCRδ (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 additional N region nucleotides can be added to the junction between the two D gene segments and to the VD and DJ junctions.

[0247] TCR diversity may be mainly due to the combination and connection diversity generated during the gene rearrangement process. Most of the variability of TCR chains is found in the junction area encoded by V, D and J gene segments and modified by P-nucleotides and N-nucleotides. This region encodes the CDR3 loop in the TCR chain forming the center of the antigen binding site. Therefore, the center of the TCR chain will be highly variable, and the periphery will undergo relatively small changes.

[0248] Interaction with the thymic stroma triggers thymocytes to undergo several developmental stages, characterized by the expression of various cell surface markers. Table 1 presents a summary of characteristic cell surface markers of various developmental stages in the thymus. The rearrangement at the TCRβ variable gene locus begins at the DN2 stage and ends at the DN4 stage, while the rearrangement of the TCRα variable gene locus occurs at the DP stage. After the TCRβ locus rearrangement is completed, the cell expresses the TCRβ chain and the alternative α chain pTα on the cell surface. See Janeway's Immunobiology, Chapter 7, 7th edition, edited by Murphy et al., Garland Science, 2008.

[0249] Table 1: Developmental stages of T cells in the thymus

[0250]

[0251] Naive CD4+ and CD8+ T cells leave the thymus and enter peripheral lymphoid organs (e.g., spleen), where they are exposed to antigens and activated to clonally expand and differentiate into many effector T cells (Teff), such as cytotoxic T cells, T REG Cells, T H 17 cells, T H 1 cells, T H 2 cells, etc. After infection, many T cells persist as memory T cells and are classified as 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 colleagues proposed that after initial infection, Tem cells represent easily accessible antigen-sensitized memory T cell pools with effector functions in peripheral tissues, while Tcm cells represent antigen-sensitized memory T cells in peripheral lymphoid organs that can become new effector T cells after secondary stimulation. Although 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+, both of which are important for binding to and signaling in peripheral lymphoid organs and lymph nodes. Same as above. Therefore, 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 many different cell surface markers, such as CD44. For a summary of various cell surface markers on T cells, see Janeway's Immunobiology, Chapter 10, supra.

[0252] Early studies led scientists to believe that α / β and γ / δ T cells develop in a sequential manner, for example, when the γ / δ TCR rearrangement is nonfunctional, T cells will develop from developing α / β thymocytes. 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 reference is incorporated herein by reference in its entirety. However, studies of genetically modified mice (KN6 mice with Vγ4Jγ1Cγ1 and Vδ5DJδ1Cδ rearranged sequences) that express integrated rearranged TCRγ and / or TCRδ sequences in the germline contradict the sequential rearrangement model because such expression does not prevent normal development of α / β T cells. It was also confirmed that knockout of the TCR Cα gene had no effect on the production of γ / δ thymocytes or the development of γ / δ T cells, but instead caused an increase in the number of γ / δ thymocytes that may be derived from non-canonical developmental pathways. Knockout of mouse TCR Cβ also did not show developmental block in γ / δ T cell development, so it can be inferred that double positive cells do not seem to be intermediates in TCRγ / δ T cell development. Kreslavasky, T. et al. (2010) Curr Opin Immunol. 22: 185-192; Mombaerts P. et al., (1992) 360: 225-231. Other extensive studies of various knockout and TCR transgenic mice have proposed several different models of requirements for lineage determination and maturation into specific T cell lineages, which are reviewed in Hahn, AM and Winkler, TH, (2020) J. Leuk. Bio. 107: 2019, which are incorporated herein by reference in their entirety. The stochastic model proposes that fate determination occurs randomly before TCR expression. Using Vγ4-Jγ1Cγ1-modified mice, it was demonstrated that cells with high IL-7Rα showed biased differentiation toward a γ / δ fate. Based on studies of Vγ6Jγ1Cγ1 and Vδ1Dδ1Jδ2Cδ-modified mice, the signal strength model proposes that the strength of TCR signaling is a key determinant of lineage fate decisions and that γ / δTCR signaling may be an essential 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.

[0253] Although the TCR variable domain plays a major role in antigen recognition, the extracellular portion of the constant domain of the TCR, as well as the transmembrane domain and the cytoplasmic domain, also play an important role. A complete TCR receptor complex requires more than α and β, or γ and δ polypeptides; additional molecules required include CD3γ, CD3δ and CD3ε, and ζ chain homodimers (ζζ). When TCRβ rearrangement is complete, when cells express TCRβ / pTα, this pre-TCR complex is present on the cell surface together with CD3. The TCRα (or pTα) on the cell surface has two basic residues in its transmembrane domain, one of which recruits CD3γε heterodimers, and the other recruits ζζ via their respective acidic residues. TCRβ has additional basic residues in its transmembrane domain, which are believed to recruit CD3δε heterodimers. See, e.g., 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. An assembled complex comprising TCRαβ heterodimers, CD3γε, CD3δε, and ζζ is expressed on the surface of T cells. It has been proposed that polar residues in the transmembrane domain serve as quality control for leaving the endoplasmic reticulum; it has been demonstrated that in the absence of CD3 subunits, TCR chains remain in the ER and are 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.

[0254] Since the TCRαβ heterodimer (or TCRγ / δ heterodimer) itself lacks signal transduction activity, the CD3 and ζ chains of the assembled complex provide components for TCR signal transduction. Each CD3 chain has an activation motif (ITAM) based on immunoreceptor tyrosine, and the ζ chain contains three tandem ITAMs. ITAM contains tyrosine residues that can be phosphorylated by related kinases. Therefore, the assembled TCR-CD3 complex contains 10 ITAM motifs. See, for example, Love and Hayes (2010) ITAM-MediatedSignaling by the T-Cell Antigen Receptor, Cold Spring Harb.Perspect.Biol.2:e002485. After TCR engagement, the ITAM motif is phosphorylated by the Src family tyrosine kinases Lck and Fyn, thereby triggering a signal cascade, leading to Ras activation, calcium mobilization, actin cytoskeleton rearrangement and transcription factor activation, all of which ultimately lead to T cell differentiation, proliferation and effector action. Ibid., see also Janeway's Immunobiology, supra, both of which are incorporated herein by reference.

[0255] In addition, the TCRβ transmembrane domain and cytoplasmic domain are believed to play a role in mitochondrial targeting and apoptosis induction; in fact, naturally occurring N-terminally truncated TCRβ molecules are present in thymocytes. Shani et al. (2009) Incomplete T-cell receptor--βpeptides target the mitochondrion and induce apoptosis, Blood 113: 3530-41. Therefore, the TCR constant domain (which, in various embodiments, comprises a portion of the extracellular domain as well as the transmembrane domain and the cytoplasmic domain) provides several important functions; and in various embodiments, when designing a humanized TCR or a genetically modified non-human animal expressing a humanized TCR, the structure of this region should be considered.

[0256] The signal strength model is related to the observation that γ / δTCRs have different signal potential CD3 complex compositions. Hayes, SM and Love PE, (2002) Immunity, see above. γ / δT cell development also begins in the immature DN stage; however, γ / δT lineage cells do not undergo developmental stages defined by the expression of pre-TCR or CD4 / CD8 co-receptors. In contrast, in-frame rearrangements of TCRγ and TCRδ genes in DN thymocytes cause surface expression of mature γ / δTCRs, and signals transduced by mature γ / δTCRs are thought to regulate the commitment of immature DN cells to the γ / δT cell lineage and are required for differentiation into mature γ / δT cells. It has been reported that the structure of γ / δTCRs is similar to that of α / βTCRs, except that γ / δTCRs can include Fc∈RIγ (FcRγ) chains. 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. Loss of CD3γ, CD3ε, or CD3ζ blocks the development of γ / δ T cells, while loss of CD3δ or FcRγ has no effect on the maturation of γ / δ TCRs. + Hayes et al. (2002) Immunity demonstrated that despite the absence of CD3δ, γ / δ TCRs signaled better than α / β TCRs as measured by their ability to induce calcium mobilization, MAP kinase activation, and cell proliferation.

[0257] Gamma / delta (γ / δ) T cells are still less characterized. T cells carrying γ / δTCR are a unique lineage of T cells and appear to be able to directly recognize antigens, just like antibodies, without the need for presentation or processing of MHC molecules, and are able to recognize multiple antigens (e.g., phosphoproteins, lipids, glycolipids) independently of MHC. Although γ / δT cells are rare in peripheral blood, they can be detected in the thymus, spleen, and lymph nodes, and are very abundant in selected tissue sites such as intestinal tissue (e.g., colon, intestine (e.g., small intestine)) and skin under continuous monitoring of microorganisms and other environmental antigens (JCRibot, N.Lopes, B.Silva-Santos, γ / δTcells in tissue physiology and surveillance.NatRev Immunol 21, 221–232 (2021); incorporated herein by reference in its entirety). Relative to α / βT cells, γ / δ cells have a more limited TCR library and acquire specific cytokine production and tissue homing properties early in development. These features, as well as the lack of MHC restriction and recognition of antigens associated with "danger" signals, describe the properties of this T lymphoid subset conventionally associated with "innate" immunity (B. Silva-Santos, S. Mensurado, SB B Cofelt, 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).

[0258] Described herein are genetically modified non-human animals (e.g., rodents, such as rats, mice) that can be used as animal models of human or humanized cellular responses involving γ / δ T cells to study such responses, develop human therapeutics, etc. Typically, a genetically modified non-human animal as described herein comprises an unrearranged human or humanized (e.g., human γ and / or δ; and / or human α and / or β) T cell variable gene locus that is capable of rearranging (or rearranging) in a T cell to form a nucleic acid sequence encoding a human T cell receptor variable domain, including animals comprising T cells containing 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, such as rats, mice) capable of producing a diverse library of human T cell receptor variable region sequences; therefore, the present invention provides TCRs and / or fully human TCRs (e.g., human TCRγ chain and / or human TCRδ chain) expressing a fully human variable domain (e.g., human TCRγ variable domain and / or human TCRδ variable domain) in response to an antigen of interest and binding to an epitope of an antigen of interest. In some embodiments, non-human animals are provided that produce a diverse library of T cell receptors (γ / δT cells and α / βT cells) capable of reacting with various antigens, including but not limited to antigens not presented by APCs (recognized by γ / δT cells) and antigens presented by APCs (recognized by α / βT cells).

[0259] In one embodiment, the present invention provides genetically modified non-human animals (e.g., rodents, such as rats, mice), which contain unrearranged human TCR variable region segments (V(D)J segments) in their genomes, wherein the unrearranged human TCR variable region segments replace endogenous non-human TCR variable region segments at endogenous non-human (e.g., rodent) TCR variable gene loci (e.g., TCRγ and / or TCRδ loci, and / or TCRα and / or TCRβ loci). In one embodiment, unrearranged human TCR variable gene loci replace endogenous non-human TCR variable gene loci.

[0260] In another embodiment, the present invention provides a genetically modified non-human animal (e.g., rodent, such as rat, mouse) comprising an unrearranged human TCR variable region segment (V (D) J segment) in its genome, wherein the unrearranged human TCR variable region segment is operably linked to a human or non-human TCR constant region gene sequence, producing a human or humanized TCR locus, respectively, wherein the human or humanized TCR locus is located at a site other than the endogenous non-human TCR locus in the genome. Therefore, in one embodiment, a non-human animal (e.g., rodent, such as mouse, rat) comprising a transgenic is also provided, the transgenic comprising an unrearranged human TCR variable region segment operably linked to a human or non-human TCR constant region gene sequence.

[0261] In some embodiments, the genetically modified non-human animal of the present invention comprises (a) human TCR variable region segments in its genome, operably connected to (b) people encoding TCR constant domains or retained non-human (e.g., rodents, such as mice, rats) TCR constant region gene sequences. As shown above, the constant domains of TCR participate in the signal cascades triggered during activation of antigen-sensitized T cells; therefore, TCR constant domains interact with a variety of anchor proteins and signal proteins in T cells. Therefore, in one aspect, the genetically modified non-human animal of the present invention expresses human or humanized T cell receptors, which retain the ability to recruit a variety of endogenous non-human anchors or signal molecules, such as CD3 molecules (e.g., CD3γ, CD3δ, CD3ε), ζ chains, Lck, Fyn, ZAP-70, etc. A non-limiting list of molecules recruited into the TCR complex is described in Janeway's Immunobiology, see above. In some embodiments, a non-human animal as described herein or a cell of the non-human animal comprises a human T cell polypeptide as described herein (e.g., a human TCRγ polypeptide and / or a human δ polypeptide), which is associated with a non-human animal (e.g., a rodent, such as a rat or mouse) TCR anchor or signaling molecule, such as a CD3 molecule (e.g., CD3γ, CD3δ, CD3ε), a ζ chain, Lck, Fyn, ZAP-70 and / or a non-human animal Fc∈RIγ (FcRγ) chain, etc.

[0262] Therefore, in various embodiments, the present invention generally provides genetically modified non-human animals, wherein these non-human animals include unrearranged humanized TCR variable gene loci in the genome, such as unrearranged human TCR variable gene regions, which are included in, for example, T cells (e.g., T cells of mice) that can be reorganized (or reorganized) to form human TCR variable gene sequences of rearranged TCR variable gene sequences. As used herein, TCR loci or TCR gene loci refer to the position of TCR coding regions included in genomic DNA, including the entire TCR coding region, including unrearranged V (D) J sequences, enhancer sequences, constant region gene sequences and any upstream or downstream (UTR, regulatory regions, etc.) or inserted DNA sequences (introns, etc.). TCR variable loci, TCR variable regions or TCR variable gene loci refer to genomic DNA including TCR variable region segments (V (D) J regions), but do not include TCR constant region gene sequences and in various embodiments, do not include the position of enhancer sequences. For the purpose of genetic manipulation, other sequences (e.g., selection boxes, restriction sites, etc.) may be included in the TCR variable gene locus, and these are all included in this article. In various aspects, non-human animals include a contiguous portion of a human genome TCR variable locus, which includes V, D and J segments, or D and J segments, or V and J segments, or V segments, such as those arranged in the human genome TCR variable locus, which are not rearranged, For example, including promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., such as those arranged in the human genome TCR variable locus. Reference to contiguous human sequences for TCRG, TCRD, TCRA and / or TCRB sequences also generally refers to fully human sequences, e.g., wherein the TCR coding sequences (e.g., TCR gene segments) and the TCR non-coding sequences (e.g., non-coding DNA that separates and flanks the TCR gene segments, such as the non-coding recombination signal sequence (RSS) and other non-coding intergenic sequences) are both human, and preferably, wherein the TCR gene segments and the TCR non-coding sequences are in the same order that they can be found in the human germline.

[0263] In various embodiments of people or humanized TCRγ loci and / or people and / or humanized TCRδ loci and optionally people or humanized TCRα and / or TCRβ loci, humanized loci may include people coding sequences (e.g., TCR gene segments) and non-human (e.g., mouse) TCR non-coding sequences. In some embodiments, human TCR gene segments replace orthologous non-human (e.g., mouse) TCR gene segments, so that the human TCR gene segments are flanked by the same non-human (e.g., mouse) TCR gene segments of the orthologous non-human (e.g., mouse) TCR gene segments that are replaced, for example, so that the order of human TCR gene segments and non-human (e.g., mouse) TCR non-coding sequences is the same as that found in non-human (e.g., mouse) germline, but the orthologous (e.g., non-human) TCR gene segments are replaced. See, for example, Figure 8C .

[0264] In other aspects, various segments are arranged in unrearranged non-human genome TCR variable loci. In various embodiments of humanized TCR α, β, δ and / or γ loci, the humanized loci may include two or more human genome segments that do not appear in parallel in the human genome, for example, a fragment of a human variable locus V segment located near a constant region in the human genome is juxtaposed with a fragment of a human variable locus V segment located in the human genome at the upstream end of the human variable locus.

[0265] Human or humanized gamma and / or delta TCR responses

[0266] In some embodiments, a non-human animal is provided that comprises an unrearranged human TCRγ variable region segment in its genome, wherein the unrearranged human TCRγ variable region segment is operably linked to a human or non-human TCRγ constant region gene sequence, producing a human or humanized TCRγ locus, respectively. In one embodiment, the human or humanized TCRγ locus is at a site in the genome other than an endogenous non-human TCRγ locus. In another embodiment, the unrearranged human TCRγ variable region segment replaces the endogenous non-human TCRγ variable region segment, and the human TCRγ constant region gene sequence replaces the endogenous non-human TCRγ constant region gene sequence, (e.g., wherein the nucleotide sequence comprising the endogenous TCRγ constant region gene sequence (e.g., the endogenous Trgc1 constant region gene sequence, the endogenous Trgc2 constant region gene sequence, the endogenous Trgc3 constant region gene sequence and / or the endogenous Trgc4 constant region gene sequence) is replaced by a nucleotide sequence comprising the human TCRγ constant region gene sequence (e.g., the human TRGC1 constant region sequence and / or the human TRGC2 constant region sequence). In one embodiment, the unrearranged human TCRγ variable gene locus replaces the endogenous non-human TCRγ variable gene locus.

[0267] In some embodiments, the unrearranged TCRγ variable gene locus comprising human variable region segments (e.g., human Vγ and Jγ segments) is located in a non-human genome, so that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, the unrearranged TCRγ variable gene locus comprising human variable region segments replaces the endogenous TCRγ variable gene locus. In one aspect, endogenous non-human Vγ and Jγ segments cannot be rearranged to form rearranged Vγ / Jγ sequences. Therefore, in one aspect, the human Vγ and Jγ segments in the unrearranged TCRγ variable gene locus can be rearranged (or rearranged) in T cells (e.g., T cells of mice) to form rearranged human Vγ / Jγ sequences.

[0268] In some embodiments, the non-human animal of the present invention comprises an unrearranged humanized TCRγ locus, for example, 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 library of functional unrearranged human Vγ segments and a complete library of functional unrearranged human Jγ variable region segments). In some embodiments, the non-human animal of the present invention comprises an unrearranged human TCRγ locus, for example, a TCRγ locus comprising at least one functional unrearranged human Vγ segment operably linked to at least one functional human TCR Cγ gene (e.g., a complete library of functional human TCR Cγ genes) and at least one functional unrearranged human Jγ segment (e.g., a complete library of functional unrearranged human Vγ segments and a complete library of functional unrearranged human Jγ segments), optionally wherein the functional unrearranged human Vγ segments, the functional unrearranged human Jγ segments and the functional human TCR Cγ genes are in the same order as found in the germline human TCRγ locus. The number and location of the various TCRγ segments can be determined from the IMGT database. The human TCRγ locus is on human chromosome 7, while the mouse TCRγ locus is on mouse chromosome 13.

[0269] The mouse TCRγ variable locus is approximately 200 kilobases and contains 7 TRGV gene segments, 4 TRGJ gene segments, and 4 TRGC genes (3 of which are functional) belonging to 5 subgroups. The human TCRγ variable locus is approximately 160 kilobases and contains 12-15 TRGVs (TRGV1, TRGV2, TRGV3, TRGV3P, TRGV4, TRGV5, TRGV5P, TRGV6, TRGV7, TRGV8, TRGV9, TRGV10, TRGV11, TRGVA, TRGVB), which belong to 6 subgroups and are located upstream of the repeated JC cluster. The first part contains 3 TRGJs (TRGJP1, TRGJP, and TRGJ1) and TRGC1 genes, and the second part contains 2 TRGJs (TRGJP2 and TRGJ2) and TRGC2 genes. Although in Figure 3BIt is not depicted in, but MAID20200 includes all 15 hTRGV segments, all 5 hTRGJ segments and two hTRGC genes of germline configuration. Unless otherwise specified, the number of human V (D) J segments mentioned in the 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, such as a mouse or a rat) includes at least one human Vγ and at least one human Jγ segment. In some embodiments, the non-human animal includes a human or humanized TCRγ locus, and the human or humanized TCRγ locus includes 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 includes at least one of a functional human Vγ gene segment. In some embodiments, the human or humanized TCRγ locus includes two of a functional human Vγ gene segment. In some embodiments, the human or humanized TCRγ locus comprises three of the functional human Vγ gene segments. In some embodiments, the human or humanized TCRγ locus comprises four of the functional human Vγ gene segments. In some embodiments, the human or humanized TCRγ locus comprises five of the functional human Vγ gene segments. In some embodiments, the human or humanized TCRγ locus comprises six of the functional human Vγ gene segments. In some embodiments, the functional human Vγ gene segments are selected from human TRGV2, human TRGV3, human TRGV4, human TRGV5, human TRGV8, human TRGV9, and variants thereof. Thus, in some embodiments, the humanized TCRγ 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% of human Vγ; in some embodiments, it may comprise about 2%, about 3%, about 15%, about 65%, about 90% or 100% of human Vγ. In some embodiments, the non-human animal comprises a human or 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, which comprises a human TRGJP1 gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus, which comprises a human TRGJP gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus, which comprises a human TRGJP gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus, which comprises a human TRGJ1 gene segment.In some embodiments, the non-human animal comprises a human or humanized TCRγ locus, which comprises a human TRGJP2 gene segment. In some embodiments, the non-human animal comprises a human or humanized TCRγ locus, which comprises a human TRGJ2 gene segment. In some embodiments, the non-human animal comprises an endogenous TCRγ locus, which comprises a human TRGC1 gene operably connected to human TRGV and TRJV segments. In some embodiments, the non-human animal comprises an endogenous TCRγ locus, which comprises a human TRGC2 gene operably connected to human TRGV and TRJV segments.

[0270] In one embodiment, the non-human animal comprises a human TCRγ locus, which comprises a DNA fragment containing contiguous human sequences from human Vγ1 (Vγ segment is also called "TRGV" or "TCRGV") to human Cγ2 ("TCRGC2"; Cγ is also called "TRGC" or "TCRGC"), and the contiguous human sequences include the human Jγ gene segment (Jγ segment is also called "TRGJ" or "TCRGJ") and the human Cγ1 ("TCGRC1") gene between the human Vγ gene segment and the human Cγ2 gene. TCRG non-coding sequences refer to contiguous non-coding sequences, including non-coding recombination signal sequences (RSS) and other non-coding intergenic sequences 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.

[0271] In some embodiments, a humanized TCRγ mouse as described herein comprises:

[0272] (I) germ cells and CD3- somatic cells comprising an unrearranged TCRγ variable region sequence, wherein the unrearranged TCRγ variable region sequence comprises an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment,

[0273] wherein the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally at an endogenous TCRγ locus (e.g., a human TCRγ constant region gene sequence at an endogenous TCRγ locus, wherein a nucleotide sequence comprising an endogenous TCRγ 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 by a nucleotide sequence comprising a human TCRγ constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence),

[0274] wherein the unrearranged human TCR Vγ segment and the unrearranged human TCR Jγ segment are capable of rearranging (or rearranging) in a T cell to form a rearranged human TCR Vγ / Jγ variable region gene operably linked to the human TCRγ constant region gene sequence, and

[0275] wherein the rearranged human TCR Vγ / Jγ variable region gene operably linked to the human TCRγ constant region gene sequence together encodes a human TCRγ polypeptide, and

[0276] (II) CD3 expressing a functional TCR comprising a human TCRγ polypeptide on its surface + T cells.

[0277] In some embodiments, 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. In some embodiments, 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.

[0278] In some humanized TCRγ mouse embodiments, germ cells and CD3 - The somatic cell also comprises an unrearranged T cell receptor (TCR) δ variable region sequence, the 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, wherein the unrearranged TCR δ variable region sequence is operably linked to a human TCR δ constant region gene sequence, optionally at an endogenous TCR δ locus, and wherein the unrearranged human TCR Vδ segment, the unrearranged human TCR Dδ segment, and the unrearranged human TCR Jδ segment are capable of rearranging (or rearranging) in the T cell to form a rearranged human TCR Vδ / Dδ / Jδ variable region gene operably linked to the human TCR δ constant region gene sequence, and wherein the rearranged human TCR δ constant region gene sequence is operably linked to the human TCR The Vδ / Dδ / Jδ variable region genes together encode a human TCRδ polypeptide, wherein the mouse further comprises a CD3 T cell expressing on its surface a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells.

[0279] In some embodiments, 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 / DV8 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 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.

[0280] In some embodiments, the non-human animal comprises in its genome (with or without a human or humanized TCRγ locus as described herein) an unrearranged human TCRδ variable region segment, wherein the unrearranged human TCRδ variable region segment is operably connected to a human or non-human TCRδ constant region gene sequence, producing a human or humanized TCRδ locus, respectively. In one embodiment, the humanized TCRδ locus is at a site other than the endogenous non-human TCRδ locus in the genome. In another embodiment, the unrearranged human TCRδ variable region segment replaces the endogenous non-human TCRδ variable region segment and the human TCRδ constant region gene sequence replaces the endogenous non-human TCRδ constant region gene sequence. In one embodiment, the unrearranged human TCRδ variable gene locus replaces the endogenous non-human TCRδ variable gene locus.

[0281] In some embodiments, the unrearranged TCRδ variable gene locus comprising a human variable region segment (e.g., human Vδ, Dδ, and Jδ segments) is located in a non-human genome, so that the human variable region segment replaces the corresponding non-human variable region segment. In one embodiment, the unrearranged TCRδ variable gene locus comprising a human variable region segment replaces the endogenous TCRδ variable gene locus. In one aspect, endogenous non-human Vδ, Dδ, and Jδ segments cannot be rearranged to form rearranged Vδ / Dδ / Jδ sequences. Therefore, in one aspect, the human Vδ, Dδ, and Jδ segments in the unrearranged TCRδ variable gene locus can be rearranged (or rearranged), for example, in T cells, to form rearranged human Vδ / Dδ / Jδ sequences.

[0282] In some embodiments, the non-human animals of the present invention comprise an unrearranged humanized 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). In some embodiments, the non-human animal of the present invention comprises an unrearranged human TCRδ locus, for example, a TCRδ locus comprising at least one functional unrearranged human Vδ segment operably linked to a functional human TCR Cδ gene, at least one functional unrearranged human Dδ segment, and at least one functional unrearranged human Jδ segment (e.g., a complete library of functional unrearranged human Vδ segments, a complete library of functional unrearranged human Dδ segments, and a complete library of functional unrearranged human Jδ segments), optionally wherein the order of functional unrearranged human Vδ segments, functional unrearranged human Dδ segments, functional unrearranged human Jδ segments, and functional human TCR Cδ genes is the same as the order found in the germline human TCRδ locus. The number and position of various TCRδ segments can be determined from the IMGT database. In mice and humans, the TCRδ gene segment is located within the TCRα locus on chromosome 14 (see 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, with the majority located between the individual Vα segments. Due to the genomic arrangement of the TCRδ gene segments within the TCRα locus, successful rearrangement at the TCRα locus can result in the deletion or inactivation of the TCRδ gene segments.

[0283] Mouse TCRδ variable locus is about 275 kilobases and comprises 6 TRDVs (16, including 10 upstream TRAV / DVs), 2 TRDDs, 2TRDJs and 1 TRDC. One TRDV is in the reverse 3' of TRDC. The human TCRδ cluster between TRAV and TRAJ gene segments comprises 1 TRDV gene segment (TRDV2), 3 TRDD gene segments (TRDD1, TRDD2, TRDD3), 4 TRDJ gene segments (TRDJ1, TRDJ4, TRDJ2 and TRDJ3), 1 TRDC gene and 1 reverse TRDV (TRDV3) of TRDC gene downstream. The cluster spans 60 kilobases. One TRDV gene segment (TRDV1) is located in the TRAV gene segment, and 5 gene segments (TRAV14 / DV4, TRAV23 / DV6, TRAV29 / DV5, TRAV36 / DV7, and TRAV38-2 / DV8) described as TRAV / DV gene segments within the TRAV gene segment can be used to synthesize TCRδ chains or TCRα chains. In one embodiment, the genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) comprises at least one human Vδ gene segment, one human Dδ gene segment, and at least one human Jδ gene segment. In one embodiment, the non-human animal comprises a human TCRδ locus containing 1 human Vδ segment. In one embodiment, the non-human animal comprises a human TCRδ locus containing 2 human Vδ segments. In one embodiment, the non-human animal comprises a human TCRδ locus containing 3 human Vδ segments. In one embodiment, the non-human animal comprises a human TCRδ locus containing 1 human TRAV / DV segment. In one embodiment, the non-human animal comprises a human TCRδ locus containing 2 human TRAV / DV segments. In one embodiment, the non-human animal comprises a human TCRδ locus containing 3 human TRAV / DV segments. In one embodiment, the non-human animal comprises a human TCRδ locus containing 4 human TRAV / DV segments. In one embodiment, the non-human animal comprises a human TCRδ locus containing 5 human TRAV / DV segments. In some embodiments, the non-human animal comprises a human TCRδ locus containing 1 human TRDJ gene segment. In some embodiments, the non-human animal comprises a human TCRδ locus containing 2 human TRDJ gene segments. In some embodiments, the non-human animal comprises a human TCRδ locus containing 3 human TRDJ gene segments. In some embodiments, the non-human animal comprises a human TCRδ locus containing 4 human TRDJ gene segments. In some embodiments, the non-human animal comprises a human TCRδ locus containing a human TRDC gene.

[0284] In one embodiment, the non-human animal comprises a humanized TCRδ locus, which comprises a DNA fragment containing contiguous human sequences, which contiguous human sequences include human Vδ1 to Vδ3 gene segments (Vδ segments are also called "TRDV" or "TCRDV"), including Dδ1, Dδ2, Dδ3 (Dδ segments are also called "TRDD" or "TCRDD") and Cδ genes (Cδ is also called "TRDC" or "TCRDC"). In some embodiments, the DNA fragment also comprises a human Jα segment. TCRD non-coding sequences refer to contiguous non-coding sequences, including non-coding recombination signal sequences (RSS) and other non-coding intergenic sequences found between any two consecutive unrearranged TRDV segments, between any unrearranged TRDV segments and unrearranged TRDD segments, between any two consecutive unrearranged TRDD segments, and between any TRDD segments and TRCD genes. In various embodiments, the DNA fragments comprising the adjacent human sequences of the human TCR δ variable region segments also comprise restriction enzyme sites, selection boxes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the humanization of the locus. In various embodiments, these additional sites do not interfere with the normal function (e.g., rearrangement, splicing, etc.) of the various genes at the TCR δ locus.

[0285] In some embodiments, a mouse as described herein comprises germ cells and CD3 - A somatic cell, the cell comprising:

[0286] (A) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence; or

[0287] (B) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or

[0288] (C)(i) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence, and

[0289] (ii) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

[0290] In some embodiments,

[0291] (A) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments; or

[0292] (B) 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; or

[0293] (C)(i) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments, and

[0294] (ii) the unrearranged human TCR Vδ segment comprises the complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segment comprises the complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segment comprises the complete repertoire of unrearranged human TCR Jδ segments.

[0295] In some embodiments, a mouse as described herein comprises:

[0296] (I) contains the following germ cells and CD3 - T cells:

[0297] (A) at the endogenous TCRγ locus:

[0298] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0299] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0300] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0301] (B) at the endogenous TCRδ locus:

[0302] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0303] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0304] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0305] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0306] (II) CD3 expressing on its surface a functional TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide + T cells.

[0307] In some embodiments, a non-human animal having a humanized TRD and / or TRG locus as described herein has γ / δ T cells (a small fraction of total T cells, as in WT mice ( Figure 5A-Figure 5B ) and the presence of γ / δ T cells in the skin and intestinal mucosa ( Figures 12A-12D ). In addition, TCR repertoire analysis of thymic and splenic mRNA revealed successful and diverse V(D)J gene usage, indicating correct recombination, expression, and thymic selection of humanized γ / δ TCRs ( Fig. 6A , Figure 6B , Fig. 7A and Figure 7B Diverse repertoire recombination of V(D)J gene segments was also observed in γ / δ T cells of the skin and intestinal mucosa (data not shown).

[0308] Because the TRD locus is located within the TRA locus, in some embodiments, germ cells and CD3 -The somatic cell further comprises an unrearranged human TCR Vα segment located upstream of an unrearranged TCRδ variable region sequence and a human TCRδ constant region gene sequence, wherein the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ and the unrearranged human TCR Jδ segment are capable of rearranging (or rearranging) in the T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to a 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 polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, and wherein the mouse further comprises a CD3+ TCR polypeptide expressing on its surface the human hybrid TCRα / δ variable domain operably linked to the human TCRδ constant domain. + T cells.

[0309] In some embodiments, humanized TCRγ and / or humanized TCRδ non-human animals as described herein may also have a human or humanized TCRα locus. Mice with a human or humanized α locus are described in U.S. Pat. No. 9,113,616, which is incorporated herein by reference. See also Fig. 8A .

[0310] Fully humanized T cell responses

[0311] Described herein are non-human animals that, along with human or humanized TRD and TRG loci, also contain human or humanized TRA and / or TRB loci and other components involved in α / β TCR rearrangement and / or activation, such as TCR co-receptors (CD4 and CD8) and MHC loci.

[0312] Human or humanized α and / or β TCR loci

[0313] In some embodiments, a non-human animal comprising an unrearranged human TCRα variable region segment in its genome is provided, wherein the unrearranged human TCRα variable region segment is operably linked to a human or non-human TCRα constant region gene sequence, producing a human or humanized TCRα locus, respectively. In one embodiment, the human or humanized TCRα locus is at a site other than the endogenous non-human TCRα locus in the genome. In another embodiment, the unrearranged human TCRα variable region segment replaces the endogenous non-human TCRα variable region segment and the human TCRα constant region gene sequence replaces the endogenous non-human TCRα constant region gene sequence. In one embodiment, the unrearranged human TCRα variable gene locus replaces the endogenous non-human TCRα variable gene locus.

[0314] In one embodiment, the unrearranged TCRα variable gene locus comprising human variable region segments (e.g., human Vα and Jα segments) is located in a non-human genome, so that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, the unrearranged TCRα variable gene locus comprising human variable region segments replaces the endogenous TCRα variable gene locus. In one aspect, the endogenous non-human Vα and Jα segments cannot be rearranged to form a rearranged Vα / Jα sequence. Therefore, in one aspect, the human Vα and Jα segments in the unrearranged TCRα variable gene locus can be rearranged (or rearranged), for example, in T cells, to form a rearranged human Vα / Jα sequence.

[0315] In some embodiments, the non-human animal of the present invention comprises an unrearranged humanized TCR α locus, for example, a TCR α locus comprising at least one functional unrearranged human V α segment and at least one functional unrearranged human J α segment (e.g., a complete library of functional unrearranged human V α segments and a complete library of functional unrearranged human J α variable region segments). In some embodiments, the non-human animal of the present invention comprises an unrearranged human TCR α locus, for example, a TCR α locus comprising at least one functional unrearranged human V α segment and at least one functional unrearranged human J α segment (e.g., a complete library of functional unrearranged human V α segments and a complete library of functional unrearranged human J α segments) operably linked to a functional human TCR C α gene (e.g., an endogenous TCR C α gene), optionally wherein the functional unrearranged human V α segment, the functional unrearranged human J α segment and the functional TCR C α gene are in the same order as found in the germline human TCR α locus. The number and location of the various TCRα segments can be determined from the IMGT database.

[0316] Mouse TCR α variable locus is about 1.5 megabases and includes a total of 110 Vα segments and 60 Jα segments. Human TCR α variable locus is about 1 megabase and includes a total of 54 Vα segments and 61 Jα segments, and it is believed that 45 Vα and 50 Jα are functional. Unless otherwise specified, the number of human V (D) J segments mentioned in the 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, such as a mouse or a rat) includes at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal includes a humanized TCR α locus, and the humanized TCR α locus includes 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 TCRα locus comprises 2, 8, 23, 35, 48, or 54 human Vα segments. Thus, in some embodiments, the humanized TCRα 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; in some embodiments, it may comprise about 2%, about 3%, about 15%, about 65%, about 90%, or 100% human Vα.

[0317] In one embodiment, the non-human animal comprises a humanized TCRα locus, which comprises a DNA fragment containing a contiguous human sequence of human Vα40 to Vα41 (the Vα segment is also referred to as "TRAV" or "TCRAV") and a DNA fragment containing a contiguous human sequence of 61 human Jα segments (the Jα segment is also referred to as "TRAJ" or "TCRAJ"). The TCRA non-coding sequence refers to a contiguous non-coding sequence, including a non-coding recombination signal sequence (RSS) and other non-coding intergenic sequences found between any two consecutive unrearranged TRAV segments, between any unrearranged TRAV segments and unrearranged TRAJ segments, and between any two consecutive unrearranged TRAJ segments. In one embodiment, the non-human animal comprises a humanized TCRα locus, which comprises a DNA fragment containing a contiguous human sequence of human TRAV35 to TRAV41 and a DNA fragment containing a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus, which comprises a DNA fragment containing a contiguous human sequence of human TRAV22 to TRAV41 and a DNA fragment containing a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus, which comprises a DNA fragment containing a contiguous human sequence of human TRAV13-2 to TRAV41 and a DNA fragment containing a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus, which comprises a DNA fragment containing a contiguous human sequence of human TRAV6 to TRAV41 and a DNA fragment containing 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus, which comprises a DNA fragment containing a contiguous human sequence of human TRAV1-1 to TRAV 41 and a DNA fragment containing 61 human TRAJs. In various embodiments, the DNA fragments comprising the adjacent human sequences of the human TCRα variable region segments also include restriction enzyme sites, selection boxes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the humanization of the locus. In various embodiments, these additional sites do not interfere with the normal function (e.g., rearrangement, splicing, etc.) of each gene at the TCRα locus.

[0318] In one embodiment, the humanized TCRα locus comprises 61 human Jα segments, or 100% human Jα segments. In a specific embodiment, the humanized TCRα locus comprises 8 human Vα segments and 61 human Jα segments; in another specific embodiment, the humanized TCRα locus comprises 23 human Vα segments and 61 human Jα segments. In another specific embodiment, the humanized TCRα locus comprises a complete library of human Vα and Jα segments, i.e., all human variable α region gene segments encoded by the α locus, or 54 human Vα substitutions 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 TCRα locus.

[0319] In some embodiments, a mouse as described herein comprises:

[0320] (I) germ cells and CD3-somatic cells, said cells comprising from 5' to 3':

[0321] Unrearranged human TCR Vα segment and

[0322] an unrearranged TCRδ variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged TCR Dδ and an unrearranged human TCR Jδ segment,

[0323] wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus,

[0324] wherein the unrearranged human TCR Vα segment, the unrearranged TCR Dδ and the unrearranged human TCR Jδ segment are capable of rearranging (or rearrangement) in a T cell to form a rearranged human TCR Vα / Dδ / Jδ variable region gene operably linked to the human TCRδ constant region gene sequence,

[0325] 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 polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, and

[0326] (II) a 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 + T cells.

[0327] In some embodiments, the germ cells and CD3 -The somatic 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 and to a TCRα constant region gene sequence, such as a mouse TCRα constant region gene sequence, and wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are capable of rearranging (or rearranging) in a 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, and wherein the mouse further comprises a CD3 T cell expressing on its surface a functional TCR comprising the TCRα polypeptide. + In some embodiments, germ cells and CD3 - The somatic cell comprises replacement of all endogenous TCR Vα segments with a complete repertoire of unrearranged human TCR Vα segments and replacement of all endogenous TCR Jα segments with a complete repertoire of unrearranged human TCR Jα segments, wherein said complete repertoire of unrearranged human TCR Vα segments and said complete repertoire of unrearranged human TCR Jα segments are operably linked to each other and to a mouse TCR α constant region gene sequence at an 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 are capable of rearranging (or rearrangement) in a T cell to form a rearranged human TCR Vα / Jα variable region gene operably linked to said mouse 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 wherein the mouse comprises a CD3 T cell expressing on its surface a functional TCR comprising the chimeric TCRα polypeptide. + T cells.

[0328] Humanized TRA / D and TRG loci can be introduced into mice with humanized TRB loci and other components of T cell immunity, including TCR co-receptors (CD4 and CD8) and MHC loci. These mice will thus carry complete humanization of both T cell lineages.

[0329] As a non-limiting example, humanized TRD and / or TRG loci can be introduced into mice comprising a human or humanized TRB locus (e.g., a TRB locus comprising humanized TCRBDJ1 and TCRBDJ2 clusters with murine TCRB non-coding sequences and human coding sequences), such as those described in U.S. Pat. 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 Figure 8B and Figure 8C .

[0330] In some embodiments, a non-human animal comprising an unrearranged human TCR β variable region segment in its genome is provided, wherein the unrearranged human TCR β variable region segment is operably connected to a non-human TCR β constant region gene sequence to produce a humanized TCR β locus. In one embodiment, the humanized TCR β locus is at a site other than the endogenous non-human TCR β locus in the genome. 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 gene locus replaces the endogenous non-human TCR β variable gene locus.

[0331] In some embodiments, the unrearranged TCRβ variable gene locus comprising a human variable region segment (e.g., human Vβ, Dβ, and Jβ segments) is located in a non-human genome, so that the human variable region segment replaces the corresponding non-human variable region segment. In one embodiment, the unrearranged TCRβ variable gene locus comprising a human variable region segment replaces the endogenous TCRβ variable gene locus. In one aspect, endogenous non-human Vβ, Dβ, and Jβ segments cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence. Therefore, in one aspect, the human Vβ, Dβ, and Jβ segments in the unrearranged TCRβ variable gene locus can be rearranged (or rearranged), for example, in T cells, to form a rearranged human Vβ / Dβ / Jβ sequence.

[0332] The mouse TCRβ variable locus is approximately 0.6 megabases and comprises a total of 33 Vβ segments, 2 Dβ segments, and 14 Jβ segments. The human TCRβ variable locus is approximately 0.6 megabases and comprises a total of 67 Vβ segments, 2 Dβ segments, and 14 Jβ segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) comprises at least one human Vβ segment, at least one human Dβ segment, and at least one human Jα segment.

[0333] In one embodiment, the non-human animal comprises a humanized TCR β 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 β segments. In some embodiments, the humanized TCR β locus comprises 8, 14, 40, 66 or 67 human V β segments. Thus, in some embodiments, the humanized TCRβ 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 Vβ; in some embodiments, it may comprise about 20%, about 60%, about 15%, about 98% or 100% human Vβ.

[0334] In some embodiments, the endogenous TCR beta variable gene locus, e.g., the endogenous TCR beta mouse variable gene locus, comprises:

[0335] replacing one or all adjacent endogenous T cell variable region Vβ gene segments, such as one or all 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 unrearranged human T cell variable region gene segments from TRBV1 to TRBV29-1, and / or

[0336] One or more non-contiguous endogenous Vβ gene segments (eg, endogenous mouse TCRBV31 gene segments) are replaced with human TCRBV gene segments (eg, mouse TCRBV31 gene segments are replaced with orthologous human TCRBV30 gene segments).

[0337] In one embodiment, the non-human animal comprises a humanized TCRβ locus, which comprises a DNA fragment containing a contiguous human sequence of human Vβ18 to Vβ29-1 (the Vβ segment is also referred to as "TRBV" or "TCRBV"). In one embodiment, the non-human animal comprises a humanized TCRβ locus, which comprises a DNA fragment containing a contiguous human sequence of human TRBV18 to TRBV29-1, a separate DNA fragment containing a contiguous human TCRBDJ1 sequence containing human Dβ1-Jβ1 (i.e., a human Dβ1-Jβ1-1-Jβ1-6 segment), and a separate DNA fragment containing a contiguous human TCRBDJ2 sequence containing human Dβ2-Jβ2 (i.e., a human Dβ2-Jβ2-1-Jβ2-7 segment). The unrearranged TCRBDJ1 sequence (also referred to as an unrearranged TCRBJD1 cluster) comprises an unrearranged TCRBD1 segment, one to all unrearranged TCRBJ1 segments (e.g., Jβ1-1, Jβ1-2, Jβ1-3, Jβ1-4, Jβ1-5, and Jβ1-6 segments), and TCRBDJ1 non-coding sequences between the unrearranged TCRBD1 segment and the unrearranged TCRBJ1 segment and between any two consecutive unrearranged TCRBJ1 gene segments. TCRB non-coding sequence refers to a contiguous non-coding sequence comprising a non-coding recombination signal sequence (RSS) and other non-coding intergenic sequences found between any two consecutive unrearranged TRBV segments, and may include a TCRBDJ1 non-coding sequence, such as a contiguous non-coding sequence comprising a non-coding recombination signal sequence (RSS) and other non-coding intergenic sequences found between unrearranged TRBD1 segments and TRBJ1 segments and between any two consecutive unrearranged TRBJ1 segments, or a TCRBDJ2 non-coding sequence, such as a contiguous non-coding sequence comprising a non-coding recombination signal sequence (RSS) and other non-coding intergenic sequences found between unrearranged TRBD2 segments and TRBJ2 segments and between any two consecutive unrearranged TRBJ2 segments. Unrearranged TCRBDJ1 sequences may be operably linked to multiple unrearranged TRBV segments and TCRBC1 constant region gene sequences (also referred to as TRBC1 region sequences). The unrearranged TCRBDJ2 sequence (also referred to as an unrearranged TCRBJD2 cluster) comprises an unrearranged TCRBD2 segment, one to all unrearranged TCRBJ2 segments (e.g., Jβ2-1, Jβ2-2, Jβ2-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments), and 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 can be operably linked to a plurality of unrearranged TRBV segments and TCRBC2 constant region gene sequences (also referred to as TRBC2 region sequences). In one embodiment, the non-human animal comprises a humanized TCRβ locus, the humanized TCR locus comprising any one or both of the following: (i) a TCRBDJ1 cluster, wherein at least all or at least one Dβ1-Jβ1 segment (i.e., Dβ1, Jβ1-1, Jβ1-2, Jβ1-3, Jβ1-4, Jβ1-5, and Jβ1-6 segments) are human, and wherein the non-coding sequences between the Dβ1-Jβ1 segments, including RSS and other intergenic sequences are non-human, such as mouse, and optionally wherein the Dβ1 and Jβ1-1 to JβJ1-6 segments are flanked by mouse Trbd1 and mouse Trbj1-1 to Trbj1-6 regions The invention relates to a method of manufacturing a TCR comprising: (i) a Dβ2-Jβ2 cluster comprising at least one or all of the Dβ2-Jβ2 segments (i.e., Dβ2, Jβ2-1, Jβ2-2, Jβ2-3, Jβ2-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments) and (ii) a TCR B DJ2 cluster, wherein at least one or all of the Dβ2-Jβ2 segments (i.e., Dβ2, Jβ2-1, Jβ2-2, Jβ2-3, Jβ2-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments) are human and wherein the non-coding sequences between the Dβ2-Jβ2 segments, including RSS and other intergenic sequences, are non-human, e.g., mouse, optionally wherein the Dβ2 and JβJ2-1 to Jβ2-7 gene segments are flanked by the same mouse TCR non-coding sequences that are typically flanked by mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments. In one embodiment, the non-human animal comprises a humanized TCRβ locus, which comprises a DNA fragment containing a contiguous human sequence of human TRBV6-5 to TRBV29-1, a separate DNA fragment containing a contiguous human sequence of human Dβ1-Jβ1 (i.e., a human Dβ1-Jβ1-1-Jβ1-6 segment), and a separate DNA fragment containing a contiguous human sequence of human Dβ2-Jβ2 (i.e., a human Dβ2-Jβ2-1-Jβ2-7 segment). In one embodiment, the non-human animal comprises a humanized TCRβ locus, which comprises a DNA fragment containing a contiguous human sequence of human TRBV1 to TRBV29-1, a separate DNA fragment containing a contiguous human sequence of human Dβ1-Jβ1, and a separate DNA fragment containing a contiguous human sequence of human Dβ2-Jβ2. In one embodiment, the non-human animal comprises a humanized TCRβ locus, which comprises a DNA fragment containing contiguous human sequences of human TRBV1 to TRBV29-1, a separate DNA fragment containing contiguous human sequences of human Dβ1-Jβ1, a separate DNA fragment containing contiguous human sequences of human Dβ2-Jβ2, and a separate DNA fragment containing the sequence of human TRBV30.In various embodiments, the DNA fragments comprising the adjacent human sequences of the human TCR β variable region segments also include restriction enzyme sites, selection boxes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the humanization of the locus. In various embodiments, these additional sites do not interfere with the normal function (e.g., rearrangement, splicing, etc.) of each gene at the TCR β locus.

[0338] In one embodiment, the humanized TCR β locus includes 14 human J β segments or 100% human J β segments, and 2 human D β segments or 100% human D β segments. In another embodiment, the humanized TCR β locus includes at least one human V β segment, such as 14 human V β segments, and all mouse βD and J β segments. In a specific embodiment, the humanized TCR β locus includes 14 human V β segments, 2 human D β segments and 14 human J β segments. In another specific embodiment, the humanized TCR β locus includes a complete library of human V β, D β and J β segments, i.e., all human variable β region gene segments or 67 human V β segments, 2 human D β segments and 14 human J β segments encoded by the β locus. In one embodiment, the non-human animal includes one (e.g., 5') non-human V β segment at the humanized TCR β locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ, Dβ, or Jβ segments at the TCRβ locus.

[0339] In one embodiment, the humanized TCR β locus includes 13 human J β segments or 100% functional human J β segments, and 2 human D β segments or 100% functional human J β segments. In another embodiment, the humanized TCR β locus includes at least one human V β segment, such as 14 human V β segments, and all functional mouse βD and J β segments. In a specific embodiment, the humanized TCR β locus includes 14 human V β segments, 2 human D β segments and 13 functional human J β segments. In another specific embodiment, the humanized TCR β locus includes a complete library of human V β, D β and J β segments, i.e., all human variable β region gene segments or 67 human V β segments, 2 human D β segments and 13 functional human J β segments encoded by the β locus. In one embodiment, the non-human animal includes one (e.g., 5') non-human V β segment at the humanized TCR β locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ, Dβ, or Jβ segments at the TCRβ locus.

[0340] In one aspect, a non-human animal (e.g., a rodent, such as a mouse or a rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., a rodent, such as a mouse or a rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 10% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ1 cluster, and at least 10% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, such as a mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., a rodent, such as a mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 15% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ1 cluster, and at least 15% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., a rodent, such as a mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., a rodent, such as a mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 20% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ1 cluster, and at least 20% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., rodent, such as mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., rodent, such as mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 30% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ1 cluster, and at least 30% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., rodent, such as mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., rodent, such as mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 40% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster.In some embodiments, a non-human animal (e.g., rodent, such as mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., rodent, such as mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 50% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., rodent, such as mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., rodent, such as mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 60% of the TCRs expressed by the spleen cell population are derived from gene segments from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., rodent, such as mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., rodent, such as mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, wherein at least 70% of the TCR expressed by the spleen cell population is derived from a gene segment from the TCRBDJ2 cluster. In some embodiments, a non-human animal (e.g., rodent, such as mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., rodent, such as mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, which expresses a TCR derived from a gene segment from the TCRBDJ1 cluster and a TCR derived from a gene segment from the TCRBDJ2 cluster, and the ratio thereof is 1:3, 3:7, 1:2, 2:3 or 1:1. In some embodiments, a non-human animal (e.g., a rodent, such as a mouse or rat) comprising a humanized TCRβ variable gene locus containing a non-human animal (e.g., a rodent, such as a mouse or rat) TCRB non-coding sequence as described herein comprises a spleen cell population, such as a CD4+ and / or CD8+ T cell population, which expresses TCRs derived from gene segments from the TCRBDJ2 cluster and TCRs derived from gene segments from the TCRBDJ1 cluster at a ratio of 1:3, 3:7, 1:2 or 2:3.

[0341] In some embodiments, germ cells and CD3 -The somatic cell comprises an unrearranged TCRβ variable region sequence, the unrearranged TCRβ 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 TCRβ variable region sequence is operably linked to a TCRβ constant region gene sequence, such as a mouse TCRβ constant region gene sequence, optionally at an endogenous TCRβ locus, and wherein the unrearranged human TCR Vβ segment, the unrearranged human TCR Dβ segment and the unrearranged human TCR Jβ segment are capable of rearranging (or rearranging) in a T cell to form a rearranged human TCR Vβ / Dβ / Jβ variable region gene operably linked to the TCRβ constant region gene sequence, and wherein the rearranged human TCR operably linked to the TCRβ constant region gene sequence The Vβ / Dβ / Jβ variable region genes together encode a TCRβ polypeptide comprising a human TCRβ variable domain; and wherein the mouse further comprises a CD3 TCR expressing a TCR comprising the TCRβ polypeptide on its surface. + In some embodiments, the unrearranged TCRβ variable region sequence comprises a mouse TCRB noncoding sequence.

[0342] In some mouse embodiments described herein,

[0343] (I) the germ cells and the CD3-somatic cells comprise:

[0344] (A) at the endogenous TCRγ locus:

[0345] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0346] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0347] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0348] (B) at the endogenous TCRδ locus:

[0349] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0350] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0351] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0352] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0353] (C) at the endogenous TCRα locus:

[0354] Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and

[0355] Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments, and

[0356] (D) at the endogenous TCRβ locus:

[0357] Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments,

[0358] Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments, and

[0359] Replace all endogenous TCR Dβ segments with the complete repertoire of unrearranged human TCR Jδ segments; and

[0360] (II) the mouse further comprises a CD3 T cell expressing 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 TCRβ polypeptide + T cells.

[0361] In some embodiments, germ cells and CD3 - The somatic cells each comprise a human CTCF binding element. In some embodiments, the germ cells and the somatic cells each comprise a human CTCF binding element upstream of the TCRα locus. In some embodiments, the germ cells and the somatic cells each comprise a human CTCF binding element upstream of the TCRγ locus.

[0362] In some embodiments, the germ cell and the somatic cell each comprise a human CTCF binding element upstream of the TCRα locus and a human CTCF binding element upstream of the TCRγ locus.

[0363] In one aspect, the non-human animal expresses a humanized T cell receptor with a non-human constant domain on the surface of the T cell, wherein the receptor is capable of interacting with a non-human molecule, such as an anchor or signal molecule expressed in a T cell (e.g., a CD3 molecule, a ζ chain, or other proteins anchored to the TCR by a CD3 molecule or a ζ chain). Therefore, in one aspect, a cell complex is provided, the cell complex comprising (a) expressing the following non-human T cells: (i) a TCR comprising a humanized TCR α chain as described herein and a humanized TCR β chain as described herein, and (ii) a chimeric auxiliary receptor as 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 as described herein. In one embodiment, human TCRγ and / or TCRδ chains are complexed with non-human, such as endogenous zeta (ζ) chain homodimers and non-human, such as endogenous CD3 heterodimers. In one embodiment, the cell complex is an in vivo cell complex.

[0364] In various embodiments, the non-human animals described herein (e.g., rodents, such as mice or rats) 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 present invention express cell surface molecules (e.g., CD25, CD44, Kit, CD3, pTα, etc.) that are typically produced by T cells at specific stages of thymic development.

[0365] In various embodiments, the non-human animals described herein produce T cells capable of undergoing T cell differentiation in the periphery. In additional embodiments, the non-human animals described herein comprise CD3+ T cells in the periphery, for example in the spleen, skin, and intestinal mucosa.

[0366] DN1 and DN2 cells that do not receive enough signals (e.g., Notch signals) can develop into B cells, bone marrow cells (e.g., dendritic cells), mast cells, and NK cells. See, e.g., 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, bone marrow cells (e.g., dendritic cells), mast cells, and NK cells. In some embodiments, the non-human animals described herein develop dendritic cell populations in the thymus.

[0367] The main type of T cell receptor expressed on the surface of T cells is TCRα / β, and a few cells express TCRδ / γ. In some embodiments, T cells of non-human animals comprising humanized TCRγ and / or δ loci show the use of TCRα / β and TCRγ / δ loci similar to wild-type animals (e.g., T cells of non-human animals described herein express TCRα / β and TCRδ / γ proteins in a ratio comparable to that expressed by wild-type animals). Therefore, in some embodiments, non-human animals comprising optional humanized TCRα / β and human TCRγδ loci show the use of all loci.

[0368] Humanized T cell coreceptor

[0369] Although antigen recognition by γ / δ T cells may not require interaction between T cell co-receptors (e.g., CD4 and CD8) and MHC, because the non-human animals described herein may also contain human or humanized TRA and TRB loci in addition to human or humanized TRD (and TRG) loci, the non-human animals described herein may also contain human or humanized CD4 loci and / or human or humanized CD8 (e.g., CD8α and CD8β) loci, see, e.g., U.S. Pat. Nos. 9,848,587 and 10,820,581, each of which is incorporated herein by reference.

[0370] Therefore, disclosed herein is a non-human animal expressing at least one human or humanized T cell co-receptor, such as CD4, CD8α and / or CD8β. Therefore, a non-human animal as disclosed herein comprises at least one of a first nucleotide sequence, a second nucleotide sequence and / or a third nucleotide sequence, wherein each nucleotide sequence encodes a different human or chimeric human / non-human T cell co-receptor polypeptide selected from the following: a human or humanized CD4 polypeptide, a human or humanized CD8α polypeptide and a human or humanized CD8β polypeptide. The use of the first name, the second name, and the third name herein should not be interpreted as limiting the non-human animals disclosed herein to requiring all three nucleotide sequences or requiring any co-receptor nucleotide sequence to be present in any order. Therefore, a non-human animal as disclosed herein may include one or more nucleic acid sequences encoding a human or humanized CD4 and / or a human or humanized CD8 (e.g., a human or humanized CD8α and / or CD8β) polypeptide.

[0371] In one embodiment, a non-human animal as disclosed herein comprises a first nucleotide sequence encoding a human or humanized CD4 polypeptide. In another embodiment, a non-human animal as disclosed herein comprises 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, a non-human animal as disclosed herein comprises a first nucleotide sequence encoding a human or humanized CD8α and CD8β polypeptide and a second nucleotide sequence, and further comprises a third nucleotide sequence encoding a human or humanized CD4 polypeptide.

[0372] In various embodiments, the present invention generally provides genetically modified non-human animals that comprise in their genome, e.g., at the endogenous CD4 locus, a nucleotide sequence encoding a human or humanized CD4 polypeptide; thus, these animals express the human or humanized CD4 polypeptide.

[0373] 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, which is encoded by exon 2 and exon 3 of the gene. The protein contains four extracellular immunoglobulin-like domains, Ig1-Ig4, also commonly referred to as 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. It is believed that the D1 domain is encoded by exon 3 (the sequence downstream of the signal peptide) and exon 4, while D2, D3 and D4 are each encoded by separate exons - exon 5, exon 6 and exon 7 (see Fig. 9A: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 contact areas 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.

[0374] The D1 domain of CD4 is similar to the immunoglobulin variable (V) domain and, together with a portion of the D2 domain, is believed to bind (associate) 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. In turn, MHC II interacts with the T cell coreceptor CD4 at 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.

[0375] It is believed that domains D3 and D4 of the CD4 co-receptor interact with the TCR-CD3 complex because substitution of these two domains eliminates 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 it is believed that the residues in the D4 domain of the molecule are responsible for CD4 dimerization. Moldovan et al. (2002) CD4 Dimers Constitute the Functional Components Required for T Cell Activation, J. Immunol. 169: 6261-68.

[0376] Exon 8 of the CD4 gene encodes a transmembrane domain, while the rest of the gene encodes a cytoplasmic domain. The CD4 cytoplasmic domain has many different functions. For example, the cytoplasmic domain of CD4 recruits tyrosine kinase Lck. Lck is a Src family kinase associated with CD4 and CD8 cytoplasmic domains and the simultaneous binding of auxiliary receptors and TCRs with the same MHC leads to an increase in the 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. Itano and colleagues have proposed that by designing and testing the expression of hybrid proteins comprising CD8 extracellular domains and CD4 cytoplasmic tails in transgenic mice, the cytoplasmic tail of CD4 also promotes CD4+CD8+T cell differentiation into CD4+ lineage. Itano et al. (1996) The Cytoplasmic Domain of CD4 Promotes the Development of CD4Lineage T Cells, J.Exp.Med.183:731-41. The expression of hybrid proteins leads to the development of MHC I specificity and CD4 lineage T cells. Same as above.

[0377] CD4 co-receptor appears to be the main receptor for HIV virus, and depletion of CD4+T cells is an indicator of disease progression. In HIV-induced apoptosis, the cytoplasmic tail of CD4 appears to be necessary for delivering apoptotic signals to CD4+T cells. Specifically, it has been shown that the interaction of CD4 and Lck enhances HIV-induced apoptosis in these cells. Corbeil et al. (1996) HIV-induced Apoptosis Requires the CD4 Receptor Cytoplasmic Tail and IsAccelerated by Interaction of CD4 with p56lck, J.Exp.Med.183:39-48.

[0378] T cells develop in the thymus, progressing from immature CD4- / CD8- (double negative or DN) thymocytes to CD4+ / CD8+ (double positive or DP) thymocytes, which eventually undergo positive selection to become CD4+ or CD8+ (single positive or SP) T cells. DP thymocytes that receive signals through MHC I restricted TCRs differentiate into CD8+ T cells, while DP thymocytes that receive signals through MHC II restricted TCRs differentiate into CD4+ T cells. The cues received by DP cells that lead to their differentiation into CD4+ or CD8+ T cells have become the subject of many studies. Various models of CD4 / CD8 lineage selection have been proposed and reviewed in the following references: 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.

[0379] Inactivation of specific T cell co-receptors due to positive selection is a 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 the expression of CD4 in cells other than CD4+ T cells. Siu et al. (1994) A transcriptional silencer control the developmental expression of the CD4 gene, EMBO J. 13: 3570-3579.

[0380] Because important transcriptional regulators (e.g., promoters, enhancers, silencers, etc.) that control CD4 lineage selection are missing in several strains of previously developed transgenic mice expressing human CD4, these mice cannot reproduce normal T cell lineage development and produce immune cells other than CD4+T cells expressing 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 beneficial to develop genetically modified animals that retain endogenous mouse promoters and / or other regulatory elements so that the animals produce T cells that are capable of undergoing T cell development and lineage selection.

[0381] Thus, in various embodiments, the present invention provides a genetically modified non-human animal, which comprises a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide, for example, at its endogenous T cell co-receptor locus (e.g., CD4 locus). In one embodiment, the human portion of the chimeric polypeptide comprises the entire or substantially the entire extracellular portion (or a portion thereof, such as one or more extracellular domains, such as at least two consecutive extracellular domains) of a human T cell co-receptor. In one embodiment, the non-human portion of the chimeric polypeptide comprises a transmembrane domain and a cytoplasmic domain 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 present invention provides a genetically modified non-human animal, which comprises a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide at its endogenous CD4 locus, wherein the human portion of the chimeric polypeptide comprises the entire or substantially the entire extracellular portion of a human CD4, wherein the non-human portion comprises at least a transmembrane domain and a cytoplasmic domain of a non-human CD4, and wherein the animal expresses a functional chimeric CD4 polypeptide. In one aspect, the non-human animal expresses only a humanized CD4 polypeptide, ie, a chimeric human / non-human CD4 polypeptide, and does not express a functional endogenous non-human CD4 protein from its endogenous CD4 locus.

[0382] In one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises the entire or substantially the entire extracellular portion of a human CD4 polypeptide. In another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises at least the entire or substantially the entire MHC II binding domain of a human CD4 polypeptide (e.g., the major portion of the human D1 and D2 domains); in one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises the entire or substantially the entire D1, D2, and D3 domains of a human CD4 polypeptide; in 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, such as the domains referred to as D1, D2, D3, and D4. In another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises in its human portion the entire or substantially the entire human CD4 sequence responsible for interacting with the extracellular portion of MHC II and / or a T cell receptor. In another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises the entire or substantially the entire extracellular portion of a human CD4 responsible for interacting with the variable domains of MHC II and / or a T cell receptor. Thus, in one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises the entire or substantially the entire coding sequence of the D1-D2 domain of human CD4 (e.g., a portion of exon 3 and exon 4-5 of the human CD4 gene); in another embodiment, it comprises the entire or substantially the entire coding sequence of D1-D3 of human CD4 (e.g., a portion of exon 3 and exon 4-6 of human CD4). Thus, in one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD4 comprises a nucleotide sequence 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 a coding sequence of the D1-D4 domain of the human CD4 gene. In another embodiment, the nucleotide sequence may comprise a nucleotide sequence encoding a mouse CD4 signal peptide, such as a region encoded by a portion of exon 2-3 of the mouse gene. In another embodiment, the nucleotide sequence may comprise 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, and the human portion of the chimeric polypeptide spans approximately amino acids 27-319 of SEQ ID NO: 1 (set forth separately in SEQ ID NO: 2).

[0383] 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 comprises one or more conservative or non-conservative modifications.

[0384] In one aspect, a non-human animal expressing a human CD4 sequence is provided, wherein the human CD4 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a human CD4 sequence. In a specific embodiment, the human CD4 sequence is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to a human CD4 sequence described in U.S. Pat. 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.

[0385] In some embodiments, a portion of a chimeric CD4, such as a human portion, may comprise substantially the entire sequence set forth herein (e.g., substantially the entire protein domain set forth herein). Substantially the entire sequence typically includes 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acids believed to represent a particular portion of a protein (e.g., a particular functional domain, etc.). One skilled in the art will appreciate that the boundaries of a functional domain may vary slightly depending on the alignment and domain prediction method used.

[0386] In one aspect, the non-human portion of the chimeric human / non-human CD4 polypeptide comprises at least the transmembrane domain and cytoplasmic domain of the non-human CD4 polypeptide. Due to the important functions provided by the CD4 cytoplasmic domain, retaining the endogenous non-human (e.g., mouse) sequence in the genetically engineered animal ensures the preservation of appropriate intracellular signaling and other functions of the auxiliary receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD4 polypeptide is a mouse CD4 polypeptide. Although specific mouse CD4 sequences are described in U.S. Patent No. 10,820,581 cited in the Examples, any suitable sequence derived therefrom is contemplated herein, such as a sequence comprising conservative / non-conservative amino acid substitutions. In one embodiment, the non-human portion of the chimeric CD4 auxiliary receptor comprises any sequence of endogenous CD4 that has not yet been humanized.

[0387] The non-human animals described herein may comprise a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide at its endogenous locus. In one aspect, this causes a nucleotide sequence encoding a portion of a human CD4 polypeptide to replace a portion of an endogenous CD4 gene. In one embodiment, such replacement is an endogenous nucleotide sequence encoding, for example, the entire or substantially entire extracellular domain of a non-human CD4, for example, a sequence encoding at least the entire or substantially entire first immunoglobulin-like domain (i.e., D1) of a non-human CD4 (e.g., a sequence encoding all or substantially all domains D1-D2 of a non-human CD4, for example, a sequence encoding all or substantially all domains D1-D3 of a non-human CD4, for example, a sequence encoding all or substantially all domains D1-D4 of a non-human CD4) is replaced with a human nucleotide sequence encoding them. In one embodiment, the replacement produces a chimeric protein comprising a human CD4 sequence responsible for interacting with the extracellular portion of MHC II and / or a T cell receptor. In another embodiment, the replacement produces a chimeric protein comprising a human CD4 sequence responsible for interacting with the variable domains of MHC II and / or a T cell receptor. In one embodiment, the replacement does not comprise replacement of the CD4 sequence encoding at least the transmembrane domain and the cytoplasmic domain of the non-human CD4 polypeptide. Thus, in one aspect, the non-human animal expresses the chimeric human / non-human CD4 polypeptide from an endogenous non-human CD4 locus. In another embodiment, the replacement produces a protein comprising the polypeptide sequence shown in SEQ ID NO: 1.

[0388] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD4 locus (e.g., a chimeric human / rodent CD4 locus, such as a chimeric human / mouse CD4 locus) as described herein is provided. In one aspect, because the chimeric human / non-human (e.g., human / rodent, such as human / mouse) CD4 sequence is located at an endogenous non-human (e.g., rodent, such as mouse) CD4 locus, it retains a CD4 enhancer element located upstream of the first CD4 exon. In one embodiment, replacement at an endogenous non-human (e.g., rodent, such as mouse) CD4 locus includes replacement of, for example, a portion of exon 3 encoding D1, and exons 4-6 of the remainder of D1 and D2-D3 encoding a CD4 polypeptide; therefore, in one aspect, the chimeric CD4 locus retains a cis-acting silencer located in intron 1 of a non-human (e.g., mouse) CD4 gene. Therefore, in one embodiment, the chimeric locus retains an endogenous non-human (e.g., rodent, such as mouse) CD4 promoter and regulatory elements. In another embodiment, the chimeric locus can contain human promoters and regulatory elements to the extent that appropriate CD4 expression, CD4+T cell development, CD4 lineage selection, and auxiliary receptor function are allowed. Thus, in some aspects, the animals of the present invention comprise genetic modifications that do not alter appropriate lineage selection and development of T cells. In one aspect, in addition to cells that normally express CD4, the animals of the present invention (e.g., rodents, such as mice) do not express chimeric CD4 polypeptides on immune cells. In one aspect, the animal does not express CD4 on B cells or mature CD8+T cells. In one embodiment, the replacement causes the retention of elements that allow appropriate spatial and temporal regulation of CD4 expression.

[0389] In various embodiments, a non-human animal (e.g., a rodent, such as a mouse or rat) expressing a functional chimeric CD4 protein from a chimeric CD4 locus as described herein displays the chimeric protein on a cell surface, such as a T cell surface. In one embodiment, the non-human animal expresses the chimeric CD4 protein on the cell surface with the same cellular distribution 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, such as an antigen presenting cell (APC).

[0390] In various embodiments, the present invention generally provides genetically modified non-human animals, which contain nucleotide sequences encoding human or humanized CD8 polypeptides in their genomes, such as at the endogenous CD8 locus; thus, these animals express human or humanized CD8 polypeptides. In various embodiments, the present invention generally provides non-human animals, which contain nucleotide sequences encoding human or humanized CD8α polypeptides and / or nucleotide sequences encoding human or humanized CD8β polypeptides in their genomes, such as at the endogenous CD8 locus. Thus, the genetically modified non-human animals of the present invention express human or humanized CD8α and / or human or humanized CD8β polypeptides.

[0391] Human CD8 protein is usually expressed on the cell surface as a heterodimer of two polypeptides, CD8α and CD8β, but disulfide-linked homodimers and homopolymers have also been detected (for example, in NK cells and intestinal γδ T cells expressing CD8αα). The genes encoding human CD8α and CD8β are located very close to each other on chromosome 2. Nakayama et al. (1992) Recent Duplication of the Two Human CD8β-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. Fig. 9B The exons / introns of the CD8α gene are schematically depicted in FIG.

[0392] The human CD8β gene is located upstream of the CD8α gene on chromosome 2. Multiple isoforms produced by alternative splicing of the CD8β gene have been reported, one of which is predicted to lack a transmembrane domain and produce a secreted protein. Norment et al. (1988) A second subunit of CD8 is expressed in human T cells, EMBO J. 7: 3433-39. Fig. 9B The exons / introns of the CD8β gene are also schematically depicted.

[0393] 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 believed to maintain its conformation and protect the protein from protease cleavage. Leahy (1995) A structural view of CD4 and CD8, FASEB J. 9: 17-25.

[0394] CD8 protein is usually expressed on cytotoxic T cells and interacts with MHC I molecules. The interaction is mediated by the binding of CD8 to the α3 domain of MHC I. Although the binding of MHC class I to CD8 is 100 times weaker than the binding of TCR to MHC class I, the binding of CD8 enhances the affinity of TCR binding. Wooldridge et al. (2010) MHC Class IMolecules with Superenhanced CD8 Binding Properties Bypass the Requirementfor Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184: 3357-3366.

[0395] CD8 binding to MHC class I molecules is species-specific; Lyt-2, the mouse homolog of CD8, has been shown to bind H-2D at the α3 domain dmolecules, but it does not bind to HLA-A molecules. 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 may be due to CDR-like determinants (CDR1-like and CDR2-like) on CD8 that are 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. It has been reported that CD8 binds to HLA-A2 in the conserved region of the α3 domain (at positions 223-229). A single substitution (V245A) in HLA-A reduces CD8 binding to HLA-A, with a substantial 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, polymorphisms in the α3 domain of HLA-A molecules also affect binding to CD8. Same as above. In mice, H-2D d Amino acid substitution at residue 227 affects the relationship between mouse Lyt-2 and H-2D d of the mutant H-2D dTransfected cells are 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. Therefore, the expression of human or humanized CD8 may be useful for studying the response of T cells to antigens presented by human or humanized MHC I.

[0396] Similar to CD4, the cytoplasmic domain of CD8 interacts with the tyrosine kinase Lck, which in turn leads to T cell activation. Although Lck appears to interact with the cytoplasmic domain of CD8α, this interaction appears to be regulated by the presence of the CD8β cytoplasmic domain, because mutations or deletions in the CD8β cytoplasmic domain cause a decrease in CD8α-related 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. The reduction in Lck activity is associated with impaired T cell development. Same as above.

[0397] The expression of CD8 on appropriate cells (e.g., cytotoxic T cells) is tightly regulated by multiple enhancer elements located throughout the CD8 locus. For example, at least 4 DNA enzyme I-hypersensitive regions have been identified at the CD8 locus, and these regions are often associated with regulatory factor binding. Hosert et al. (1997) A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice, J. Immunol. 158: 4270-81. Since these DNA enzyme I hypersensitive regions were found at the CD8 locus, at least 5 enhancer elements have been identified, spread throughout the CD8 locus, which regulate the expression of CD8 α and / or β in T cells of various lineages, including DP, CD8 SP T cells, or cells expressing γδTCR. See, e.g., Kioussis et al. (2002) Chromatin and CD4, CD8A, and CD8Bgene expression during thymic differentiation, Nature Rev. 2:909-919 and OnlineErratum; Ellmeier et al. (1998) Multiple Development Stage-Specific EnhancersRegulate CD8 Expression in Developing Thymocytes and in Thymus-Independent Tcells, Immunity 9:485-96.

[0398] Thus, similar to the benefits that human or humanized CD4 genetically modified animals derive from retaining the endogenous CD4 promoter and regulatory elements, in some embodiments, there may be benefits in developing genetically modified non-human animals that retain the endogenous mouse promoter and regulatory elements that will control the expression of human or humanized CD8. As described herein, there may be particular benefits in creating genetically modified animals that include replacement of endogenous non-human sequences encoding CD8 alpha and / or beta proteins with sequences encoding human or humanized CD8 alpha and / or beta proteins.

[0399] In various embodiments, the present invention provides a genetically modified non-human animal, which comprises in its genome, e.g., at its 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 the entire or substantially the entire extracellular portion (or a portion thereof, e.g., the extracellular domain) of a human CD8 polypeptide (e.g., CD8α and / or β), wherein the non-human portion comprises at least the transmembrane domain and the cytoplasmic domain of non-human CD8 (e.g., CD8α and / or β), and wherein the animal expresses the chimeric CD8 polypeptide (e.g., CD8α and / or β polypeptide). Thus, in one embodiment, the present invention provides a genetically modified non-human animal comprising a first nucleotide sequence encoding a chimeric human / non-human CD8 alpha polypeptide and a second nucleotide sequence encoding a chimeric human / non-human CD8 beta polypeptide at its endogenous non-human CD8 locus, wherein the first nucleotide sequence comprises a sequence encoding the entire or substantially the entire extracellular portion of a human CD8 alpha polypeptide and at least the transmembrane domain and the cytoplasmic domain of a non-human CD8 alpha polypeptide, and wherein the second nucleotide sequence comprises a sequence encoding the entire or substantially the entire extracellular portion of a human CD8 beta polypeptide and at least the transmembrane domain and the cytoplasmic domain of a non-human CD8 beta polypeptide, wherein the animal expresses a functional chimeric human / non-human CD8 protein. In one aspect, the non-human animal expresses only humanized CD8 polypeptides (e.g., chimeric human / non-human CD8 alpha and / or beta polypeptides) and does not express corresponding functional non-human CD8 polypeptides from the endogenous CD8 locus.

[0400] In one embodiment, the chimeric human / non-human CD8α polypeptide comprises the entire or substantially the entire extracellular portion of a human CD8α polypeptide in its human portion. In one embodiment, the human portion of the chimeric CD8α polypeptide comprises at least the MHC I binding domain of a human CD8α polypeptide. In one embodiment, the human portion of the chimeric CD8α polypeptide comprises at least the entire or substantially the entire immunoglobulin V-like domain sequence of human CD8α. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8α polypeptide comprises at least the exon encoding the extracellular portion of the human CD8α polypeptide. In one embodiment, the nucleotide sequence comprises at least the exon encoding the Ig V-like domain. In one embodiment, the extracellular portion of the human CD8α polypeptide is a region covering the polypeptide portion of a non-transmembrane domain or a cytoplasmic domain. In one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD8α polypeptide comprises a sequence encoding a non-human (e.g., rodent, such as a 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 CD8a polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the human portion of the chimeric polypeptide is set forth as amino acids 28-179 of SEQ ID NO:3 (represented separately in SEQ ID NO:4).

[0401] Similarly, in one embodiment, the chimeric human / non-human CD8β polypeptide comprises the entire or substantially the entire extracellular portion of a human CD8β polypeptide in its human portion. In one embodiment, the human portion of a chimeric CD8β polypeptide comprises the entire or substantially the entire immunoglobulin V-like domain sequence of human CD8β. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric CD8β polypeptide comprises at least an exon encoding the extracellular portion of a human CD8β polypeptide. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric human / non-human CD8β polypeptide comprises at least an exon encoding the IgG V-like domain of human CD8β. In one embodiment, the nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide comprises a sequence encoding a non-human (e.g., rodent, such as a mouse) CD8β signal peptide. Alternatively, the nucleotide sequence may comprise a sequence encoding a human CD8β signal sequence. In one embodiment, the chimeric human / non-human CD8 beta polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5, and the human portion of the chimeric polypeptide is set forth as amino acids 15-165 of SEQ ID NO:5 (represented separately in SEQ ID NO:6).

[0402] In one embodiment, the non-human animal expresses a chimeric human / non-human CD8 alpha and / or CD8 beta polypeptide. In some embodiments, the human portion of the chimeric human / non-human CD8 alpha and / or beta polypeptide comprises one or more conservative or non-conservative modifications.

[0403] In one aspect, a non-human animal expressing a human CD8α and / or β polypeptide sequence is provided, 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 corresponding 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.

[0404] In some embodiments, a portion of a chimeric CD8, such as a human portion, may comprise substantially the entire sequence set forth herein (e.g., substantially the entire protein domain set forth herein). Substantially the entire sequence typically includes 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids believed to represent a particular portion of a protein (e.g., a particular functional domain, etc.). One skilled in the art will appreciate that the boundaries of a functional domain may vary slightly depending on the alignment and domain prediction methods used.

[0405] In one aspect, the non-human part of the chimeric human / non-human CD8 α and / or β polypeptide comprises at least the transmembrane domain and / or cytoplasmic domain of the non-human CD8 α and / or β polypeptide, respectively. Due to the important functions provided by the CD8 cytoplasmic domain, retaining the endogenous non-human (e.g., mouse) sequence in genetically engineered animals ensures the preservation of the appropriate intracellular signaling and other functions of the auxiliary receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD8 α and / or β polypeptides are mouse CD8 α and / or β polypeptides, respectively. Although specific mouse CD8 α and β sequences are described in U.S. Patent No. 9,848,587 cited in the examples, any suitable sequence derived therefrom is contemplated herein, such as a sequence comprising conservative / non-conservative amino acid substitutions. In one embodiment, the non-human animal (e.g., a rodent, such as a mouse) retains any endogenous sequence that has not yet been humanized.

[0406] The non-human animals described herein may include nucleotide sequences encoding chimeric human / non-human CD8α and / or β polypeptides at their endogenous loci. In one aspect, this results in a nucleotide sequence encoding a portion of a human CD8α polypeptide replacing a portion of an endogenous CD8α gene, and / or a nucleotide sequence encoding a portion of a human CD8β polypeptide replacing a portion of an endogenous CD8β gene. In one embodiment, such replacement is the replacement of an endogenous nucleotide sequence encoding the entire or substantially entire extracellular portion of non-human CD8α and / or β with a human nucleotide sequence encoding the extracellular portion. In one embodiment, such replacement is the replacement of a sequence encoding at least the entire or substantially entire immunoglobulin V-like domain of non-human CD8α and / or β with a human nucleotide sequence encoding the domain. In one embodiment, the replacement does not include the replacement of CD8α and / or β sequences encoding the transmembrane domain and cytoplasmic domain of non-human CD8α and / or β polypeptides. Therefore, the non-human animal expresses chimeric human / non-human CD8α and / or β polypeptides from an endogenous non-human CD8 locus. In another embodiment, the replacement produces a CD8 alpha and / or beta protein comprising the polypeptide sequence shown in SEQ ID NO: 3 and / or 5, respectively.

[0407] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD8 locus (e.g., a chimeric rodent CD8 locus, such as a chimeric mouse CD8 locus) is provided. In one aspect, because a chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD8 α and / or β sequence is located at a corresponding endogenous non-human (e.g., rodent, such as mouse) CD8 α and / or β locus, it retains endogenous CD8 α and / or β promoters and regulatory elements. In another embodiment, the chimeric locus may contain human CD8 α and / or β promoters and regulatory elements to an extent that allows appropriate CD8 α and / or β expression (appropriate spatial and temporal protein expression), CD8+T cell development, CD8 lineage selection, and auxiliary receptor function. Therefore, in one aspect, the animal of the present invention comprises a genetic modification that does not change the appropriate lineage selection and development of T cells. In one aspect, the animal (e.g., rodent, such as mouse) of the invention does not express the chimeric CD8 protein on immune cells other than cells 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 the retention of elements that allow for appropriate spatial and temporal regulation of CD8 alpha and / or beta expression.

[0408] In various embodiments, a non-human animal (e.g., a rodent, such as a mouse or rat) expressing 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 with the same cellular distribution observed in humans. In one aspect, the CD8 protein of the present invention is capable of interacting with an MHC I protein expressed on the surface of a second cell.

[0409] Human or humanized MHC molecules

[0410] Although antigen recognition by γ / δ T cells may not require interaction between T cell co-receptors (e.g., CD4 and CD8) and MHC, because the non-human animals described herein may also contain human or humanized TRA, TRB, CD4 and / or CD8 (e.g., CD8α and CD8β) loci in addition to human or humanized TRD (and TRG) loci, the non-human animals described herein may also contain one or more human or humanized MHC loci.

[0411] In various embodiments, provided herein are genetically modified non-human animals that co-express at least one humanized T cell co-receptor, at least one humanized MHC associated with the humanized T cell co-receptor, and a human or humanized α / βTCR, which, after recognizing and binding to a peptide presented by the humanized MHC, together with the humanized co-receptor, provides an activation signal to a cell expressing the humanized TCR and a chimeric T cell co-receptor polypeptide. Thus, a non-human animal as disclosed herein comprises at least one of a first nucleic acid sequence, a second nucleic acid sequence, and / or a third nucleic acid sequence, wherein each nucleic acid sequence encodes a different human or humanized MHC polypeptide selected from the following: a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, and a human or humanized MHC Iα polypeptide; the non-human animal optionally also comprises a human or humanized β2 microglobulin. The use of the first, second, and third designations herein should not be construed as limiting the non-human animals disclosed herein to requiring all three nucleic acid sequences or requiring any human or humanized MHC polypeptides to be present in any particular order.

[0412] Thus, in some embodiments, a non-human animal as disclosed herein may comprise, for example, a first nucleotide sequence and a second nucleotide sequence encoding, for example, a human or chimeric CD8α polypeptide and a human or chimeric CD8β polypeptide, 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 TCRα constant region gene sequence, 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 a first nucleic acid sequence and a second nucleic acid sequence encoding, for example, a human or humanized MHC Iα polypeptide and a human or humanized β2 microglobulin polypeptide. In other embodiments, a non-human animal as disclosed herein may comprise, for example, a first nucleotide sequence encoding, for example, a chimeric CD4 polypeptide; 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 TCRα constant region gene sequence, 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 a first nucleic acid sequence and a second nucleic acid sequence encoding, for example, a human or humanized MHC IIα polypeptide and a human or humanized MHC IIβ polypeptide. In some embodiments, a non-human animal as disclosed herein may comprise, for example, a first nucleotide sequence, a second nucleotide sequence, and a third nucleotide sequence encoding, for example, a chimeric CD4 polypeptide, a chimeric CD8α polypeptide, and a chimeric CD8β polypeptide; 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 TCRα constant region gene sequence, 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 a first nucleic acid sequence, a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence encoding, for example, 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 polypeptide.

[0413] In various embodiments, provided herein is a genetically modified non-human animal, such as a rodent (e.g., a mouse or rat), comprising in its genome a nucleic acid sequence encoding a human or humanized MHC I polypeptide and / or a nucleic acid sequence encoding a human or humanized MHC II protein. The MHC I nucleic acid sequence may encode a partially human and partially non-human MHC I polypeptide, such as a chimeric human / non-human MHC I polypeptide, and the MHC II nucleic acid sequence may encode a partially human and partially non-human MHC II protein, such as a chimeric human / non-human MHC II protein (e.g., comprising a chimeric human / non-human MHC II α and β polypeptide). In some aspects, the animal does not express endogenous MHC I and / or endogenous MHC II polypeptides, such as functional endogenous MHC I and / or MHC II polypeptides, on the 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.

[0414] A genetically modified non-human animal comprising a nucleic acid sequence encoding a chimeric human / non-human MHC I polypeptide in its genome, e.g., at an endogenous locus, is disclosed in U.S. Pat. No. 9,615,550 and U.S. Pat. No. 9,591,835; each is incorporated herein by reference in its entirety. A genetically modified non-human animal comprising a nucleic acid sequence encoding a humanized, e.g., chimeric human / non-human MHC II polypeptide in its genome, e.g., at an endogenous locus, is disclosed in U.S. Pat. No. 8,847,005 and U.S. Pat. No. 9,043,966; each is incorporated herein by reference in its entirety. A genetically modified non-human animal comprising in its genome, e.g., at an endogenous locus, a nucleic acid sequence encoding a chimeric human / non-human MHC I polypeptide and in its genome, e.g., at an endogenous locus, a nucleic acid sequence encoding a humanized, e.g., chimeric human / non-human MHC II polypeptide is disclosed in U.S. Patent Publication No. 20140245467, which is incorporated herein by reference in its entirety.

[0415] In various embodiments, provided herein is a genetically modified non-human animal comprising 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 a 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 a 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 a portion thereof, e.g., one or more extracellular domains) of a human MHC II β polypeptide; wherein the non-human animal expresses functional chimeric human / non-human MHC I and MHC II proteins from its endogenous non-human MHC loci. In one embodiment, the first nucleic acid sequence, the second nucleic acid sequence and / or the third nucleic acid sequence are located at endogenous non-human MHC I, MHC IIα and MHC IIβ loci, respectively. In one embodiment, wherein the non-human animal is a mouse, the first nucleic acid sequence, the second nucleic acid sequence and / or the third nucleic acid sequence are 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.

[0416] In one embodiment, the non-human animal expresses only chimeric human / non-human MHC I, MHC IIα and / or MHC βII polypeptides, and does not express endogenous non-human MHC polypeptides (e.g., functional endogenous MHC I, IIα and / or IIβ polypeptides) from endogenous non-human MHC loci. In one embodiment, the animals described herein express functional chimeric MHC I and functional chimeric MHC II on the surface of their cells (e.g., antigen presenting cells, etc.). In one embodiment, the only MHC I and MHC II expressed by the animal on the cell surface are chimeric MHC I and chimeric MHC II, and the animal does not express any endogenous MHC I and MHC II on the cell surface.

[0417] In one embodiment, the chimeric human / non-human MHC I polypeptide comprises a peptide binding cleft in its human portion, such as a peptide binding groove of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises an extracellular portion of a human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises an extracellular domain of the α chain of human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises an α1 domain and an α2 domain of human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises an α1 domain, an α2 domain, and an α3 domain of human MHC I.

[0418] In one aspect, the human portion of the chimeric MHC II α polypeptide and / or the human portion of the chimeric MHC II β polypeptide comprises a peptide binding domain of a human MHC II α polypeptide and / or a human MHC II β polypeptide, respectively. In one aspect, the human portion of the chimeric MHC II α and / or β polypeptide comprises an extracellular portion of a human MHC II α and / or β polypeptide, respectively. In one embodiment, the human portion of the chimeric MHC II α polypeptide comprises the α1 domain of a human MHC II α polypeptide; in another embodiment, the human portion of the chimeric MHC II α polypeptide comprises the α1 domain and the α2 domain of a human MHC II α polypeptide. In another embodiment, the human portion of the chimeric MHC II β polypeptide comprises the β1 domain of a human MHC II β polypeptide; in another embodiment, the human portion of the chimeric MHC II β polypeptide comprises the β1 domain and the β2 domain of a human MHC II β polypeptide.

[0419] In some embodiments, human or humanized MHC I polypeptides may be derived from functional human HLA molecules encoded by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G loci. Human or humanized MHC II polypeptides may be derived from functional human HLA molecules encoded by HLA-DP, HLA-DQ and HLA-DR loci. A list of commonly used HLA antigens and alleles is described 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 presents a brief description of the HLA nomenclature used in the art. Additional information on 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 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, the human or humanized MHC I and / or II polypeptide can be derived from any functional human HLA molecule described herein. In some embodiments, all of the MHC I and MHC II polypeptides expressed on the surface of a cell comprise portions derived from human HLA molecules.

[0420] Of particular interest are human HLA molecules, i.e., specific polymorphic HLA alleles, which are known to be associated with many human diseases, such as human autoimmune diseases. In fact, specific polymorphisms in the HLA loci have been identified that are associated 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 diseases. See, e.g., 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 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. Therefore, human or humanized MHC I and / or II polypeptides can be derived from human HLA molecules known to be associated with specific diseases, such as autoimmune diseases.

[0421] In a specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific 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, such as an HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is commonly used in North American populations. Although the present invention provides US Patent Nos. 9,615,550 and 10,154,658 to describe this specific HLA sequence, any suitable HLA-A sequence is contemplated herein, such as polymorphic variants of HLA-A2 expressed in human populations, sequences having one or more conservative or non-conservative amino acid modifications, nucleic acid sequences that differ from the sequences described herein due to the degeneracy of the genetic code, and the like.

[0422] In another specific aspect, 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 aspect, it is derived from HLA-B, such as HLA-B27. In another aspect, it is derived from HLA-A3, HLA-B7, HLA-Cw6, etc.

[0423] In a specific aspect, the human portion of the humanized MHC II α and β polypeptides described herein is derived from human HLA-DR, such as HLA-DR2. Typically, the HLA-DR α chain is monomorphic, for example, the α chain of the HLA-DR complex is encoded by the HLA-DRA gene (e.g., the HLA-DR α * 01 gene). In another aspect, the HLA-DR β chain is polymorphic. Thus, HLA-DR2 comprises an α chain encoded by the HLA-DRA gene and a β chain encoded by the HLA-DR1 β * 1501 gene. Although the embodiments of the present invention cite U.S. Pat. Nos. 8,847,005 and 9,043,996 to describe these specific HLA sequences, any suitable HLA-DR sequence is contemplated herein, such as polymorphic variants exhibited in the human population, sequences having one or more conservative or non-conservative amino acid modifications, nucleic acid sequences that differ from the sequences described herein due to the degeneracy of the genetic code, and the like.

[0424] The human portion of the chimeric MHC II α and / or β polypeptide can be encoded by a nucleic acid sequence of an HLA allele known to be associated with common human diseases. 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, incorporated herein by reference.

[0425] In one aspect, the non-human portion of the chimeric human / non-human MHC I, MHC IIα and / or MHC IIβ polypeptide comprises a transmembrane and / or cytoplasmic domain of an endogenous non-human (e.g., rodent, such as 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 a 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 a 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 a transmembrane and / or cytoplasmic domain of an endogenous non-human MHC IIβ polypeptide. In one aspect, 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 polypeptide is derived from a mouse H-2E protein. Thus, the non-human portion of the chimeric MHC I polypeptide may comprise a transmembrane domain and a cytoplasmic domain derived from mouse H-2K, and the non-human portion of the chimeric MHC II α and β polypeptide may comprise a transmembrane domain and a cytoplasmic domain derived from mouse H-2E protein. Although specific H-2K and H-2E sequences are contemplated in U.S. Patents 9,615,550 and 10,154,658 cited in the Examples, any suitable sequence is contemplated herein, such as polymorphic variants, conservative / non-conservative amino acid substitutions, etc. In one aspect, the non-human animal is a mouse, and the mouse does not express functional endogenous MHC polypeptides 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 aspects, the mouse does not express any functional endogenous mouse MHC I and MHC II on the cell surface.

[0426] The chimeric human / non-human polypeptide may be such that it comprises a human or non-human leader (signal) sequence. In one embodiment, the chimeric MHC I polypeptide comprises a non-human leader sequence of an endogenous MHC I polypeptide. In one embodiment, the chimeric MHC IIα polypeptide comprises a non-human leader sequence of an endogenous MHC IIα polypeptide. In one embodiment, the chimeric MHC IIβ polypeptide comprises a non-human leader sequence of an endogenous MHC IIβ polypeptide. In an alternative embodiment, the chimeric MHC I, MHC IIα and / or MHC IIβ polypeptide comprises a non-human leader sequence of an MHC I, MHC IIα and / or MHC IIβ polypeptide from another non-human animal, such as another rodent or another mouse strain, respectively. Thus, the nucleic acid sequence encoding the chimeric MHC I, MHC IIα and / or MHC IIβ polypeptide may be operably linked to the nucleic acid sequence encoding the non-human MHC I, MHC IIα and / or MHC IIβ leader sequence, respectively. In another embodiment, the chimeric MHC I, MHC IIα and / or MHC IIβ polypeptide comprises a human leader sequence of a human MHC I, human MHC IIα and / or human MHC IIβ polypeptide, respectively (e.g., the leader sequence of human HLA-A2, human HLA-DRα and / or human HLA-DRβ1*1501, respectively).

[0427] The chimeric human / non-human MHC I, MHC IIα and / or MHC IIβ polypeptide may comprise a complete or substantially complete extracellular domain of a human MHC I, human MHC IIα and / or human MHC IIβ polypeptide, respectively, in its human portion. Thus, the human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, such as 95% or more of the amino acids encoding the extracellular domain of a human MHC I, human MHC IIα and / or human MHC IIβ polypeptide (e.g., human HLA-A2, human HLA-DRα and / or human HLA-DRβ1*1501). In one example, the substantially complete extracellular domain of a human MHC I, human MHC IIα and / or human MHC IIβ polypeptide lacks a human leader sequence. In another example, the chimeric human / non-human MHC I, chimeric human / non-human MHC IIα and / or chimeric human / non-human MHC IIβ polypeptide comprises a human leader sequence.

[0428] In addition, the chimeric MHC I, MHC IIα and / or MHC IIβ polypeptides can be operably linked to (e.g., expressed under the regulatory control of) endogenous non-human promoters and regulatory elements, such as mouse MHC I, MHC IIα and / or MHC IIβ regulatory elements, respectively. Such an arrangement will facilitate appropriate expression of the chimeric MHC I and / or MHC II polypeptides in a non-human animal, such as during an immune response in a non-human animal.

[0429] In another embodiment, the non-human animal of the present invention, such as a rodent, such as a mouse, comprises (e.g., at an endogenous β2 microglobulin locus) a nucleic acid sequence encoding a human or humanized β2 microglobulin. The light chain of the β2 microglobulin or MHC class I complex (also abbreviated as "β2M") is a small (12 kDa) non-glycosylated protein that primarily functions to stabilize the MHC I α chain. The production of human or humanized β2 microglobulin animals is described in detail in U.S. Pat. No. 9,615,550 and is incorporated herein by reference.

[0430] The nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide may include nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may include nucleic acid residues encoding the amino acid sequence shown in amino acids 21-119 of the human β2 microglobulin protein (i.e., corresponding to the amino acid residues of mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may include nucleic acid residues encoding the amino acid sequence shown in amino acids 23-115 of the human β2 microglobulin protein, such as the amino acid sequence shown in amino acids 23-119 of the human β2 microglobulin protein. The nucleic acid sequence and amino acid sequence of human β2 microglobulin are described in Gussow et al., supra (incorporated herein by reference).

[0431] Thus, a human or humanized β2 microglobulin polypeptide may comprise the amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin polypeptide, such as the amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin polypeptide, such as the amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin polypeptide. Alternatively, a human β2 microglobulin may comprise amino acids 1-119 of a human β2 microglobulin polypeptide.

[0432] In some embodiments, the nucleotide sequence encoding human or humanized β2 microglobulin comprises the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises the nucleotide sequence shown in exons 2, 3, and 4 of the human β2 microglobulin gene. In this embodiment, the nucleotide sequences shown in exons 2, 3, and 4 are operably linked to allow normal transcription and translation of the gene. Therefore, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to exon 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a human β2 microglobulin gene of about 2.8 kb.

[0433] Therefore, the human or humanized β2 microglobulin polypeptide can be encoded by a nucleotide sequence comprising the nucleotide sequence shown in exon 2 to exon 4 of human β2 microglobulin, for example, a nucleotide sequence corresponding to the nucleotide sequence of exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the polypeptide can be encoded by a nucleotide sequence comprising the nucleotide sequence shown in exons 2, 3 and 4 of the human β2 microglobulin gene. In a specific embodiment, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to about 267 bp after exon 2 to exon 4 of the human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising about 2.8 kb of the human β2 microglobulin gene. Since exon 4 of the β2 microglobulin gene contains a 5' untranslated region, the human or humanized polypeptide can be encoded by a nucleotide sequence comprising exon 2 and exon 3 of the β2 microglobulin gene.

[0434] It will be understood by those of ordinary skill in the art that although specific nucleic acid sequences and amino acid sequences for producing genetically engineered animals are described herein, sequences having 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.

[0435] Thus, a non-human animal expressing a human β2 microglobulin sequence is provided, 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 a specific embodiment, 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.

[0436] In addition, a non-human animal is provided, wherein the nucleotide sequence encoding the human or humanized β2 microglobulin protein further comprises the nucleotide sequence set forth in exon 1 of a non-human β2 microglobulin gene. Thus, in a specific embodiment, the non-human animal comprises in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin, wherein the nucleotide sequence comprises exon 1 of a non-human β2 microglobulin and exons 2, 3, and 4 of a human β2 microglobulin gene. Thus, a human or humanized β2 microglobulin polypeptide is encoded by exon 1 of a non-human β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene (e.g., exons 2 and 3 of a human β2 microglobulin gene).

[0437] In one embodiment, the non-human animal (e.g., rodent, such as mouse) of the present invention comprises, in addition to the nucleotide sequence encoding the chimeric CD8 protein, a nucleic acid sequence encoding a human or humanized MHC I protein, such that the chimeric CD8 protein expressed on the surface of the animal's T cells is able to associate, bind and / or interact 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 an 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. Pat. Nos. 9,615,550 and 9,591,835, both of which are incorporated herein by reference in their entirety. 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., wherein the humanized MHC I polypeptide comprises a human MHC I extracellular domain and a transmembrane domain and a cytoplasmic domain of an endogenous (e.g., mouse) MHC I, e.g., wherein 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 a nucleotide sequence set forth in exons 2, 3, and 4 of human β2 microglobulin). In one aspect, both the humanized MHC I and the human or humanized β2 microglobulin polypeptide are encoded by nucleotide sequences located at endogenous MHC I and β2 microglobulin loci, respectively; in one aspect, the animal does not express functional endogenous MHC I and β2 microglobulin polypeptides. Thus, the MHC I expressed by these animals can be a chimeric human / non-human, e.g., human / rodent (e.g., human / mouse) MHC I polypeptide. The human portion of the chimeric MHC I polypeptide can 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 can be derived from a mouse MHC I protein selected from the group consisting of H-2D, H-2K, and H-2L.

[0438] In one embodiment, the non-human animal (e.g., rodent, such as mouse) of the present invention further comprises a nucleotide sequence encoding a human or humanized MHC II protein, such that the chimeric CD4 protein expressed on the surface of the T cells of the animal is capable of interacting with the 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 II α polypeptide and the extracellular domain of a human MHC II β polypeptide. Exemplary genetically modified animals expressing human or humanized MHC II polypeptides are described in U.S. Pat. Nos. 8,847,005 and 9,043,996, issued on September 30, 2014, which are incorporated herein by reference in their entirety. Thus, in one embodiment, an animal comprising a chimeric CD4 protein as described herein may further comprise a humanized MHC II protein, wherein the humanized MHC II protein comprises: (1) a humanized MHC II α polypeptide comprising a human MHC II α extracellular domain and a transmembrane domain and a cytoplasmic domain of an endogenous (e.g., mouse) MHC II, wherein the human MHC II α extracellular domain comprises an α1 domain and an α2 domain of human MHC II α, and (2) a humanized MHC II β polypeptide comprising a human MHC II β extracellular domain and a transmembrane domain and a cytoplasmic domain of an endogenous (e.g., mouse) MHC II, wherein the human MHC II β extracellular domain comprises a β1 domain and a β2 domain of human MHC II β. In one aspect, the humanized MHC II α and β polypeptides are encoded by nucleic acid sequences located at endogenous MHC II α and β loci, respectively; in one aspect, the animal does not express functional endogenous MHC II α and β polypeptides. Thus, the MHC II expressed by these animals 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 polypeptide 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 where the animal is a mouse, the non-human (i.e., mouse) portion of the chimeric MHC II polypeptide can be derived from a mouse MHC II protein selected from the group consisting of H-2E and H-2A.

[0439] Various other embodiments of genetically modified non-human animals (e.g., rodents, such as rats or mice) will be apparent to those skilled in the art from this 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; each of which is incorporated herein by reference.

[0440] In various embodiments, the genetically modified non-human animals described herein produce cells, such as APCs, having human or humanized MHC I and II on the cell surface, and thus present peptides as epitopes for T cells in a human-like manner, because substantially all components of the complex are human or humanized. The genetically modified non-human animals of the present invention can be used to study the function of the human immune system in humanized animals; to identify antigens and antigenic epitopes (e.g., T cell epitopes, such as unique human cancer epitopes) that elicit immune responses, such as for vaccine development; to evaluate vaccine candidates and other vaccine strategies; to study human autoimmunity; to study human infectious diseases; and in addition to designing better therapeutic strategies based on human MHC expression.

[0441] In some embodiments, a mouse as described herein comprises:

[0442] (I) contains the following germ cells and CD3 - Somatic cells:

[0443] (A) at the endogenous TCRγ locus:

[0444] Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments,

[0445] Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and

[0446] Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and

[0447] (B) at the endogenous TCRδ locus:

[0448] Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments,

[0449] Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments,

[0450] Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and

[0451] Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and

[0452] (C) at the endogenous TCRα locus:

[0453] Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and

[0454] Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments, and

[0455] (D) at the endogenous TCRβ locus:

[0456] Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments,

[0457] Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments,

[0458] Replace all endogenous TCR Jβ segments with the full repertoire of unrearranged human TCR Jβ segments,

[0459] (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 coreceptor 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;

[0460] (F) a second nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide,

[0461] 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 wherein 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;

[0462] (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC II alpha polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC II beta polypeptide,

[0463] wherein the chimeric human / mouse MHC II alpha polypeptide comprises an alpha 1 domain and an alpha 2 domain of a human HLA class II alpha polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II alpha polypeptide, and wherein the chimeric human / mouse MHC II beta polypeptide comprises a beta 1 domain and a beta 2 domain of a human HLA class II beta polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II beta polypeptide;

[0464] (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 the transmembrane domain and cytoplasmic domain of a mouse MHC class I polypeptide; and

[0465] (I) a polynucleotide sequence encoding a human or humanized β2 microglobulin polypeptide and comprising a nucleotide sequence comprising the nucleotide sequence set forth in exon 1 of a mouse β2 microglobulin gene operably linked to the nucleotide sequences set forth in exon 2, exon 3, and exon 4 of a human β2 microglobulin gene,

[0466] (II) wherein the mouse further comprises a CD3 T cell expressing on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells, and CD3 T cells 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 + T cells, and optionally wherein the mouse expresses:

[0467] (e) the chimeric human / mouse CD4 coreceptor,

[0468] (f) a chimeric CD8 coreceptor comprising said chimeric human / mouse CD8 alpha polypeptide and said chimeric human / mouse CD8 beta polypeptide,

[0469] (h) a chimeric MHC II complex comprising the chimeric human / mouse MHC II α polypeptide and the chimeric human / mouse MHC II β polypeptide, wherein the chimeric MHC II complex is capable of binding to the chimeric human / mouse CD4 coreceptor,

[0470] (i) the chimeric human / mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding to the chimeric CD8 coreceptor, and

[0471] (j) the human or humanized β2 microglobulin polypeptide.

[0472] In some embodiments, non-human animals as described herein include two copies of one or more modified loci as described herein.In some embodiments, non-human animals as described herein include two copies of unrearranged people or humanized TCRγ loci, two copies of unrearranged people or humanized TCRδ loci, two copies of unrearranged people or humanized TCRα loci, two copies of unrearranged people or humanized TCRβ loci, two copies of people or humanized CD4 loci, two copies of people or humanized CD8α loci, two copies of people or humanized CD8β loci, two copies of people or humanized MHC I loci and / or two copies of people or humanized MHC IIα and / or MHC IIβ loci.Therefore, for one or more unrearranged people or humanized TCRγ, TCRδ, TCRα and / or TCRβ loci, one or more people or humanized auxiliary receptor loci and / or one or more MHC loci, the non-human animal is homozygous. In some embodiments of the invention, the non-human animal comprises one copy of an unrearranged human or humanized TCRγ locus, one copy of an unrearranged human or humanized TCRδ variable locus, one copy of an unrearranged human or humanized TCRα variable locus, one copy of an unrearranged human or humanized TCRβ variable locus, one copy of a human or humanized CD4 locus, one copy of a human or humanized CD8α locus, one copy of a human or humanized CD8β locus, one copy of a human or humanized MHC I locus, and / or one copy of a human or humanized MHC IIα and / or MHC IIβ locus. Thus, the non-human animal may be heterozygous for unrearranged human or humanized TCRγ, TCRδ, TCRα, and / or TCRβ loci, one or more human or humanized auxiliary receptor loci, and / or one or more human or humanized MHC loci. In some embodiments, for unrearranged human TCRγ loci, non-human animals (e.g., mice) are heterozygous or homozygous, for example, wherein the non-human animal comprises an unrearranged human TCR Vγ segment operably linked to a human TCR Cγ gene and an unrearranged human Jγ segment. In some embodiments, for unrearranged human TCRδ loci, non-human animals (e.g., mice) are heterozygous or homozygous, for example, wherein the non-human animal comprises an unrearranged human TCR Vδ segment operably linked to a human TCR Cδ gene, an unrearranged human TCR Dδ segment, and an unrearranged human Jδ segment.

[0473] In some embodiments, a non-human animal that is heterozygous or homozygous for human TRD and TRG loci and humanized TRA, TRB, MHC I, MHC II, CD4, CD8, and β2 microglobulin loci as described herein comprises a population of CD45+CD3+T cells in its spleen, thymus, mesenteric lymph nodes (MLN), skin, intestinal mucosa, and / or in intraepithelial lymphocytes (IEL) isolated from its colon or small intestine, wherein a certain percentage of the population of CD45+CD3+T cells expresses human γ / δTCR. In some embodiments, the percentage of spleen, thymus, MLN, and / or IEL CD45+CD3+T cells expressing human γ / δTCR in the γ and / or δTCR mouse embodiments as described herein is comparable to the percentage of CD45+CD3+T cells expressing mouse γ / δTCR in wild-type mice (e.g., not significantly different, any differences are statistically insignificant, within 10 percentage points of each other, etc.). In some embodiments, in the γ and / or δTCR mouse embodiments as described herein, the percentage of spleen, thymus, MLN and / or IEL CD45+CD3+ T cells expressing human γ / δTCR is greater than the percentage of CD45+CD3+ T cells expressing mouse γ / δTCR in wild-type mice (e.g., 1.5-fold to 3-fold).

[0474] The genetically modified non-human animals of the present invention can be selected from mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For those non-human animals where it is not easy to obtain suitable genetically modified ES cells, other methods are used to prepare non-human animals comprising genetic modifications. Such methods include, for example, modifying non-ES cell genomes (e.g., fibroblasts or induced pluripotent cells) and using nuclear transfer to transfer the genetically modified genome to suitable cells, such as oocytes, and gestating modified cells (e.g., modified oocytes) in non-human animals under conditions suitable for forming embryos.

[0475] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal such as Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats and hamsters. In one embodiment, the rodent is selected from Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., hamsters of the mouse class), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (Muridae) (true mice and rats, gerbils, spiny mice, crown mice), Nesomyidae (climbing mice, rock mice, white-tailed rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormouse) and Spalacidae (e.g., moles, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muridae), gerbils, spiny mice, and crested mice. In one embodiment, the genetically modified mouse is a member from the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0476] In certain embodiments, the non-human animal is a rodent that is a mouse of the C57BL strain selected from the group consisting of 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 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, also see, 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 specific embodiment, the mouse is a mixture of the aforementioned 129 strain, or a mixture of the aforementioned BL / 6 strain. In a specific embodiment, the 129 strain in the mixture is a 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 the BALB strain and another aforementioned strain. As provided herein, the non-human animal can be a mouse derived from any combination of the aforementioned strains.

[0477] 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.

[0478] In some aspects of the invention, non-human animals comprising unrearranged human or humanized TCRγ, TCRδ, TCRα and / or TCRβ gene loci (comprising unrearranged human TCRγ, TCRδ, TCRα and / or TCRβ variable V, (D) and J segments, respectively) retain endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ gene segments or gene loci. In one embodiment, the retained endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ segments are functional gene segments, and, for example, in T cells, can be rearranged (rearranged) according to the 12 / 23 recombination rules with human TCRγ, TCRδ, TCRα and / or TCRβ segments found at the same locus, respectively. See Olaru A., supra. In one embodiment, endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ are non-functional gene segments or gene loci. In one embodiment, the non-functional locus is an inactivated locus, such as a reverse locus (e.g., the encoding nucleic acid sequence of the variable gene locus is in the reverse direction relative to the constant region gene sequence, making it impossible to successfully rearrange the variable region segment using the reverse locus). In one embodiment, humanized TCRγ, TCRδ, TCRα and / or TCRβ gene segments or variable gene loci are respectively located between endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ variable gene loci and endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ constant region gene loci. In one embodiment, humanized TCRγ, TCRδ, TCRα and / or TCRβ gene segments or variable gene loci are respectively located between endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ variable gene loci and endogenous non-human TCRγ, TCRδ, TCRα and / or TCRβ constant region gene loci.

[0479] In various embodiments of the present invention, unrearranged human or humanized TCR variable gene loci (e.g., TCRαTCRβ, TCRγ and / or TCRδ variable gene loci) are included in the germline of a non-human animal (e.g., a rodent, e.g., a mouse or rat), e.g., the non-human animal comprises germ cells (sperm and oocytes) containing modified loci 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 located at one (or more) endogenous non-human TCR variable loci, wherein unrearranged human V and J and / or V and D and J segments are operably linked to human or non-human TCR constant region gene sequences.

[0480] In one aspect, non-human animals (e.g., rodents, such as mice or rats) comprising people or humanized TCRγ and / or TCRδ loci as described herein and optionally TCRα and / or TCRβ loci express humanized T cell receptors comprising human variable regions and non-human (e.g., rodents, such as mice or rats) constant regions on the surface of T cells. In some aspects, the non-human animal can express a diversity library of humanized T cell receptors that recognize multiple presented antigens.

[0481] In addition to genetically engineered non-human animals, non-human embryonic stem (ES) cell lines or germ cell lines are also provided, as well as embryos (e.g., rodent embryos, such as mouse or rat embryos) comprising and / or derived from ES cells. ES cells, germ cells and / or embryos as described herein comprise genetically modified loci as described herein, such as 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.

[0482] Also provided is a tissue, wherein the tissue is derived from a non-human animal (e.g., a rodent, such as a mouse or rat) as described herein and comprises cells expressing human TCRγ and / or TCRδ proteins from human or humanized TCRG and / or TCRD loci, respectively.

[0483] In some embodiments, a method for preparing human TCRγ and / or human TCRδ protein is provided, the method comprising expressing human TCRγ and / or human TCRδ protein from a nucleotide construct as described herein in a single cell. In one embodiment, the nucleotide construct is a viral vector; 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 expressing viral nucleic acid sequences (e.g., PERC.6 TM cell).

[0484] In one aspect, a cell expressing human TCRγ and / or human TCRδ protein is provided. In one embodiment, the cell comprises an expression vector comprising human TCRγ and / or human TCRδ protein as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells expressing viral nucleic acid sequences (e.g., PERC.6 TM cell).

[0485] Also provided are human TCRγ and / or human TCRδ proteins produced by non-human animals as described herein. Therefore, human TCR proteins are included in the human complementary determining regions (i.e., human CDR1, CDR2 and CDR3) and human constant regions in their variable domains. Also provided are nucleic acids encoding human TCR variable domains produced by non-human animals as described herein.

[0486] In addition, non-human cells isolated from non-human animals as described herein are provided. In one embodiment, the cells are ES cells. In one embodiment, the cells are T cells, such as γ / δ T cells. Non-human cells expressing TCR proteins comprising human TCRγ proteins and / or human TCRδ proteins are also provided.

[0487] Also provided is a non-human cell comprising a chromosome or a fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a cell nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises a chromosome or a fragment thereof as a result of nuclear transfer.

[0488] In one aspect, a hybridoma or quadroma derived from a cell of a non-human animal as described herein is provided. In one embodiment, the non-human animal is a mouse or a rat.

[0489] Preparation of genetically modified non-human animals that produce a substantially humanized T cell immune response

[0490] Provided is a method for preparing a genetically engineered non-human animal as described herein (e.g., a genetically engineered rodent, such as a mouse or rat). Typically, the method includes inserting into the genome of a non-human animal an unrearranged T cell receptor (TCR) γ variable gene locus comprising at least one human Vγ segment and at least one human Jγ segment operably linked to a human or non-human TCRγ constant region gene sequence, and / or an unrearranged TCRδ variable gene 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 TCRδ constant region gene sequence. In some embodiments, the method may also optionally include any one or a combination of the following: (a) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment operably linked to a non-human TCRα constant region gene sequence, and / or an unrearranged TCRβ variable gene 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; (b) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human / non-human T cell auxiliary receptor polypeptide, a second nucleotide sequence encoding a second chimeric human / non-human T cell auxiliary receptor polypeptide, and / or a second nucleotide sequence encoding a chimeric human / non-human T cell auxiliary receptor polypeptide. (c) placing a first nucleic acid sequence encoding a first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human / non-human MHC polypeptide, and / or a third nucleic acid sequence encoding a third chimeric human / non-human T cell co-receptor polypeptide, wherein the non-human portion of each chimeric T cell co-receptor polypeptide comprises at least a transmembrane domain and a cytoplasmic domain of a non-human T cell co-receptor, and wherein the human portion of each chimeric polypeptide comprises an extracellular portion (or a portion thereof) of a human T cell co-receptor; (c) placing a first nucleic acid sequence encoding a first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human / non-human MHC polypeptide, and / or a third nucleic acid sequence encoding a third chimeric human / non-human MHC polypeptide into a genome, and / or (d) adding a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide to the genome of a non-human animal.In some embodiments, the method may further include: (a) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment operably linked to a non-human TCRα constant region gene sequence, and / or an unrearranged TCRβ variable gene 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 the method may further include (b) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human / non-human T cell auxiliary receptor polypeptide, a second nucleotide sequence encoding a second chimeric human / non-human T cell auxiliary receptor polypeptide; The invention relates to a method of producing an animal comprising: (a) producing a chimeric human / non-human MHC polypeptide comprising: a first nucleic acid sequence encoding a first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human / non-human MHC polypeptide, and / or a third nucleic acid sequence encoding a third chimeric human / non-human T cell co-receptor polypeptide, wherein the non-human portion of each chimeric T cell co-receptor polypeptide comprises at least a transmembrane domain and a cytoplasmic domain of a non-human T cell co-receptor, and wherein the human portion of each chimeric polypeptide comprises an extracellular portion (or a portion thereof) of a human T cell co-receptor; (c) placing a first nucleic acid sequence encoding a first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human / non-human MHC polypeptide, and / or a third nucleic acid sequence encoding a third chimeric human / non-human MHC polypeptide into the genome, and (d) adding a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide to the genome of the non-human animal. In some embodiments, the steps of introducing, inserting and / or placing include targeting a sequence encoding an extracellular domain of a T cell co-receptor, a variable domain of a TCR, and optionally a TCR constant region gene sequence, an extracellular domain of an MHC polypeptide, or a portion of β2 microglobulin, and replacing it with a sequence encoding an extracellular domain of a human T cell co-receptor, a human TCR variable domain, and optionally a human TCR constant domain, a human MHC extracellular domain, and / or a human portion of β2 microglobulin, respectively.

[0491] In other embodiments, introducing, inserting, placing and / or adding may include breeding of animals of the same species, such as mating. In other embodiments, introducing, inserting, placing and / or adding include sequential homologous recombination in ES cells. In some embodiments, ES cells are derived from non-human animals that are genetically modified to contain one or more (but not all) of the desired genetic modifications, and homologous recombination in such ES cells completes genetic modification. In other embodiments, introducing, inserting, placing and / or adding may include a combination of breeding and homologous recombination in ES cells, for example, breeding an animal with another (or more) animal of the same species, some or all of which may be produced by ES cells genetically modified by a single homologous recombination or sequential homologous recombination events, and some of which may be separated from non-human animals comprising one or more genetic modifications disclosed herein.

[0492] In some embodiments, as described in the Examples, the method utilizes The targeting construct was prepared using the technique and introduced into ES cells and used Technology introduces targeted ES cell clones into mouse embryos. The targeting construct may include 5' and / or 3' homology arms, insertion sequences (replacing endogenous sequences) and one or more selection cassettes of the endogenous sequence to be replaced by the targeting construct. The selection cassette is a nucleotide sequence inserted into the targeting construct to promote the selection of cells (e.g., ES cells) that integrate the related constructs. A variety of suitable selection cassettes are known in the art. Typically, the selection cassette is positively selected in the presence of a specific antibiotic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). In addition, the selection cassette can be flanked by a recombination site that allows the selection cassette to be deleted when treated with a recombinase. Commonly used recombination sites are loxP and Frt, which are recognized by Cre and Flp enzymes, respectively, but other recombination sites are known in the art. The selection cassette may be located at any position outside the coding region in the construct. In one embodiment, the selection cassette is located in 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 the intron of the human DNA fragment. In another embodiment, the selection cassette is located at the junction of the human and mouse DNA fragments.

[0493] In some embodiments, methods for preparing a genetically modified non-human animal produce an animal whose genome comprises a human or humanized unrearranged TCR locus (e.g., a human or humanized unrearranged TCRγ and / or TCRδ locus, and optionally a human or humanized TCRα and / or TCRβ locus). In one embodiment, a method is provided for preparing a genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) that expresses a T cell receptor comprising a human variable region and a human or endogenous TCR constant domain on the surface of a T cell, wherein the method comprises inserting an unrearranged humanized TCRγ variable gene locus comprising at least one human Vγ segment and at least one human Jγ segment into a first non-human animal, such as replacing an endogenous non-human TCRγ variable gene locus, wherein the humanized TCRγ variable gene locus is operably linked to a human or endogenous TCRγ constant region gene sequence; in a second non-human animal, inserting an unrearranged humanized TCRδ variable gene locus comprising at least one human Vδ segment, a human Dδ segment and a human Jδ segment, such as replacing an endogenous non-human TCRδ variable gene locus, wherein the humanized TCRδ variable gene locus is operably linked to a human or endogenous TCRδ constant region gene sequence; and breeding the first non-human animal and the second non-human animal to obtain a non-human animal that expresses a T cell receptor comprising a human or humanized γ / δTCR. In some embodiments, methods for making a genetically modified non-human animal produce an animal whose genome comprises an unrearranged human TCR locus (e.g., an unrearranged human TCRγ and / or TCRδ locus, and optionally an unrearranged human TCRα and / or TCRβ locus).In one embodiment, a method for preparing a genetically modified non-human animal (e.g., a rodent, such as a mouse or a rat) that expresses a T cell receptor comprising a human variable region and a human constant domain on the surface of a T cell is provided, wherein the method comprises inserting an unrearranged human TCRγ gene locus into a first non-human animal, such as replacing an endogenous non-human TCRγ gene locus, the unrearranged human TCRγ gene locus comprising at least one human Vγ segment and at least one human Jγ segment operably linked to a human TCRγ constant region gene sequence (e.g., wherein the method further comprises replacing a nucleotide sequence comprising a human TCRγ constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence) with a nucleotide sequence comprising a human TCRγ constant region gene sequence (e.g., a human TRGC1 constant region sequence and / or a human TRGC2 constant region sequence) sequence, endogenous Trgc2 constant region gene sequence, endogenous Trgc3 constant region gene sequence and / or endogenous Trgc4 constant region gene sequence); inserting an unrearranged human TCRδ variable gene locus comprising at least one human Vδ segment, one human Dδ segment and one human Jδ segment operably linked to a human TCRδ constant region gene sequence into a second non-human animal, for example, replacing an endogenous non-human TCRδ gene locus (for example, wherein the method further comprises replacing an endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence); and breeding the first non-human animal and the second non-human animal to obtain a non-human animal expressing a T cell receptor comprising a human γ / δTCR. In some embodiments, the second non-human animal comprises a human or humanized TCRα locus before and / or in addition to the human TCRδ locus.

[0494] In some embodiments, the method for preparing a genetically modified non-human animal produces an animal whose genome comprises an unrearranged human TCR locus (e.g., an unrearranged human TCRγ and / or TCRδ locus, and optionally a human or humanized unrearranged TCRα and / or TCRβ locus). In one embodiment, a method for preparing a genetically modified non-human animal (e.g., a rodent, such as a mouse or a rat) that expresses a T cell receptor comprising a human variable domain and a human constant domain on the surface of a T cell is provided, wherein the method comprises inserting an unrearranged human TCRγ variable gene locus 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) into a first non-human animal, for example, replacing 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 gene);In the second non-human animal, insert an unrearranged humanized TCRδ gene locus comprising 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 human Cδ genes, for example, replace an endogenous non-human TCRδ sequence comprising 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; and breed the first non-human animal and the second non-human animal to obtain a non-human animal expressing a T cell receptor comprising a human γ / δTCR. In some embodiments, the second non-human animal comprises a human or humanized TCRα locus before and / or in addition to the human TCRδ locus.

[0495] In some embodiments, the method further comprises inserting an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment into a first non-human animal, such as replacing an endogenous non-human TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to an endogenous TCRα constant region gene sequence; inserting an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, one human Dβ segment, and one human Jβ segment into a second non-human animal, such as replacing an endogenous non-human TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to an endogenous TCRβ constant region gene sequence; and breeding the first non-human animal and the second non-human animal to obtain a non-human animal expressing a T cell receptor comprising a human variable region and a non-human constant region gene sequence. In other embodiments, the present invention provides a method for preparing a genetically modified non-human animal whose genome comprises a humanized unrearranged TCRα locus, or a non-human animal whose genome comprises a humanized unrearranged TCRβ locus.

[0496] In various embodiments, the replacement is performed at the endogenous locus. In various embodiments, the method comprises a progressive humanization strategy, wherein constructs comprising additional variable region segments are introduced into ES cells in each subsequent step of humanization, ultimately producing mice comprising a complete repertoire of human variable region segments and full human constant region gene sequences (see, e.g., Figure 2 and Figure 3).

[0497] Some method embodiments described herein may also include (1) replacing an endogenous non-human (e.g., mouse) tcrbdj1 sequence with a nucleic acid sequence comprising human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments and non-human (e.g., mouse) tcrbdj1 non-coding sequences (including a non-coding recombination signal sequence (RSS) and other non-intergenic sequences), wherein the human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments are flanked by the same non-human (e.g., mouse) tcrbdj1 gene segments that are typically flanked by non-human (e.g., mouse) Trbd1 and non-human (e.g., mouse) Trbj1-1 to Trbj1-6 gene segments; TCR non-coding sequences and / or (2) replacing endogenous non-human (e.g., mouse) Tcrbdj2 sequences with a nucleic acid sequence comprising human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments and mouse Tcrbdj2 non-coding sequences, wherein human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments are flanked by the same mouse Tcrbdj2 non-coding sequences that are typically flanked by mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments. In some embodiments, such replacements result in a nucleic acid sequence comprising human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments and a non-human (e.g., mouse) TCRBDJ1 non-coding sequence (including a non-coding recombination signal sequence (RSS) and other non-genic intergenic sequences) being operably linked to a non-human (e.g., mouse) TCRBC1 constant region gene sequence and / or a sequence comprising human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments and a mouse TCRBDJ2 non-coding sequence (including a non-coding recombination signal sequence (RSS) and other non-genic intergenic sequences) being operably linked to a non-human (e.g., mouse) TCRBC2 constant region gene sequence (see, Figure 8C In such embodiments, the resulting mouse may comprise one to a complete repertoire of human TCRB variable region segments operably linked to the TCRBDJ1 and TCRBDJ2 clusters, wherein endogenous TCRB non-coding sequences, such as non-coding DNA (e.g., non-coding recombination signal sequences (RSS) and other non-coding intergenic sequences) that space the gene segments may be retained.

[0498] The present disclosure also provides a method for modifying the TCR variable gene loci (e.g., TCRα, TCRβ, TCRδ and / or TCRγ gene loci) of non-human animals to express people or humanized TCR proteins as described herein. In one embodiment, the present invention provides a method for modifying TCR variable gene loci to express people or humanized TCR proteins on the surface of T cells, wherein the method includes inserting unrearranged humanized TCR variable gene loci in non-human animals, such as replacing endogenous non-human TCR variable gene loci. In one embodiment, wherein the TCR variable gene loci are TCRγ variable gene loci, unrearranged humanized TCR variable gene loci include at least one human Vγ segment and at least one human Jγ segment optionally operably connected to human Cγ genes. In one embodiment, wherein the TCR variable gene loci are TCRδ variable gene loci, unrearranged humanized TCR variable gene loci include at least one human Vδ segment, at least one human Dδ segment and at least one human Jδ segment. In various aspects, the unrearranged humanized TCR variable gene loci are operably linked to corresponding human TCR constant region gene sequences.

[0499] Therefore, also provided is a nucleotide construct for producing a genetically modified animal comprising a humanized TCR variable region gene. In one aspect, the nucleotide construct comprises: a 5' homology arm and a 3' homology arm, a human DNA fragment comprising a human TCR variable region gene segment and a human TCR constant region gene sequence, and a selection box flanked by a recombination site.

[0500] In one aspect, at least one homology arm is a non-human homology arm, and it is homologous to a non-human TCR locus (e.g., non-human TCRγ). In one aspect, one or more homology arms are human homology arms, and it is homologous to a human TCR locus (e.g., human TCRα locus) in a non-human animal genome.

[0501] Various exemplary embodiments of the humanized loci described herein are presented in the Figures and described in the Examples.

[0502] After gene targeting is completed, ES cells or genetically modified non-human animals are screened to confirm successful incorporation of the exogenous nucleotide sequence of interest or expression of the exogenous polypeptide. A variety of techniques are known to those skilled in the art and include, but are not limited to, Southern blotting, long PCR, quantitative PCR (e.g., using Real-time PCR), fluorescent in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (e.g., mice) carrying a genetic modification of interest can be identified by screening for loss of mouse alleles and / or gain of human alleles using the allele modification assay described in Valenzuela et al. (2003) 21 (6): 652-659. Other analytical methods for identifying specific nucleotide or amino acid sequences in genetically modified animals are known to those skilled in the art.

[0503] In some embodiments, animals are produced herein by breeding. For example, in some embodiments, mice are produced by a method comprising:

[0504] (a) obtaining a first mouse, said first mouse comprising homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with said heterologous sequence comprising said unrearranged human TCR Vγ segment and said unrearranged human TCR Jγ segment operably linked to said human TCRγ constant region gene sequence,

[0505] (b) obtaining a second mouse, said second mouse comprising homozygous replacement of an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ gene with said heterologous sequence comprising said unrearranged human TCR Vδ segment, said unrearranged human TCR Dδ segment, and said unrearranged human TCR Jδ segment operably linked to said human TCR δ constant region gene sequence, and

[0506] (c) breeding the first mouse and the second mouse to obtain a genetically modified mouse,

[0507] The genetically modified mouse comprises:

[0508] (i) replacing an endogenous genomic sequence comprising endogenous TCR Vγ and Jγ gene segments and an endogenous TCR Cγ gene with said heterologous sequence comprising said unrearranged human TCR Vγ segment and said unrearranged human TCR Jγ segment operably linked to said human TCRγ constant region gene sequence, and

[0509] (ii) replacing an endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ gene with said heterologous sequence comprising said unrearranged human TCR Vδ segment, said unrearranged human TCR Dδ segment, and said unrearranged human TCR Jδ segment operably linked to said human TCR δ constant region gene sequence, and

[0510] The genetically modified mouse expresses human TCRγ polypeptide and human TCRδ polypeptide.

[0511] Use of genetically modified non-human animals that produce a substantially humanized T cell immune response

[0512] The role of γ / δT cells in immune defense remains poorly understood. See, e.g., Vermijlen, D. et al. (2017) Seminars in Cell and Developmental Biol. 84: 75-86. However, several features outlined above offer promise for therapeutic applications. The main obstacle to T cell therapy derived from α / βT cells is the MHC restriction of the target antigen, which limits the applicability of any engineered α / βTCR to patients with the appropriate MHC haplotype. In addition, many α / βT cells have “allogeneic reactivity” to foreign MHC, which limits their use as allogeneic cell therapy due to the risk of graft-versus-host disease (GVHD). γ / δT cells do not have these caveats and may be a “ready-made” allogeneic therapy for rapid access, requiring less extensive engineering and manipulation to mitigate the risk of GVHD. In addition, the observed inherent anti-tumor and anti-microbial properties of γ / δT cells—similar to other “innate-like” cells such as natural killer cells—may have further clinical benefits that are being evaluated in various trials. See, e.g., Ferry GM and Anderson J. (2002) Exp. Immunol 2:168-79; Park JH and Lee HK (2021) Experimental & Molecular Medicine 53:318-327.

[0513] The TRG / TRD humanized mice described herein provide several new areas of research for γ / δT cell biology and potential therapies. Although published mice have effectively modeled infectious and autoimmune disease responses dominated by α / βT cells (Moore, M. et al. Sci Immunol 6 (2021); doi: 10.1126 / sciimmunol.abj4026; incorporated by reference as a whole), further humanization of the γ / δ lineage can facilitate modeling of skin and mucosal immune responses. This includes, but is not limited to, studies on the role of γ / δT cells and antigens in inflammatory skin diseases, interactions with intestinal microbiota, and barrier tissue repair and homeostasis. TRG / TRD humanized mice also provide new tools for studying and identifying γ / δT cell antigens and cognate TCRs that are poorly characterized compared to their α / β counterparts. For example, identifying antigen / TCR interactions enriched on tumor cells may lead to γ / δ-based anti-tumor cell therapies without the MHC restrictions that limit existing TCR therapies. In addition, since graft-versus-host disease is caused by alloreactive α / β T cell receptors, γ / δ T cells are becoming increasingly interesting in allogeneic hematopoietic stem cell transplantation, and clinical strategies that utilize the full functionality of these lymphocytes have been and are being developed, which can include in vivo activation of γ / δ T cells or subpopulations by certain drugs or antibodies after transplantation, or ex vivo expansion and manipulation of patient-derived or donor-derived γ / δ T cells and their subpopulations, and adoptive transfer of ex vivo activated lymphocytes. Handgretinger, R. and Schilbach, K. (2018) Blood 131: 1063-72, incorporated herein by reference. Therefore, TRG / TRD humanized mice as described herein can be useful tools in preclinical studies of new methods for expanding γ / δ T cells, directing them to tumors, and / or expanding γ / δ T cells in vivo or ex vivo.

[0514] Embodiments are also described herein in which humanized TRA / D and TRG loci can be introduced into mice with humanized TRB loci and other components of T cell immunity, including TCR co-receptors (CD4 and CD8) and MHC loci. These mice thus carry complete humanization of both T cell lineages to aid in studies involving cellular immunity.

[0515] The genetically modified non-human animals described herein, e.g., rodents, e.g., mice or rats, provide a non-MHC restricted γ / δ response and, optionally, when substantially all components of the complex are human or humanized, humanized CD4 and MHC II or humanized CD8 and MHC I (and β2 microglobulin), or both, present peptides to α / β T cells (CD4+ or CD8+ T cells, respectively) in a human-like manner. Thus, 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; to identify antigens and antigenic epitopes (e.g., T cell epitopes, such as unique human cancer epitopes) that elicit an immune response, e.g., for vaccine development; to identify high-affinity T cells for human pathogens or cancer antigens (i.e., T cells that bind with high affinity to antigens in the context of a human MHC I complex), e.g., for adaptive T cell therapy and "innate" T cell therapy; to evaluate vaccine candidates and other vaccine strategies; to study human autoimmunity; to study human infectious diseases; and additionally to design better therapeutic strategies based on human TCR expression.

[0516] Thus, in various embodiments, the genetically engineered animals of the invention are particularly useful for evaluating the ability of antigens to elicit an immune response in humans, as well as for generating multiple antigens and identifying specific antigens that can be used for human vaccine development.

[0517] In one aspect, a method for determining whether a peptide will stimulate a cellular immune response in a human is provided, the method comprising exposing a genetically modified non-human animal as described herein to the peptide, allowing the non-human animal to generate an immune response, and detecting in the non-human animal a TCR (e.g., a γ / δ TCR) that binds to the peptide sequence itself (or for an α / β TCR presented by a chimeric human / non-human MHC I or II molecule) as described herein.

[0518] In one aspect, a method for identifying a candidate agent that expands and / or activates γ / δT cells is described, the method comprising administering a candidate agent (or an in vitro composition comprising γ / δT cells isolated from a non-human animal as described herein) to a non-human animal as described herein, and measuring the level of γ / δT cell expansion and / or activation, wherein an increased level of γ / δT cell expansion and / or activation identifies the candidate agent as an agent that can expand and / or activate γ / δT cells. In some embodiments, the candidate agent is a tumor-associated antigen. In some embodiments, the candidate agent is an antibody.

[0519] In one aspect, the method of stimulating and / or activating γ / δT cells (e.g., Vδ2γ / δT cells) includes administering aminobisphosphonates (e.g., zoledronate, pamidronate, risedronate) to non-human animals as described herein. See, e.g., Latha et al. (2014) Front.Immunol.Vol.5DOI=10.3389 / fimmu.2014.00571, incorporated herein by reference in its entirety. Aminobisphosphonates act as inhibitors of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, stimulating the accumulation of isopentenyl pyrophosphate (IPP), and thus activating γ / δT cells in vivo. See, e.g., Park et al. (2021) Vol.8doi.org / 10.3389 / fchem.2020.612728.

[0520] 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 a putative T cell epitope, allowing the non-human animal to generate an immune response, isolating T cells from the non-human animal that bind to the epitope, which may or may not be MHC class I or MHC class II restricted, and identifying the epitope bound by the T cells.

[0521] In one aspect, a method for identifying an antigen that generates a T cell response in humans is provided, the method comprising exposing a putative antigen to a mouse as described herein, allowing the mouse to generate an immune response, and identifying an antigen recognized by T cells, which antigen may or may not be presented by an HLA class I or class II restricted molecule.

[0522] In one aspect, a method is provided for determining whether a putative antigen contains epitopes that may require or generate an HLA class I or class II restricted immune response when exposed to the human immune system, the method comprising exposing a mouse as described herein to the putative antigen and measuring the γ / δ or antigen-specific HLA class I or HLA class II restricted immune response in the mouse, respectively.

[0523] In addition, the genetically engineered non-human animals described herein can be used to identify T cell receptors, such as high affinity T cell receptors, that recognize an antigen of interest, such as a tumor or another disease antigen. The method may include exposing the non-human animal described herein to an antigen, allowing the non-human animal to generate an immune response to the antigen, isolating from the non-human animal T cells comprising a T cell receptor that binds to the antigen (whether or not presented by a human or humanized MHC I or MHC II), and determining the sequence of the T cell receptor. In some embodiments, the method includes exposing the non-human animal described herein to an antigen, allowing the non-human animal to generate an immune response to the antigen, and isolating from the non-human animal γ / δ T cells comprising a γ / δ T cell receptor that binds to the antigen in the absence of MHC. In addition to exposure to antigens, γ / δ T cells, similar to 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 NKG2D receptors expressed on activated γ / δ T cells (via NKG2D ligands) and via Vδ1 receptors. In addition, γ / δ T cells also express pattern recognition receptors, such as toll-like receptors, that enhance their anti-tumor activity. It has also been demonstrated that γ / δ T cells express the natural cytotoxicity receptors NKp30 and NKp44, and that γ / δ T cells can kill lymphocytic leukemia cell lines and leukemic blasts from patients with chronic myeloid leukemia via NKp30.18. Finally, through the activating receptor DNAM-1, γ / δ T cells effectively target acute myeloid leukemia and multiple myeloma cells that are positive for nectin-2 (CD112) and poliovirus receptor (CD155).

[0524] Non-human animals expressing a diverse library of functional human TCR V (D) J gene segments can be used for the study of human diseases. Therefore, in one embodiment, the genetically engineered non-human animals described herein can express a TCR library substantially similar to the TCR library expressed in humans, for example, the TCR library of the non-human animal disclosed herein can 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 at least about 99% of all functional human TCR α, TCR β, TCR γ and / or TCR δ gene segments.

[0525] 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. In addition, the genetically modified animals of the present invention can be used to study various aspects of human autoimmune diseases and can be used as autoimmune disease models.

[0526] In various embodiments, the genetically modified non-human animals of the present invention produce T cells with human or humanized TCR molecules on their surface, and thus recognize peptides in a human-like manner, optionally when the peptides are presented to them through MHC complexes. The genetically modified non-human animals described herein can be used to study the development and function of human T cells and the immune tolerance process; test human vaccine candidates; produce TCRs with certain specificities for TCR gene therapy; produce TCR libraries against disease-associated antigens (e.g., tumor-associated antigens (TAA)); and the like.

[0527] Since T cells (e.g., cytotoxic T cells) can be directed to attack an antigen of interest (e.g., a viral antigen, a bacterial antigen, a tumor antigen, etc.) or a cell presenting the antigen or cause their destruction, there is an increasing interest in T cell therapy in the art. Initial studies of cancer T cell therapy aimed to isolate tumor infiltrating lymphocytes (TILs; a population of lymphocytes in a tumor mass that presumably contains T cells reactive to tumor antigens), expand them in vitro using T cell growth factors, and transfer them back to the patient in a process called adoptive T cell transfer. See, e.g., 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 is not specifically directed against defined tumor-associated antigens (TAAs). Linnermann et al., supra.

[0528] Attempts have been made to start TCR gene therapy, in which T cells are selected or programmed to target an antigen of interest, such as TAA. Current TCR gene therapy relies on the identification of TCR sequences for specific antigens (e.g., tumor-associated antigens). For example, Rosenberg and colleagues have published several studies in which they transduced peripheral blood lymphocytes from melanoma patients with genes encoding TCR α and β chains specific to melanoma-associated antigen MART-1 epitopes, and used the resulting amplified 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 were isolated from patients who experienced tumor regression after TIL therapy. However, the identification of such TCRs, especially high-affinity TCRs (which are most likely to be therapeutically useful), is complicated by the fact that most tumor antigens are self-antigens and that TCRs targeting these antigens are often missing or have suboptimal affinity, primarily due to immune tolerance.

[0529] In various embodiments, the present invention solves this problem by providing a genetically engineered non-human animal comprising an unrearranged human TCR variable gene locus in its genome. The non-human animals described herein are capable of producing T cells with a diverse library of humanized T cell receptors. Therefore, the non-human animals described herein can be a diverse library of humanized T cell receptors, such as a source of high-affinity humanized T cell receptors for adoptive T cell transfer.

[0530] Thus, in one embodiment, the invention provides a method for producing a T cell receptor for a human antigen, the method comprising immunizing a non-human animal described herein (e.g., a rodent, such as a mouse or rat) with an antigen of interest, allowing the animal to generate an immune response, isolating activated T cells specific 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.

[0531] In one embodiment, the present invention provides a method for producing a human T cell receptor specific for an antigen of interest (e.g., a disease-associated antigen), the method comprising immunizing a non-human animal described herein with an antigen of interest; allowing the animal to generate an immune response; isolating T cells reactive to the antigen of interest from the animal; determining the nucleic acid sequence of the human TCR variable region expressed by the T cell; cloning (a) the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region gene sequence, so that the human TCR variable region is operably linked to the human TCR constant region gene sequence, or, when the non-human animal comprises a full human TCR locus, cloning (b) the human TCR variable region operably linked to the human TCR constant region sequence into a nucleotide construct; and expressing a human T cell receptor specific for the antigen of interest from the construct. 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 skilled in the art.

[0532] In one embodiment, a nucleotide sequence encoding a T cell receptor specific for an antigen of interest is expressed in a cell. In one embodiment, the cell expressing the TCR is selected from CHO, COS, 293, HeLa, PERC.6 TM Cells, etc.

[0533] The antigen of interest can be any antigen known to cause or be associated with a disease or condition, such as a tumor-associated antigen; an antigen of viral, bacterial or other pathogenic origin; etc. 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

[0534] (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the invention, the antigen of interest is a human antigen, such as a human tumor-associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and the T cell receptor is used to kill cells expressing the antigen.

[0535] In one embodiment, provided herein is a method for identifying T cells specific for an antigen of interest (e.g., a tumor-associated antigen), the method comprising immunizing a non-human animal described herein with the antigen of interest, allowing the animal to generate an immune response, and isolating T cells specific for the antigen from the non-human animal.

[0536] The present invention provides a new method for adoptive T cell therapy. Therefore, a method for treating or improving a disease or condition (e.g., cancer) of a subject (e.g., a mammalian subject, e.g., a human subject) is provided herein, the method comprising immunizing a non-human animal described herein with an antigen associated with the disease or condition, allowing the animal to produce an immune response, isolating a population of antigen-specific T cells from the animal, and infusing the separated antigen-specific T cells into the subject. In one embodiment, the present invention provides a method for treating or improving 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-related antigen, such as a tumor-associated antigen), allowing the animal to produce an immune response, isolating an antigen-specific T cell population from the animal, determining the nucleic acid sequence of a T cell receptor expressed by an antigen-specific T cell (e.g., a first nucleic acid sequence and / or a second nucleic acid sequence encoding a rearranged human TCRδ variable region gene or a TCRγ variable region gene, or a third nucleic acid sequence and / or a fourth nucleic acid sequence encoding a rearranged human TCRα variable region gene and / or a rearranged human TCRβ variable region gene), cloning the nucleic acid sequence of the T cell receptor, such as the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and / or the fourth nucleic acid sequence into an expression vector (e.g., a retroviral vector), introducing the vector into a T cell derived from a subject, so that the T cell expresses an antigen-specific T cell receptor, and infusing the T cell into the subject. In one embodiment, the T cell receptor nucleic acid sequence does not need to be further humanized (e.g., because some animal embodiments herein contain full human TRD and TRG loci). In one embodiment, the T cell receptor nucleic acid sequence is further humanized before being introduced into the T cell derived from the subject, for example, any sequence encoding the non-human constant region gene sequence is modified to be further similar to the human TCR constant region gene sequence (for example, the non-human constant region gene sequence is ...

Claims

1. A mouse, comprising: (I) Germ cells containing unrearranged TCRγ variable region sequences and CD3 - somatic cell, the unrearranged TCRγ variable region sequence comprises an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment, wherein the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally at an endogenous TCRγ locus, wherein the unrearranged human TCR Vγ segment and the unrearranged human TCR Jγ segment are rearranged in the T cells 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, and wherein the rearranged human TCR Vγ / Jγ variable region gene sequence operably linked to the human TCRγ constant region gene sequence together encodes a human TCRγ polypeptide, and (II) a CD3 cell expressing on its surface a functional TCR comprising the human TCRγ polypeptide + T cells.

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

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

4. A mouse, comprising: (I) Germ cells and CD3 - A somatic cell, said cell comprising from 5' to 3': Unrearranged human TCR Vα segment and an unrearranged TCR delta variable region sequence comprising an unrearranged human TCR Vδ segment, an unrearranged human TCR Dδ segment, and an unrearranged human TCR Jδ segment, wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus, wherein the unrearranged human TCR Vα segment, the unrearranged human TCR Dδ and the unrearranged human TCR Jδ segment are rearranged in the T cells 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, 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 polypeptide comprising a human hybrid TCRα / δ variable domain and a human TCRδ constant domain, (II) a CD3 cell expressing on its surface a functional TCR comprising the human hybrid TCR + T cell, the human hybrid TCR comprises the human hybrid α / δ variable domain and the human TCRδ constant domain.

5. The mouse of claim 4, wherein the germ cells and the 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, 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, such as a mouse TCRα constant region gene sequence, and wherein the unrearranged human TCR Vα segment and the unrearranged human TCR Jα segment are rearranged in the T cells of the mouse to form a rearranged TCR Vα / Jα variable region gene sequence operably linked to the TCRα constant region gene sequence, wherein 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, and wherein the mouse comprises a CD3 T cell expressing on its surface a functional TCR comprising the TCRα polypeptide + T cells.

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

7. The mouse of any one of claims 1 to 6, wherein the germ cells and the CD3 - Somatic cells contain: (A) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence; or (B) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or (C)(i) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence, and (ii) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

8. The mouse of claim 7, wherein: (A) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments; (B) 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; or (C)(i) the unrearranged TCR Vγ segments comprise the complete repertoire of unrearranged human TCR Vγ segments, and the unrearranged human TCR Jγ segments comprise the complete repertoire of unrearranged human TCR Jγ segments, and (ii) the unrearranged human TCR Vδ segment comprises the complete repertoire of unrearranged human TCR Vδ segments, the unrearranged human TCR Dδ segment comprises the complete repertoire of unrearranged human TCR Dδ segments, and the unrearranged human TCR Jδ segment comprises the complete repertoire of unrearranged human TCR Jδ segments.

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

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

11. A mouse as described in claim 10, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence.

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

13. The mouse of any one of claims 1 to 12, wherein: (I) the germ cells and the CD3 - Somatic cells contain: (A) at the endogenous TCRγ locus: Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments, Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus: Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments, Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments, Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and (C) at the endogenous TCRα locus: Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments; (D) at the endogenous TCRβ locus: Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments, Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments, Replace all endogenous TCR Jβ segments with the complete repertoire of unrearranged human TCR Jβ segments; and (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 coreceptor 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; and (F) a second nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide, 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 wherein 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; and (G) a first nucleic acid sequence encoding a chimeric human / mouse MHC II alpha polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC II beta polypeptide, wherein the chimeric human / mouse MHC II alpha polypeptide comprises an alpha 1 domain and an alpha 2 domain of a human HLA class II alpha polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II alpha polypeptide, and wherein the chimeric human / mouse MHC II beta polypeptide comprises a beta 1 domain and a beta 2 domain of a human HLA class II beta polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II beta 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 the transmembrane domain and cytoplasmic domain of a mouse MHC class I polypeptide; and (I) a polynucleotide sequence encoding a human or humanized β2 microglobulin polypeptide and comprising a nucleotide sequence comprising the nucleotide sequence set forth in exon 1 of a mouse β2 microglobulin gene operably linked to the nucleotide sequences set forth in exon 2, exon 3, and exon 4 of a human β2 microglobulin gene, (II) wherein the mouse further comprises a CD3 T cell expressing on its surface a functional human TCR comprising a human TCRγ polypeptide and a human TCRδ polypeptide. + T cells, and CD3 T cells 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 + T cells, and optionally wherein the mouse expresses: (e) the chimeric human / mouse CD4 coreceptor, (f) a chimeric CD8 coreceptor comprising said chimeric human / mouse CD8 alpha polypeptide and said chimeric human / mouse CD8 beta polypeptide, (h) a chimeric MHC II complex comprising the chimeric human / mouse MHC II α polypeptide and the chimeric human / mouse MHC II β polypeptide, wherein the chimeric MHC II complex is capable of binding to the chimeric human / mouse CD4 coreceptor, (i) the chimeric human / mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding to the chimeric CD8 coreceptor, and (j) the human or humanized β2 microglobulin polypeptide.

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

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

16. The mouse of any one of claims 1 to 15, wherein the mouse comprises: (a) gamma / delta T cells in their thymus, their spleen, their skin and / or their intestinal mucosa, and / or (b) a population of CD45+CD3+ T cells expressing human γδTCR in its thymus, spleen, mesenteric lymph nodes, skin, intestinal mucosa and / or in intraepithelial lymphocytes in its colon and / or small intestine, optionally wherein the percentage of the population of CD45+CD3+ T cells expressing human γδTCR in its thymus, spleen, mesenteric lymph nodes, skin, intestinal mucosa and / or in intraepithelial lymphocytes in its colon and / or small intestine is equivalent to or greater than the percentage of the population of CD45+CD3+ T cells expressing human γδTCR in the thymus, spleen, mesenteric lymph nodes, skin, intestinal mucosa and / or in intraepithelial lymphocytes in the colon and / or small intestine of wild-type mice.

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. 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 / DV8 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 claim 1 , 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, wherein the unrearranged TCRγ variable region sequence comprises an unrearranged human TCR Vγ segment and an unrearranged human TCR Jγ segment, wherein the unrearranged TCRγ variable region sequence is operably linked to a human TCRγ constant region gene sequence, optionally at an endogenous TCRγ locus.

22. Mouse ES cells or germ cells as claimed in claim 21, wherein the ES cells or germ cells also comprise unrearranged T cell receptor (TCR) δ variable region sequences, and the unrearranged T cell receptor (TCR) δ variable region sequences comprise unrearranged human TCR V δ segments, unrearranged human TCR D δ segments and unrearranged human TCR J δ segments, wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus.

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 located upstream of the unrearranged TCRδ variable region sequence and the human TCRδ constant region gene sequence.

24. A mouse ES cell or germ cell, comprising from 5' to 3': 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, wherein the unrearranged TCRδ variable region sequence is operably linked to a human TCRδ constant region gene sequence, optionally at an endogenous TCRδ locus.

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

26. The mouse ES cell or germ cell of claim 24 or claim 25, comprising replacing all endogenous TCR Vα segments with a complete library of unrearranged human TCR Vα segments and replacing all endogenous TCR Jα segments with a complete library of unrearranged human TCR Jα segments, wherein said complete repertoire of unrearranged human TCR Vα segments and said complete repertoire of unrearranged human TCR Jα segments are operably linked to each other and to a mouse TCRα constant region gene sequence at an endogenous TCRα locus.

27. The mouse ES cell or germ cell of any one of claims 21 to 26, wherein the ES cell or germ cell comprises: (A) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence; or (B) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence; or (C)(i) replacing the endogenous TCR Vγ segment with an unrearranged human TCR Vγ segment, replacing the endogenous TCR Jγ segment with an unrearranged human TCR Jγ segment, and replacing the endogenous TCR γ constant region gene sequence with a human TCR γ constant region gene sequence, and (ii) replacing the endogenous TCR Vδ segment with an unrearranged human TCR Vδ segment, replacing the endogenous TCR Dδ segment with an unrearranged human TCR Dδ segment, replacing the endogenous TCR Jδ segment with an unrearranged human TCR Jδ segment, and replacing the endogenous TCR δ constant region gene sequence with a human TCR δ constant region gene sequence.

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

29. The mouse ES cell or germ cell according to any one of claims 21 to 28, wherein: (I) The ES cell or germ cell comprises: (A) at the endogenous TCRγ locus: Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments, Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences, Optionally, replacing the 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); and (B) at the endogenous TCRδ locus: Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments, Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments, Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Jδ segments, and The endogenous TCRδ constant region gene sequence was replaced with the human TCRδ constant region gene sequence.

30. The mouse ES cell or germ cell of any one of claims 21 to 29, wherein the ES cell or germ cell further comprises an unrearranged TCR β variable region sequence, the unrearranged TCR β variable region sequence comprising at least one unrearranged human T cell variable region V β segment, at least one unrearranged human T cell variable region D β segment, and at least one unrearranged human T cell variable region J β segment, wherein the unrearranged TCRβ variable region sequence is operably linked to a TCRβ constant region gene sequence (eg, a mouse TCRβ constant region gene sequence), optionally at an endogenous TCRβ locus.

31. An ES cell or germ cell as described in claim 30, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence.

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

33. The mouse ES cell or germ cell of any one of claims 21 to 32, wherein the ES cell or germ cell comprises: (A) at the endogenous TCRγ locus: Replace all endogenous TCR Vγ segments with the full repertoire of unrearranged human TCR Vγ segments, Replace all endogenous TCR Jγ segments with the full repertoire of unrearranged human TCR Jγ segments, and Replace all TCRγ constant region gene sequences with the complete library of human TCRγ constant region gene sequences; and (B) at the endogenous TCRδ locus: Replace all endogenous TCR Vδ segments with the full repertoire of unrearranged human TCR Vδ segments, Replace all endogenous TCR Dδ segments with the full repertoire of unrearranged human TCR Dδ segments, Replace all endogenous TCR Jδ segments with the full repertoire of unrearranged human TCR Jδ segments, and Replace the endogenous TCRδ constant region gene sequence with a human TCRδ constant region gene sequence; and (C) at the endogenous TCRα locus: Replace all endogenous TCR Vα segments with the full repertoire of unrearranged human TCR Vα segments, and Replace all endogenous TCR Jα segments with the full repertoire of unrearranged human TCR Jα segments, and (D) at the endogenous TCRβ locus: Replace all endogenous TCR Vβ segments with the full repertoire of unrearranged human TCR Vβ segments, Replace all endogenous TCR Dβ segments with the full repertoire of unrearranged human TCR Dβ segments, Replace all endogenous TCR Jβ segments with the full repertoire of unrearranged human TCR Jβ segments, (E) a first nucleotide sequence encoding a chimeric human / mouse CD4 coreceptor 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, 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 wherein 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 II alpha polypeptide and a second nucleic acid sequence encoding a chimeric human / mouse MHC II beta polypeptide, wherein the chimeric human / mouse MHC II alpha polypeptide comprises an alpha 1 domain and an alpha 2 domain of a human HLA class II alpha polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II alpha polypeptide, and wherein the chimeric human / mouse MHC II beta polypeptide comprises a beta 1 domain and a beta 2 domain of a human HLA class II beta polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of a mouse MHC II beta 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 the transmembrane domain and cytoplasmic domain of a mouse MHC class I polypeptide; and (I) A polynucleotide sequence encoding a human or humanized β2 microglobulin polypeptide and 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. 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. The mouse ES cell or germ cell of any one of claims 21 to 35, wherein the cell comprises the human nucleotide sequence shown in chr7:38383439-38230960 (GRCh38 coordinates).

37. The mouse ES cell or germ cell of any one of claims 22 to 35, wherein the cell comprises the human nucleotide sequence shown in Chr14:22421820-22464666 (GRCh38 coordinates).

38. A targeting vector, comprising from 5' to 3': (a) 5' mouse homology arm, (b) the human nucleotide sequence shown at chr7:38383439-38230960 (GRCh38 coordinates), and (c) 3' mouse homology arm.

39. A targeting vector comprising (i) a selection box and (ii) a human nucleotide sequence shown in Chr14:22421820-22464666 (GRCh38 coordinates).

40. A method for producing a nucleic acid sequence encoding a human T cell receptor (TCR) gamma variable domain, the method comprising: immunizing the genetically modified mouse according to any one of claims 1 to 20 with an antigen of interest, allowing the mouse to develop an immune response to the antigen of interest, and A nucleic acid sequence encoding a human TCRγ variable domain that binds to the antigen of interest is obtained therefrom.

41. A method for producing a nucleic acid sequence encoding a human TCRγ polypeptide, the method comprising: immunizing the genetically modified mouse according to any one of claims 1 to 20 with an antigen of interest, allowing the mouse to develop an immune response to the antigen of interest, A nucleic acid sequence encoding a human TCRγ polypeptide of the TCR that binds to the antigen of interest is obtained therefrom.

42. A method for producing a nucleic acid sequence encoding a human T cell receptor (TCR) delta variable domain, the method comprising: immunizing the genetically modified mouse according to any one of claims 2 to 20 with an antigen of interest, allowing the mouse to develop an immune response to the antigen of interest, and A nucleic acid sequence encoding a human TCRδ variable domain that binds to the antigen of interest is obtained therefrom.

43. A method for producing a nucleic acid sequence encoding a human TCRδ polypeptide, the method comprising: immunizing the genetically modified mouse according to any one of claims 2 to 20 with an antigen of interest, allowing the rodent to mount an immune response to the antigen of interest, A nucleic acid sequence encoding a human TCRδ polypeptide of a TCR that binds to the antigen of interest is obtained therefrom.

44. A method for producing a nucleic acid sequence encoding a human hybrid T cell receptor (TCR) α / δ variable domain, the method comprising: immunizing the genetically modified mouse according to any one of claims 4 to 20 with an antigen of interest, allowing the mouse to develop an immune response to the antigen of interest, and A nucleic acid sequence encoding a human hybrid TCR α / δ variable domain that binds to the antigen of interest is obtained therefrom.

45. A method for preparing a human therapeutic agent, the method comprising: immunizing the genetically modified mouse according to any one of claims 1 to 20 with an antigen of interest, allowing the mouse to develop an immune response, obtaining from the mouse T cells reactive to an antigen of interest from the mouse, A T cell receptor that binds to an antigen of interest and / or a nucleic acid sequence encoding the T cell receptor is obtained from the T cell, wherein the T cell receptor comprises a human TCR variable domain.

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

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

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

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

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

51. The method of claim 46, wherein the soluble T cell receptor is fused to an immunosuppressive molecule, such as a molecule that inhibits T cells from killing other cells bearing an antigen recognized by the T cell.

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

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

54. The method of claim 53, wherein modifying comprises: (a) 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 segment and said unrearranged human TCR Jγ segment operably linked to said human TCRγ constant region gene sequence, and / or (b) replacing the endogenous genomic sequence comprising endogenous TCR Vδ, Dδ, Jδ gene segments and endogenous TCR Cδ gene with a heterologous sequence comprising said unrearranged human TCR Vδ segment, said unrearranged human TCR Dδ segment, said unrearranged human TCR Jδ segment and said human TCRδ constant region gene sequence.

55. The method of claim 54, wherein: (a) said 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 and a TCR Cδ gene located between the TCR Vα and TCR Jα gene segments.

56. A method as claimed in claim 54 or claim 55, wherein: (a) said heterologous sequence comprising said unrearranged human TCR Vγ segment and said unrearranged human TCR Jγ segment operably linked to said 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 library of unrearranged TCR Vδ, unrearranged TCR Dδ and unrearranged human TCR Jδ segments, and a human TCR Cδ gene sequence located between the human TCR Vα gene segment and the human TCR Jα gene segment on chromosome 14 of the human genome.

57. A method as described in any one of claims 53 to 56, wherein the modification includes performing homologous recombination in one or more ES cells, so that a 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, and 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 are added to the genome of the one or more ES cells in any order.

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

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

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