Humanized T cell-mediated immune responses in non-human animals

Abstract

To provide genetically engineered non-human animals (e.g., rodents, e.g., mice or rats).SOLUTION: Non-human animals genetically engineered may express a humanized T cell co-receptor (e.g., humanized CD4 and / or CD8 (e.g., CD8α and / or CD8β)), a human or humanized T cell receptor (TCR) comprising a variable domain encoded by at least one human TCR variable region gene segment, and / or a human or humanized major histocompatibility complex that binds the humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., MHC II α and / or MHC II β chains) and / or MHC I (e.g., MHC I α) respectively, and optionally human or humanized β2 microglobulin).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Serial Nos. 62 / 143,687 (filed April 6, 2015), 62 / 158,804 (filed May 8, 2015), and 62 / 186,935 (filed June 30, 2015), each of which is incorporated herein by reference.

[0002] Sequence Listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII Sequence Listing with the file name "2016-04-06-10145WO01-SEQ-LIST_ST25.txt," created on April 6, 2016, and having a size of 56.7 kilobytes, and is being submitted herewith. The Sequence Listing contained in this ASCII document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to non-human animals (e.g., rodents, e.g., mice or rats) that are capable of initiating a substantially human(ized) T cell-mediated immune response and that are capable of expressing (i) one or more human(ized) T cell co-receptors (e.g., CD4 and / or CD8 (e.g., CD8α and / or CD8β)), (ii) one or more human(ized) major histocompatibility complexes (e.g., MHC II (e.g., MHC IIα and / or MHC IIβ) and / or MHC I (e.g., MHC Iα and / or β2 microglobulin) that associate with one or more human(ized) T cell co-receptors, and / or (iii) human(ized) T cell receptors (TCRs) (e.g., TCRα and / or TCRβ); embryos, tissues, cells, and / or nucleic acids isolated from the non-human animals; methods of producing the non-human animals; and methods of using the non-human animals for the development of human therapeutics. [Background technology]

[0004] In adaptive immune responses, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptors, also called TCRs). These foreign antigens are presented as peptide fragments on the surface of cells by specialized proteins commonly called major histocompatibility complex (MHC) molecules, specifically human leukocyte antigens (HLA) in humans. During T cell-mediated responses, antigens presented by MHC molecules are recognized by T cell receptors. However, more T cell receptor recognition of MHC-antigen complexes is required for an effective immune response. Binding of T cell coreceptor molecules (e.g., CD4 or CD8) to the invariant portion of the MHC is also required.

[0005] T cells are diverse, including helper T cells and cytotoxic T cells. Helper T cells express the co-receptor CD4 and recognize antigens bound to MHC II molecules. CD4+ T cells activate other effector cells in the immune system, such as MHC II-expressing B cells that produce antibodies and MHC II-expressing macrophages that destroy pathogens. Binding of CD4 and the T cell receptor to a foreign antigen presented by the same MHC II molecule makes the T cell significantly more sensitive to that antigen.

[0006] In contrast, cytotoxic T cells (CTLs) express the co-receptor CD8 and recognize foreign antigens bound to MHC I molecules. CTLs are specialized to kill any cell bearing an MHC I-bound peptide recognized by its own membrane-bound TCR. When a cell presents a peptide derived from a normally absent cellular protein (e.g., of viral, tumor, or other non-self origin), such a peptide is recognized by the CTL, which becomes activated and kills the cell presenting the peptide. Like CD4, binding of CD8 makes CTLs more sensitive to antigens presented by MHC I.

[0007] Due to tolerance mechanisms, not all antigens trigger T cell activation. On the other hand, in some diseases (e.g., cancer, autoimmune diseases), peptides derived from self-proteins are targeted by cellular components of the immune system. This targeting results in the destruction of cells presenting such peptides. Significant progress has been made in recognizing clinically relevant antigens (e.g., antigens associated with various types of cancer) and / or TCR sequences that bind to clinically relevant antigens. However, there remains a need for in vivo and in vitro systems that mimic aspects of the human immune system to improve the identification and selection of clinically relevant peptides and / or TCRs capable of binding to clinically relevant antigens that will elicit appropriate responses in human T cells (e.g., for adaptive immunotherapy of cancer, T cell vaccines for autoimmunity, etc.). Therefore, there is a need for biological systems (e.g., genetically modified non-human animals and cells) that can present components of the human immune system, particularly components of the T cell immune response. Summary of the Invention [Means for solving the problem]

[0008] As disclosed herein, the thymus of a genetically modified non-human animal comprising a substantially humanized T cell immune system has absolute numbers of thymocytes and CD3+ T cells similar to those of a control animal. In addition, these cells exhibit comparable development into single-positive T cells relative to control animals and are capable of generating robust human cellular responses to antigens, e.g., viral antigens. The human cellular response of the non-human animal generally comprises activated non-human T cells expressing human or humanized T cell receptor (TCR) variable domains that recognize antigens presented in peptide-binding clefts formed by human leukocyte antigen (HLA) extracellular domains. The extracellular domains may be expressed on the surface of non-human antigen-presenting cells. In some embodiments, the substantially humanized T cell immune system comprises: (A) A non-human T cell, (i) A T cell coreceptor polypeptide comprising part or all of the extracellular portion of a human T cell coreceptor, for example, a T cell coreceptor polypeptide comprising one or more human T cell coreceptor extracellular domains, wherein the T cell coreceptor polypeptide is (a) one or more extracellular domains of a human or humanized HLA molecule (e.g., a first human HLA extracellular domain that is a binding site for a T-cell co-receptor polypeptide, and / or a second human HLA extracellular domain that forms a peptide-binding cleft and, e.g., a third human HLA extracellular domain); (b) the extracellular domain of a human or humanized TCR variable domain (e.g., a human or humanized TCR α variable domain and / or a human or humanized TCR β variable domain encoded by at least one human TCR α and / or TCR β variable region gene segment, respectively), and / or (c) capable of associating with and / or expressed to associate with the extracellular domain of a human TCR constant domain; (ii) a non-human T cell expressing a T cell receptor (TCR) comprising at least a human TCR variable domain; and, optionally, (B) Non-human antigen-presenting cells that present antigens in the context of human HLA, for example, non-human antigen-presenting cells that contain, on their cell surface, a peptide-binding cleft formed by two HLA extracellular domains and that express at least one MHC molecule that can activate and / or activate non-human T cells.

[0009] In one embodiment, the non-human T cells and the non-human antigen presenting cells are found in or isolated from the same non-human animal.

[0010] Therefore, in this specification, (A) a human or humanized T cell co-receptor (e.g., human or humanized CD4 and / or human or humanized CD8 (e.g., human or humanized CD8α and / or human or humanized CD8β)); (B) a human or humanized major histocompatibility complex (MHC) complex associated with a human or humanized T cell co-receptor (e.g., a human or humanized MHC II (e.g., a human or humanized MHC IIα and / or a human or humanized MHC IIβ) that binds to human or humanized CD4, and / or a human or humanized MHC I (e.g., a human or humanized MHC Iα and, optionally, a human or humanized β2 microglobulin) that binds to human or humanized CD8, and / or (C) a non-human animal (e.g., a rodent, e.g., a mouse or rat) that has been genetically engineered to express a human or humanized T cell receptor (TCR); As well as embryos, tissues, and cells expressing them, and nucleic acids encoding them, are provided. Also provided are methods of producing and using the disclosed non-human animals.

[0011] In one embodiment, there is provided a genetically modified non-human animal, comprising: (A) a humanized CD4 co-receptor and / or a humanized CD8 co-receptor, including a humanized CD8α polypeptide and a humanized CD8β polypeptide (e.g., a non-human animal comprising, e.g., in its germline genome, a first nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide, and / or a second nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide, and a third nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide), each humanized T cell co-receptor polypeptide comprises at least the transmembrane and cytoplasmic domains of a non-human T cell co-receptor, e.g., the humanized CD4 co-receptor comprises at least the transmembrane and cytoplasmic domains of a non-human CD4 co-receptor, and / or the humanized CD8 co-receptor comprises at least the transmembrane and cytoplasmic domains of a non-human CD8α and a non-human CD8β polypeptide; a humanized CD4 co-receptor and / or a humanized CD8 co-receptor, wherein each chimeric T cell co-receptor polypeptide comprises part or all of the extracellular portion of a human T cell co-receptor, e.g., one or more extracellular domains of a human T cell co-receptor, e.g., at least the extracellular domain of a human T cell co-receptor that associates with an HLA molecule, e.g., a humanized CD4 co-receptor comprising the extracellular portion of human CD4 (or a portion thereof, e.g., the extracellular domain) responsible for interaction with MHC II, a T cell receptor variable domain, a T cell receptor constant domain, or a combination thereof, and / or a humanized CD8 co-receptor, wherein, e.g., a humanized CD8 co-receptor comprises the extracellular portion of human CD8α and / or human CD8β (or a portion thereof, e.g., the extracellular domain) responsible for interaction with MHC I, a T cell receptor variable domain, a T cell receptor constant domain, or a combination thereof; (B) a human(ized) TCR (e.g., a non-human animal, e.g., comprising, in its germline genome, an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCR α constant gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence), and optionally (C) a human(ized) MHC II complex associated with a humanized CD4 co-receptor and / or a human(ized) MHC I complex associated with a humanized CD8 co-receptor (e.g., a non-human animal comprising, e.g., in its germline genome, a first nucleic acid sequence encoding a chimeric human / non-human MHC IIα polypeptide, and a second nucleic acid sequence encoding a chimeric human / non-human MHC IIβ polypeptide, and / or a third nucleic acid sequence encoding a chimeric human / non-human MHC I polypeptide), Each chimeric MHC polypeptide comprises at least the extracellular portion (or a portion thereof) of a human MHC polypeptide (e.g., an HLA polypeptide), and each chimeric MHC polypeptide, either alone (e.g., MHC I) or when complexed with another chimeric MHC polypeptide (e.g., MHC IIα and MHC IIβ), is capable of associating with a human(ized) CD8 co-receptor or a human(ized) CD4 co-receptor, respectively, and presenting peptides in the context of an HLA, e.g., a humanized MHC II complex comprises (i) a chimeric human / non-human MHC IIα polypeptide comprising the α1 and α2 domains of a human HLA class IIα polypeptide and the transmembrane and cytoplasmic domains of a non-human HLA class IIα polypeptide, (ii) a chimeric human / non-human MHC IIβ polypeptide comprising the β1 and β2 domains of a human HLA class IIβ polypeptide, the transmembrane and cytoplasmic domains of a non-human HLA class IIβ polypeptide, and / or a humanized MHC I complex comprises a human MHC Genetically modified non-human animals are provided that comprise a human(ized) MHC II complex and / or a human(ized) MHC I complex, comprising the α1, α2, and α3 domains of an MHC I polypeptide and, optionally, human(ized) β2 microglobulin.

[0012] In some embodiments, the non-human animal is (A) a humanized CD4 co-receptor, and a humanized CD8 co-receptor, including a humanized CD8α polypeptide and a humanized CD8β polypeptide (e.g., a non-human animal comprising, e.g., in its germline genome, a first nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide, a second nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide, and a third nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide), each humanized T cell co-receptor polypeptide comprises at least the transmembrane and cytoplasmic domains of a non-human T cell co-receptor, e.g., the humanized CD4 co-receptor comprises at least the transmembrane and cytoplasmic domains of a non-human CD4 co-receptor, and the humanized CD8 co-receptor comprises at least the transmembrane and cytoplasmic domains of a non-human CD8α and a non-human CD8β polypeptide; a humanized CD4 co-receptor and / or a humanized CD8 co-receptor, each chimeric T cell co-receptor polypeptide comprising part or all of the extracellular portion of a human T cell co-receptor, e.g., one or more extracellular domains of a human T cell co-receptor, e.g., at least the extracellular domain of a human T cell co-receptor that associates with an HLA molecule, e.g., a humanized CD4 co-receptor comprising the extracellular portion of human CD4 (or a portion thereof, e.g., the extracellular domain) responsible for interaction with MHC II, a T cell receptor variable domain, a T cell receptor constant domain, or a combination thereof, and / or a humanized CD8 co-receptor, e.g., a humanized CD8 co-receptor comprising the extracellular portion of human CD8α and / or human CD8β (or a portion thereof, e.g., the extracellular domain) responsible for interaction with MHC I, a T cell receptor variable domain, a T cell receptor constant domain, or a combination thereof; (B) a human(ized) TCR (e.g., a non-human animal that, e.g., comprises in its germline genome, an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCR α constant gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence); (C) a humanized MHC II complex associated with a humanized CD4 co-receptor and a humanized MHC I complex associated with a humanized CD8 co-receptor (e.g., the non-human animal comprises, e.g., in its germline genome, a first nucleic acid sequence encoding a chimeric human / non-human MHC IIα polypeptide, a second nucleic acid sequence encoding a chimeric human / non-human MHC IIβ polypeptide, and a third nucleic acid sequence encoding a chimeric human / non-human MHC I polypeptide), each chimeric MHC polypeptide comprises at least the extracellular portion (or a portion thereof) of a human MHC polypeptide (e.g., an HLA polypeptide); each chimeric MHC polypeptide, either alone (e.g., MHC I) or when complexed with another chimeric MHC polypeptide (e.g., MHC IIα and MHC IIβ), is capable of associating with a humanized CD8 co-receptor or a humanized CD4 co-receptor, respectively, and presenting peptides in the context of an HLA; the humanized MHC II complex comprises (i) a chimeric human / non-human MHC IIα polypeptide comprising the α1 and α2 domains of a human HLA class IIα polypeptide and the transmembrane and cytoplasmic domains of a non-human HLA class IIα polypeptide; (ii) a chimeric human / non-human MHC IIβ polypeptide comprising the β1 and β2 domains of a human HLA class IIβ polypeptide and the transmembrane and cytoplasmic domains of a non-human HLA class IIβ polypeptide; and the humanized MHC I complex comprises a human MHC and optionally humanized β2 microglobulin (e.g., the non-human animal further comprises a β2 microglobulin locus encoding a polypeptide comprising a human β2 microglobulin amino acid sequence or a portion thereof), a humanized(ized) MHC II complex, and a humanized(ized) MHC I complex.

[0013] In some embodiments, a first nucleotide sequence encoding a chimeric T cell CD4 co-receptor polypeptide is present at the endogenous CD4 T cell co-receptor locus, and / or a second nucleotide sequence encoding a chimeric T cell CD8α co-receptor polypeptide is present at the endogenous CD8α T cell co-receptor locus, and a third nucleotide sequence encoding a chimeric T cell CD8β co-receptor polypeptide is present at the endogenous CD8β T cell co-receptor locus. Further embodiments include chimeric human / non-human CD4 polypeptides encoded by the genes shown in FIG. 5A (e.g., the human portion of the resulting chimeric human / non-human CD4 T-cell co-receptor polypeptide includes at least the human Ig1, human Ig2, and human Ig3 domains (otherwise referred to as the D1, D2, and D3 domains, respectively)), and / or chimeric CD8 co-receptors encoded by the genes shown in FIG. 5B (e.g., the human portion of the chimeric CD8 co-receptor includes all or substantially all of the extracellular portion of a human CD8 polypeptide (e.g., CD8α and / or CD8β), including the human immunoglobulin V (IgV)-like α and β domains). In some embodiments, the human portion of the chimeric CD4 T-cell co-receptor polypeptide comprises one or more extracellular domains of a human CD4 polypeptide (e.g., D1, D2, D3, D4, or any combination thereof), the non-human portion of the chimeric CD4 T-cell co-receptor polypeptide comprises a transmembrane domain and a cytoplasmic domain of a non-human CD4 T-cell co-receptor, the human portion of the chimeric CD8α polypeptide comprises an extracellular domain of a human CD8α polypeptide (e.g., an IgV-like domain), the non-human portion of the chimeric CD8α polypeptide comprises a transmembrane domain and a cytoplasmic domain of a non-human CD8α polypeptide, and / or the human portion of the CD8β polypeptide comprises an extracellular domain of a human CD8β polypeptide (e.g., an IgV-like domain). The non-human portion of the T cell co-receptor polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD8β polypeptide.

[0014] In some embodiments, a first nucleic acid sequence encoding human(ized) MHC IIα is present at an endogenous non-human MHC IIα locus, a second nucleic acid sequence encoding human(ized) MHC IIβ is present at an endogenous non-human MHC IIβ locus, and / or a third nucleic acid sequence encoding human(ized) MHC I is present at an endogenous non-human MHC I locus. In one aspect, the human(ized) MHC IIα polypeptide comprises the extracellular portion (or a portion thereof) of a human MHC IIα polypeptide (e.g., an HLA class IIα polypeptide), the human(ized) MHC IIβ polypeptide comprises the extracellular portion (or a portion thereof) of a human MHC IIβ polypeptide (e.g., an HLA class IIβ polypeptide), and / or the human(ized) MHC I polypeptide comprises the extracellular portion (or a portion thereof) of a human MHC I polypeptide (e.g., an HLA class I polypeptide). In some embodiments, the humanized MHC The IIα polypeptide comprises human MHC II α1 and α2 domains, the humanized MHC IIβ polypeptide comprises human MHC II β1 and β2 domains, and / or the humanized MHC I polypeptide comprises human MHC I α1, α2, and α3 domains. In some embodiments, a first nucleic acid sequence encoding a chimeric human / non-human MHC IIα polypeptide is expressed under the control of an endogenous non-human MHC IIα promoter and regulatory elements, a second nucleic acid sequence encoding a chimeric human / non-human MHC IIβ polypeptide is expressed under the control of an endogenous non-human MHC IIβ promoter and regulatory elements, and / or a third nucleic acid sequence encoding a chimeric human / non-human MHC I polypeptide is expressed under the control of an endogenous non-human MHC I promoter and regulatory elements. In further embodiments, the non-human portion of the chimeric human / non-human MHC IIα polypeptide comprises the transmembrane and cytoplasmic domains of an endogenous non-human MHC IIα polypeptide, the non-human portion of the chimeric human / non-human MHC IIβ polypeptide comprises the transmembrane and cytoplasmic domains of an endogenous non-human MHC IIβ polypeptide, and / or the non-human portion of the chimeric human / non-human MHC I polypeptide comprises the transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide. Embodiments include a non-human animal wherein the human portions of the proteins in the chimeric human / non-human MHC II complex are derived from a corresponding human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, and / or the human portion of the third chimeric human / non-human MHC I polypeptide is derived from human HLA-A, human HLA-B, or human HLA-C.As non-limiting examples, in some embodiments, a chimeric MHCIIα polypeptide comprises the extracellular portion or a portion thereof of an HLA-DRα protein, an HLA-DQα protein, or an HLA-DPα protein; a chimeric MHCIIβ polypeptide comprises the extracellular portion or a portion thereof of an HLA-DRβ protein, an HLA-DQβ protein, or an HLA-DPβ protein; and / or a chimeric MHCII polypeptide comprises the extracellular portion or a portion thereof of a human HLA-A protein, a human HLA-B protein, or a human HLA-C protein. Also provided are non-human animals in which the human portion of the chimeric human / non-human MHC II protein is derived from the corresponding human HLA-DR protein, e.g., the human portion of the human / non-human MHC IIα polypeptide comprises the α1 and α2 domains of the α chain of HLA-DR2, the human portion of the human / non-human MHC IIβ polypeptide comprises the β1 and β2 domains of the β chain of HLA-DR2, and the human portion of the MHC I polypeptide is derived from a human HLA-A polypeptide, e.g., the human portion of the human / non-human MHC I polypeptide comprises the α1, α2, and α3 domains of a human HLA-A2 polypeptide, e.g., the α1, α2, and α3 domains of a human HLA-A2.1 polypeptide. Also provided are non-human animals in which the non-human portion of the MHC II complex is derived from a mouse H-2E coding sequence and / or the non-human portion of the MHC I polypeptide is derived from a mouse H-2K coding sequence. For example, a chimeric MHC IIα polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2Eα polypeptide, a chimeric MHC IIβ polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2Eβ polypeptide, and a chimeric MHC I polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2K polypeptide.

[0015] In some embodiments, the unrearranged TCR alpha variable locus is present at an endogenous TCR alpha variable locus and the unrearranged TCR beta variable locus is present at an endogenous TCR beta variable locus. In some aspects, the unrearranged TCR alpha variable locus comprises a complete repertoire of human unrearranged V alpha gene segments and a complete repertoire of human unrearranged J alpha gene segments, and / or the unrearranged TCR beta variable locus comprises a complete repertoire of human unrearranged V beta gene segments, a complete repertoire of human unrearranged D beta gene segments, and a complete repertoire of human unrearranged J beta gene segments. In some embodiments, the unrearranged human V alpha and J alpha gene segments have rearranged to form a rearranged human V alpha / J alpha sequence, and / or the unrearranged human V beta, D beta, and J beta gene segments have rearranged to form a rearranged human V beta / D beta / J beta sequence. In some embodiments, the non-human animals disclosed herein express T cell receptors comprising a human TCR alpha variable region and / or a human TCR beta variable region on the surface of the T cells. In some embodiments, the endogenous non-human Vα and Jα segments are incapable of rearranging to form rearranged Vα / Jα sequences, and / or the endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form rearranged Vβ / Dβ / Jβ sequences. For example, the animal may lack a functional endogenous non-human TCR alpha variable locus, and / or the animal may lack a functional endogenous non-human TCR beta variable locus, e.g., the animal comprises (a) a deletion of all or substantially all functional endogenous V alpha gene segments, (b) a deletion of all or substantially all functional endogenous J alpha gene segments, (c) a deletion of all or substantially all functional endogenous V beta gene segments, (d) a deletion of all or substantially all functional endogenous D beta gene segments, (e) a deletion of all or substantially all functional endogenous J beta gene segments, and / or (f) a combination thereof.In some embodiments, the endogenous non-human TCR alpha variable locus lacks all or substantially all functional endogenous Valpha gene segments, and / or lacks all or substantially all functional endogenous Jalpha gene segments, and / or the endogenous non-human TCR beta variable locus (a) lacks all or substantially all functional endogenous Vbeta gene segments, (b) lacks all or substantially all functional endogenous Dbeta gene segments, (c) lacks all or substantially all functional endogenous Jbeta gene segments, or (d) any combination of (a), (b), and (c).

[0016] In some embodiments, the first, second, and / or third nucleotide sequences encoding the chimeric T cell CD4, CD8α, and / or CD8β co-receptor polypeptides, respectively, are present at an endogenous T cell co-receptor locus, e.g., an endogenous CD4, CD8α, and / or CD8β co-receptor locus, respectively. The unrearranged TCR α variable locus is present at an endogenous TCR α variable locus, the unrearranged TCR β variable locus is present at an endogenous TCR β variable locus, and / or the first, second, and / or third nucleic acid sequences encoding the chimeric MHC IIα, MHC IIβ, and / or MHC I polypeptides, respectively, are present at an endogenous MHC locus, e.g., an MHC IIα, MHC IIβ, and / or MHC I locus, respectively. In some embodiments, the nucleotide sequence encoding the chimeric T-cell co-receptor, the unrearranged TCR alpha variable locus, the unrearranged TCR beta variable locus, and / or the nucleic acid sequence encoding the chimeric MHC molecule may be operably linked to non-human promoters and regulatory sequences. For example, a first nucleotide sequence may be expressed under the regulatory control of the endogenous non-human CD4 promoter and regulatory elements, a second nucleotide sequence may be expressed under the regulatory control of the endogenous non-human CD8 alpha promoter and regulatory elements, and / or a third nucleotide sequence may be expressed under the regulatory control of the endogenous non-human CD8 beta promoter and regulatory elements. The unrearranged TCR alpha variable locus may be expressed under the regulatory control of the endogenous TCR alpha (variable) regulatory and promoter elements, and the unrearranged TCR beta variable locus may be expressed under the regulatory control of the endogenous TCR beta (variable) regulatory and promoter elements. The first nucleic acid sequence may be expressed under the control of an endogenous non-human MHC IIα promoter and regulatory elements, the second nucleic acid sequence may be expressed under the control of an endogenous non-human MHC IIβ promoter and regulatory elements, and the third nucleic acid sequence may be expressed under the control of an endogenous non-human MHC I promoter and regulatory elements.

[0017] In some embodiments, a nucleotide sequence encoding the extracellular portion of a human CD4 polypeptide (or a portion thereof, e.g., D1, D2, D3, and / or D4) replaces sequence encoding the extracellular portion of an endogenous non-human (mouse) CD4 co-receptor polypeptide and may be operably linked to endogenous non-human (mouse) CD4 transmembrane and cytoplasmic domain-encoding sequence at the endogenous non-human (mouse) CD4 co-receptor locus; and a nucleotide sequence encoding all or a portion of the extracellular portion of a human CD8α polypeptide replaces sequence encoding all or a portion of the extracellular portion of an endogenous non-human (mouse) T-cell CD8α polypeptide and may be operably linked to endogenous non-human (mouse) CD8α transmembrane and cytoplasmic domain-encoding sequence at the endogenous non-human (mouse) CD8α locus. the nucleotide sequence encoding all or a portion of the extracellular portion of a human CD8β polypeptide replaces sequence encoding all or a portion of the extracellular domain of an endogenous non-human (mouse) T-cell CD8β polypeptide and may be operably linked to coding sequence for the transmembrane and cytoplasmic domains at the endogenous CD8β locus; the unrearranged TCR α variable locus replaces one or more endogenous Vα and / or Jα gene segments at the endogenous non-human (mouse) TCR α variable locus; the unrearranged TCR β variable locus replaces one or more endogenous Vβ, Dβ, and / or Jβ gene segments at the endogenous non-human (mouse) TCR β variable locus; and the nucleic acid sequence encoding the extracellular portion of a human MHC IIα polypeptide (or a portion thereof, e.g., the α1 and α2 domains) replaces sequence encoding all or a portion of the extracellular domain of an endogenous non-human (mouse) MHC The nucleic acid sequence encoding the extracellular portion of a human MHC IIβ polypeptide (or a portion thereof, e.g., the β1 and β2 domains) may replace a sequence encoding the extracellular portion of a human MHC IIα polypeptide (or a portion thereof, e.g., the α1 and α2 domains) and may be operably linked to a coding sequence for the endogenous non-human (mouse) MHC IIα transmembrane and cytoplasmic domains at the endogenous non-human (mouse) MHC IIα locus; the nucleic acid sequence encoding the extracellular portion of a human MHC IIβ polypeptide (or a portion thereof, e.g., the β1 and β2 domains) may replace a sequence encoding the extracellular portion of a human MHC IIβ polypeptide (or a portion thereof, e.g., the β1 and β2 domains) and may be operably linked to a coding sequence for the endogenous non-human (mouse) MHC IIβ transmembrane and cytoplasmic domains at the endogenous non-human (mouse) MHC IIβ locus; and / or the nucleic acid sequence encoding the extracellular portion of a human MHC Iβ polypeptide (or a portion thereof, e.g., the α1, α2, and / or α3 domains) may replace a sequence encoding the extracellular portion of a human MHC Iβ polypeptide (or a portion thereof, e.g., the α1, α2, and / or α3 domains) at the endogenous non-human (mouse) MHC IIβ locus. The sequence encoding the extracellular portion of the MHC I polypeptide (or a portion thereof, e.g., the α1, α2, and / or α3 domains) may be replaced and operably linked to the coding sequence for the endogenous non-human (mouse) MHC I transmembrane and cytoplasmic domains at the endogenous non-human (mouse) MHC I locus.

[0018] In some embodiments, a genetically modified non-human animal disclosed herein does not express a functional endogenous non-human T-cell CD4 co-receptor from its endogenous locus, does not express a functional endogenous non-human T-cell CD8 co-receptor from its endogenous CD8 locus, does not express a functional TCR alpha variable domain from its endogenous TCR alpha variable locus, does not express a functional TCR beta variable domain from its endogenous TCR beta variable locus, does not express (e.g., on the cell surface) the extracellular domain of an endogenous MHC II complex from its endogenous MHC II locus, and / or does not express (e.g., on the cell surface) the extracellular domain of an endogenous MHC I polypeptide from its endogenous MHC I locus.

[0019] Any of the non-human animals disclosed herein may further comprise a β2 microglobulin locus encoding a polypeptide comprising the amino acid sequence of a human or humanized β2 microglobulin, wherein the non-human animal expresses a human or humanized β2 microglobulin polypeptide. In some embodiments, the non-human animal does not express a functional endogenous non-human animal β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In some embodiments, the β2 microglobulin locus is operably linked to an endogenous non-human β2 microglobulin regulatory element. In one embodiment, the β2 microglobulin locus comprises the nucleotide sequence represented by exon 2, exon 3, and exon 4 (e.g., exon 2-exon 4) of the human β2 microglobulin gene, and optionally, the β2 microglobulin locus further comprises the nucleotide sequence represented by exon 1 of a non-human, e.g., rodent, β2 microglobulin gene.

[0020] The non-human animals provided herein can be rodents, such as mice or rats.

[0021] Also provided herein are mice expressing, on the surface of the T cells, chimeric human / mouse T cell CD4, CD8α, and CD8β co-receptor polypeptides, each comprising mouse CD4, CD8α, and CD8β transmembrane and cytoplasmic domains, respectively; a T cell receptor comprising a human TCRα variable region and a human TCRβ variable region; chimeric human / mouse MHC IIα, MHC IIβ, and MHC I polypeptides, each comprising the extracellular domains of human MHC IIα (e.g., human HLA class II α1 and α2 domains), MHC IIβ (human HLA class II β1 and β2 domains), and MHC I polypeptides (e.g., human HLA class I α1, α2, and α3 domains), respectively; and, optionally, a human or humanized β2 microglobulin polypeptide. In one embodiment, a non-human animal, e.g., a mouse, is provided, wherein a first nucleic acid sequence encodes the alpha chain of a chimeric human / mouse HLA-DR / H-2E polypeptide, a second nucleotide sequence encodes the beta chain of the chimeric HLA-DR / H-2E polypeptide, and a third nucleic acid sequence encodes a chimeric human / mouse HLA-A / H-2K polypeptide, and the mouse expresses HLA-A / H-2K and HLA-DR / H-2E proteins.

[0022] Also provided herein are non-human animals comprising a substantially humanized T cell immune system, e.g., the substantially humanized T cell immune system mounts a substantially humanized T cell immune response to an antigen. In some embodiments, the substantially humanized T cell immune response comprises activated T cells expressing a human T cell receptor (TCR) variable domain that recognizes an antigen presented in the context of a human leukocyte antigen (HLA) extracellular domain and / or an antigen-presenting cell presenting the antigen in the context of an HLA extracellular domain. In some embodiments, the substantially humanized T cell immune system comprises (a) non-human T cells that express a T cell co-receptor polypeptide comprising a human T cell co-receptor domain that binds to a human HLA molecule and / or a T cell receptor (TCR) comprising a TCR variable domain encoded by at least one human TCR variable region gene segment, and (b) non-human antigen-presenting cells that present the antigen in the context of a human HLA and activate the non-human T cells.

[0023] Also provided are methods of producing and using the non-human animals disclosed herein. Generally, a method of producing a genetically modified non-human animal disclosed herein comprises: (a) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide (e.g., a chimeric CD4 polypeptide), and / or a second nucleotide sequence encoding a second chimeric human / non-human T cell co-receptor polypeptide (e.g., a chimeric CD8α polypeptide), and a third nucleotide sequence encoding a third chimeric human / non-human T cell co-receptor polypeptide (e.g., a chimeric CD8β polypeptide), wherein the non-human portion of each chimeric T cell co-receptor polypeptide comprises at least the transmembrane and cytoplasmic domains of a non-human T cell co-receptor; the human portion comprises an extracellular portion (or a portion thereof, e.g., one or more domains) of a human T-cell co-receptor; (b) inserting into the genome of the non-human animal an unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCR α constant gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence; and optionally, (c) inserting into the genome a first chimeric human / non-human MHC polypeptide (e.g., a chimeric MHC and / or (d) placing into the genome of the non-human animal a first nucleic acid sequence encoding a human or humanized β2 microglobulin polypeptide, a second nucleic acid sequence encoding a second chimeric human / non-human MHC polypeptide (e.g., a chimeric MHC IIβ polypeptide), and / or a third nucleic acid sequence encoding a third chimeric human / non-human MHC polypeptide (e.g., a chimeric MHC I polypeptide).In some embodiments, the first nucleotide sequence encodes the extracellular portion of human CD4 or a portion thereof operably linked to at least the transmembrane and cytoplasmic domains of a non-human CD4 co-receptor; the second nucleotide sequence encodes the extracellular portion of human CD8α and at least the transmembrane and cytoplasmic domains of the non-human CD8α; the third nucleotide sequence encodes the extracellular portion of human CD8β and at least the transmembrane and cytoplasmic domains of the non-human CD8β; the first nucleic acid sequence encodes the extracellular portion (or portion) of a human HLA class IIα polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC IIα polypeptide; the second nucleic acid sequence encodes the extracellular portion (or portion) of a human HLA class IIβ polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC IIβ polypeptide; and the third nucleic acid sequence encodes the extracellular portion (or portion) of a human HLA class I polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC IIβ polypeptide. The β2 microglobulin locus encodes the transmembrane and cytoplasmic domains of the I polypeptide, and includes the nucleotide sequence represented by exons 2 to 4 of the human β2 microglobulin gene, for example, the nucleotide sequence represented by exons 2, 3, and 4 of the human β2 microglobulin gene.

[0024] The method for producing a non-human animal includes: (a) introducing into the genome of the non-human animal first, second, and / or third nucleotide sequences encoding chimeric T cell co-receptor polypeptides comprises, at the endogenous CD4 locus, replacing a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide, and / or, at the endogenous CD8α locus, replacing a nucleotide sequence encoding an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide, and, at the endogenous CD8β locus, replacing a nucleotide sequence encoding an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide; and (b) inserting into the genome of the animal an unrearranged TCR α locus and / or an unrearranged TCR β locus comprises, (c) replacing an endogenous non-human TCR α variable locus with an unrearranged humanized TCR α variable locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCR α variable locus, wherein the humanized TCR α variable locus is operably linked to an endogenous non-human TCR α constant region; and / or replacing an endogenous non-human TCR β variable locus with an unrearranged humanized TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCR β variable locus, wherein the humanized TCR β variable locus is operably linked to an endogenous non-human TCR β constant region; replacing a nucleotide sequence encoding a non-human MHC II complex at the endogenous non-human MHC I locus with a nucleotide sequence encoding a chimeric human / non-human MHC II complex; replacing a nucleotide sequence encoding a non-human MHC I polypeptide at the endogenous non-human MHC I locus with a nucleotide sequence encoding a chimeric human / non-human MHC II complex;and / or (d) adding a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide into the genome of the non-human animal comprises replacing the nucleotide sequence encoding the non-human β2 microglobulin polypeptide at the endogenous non-human β2 microglobulin locus with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide.

[0025] In some embodiments, (a) introducing each of the first, second, and / or third nucleotide sequences into the genome of the non-human animal comprises (i) replacing, at the endogenous CD4 locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD4 polypeptide in operably linked relation to a sequence encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains; (ii) replacing, at the endogenous CD8α locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8α polypeptide in operably linked relation to a sequence encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains; and / or (iii) replacing, at the endogenous CD8β locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8β polypeptide in operably linked relation to a sequence encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains. (b) inserting into the genome of the animal each of the unrearranged TCR α locus and / or the unrearranged TCR β locus comprises (i) replacing the endogenous non-human TCR α variable locus with an unrearranged humanized TCR α variable locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCR α variable locus. wherein the humanized TCR alpha variable locus is operably linked to an endogenous non-human TCR alpha constant region; and / or (ii) replacing the endogenous non-human TCR beta variable locus with an unrearranged humanized TCR beta variable locus comprising at least one human V beta segment, at least one human D beta segment, and at least one human J beta segment to generate a humanized TCR beta variable locus, wherein the humanized TCR beta variable locus is operably linked to an endogenous non-human TCR beta constant region;and / or placing each of the third nucleic acid sequences in the genome of the non-human animal by (i) replacing, at the endogenous non-human MHC IIα locus, a nucleotide sequence encoding the extracellular portion (or a portion thereof) of a non-human MHC IIα polypeptide with a nucleotide sequence encoding the extracellular portion (or a portion thereof) of a human HLA class IIα polypeptide, such that the nucleotide sequence is operably linked to a sequence encoding the endogenous non-human MHC IIα transmembrane and cytoplasmic domains; (ii) replacing, at the endogenous non-human MHC IIβ locus, a nucleotide sequence encoding the extracellular portion (or a portion thereof) of a non-human MHC IIβ polypeptide with a nucleotide sequence encoding the extracellular portion (or a portion thereof) of a human HLA class IIβ polypeptide, such that the endogenous non-human MHC IIα transmembrane and cytoplasmic domains are operably linked to a sequence encoding the endogenous non-human MHC IIα transmembrane and cytoplasmic domains; and / or (iii) at the endogenous non-human MHC I locus, replacing the nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class I polypeptide, so that the nucleotide sequence is operably linked to the sequence encoding the endogenous non-human MHC I transmembrane domain and cytoplasmic domain, and / or at the endogenous β2 microglobulin locus, replacing the nucleotide sequence set forth in exons 2 through 4 with a nucleotide sequence comprising exons 2, 3, and 4 of the human β2 microglobulin gene.

[0026] In one embodiment, the introducing step comprises replacing, in the first non-human animal, a nucleotide sequence encoding an endogenous non-human CD4 polypeptide at the endogenous CD4 locus with a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide; replacing, in the second non-human animal, a nucleotide sequence encoding an endogenous non-human CD8α polypeptide at the endogenous CD8α locus with a nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide; and replacing, in the second non-human animal, a nucleotide sequence encoding an endogenous non-human CD8β polypeptide at the endogenous CD8β locus with a nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide. In some embodiments, the introducing step comprises replacing, at the endogenous CD4 locus of the first non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD4 polypeptide so that the nucleotide sequence is operably linked to a sequence encoding the endogenous non-human CD4 transmembrane and cytoplasmic domain; and replacing, at the endogenous CD8α locus of the second non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8α polypeptide. and replacing, at the endogenous CD8β locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8β polypeptide, so that the nucleotide sequence is operably linked to a sequence encoding the endogenous non-human CD8β transmembrane and cytoplasmic domain. In some embodiments, the replacing steps are performed simultaneously or in any order.

[0027] In some embodiments, the inserting step comprises: replacing, in a third non-human animal, an endogenous non-human TCR α variable locus with an unrearranged humanized TCR α variable locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCR α variable locus, wherein the humanized TCR α variable locus is operably linked to an endogenous non-human TCR α constant region; and replacing, in a fourth non-human animal, an endogenous non-human TCR β variable locus with an unrearranged humanized TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCR β variable locus, wherein the humanized TCR β variable locus is operably linked to an endogenous non-human TCR β constant region. In some embodiments, the replacing steps are performed simultaneously or in any order.

[0028] In some embodiments, the placing step comprises, in no specified order, replacing, at an endogenous non-human MHC II locus of the fifth non-human animal, one or more nucleotide sequences encoding a non-human MHC II complex with one or more nucleotide sequences encoding a chimeric human / non-human MHC II complex, and replacing, at an endogenous non-human MHC I locus of the fifth non-human animal, a nucleotide sequence encoding a non-human MHC I polypeptide with a nucleotide sequence encoding a chimeric human / non-human MHC I polypeptide. In some embodiments, the placing step includes replacing, at the endogenous non-human MHC IIα locus in the fifth non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC IIα polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human MHC IIα polypeptide so that the nucleotide sequence is operably linked to a sequence encoding the endogenous non-human MHC IIα transmembrane and cytoplasmic domains; replacing, at the endogenous non-human MHC IIβ locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC IIβ polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human MHC IIβ polypeptide so that the nucleotide sequence is operably linked to a sequence encoding the endogenous non-human MHC IIβ transmembrane and cytoplasmic domains; and replacing, at the endogenous non-human MHC I locus of the fifth non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human MHC Iβ polypeptide. and replacing, in operably linked relation to, the sequences encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains with a nucleotide sequence encoding the extracellular portion (or portion thereof) of the endogenous non-human MHC I polypeptide. In some embodiments, the replacing steps are performed simultaneously or in any order.

[0029] In some embodiments, the adding step comprises replacing a nucleotide sequence encoding a non-human β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide at an endogenous non-human β2 microglobulin locus of the sixth non-human animal. In some embodiments, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.

[0030] Methods disclosed herein include embodiments in which a non-human animal containing one or more of the genetic modifications described herein is bred to another (or multiple) non-human animals of the same species containing the remaining genetic modifications, thereby introducing a first, second, and / or third nucleotide sequence encoding a chimeric T cell co-receptor polypeptide, inserting a TCR α locus and / or an unrearranged TCR β locus, arranging a first, second, and / or third nucleic acid sequence encoding a chimeric MHC polypeptide, and / or adding a β2 microglobulin locus. Non-limiting embodiments include breeding the above-described first, second, third, fourth, fifth, and sixth non-human animals in any order.

[0031] The methods disclosed herein may include homologous recombination in non-human embryonic stem (ES) cells. The methods disclosed herein may be used to generate the mice disclosed herein. Non-human animals expressing chimeric human / non-human CD4, CD8α and / or CD8β T-cell co-receptor polypeptides, human(ized) TCRα / β proteins, and chimeric MHC II complexes and MHC I (plus human or humanized β2 microglobulin) may be generated by (a) first introducing each individual human(ized) gene into an individual ES cell by homologous recombination, generating each individual non-human animal from such ES cell, and then breeding the resulting non-human animals in any order; (b) introducing all human(ized) genes into a single ES cell by sequential homologous recombination, and then generating a non-human animal from such ES cell; or (c) a combination of sequential homologous recombination at several loci in ES cells and breeding. The animals disclosed herein may also be generated by breeding the progeny of the initial breeding with other animals, if desired. Breeding and / or homologous recombination may be accomplished in any preferred order.

[0032] Also provided are methods for isolating a human TCR variable domain specific for an antigen from a non-human animal, the method comprising isolating a T cell or TCR protein that binds the antigen from a non-human animal provided herein or produced by a method disclosed herein. In some embodiments, the method may further comprise identifying a first and / or second nucleic acid encoding a TCR alpha and / or TCR beta variable domain that binds the antigen, and / or culturing a cell comprising one or more vectors under conditions sufficient for expression of the vectors, wherein the vectors comprise a third and / or fourth nucleic acid identical or substantially identical to the first and / or second nucleic acid, respectively, wherein the third and / or fourth nucleic acid is cloned in-frame with, for example, a human TCR constant region gene, e.g., the TCR alpha constant region gene and / or the TCR beta constant gene region, respectively. Also provided are tissues and cells comprising the genetic modifications disclosed herein (which may include rearranged human TCR alpha and / or TCR beta variable region genes), and nucleic acids encoding such human TCR variable domains expressed by such tissues or cells isolated from non-human animals modified as described herein. Also included are (1) recombinant nucleic acids, e.g., expression vectors, comprising nucleic acid sequences encoding human TCR variable domains disclosed herein, e.g., human rearranged TCR alpha or human rearranged TCR beta variable region genes, cloned in-frame into appropriate human TCR constant region genes, e.g., TCR alpha constant region genes or TCR beta constant region genes, respectively; (2) host cells comprising such nucleic acids (e.g., expression vectors); and (3) TCRs expressed by host cells. In some embodiments, the recombinant nucleic acids provided herein comprise a human rearranged TCR delta variable region gene or a TCR gamma variable region gene, e.g., derived from a non-human animal that has been genetically modified as disclosed herein or tissue isolated therefrom, cloned in-frame with a human TCR delta constant region gene or a TCR gamma constant region gene, respectively.

[0033] Also provided are methods of generating a humanized T cell response in a non-human animal. The methods generally involve immunizing a non-human animal that has been genetically modified as described herein or that has a substantially humanized T cell immune system as described herein with an antigen, e.g., a human antigen, e.g., a human tumor antigen, a human bacterial pathogen, a human viral pathogen, etc. In some embodiments, the immunized non-human animal expresses at least 50% of all functional human TCR Vα gene segments and / or at least 50% of all functional human TCR Vβ gene segments and / or comprises all or substantially all functional human TCR Vα gene segments and / or all or substantially all functional human TCR Vβ gene segments.

[0034] Also provided is an in vitro method for isolating a human TCR specific for an antigen, the method generally comprising (a) contacting a first cell from a non-human animal with a second cell from a non-human animal and (b) detecting activation of the first cell from the non-human animal after incubation with the antigen, wherein the first cell expresses a chimeric human / non-human T cell co-receptor and either or both of (i) a chimeric human / non-human TCR α chain and (ii) a chimeric human / non-human TCR β chain, and the second cell expresses a chimeric human / non-human MHC polypeptide. The method may further comprise isolating the TCR or a nucleic acid encoding it from the first cell.

[0035] In the in vitro methods disclosed herein, the antigen can be a tumor antigen, a viral antigen, an autoantigen, or a bacterial antigen. In some embodiments, the non-human animal is a rodent, e.g., a rat or a mouse. Also provided herein are tissues, T cells, TCRs (e.g., soluble TCRs), or nucleic acids encoding all or a portion of a TCR, hybridomas, or quadromas derived from such T cells, isolated from a non-human animal that has been genetically modified as described herein or that has a substantially humanized T cell immune system as described herein.

[0036] Also provided are compositions, for example, comprising first and second cells of a non-human animal, wherein the first cell expresses a chimeric human / non-human T cell co-receptor and, optionally, either or both of (i) a chimeric human / non-human TCR α chain and (ii) a chimeric human / non-human TCR β chain, and the second cell expresses a chimeric human / non-human MHC polypeptide that associates with the chimeric human / non-human T cell co-receptor. In some embodiments, the first cell is a non-human T cell. In other embodiments, the second cell is a non-human antigen-presenting cell. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A genetically modified non-human animal, comprising: (a) a chimeric CD4 co-receptor and / or a chimeric CD8 co-receptor comprising a chimeric CD8α polypeptide and a chimeric CD8β polypeptide, the chimeric CD4 co-receptor is encoded by a first nucleotide sequence, the chimeric CD8α polypeptide is encoded by a second nucleotide sequence, and the chimeric CD8β polypeptide is encoded by a third nucleotide sequence; the chimeric CD4 co-receptor comprises the extracellular portion of human CD4 or a portion thereof and at least the transmembrane and cytoplasmic domains of a non-human CD4 co-receptor; the chimeric CD8α polypeptide comprises the extracellular portion of human CD8α or a portion thereof and at least the transmembrane and cytoplasmic domains of non-human CD8α; a chimeric CD4 co-receptor and / or a chimeric CD8 co-receptor, wherein the chimeric CD8 β polypeptide comprises the extracellular portion or a portion thereof of human CD8 β and at least the transmembrane and cytoplasmic domains of non-human CD8 β; (b) a humanized TCR α chain and a humanized TCR β chain, a humanized TCR α chain and a humanized TCR β chain, wherein the humanized TCR α chain is derived from an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment and is operably linked to a non-human TCR α constant gene sequence, and the humanized TCR β chain is derived from an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment and is operably linked to a non-human TCR β constant gene sequence; and optionally (c) a chimeric MHC II complex comprising a chimeric MHC IIα polypeptide and a chimeric MHC IIβ polypeptide, and / or a chimeric MHC I polypeptide, the chimeric MHC IIα polypeptide is encoded by a first nucleic acid sequence, the chimeric MHC IIβ polypeptide is encoded by a second nucleic acid sequence, and the chimeric MHC I polypeptide is encoded by a third nucleic acid sequence; the chimeric MHC IIα polypeptide comprises the extracellular portion (or a portion thereof) of a human HLA class IIα polypeptide and at least the transmembrane domain and the cytoplasmic domain of a non-human MHC IIα polypeptide; the chimeric MHC II β polypeptide comprises the extracellular portion (or a portion thereof) of a human HLA class II β polypeptide and at least the transmembrane domain and the cytoplasmic domain of a non-human MHC II β polypeptide; the chimeric MHC I polypeptide comprises the extracellular portion (or a portion thereof) of a human HLA class I polypeptide and the transmembrane and cytoplasmic domains of a non-human MHC I polypeptide; a chimeric MHC II complex, wherein the chimeric MHC II complex is associated with the chimeric CD4 co-receptor and / or the chimeric MHC I polypeptide is associated with the chimeric CD8 co-receptor. (Item 2) In the germline genome, (a) the first, second, and third nucleotide sequences; (b) the unrearranged T cell receptor (TCR) alpha variable locus operably linked to a non-human TCR alpha constant gene sequence, and the unrearranged TCR beta variable locus operably linked to a non-human TCR beta constant gene sequence; (c) the first, second, and third nucleic acid sequences. (Item 3) 3. The genetically modified non-human animal of item 1 or 2, wherein the first nucleotide sequence is present in an endogenous CD4 T-cell co-receptor locus and / or the second nucleotide sequence is present in an endogenous CD8α T-cell co-receptor locus and the third nucleotide sequence is present in an endogenous CD8β T-cell co-receptor locus. (Item 4) 4. The genetically modified non-human animal of any one of items 1 to 3, wherein the chimeric CD4 polypeptide comprises the D1, D2, and D3 domains of a human CD4 polypeptide, and / or the chimeric CD8α polypeptide comprises the IgV-like domain of a human CD8α polypeptide and the chimeric CD8β polypeptide comprises the IgV-like domain of a human CD8β polypeptide. (Item 5) 5. The genetically modified non-human animal according to any one of items 1 to 4, wherein the first nucleic acid sequence is present at an endogenous non-human MHC IIα locus, the second nucleic acid sequence is present at an endogenous non-human MHC IIβ locus, and / or the third nucleic acid sequence is present at an endogenous non-human MHC I locus. (Item 6) 6. The genetically modified non-human animal according to any one of items 1 to 5, wherein the chimeric MHC IIα polypeptide comprises human HLA class II α1 and α2 domains, the chimeric MHC IIβ polypeptide comprises human HLA class II β1 and β2 domains, and / or the MHC I polypeptide comprises human HLA class I α1, α2, and α3 domains. (Item 7) 7. The genetically modified non-human animal according to any one of items 1 to 6, wherein the first nucleic acid sequence is expressed under the control of an endogenous non-human MHC IIα promoter and regulatory elements, the second nucleic acid sequence is expressed under the control of an endogenous non-human MHC IIβ promoter and regulatory elements, and / or the third nucleic acid sequence is expressed under the control of an endogenous non-human MHC I promoter and regulatory elements. (Item 8) 8. The genetically modified non-human animal according to any one of items 1 to 7, wherein the chimeric MHC IIα polypeptide comprises the transmembrane domain and the cytoplasmic domain of an endogenous non-human MHC IIα polypeptide, the chimeric MHC IIβ polypeptide comprises the transmembrane domain and the cytoplasmic domain of an endogenous non-human MHC IIβ polypeptide, and / or the chimeric MHC I polypeptide comprises the transmembrane domain and the cytoplasmic domain of an endogenous non-human MHC I polypeptide. (Item 9) the chimeric MHC IIα polypeptide comprises the extracellular portion or a portion thereof of a human HLA-DRα protein, a human HLA-DQα protein, or a human HLA-DPα protein; the chimeric MHC IIβ polypeptide comprises the extracellular portion of a human HLA-DRβ protein, a human HLA-DQβ protein, or a human HLA-DPβ protein, or a portion thereof; and / or 9. The genetically modified non-human animal according to any one of items 1 to 8, wherein the chimeric MHC I polypeptide comprises the extracellular portion or a part thereof of a human HLA-A protein, a human HLA-B protein, or a human HLA-C protein. (Item 10) the chimeric MHC IIα polypeptide comprises the extracellular portion of a human HLA-DRα protein or a portion thereof; the chimeric MHC IIβ polypeptide comprises the extracellular portion of a human HLA-DRβ protein or a portion thereof; and / or 10. The genetically modified non-human animal according to any one of items 1 to 9, wherein the chimeric MHC I polypeptide comprises the extracellular portion of a human HLA-A polypeptide or a part thereof. (Item 11) 12. The genetically modified non-human animal according to any one of items 1 to 10, wherein the chimeric MHC I polypeptide comprises the α1, α2, and α3 domains of a human HLA-A2 polypeptide. 12. The genetically modified non-human animal according to any one of items 1 to 11, wherein the chimeric MHC I polypeptide comprises the α1, α2, and α3 domains of a human HLA-A2.1 polypeptide. (Item 13) the chimeric MHC IIα polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2Eα polypeptide; the chimeric MHC IIβ polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2Eβ polypeptide; and / or 13. The genetically modified non-human animal of any one of items 1 to 12, wherein the chimeric MHC I polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2K polypeptide. (Item 14) 14. The genetically modified non-human animal of any one of items 1 to 13, wherein the unrearranged TCR alpha variable locus comprises a complete repertoire of human Valpha gene segments and a complete repertoire of human Jalpha gene segments, and / or the unrearranged TCR beta variable locus comprises a complete repertoire of human Vbeta gene segments, a complete repertoire of human Dbeta gene segments, and a complete repertoire of human Jbeta gene segments. (Item 15) 15. The genetically modified non-human animal of any one of items 1 to 14, wherein the human Vα and Jα gene segments have rearranged to form a rearranged human Vα / Jα sequence, and / or the human Vβ, Dβ, and Jβ gene segments have rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. (Item 16) 16. The genetically modified non-human animal according to any one of items 1 to 15, wherein the non-human animal expresses a T cell receptor comprising a human TCR α variable region and / or a human TCR β variable region on the surface of the T cell. (Item 17) 17. The genetically modified non-human animal of any one of items 1 to 16, wherein the endogenous non-human Vα and Jα segments are incapable of rearranging to form rearranged Vα / Jα sequences and / or the endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form rearranged Vβ / Dβ / Jβ sequences. (Item 18) 18. The genetically modified non-human animal of any one of items 1 to 17, wherein the animal lacks a functional endogenous non-human TCR alpha variable locus and / or lacks a functional endogenous non-human TCR beta variable locus. (Item 19) the endogenous non-human TCR alpha variable locus lacks all or substantially all functional endogenous V alpha gene segments, and / or lacks all or substantially all functional endogenous J alpha gene segments, and / or 19. The genetically modified non-human animal of any one of Items 1 to 18, wherein the endogenous non-human TCR β variable locus (a) lacks all or substantially all functional endogenous Vβ gene segments, (b) lacks all or substantially all functional endogenous Dβ gene segments, (c) lacks all or substantially all functional endogenous Jβ gene segments, or (d) any combination of (a), (b), and (c). (Item 20) the first nucleotide sequence is present in an endogenous CD4 T-cell co-receptor locus, the second nucleotide sequence is present in an endogenous CD8α T-cell co-receptor locus, and the third nucleotide sequence is present in an endogenous CD8β T-cell co-receptor locus; the unrearranged TCR alpha variable locus is present at an endogenous TCR alpha variable locus and the unrearranged TCR beta variable locus is present at an endogenous TCR beta variable locus; 20. The genetically modified non-human animal according to any one of Items 1 to 19, wherein the first nucleic acid sequence is present at an endogenous non-human MHC IIα locus, the second nucleic acid sequence is present at an endogenous non-human MHC IIβ locus, and the third nucleic acid sequence is present at an endogenous non-human MHC I locus. (Item 21) the first nucleotide sequence is expressed under the control of an endogenous non-human CD4 promoter and regulatory elements, the second nucleotide sequence is expressed under the control of an endogenous non-human CD8α promoter and regulatory elements, and the third nucleotide sequence is expressed under the control of an endogenous non-human CD8β promoter and regulatory elements; the unrearranged TCR alpha variable locus is expressed under the control of the endogenous TCR alpha promoter and regulatory elements, and the unrearranged TCR beta variable locus is expressed under the control of the endogenous TCR beta promoter and regulatory elements; 21. The genetically modified non-human animal of claim 20, wherein the first nucleic acid sequence is expressed under the control of an endogenous non-human MHC IIα promoter and regulatory elements, the second nucleic acid sequence is expressed under the control of an endogenous non-human MHC IIβ promoter and regulatory elements, and the third nucleic acid sequence is expressed under the control of an endogenous non-human MHC I promoter and regulatory elements. (Item 22) (a) a sequence encoding the extracellular portion (or portion thereof) of the human CD4 polypeptide replaces a sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD4 co-receptor polypeptide and is operably linked to endogenous non-human CD4 transmembrane and cytoplasmic domain-encoding sequences at the endogenous non-human CD4 co-receptor locus to form the first nucleotide sequence; a sequence encoding the extracellular portion (or portion thereof) of a human CD8α polypeptide replaces a sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human T-cell CD8α polypeptide and is operably linked to an endogenous non-human CD8α transmembrane and cytoplasmic domain-encoding sequence in the endogenous non-human CD8α locus to form said second nucleotide sequence; and / or a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8β polypeptide replaces the sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human T-cell CD8β polypeptide and is operably linked to the endogenous non-human CD8β transmembrane and cytoplasmic domain-encoding sequence in the endogenous CD8β locus to form said third nucleotide sequence; (B) the unrearranged TCR alpha variable locus replaces one or more endogenous Vα and / or Jα gene segments at the endogenous TCR alpha variable locus, and the unrearranged TCR beta variable locus replaces one or more endogenous Vβ, Dβ, and / or Jβ gene segments at the endogenous TCR beta variable locus, and / or (C) a sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIα polypeptide replaces a sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human MHC IIα polypeptide and is operably linked to endogenous MHC IIα polypeptide transmembrane and cytoplasmic domain-encoding sequences in the endogenous non-human MHC IIα locus to form the first nucleic acid sequence; a sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIβ polypeptide replaces a sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human MHC IIβ polypeptide and is operably linked to endogenous MHC IIβ polypeptide transmembrane and cytoplasmic domain-encoding sequences in the endogenous non-human MHC IIβ locus to form said second nucleic acid sequence; and / or 22. The genetically modified non-human animal of any one of items 1 to 21, wherein a sequence encoding the extracellular portion (or portion thereof) of a human HLA class I polypeptide replaces a sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human MHC I polypeptide and is operably linked to an endogenous MHC I polypeptide transmembrane and cytoplasmic domain-encoding sequence at the endogenous non-human MHC I locus to form the third nucleic acid sequence. (Item 23) 23. The genetically modified non-human animal of any one of items 1 to 22, wherein the animal does not express on its cell surface: (a) a functional endogenous non-human CD4 and / or CD8 co-receptor from the endogenous CD4 and / or CD8 co-receptor locus, respectively; (b) an endogenous TCR alpha variable domain from the endogenous TCR alpha locus; (c) an endogenous TCR beta variable domain from the endogenous TCR beta locus; and / or (d) an extracellular domain of an endogenous MHC polypeptide from the endogenous MHC locus. (Item 24) 25. The genetically modified non-human animal according to any one of Items 1 to 23, further comprising a β2 microglobulin locus encoding a polypeptide comprising the amino acid sequence of human β2 microglobulin, wherein the non-human animal expresses a human or humanized β2 microglobulin polypeptide. 25. The genetically modified non-human animal of any one of items 1 to 24, wherein the non-human animal does not express a functional endogenous non-human animal β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. (Item 26) 26. The genetically modified non-human animal of item 24 or 25, wherein the β2 microglobulin locus is operably linked to an endogenous non-human β2 microglobulin regulatory element. (Item 27) 27. The genetically modified non-human animal of any one of items 24 to 26, wherein the β2 microglobulin locus comprises the nucleotide sequence represented by exon 2, exon 3, and exon 4 of the human β2 microglobulin gene. (Item 28) 28. The genetically modified non-human animal of any one of items 24 to 27, wherein the β2 microglobulin locus further comprises a nucleotide sequence represented by exon 1 of the non-human β2 microglobulin gene. (Item 29) 29. The genetically modified non-human animal according to any one of items 24 to 28, wherein the nucleotide sequence further comprises a nucleotide sequence represented by exon 1 of a rodent β2 microglobulin gene. (Item 30) 30. The genetically modified non-human animal according to any one of items 1 to 29, wherein the animal is a rodent. (Item 31) 31. The genetically modified non-human animal according to any one of items 1 to 30, wherein the animal is a mouse. (Item 32) The animal is a mouse, and the mouse is chimeric T cell CD4, CD8α, and CD8β co-receptor polypeptides, each comprising a CD4, CD8α, and CD8β murine transmembrane and cytoplasmic domain, respectively; a T cell receptor on the surface of a T cell, the T cell receptor comprising a human TCR alpha variable region and a human TCR beta variable region; 32. The genetically modified non-human animal according to any one of Items 24 to 31, wherein the genetically modified non-human animal expresses chimeric MHC IIα, MHC IIβ, and MHC I polypeptides, each comprising the extracellular portion of a human HLA class IIα, HLA class IIβ, and HLA class I polypeptide, respectively, and a humanized β2 microglobulin polypeptide. (Item 33) 33. The genetically modified mouse of claim 31, wherein the first nucleic acid sequence encodes the alpha chain of a chimeric human / mouse HLA-DR / H-2E polypeptide, the second nucleotide sequence encodes the beta chain of a chimeric human / mouse HLA-DR / H-2E polypeptide, and the third nucleic acid sequence encodes a chimeric human / mouse HLA-A / H-2K polypeptide, and the mouse expresses HLA-A / H-2K and HLA-DR / H-2E proteins. (Item 34) (a) introducing into the genome of said non-human animal a first nucleotide sequence encoding a chimeric CD4 co-receptor, and / or a second nucleotide sequence encoding a chimeric CD8α co-receptor, and a third nucleotide sequence encoding a chimeric CD8β polypeptide; the first nucleotide sequence encodes the extracellular portion of human CD4 or a portion thereof operably linked to at least the transmembrane and cytoplasmic domains of a non-human CD4 co-receptor; the second nucleotide sequence encodes the extracellular portion or a portion thereof of human CD8α and at least the transmembrane and cytoplasmic domains of non-human CD8α; the third nucleotide sequence encodes the extracellular portion or a portion thereof of human CD8β and at least the transmembrane and cytoplasmic domains of non-human CD8β; (b) inserting into the genome of said non-human animal an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCR α constant gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence; and optionally (c) placing within said genome a first nucleic acid sequence encoding a chimeric MHC IIα polypeptide, a second nucleic acid sequence encoding a chimeric MHC IIβ polypeptide, and / or a third nucleic acid sequence encoding a chimeric MHC I polypeptide; the first nucleic acid sequence encodes the extracellular portion (or a portion thereof) of a human HLA class IIα polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC IIα polypeptide; the second nucleic acid sequence encodes the extracellular portion (or a portion thereof) of a human HLA class II β polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC II β polypeptide; the third nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class I polypeptide and the transmembrane and cytoplasmic domains of a non-human MHC I polypeptide; and / or (d) a method for producing a genetically modified non-human animal according to any one of items 1 to 33, comprising adding a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide into the genome of the non-human animal. (Item 35) (a) introducing each of the first, second, and / or third nucleotide sequences into the genome of the non-human animal comprises (i) replacing, at the endogenous CD4 locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD4 polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human CD4 polypeptide is operably linked to a sequence encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains; (ii) replacing, at the endogenous CD8α locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8α polypeptide. and / or (iii) replacing, at the endogenous CD8β locus, a nucleotide sequence encoding the extracellular portion (or portion) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or portion) of a human CD8β polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion) of the human CD8β polypeptide is operably linked to sequences encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains; and / or (iv) replacing, at the endogenous CD8β locus, a nucleotide sequence encoding the extracellular portion (or portion) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or portion) of a human CD8β polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion) of the human CD8β polypeptide is operably linked to sequences encoding the endogenous non-human CD8β transmembrane and cytoplasmic domains; (b) inserting the unrearranged TCR α locus and / or the unrearranged TCR β locus, respectively, into the genome of the animal comprises: (i) replacing an endogenous non-human TCR α variable locus with an unrearranged TCR α variable locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCR α variable locus, wherein the humanized TCR α variable locus is operably linked to an endogenous non-human TCR α constant region; and / or (ii) replacing an endogenous non-human TCR β variable locus with an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCR β variable locus, wherein the humanized TCR β variable locus is operably linked to an endogenous non-human TCR β constant region; (c) placing the first, second, and / or third nucleic acid sequences, respectively, in the genome of the non-human animal comprises (i) replacing, at the endogenous non-human MHC IIα locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC IIα polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIα polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human HLA class IIα polypeptide is operably linked to a sequence encoding the endogenous non-human MHC IIα transmembrane and cytoplasmic domains; (ii) replacing, at the endogenous non-human MHC IIβ locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC IIβ polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIβ polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human HLA class IIβ polypeptide is operably linked to a sequence encoding the endogenous non-human MHC IIα transmembrane and cytoplasmic domains; and / or (iii) replacing the non-human MHC I locus at the endogenous non-human MHC I locus so that the non-human MHC I locus is in operably linked with sequences encoding the IIβ transmembrane and cytoplasmic domains. and / or replacing a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class I polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class I polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human HLA class I polypeptide is operably linked to a sequence encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains; (d) The method of item 34, wherein adding the β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide into the genome of the non-human animal comprises replacing a nucleotide sequence encoding a non-human β2 microglobulin polypeptide in the endogenous non-human β2 microglobulin locus with a nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide. (Item 36) 36. The method of claim 35, wherein the replacing step comprises homologous recombination in a non-human ES cell, whereby the first, second, and third nucleotide sequences are introduced, in any order, into the genome of the non-human ES cell, inserting the unrearranged TCR α locus and unrearranged TCR β locus, positioning the first, second, and third nucleic acid sequences, and adding the β2 microglobulin locus. (Item 37) 37. The method of claim 36, further comprising generating a non-human animal from the non-human ES cells. (Item 38) The introducing step comprises, in any order: replacing, at the endogenous CD4 locus in a first non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD4 polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human CD4 polypeptide is operably linked to sequences encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains; replacing, at the endogenous CD8α locus in the second non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8α polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human CD8α polypeptide is operably linked to a sequence encoding the endogenous non-human CD8α transmembrane and cytoplasmic domain; and replacing, at the endogenous CD8β locus, a nucleotide sequence encoding the extracellular portion (or portion thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human CD8β polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human CD8β polypeptide is operably linked to a sequence encoding the endogenous non-human CD8β transmembrane and cytoplasmic domain; The inserting step may, in any order, replacing an endogenous non-human TCR alpha variable locus in a third non-human animal with an unrearranged humanized TCR alpha variable locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCR alpha variable locus, wherein the humanized TCR alpha variable locus is operably linked to an endogenous non-human TCR alpha constant region; replacing an endogenous non-human TCR β variable locus in a fourth non-human animal with an unrearranged humanized TCR β variable locus comprising at least one human V β segment, at least one human D β segment, and at least one human J β segment to generate a humanized TCR β variable locus, wherein the humanized TCR β variable locus is operably linked to an endogenous non-human TCR β constant region; The step of placing includes, in any order: replacing, at the endogenous non-human MHC IIα locus in a fifth non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC IIα polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIα polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human HLA class IIα polypeptide is operably linked to a sequence encoding the endogenous non-human MHC IIα transmembrane and cytoplasmic domains; replacing, at the endogenous non-human MHC IIβ locus in the fifth non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC IIβ polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIβ polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class IIβ polypeptide is operably linked to a sequence encoding the endogenous non-human MHC IIβ transmembrane and cytoplasmic domains, to produce a seventh non-human animal; and / or replacing, at the endogenous non-human MHC I locus in the fifth non-human animal, a nucleotide sequence encoding the extracellular portion (or portion thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or portion thereof) of a human HLA class I polypeptide, such that the nucleotide sequence encoding the extracellular portion (or portion thereof) of the human HLA class I polypeptide is operably linked to a sequence encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains, to produce an eighth non-human animal; and / or The adding step comprises: 36. The method of claim 34 or 35, comprising replacing a nucleotide sequence encoding a non-human β2 microglobulin polypeptide at an endogenous non-human β2 microglobulin locus in a sixth non-human animal with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, by breeding the first, second, third, fourth, fifth, and sixth non-human animals in any order, thereby introducing the first, second, and third nucleotide sequences into the genome of the non-human animal, inserting the unrearranged TCR α locus and unrearranged TCR β locus, positioning the first, second, and third nucleic acid sequences, and / or adding the β2 microglobulin locus. (Item 39) the replacement at the endogenous CD4 locus comprises homologous recombination at the endogenous CD4 locus of the first non-human ES cell; the replacement at the endogenous CD8α and CD8β loci comprises sequential homologous recombination at the endogenous CD8α and CD8β loci in the second non-human ES cell, in any order; the replacement at the endogenous TCR alpha variable locus comprises homologous recombination at the endogenous TCR alpha variable locus in a third non-human ES cell; the replacement at the endogenous TCR β variable locus comprises homologous recombination at the endogenous TCR β variable locus in a fourth non-human ES cell; the replacement at the endogenous MHC IIα, MHC IIβ, and MHC I loci comprises, in any order, homologous recombination at the endogenous MHC IIα, MHC IIβ, and MHC I loci in the fifth non-human ES cell; 39. The method of claim 38, wherein the replacement at the β2 microglobulin locus comprises homologous recombination at the endogenous β2 microglobulin locus in the sixth non-human ES cell. (Item 40) 39. The method of claim 38, further comprising generating the first, second, third, fourth, fifth, and sixth non-human animals from the first, second, third, fourth, fifth, and sixth non-human ES cells, respectively, prior to breeding. (Item 41) 41. The method according to any one of items 34 to 40, wherein the non-human animal is a mouse. (Item 42) A method for obtaining a human TCR variable domain specific to an antigen from a non-human animal, the method comprising isolating T cells or TCR proteins that bind to the antigen from the non-human animal according to any one of Items 1 to 33 or a non-human animal produced by the method according to any one of Items 34 to 41. (Item 43) 43. The method of claim 42, further comprising identifying a first nucleic acid encoding a TCR alpha variable domain and / or a second nucleic acid encoding a TCR beta variable domain, each of which variable domains is expressed by a T cell or forms part of the antigen-binding site of the TCR protein. (Item 44) 45. The method of claim 43, further comprising culturing the cells under conditions sufficient for expression of a third nucleic acid identical or substantially identical to the first nucleic acid identified in claim 43, and / or a fourth nucleic acid identical or substantially identical to the second nucleic acid identified in claim 43, wherein the third and fourth nucleic acids are present in the same or different expression vectors. 1. An in vitro method for generating a human TCR variable domain specific for an antigen, the method comprising: (a) contacting a non-human animal with a non-human antigen-presenting cell; and (b) detecting activation of the non-human T cell after incubation with the antigen, wherein the non-human T cell expresses a chimeric human / non-human T cell co-receptor and either or both of (i) a chimeric human / non-human TCR α chain and (ii) a chimeric human / non-human TCR β chain, and the non-human antigen-presenting cell expresses a chimeric human / non-human MHC polypeptide. (Item 46) 46. ​​The method of claim 45, further comprising isolating a human TCR alpha variable domain and / or a human TCR beta variable domain, or a first and / or a second nucleic acid encoding the human TCR alpha variable domain and / or the human TCR beta variable domain, respectively, from the T cell. (Item 47) 47. The method according to any one of items 42 to 46, wherein the antigen is a tumor antigen. (Item 48) 48. The method according to any one of items 42 to 47, wherein the antigen is a viral antigen. (Item 49) 49. The method according to any one of items 42 to 48, wherein the non-human animal is a mouse. (Item 50) A hybridoma produced from the T cells isolated or detected by the method according to any one of Items 42 to 49. (Item 51) A human T cell receptor variable domain obtained or produced by the method according to any one of items 42 to 49. (Item 52) A nucleic acid isolated by the method according to any one of Items 43 to 44 and Items 46 to 49. (Item 53) 53. A cell containing a nucleic acid identical or substantially identical to the nucleic acid of item 52. (Item 54) 53. An expression vector comprising the nucleic acid of Item 52, wherein the nucleic acid comprises a sequence encoding a human TCR β variable domain. (Item 55) 55. The expression vector of claim 54, wherein the expression vector further comprises a sequence encoding a TCR alpha constant gene operably linked to the nucleic acid encoding a human TCR alpha variable domain. (Item 56) 53. An expression vector comprising the nucleic acid of Item 52, wherein the nucleic acid comprises a sequence encoding a human TCR β variable domain. (Item 57) 57. The expression vector of claim 56, wherein the expression vector further comprises a sequence encoding a TCR β constant gene operably linked to the nucleic acid encoding the human TCR β variable domain. (Item 58) 1. A composition comprising first and second cells of a non-human animal, wherein the first cell expresses a chimeric human / non-human T-cell co-receptor and, optionally, either one or both of (i) a chimeric human / non-human TCR α chain and (ii) a chimeric human / non-human TCR β chain, and the second cell expresses a chimeric human / non-human MHC polypeptide that associates with the chimeric human / non-human T-cell co-receptor. (Item 59) 59. The composition of claim 58, wherein the first cell is a non-human T cell. (Item 60) 60. The composition of item 58 or 59, wherein the second cell is a non-human antigen-presenting cell. (Item 61) 61. The composition according to any one of items 58 to 60, further comprising an antigen. (Item 62) A cell isolated from the non-human animal according to any one of Items 1 to 33, or the non-human animal produced by the method according to any one of Items 34 to 41. (Item 63) 63. The cell of item 62, wherein the cell is a T cell. (Item 64) 63. The cell of item 62, wherein the cell is an antigen-presenting cell. (Item 65) 65. A nucleic acid sequence isolated from the cell of any one of items 62 to 64, wherein the nucleic acid sequence comprises a sequence encoding a TCR variable domain or an MHC extracellular domain. (Item 66) A method for generating a humanized T cell response in a non-human animal, the method comprising immunizing the non-human animal according to any one of Items 1 to 33 or the non-human animal produced by the method according to any one of Items 34 to 41 with an antigen. (Item 67) 67. The method of item 66, wherein the antigen is a human antigen or a human tumor antigen. (Item 68) 68. The method of item 66 or 67, wherein the non-human animal expresses at least 50% of all functional human TCR Vα gene segments and / or at least 50% of all functional human TCR Vβ gene segments. (Item 69) 69. The method of any one of items 66 to 68, wherein the T cell receptor repertoire of the non-human animal comprises all or substantially all functional human TCR Vα gene segments and / or all or substantially all functional human TCR Vβ gene segments. (Item 70) 58. The expression vector according to Item 55 or 57, wherein the TCR alpha constant region gene or the TCR beta constant region gene is a human TCR alpha constant region gene or a human TCR beta constant region gene, respectively. [Brief explanation of the drawings]

[0037] [Figure 1] Schematic diagrams (not to scale) of a humanized T cell receptor complex comprising humanized TCR alpha and beta proteins, humanized MHC class I complexed with humanized β2 microglobulin, and a humanized CD8 heterodimer (left panel), and a T cell receptor complex comprising humanized TCR alpha and beta proteins, a humanized MHC class II heterodimer, and a humanized CD4 (right panel). Antigens presented by the humanized MHC are shown as circles. Mouse regions are shown as solid shapes, while human regions are shown as striped shapes.

[0038] [Figure 2A]2A-C are schematic diagrams (not to scale) of exemplary chimeric MHC I and MHC II loci, e.g., the chimeric HLA-A2 / H-2K locus (FIG. 2A), the chimeric HLA-DR2 / H-2E locus (FIG. 2B), and the humanized β2M locus (FIG. 2C). Unless otherwise indicated, human sequences are shown as open shapes and mouse sequences are shown as filled shapes. The striped shapes represent exon 1 of H-2E obtained from a mouse strain that is not the endogenous locus (see Example 1.3 and FIG. 3B). The loxP-introduced neomycin phosphotransferase cassette is indicated by an arrowhead. [Figure 2B] 2A-C are schematic diagrams (not to scale) of exemplary chimeric MHC I and MHC II loci, e.g., the chimeric HLA-A2 / H-2K locus (FIG. 2A), the chimeric HLA-DR2 / H-2E locus (FIG. 2B), and the humanized β2M locus (FIG. 2C). Unless otherwise indicated, human sequences are shown as open shapes and mouse sequences are shown as filled shapes. The striped shapes represent exon 1 of H-2E obtained from a mouse strain that is not the endogenous locus (see Example 1.3 and FIG. 3B). The loxP-introduced neomycin phosphotransferase cassette is indicated by an arrowhead. [Figure 2C] 2A-C are schematic diagrams (not to scale) of exemplary chimeric MHC I and MHC II loci, e.g., the chimeric HLA-A2 / H-2K locus (FIG. 2A), the chimeric HLA-DR2 / H-2E locus (FIG. 2B), and the humanized β2M locus (FIG. 2C). Unless otherwise indicated, human sequences are shown as open shapes and mouse sequences are shown as filled shapes. The striped shapes represent exon 1 of H-2E obtained from a mouse strain that is not the endogenous locus (see Example 1.3 and FIG. 3B). The loxP-introduced neomycin phosphotransferase cassette is indicated by an arrowhead.

[0039] [Figure 3A]Figure 3A shows a strategy for generating a humanized MHC locus containing humanized MHC I and MHC II genes. In the specific embodiment shown in Figure 3A, the resulting mouse MHC locus contains chimeric HLA-A2 / H-2K and HLA-DR2 / H-2E sequences (H2-K+ / 1666 MHC-II+ / 6112) and lacks H2-D sequences (H2-D+ / delete) and H-2A sequences (the genetic engineering scheme also results in deletion of H-2A; see Example 1.2). The large targeting vector (LTVEC) or Cre recombinase construct introduced into ES cells at each stage of humanization is shown to the right of the arrow. MAID, or four-digit number, refers to the modified allele ID number. Figure 3B is a schematic diagram (not to scale) of an exemplary HLA-DR2 / H-2E large targeting vector. Unless otherwise noted, human sequences are shown as open shapes and mouse sequences are shown as filled shapes. The striped shapes represent exon 1 of H-2E obtained from a mouse strain that is not the endogenous locus (see Example 1.3). The loxP-introduced hygromycin cassette is shown as an arrowhead. Figure 3C is a schematic (not to scale) of an exemplary genotype of a chimeric human / mouse MHC locus (** represents the H-2L gene, which is absent from all mouse strains, e.g., C57BL / 6 or 129 mouse strains). In this case, 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, dashed boxes), and the remaining loci are endogenous mouse genes (solid boxes, solid shapes). [Figure 3B]Figure 3A shows a strategy for generating a humanized MHC locus containing humanized MHC I and MHC II genes. In the specific embodiment shown in Figure 3A, the resulting mouse MHC locus contains chimeric HLA-A2 / H-2K and HLA-DR2 / H-2E sequences (H2-K+ / 1666 MHC-II+ / 6112) and lacks H2-D sequences (H2-D+ / delete) and H-2A sequences (the genetic engineering scheme also results in deletion of H-2A; see Example 1.2). The large targeting vector (LTVEC) or Cre recombinase construct introduced into ES cells at each stage of humanization is shown to the right of the arrow. MAID, or four-digit number, refers to the modified allele ID number. Figure 3B is a schematic diagram (not to scale) of an exemplary HLA-DR2 / H-2E large targeting vector. Unless otherwise noted, human sequences are shown as open shapes and mouse sequences are shown as filled shapes. The striped shapes represent exon 1 of H-2E obtained from a mouse strain that is not the endogenous locus (see Example 1.3). The loxP-introduced hygromycin cassette is shown as an arrowhead. Figure 3C is a schematic (not to scale) of an exemplary genotype of a chimeric human / mouse MHC locus (** represents the H-2L gene, which is absent from all mouse strains, e.g., C57BL / 6 or 129 mouse strains). In this case, 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, dashed boxes), and the remaining loci are endogenous mouse genes (solid boxes, solid shapes). [Figure 3C]Figure 3A shows a strategy for generating a humanized MHC locus containing humanized MHC I and MHC II genes. In the specific embodiment shown in Figure 3A, the resulting mouse MHC locus contains chimeric HLA-A2 / H-2K and HLA-DR2 / H-2E sequences (H2-K+ / 1666 MHC-II+ / 6112) and lacks H2-D sequences (H2-D+ / delete) and H-2A sequences (the genetic engineering scheme also results in deletion of H-2A; see Example 1.2). The large targeting vector (LTVEC) or Cre recombinase construct introduced into ES cells at each stage of humanization is shown to the right of the arrow. MAID, or four-digit number, refers to the modified allele ID number. Figure 3B is a schematic diagram (not to scale) of an exemplary HLA-DR2 / H-2E large targeting vector. Unless otherwise noted, human sequences are shown as open shapes and mouse sequences are shown as filled shapes. The striped shapes represent exon 1 of H-2E obtained from a mouse strain that is not the endogenous locus (see Example 1.3). The loxP-introduced hygromycin cassette is shown as an arrowhead. Figure 3C is a schematic (not to scale) of an exemplary genotype of a chimeric human / mouse MHC locus (** represents the H-2L gene, which is absent from all mouse strains, e.g., C57BL / 6 or 129 mouse strains). In this case, 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, dashed boxes), and the remaining loci are endogenous mouse genes (solid boxes, solid shapes).

[0040] [Figure 4A]Figure 1 shows the incremental strategy for humanization of the mouse TCR alpha locus (not to scale). In this case, TCR alpha variable region gene segments are added sequentially upstream of the initial humanization of the deleted mouse locus (MAID1540). Mouse sequences are shown by filled shapes. Human sequences are shown by open shapes. MAID refers to the modified allele ID number. TRAV = TCR V alpha segment, TRAJ = TCR J alpha segment (hTRAJ = human TRAJ), TRAC = TCR C alpha domain, TCRD = TCR delta. [Figure 4B] Figure 1 shows (not to scale) the incremental strategy for humanization of the mouse TCR β locus. In this case, TCR β variable region gene segments are sequentially added to a deleted mouse TCR β variable locus. Mouse sequences are shown by filled shapes. Human sequences are shown by open shapes. MAID means modified allele ID number. TRBV or TCRBV = TCR β V segment.

[0041] [Figure 5A] Figure 1 shows a schematic diagram (not to scale) of the chimeric CD4 locus. Human coding exons are represented by striped shapes. Mouse coding exons are represented by filled shapes. Non-coding exons are represented by open shapes. The immunoglobulin-like domain (Ig), transmembrane (TM), cytoplasmic (CYT), and signal peptide (signal) coding exons, as well as the 3' untranslated region (UTR), are indicated. The loxP-inserted (loxP) neomycin phosphotransferase (Pgk-neo) cassette is indicated by an arrowhead. [Figure 5B]Figure 1 shows a schematic diagram (not to scale) of the chimeric CD8a and CD8b loci. Human coding exons are represented by striped shapes. Mouse coding exons are represented by filled shapes. Non-coding exons are represented by open shapes. The immunoglobulin-like domain (IgV), transmembrane (TM), cytoplasmic (CYT), and signal peptide (signal) coding exons, as well as the 3' untranslated region (UTR), are indicated. The loxP-transfected (loxP) hygromycin (Hyg) and neomycin phosphotransferase (Pgk-neo) cassettes are indicated by arrowheads.

[0042] [Figure 6A] Figure 6 shows FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on singlets, and stained with (Figure 6A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 6B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, or (Figure 6C) anti-mouse CD8α and anti-mouse CD4 antibodies (left panel) or anti-human CD8α and anti-human CD4 antibodies (right panel). [Figure 6B] Figure 6 shows FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on singlets, and stained with (Figure 6A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 6B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, or (Figure 6C) anti-mouse CD8α and anti-mouse CD4 antibodies (left panel) or anti-human CD8α and anti-human CD4 antibodies (right panel). [Figure 6C]Figure 6 shows FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on singlets, and stained with (Figure 6A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 6B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, or (Figure 6C) anti-mouse CD8α and anti-mouse CD4 antibodies (left panel) or anti-human CD8α and anti-human CD4 antibodies (right panel).

[0043] [Figure 7A] Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 7B]Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 7C] Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 7D]Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 7E] Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 7F]Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 7G] Figures 7A and 7B show FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 7A and 7B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 7C and 7D) anti-HLA-A2 or anti-HLA-DR antibodies, (Figures 7E and 7F) anti-H-2D and anti-IAIE antibodies, or (Figure 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom).

[0044] [Figure 8] FIG. 1 shows FACS contour plots of thymocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8), gated on CD3+CD4+ cells, and stained with anti-mouse FoxP3 and anti-mouse CD25 antibodies.

[0045] [Figure 9A]Figure 9 shows FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD3+ cells, CD4+ T cells, or CD8+ T cells in singlets, and stained with (Figure 9A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 9B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, (Figure 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (Figures 9D and 9E) anti-mouse CD44 and anti-mouse CD62L antibodies. [Figure 9B] Figure 9 shows FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD3+ cells, CD4+ T cells, or CD8+ T cells in singlets, and stained with (Figure 9A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 9B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, (Figure 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (Figures 9D and 9E) anti-mouse CD44 and anti-mouse CD62L antibodies. [Figure 9C] Figure 9 shows FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD3+ cells, CD4+ T cells, or CD8+ T cells in singlets, and stained with (Figure 9A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 9B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, (Figure 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (Figures 9D and 9E) anti-mouse CD44 and anti-mouse CD62L antibodies. [Figure 9D]Figure 9 shows FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD3+ cells, CD4+ T cells, or CD8+ T cells in singlets, and stained with (Figure 9A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 9B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, (Figure 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (Figures 9D and 9E) anti-mouse CD44 and anti-mouse CD62L antibodies. [Figure 9E] Figure 9 shows FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD3+ cells, CD4+ T cells, or CD8+ T cells in singlets, and stained with (Figure 9A) anti-mouse CD19 and anti-mouse CD3 antibodies, (Figure 9B) anti-mouse CD19 and anti-mouse F4 / 80 antibodies, (Figure 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (Figures 9D and 9E) anti-mouse CD44 and anti-mouse CD62L antibodies.

[0046] [Figure 10A]Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 10B] Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 10C]Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 10D] Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 10E]Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 10F] Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). [Figure 10G]Figures 10A and 10B show FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci, gated on CD19+ cells, F4 / 80+ cells, or CD3+ cells, and stained with (Figures 10A and 10B) anti-human B2M or anti-mouse H-2D antibodies, (Figures 10C and 10D) anti-HLA-2A or anti-HLA-DR antibodies, (Figures 10E and 10F) anti-H-2D and IAIE antibodies, or (Figure 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8β antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom).

[0047] [Figure 11] FIG. 1 shows FACS contour plots of splenocytes isolated from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8), gated on CD3+CD4+ cells, and stained with anti-mouse FoxP3 and anti-mouse CD25 antibodies.

[0048] [Figure 12] Figure 1 shows the number of IFN-γ-producing splenocytes (spots per well (mean + SD); y-axis) in an enzyme-linked immunosorbent spot assay after isolation from control mice or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci and incubation in the absence of peptide (200k cells only; x-axis) or in the presence of 10 μg / mL or 1 μg / mL MAGE-A3 peptide (x-axis).

[0049] [Figure 13A]1 shows the progression of acute Armstrong strain virus infection in either control or mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci. The experimental timeline is shown at the top of the figure, and viral titer measurements at various days post-infection for both mouse strains are shown in the lower graph. [Figure 13B] Figure 13C shows the progression of viral infection with chronic clone 13 strains in either control or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci. The experimental timeline is shown in the upper part of the figure, and viral titer measurements at day 21 post-infection for both mouse strains are shown in the lower graph. T cells from uninfected or chronically infected TM I / II B C4 / 8 or control B6 mice were stained with anti-PD1, anti-Lag3, and anti-Tim3 antibodies (Figure 13C; x-axis). The figure shows quantification of positively stained cells (% positive cells; y-axis). [Figure 13C] Figure 13C shows the progression of viral infection with chronic clone 13 strains in either control or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci. The experimental timeline is shown in the upper part of the figure, and viral titer measurements at day 21 post-infection for both mouse strains are shown in the lower graph. T cells from uninfected or chronically infected TM I / II B C4 / 8 or control B6 mice were stained with anti-PD1, anti-Lag3, and anti-Tim3 antibodies (Figure 13C; x-axis). The figure shows quantification of positively stained cells (% positive cells; y-axis).

[0050] [Figure 14]Figure 1 shows the progression of chronic Clone 13 strain virus infection in either control or TM I / II B C4 / 8 mice after a prior acute Armstrong strain infection. The experimental timeline is shown in the upper part of the figure, and viral titer measurements at day 31 post-infection are shown in the lower graph. Mock-infected mice were included in the experiment as an additional control.

[0051] [Figure 15A] Figure 15 shows the number of CD8+ cells (y-axis; IFN-γ-positive cells) isolated from either control animals (Figure 15A) or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci (Figure 15B) that produced IFN-γ in response to HLA-A2-restricted (GPC10-18; N69-77; Z49-58), H2Db-restricted (GP33-41), ovalbumin, or incubation-only LCMV peptides. Mice were mock-infected (mock; n = 1 per group) or acutely infected with the Armstrong strain (Arm; n = 3 per group). The percentage (y-axis) of IFNγ+CD8+ lymphocytes after stimulation with the indicated peptides (OVA, GP33, NP69, GPC10, GPC447, or Z49) during the time course of infection (days post-infection; x-axis) in mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci or control B6 animals is shown in Figures 15C and 15D, respectively. [Figure 15B]Figure 15 shows the number of CD8+ cells (y-axis; IFN-γ-positive cells) isolated from either control animals (Figure 15A) or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci (Figure 15B) that produced IFN-γ in response to HLA-A2-restricted (GPC10-18; N69-77; Z49-58), H2Db-restricted (GP33-41), ovalbumin, or incubation-only LCMV peptides. Mice were mock-infected (mock; n = 1 per group) or acutely infected with the Armstrong strain (Arm; n = 3 per group). The percentage (y-axis) of IFNγ+CD8+ lymphocytes after stimulation with the indicated peptides (OVA, GP33, NP69, GPC10, GPC447, or Z49) during the time course of infection (days post-infection; x-axis) in mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci or control B6 animals is shown in Figures 15C and 15D, respectively. [Figure 15C]Figure 15 shows the number of CD8+ cells (y-axis; IFN-γ-positive cells) isolated from either control animals (Figure 15A) or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci (Figure 15B) that produced IFN-γ in response to HLA-A2-restricted (GPC10-18; N69-77; Z49-58), H2Db-restricted (GP33-41), ovalbumin, or incubation-only LCMV peptides. Mice were mock-infected (mock; n = 1 per group) or acutely infected with the Armstrong strain (Arm; n = 3 per group). The percentage (y-axis) of IFNγ+CD8+ lymphocytes after stimulation with the indicated peptides (OVA, GP33, NP69, GPC10, GPC447, or Z49) during the time course of infection (days post-infection; x-axis) in mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci or control B6 animals is shown in Figures 15C and 15D, respectively. [Figure 15D]Figure 15 shows the number of CD8+ cells (y-axis; IFN-γ-positive cells) isolated from either control animals (Figure 15A) or mice containing humanized MHC I, MHC IIα and β, TCRα and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci (Figure 15B) that produced IFN-γ in response to HLA-A2-restricted (GPC10-18; N69-77; Z49-58), H2Db-restricted (GP33-41), ovalbumin, or incubation-only LCMV peptides. Mice were mock-infected (mock; n = 1 per group) or acutely infected with the Armstrong strain (Arm; n = 3 per group). The percentage (y-axis) of IFNγ+CD8+ lymphocytes after stimulation with the indicated peptides (OVA, GP33, NP69, GPC10, GPC447, or Z49) during the time course of infection (days post-infection; x-axis) in mice containing humanized MHC I, MHC IIα and β, TCR α and β, CD4, CD8α and β, and β2M (TM I / II B C4 / 8) loci or control B6 animals is shown in Figures 15C and 15D, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0052] Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) that have been genetically engineered to express humanized T cell co-receptors (e.g., humanized CD4 and / or CD8 (e.g., CD8α and / or CD8β)), human or humanized major histocompatibility complexes that bind to the humanized T cell co-receptors (e.g., human or humanized MHC II (e.g., MHC IIα and / or MHC IIβ chains) and / or MHC I (e.g., MHC Iα), and optionally human or humanized β2 microglobulin), as well as embryos, tissues, and cells expressing them. The development of the cellular arm of the immune system of the non-human animals disclosed herein is comparable to that of control animals. For example, the thymus and spleen contain similar absolute numbers of thymocytes and CD3+ cells. This is in stark contrast to other non-human animals that have been engineered to contain both a human TCR (α and β) and a chimeric human / mouse MHC I molecule. See, e.g., Li (2010) Nature Medicine 16:1029-1035 and supplemental materials. Such animals exhibited reduced T cell populations compared to wild-type control animals as well as animals modified with only a human TCR and animals modified with only a chimeric human / mouse MHC I molecule (ibid.). Accordingly, provided herein are non-human animals engineered to co-express a humanized CD4 co-receptor and a humanized MHC II and / or a humanized CD8 co-receptor and a humanized MHC I, and optionally a humanized TCR. Also provided are methods for producing genetically engineered animals that express at least one humanized T cell co-receptor (e.g., humanized CD4 and / or CD8), at least one humanized MHC associated with the humanized T cell co-receptor (e.g., humanized MHC II and / or MHC I associated with humanized CD4 and / or CD8, respectively), and / or a humanized TCR. Also provided are methods for using genetically engineered animals that mount a substantially humanized T cell immune response to develop human therapeutics. Substantially humanized T cell immune responses

[0053] Disclosed herein are non-human animals genetically modified to mount a substantially humanized T cell immune response. The disclosed mice express at least one human or humanized T cell co-receptor, at least one human or humanized major histocompatibility complex (MHC) capable of associating with the at least one human or humanized T cell co-receptor, and / or a human or humanized T cell receptor (TCR), wherein the human or humanized T cell receptor is preferably capable of recognizing an antigen presented in the context of the human or humanized MHC in association with the human or humanized T cell co-receptor and capable of providing an activation signal to a non-human cell, e.g., a non-human T cell, that expresses the human or humanized TCR. The human or humanized T cell co-receptor, the human or humanized TCR, and / or the human or humanized MHC may be encoded by the genome of the non-human animal. In a preferred embodiment, upon immunization with an antigen, the non-human animal presents an HLA-restricted epitope of the antigen to a TCR derived from a human TCR gene segment, e.g., a human TCRαV segment, a human TCRαJ segment, a human TCRβV segment, a human TCRβD segment, and / or a human TCRβJ segment.

[0054] Thus, the present invention includes genetically modified non-human animals whose genome comprises (e.g., at an endogenous locus) a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., a CD4 or CD8 polypeptide), wherein the chimeric T cell co-receptor polypeptide comprises conservative amino acid substitutions of the amino acid sequences described herein, and / or a nucleic acid sequence encoding a humanized MHC polypeptide that associates with a humanized T cell co-receptor polypeptide, wherein the humanized MHC polypeptide comprises conservative amino acid substitutions of the amino acid sequences described herein.

[0055] Conservative amino acid substitutions involve the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying the nucleotide sequence to introduce a nucleotide change that encodes the conservative substitution. Generally, conservative amino acid substitutions will not substantially alter the intended functionality of the protein, e.g., the ability of CD4 or CD8 to associate with, e.g., bind to, MHC II or MHC I, respectively, and e.g., enhance the sensitivity of TCRs to MHC-presented antigens. Examples of amino acid groups with side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Conservative amino acid substitutions include, 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 alanine substitutions for any naturally occurring residue in a protein, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), which is incorporated herein by reference. In some embodiments, the substitution is a moderately conservative substitution, which has a non-negative value in the PAM250 log-likelihood matrix.

[0056] Those skilled in the art will understand that, in addition to the nucleic acid residues encoding the humanized T cell co-receptor polypeptides, humanized MHC polypeptides, and / or TCR variable regions described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the polypeptides of the invention. Thus, in addition to genetically modified non-human animals comprising in their genomes nucleotide sequences encoding humanized T cell co-receptor polypeptides (e.g., CD4 or CD8 polypeptides), unrearranged T cell receptor variable loci (e.g., TCRα and / or TCRβ) comprising human unrearranged gene segments, and / or nucleic acid sequences encoding humanized MHC polypeptides capable of associating with humanized T cell co-receptor polypeptides comprising conservative amino acid substitutions, also provided are non-human animals whose genomes comprise nucleotide sequences encoding humanized T cell co-receptor polypeptides (e.g., CD4 or CD8 polypeptides), unrearranged T cell receptor variable loci (e.g., TCRα and / or TCRβ) comprising human unrearranged gene segments, and / or nucleic acid sequences encoding humanized MHC polypeptides capable of associating with humanized T cell co-receptor polypeptides that differ due to the degeneracy of the genetic code.

[0057] Sequence identity may be determined by a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments described herein, identity is determined using ClustalW v.1.83 (slow) alignment with an open gap penalty of 10.0, an extend gap penalty of 0.1, and a Gonnet similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared for sequence identity depends on the particular sequence. In various embodiments, identity is determined by comparing the sequence of the mature protein from N-terminus to C-terminus. In various embodiments, when comparing a chimeric human / non-human sequence to a human sequence, the human portion (but not the non-human portion) of the chimeric human / non-human sequence is used to ascertain the level of identity between the human sequence and the human portion of the chimeric human / non-human sequence (e.g., comparing the human ectodomain of a chimeric human / mouse protein to the human ectodomain of a human protein).

[0058] The term "homology" or "homologous," in reference to a sequence, e.g., a sequence of nucleotides or amino acids, means that, upon optimal alignment and comparison, the two sequences are identical, e.g., in at least about 75% of the nucleotides or amino acids, e.g., at least about 80% of the nucleotides or amino acids, e.g., at least about 90-95% of the nucleotides or amino acids, e.g., greater than 97% of the nucleotides or amino acids. Those skilled in the art will understand that for optimal gene targeting, the targeting construct should contain arms homologous to the endogenous DNA sequence (i.e., "homology arms") so that homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

[0059] The term "operably linked" refers to a juxtaposition in a relationship permitting the components so described to function in their intended manner. Thus, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoters, enhancers, silencer sequences, etc.) so that proper transcriptional regulation is maintained. In addition, various portions of a chimeric or humanized protein of the invention may be operably linked so as to maintain proper folding, processing, targeting, expression, and other functionalities of the protein in the cell. Unless otherwise specified, the various domains of a chimeric or humanized protein of the invention are operably linked to each other.

[0060] The term "replacement," in reference to gene replacement, means the placement of foreign genetic material at the endogenous locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. As demonstrated in the Examples below, in one embodiment, a nucleic acid sequence at the endogenous locus encoding a portion of a mouse CD4 or CD8 (CD8α and / or CD8β) polypeptide was replaced with a nucleotide sequence encoding a portion of a human CD4 or CD8 (CD8α and / or CD8β) polypeptide, respectively.

[0061] For example, as used herein in reference to a functional polypeptide, "functional" means that the polypeptide maintains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, replacement at an endogenous locus (e.g., replacement with an endogenous non-human CD4 or CD8 locus) results in the locus being incapable of expressing a functional endogenous polypeptide. Humanized T cell co-receptors

[0062] Disclosed herein are non-human animals expressing at least one human or humanized T cell receptor, e.g., CD4, CD8α, and / or CD8β. Accordingly, the non-human animals disclosed herein comprise at least one of a first, second, and / or third nucleotide sequence, each encoding a different human or chimeric human / non-human T cell co-receptor polypeptide selected from a human or humanized CD4 polypeptide, a human or humanized CD8α polypeptide, and a human or humanized CD8β polypeptide. The use of the first, second, and third designations herein should not be construed as limiting the non-human animals disclosed herein to the requirement that all three nucleotide sequences be present, or that any of the co-receptor nucleotide sequences be present in any order. Accordingly, the non-human animals disclosed herein may comprise one or more nucleic acid sequences encoding human or humanized CD4 and / or human or humanized CD8 (e.g., human or humanized CD8α and / or CD8β) polypeptides.

[0063] In one embodiment, the non-human animals disclosed herein comprise a first nucleotide sequence encoding a human or humanized CD4 polypeptide. In another embodiment, the non-human animals disclosed herein comprise a first nucleotide sequence encoding a human or humanized CD8α polypeptide and a second nucleotide sequence encoding a human or humanized CD8β polypeptide. In another embodiment, the non-human animals disclosed herein comprise first and second nucleotide sequences encoding human or humanized CD8α and CD8β polypeptides, and further comprise a third nucleotide sequence encoding a human or humanized CD4 polypeptide. Human or humanized CD4

[0064] In various embodiments, the present invention generally provides a genetically modified non-human animal that includes in its genome, e.g., at the endogenous CD4 locus, a nucleotide sequence encoding a human or humanized CD4 polypeptide, such that the animal expresses the human or humanized CD4 polypeptide.

[0065] The human CD4 gene is located on chromosome 12 and is thought to contain 10 exons. The CD4 gene encodes a protein with an amino-terminal hydrophobic signal sequence encoded by exons 2 and 3 of the gene. The protein contains four extracellular immunoglobulin-like domains, Ig1 to Ig4, commonly referred to as the D1 to D4 domains, respectively. Maddon et al. (1987) Structure and expression of the human and mouse T4 genes, Proc. Natl. Acad. Sci. USA 84:9155-59. The D1 domain is thought to be encoded by exon 3 (the sequence downstream of the signal peptide) and exon 4, while D2, D3, and D4 are encoded by separate exons—exons 5, 6, and 7, respectively (see Figure 5A; the D1, D2, D3, and D4 domains are encoded by sequences designated Ig1, Ig2, Ig3, and Ig4, respectively). Littman (1987) The Structure of the CD4 and CD8 Genes,Ann.Rev.Immunol.5:561~84;Hanna et al. (1994)Specific Expression of the Human CD4 Gene in Mature CD4+CD8- and Immature CD4+CD8+ T cells and in Macrophages of Transgenic Mice, Mol. Cell. Biol. 14(2):1084-94; Maddon et al. (see above). In areas of high protein concentration, such as the contact area between T cells and antigen-presenting cells, the molecules tend to homodimerize due to 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 Components Required for T Cell Activation, J. Immunol. 169:6261~68.

[0066] The D1 domain of CD4 is similar to the immunoglobulin variable (V) domain and, together with part of the D2 domain, is thought to bind (associate) with MHC II, for example, at the MHC II coreceptor binding site. Huang et al. (1997) Analysis of the contact sites on the CD4 molecule with Class II MHC molecule, J. Immunol. 158:216-25. MHC II then interacts with the T cell coreceptor CD4 at the hydrophobic crevice 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.

[0067] Domains D3 and D4 of the CD4 coreceptor are thought to interact with the TCR-CD3 complex, as substitutions in these two domains inhibit 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 the remaining portion of the D4 domain of this molecule is thought to be responsible for CD4 dimerization. Moldovan et al. (2002) CD4 Dimers Constitute the Functional Components Required for T Cell Activation, J. Immunol. 169:6261~68.

[0068] Exon 8 of the CD4 gene encodes the transmembrane domain, while the remainder of the gene encodes the cytoplasmic domain. The CD4 cytoplasmic domain has many additional functions. For example, the CD4 cytoplasmic domain recruits the tyrosine kinase Lck. Lck is a Src family kinase that associates with the CD4 and CD8 cytoplasmic domains. Simultaneous binding of a co-receptor and TCR to the same MHC leads to increased tyrosine phosphorylation of the CD3 and ζ chains of the TCR complex. This increase then leads to the recruitment of other factors that play a role in T cell activation. Itano et al. proposed that the CD4 cytoplasmic tail also promotes the differentiation of CD4+CD8+ T cells into the CD4+ lineage by designing a hybrid protein containing the CD8 extracellular domain and the CD4 cytoplasmic tail and testing the expression of the protein in transgenic mice. Itano et al. (1996) The Cytoplasmic Domain of CD4 Promotes the Development of CD4 Lineage T Cells, J. Exp. Med. 183:731-41. Expression of the hybrid protein led to the development of MHC I-specific CD4 lineage T cells (ibid.).

[0069] The CD4 coreceptor is thought to be the primary receptor for the HIV virus, and CD4+ T cell depletion is an indicator of disease progression. The cytoplasmic tail of CD4 is thought to be essential for transmitting the apoptotic signal to CD4+ T cells during HIV-induced apoptosis. Specifically, interaction of CD4 with Lck has been shown to enhance HIV-induced apoptosis in these cells. Corbeil et al. (1996) HIV-induced Apoptosis Requires the CD4 Receptor Cytoplasmic Tail and Is Accelerated by Interaction of CD4 with p56lck, J. Exp. Med. 183:39-48.

[0070] T cells develop in the thymus by progressing from immature CD4- / CD8- (double-negative or DN) thymocytes to CD4+ / CD8+ (double-positive or DP) thymocytes. These thymocytes ultimately undergo positive selection to become either CD4+ or CD8+ (single-positive or SP) T cells. DP thymocytes receive signals from an MHC I-restricted TCR to differentiate into CD8+ T cells. DP thymocytes, on the other hand, receive signals from an MHC II-restricted TCR to differentiate into CD4+ T cells. The cues received by DP cells that lead to their differentiation into either CD4+ or CD8+ T cells have been the subject of numerous studies. Various models for CD4 / CD8 lineage selection have been proposed, including Singer et al. (2008) Lineage fate and The intense debate: myths, models, and mechanisms of CD4-versus CD8-lineage choice, reviewed in Nat. Rev. Immunol. 8:788-801.

[0071] Inactivation of specific T cell coreceptors as a result of positive selection is the product of transcriptional regulation. For CD4, an enhancer located 13 kb upstream of exon 1 of CD4 has been shown to upregulate CD4 expression in CD4+ and CD8+ T cells. Killeen et al. (1993) Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4, EMBO J. 12:1547-53. A cis-acting transcriptional silencer located within the first intron of the mouse CD4 gene functions to silence CD4 expression in cells other than CD4+ T cells. et al. (1994) A transcriptional silencer control the developmental expression of the CD4 gene, EMBO J.13:3570~3579.

[0072] Key transcriptional regulators (e.g., promoters, enhancers, silencers) controlling CD4 lineage selection were impaired in several previously developed lines of transgenic mice expressing human CD4, resulting in the failure of these mice to recapitulate normal T cell lineage development and the generation of immune cells other than CD4+ T cells that express CD4. See, e.g., Law et al. (1994) Human CD4 Restores Normal T Cell Development and Function in Mice Deficient in CD4, J. Exp. Med. 179:1233-42 (CD4 expression in CD8+ T cells and B cells); Fugger et al. (1994) Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region β-chain T-cell repertoire and mediates an HLA-D-restricted immune response, Proc. Natl. Acad. Sci. USA, 91:6151-55 (CD4 expression in all CD3+ thymocytes and B cells). Thus, in one embodiment, there may be an advantage to developing genetically modified animals that retain the endogenous mouse promoter and other regulatory elements to generate T cells in the animals that can undergo T cell development and lineage selection.

[0073] Thus, in various embodiments, the present invention provides a genetically modified non-human animal that comprises, for example, in its endogenous T cell co-receptor locus (e.g., CD4 locus), a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide. In one embodiment, the human portion of the chimeric polypeptide comprises all or substantially all of the extracellular portion (or a portion thereof, e.g., one or more extracellular domains, e.g., at least two contiguous extracellular domains) of a human T cell co-receptor. In one embodiment, the non-human portion of the chimeric polypeptide comprises the transmembrane and cytoplasmic domains of a non-human T cell co-receptor. In one embodiment, the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. Thus, in one aspect, the invention provides a genetically modified non-human animal comprising, at its endogenous CD4 locus, a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide, wherein the human portion of the chimeric polypeptide comprises all or substantially all of the extracellular portion of human CD4 and the non-human portion comprises at least the transmembrane and cytoplasmic domains of the non-human CD4, and wherein the animal expresses a functional chimeric CD4 polypeptide. In one aspect, the non-human animal expresses only the humanized CD4 polypeptide, i.e., the chimeric human / non-human CD4 polypeptide, and does not express a functional endogenous non-human CD4 protein from its endogenous CD4 locus.

[0074] In one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of a human CD4 polypeptide. In another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises at least all or substantially all of the MHC II binding domains of a human CD4 polypeptide (e.g., a substantial portion of the human D1 and D2 domains). In one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the D1, D2, and D3 domains of a human CD4 polypeptide. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the immunoglobulin-like domains of CD4, e.g., the domains designated D1, D2, D3, and D4. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the human CD4 sequences responsible for interaction with MHC II and / or the extracellular portion of the T-cell receptor. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of human CD4 responsible for interacting with MHC II and / or the variable domains of the T-cell receptor. Thus, in one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises the coding sequence for all or substantially all of the D1-D2 domains of human CD4 (e.g., exon 3 and a portion of exons 4-5 of the human CD4 gene). In another embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises the coding sequence for all or substantially all of the D1-D3 domains of human CD4 (e.g., exon 3 and a portion of exons 4-6 of human CD4). Thus, in one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD4 comprises the 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 the coding sequence for D1-D4 of the human CD4 gene. In another embodiment, the nucleotide sequence may include a nucleotide sequence encoding a mouse CD4 signal peptide, for example, a region encoded by part of exons 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:78, with the human portion of the chimeric polypeptide spanning approximately amino acids 27-319 of SEQ ID NO:78 (set forth separately in SEQ ID NO:79).

[0075] In one embodiment, the non-human animal expresses a chimeric human / non-human CD4 polypeptide sequence. In one embodiment, the human portion of the chimeric CD4 sequence contains one or more conservative or non-conservative modifications.

[0076] In one aspect, a non-human animal is provided that expresses a human CD4 sequence, wherein the human CD4 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence. In a specific embodiment, the human CD4 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to a human CD4 sequence described in the Examples. In one embodiment, the human CD4 sequence contains one or more conservative substitutions. In one embodiment, the human CD4 sequence contains one or more non-conservative substitutions.

[0077] In some embodiments, a portion of a chimeric CD4, e.g., a human portion, can include substantially all of the sequences set forth herein (e.g., substantially all of the protein domains set forth herein). Substantially all of the sequences include approximately 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids that are believed to represent a particular portion of the protein (e.g., a particular functional domain, etc.). Those skilled in the art will appreciate that the limits of a functional domain may vary slightly depending on the alignment and domain prediction methods used.

[0078] In one aspect, the non-human portion of the chimeric human / non-human CD4 polypeptide comprises at least the transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Due to the important functions provided by the CD4 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequence in the genetically engineered animal ensures preservation of proper intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD4 polypeptide is a mouse CD4 polypeptide. Specific mouse CD4 sequences are described in the Examples, but any suitable sequence derived therefrom, e.g., sequences containing conservative / non-conservative amino acid substitutions, is encompassed herein. In one embodiment, the non-human portion of the chimeric CD4 co-receptor comprises any sequence of endogenous CD4 that has not been humanized.

[0079] The non-human animals described herein may comprise, at their endogenous loci, nucleotide sequences encoding chimeric human / non-human CD4 polypeptides. In one aspect, this results in the replacement of a portion of an endogenous CD4 gene with a nucleotide sequence encoding a portion of a human CD4 polypeptide. In one embodiment, such a replacement is, for example, the replacement of an endogenous nucleotide sequence encoding all or substantially all of the extracellular domains of non-human CD4, e.g., a sequence encoding at least all or substantially all of the first immunoglobulin-like domain (i.e., D1) of non-human CD4 (e.g., a sequence encoding all or substantially all domains D1-D2 of non-human CD4, e.g., a sequence encoding all or substantially all domains D1-D3 of non-human CD4, e.g., a sequence encoding all or substantially all domains D1-D4 of non-human CD4), with a human nucleotide sequence encoding the same domain. In one embodiment, the replacement results in the replacement of an endogenous nucleotide sequence encoding all or substantially all of the extracellular domains of non-human CD4, e.g., a sequence encoding at least all or substantially all of the first immunoglobulin-like domain (i.e., D1) of non-human CD4 (e.g., a sequence encoding all or substantially all of domains D1-D2 of non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D3 of non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D4 of non-human CD4). In yet another embodiment, the replacement results in a chimeric protein comprising a human CD4 sequence responsible for interaction with MHC II and / or the extracellular portion of the T cell receptor. In yet another embodiment, the replacement results in a chimeric protein comprising a human CD4 sequence responsible for interaction with MHC II and / or the variable domain of the T cell receptor. In one embodiment, the replacement does not involve replacement of CD4 sequences encoding at least the transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Thus, in one aspect, the non-human animal expresses a chimeric human / non-human CD4 polypeptide from an endogenous non-human CD4 locus. In yet another embodiment, the replacement results in a protein comprising the polypeptide sequence set forth in SEQ ID NO:78.

[0080] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD4 locus (e.g., a chimeric human / rodent CD4 locus, e.g., a chimeric human / mouse CD4 locus) described herein is provided. In one aspect, the chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD4 sequence retains the CD4 enhancer element located upstream of the first CD4 exon as it is located in an endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus. In one embodiment, the replacement in the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus includes, for example, replacement of exon 3, which encodes D1 of the CD4 polypeptide, and portions of exons 4-6, which encode the remainder of D1 and D2-D3. Thus, in one aspect, the chimeric CD4 locus retains a cis-acting silencer located in intron 1 of the non-human (e.g., mouse) CD4 gene. Thus, in one embodiment, the chimeric locus retains the endogenous non-human (e.g., rodent, e.g., mouse) CD4 promoter and regulatory elements. In another embodiment, the chimeric locus may contain human promoter and regulatory elements to the extent that they allow for proper CD4 expression, CD4+ T cell development, CD4 lineage selection, and coreceptor function. Thus, in some aspects, animals of the invention comprise genetic modifications that do not alter proper lineage selection and T cell development. In one aspect, animals of the invention (e.g., rodents, e.g., mice) do not express the chimeric CD4 polypeptide on immune cells other than those cells that normally express CD4. In one aspect, the animals do not express CD4 on B cells or mature CD8+ T cells. In one embodiment, the replacement results in retention of elements that allow for proper spatial and temporal regulation of CD4 expression.

[0081] In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) expressing a functional chimeric CD4 protein from a chimeric CD4 locus described herein displays the chimeric protein on the cell surface, e.g., on the T cell surface. In one embodiment, the non-human animal expresses the chimeric CD4 protein on the cell surface in the same cellular distribution as observed in humans. In one aspect, the CD4 protein of the invention is coupled to an MHC expressed on the surface of a second cell, e.g., an antigen-presenting cell (APC). II protein. Human or humanized CD8

[0082] In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome, e.g., at their endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8 polypeptide, such that the animal expresses the human or humanized CD8 polypeptide. In various embodiments, the invention provides non-human animals that comprise in their genome, e.g., at their endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8α polypeptide and / or a nucleotide sequence encoding a human or humanized CD8β polypeptide. Thus, the genetically modified non-human animals of the invention express human or humanized CD8α and / or CD8β polypeptides.

[0083] Human CD8 protein is typically expressed on the cell surface as a heterodimer of two polypeptides, CD8α and CD8β. However, disulfide-linked homodimers and homomultimers have also been detected (e.g., in NK cells and intestinal γδ T cells, which express CD8αα). The genes encoding human CD8α and CD8β are located close to each other on chromosome 2. Nakayama et al. (1992) Recent Duplication of the Two Human 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. The exons / introns of the CD8α gene are shown schematically in Figure 5B.

[0084] The human CD8β gene is located upstream of the CD8α gene on chromosome 2. Multiple isoforms have been reported, generated by alternative splicing of the CD8β gene. One isoform lacks the transmembrane domain and is predicted to produce a secreted protein. Norment et al. (1988) A second subunit of CD8 is expressed in human T cells, EMBO J. 7:343-39. The exons and introns of the CD8β gene are also shown diagrammatically in Figure 5B.

[0085] 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 an extended glycosylation site, which is thought to maintain its conformation and protect the protein from cleavage by proteases. Leahy (1995) A structural view of CD4 and CD8, FASEB J. 9:17-25.

[0086] The CD8 protein is typically expressed on cytotoxic T cells and interacts with MHC I molecules. This interaction is mediated by CD8 binding to the α3 domain of MHC I. However, MHC class I binding to CD8 is approximately 100-fold weaker than TCR binding to MHC class I, and CD8 binding improves the affinity of TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366.

[0087] CD8 binding to MHC class I molecules is species-specific. Lyt-2, the mouse homolog of CD8, binds to H-2D in the α3 domain. dIt has been shown to bind to HLA-specific antibodies, but not 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 was probably due to CDR-like determinants (CDR1-like and CDR2-like) in 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 CD8αα 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 (positions 223-229). A single substitution in HLA-A (V245A) reduced the binding of CD8 to HLA-A, accompanied by a significant reduction in T cell-mediated lysis. Salter et al. (1989), Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8, Nature 338:345-348. In general, polymorphism in the α3 domain of HLA-A molecules also affects binding to CD8 (see above). In mice, H-2D d The amino acid substitution at residue 227 in d Mutant H-2D affects binding to d Cells transfected with human CD8 were not lysed by CD8+ T cells. Potter et al. (1989) Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73-75. Therefore, expression of human or humanized CD8 may be useful for studying T cell responses to antigens presented by human or humanized MHC I.

[0088] Like CD4, the cytoplasmic domain of CD8 interacts with the tyrosine kinase Lck, which subsequently leads to T cell activation. Lck is thought to interact with the cytoplasmic domain of CD8α, and this interaction is thought to be regulated by the presence of the cytoplasmic domain of CD8β. Mutation or deletion of the CD8β cytoplasmic domain results in reduced CD8α-associated Lck activity. Irie et al. (1998) The cytoplasmic domain of CD8β regulates Lck kinase activation and CD8 T cell development, J. Immunol. 161:183-91. Reduced Lck activity has been associated with impaired T cell development (ibid.).

[0089] Expression of CD8 on appropriate cells, e.g., cytotoxic T cells, is tightly regulated by various enhancer elements located throughout the CD8 locus. For example, at least four regions of DNAse I hypersensitivity, often associated with regulator binding, have been identified in the CD8 locus. Hosert et al. (1997) A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice, J. Immunol. 158:4270-81. The discovery of these DNAse I hypersensitive regions in the CD8 locus led to the identification of at least five enhancer elements spread throughout the CD8 locus that regulate expression of CD8α and / or CD8β in various lineages of T cells, including DP, CD8 SP T cells, or cells expressing the γδ TCR. For example, Kioussis et al. (2002) Chromatin and CD4, CD8A, and CD8B gene expression during See thymic differentiation, Nature Rev. 2:909-919 and online Erratum; Ellmeier et al. (1998) Multiple Development Stage-Specific Enhancers Regulate CD8 Expression in Developing Thymocytes and in Thymus-Independent T cells, Immunity 9:485-96.

[0090] Thus, similar to the benefits of maintaining the endogenous CD4 promoter and regulatory elements for a human or humanized CD4 genetically modified animal, in some embodiments, there may be an advantage to developing genetically modified non-human animals that retain the endogenous mouse promoter and regulatory elements that would control expression of human or humanized CD8. As described herein, there may be a particular advantage to generating genetically modified animals that include replacement of endogenous non-human sequences encoding CD8 α and / or β proteins with sequences encoding human or humanized CD8 α and / or β proteins.

[0091] In various embodiments, the invention provides genetically modified non-human animals that comprise in their genome, e.g., in their endogenous CD8 locus, at least one nucleotide sequence encoding a chimeric human / non-human CD8 polypeptide (e.g., a CD8α and / or β polypeptide), wherein the human portion of the polypeptide comprises all or substantially all of the extracellular portion (or a portion thereof, e.g., the extracellular domain) of the human CD8 polypeptide (e.g., CD8α and / or β), and the non-human portion comprises at least the transmembrane and cytoplasmic domains of the non-human CD8 (e.g., CD8α and / or β), and wherein the animal expresses the chimeric CD8 polypeptide (e.g., a CD8α and / or β polypeptide). Thus, in one embodiment, the invention provides a genetically modified non-human animal comprising, at its endogenous non-human CD8 locus, a first nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide, wherein the first nucleotide sequence comprises a sequence encoding all or substantially all of the extracellular portion of the human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of the non-human CD8α polypeptide, and the second nucleotide sequence comprises a sequence encoding all or substantially all of the extracellular portion of the human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of the non-human CDβ polypeptide, and 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 CD8α and / or β polypeptides) and does not express a corresponding functional non-human CD8 polypeptide from the endogenous CD8 locus.

[0092] In one embodiment, the chimeric human / non-human CD8α polypeptide comprises, in its human portion, all or substantially all of the extracellular portion of a human CD8α polypeptide. In one embodiment, the human portion of 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 sequence of all or substantially all of the immunoglobulin V-like domain of a human CD8α. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8α polypeptide comprises at least an exon encoding the extracellular portion of the human CD8α polypeptide. In one embodiment, the nucleotide sequence comprises at least an exon encoding an Ig V-like domain. In one embodiment, the extracellular portion of the human CD8α polypeptide is a region encompassing a portion of the polypeptide that is not the transmembrane domain or the 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, e.g., mouse) CD8α signal peptide. Alternatively, the nucleotide sequence may comprise a sequence encoding a human CD8β signal sequence. In one embodiment, the chimeric human / non-human CD8α polypeptide comprises the amino acid sequence set forth in SEQ ID NO:88, with the human portion of the chimeric polypeptide set forth in amino acids 28-179 of SEQ ID NO:88 (represented separately in SEQ ID NO:89).

[0093] Similarly, in one embodiment, a chimeric human / non-human CD8 β polypeptide comprises, in its human portion, all or substantially all of the extracellular portion of a human CD8 β polypeptide. In one embodiment, the human portion of a chimeric CD8 β polypeptide comprises the sequence of all or substantially all of the immunoglobulin V-like domain of human CD8 β. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric CD8 β polypeptide comprises at least an exon encoding the extracellular portion of a human CD8 β polypeptide. In one embodiment, the nucleotide sequence encoding the human portion of a chimeric human / non-human CD8 β polypeptide comprises at least an exon encoding an IgG V-like domain of human CD8 β. In one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD8 β polypeptide comprises a sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8 β signal peptide. Alternatively, the nucleotide sequence may comprise a sequence encoding a human CD8 β signal sequence. In one embodiment, the chimeric human / non-human CD8β polypeptide comprises the amino acid sequence set forth in SEQ ID NO:83, with the human portion of the chimeric polypeptide set forth in amino acids 15-165 of SEQ ID NO:83 (alternatively represented in SEQ ID NO:84).

[0094] In one embodiment, the non-human animal expresses a chimeric human / non-human CD8α and / or CD8β polypeptide. In one embodiment, the human portion of the chimeric human / non-human CD8α and / or β polypeptide comprises one or more conservative or non-conservative modifications.

[0095] In one aspect, a non-human animal is provided that expresses a human CD8α and / or β polypeptide sequence, wherein the human CD8α and / or β polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the respective human CD8α and / or β polypeptide sequences. 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 each of the human CD8α and / or β polypeptide sequences described in the Examples. 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.

[0096] In some embodiments, a portion of a chimeric CD8, e.g., a human portion, can include substantially all of the sequences set forth herein (e.g., substantially all of the protein domains set forth herein). Substantially all of the sequences generally include 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.). Those skilled in the art will appreciate that the limits of a functional domain may vary slightly depending on the alignment and domain prediction methods used.

[0097] In one aspect, the non-human portion of the chimeric human / non-human CD8α and / or β polypeptide comprises at least the transmembrane and / or cytoplasmic domains of the non-human CD8α and / or β polypeptide, respectively. Due to the important functions provided by the CD8 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequences in the genetically engineered animal ensures preservation of proper intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD8α and / or β polypeptides are mouse CD8α and / or β polypeptides, respectively. Specific mouse CD8α and / or β sequences are described in the Examples, but any suitable sequences derived therefrom, e.g., sequences containing conservative / non-conservative amino acid substitutions, are encompassed herein. In one embodiment, the non-human animal (e.g., a rodent, e.g., a mouse) retains any endogenous sequences that have not been humanized.

[0098] The non-human animals described herein may comprise, at their endogenous loci, nucleotide sequences encoding chimeric human / non-human CD8α and / or β polypeptides. In one aspect, this results in the replacement of a portion of the endogenous CD8α gene with a nucleotide sequence encoding a portion of a human CD8α polypeptide and / or the replacement of a portion of the endogenous CD8β gene with a nucleotide sequence encoding a portion of a human CD8β polypeptide. In one embodiment, such a replacement is the replacement of an endogenous nucleotide sequence encoding all or substantially all of the extracellular portion of a non-human CD8α and / or β with a human nucleotide sequence encoding the same. In one embodiment, such a replacement is the replacement of an endogenous nucleotide sequence encoding at least all or substantially all of the immunoglobulin V-like domains of a non-human CD8α and / or β with a human nucleotide sequence encoding the same. In one embodiment, the replacement does not include the replacement of CD8α and / or β sequences encoding the transmembrane and cytoplasmic domains of the non-human CD8α and / or β polypeptide. Thus, the non-human animal expresses chimeric human / non-human CD8 α and / or β polypeptides from the endogenous non-human CD8 locus. In yet another embodiment, the replacement results in a CD8 α and / or β protein comprising the polypeptide sequence set forth in SEQ ID NO: 88 and / or 84, respectively.

[0099] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD8 locus (e.g., a chimeric rodent CD8 locus, e.g., a chimeric mouse CD8 locus) is provided. In one aspect, the chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD8α and / or β sequences are located at the respective endogenous non-human (e.g., rodent, e.g., mouse) CD8α and / or β loci and thus retain the endogenous CD8α and / or β promoter and regulatory elements. In another embodiment, the chimeric locus may contain human CD8α and / or β promoter and regulatory elements to the extent that they allow proper CD8α and / or β expression (proper spatial and temporal protein expression), CD8+ T cell development, CD8 lineage selection, and coreceptor function. Thus, in one aspect, the animals of the invention comprise genetic modifications that do not alter proper lineage selection and T cell development. In one aspect, an animal (e.g., a rodent, e.g., a mouse) of the invention does not express the chimeric CD8 protein on immune cells other than those cells that normally express CD8. For example, the animal does not express CD8 on B cells or mature CD4+ T cells. In one embodiment, the replacement results in retention of elements that allow for proper spatial and temporal regulation of CD8α and / or β expression.

[0100] In various embodiments, a non-human animal (e.g., a rodent, e.g., 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 its cell surface. In one embodiment, the non-human animal expresses the chimeric CD8 protein on its cell surface in the same cellular distribution as observed in humans. In one aspect, the CD8 protein of the invention can interact with an MHC I protein expressed on the surface of a second cell. Human or humanized T cell receptors

[0101] Disclosed herein are genetically modified non-human animals comprising a substantially humanized T cell immune system. In some embodiments, the non-human animals disclosed herein comprise, for example, an animal having in its genome: (a) a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor, wherein the human portion of the chimeric T cell co-receptor polypeptide is encoded by a sequence encoding an extracellular domain of a human T cell co-receptor, the sequence encoding the extracellular domain of the human T cell co-receptor being operably linked to nucleotides comprising a sequence encoding a transmembrane domain and / or a cytoplasmic domain of a non-human T cell co-receptor; and (b) at least one human V segment, optionally at least one and at least one human J segment, wherein the unrearranged V, and optionally the D, and J segments of the TCR variable region gene have been recombined to form a rearranged gene that is operably linked to a non-human TCR constant gene sequence, and (c) a nucleic acid sequence encoding a chimeric human / non-human MHC polypeptide, wherein the human portion of the chimeric MHC polypeptide comprises an extracellular domain of a human MHC polypeptide that associates with the human portion of a chimeric T-cell co-receptor polypeptide. Optionally, the non-human animal also comprises a human or humanized β2-microglobulin polypeptide.

[0102] Thus, in various embodiments, the invention generally provides genetically modified non-human animals, wherein the non-human animal comprises in its genome an unrearranged humanized TCR variable locus, e.g., an unrearranged human TCR variable gene region comprising human TCR variable segments that can recombine to form a rearranged TCR variable gene sequence. As used herein, a TCR locus or TCR locus (e.g., a TCR alpha locus or a TCR beta locus) refers to genomic DNA that comprises a TCR coding region (including the entire TCR coding region, including unrearranged V(D)J sequences, enhancer sequences, constant sequences, and any upstream or downstream (e.g., UTRs, regulatory regions), or inverted DNA sequences (e.g., introns)). A TCR variable locus, TCR variable region, or TCR variable locus (e.g., a TCR alpha variable locus or a TCR beta variable locus) refers to genomic DNA that comprises a TCR variable region segment (V(D)J region), but excluding the TCR constant sequences and, in various embodiments, enhancer sequences. Other sequences may be included in the TCR variable loci for purposes of genetic manipulation (eg, selection cassettes, restriction sites, etc.) and these are encompassed herein.

[0103] T cells bind to epitopes in small antigenic determinants on the surface of antigen-presenting cells in association with major histocompatibility complex (MHC; mouse) or human leukocyte antigen (HLA; human) complexes. T cells bind to these epitopes via the T cell receptor (TCR) complex on the T cell surface. T cell receptors are heterodimeric structures composed of two types of chains: α (alpha) and β (beta) chains or γ (gamma) and δ (delta) chains. The α chain is encoded by a nucleic acid sequence located within the α locus (on chromosome 14 in humans or mice). The same locus also encompasses the entire δ locus. The β chain is encoded by a nucleic acid sequence located within the β locus (on chromosome 6 in mice or chromosome 7 in humans). The majority of T cells have αβ TCRs, while a minority have γδ TCRs. The interaction of TCRs with MHC class I (presented to CD8+ T cells) and MHC class II (presented to CD4+ T cells) molecules is shown in FIG. 1 (solid symbols represent non-human sequences, striped symbols represent human sequences; one particular embodiment of a TCR protein of the invention is shown).

[0104] T cell receptor α and β polypeptides (and similarly, γ and δ polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides constituting a TCR contains an extracellular domain including constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable region of a TCR determines its antigen specificity and, like immunoglobulins, contains three complementarity-determining regions (CDRs). Also, like immunoglobulin genes, T cell receptor variable loci (e.g., TCRα and TCRβ loci) contain numerous unrearranged V(D)J segments (variable (V), joining (J), and, in TCRβ and δ, diversity (D) segments). During T cell development in the thymus, the TCRα variable locus undergoes rearrangement. The resulting TCRα chain is encoded by a specific combination of VJ segments (Vα / Jα sequences). The TCRβ variable locus then undergoes rearrangement. The resulting TCR β chain is encoded by a specific combination of VDJ segments (Vβ / Dβ / Jβ sequences).

[0105] Interaction with the thymic stroma triggers thymocytes to undergo multiple developmental stages, characterized by the expression of various cell surface markers. A summary of the characteristic cell surface markers at various developmental stages in the thymus is presented in Table 1. Rearrangement at the TCRβ variable locus begins at the DN2 stage and is completed during the DN4 stage. Meanwhile, rearrangement at the TCRα variable locus occurs at the DP stage. After completion of TCRβ locus rearrangement, cells express the TCRβ chain along with a surrogate α chain, pTα, on the cell surface. See Janeway's Immunobiology, Chapter 7, 7th Ed., Murphy et al. eds., Garland Science, 2008. [Table 1]

[0106] Naive CD4+ and CD8+ T cells exit the thymus and enter peripheral lymphoid organs (e.g., the spleen), where they are exposed to antigen, become activated, clonally expand, and produce large numbers of effector T cells (Teff), e.g., cytotoxic T cells, T REG cell, T H 17 cells, T H 1 cell, T H After infection, many T cells persist as memory T cells and are classified as either central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708-12, and commentary by Mackay (1999) Dual personality of memory T cell, Nature 401:659-60. Sallusto and colleagues proposed that after initial infection, Tem cells provide a readily available pool of antigen-primed memory T cells in peripheral tissues with effector function, while Tcm cells provide antigen-primed memory T cells in peripheral lymphoid organs that can become new effector T cells upon secondary challenge. All memory T cells express the CD45RO isoform of CD45 (naive T cells express the CD45RA isoform), while Tcm are characterized by expression of L-selectin (also known as CD62L) and CCR7+, which are important for binding to and signaling within peripheral lymphoid organs and lymph nodes. Ibid. Thus, all T cells found in peripheral lymphoid organs (e.g., naive T cells, Tcm cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express a number of different cell surface markers, e.g., CD44. For a review of the various cell surface markers on T cells, see Janeway's Immunobiology, Chapter 10, supra.

[0107] Although the TCR variable domains primarily function in antigen recognition, the extracellular portions of the constant domains and the transmembrane and cytoplasmic domains of the TCR also play important roles. A complete TCR receptor complex requires more than just the α and β or γ and δ polypeptides. The additional molecules required include CD3γ, CD3δ, and CD3ε, as well as the ζ chain homodimer (ζζ). Upon completion of TCRβ rearrangement, if the cell expresses TCRβ / pTα, this pre-TCR complex is present on the cell surface along with CD3. TCRα (or pTα) on the cell surface has two basic residues in its transmembrane domain. One of these residues recruits the CD3γε heterodimer, and the other, through its respective acidic residue, recruits ζζ. TCRβ has an additional basic residue in its transmembrane domain that is thought to recruit the CD3δε heterodimer. See, for example, Kuhns et al. (2006) Deconstructing the Form and Function of the TCR / CD3 Complex, Immunity 24:133-39; Wucherpfennig et al. (2009) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harbor. Perspective. Biol. 2:a005140. An assembly complex containing the TCRαβ heterodimer, CD3γε, CD3δε, and ζζ is expressed on the surface of T cells. Polar residues in the transmembrane domain have been suggested to function as quality control for the endoplasmic reticulum (ER). In the absence of the CD3 subunit, TCR chains have been shown to be retained in the ER and targeted for degradation. See, e.g., Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Function, Annu. Rev. Immunol. 23:101-25.

[0108] Because the TCRαβ heterodimer (or TCRγδ heterodimer) itself lacks signaling activity, the CD3 and ζ chains of the assembled complex provide the components for TCR signaling. Each CD3 chain has one immunoreceptor tyrosine-based activation motif (ITAM), while the ζ chain contains three tandem ITAMs. ITAMs contain tyrosine residues that can be phosphorylated by associated kinases. Thus, the assembled TCR-CD3 complex contains 10 ITAM motifs. See, for example, Love and Hayes (2010) ITAM-Mediated Signaling See, by the T-Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. 2:e002485. After TCR binding, ITAM motifs are phosphorylated by the Src family tyrosine kinases Lck and Fyn. This phosphorylation initiates a signaling cascade that leads to Ras activation, calcium mobilization, actin cytoskeleton rearrangement, and transcription factor activation, all of which ultimately result in T cell differentiation, proliferation, and effector functions. Id. See also Janeway's Immunobiology, supra, both of which are incorporated herein by reference.

[0109] In addition, the TCRβ transmembrane and cytoplasmic domains are thought to play a role in mitochondrial targeting and apoptosis induction. In fact, natural N-terminal truncated TCRβ molecules are present in thymocytes. Shani et al. (2009) Incomplete T-cell receptor-β peptides target the mitochondrion and induce apoptosis, Blood 113:3530-41. Thus, several important functions are provided by the TCR constant region (which in various embodiments includes the extracellular domain and portions of the transmembrane and cytoplasmic domains), and in various embodiments, the structure of this region should be considered when designing a humanized TCR or a genetically modified non-human animal that expresses it.

[0110] Mouse transgenics for rearranged T cell receptor sequences are known in the art. The present invention relates to genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that contain unrearranged human or humanized T cell variable loci that can be rearranged to form nucleic acid sequences encoding human T cell receptor variable domains, including animals that contain T cells that contain rearranged human variable domains and non-human (e.g., mouse or rat) constant regions. The present invention also provides non-human animals (e.g., rodents, e.g., rats, mice) that are capable of generating a diverse repertoire of human T cell receptor variable region sequences. Thus, the present invention provides non-human animals that, in response to an antigen of interest, express TCRs with fully human variable domains that bind to epitopes of the antigen of interest. In some embodiments, non-human animals are provided that generate a diverse T cell receptor repertoire that can react with a variety of antigens (including, but not limited to, antigens presented by APCs).

[0111] In one embodiment, the invention provides a genetically modified non-human animal (e.g., a rodent, e.g., a rat, a mouse) that comprises in its genome unrearranged human TCR variable region segments (V(D)J segments), where the unrearranged human TCR variable region segments replace endogenous non-human TCR variable region segments at endogenous non-human (e.g., rodent) TCR variable loci (e.g., TCR α, β, δ, and / or γ variable loci). In one embodiment, the unrearranged human TCR variable loci replace the endogenous non-human TCR variable loci.

[0112] In another embodiment, the present invention provides a genetically modified non-human animal (e.g., a rodent, e.g., a rat, a mouse) comprising in its genome an unrearranged human TCR variable region segment (V(D)J segment), which is operably linked to a non-human TCR constant region gene sequence to provide a humanized TCR locus, which is present at a genomic site other than the endogenous non-human TCR locus. Thus, in one embodiment, a non-human animal (e.g., a rodent, e.g., a mouse, a rat) is also provided that comprises a transgene comprising a rearranged human TCR variable region segment operably linked to a non-human TCR constant region gene sequence.

[0113] In one aspect, the genetically modified non-human animal of the present invention comprises a human TCR variable region segment in its genome while retaining a non-human (e.g., rodent, e.g., mouse, rat) TCR constant gene sequence encoding a TCR constant region. In various embodiments, the TCR constant domain comprises the transmembrane domain and cytoplasmic tail of the TCR. Thus, in various embodiments of the present invention, the genetically modified non-human animal retains the endogenous non-human TCR transmembrane domain and cytoplasmic tail. In other embodiments, the non-human animal comprises a non-human, non-endogenous TCR constant gene sequence, e.g., encoding the transmembrane domain and cytoplasmic tail of the non-human, non-endogenous TCR. As noted above, the constant domain of the TCR is involved in the signaling cascade initiated during antigen-stimulated T cell activation. Thus, the endogenous TCR constant domain interacts with various non-human anchor and signaling proteins in T cells. Thus, in one embodiment, the genetically modified non-human animals of the present invention express humanized T cell receptors that retain the ability to recruit various endogenous non-human anchor or signaling molecules, such as CD3 molecules (e.g., CD3γ, CD3δ, CD3ε), zeta chain, Lck, Fyn, ZAP-70, etc. A non-limiting list of molecules recruited to the TCR complex is described in Janeway's Immunobiology, supra. It is believed that the ability of T cell development and differentiation processes in non-human animals to mount and enable a robust immune response may be due, at least in part, to the placement of variable regions at the endogenous mouse locus and the maintenance of mouse constant domains.

[0114] In some embodiments, a non-human animal is provided, the non-human animal comprising in its genome unrearranged human TCR α variable region segments operably linked to non-human TCR α constant region gene segments, resulting in a humanized TCR α locus. In one embodiment, the humanized TCR α locus is present at a genomic site other than the endogenous non-human TCR α locus. In another embodiment, the unrearranged human TCR α variable region segments replace the endogenous non-human TCR α variable region segments while retaining the endogenous non-human TCR α constant region gene sequence. In one embodiment, the unrearranged human TCR α variable locus replaces the endogenous non-human TCR α variable locus. In some embodiments, the replacement of the endogenous non-human TCR α variable region locus with the unrearranged human TCR α variable locus comprises deletion or inactivation of the TCR δ variable locus. In other embodiments, the replacement of the endogenous non-human TCR alpha variable region gene with an unrearranged human TCR alpha locus comprises replacing the endogenous TCR delta variable locus with an unrearranged human TCR delta variable region segment. In some embodiments, the animal retains the endogenous non-human TCR beta variable and constant region gene sequences. Thus, the animal expresses a TCR comprising a chimeric human / non-human (i.e., humanized) TCR alpha chain and a non-human TCR beta chain.

[0115] In some embodiments, a non-human animal is provided, the non-human animal comprising in its genome an unrearranged human TCRδ variable region segment operably linked to a non-human TCRδ constant region gene sequence to provide a humanized TCRδ locus. In one embodiment, the humanized TCRδ locus is present at a genomic site other than the endogenous non-human TCRδ locus. In another embodiment, the unrearranged human TCRδ variable region segment replaces the endogenous non-human TCRδ variable region segment while retaining the endogenous non-human TCRδ constant region gene sequence. In one embodiment, the unrearranged human TCRδ variable locus replaces the endogenous non-human TCRδ variable locus.

[0116] In other embodiments, a non-human animal is provided, the non-human animal comprising in its genome an unrearranged human TCR β variable region segment operably linked to a non-human TCR β constant region gene sequence, resulting in a humanized TCR β locus. In one embodiment, the humanized TCR β locus is present at a genomic site other than the endogenous non-human TCR β locus. In another embodiment, the unrearranged human TCR β variable region segment replaces the endogenous non-human TCR β variable region segment while retaining the endogenous non-human TCR β constant region gene sequence. In one embodiment, the unrearranged human TCR β variable locus replaces the endogenous non-human TCR β variable locus. In some embodiments, the animal retains the endogenous non-human TCR α variable and constant region gene sequences. Thus, the animal expresses a TCR comprising a chimeric human / non-human (i.e., humanized) TCR β chain and a non-human TCR α chain.

[0117] In some specific embodiments, the present invention provides a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) having in its genome (a) 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 an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR α constant gene sequence; (b) at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment; Provided are genetically modified non-human animals comprising: (a) an unrearranged TCR β variable locus comprising a β segment and operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR β constant region gene sequence; and / or (c) an unrearranged TCR δ variable locus comprising at least one human V δ segment, at least one human D δ segment, and at least one human J δ segment and operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR δ constant region gene sequence. Another non-human animal provided herein has in its genome (a) an unrearranged T cell receptor (TCR) α variable locus that comprises at least one human Vα segment and at least one human Jα segment and is operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR α constant region gene sequence; (b) an unrearranged T cell receptor (TCR) α variable locus that comprises at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment and is operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR β constant region gene sequence; (c) an unrearranged TCR β variable locus comprising at least one human V δ segment, at least one human D δ segment, and at least one human J δ segment, and operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR δ constant region gene sequence; and / or (d) an unrearranged TCR γ variable locus comprising at least one human V γ segment and at least one human J γ segment, and operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR γ constant region gene sequence.

[0118] In various embodiments of the invention, unrearranged human or humanized TCR variable loci (e.g., TCR alpha, TCR beta, and / or TCR delta variable loci) are comprised in the germline of a non-human animal (e.g., a rodent, e.g., a mouse or rat). In various embodiments, the replacement of TCR V(D)J segments with unrearranged human TCR V(D)J segments (e.g., Valpha and Jalpha; Vbeta and Dbeta and Jbeta; Vdelta and Ddelta and Jdelta; Vgamma and Jgamma segments) is at an endogenous non-human TCR variable locus (or multiple loci). In this case, the unrearranged human V and J and / or V and D and J segments are operably linked to non-human TCR constant region gene sequences.

[0119] In some embodiments of the invention, the non-human animal comprises two copies of unrearranged human or humanized TCR alpha variable loci, two copies of unrearranged human or humanized TCR beta variable loci, and / or two copies of unrearranged human or humanized TCR delta variable loci. Thus, the non-human animal is homozygous for one or more unrearranged human or humanized TCR alpha, beta, and / or TCR delta variable loci. In some embodiments of the invention, the non-human animal comprises one copy of unrearranged human or humanized TCR alpha variable loci, one copy of unrearranged human or humanized TCR beta variable loci, and / or one copy of unrearranged human or humanized TCR delta variable loci. Thus, the non-human animal is heterozygous for the unrearranged human or humanized TCR alpha, beta, and / or TCR delta variable loci. In other embodiments, the non-human animal is heterozygous or homozygous for unrearranged human or humanized TCR gamma variable loci.

[0120] In one embodiment, an unrearranged TCR α variable locus comprising human variable region segments (e.g., human Vα and Jα segments) is positioned in a non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCR α variable locus comprising human variable region segments replaces an endogenous TCR α variable locus. In one aspect, the endogenous non-human Vα and Jα segments cannot rearrange to form a rearranged Vα / Jα sequence. Thus, in one aspect, the human Vα and Jα segments at the unrearranged TCR α variable locus can rearrange to form a rearranged human Vα / Jα sequence.

[0121] Similarly, in one embodiment, an unrearranged TCR β variable locus comprising human variable region segments (e.g., human Vβ, Dβ, and Jβ segments) is positioned in a non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, the unrearranged TCR β variable locus comprising human variable region segments replaces an endogenous TCR β variable locus. In one aspect, the endogenous non-human Vβ, Dβ, and Jβ segments cannot rearrange to form rearranged Vβ / Dβ / Jβ sequences. Thus, in one aspect, the human Vβ, Dβ, and Jβ segments in the unrearranged TCR β variable locus can rearrange to form rearranged human Vβ / Dβ / Jβ sequences.

[0122] In one embodiment, an unrearranged TCRδ variable locus comprising human variable region segments (e.g., human Vδ, Dδ, and Jδ segments) is located in a non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, the unrearranged TCRδ variable locus comprising human variable region segments replaces an endogenous TCRδ variable locus. In one aspect, the endogenous non-human Vδ, Dδ, and Jδ segments cannot rearrange to form a rearranged Vδ / Dδ / Jδ sequence. Thus, in one aspect, the human Vδ, Dδ, and Jδ segments at the unrearranged TCRδ variable locus can rearrange to form a rearranged human Vδ / Dδ / Jδ sequence.

[0123] In one embodiment, an unrearranged TCR gamma variable locus comprising human variable region segments (e.g., human Vγ and Jγ segments) is positioned in a non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCR gamma variable locus comprising human variable region segments replaces an endogenous TCR gamma variable locus. In one aspect, the endogenous non-human Vα and Jα segments cannot rearrange to form rearranged Vγ / Jγ sequences. Thus, in one aspect, the human Vγ and Jγ segments in the unrearranged TCR gamma variable locus can rearrange to form rearranged human Vγ / Jγ sequences.

[0124] In yet another embodiment, both unrearranged TCR α, β, δ, and / or γ variable loci comprising human variable region segments replace the respective endogenous TCR α, β, δ, and γ variable loci. In one aspect, endogenous non-human Vα and Jα segments cannot rearrange to form rearranged Vα / Jα sequences, endogenous non-human Vβ, Dβ, and Jβ segments cannot rearrange to form rearranged Vβ / Dβ / Jβ sequences, endogenous non-human Vδ, Dδ, and Jδ segments cannot rearrange to form rearranged Vδ / Dδ / Jδ sequences, and / or endogenous non-human Vγ and Jγ segments cannot rearrange to form rearranged Vγ / Jγ sequences. Thus, in one aspect, human Vα and Jα segments at a non-rearranged TCR alpha variable locus can rearrange to form a rearranged human Vα / Jα sequence; human Vβ, Dβ, and Jβ segments at a non-rearranged TCR beta variable locus can rearrange to form a rearranged human Vβ / Dβ / Jβ sequence; human Vδ, Dδ, and Jδ segments at a non-rearranged TCR delta variable locus can rearrange to form a rearranged human Vδ / Dδ / Jδ sequence; and / or human Vγ and Jγ segments at a non-rearranged TCR alpha variable locus can rearrange to form a rearranged human Vγ / Jγ sequence.

[0125] In some aspects of the invention, the non-human animal comprising a humanized TCR alpha, TCR beta, and / or TCR delta locus (including a non-rearranged human TCR alpha, TCR beta, and / or TCR delta variable locus) retains an endogenous non-human TCR alpha, TCR beta, and / or TCR delta variable locus. In one embodiment, the endogenous non-human TCR alpha, TCR beta, and / or TCR delta variable locus is a non-functional locus. In one embodiment, the non-functional locus is an inactivated locus, e.g., an inverted locus (e.g., the coding nucleic acid sequence of the variable locus is in an inverted orientation relative to the constant region sequence, such that variable region segments from the inverted locus cannot be utilized for successful rearrangement). In one embodiment, the humanized TCR alpha, TCR beta, and / or TCR delta variable loci are located between the endogenous non-human TCR alpha, TCR beta, and / or TCR delta variable loci and the endogenous non-human TCR alpha, TCR beta, and / or TCR delta constant loci, respectively. Similar chromosomal rearrangements may be performed to place a human or non-human TCR gamma into the genome of a non-human animal, for example, at the TCR gamma locus.

[0126] The number, nomenclature, location, and other aspects of V and J and / or V, D, and J segments of human and mouse TCR loci may be ascertained using the IMGT database available at the International Immunogenetic Information System (IMGT) website. The mouse TCR α variable locus is approximately 1.5 megabase pairs and contains a total of 110 Vα and 60 Jα segments. The human TCR α variable locus is approximately 1 megabase pair and contains a total of 54 Vα and 61 Jα segments, with 45 Vα and 50 Jα segments considered functional. Unless otherwise specified, the number of human V(D)J segments referred to throughout 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, e.g., a mouse or rat) comprises at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal comprises a humanized TCR α locus that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Vα segments. In some embodiments, the humanized TCR α locus comprises 2, 8, 23, 35, 48, or 54 human Vα segments. Thus, in some embodiments, the humanized TCR alpha locus in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of human Va, and in some embodiments, about 2%, about 3%, about 15%, about 65%, about 90%, or 100% of human Va.

[0127] In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human Vα40 to Vα41 (Vα segments are also referred to as "TRAV" or "TCRAV") and a DNA fragment comprising the contiguous human sequence of 61 human Jα segments (Jα segments are also referred to as "TRAJ" or "TCRAJ"). In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV35 to TRAV41 and a DNA fragment comprising the contiguous human sequence of 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV22 to TRAV41 and a DNA fragment comprising the contiguous human sequence of 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV13-2 to TRAV41 and a DNA fragment comprising the contiguous human sequence of 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV6 to TRAV41 and 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR α locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV1-1 to TRAV41 and 61 human TRAJ. In various embodiments, the DNA fragment comprising the contiguous human sequence of a human TCR α variable region segment also comprises restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the humanization process of the locus. In various embodiments, these additional sites do not interfere with the proper function (e.g., rearrangement, splicing, etc.) of the various genes in the TCR α locus.

[0128] In one embodiment, the humanized TCR α locus comprises 61 human Jα segments or 100% of the human Jα segments. In a specific embodiment, the humanized TCR α locus comprises 8 human Vα segments and 61 Jα segments. In another specific embodiment, the humanized TCR α locus comprises 23 human Vα segments and 61 Jα segments. In another specific embodiment, the humanized TCR α locus comprises the complete repertoire of human Vα and Jα segments, i.e., all human variable α region gene segments encoded by the α locus, or 54 human Vα and 61 Jα segments. In various embodiments, the non-human animal does not comprise any endogenous non-human Vα or Jα segments in the TCR α locus.

[0129] The mouse TCR β variable locus is approximately 0.6 megabase pairs and contains a total of 33 Vβ, 2 Dβ, and 14 Jβ segments. The human TCR β variable locus is approximately 0.6 megabase pairs and contains a total of 67 Vβ, 2 Dβ, and 14 Jβ segments. In one embodiment of the invention, a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) contains at least one human Vβ, at least one human Dβ, and at least one human Jα segment. In one embodiment, the non-human animal contains a humanized TCR β locus containing 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 Vβ segments. In some embodiments, the humanized TCR β locus contains 8, 14, 40, 66, or 67 human Vβ segments. Thus, in some embodiments, a humanized TCR beta locus in a non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of human Vβs, and in some embodiments, about 20%, about 60%, about 15%, about 98%, or 100% of human Vβs.

[0130] In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising a contiguous human sequence of human Vβ18 to Vβ29-1 (Vβ segments are also referred to as "TRBV" or "TCRBV"). In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising a contiguous human sequence of human TRBV18 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 segment), and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2 (i.e., the human Dβ2-Jβ2-1-Jβ2-7 segment). In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising a contiguous human sequence of human TRBV6-5 through TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 segment), and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2 (i.e., the human Dβ2-Jβ2-1-Jβ2-7 segment). In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising a contiguous human sequence of human TRBV1 through TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1, and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2. In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising a contiguous human sequence of human TRBV1 through TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1, a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2, and a separate DNA fragment comprising a sequence of human TRBV30. In various embodiments, the DNA fragment comprising the contiguous human sequence of a human TCR β variable region segment also comprises restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the humanization process of the locus. In various embodiments, these additional sites do not interfere with the proper function (e.g., rearrangement, splicing, etc.) of the various genes in the TCR β locus.

[0131] In one embodiment, the humanized TCR β locus comprises 14 human Jβ segments or 100% of the human Jβ segments and two human Dβ segments or 100% of the human Jβ segments. In another embodiment, the humanized TCR β locus comprises at least one human Vβ segment, e.g., 14 human Vβ segments, and all mouse Dβ and Jβ segments. In a specific embodiment, the humanized TCR β locus comprises 14 human Vβ segments, two human Dβ segments, and 14 Jβ segments. In another specific embodiment, the humanized TCR β locus comprises the complete repertoire of human Vβ, Dβ, and Jβ segments, i.e., all human variable β region gene segments encoded by the β locus, or 67 human Vβ, two human Dβ, and 14 Jβ segments. In one embodiment, the non-human animal comprises one (e.g., the 5') non-human Vβ segment in the humanized TCR β locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ, Dβ, and Jβ segments in the TCRβ locus.

[0132] In various embodiments in which a non-human animal (e.g., a rodent) comprises a repertoire of human TCR alpha and TCR beta (and optionally human TCR delta and TCR gamma) variable region segments (e.g., a complete repertoire of variable region segments), the repertoire of different segments (e.g., a complete repertoire of different segments) is utilized by the animal to generate a diverse repertoire of TCR molecules against different antigens.

[0133] In various aspects, the non-human animal comprises a contiguous portion of a human genomic TCR variable locus, including the V, D, and J or D and J or V and J or V segments, arranged as in an unrearranged human genomic variable locus, including, e.g., promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., and arranged as in a human genomic TCR variable locus. In other aspects, the various segments are arranged as in an unrearranged non-human genomic TCR variable locus. In various embodiments of the humanized TCR α, β, δ, and / or γ loci, the humanized locus can comprise two or more human genomic segments not represented in the human genome, e.g., juxtaposed with a fragment of a V segment of the human variable locus located in the human genome adjacent to the constant region and juxtaposed with a fragment of a V segment of the human variable locus located in the human genome at the upstream end of the human variable locus.

[0134] In both mice and humans, TCRδ gene segments are located with the TCRα locus (see Figure 4A, top; boxed TCRD region). TCRδ J and D segments are located between the Vα and Jα segments. Meanwhile, TCRδ V segments are spread throughout the TCRα locus and are primarily located within the various Vα segments. The number and location of the various TCRδ segments can be determined from the IMGT database. Due to the genomic arrangement of TCRδ gene segments within the TCRα locus, successful rearrangements in the TCRα locus can result in deletion or inactivation of TCRδ gene segments.

[0135] In some embodiments of the invention, the non-human animal comprising an unrearranged human TCR α variable locus also comprises at least one human Vδ segment, for example, up to a complete repertoire of human Vδ segments. Thus, in some embodiments, replacement of the endogenous TCR α variable locus results in replacement of at least one non-human Vδ segment with a human Vδ segment. In other embodiments, the non-human animal of the invention comprises a complete repertoire of human Vδ, Dδ, and Jδ segments in an unrearranged humanized TCR α locus. In yet other embodiments, the non-human animal comprises a complete unrearranged human TCR δ locus in an unrearranged humanized TCR α locus (i.e., a TCR δ locus comprising human variable region segments and human enhancers and constant regions). Exemplary embodiments for constructing an unrearranged humanized TCR α locus, including a complete unrearranged TCR δ locus, are set forth in U.S. Patent No. 9,113,616, which is incorporated herein by reference.

[0136] In yet another embodiment, the non-human animal of the invention comprises an unrearranged humanized TCRγ locus, e.g., a TCRγ locus comprising at least one human Vγ and at least one human Jγ segment (e.g., a complete repertoire of human Vγ and human Jγ variable region segments). The human TCRγ locus is located on chromosome 7 in humans, while the murine TCRγ locus is located on chromosome 13 in mice. For further details on TCRγ loci, see the IMGT database.

[0137] In one embodiment, a non-human animal (e.g., a rodent, e.g., a mouse or rat) comprising the humanized TCR alpha and beta variable loci (and optionally, humanized TCR delta / gamma variable loci) described herein expresses a humanized T cell receptor on the surface of the T cell comprising a human variable region and a non-human (e.g., a rodent, e.g., a mouse or rat) constant region. In some embodiments, the non-human animal can express a diverse repertoire of humanized T cell receptors that recognize a variety of presented antigens.

[0138] In various embodiments of the invention, the humanized T cell receptor polypeptides described herein comprise a human leader sequence. In alternative embodiments, the nucleic acid sequence of the humanized TCR receptor is engineered such that the humanized TCR polypeptide comprises a non-human leader sequence.

[0139] The humanized TCR polypeptides described herein may be expressed under the control of endogenous non-human regulatory elements (e.g., rodent regulatory elements), such as promoters, silencers, enhancers, etc. The humanized TCR polypeptides described herein may alternatively be expressed under the control of human regulatory elements. In various embodiments, the non-human animals described herein further comprise all regulatory and other sequences normally found in situ in the human genome.

[0140] In various embodiments, the human variable region of the humanized TCR protein can interact with various proteins on the surface of the same cell or another cell. In one embodiment, the human variable region of the humanized TCR interacts with an MHC protein (e.g., an MHC class I or II protein) that presents an antigen on the surface of a second cell, e.g., an antigen-presenting cell (APC). In some embodiments, the MHC I or II protein is a non-human (e.g., rodent, e.g., mouse or rat) protein. In other embodiments, the MHC I or II protein is a human(ized) protein. In one aspect, the second cell, e.g., an APC, is an endogenous non-human cell expressing a human or humanized MHC molecule. In various embodiments, the second cell is a human cell expressing a human MHC molecule.

[0141] In one aspect, the non-human animal expresses a humanized TCR cell receptor having a non-human constant region on the surface of the T cell, and the receptor can interact with a non-human molecule expressed in the T cell, e.g., an anchor or signaling molecule (e.g., a CD3 molecule, a zeta chain, or another protein that anchors to the TCR via a CD3 molecule or a zeta chain). Thus, in one aspect, a cell complex is provided, comprising: (a) a non-human T cell expressing (i) a TCR comprising a humanized TCR α chain described herein and a humanized TCR β chain described herein, and (ii) a chimeric co-receptor described herein; and (b) a non-human antigen-presenting cell comprising an antigen bound to a chimeric MHC I and / or a chimeric MHC II described herein. In one embodiment, the non-human constant TCR α and TCR β chains are complexed with a non-human zeta (ζ) chain homodimer and a CD3 heterodimer. In one embodiment, the cell complex is an in vivo cell complex. In one embodiment, the cell complex is an in vitro cell complex.

[0142] In various embodiments, the non-human animals described herein (e.g., rodents, e.g., mice or rats) undergo thymic development to give rise to T cells that can progress from DN1 to DN2 to DN3 to DN4 to DP and then to CD4 or CD8 SP T cells. Such T cells of the non-human animals of the invention express cell surface molecules (e.g., CD25, CD44, Kit, CD3, pTα, etc.) typically produced by T cells during specific stages of thymic development. Thus, in one embodiment, the non-human animals described herein may express pTα complexed with TCRβ at the DN3 stage of thymic development. The non-human animals described herein undergo thymic development to give rise to T cells that can give rise to CD4+ and CD8+ T cells.

[0143] In various embodiments, the non-human animals described herein generate T cells that can undergo T cell differentiation in the periphery. In some embodiments, the non-human animals described herein generate effector T cells, e.g., CTLs (cytotoxic T lymphocytes), T H 1. T H 2. T REG , T H 17, etc. Thus, in these embodiments, the non-human animals described herein can produce effector T cells that fulfill various functions typical of particular T cell types, e.g., recognize, bind to, and respond to foreign antigens. In various embodiments, the non-human animals described herein produce effector T cells that kill cells presenting peptide fragments of cytosolic pathogens expressed in the context of MHC I molecules, recognize peptides from antigens that are degraded in intracellular vesicles and presented by MHC II molecules on the surface of macrophages, attract macrophages to kill microorganisms, produce cytokines that drive B cell differentiation, activate B cells to produce opsonizing antibodies, attract epithelial cells to produce chemokines that recruit neutrophils to sites of infection, etc.

[0144] In further embodiments, the non-human animals described herein comprise CD3+ T cells in the periphery, e.g., in the spleen. In other aspects, the non-human animals described herein can generate a population of memory T cells in response to an antigen of interest. For example, the non-human animals generate both central memory T cells (Tcm) and effector memory T cells (Tem) against an antigen, e.g., an antigen of interest (e.g., an antigen being tested for vaccine development).

[0145] DN1 and DN2 cells that do not receive sufficient signals (e.g., Notch signals) can develop into B cells, myeloid cells (e.g., dendritic cells), mast cells, and NK cells. See, 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, myeloid cells (e.g., dendritic cells), mast cells, and NK cells. In some embodiments, the non-human animals described herein develop a dendritic cell population in the thymus.

[0146] The predominant species of T cell receptor expressed on the surface of T cells is TCRα / β, with a minority of cells expressing TCRδ / γ. In some embodiments of the invention, T cells of a non-human animal comprising a humanized TCRα and / or β locus exhibit TCRα / β and TCRδ / γ locus usage, e.g., TCRα / β and TCRδ / γ locus usage similar to that of a wild-type animal (e.g., T cells of a non-human animal described herein express TCRα / β and TCRδ / γ proteins in proportions comparable to those expressed by a wild-type animal). Thus, in some embodiments, a non-human animal comprising a humanized TCRα / β and an endogenous non-human TCRδ / γ locus exhibits usage of all loci. Human or humanized MHC molecules

[0147] 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 co-receptor, and optionally a humanized TCR, which, together with the humanized co-receptor, provide an activation signal to cells expressing the humanized TCR and chimeric T cell co-receptor polypeptide based on recognition and binding of a peptide presented by the humanized MHC. Thus, the non-human animals disclosed herein comprise at least one of a first, second, and / or third nucleic acid sequence, each encoding a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, and a human or humanized MHC Iα polypeptide. The non-human animals optionally also comprise human or humanized β2 microglobulin. The use of the designations first, second, and third herein should not be construed as limiting the non-human animals disclosed herein to requiring all three nucleic acid sequences, or as limiting the presence of any of the human or humanized MHC polypeptides to any particular order.

[0148] Thus, in some embodiments, the non-human animals disclosed herein may comprise, for example, first and second nucleotide sequences encoding 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 TCR α constant gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence, and optionally, first and second nucleic acid sequences encoding, for example, a human or humanized MHC Iα polypeptide and a human or humanized β2 microglobulin polypeptide. In other embodiments, the non-human animals disclosed herein may comprise, for example, a first nucleotide sequence encoding 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 TCR α constant gene sequence, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence, and optionally first and second nucleic acid sequences encoding, for example, a human or humanized MHC IIα polypeptide and a human or humanized MHC IIβ polypeptide.In some embodiments, the non-human animals disclosed herein may comprise first, second, and third nucleotide sequences encoding, for example, a chimeric CD4 polypeptide, a chimeric CD8α polypeptide, and a chimeric CD8β polypeptide; an unrearranged T cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCR α constant gene sequence; and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence; and optionally first, second, third, and fourth nucleic acid sequences 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.

[0149] In various embodiments, provided herein are genetically modified non-human animals, e.g., rodents (e.g., mice or rats), comprising within their 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 an MHC I polypeptide that is partially human and partially non-human, e.g., a chimeric human / non-human MHC I polypeptide. The MHC II nucleic acid sequence may encode an MHC II protein that is partially human and partially non-human, e.g., a chimeric human / non-human MHC II protein (e.g., comprising chimeric human / non-human MHC II α and β polypeptides). In some aspects, the animal does not express endogenous MHC I and / or MHC II polypeptides, e.g., functional endogenous MHC I and / or MHC II polypeptides, on its cell surface. In some embodiments, the only MHC I and / or MHC II molecules expressed on the cell surface of the animal are chimeric MHC I and / or MHC II molecules.

[0150] Genetically modified non-human animals comprising nucleic acid sequences in their genomes, e.g., at endogenous loci, encoding chimeric human / non-human MHC I polypeptides, are disclosed in U.S. Patent Application Publication Nos. 20130111617 and 20130185819, the contents of which are incorporated herein by reference in their entireties. Genetically modified non-human animals comprising nucleic acid sequences in their genomes, e.g., at endogenous loci, encoding humanized, e.g., chimeric human / non-human MHC II polypeptides, are disclosed in U.S. Patent Application Publication No. 8,847,005 and U.S. Patent Application Publication No. 20130185820, the contents of each of which are incorporated herein by reference in their entireties. Genetically modified non-human animals comprising in their genomes, e.g., at endogenous loci, nucleic acid sequences encoding chimeric human / non-human MHC I polypeptides and comprising in their genomes, e.g., at endogenous loci, nucleic acid sequences encoding humanized, e.g., chimeric human / non-human MHC II polypeptides are disclosed in U.S. Patent Application Publication No. 20140245467, the entire contents of which are incorporated herein by reference.

[0151] In various embodiments, the present disclosure provides a genetically modified non-human animal that contains in its genome, for example, a chimeric human / non-human MHC locus at one or more endogenous MHC loci. and / or a third nucleic acid sequence encoding a chimeric human / non-human MHC IIβ polypeptide, wherein the human portion of the chimeric MHC IIβ polypeptide comprises the extracellular portion (or a portion thereof, e.g., one or more extracellular domains) of a human MHC IIβ polypeptide; wherein the non-human animal expresses functional chimeric human / non-human MHC I and MHC II proteins from its endogenous non-human MHC locus. In one embodiment, the first, second, and / or third nucleic acid sequences are located at endogenous non-human MHC I, MHC IIα, and MHC IIβ loci, respectively. In one embodiment, the non-human animal is a mouse, and the first, second, and / or third nucleic acid sequences are located at endogenous mouse MHC loci 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.

[0152] In one embodiment, the non-human animal expresses only chimeric human / non-human MHC I, MHC IIα, and / or MHC IIβ 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 animal described herein expresses a functional chimeric MHC I and a functional chimeric MHC II on the surface of its cells, such as antigen-presenting cells. In one embodiment, the MHC I and MHC II expressed on the cell surface by the animal are only chimeric MHC I and chimeric MHC II, and the animal does not express any endogenous MHC I or MHC II on the cell surface.

[0153] In one embodiment, the chimeric human / non-human MHC I polypeptide comprises, in its human portion, for example, the peptide-binding cleft of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises the extracellular portion of human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises the extracellular domain of the α chain of human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises the α1 and α2 domains of human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises the α1, α2, and α3 domains of human MHC I.

[0154] In one aspect, the human portion of the chimeric MHC IIα polypeptide and / or the human portion of the chimeric MHC IIβ polypeptide comprises the peptide-binding domain of a human MHC IIα polypeptide and / or a human MHC IIβ polypeptide, respectively. In one aspect, the human portion of the chimeric MHC IIα and / or β polypeptide comprises the extracellular portion of a human MHC IIα and / or β polypeptide, respectively. In one embodiment, the chimeric MHC In another embodiment, the human portion of the chimeric MHC IIα polypeptide comprises the α1 domain of a human MHC IIα polypeptide. In a further embodiment, the human portion of the chimeric MHC IIβ polypeptide comprises the β1 domain of a human MHC IIβ polypeptide. In another embodiment, the human portion of the chimeric MHC IIβ polypeptide comprises the β1 and β2 domains of a human MHC IIβ polypeptide.

[0155] In some embodiments, human or humanized MHC I polypeptides may be derived from functional human HLA molecules encoded by any of the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci. Human or humanized MHC II polypeptides may be derived from functional human HLA molecules encoded by any of the HLA-DP, -DQ, and -DR loci. A list of commonly used HLA antigens and alleles is provided in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), which is incorporated herein by reference. Shankarkumar et al. also provides a brief description of HLA terminology used in the art. Further information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) "The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1 / 3 / 4 / 5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens," Tissue Antigens 73:95-170, and the recent update by Marsh et al. (2010) "Nomenclature for factors of the HLA system," 2010, Tissue Antigens 75:291-455, both of which are incorporated herein by reference. In some embodiments, the MHC I or MHC II polypeptide may be derived from any functional human HLA-A, B, C, DR, or DQ molecule. Thus, human or humanized MHC I and / or II polypeptides may be derived from any functional human HLA molecule described herein, hi some embodiments, all MHC I and / or MHC II polypeptides expressed on the cell surface comprise a portion derived from a human HLA molecule.

[0156] Of particular interest are human HLA molecules, which are specific polymorphic HLA alleles known to be associated with numerous human diseases, such as human autoimmune diseases. Indeed, specific polymorphisms at HLA loci have been identified that are associated with the development of rheumatoid arthritis, type I diabetes, Hashimoto's thyroid disease, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune diseases. For example, Wong and Wen (2004) What can the HLA transgenic mouse tell us about autoimmune diabetes?, Diabetologia 47:1476~87; Taneja and David (1998) HLA Transgenic Mice as Humanized Mouse Models of Disease and Immunity, J.Clin.Invest.101:921~26; Bakker et al. (2006), A high-resolution 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 See, e.g., "The MHC region for 7 immune-mediated diseases," Proc. Natl. Acad. Sci. USA 106:18680-85. Thus, human or humanized MHC I and / or II polypeptides may be derived from human HLA molecules known to be associated with particular diseases, e.g., autoimmune diseases.

[0157] In one specific aspect, the human or humanized MHC I polypeptide is derived from HLA-A. In a particular embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., an HLA-A3.1 polypeptide). In one embodiment, the HLA-A polypeptide is an HLA-A4.1 polypeptide. * 0201 allele, e.g., HLA-A * A polypeptide encoded by the 02:01:01:01 allele. HLA-A * The 0201 allele is commonly used among North American populations. While this particular HLA sequence is described in this example, any suitable HLA-A sequence is encompassed herein, including, for example, polymorphic variants of HLA-A2 present in the human population, sequences containing 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, etc.

[0158] In another specific embodiment, the human portion of the chimeric MHC I polypeptide is derived from a human MHC I selected from HLA-B and HLA-C. In one embodiment, the human portion is derived from HLA-B, e.g., HLA-B27. In another embodiment, the human portion is derived from HLA-A3, -B7, -Cw6, etc.

[0159] In one specific embodiment, the human portion of the humanized MHC II α and β polypeptides described herein is derived from HLA-DR, e.g., HLA-DR2. Typically, the HLA-DR α chain is monomorphic, e.g., the α chain of the HLA-DR complex is monomorphic, e.g., the α chain of the HLA-DR complex is monomorphic, e.g., the HLA-DRA gene (e.g., HLA-DRα * On the other hand, the HLA-DRβ chain is polymorphic. Therefore, HLA-DR2 is composed of the α chain encoded by the HLA-DRA gene and the HLA-DR1β *and a β chain encoded by the 1501 gene. While these particular HLA sequences are described in this example, any suitable HLA-DR sequence is encompassed herein, including polymorphic variants present in the human population, sequences containing 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, etc.

[0160] The human portion of the chimeric MHC II α and / or β polypeptide may be encoded by the nucleic acid sequence of an HLA allele known to be associated with a common human disease. Such an HLA allele may be 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 review of HLA allele / disease associations, see Bakker et al. (2006), supra, which is incorporated herein by reference.

[0161] In one embodiment, the non-human portion of the chimeric human / non-human MHC1, MHCIIα, and / or MHCIIβ polypeptide comprises the transmembrane and / or cytoplasmic domain of an endogenous non-human (e.g., rodent, e.g., mouse, rat, etc.) MHC1, MHCIIα, and / or MHCIIβ polypeptide, respectively. Thus, the non-human portion of the chimeric human / non-human MHC1 polypeptide may comprise the transmembrane and / or cytoplasmic domain of an endogenous non-human MHC1 polypeptide. The non-human portion of the chimeric MHCIIα polypeptide may comprise the transmembrane and / or cytoplasmic domain of an endogenous non-human MHC1 polypeptide. The non-human portion of the chimeric human / non-human MHCIIβ polypeptide may comprise the transmembrane and / or cytoplasmic domain of an endogenous non-human MHC1 polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human portion of the chimeric MHC1 polypeptide is derived from a mouse H-2K protein. In one aspect, the animal is a mouse, and the non-human portions of the chimeric MHC II α and β polypeptides are derived from a mouse H-2E protein. Thus, the non-human portions of the chimeric MHC II α and β polypeptides may comprise transmembrane and cytoplasmic domains derived from a mouse H-2K protein, and the non-human portions of the chimeric MHC II α and β polypeptides may comprise transmembrane and cytoplasmic domains derived from a mouse H-2E protein. While specific H-2K and H-2E sequences are contemplated in the examples, any suitable sequences, e.g., polymorphic variants, conservative / non-conservative amino acid substitutions, etc., are encompassed herein. In one aspect, the non-human animal is a mouse, and the mouse does not express a functional endogenous MHC polypeptide from its H-2D locus. In some embodiments, the mouse has been 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.

[0162] Chimeric human / non-human polypeptides may comprise a human or non-human leader (signal) sequence. In one embodiment, a chimeric MHC1 polypeptide comprises the non-human leader sequence of an endogenous MHC1 polypeptide. In one embodiment, a chimeric MHC1Iα polypeptide comprises the non-human leader sequence of an endogenous MHC1Iα polypeptide. In one embodiment, a chimeric MHC1Iβ polypeptide comprises the non-human leader sequence of an endogenous MHC1Iβ polypeptide. In alternative embodiments, chimeric MHC1I, MHC1Iα, and / or MHC1Iβ polypeptides comprise the non-human leader sequence of the respective MHC1I, MHC1Iα, and / or MHC1Iβ polypeptides from another non-human animal, e.g., another rodent or another mouse strain. Thus, a nucleic acid sequence encoding a chimeric MHC1I, MHC1Iα, and / or MHC1Iβ polypeptide may be operably linked to a nucleic acid sequence encoding the non-human MHC1I, MHC1Iα, and / or MHC1Iβ leader sequence, respectively. In yet another embodiment, the chimeric MHC1, MHCIIα, and / or MHCIIβ polypeptides comprise a human leader sequence (e.g., human HLA-A2, human HLA-DRα, and / or human HLA-DRβ1) of a human MHC1, human MHCIIα, and / or human MHCIIβ polypeptide, respectively. * 1501 each containing the leader sequence).

[0163] A chimeric human / non-human MHC1, MHCIIα, and / or MHCIIβ polypeptide may comprise, in its human portion, the complete or substantially complete extracellular domain of a human MHC1, human MHCIIα, and / or human MHCIIβ polypeptide, respectively. Thus, the human portion may comprise a human MHC1, human MHCIIα, and / or human MHCIIβ polypeptide (e.g., human HLA-A2, human HLA-DRα, and / or human HLA-DRβ1). *The human / non-human MHC1 polypeptide may comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g., 95% or more of the amino acid sequence encoding the extracellular domain of human MHC1, human MHCIIα, and / or human MHCIIβ polypeptide. In one example, the substantially complete extracellular domain of a human MHC1, human MHCIIα, and / or human MHCIIβ polypeptide lacks a human leader sequence. In another example, the chimeric human / non-human MHC1, chimeric human / non-human MHCIIα, and / or chimeric human / non-human MHCIIβ polypeptide comprises a human leader sequence.

[0164] Furthermore, the chimeric MHC1, MHCIIα, and / or MHCIIβ polypeptides may be operably linked to (e.g., expressed under the regulatory control of) endogenous non-human promoters and regulatory elements, e.g., mouse MHC1, MHCIIα, and / or MHCIIβ regulatory elements, respectively. Such an arrangement will facilitate appropriate expression of the chimeric MHC1 and / or MHCII polypeptides in the non-human animal, e.g., during an immune response in the non-human animal.

[0165] In further embodiments, the non-human animal of the invention, e.g., a rodent, e.g., a mouse, comprises a nucleic acid sequence encoding human or humanized β2 microglobulin (e.g., at the endogenous β2 microglobulin locus). The β2 microglobulin or light chain (also abbreviated as "β2M") of the MHC class I complex is a small (12 kDa), non-glycosylated protein that primarily functions to stabilize the MHC I α chain. The development of human or humanized β2 microglobulin animals is described in detail in U.S. Patent Application Publication No. 20130111617, which is incorporated herein by reference.

[0166] A nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide may contain nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may contain nucleic acid residues encoding the amino acid sequence set forth in amino acids 21-119 of the human β2 microglobulin protein (i.e., the amino acid residues corresponding to mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may contain nucleic acid residues encoding the amino acid sequence set forth in amino acids 23-115 of the human β2 microglobulin protein, e.g., the amino acid sequence set forth in amino acids 23-119 of the human β2 microglobulin protein. The nucleic acid and amino acid sequences of human β2 microglobulin are described in Gussow et al., supra, which is incorporated herein by reference.

[0167] Thus, a human or humanized β2 microglobulin polypeptide may comprise the amino acid sequence represented by amino acids 23 to 115 of a human β2 microglobulin polypeptide, for example, the amino acid sequence represented by amino acids 23 to 119 of a human β2 microglobulin polypeptide, for example, the amino acid sequence represented by amino acids 21 to 119 of a human β2 microglobulin polypeptide. Alternatively, human β2 microglobulin may comprise amino acids 1 to 119 of a human β2 microglobulin polypeptide.

[0168] In some embodiments, the nucleotide sequence encoding human or humanized β2 microglobulin comprises the nucleotide sequence set forth in exons 2 through 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises the nucleotide sequence set forth in exons 2, 3, and 4 of the human β2 microglobulin gene. In this embodiment, the nucleotide sequences set forth in exons 2, 3, and 4 are operably linked to permit normal transcription and translation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exons 2 through 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to approximately 267 bp after exons 2 through 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises approximately 2.8 kb of the human β2 microglobulin gene.

[0169] Thus, a human or humanized β2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising the nucleotide sequence set forth in exons 2 through 4 of human β2 microglobulin, e.g., a nucleotide sequence corresponding to exons 2 through 4 of the human β2 microglobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising the nucleotide sequence set forth in exons 2, 3, and 4 of the human β2 microglobulin gene. In a specific embodiment, a human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to approximately 267 bp from exons 2 through 4 of the human β2 microglobulin gene. In another specific embodiment, a human or humanized polypeptide is encoded by a nucleotide sequence comprising approximately 2.8 kb of the human β2 microglobulin gene. In cases where exon 4 of the β2 microglobulin gene contains a 5' untranslated region, the human or humanized polypeptide may be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.

[0170] While specific nucleic acid and amino acid sequences giving rise to genetically engineered animals are described herein, it will be understood by those skilled in the art that sequences that differ from those described herein by one or more conservative or non-conservative amino acid substitutions or by degeneracy of the genetic code are also provided.

[0171] Thus, provided are non-human animals expressing a human β2 microglobulin sequence, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence. In certain embodiments, the β2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence described herein. In one embodiment, the human β2 microglobulin sequence contains one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence contains one or more non-conservative substitutions.

[0172] Additionally, a non-human animal is provided, wherein the nucleotide sequence encoding the human or humanized β2 microglobulin protein also comprises the nucleotide sequence set forth in exon 1 of the non-human β2 microglobulin gene. Thus, in certain embodiments, the non-human animal comprises in its genome a nucleotide sequence encoding human or humanized β2 microglobulin, the nucleotide sequence comprising exon 1 of the non-human β2 microglobulin and exons 2, 3, and 4 of the human β2 microglobulin gene. Thus, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of the non-human β2 microglobulin gene and exons 2, 3, and 4 of the human β2 microglobulin gene (e.g., exons 2 and 3 of the human β2 microglobulin gene).

[0173] In one embodiment, a non-human animal (e.g., a rodent, e.g., a mouse) of the invention further comprises a nucleic acid sequence encoding a human or humanized MHCI protein in addition to a nucleotide sequence encoding the chimeric CD8 protein. This allows the chimeric CD8 protein expressed on the surface of T cells from the animal to associate, bind, and / or interact with human or humanized MHCI expressed on the surface of a second cell, e.g., an antigen-presenting cell. In one embodiment, the MHCI protein comprises the extracellular domain of a human MHCI polypeptide. In one embodiment, the animal further comprises a human or humanized β2 microglobulin polypeptide. Exemplary genetically modified animals expressing human or humanized MHCI polypeptides and / or β2 microglobulin polypeptides are described in U.S. Patent Application Publication Nos. 20130111617 and 20130185819, both of which are incorporated herein by reference in their entireties. Thus, in one embodiment, an animal comprising a chimeric CD8 protein described herein may further comprise a humanized MHCI complex, the humanized MHCI complex comprising: (1) a humanized MHCI polypeptide, e.g., the humanized MHCI polypeptide comprises the extracellular domain of human MHCI and the transmembrane and cytoplasmic domains of endogenous (e.g., murine) MHCI, e.g., the humanized MHCI polypeptide comprises the α1, α2, and α3 domains of the human MHCI polypeptide; and (2) a human or humanized β2 microglobulin polypeptide (e.g., the animal comprises in its genome nucleotide sequences set forth in exons 2, 3, and 4 of human β2 microglobulin). In one aspect, both the humanized MHCI and the human or humanized β2 microglobulin polypeptide are encoded by nucleotide sequences located in the endogenous MHCI and β2 microglobulin loci, respectively. In one aspect, the animal does not express functional endogenous MHCI and β2 microglobulin polypeptides. Thus, the MHCI expressed by the animal may be a chimeric human / non-human, eg, human / rodent (eg, human / mouse) MHCI polypeptide.The human portion of the chimeric MHCI polypeptide may be derived from a human HLA class I protein selected from the group consisting of HLA-A, HLA-B, and HLA-C, e.g., HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecule present in the human population. In embodiments, when the animal is a mouse, the non-human (i.e., mouse) portion of the chimeric MHCI polypeptide may be derived from a mouse MHCI protein selected from H-2D, H-2K, and H-2L.

[0174] In one embodiment, the non-human animal (e.g., a rodent, e.g., a mouse) of the invention further comprises a nucleotide sequence encoding a human or humanized MHC II protein, thereby enabling the chimeric CD4 protein expressed on the surface of T cells of the animal to interact with 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. Patent No. 8,847,005, issued September 30, 2014, and U.S. Patent Application Publication No. 20130185820, the contents of which are incorporated herein by reference in their entireties. Thus, in one embodiment, an animal comprising the chimeric CD4 protein described herein may further comprise a humanized MHCII protein, including (1) a humanized MHCIIα polypeptide comprising an extracellular domain of human MHCIIα and the transmembrane and cytoplasmic domains of endogenous, e.g., mouse MHCII, wherein the extracellular domain of human MHCIIα comprises the α1 and α2 domains of human MHCIIα, and (2) a humanized MHCIIβ polypeptide comprising a human MHCIIβ extracellular domain and the transmembrane and cytoplasmic domains of endogenous, e.g., mouse MHCII, wherein the human MHCIIβ extracellular domain comprises the β1 and β2 domains of human MHCIIβ. In one aspect, both the humanized MHCIIα and β polypeptides are encoded by nucleic acid sequences located in the endogenous MHCIIα and β loci, respectively. In one aspect, the animal does not express functional endogenous MHCIIα and β polypeptides. Thus, the MHCII expressed by the animal may be a chimeric human / non-human, eg, human / rodent (eg, human / mouse) MHCII protein.The human portion of the chimeric MHC II protein may be derived from a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g., HLA-DR4, HLA-DR2, HLA-DQ2.5, HLA-DQ8, or any other HLA class II molecule present in the human population. In embodiments in which the animal is a mouse, the non-human (i.e., mouse) portion of the chimeric MHC II polypeptide may be derived from a mouse MHC II protein selected from H-2E and H-2A.

[0175] Various other embodiments of genetically modified non-human animals, e.g., rodents, e.g., rats or mice, will be apparent to those skilled in the art from the present disclosure and the disclosures of U.S. Patent Application Publication Nos. 20130111617, 20130185819, and 20130185820 and U.S. Patent No. 8,847,005, which are incorporated herein by reference.

[0176] In various embodiments, the genetically modified non-human animals described herein produce cells, e.g., APCs, that have human or humanized MHC I and II on their cell surface and, as a result, 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 invention can be used to study the function of the human immune system in humanized animals, for example, to identify antigens and antigenic epitopes that elicit an immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), for use in vaccine development, for evaluation of vaccine candidates and other vaccine strategies, for studying human autoimmunity, for studying human infectious diseases, and even to devise better treatment strategies based on human MHC expression. Non-human animals, tissues, and cells

[0177] The genetically modified non-human animals of the present invention may be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets and rhesus monkeys). For non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are utilized to generate non-human animals containing genetic modifications. Such methods include, for example, modifying a non-ES cell germline (e.g., fibroblasts or induced pluripotent cells) and using nuclear transfer to implant the modified genome into a suitable cell, such as an oocyte, and maturing the modified cell (e.g., modified oocyte) within the non-human animal under suitable conditions to form an embryo.

[0178] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, for example, of the Jerboidea or Muridea superfamily. 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 the superfamily Muridea. In one embodiment, the genetically modified animal belongs to a family selected from the family Macrocheonidae (e.g., mouse-like hamsters), family Cricetidae (e.g., hamsters, New World rats and mice, and voles), family Muridae (mice and rats, Mongolian gerbils, spiny mice, and maned mice), family Tetranychus (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), family Spiny Dormouse (e.g., spiny dormouse), and family Mole-Ratidae (e.g., mole rats, bamboo rats, and mole rats). In certain embodiments, the genetically modified rodent is selected from a true mouse or rat (Muridae), a gerbil, a spiny mouse, and a maned mouse. In one embodiment, the genetically modified mouse belongs to the Muridae family. In one embodiment, the animal is a rodent. In certain embodiments, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0179] In one embodiment, the non-human animal is a rodent that is a C57BL strain mouse selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836; see Auerbach et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836). (See also [PubMed] et al. (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mixture of the 129 strain and the C57BL / 6 strain. In another specific embodiment, the mouse is a mixture of the 129 strains described above, or a mixture of the BL / 6 strains described above. In a specific embodiment, the mixed 129 strain is the 129S6 (129 / SvEvTac) strain. In another embodiment, the mouse is a BALB strain, e.g., a BALB / c strain. In yet another embodiment, the mouse is a mixture of the BALB strain and another of the above strains. The non-human animals provided herein may be mice derived from any combination of the above strains.

[0180] 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.

[0181] Thus, in one embodiment of the present invention, there is provided a genetically modified mouse, wherein the mouse contains, e.g., in its genome, e.g., in its germline genome, (a) a first nucleotide sequence encoding a first chimeric human / mouse T cell co-receptor polypeptide (e.g., CD4), a second nucleotide sequence encoding a second chimeric human / mouse T cell co-receptor polypeptide (e.g., CD8α), and / or a third nucleotide sequence encoding a third chimeric human / mouse T cell co-receptor polypeptide (e.g., CD8β), wherein each chimeric T cell co-receptor polypeptide (b) a first, second, and / or third nucleotide sequence, wherein the murine portion of the peptide comprises at least the transmembrane and cytoplasmic domains of a murine T-cell coreceptor, and the human portion of each chimeric polypeptide comprises the extracellular portion (or a portion thereof, e.g., one or more extracellular domains) of a human T-cell coreceptor, and the mouse is expressing the first, second, and / or third chimeric T-cell coreceptor polypeptide; (b) a non-recombinant TCR alpha constant gene sequence comprising at least one human Vα segment and at least one human Jα segment, operably linked to a murine TCR alpha constant gene sequence. a T cell receptor (TCR) α variable locus, and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a murine TCR β constant gene sequence, and optionally (c) a first nucleic acid sequence encoding a first chimeric human / mouse MHC polypeptide (e.g., MHCIIα), a second nucleic acid sequence encoding a second chimeric human / mouse MHC polypeptide (e.g., MHCIIβ), and / or a third chimeric human / mouse MHC polypeptide (e.g., MHCIIβ). a third nucleic acid sequence encoding an MHC polypeptide (e.g., MHC1), and a β2 microglobulin locus encoding human or humanized β2 microglobulin, wherein the human portion of each chimeric MHC polypeptide comprises an extracellular domain of a human MHC polypeptide associated with a first, second, and / or third chimeric T cell co-receptor polypeptide (e.g., the human portion of a chimeric MHCII complex (e.g., humanized MHCII α and β polypeptides) associated with a chimeric CD4 polypeptide, and / or the human portion of a chimeric MHC1 polypeptide (or MHC1 complex,For example, genetically modified mice are provided that contain a chimeric CD8 co-receptor (e.g., associated with humanized CD8 α and β polypeptides), a first, second, and / or third nucleic acid sequence, and a β2 microglobulin locus.

[0182] Provided herein is a genetically modified mouse, the mouse comprising, in its genome, for example, at its endogenous CD4 locus, a nucleotide sequence encoding a chimeric human / mouse CD4 polypeptide, wherein the mouse portion of the chimeric polypeptide comprises at least the transmembrane and cytoplasmic domains of the mouse CD4 polypeptide, and the mouse expresses the chimeric human / mouse CD4. In one embodiment, the human portion of the chimeric polypeptide comprises at least all or substantially all of the extracellular domain of the human CD4 polypeptide. In one embodiment, the human portion of the chimeric polypeptide comprises at least all or substantially all of the D1 domain of the human CD4 protein. In one embodiment, the human portion of the chimeric polypeptide comprises at least all or substantially all of the D1-D2 domains of the human CD4 protein, for example, at least all or substantially all of the D1-D3 domains of the human CD4 protein, for example, all or substantially all of the D1-D4 domains of the human CD4 protein. Thus, in one embodiment, the mouse comprises, at its endogenous CD4 locus, a nucleotide sequence comprising at least all or substantially all of exons 4, 5, and 6 of the human CD4 gene, e.g., exon 3 of the human CD4 gene encoding a portion of the D1 domain of human CD4 and the sequence of exons 4-6 of the human CD4 gene. In one embodiment, the mouse comprises, at its endogenous CD4 locus, a chimeric human / mouse CD4 comprising human CD4 sequences responsible for interaction with MHCII and / or the extracellular portion of the T cell receptor. In another embodiment, the mouse comprises, at its endogenous CD4 locus, a chimeric human / mouse CD4 comprising human CD4 sequences responsible for interaction with MHCII and / or the variable domain of the T cell receptor. In one embodiment, the nucleotide sequence comprises a sequence encoding a mouse CD4 signal peptide. In one embodiment, the mouse comprises a replacement of the nucleotide sequence encoding the extracellular domain of mouse CD4 with a nucleotide sequence encoding the extracellular domain of human CD4.In another embodiment, the mouse comprises a replacement of a nucleotide sequence encoding at least all or substantially all of the mouse CD4 D1 domain, e.g., a nucleotide sequence encoding at least all or substantially all of the mouse CD4 D1-D2 domain, e.g., a nucleotide sequence encoding at least all or substantially all of the mouse CD4 D1-D3 domain, with a human nucleotide sequence encoding them. In one embodiment, the domains of the chimeric CD4 polypeptide are encoded by the nucleotide sequence schematically represented in Figure 5A.

[0183] In one embodiment, the mouse does not express functional endogenous mouse CD4 from its endogenous mouse CD4 locus. In one embodiment, the mouse described herein comprises a chimeric human / mouse CD4 nucleotide sequence in the mouse germline.

[0184] In one embodiment, the mouse retains any endogenous sequences that have not been humanized; for example, in an embodiment, the mouse includes replacement of all or substantially all of the nucleotide sequences encoding the D1-D3 domains, and the mouse retains the endogenous nucleotide sequence encoding the mouse CD4 D4 domain, as well as the nucleotide sequences encoding the transmembrane and cytoplasmic domains of mouse CD4.

[0185] In one embodiment, the mouse expressing the chimeric human / mouse CD4 protein retains mouse CD4 promoter and regulatory sequences, e.g., the nucleotide sequence in the mouse encoding the chimeric human / mouse CD4 is operably linked to the endogenous mouse CD4 promoter and regulatory sequences in the mouse. In one embodiment, these mouse regulatory sequences retained in the genetically engineered animal of the invention include sequences that regulate expression of the chimeric protein at the appropriate stage during T cell development. Thus, in one embodiment, the mouse expresses the chimeric human / mouse CD4 protein in B cells or mature CD8 cells. +The mouse does not express the chimeric CD4 on T cells. In one embodiment, the mouse also does not express the chimeric CD4 on any cell type that does not normally express endogenous CD4, e.g., any immune cell type.

[0186] The genetically modified mouse disclosed herein may comprise, in its genome, for example, at its endogenous CD8 locus, a first nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide. In one embodiment, the first nucleotide sequence comprises a sequence encoding all or substantially all of the extracellular portion of the human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of the mouse CD8α polypeptide, and the second nucleotide sequence comprises a sequence encoding all or substantially all of the extracellular portion of the human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of the mouse CD8β polypeptide, and the mouse expresses functional chimeric human / mouse CD8 proteins. In one embodiment, the first nucleotide sequence comprises a sequence encoding at least the immunoglobulin V-like domain of the human CD8α polypeptide and the remaining portion of the mouse CD8α polypeptide, and the second nucleotide sequence comprises a sequence encoding at least the immunoglobulin V-like domain of the human CD8β polypeptide and the remaining portion of the mouse CD8β polypeptide. In one embodiment, the first nucleotide sequence comprises at least the MHCI-binding domain of a human CD8α polypeptide. In one embodiment, the first and second nucleotide sequences comprise at least an exon encoding the extracellular portion of a human CD8α polypeptide and / or a CD8β polypeptide, respectively. In one embodiment, the extracellular portion of a human CD8α polypeptide and / or a human CD8β polypeptide is a region encompassing a portion of a human CD8α polypeptide and / or a CD8β polypeptide that is not the transmembrane domain or the cytoplasmic domain. In one embodiment, a domain of a chimeric CD8α polypeptide is encoded by a nucleotide sequence schematically represented in FIG. 5B. In one embodiment, a domain of a chimeric CD8β polypeptide is encoded by a nucleotide sequence schematically represented in FIG. 5B. In one embodiment, the nucleotide sequence encoding the chimeric human / mouse CD8α polypeptide and / or CD8β polypeptide comprises a sequence encoding a mouse CD8α and / or CD8β signal peptide, respectively.Alternatively, the nucleotide sequence may comprise a sequence encoding a human CD8α and / or CD8β signal peptide. In one embodiment, the mouse comprises a replacement of the nucleotide sequence encoding all or substantially all of the mouse CD8α and / or CD8β extracellular domains with a nucleotide sequence encoding all or substantially all of the human CD8α and / or CD8β extracellular domains, respectively.

[0187] In one embodiment, the mouse does not express functional endogenous mouse CD8α and / or CD8β polypeptides from its endogenous CD8 locus. In one embodiment, the mouse described herein comprises a chimeric human / mouse CD8 sequence in its germline.

[0188] In one embodiment, the mouse expressing the chimeric human / mouse CD8α and / or CD8β polypeptide retains the mouse CD8α and / or CD8β promoter and regulatory sequences, e.g., in the mouse, the nucleotide sequence encoding the chimeric human / mouse CD8 is operably linked to the endogenous mouse CD8 promoter and regulatory sequences. In one embodiment, these regulatory sequences retained in the mouse include sequences that regulate CD8 protein expression at the appropriate stage of T cell development. In one embodiment, the genetically modified mouse expresses the chimeric human / mouse CD8α and / or CD8β polypeptide in B cells or mature CD4 + The chimeric CD8 is not expressed on T cells or on any cells that do not normally express endogenous CD8, eg, immune cells.

[0189] The present invention also provides a genetically modified mouse comprising unrearranged human or humanized TCR variable loci, e.g., TCRα, TCRβ, TCRδ, and / or TCRγ variable loci, in its genome. In some embodiments, the unrearranged human or humanized TCR variable loci replace endogenous mouse TCR variable loci. In other embodiments, the unrearranged human or humanized TCR variable loci are present at a site in the genome other than the corresponding endogenous mouse TCR loci. In some embodiments, the human or humanized unrearranged TCR variable loci are operably linked to mouse TCR constant regions.

[0190] In one embodiment, a genetically modified mouse is provided, wherein the mouse comprises in its genome 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 mouse TCR α constant gene sequence, and an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a mouse TCR β constant gene sequence. In certain embodiments, the mouse comprises in its genome an unrearranged TCR α variable locus comprising a full repertoire of human Vα segments and a full repertoire of human Jα segments, operably linked to a mouse TCR α constant gene sequence, and an unrearranged TCR β variable locus comprising a full repertoire of human Vβ segments, a full repertoire of human Dβ segments, and a full repertoire of human Jβ segments, operably linked to a mouse TCR β constant gene sequence.

[0191] In some embodiments, an unrearranged TCR α variable locus comprising human TCR α variable region segments replaces an endogenous mouse TCR α variable locus, and an unrearranged TCR β variable locus comprising human TCR β variable region segments replaces an endogenous mouse TCR β variable locus. In some embodiments, the endogenous mouse Vα and Jα segments cannot rearrange to form a rearranged Vα / Jα sequence, and the endogenous mouse Vβ, Dβ, and Jβ segments cannot rearrange to form a rearranged Vβ / Dβ / Jβ sequence. In some embodiments, the human Vα and Jα segments rearrange to form a rearranged human Vα / Jα sequence, and the human Vβ, Dβ, and Jβ segments rearrange to form a rearranged human Vβ / Dβ / Jβ sequence.

[0192] The present invention also relates to a genetically modified mouse comprising in its genome a nucleic acid sequence encoding a chimeric MHC polypeptide, wherein the human portion of the chimeric MHC polypeptide is associated with a human extracellular domain of a chimeric T cell co-receptor disclosed herein. The genetically modified mouse disclosed herein may comprise a first nucleic acid sequence encoding a chimeric human / mouse MHC1, a second nucleic acid sequence encoding a chimeric human / mouse MHC1Iα, and / or a third nucleic acid sequence encoding a chimeric human / mouse MHC1Iβ polypeptide. The human portions of the chimeric MHC1I, MHC1Iα, and / or MHC1Iβ may comprise the extracellular domains of human MHC1I, MHC1Iα, and MHC1Iβ, respectively. In one embodiment, the mouse expresses functional chimeric human / mouse MHC1I, MHC1Iα, and MHC1Iβ polypeptides from its endogenous mouse MHC locus. In one embodiment, the mouse does not express functional mouse MHC polypeptides, e.g., functional mouse MHC1, MHCIIα, and MHCIIβ polypeptides, from its endogenous mouse MHC locus, hi other embodiments, the only MHC1 and MHCII expressed on the cell surface by the mouse are chimeric MHC1 and II.

[0193] In one embodiment, the human portion of the chimeric human / mouse MHCI polypeptide comprises the peptide-binding domain or extracellular domain of human MHCI (e.g., human HLA-A, e.g., human HLA-A2, e.g., human HLA-A2.1). In some embodiments, the mouse does not express the peptide-binding domain or extracellular domain of an endogenous mouse MHCI polypeptide from its endogenous mouse MHCI locus. The peptide-binding domain of human MHCI may comprise the α1 and α2 domains. Alternatively, the peptide-binding domain of human MHCI may comprise the α1, α2, and α3 domains. In one aspect, the extracellular domain of human MHCI comprises the extracellular domain of the human MHCI α chain. In one embodiment, the endogenous mouse MHCI locus is the H-2K (e.g., H-2Kb) locus, and the mouse portion of the chimeric MHCI polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. Thus, in one embodiment, a mouse of the present invention comprises, at its endogenous mouse H-C1 locus, a nucleic acid sequence encoding a chimeric human / mouse H-C1 polypeptide, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide and the mouse portion comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide, and the mouse expresses the chimeric human / mouse HLA-A2 / H-2Kb protein. In other embodiments, the mouse portion of the chimeric MHC1 polypeptide may be derived from other mouse MHC1s, e.g., H-2D, H-2L, etc. The human portion of the chimeric MHC1 polypeptide may be derived from other human MHC1s, e.g., HLA-B, HLA-C, etc. In one aspect, the mouse does not express a functional endogenous H-2K polypeptide from its endogenous mouse H-2K locus. In one embodiment, the mouse does not express a functional endogenous MHC polypeptide from its H-2D locus. In some embodiments, the mouse has been engineered to lack all or part of the endogenous H-2D locus, hi other embodiments, the only MHCI polypeptide expressed on the cell surface by the mouse is a chimeric human / mouse MHCI polypeptide.

[0194] In one embodiment, the human portion of the chimeric human / mouse MHCIIα polypeptide comprises the peptide-binding domain or extracellular domain of human MHCIIα, and the human portion of the chimeric human / mouse MHCIIβ polypeptide comprises the peptide-binding domain or extracellular domain of human MHCIIβ. In some embodiments, the mouse does not express the peptide-binding domain or extracellular domain of endogenous mouse α and / or β polypeptides from endogenous mouse loci (e.g., H-2A and / or H-2E loci). In some embodiments, the mouse comprises a genome lacking genes encoding functional MHC class II molecules, including H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, H-2Ea, and combinations thereof. In some embodiments, the only MHCII polypeptides expressed on the cell surface by the mouse are the chimeric human / mouse MHCII polypeptides. The peptide-binding domain of the human MHCIIα polypeptide may comprise the α1 domain, and the peptide-binding domain of the human MHCIIβ polypeptide may comprise the β1 domain. Thus, the peptide-binding domain of the chimeric MHCII complex may comprise human α1 and β1 domains. The extracellular domain of the human MHCIIα polypeptide may comprise α1 and α2 domains, and the extracellular domain of the human MHCIIβ polypeptide may comprise β1 and β2 domains. Thus, the extracellular domain of the chimeric MHCII complex may comprise human α1, α2, β1, and β2 domains. In one embodiment, the mouse portion of the chimeric MHCII complex comprises the transmembrane and cytoplasmic domains of mouse MHCII, e.g., mouse H-2E (e.g., the transmembrane and cytoplasmic domains of mouse H-2E α and β chains).Thus, in one embodiment, a mouse of the invention comprises, at its endogenous mouse MHCII locus, a nucleic acid sequence encoding a chimeric human / mouse MHCIIα, wherein the human portion of the chimeric MHCIIα polypeptide comprises an extracellular domain obtained from the α chain of human MHCII (e.g., the α chain of HLA-DR2), and the mouse portion comprises a transmembrane and cytoplasmic domain obtained from the α chain of mouse MHCII (e.g., H-2E); and the mouse comprises, at its endogenous mouse MHCII locus, a nucleic acid sequence encoding a chimeric human / mouse MHCIIβ, wherein the human portion of the chimeric MHCIIβ polypeptide comprises an extracellular domain obtained from the β chain of human MHCII (e.g., the β chain of HLA-DR2), and the mouse portion comprises a transmembrane and cytoplasmic domain obtained from the β chain of mouse MHC (e.g., H-2E); for example, the mouse expresses a chimeric human / mouse HLA-DR2 / H-2E protein. In other embodiments, the mouse portion of the chimeric MHCII protein may be derived from other mouse MHCIIs, such as H-2A, and the human portion of the chimeric MHCII protein may be derived from other human MHCIIs, such as HLA-DQ. In one aspect, the mouse does not express functional endogenous H-2A and H-2E polypeptides from its endogenous mouse locus (e.g., the mouse does not express H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea polypeptides). In some embodiments, the mouse lacks expression of any endogenous MHC1 or MHCII molecules on the cell surface.

[0195] In various embodiments, the human or humanized β2 microglobulin expressed by the genetically modified non-human animal or cells, embryos, or tissues obtained from the non-human animal preserves all functional aspects of endogenous and / or human β2 microglobulin. For example, the human or humanized β2 microglobulin preferably binds to the α chain of an MHCI polypeptide (e.g., an endogenous non-human or human MHCI polypeptide). The human or humanized β2 microglobulin polypeptide may also bind to, recruit, or associate with any other molecule, such as a receptor, anchor, or signaling molecule that associates with endogenous non-human and / or human β2 microglobulin (e.g., HFE).

[0196] In addition to genetically modified animals (e.g., rodents, e.g., mice or rats), tissues or cells obtained from non-human animals as described herein and containing a heterologous β2 microglobulin gene or β2 microglobulin sequence, i.e., a nucleic acid sequence and / or an amino acid sequence, are also provided. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or a human or humanized β2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express the MHCI complex, e.g., an antigen-presenting cell. The human or humanized β2 microglobulin polypeptide expressed by the cell may interact with endogenous non-human MHCI (e.g., rodent MHCI) to form a functional MHCI complex. The resulting MHCI complex may be capable of interacting with T cells, e.g., cytotoxic T cells. Thus, in vitro complexes of cells from non-human animals as described herein with T cells are also provided.

[0197] Also provided are non-human cells comprising a human or humanized β2 microglobulin gene or sequence and an additional human or humanized sequence, such as a presently disclosed chimeric MHCI polypeptide. In such instances, the human or humanized β2 microglobulin polypeptide may interact with, for example, a chimeric human / non-human MHCI polypeptide, and a functional MHCI complex may be formed. In some embodiments, such a complex can interact with a TCR on a T cell, e.g., a human or non-human T cell. Thus, in vitro complexes of cells from a non-human animal and human or non-human T cells as described herein are also provided.

[0198] Another aspect of the present disclosure is a rodent embryo (e.g., a mouse or rat embryo) comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence described herein. In one embodiment, the embryo comprises an ES donor cell comprising the heterologous β2 microglobulin gene or β2 microglobulin sequence and a host embryonic cell. The heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or β2 microglobulin sequence.

[0199] The invention also encompasses non-human cells comprising a chromosome or fragment thereof of a non-human animal as described herein (e.g., the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide). The non-human cell may comprise a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises a chromosome or fragment thereof as a result of nuclear transfer.

[0200] In one aspect, a non-human induced pluripotent cell is provided that comprises a heterologous β2 microglobulin gene or β2 microglobulin sequence. In one embodiment, the induced pluripotent cell is obtained from a non-human animal as described herein. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized gene or sequence.

[0201] In some embodiments of the invention, the mice described herein express chimeric human / mouse MHC II only on professional antigen-presenting cells of the mice, e.g., B cells, monocytes / macrophages, and / or dendritic cells. In some embodiments, the mice described herein mount an immune response, e.g., a cellular immune response, against one or more human antigens. In some embodiments, the mice described herein mount a humanized T cell response against one or more human antigens.

[0202] In addition to genetically modified non-human animals, non-human embryos (e.g., rodent, e.g., mouse or rat embryos) are also provided, which comprise donor ES cells derived from a non-human animal (e.g., rodent, e.g., mouse or rat) as described herein. In one embodiment, the embryo comprises an ES donor cell that comprises a chimeric CD4 gene, a chimeric CD8 (e.g., CD8α and / or CD8β) gene, a humanized MHC1 (e.g., MHC1α) nucleic acid sequence, a humanized MHCII (e.g., MHCIIα and / or MHCIIβ) nucleic acid sequence, a non-rearranged humanized TCR (e.g., TCRα and / or TCRβ, or TCRδ and / or TCRγ) locus, and / or a human or humanized β2 microglobulin gene sequence, and a host embryonic cell.

[0203] Also provided is tissue obtained from a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein, and expressing a chimeric CD4 protein, a chimeric CD8 protein (e.g., a chimeric CD8α and / or CD8β protein), a humanized TCR polypeptide (e.g., a TCRα and / or TCRβ, or a TCRδ and / or TCRγ polypeptide), a humanized MHC1 polypeptide (e.g., MHC1α), a humanized MHCII polypeptide (e.g., an MHCIIα and / or an MHCIIβ polypeptide), and / or a human or humanized β2 microglobulin.

[0204] In one aspect, a method for producing a chimeric human / non-human CD4 molecule is provided, comprising expressing a chimeric CD4 protein in a single cell from a nucleotide construct as described herein. 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 (e.g., PERC.6™ cells) that express a viral nucleic acid sequence.

[0205] In one aspect, a cell expressing a chimeric CD4 protein is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric CD4 sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells (e.g., PERC.6™ cells) that express a viral nucleic acid sequence.

[0206] Also provided is a chimeric CD4 molecule produced by a non-human animal as described herein, which in one embodiment comprises all or substantially all of the amino acid sequence of the extracellular domain of a human CD4 protein and at least the transmembrane and cytoplasmic domains from a non-human CD4 protein, such as, for example, a mouse CD4 protein. In another embodiment, a chimeric CD4 molecule is provided that is produced by a non-human animal as described herein, wherein the chimeric CD4 molecule comprises at least all or substantially all of the D1 domain of human CD4, e.g., at least all or substantially all of the D1-D2 domains of human CD4, e.g., at least all or substantially all of the D1-D3 domains of human CD4, e.g., the amino acid sequence of human CD4 responsible for binding to the extracellular domain of MHCII and / or TCR, e.g., the amino acid sequence of human CD4 responsible for binding to the variable domain of MHCII and / or TCR, and the remainder of the protein (e.g., the transmembrane domain, the cytoplasmic domain, any portion of the extracellular domain that is not humanized) is derived from the endogenous non-human protein sequence. An exemplary chimeric human / non-human CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:78, wherein the human portion of the chimeric polypeptide spans approximately amino acids 27-319 of SEQ ID NO:78 (separately set forth in SEQ ID NO:79).

[0207] In one aspect, a method is provided for producing a chimeric human / non-human CD8 molecule (e.g., CD8α and / or CD8β), comprising expressing the chimeric CD8 polypeptide in a single cell from a nucleotide construct as described herein. 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 (e.g., PERC.6™ cells) that express a viral nucleic acid sequence.

[0208] In one aspect, a cell expressing a chimeric CD8 protein is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric CD8 sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells (e.g., PERC.6™ cells) expressing a viral nucleic acid sequence.

[0209] Also provided are chimeric CD8 molecules produced by a non-human animal as described herein, which chimeric CD8 molecules comprise at least all or substantially all of the extracellular domain from a human CD8 protein (e.g., CD8α and / or CD8β) and the transmembrane and cytoplasmic domains from a non-human CD8 protein, e.g., a murine CD8 protein. An exemplary chimeric CD8α polypeptide is set forth in SEQ ID NO:88, and an exemplary chimeric CD8β protein is set forth in SEQ ID NO:83.

[0210] Also provided are humanized TCR proteins produced by a non-human animal (e.g., a rodent, e.g., a mouse or rat) as described herein, which comprise a human variable region and a non-human constant region. Thus, the humanized TCR protein comprises human complementarity-determining regions (i.e., human CDR1, 2, and 3) in its variable domain and non-human constant region. Also provided are nucleic acids encoding human TCR variable domains produced by the non-human animals described herein.

[0211] Additionally, provided are non-human cells isolated from a non-human animal as described herein. In one embodiment, the cells are ES cells. In one embodiment, the cells are T cells, e.g., CD4+ T cells. In one embodiment, the cells are helper T cells (T H In one embodiment, T H effector T cells H Cells, e.g., T H 1 cell or T H2 cells. In one embodiment, the cell is a CD8+ T cell. In one embodiment, the cell is a cytotoxic T cell. Non-human cells expressing a TCR protein comprising a human variable region and a non-human constant region are also provided. The TCR protein may comprise TCRα, TCRβ, or a combination thereof. In one embodiment, the cell is a T cell, e.g., a CD4+ or CD8+ T cell. Additionally, the non-human T cells provided herein may express on their cell surface (a) a chimeric human / non-human T cell co-receptor, e.g., a chimeric CD4 polypeptide or a chimeric CD8 polypeptide, comprising the extracellular domain of a human T cell co-receptor and operably linked to the transmembrane and / or intracellular domain of a non-human T cell co-receptor, and (b) a TCR protein comprising a human variable region and a non-human constant region.

[0212] In another embodiment, the cell is an antigen-presenting cell. In one embodiment, the antigen-presenting cell presents the antigen on a humanized MHC1 molecule. In another embodiment, the antigen-presenting cell is a professional antigen-presenting cell, such as a B cell, a dendritic cell, or a macrophage. In another embodiment, the antigen-presenting cell presents the antigen on a humanized MHC1 and / or a humanized MHCII molecule.

[0213] In one aspect, a cell expressing chimeric human / non-human MHC1 and MHCII proteins (e.g., HLA-A2 / H-2K and HLA-DR2 / H-2E proteins) is provided. In one aspect, the cell is a mouse cell that does not express a functional endogenous MHC polypeptide from its H-2D locus. In some embodiments, the cell is a mouse cell engineered to lack all or a portion of the endogenous H-2D locus. In some embodiments, the cell is a mouse cell that does not express any functional endogenous MHC1 and MHCII polypeptides on its surface. In one embodiment, the cell comprises an expression vector comprising a chimeric MHC class I sequence and a chimeric MHC class II sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells (e.g., PERC.6™ cells) that express viral nucleic acid sequences.

[0214] Chimeric MHCII complexes containing the extracellular domain of HLA-DR2 described herein can be detected with an anti-HLA-DR antibody. Thus, cells presenting chimeric human / non-human MHCII polypeptides can be detected and / or selected using an anti-HLA-DR antibody. Chimeric MHCII complexes containing the extracellular domain of HLA-A2 described herein can be detected with an anti-HLA-A, e.g., an anti-HLA-A2, antibody. Thus, cells presenting chimeric human / non-human MHCII polypeptides can be detected and / or selected using an anti-HLA-A antibody. Antibodies recognizing other HLA alleles are commercially available or can be produced and used for detection / selection.

[0215] Although the following examples describe genetically engineered animals whose genomes comprise replacement of nucleic acid sequences encoding mouse H-2K, H-2A, and H-2E proteins with nucleic acid sequences encoding chimeric human / mouse HLA-A2 / H-2K and HLA-DR2 / H-2E proteins, respectively, those skilled in the art will appreciate that similar strategies can be used to introduce chimeras comprising other human MHC I and II genes (other HLA-A, HLA-B, and HLA-C, and other HLA-DR, HLA-DP, and HLA-DQ genes). Also provided are such animals that comprise multiple chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) MHC I and MHC II genes at endogenous MHC loci. Examples of such chimeric MHC1 and MHCII proteins are described in U.S. Patent Application Publication Nos. 20130111617, 20130185819, 20130185820, and 20140245467, and U.S. Patent No. 8,847,005, each of which is incorporated herein by reference.

[0216] Also provided is a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as a result of nuclear transfer.

[0217] In one aspect, there is provided a non-human induced pluripotent cell comprising a gene encoding a chimeric CD4 polypeptide, a gene encoding a chimeric CD8 polypeptide (e.g., a CD8α and / or CD8β polypeptide), a gene encoding a humanized MHC1 polypeptide (e.g., MHC1α and / or β2 microglobulin), a gene encoding a humanized MHCII polypeptide (e.g., MHCIIα and / or MHCIIβ), and / or an unrearranged humanized TCR locus encoding a humanized TCRα and / or TCRβ polypeptide, as described herein. In one embodiment, the induced pluripotent cell is obtained from a non-human animal as described herein.

[0218] In one aspect, there is provided a hybridoma or quadroma obtained from a cell of a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or a rat. Production of genetically modified non-human animals that mount substantially humanized T cell immune responses

[0219] Also provided are methods for producing the genetically engineered non-human animals (e.g., genetically engineered rodents, e.g., mice or rats) described herein. Generally, the methods include: (a) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide, a second nucleotide sequence encoding a second chimeric human / non-human T cell co-receptor polypeptide, and / or a third nucleotide 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 the transmembrane and cytoplasmic domains of a non-human T cell co-receptor, and the human portion of each chimeric polypeptide comprises the extracellular portion (or a portion thereof) of a human T cell co-receptor; and (b) introducing into the genome of the non-human animal a nucleotide sequence comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCR α constant gene sequence. and / or an unrearranged TCR β variable locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR β constant gene sequence; and optionally, (c) placing within the genome 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; and / or (d) adding within the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the introducing, inserting, and / or positioning step comprises targeting a sequence encoding an extracellular domain of a T cell co-receptor, a variable domain of a TCR, an extracellular domain of an MHC polypeptide, or a portion of β2 microglobulin, and replacing it with the extracellular domain of a human T cell co-receptor, the variable domain of a human TCR, the extracellular domain of a human MHC, and / or a human portion of β2 microglobulin, respectively.

[0220] In other embodiments, the introducing, inserting, placing, and / or adding may involve crossbreeding, e.g., mating, animals of the same species. In other embodiments, the introducing, inserting, placing, and / or adding involves sequential homologous recombination in ES cells. In some embodiments, ES cells are obtained from a non-human animal that has been genetically modified to contain one or more, but not all, of the desired genetic modifications, and homologous recombination in such ES cells completes the genetic modifications. In other embodiments, the introducing, inserting, placing, and / or adding may involve a combination of crossbreeding and homologous recombination in ES cells, e.g., crossbreeding an animal with another animal(s) of the same species, and some or all of the animal may arise from genetically modified ES cells by a single homologous recombination or sequential homologous recombination events, and some ES cells may be isolated from a non-human animal containing one or more of the genetic modifications disclosed herein.

[0221] In some embodiments, the method utilizes a targeting construct generated using VELOCIGENE® technology, as described in the Examples, introducing the construct into ES cells, and introducing the targeted ES cell clones into mouse embryos using VELOCIMOUSE® technology. The targeting construct may include 5' and / or 3' homology arms that target the endogenous sequence to be replaced, an insert sequence (that replaces the endogenous sequence), and one or more selection cassettes. A selection cassette is a nucleotide sequence inserted into the targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. Many suitable selection cassettes are known in the art. Generally, the selection cassette allows for positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). Additionally, the selection cassette may be flanked by recombination sites, which allow for deletion of the selection cassette upon treatment with a recombinase enzyme. Commonly used recombination sites are loxP and Frt, which are recognized by the Cre and Flp enzymes, respectively, but others are well known in the art. The selection cassette may be located anywhere within the construct outside of the coding region. In one embodiment, the selection cassette is located at the 5' end of the human DNA fragment. In another embodiment, the selection cassette is located at the 3' end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment. In another embodiment, the selection cassette is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located at the junction of the human and mouse DNA fragments.

[0222] In one embodiment, a method for producing a genetically engineered non-human animal results in the animal comprising, in its endogenous CD4 locus, a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide. In one embodiment, the invention includes a method for modifying the CD4 locus of a non-human animal to express a chimeric human / non-human CD4 polypeptide described herein. In one embodiment, the invention provides a method for modifying the CD4 locus of a non-human animal, e.g., a mouse, to express a chimeric human / mouse CD4 polypeptide, comprising introducing a nucleotide sequence encoding an endogenous non-human CD4 polypeptide into the endogenous CD4 locus of the non-human animal, e.g., a mouse, by replacing the nucleotide sequence encoding the endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the chimeric human / mouse CD4 polypeptide. In one aspect of the method, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the extracellular domain of a human CD4 polypeptide and at least the transmembrane and cytoplasmic domains of an endogenous mouse CD4 polypeptide. In another aspect of the method, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the D1-D2 domain of a human CD4 polypeptide. In yet another embodiment, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the D1-D3 domains of a human CD4 polypeptide. In yet another embodiment, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the amino acid sequence of human CD4 responsible for interacting with MHCII and / or the extracellular domain of a T-cell receptor. In yet another embodiment, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the amino acid sequence of human CD4 responsible for interacting with MHCII and / or the variable domain of a T-cell receptor.

[0223] Thus, a nucleotide construct for generating a genetically modified animal comprising a chimeric human / non-human CD4 is provided. In one aspect, the nucleotide sequence comprises 5' and 3' homology arms, a DNA fragment comprising a human CD4 gene sequence (e.g., a human CD4 extracellular domain gene sequence, e.g., the gene sequence for all or substantially all of domains D1-D2 of human CD4, e.g., the gene sequence for all or substantially all of domains D1-D3 and / or D2-D3 of human CD4, e.g., the gene sequence for all or substantially all of domains D1-D4 of human CD4), and a selection cassette flanked by recombination sites. In one embodiment, the human CD4 gene sequence is a genomic sequence comprising introns and exons of human CD4. In one embodiment, the homology arms are homologous to a non-human (e.g., mouse) CD4 genomic sequence. An exemplary construct of the invention is shown in Figure 5A.

[0224] In some embodiments, the methods result in animals comprising, at their endogenous CD8 locus, a nucleotide sequence encoding a chimeric human / non-human CD8α and / or CD8β polypeptide. In one embodiment, the invention provides a method for modifying the CD8 locus of a non-human animal to express a chimeric human / non-human CD8 polypeptide described herein. In one aspect, a method for modifying the CD8 locus of a non-human animal, e.g., a mouse, to express a chimeric human / mouse CD8 polypeptide is provided, comprising introducing a nucleotide sequence encoding an endogenous non-human CD8 polypeptide into the endogenous CD8 locus of the non-human animal, e.g., a mouse, e.g., replacing the nucleotide sequence encoding the endogenous ...

Claims

1. A genetically modified mouse, the mouse having in its genome: (a) comprising a first nucleotide sequence encoding a chimeric CD8α polypeptide and a second nucleotide sequence encoding a chimeric CD8β polypeptide; the chimeric CD8α polypeptide comprises the extracellular portion of a human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of a murine CD8α polypeptide; the chimeric CD8β polypeptide comprises an extracellular portion of a human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of a murine CD8β polypeptide; the mouse expresses a chimeric CD8 co-receptor comprising the chimeric CD8α polypeptide and the chimeric CD8β polypeptide; (b) a third nucleotide sequence encoding a human or humanized MHC class I polypeptide; the mouse expresses the human or humanized MHC class I polypeptide; the human or humanized MHC class I polypeptide associates with the chimeric CD8 co-receptor; and (c) an unrearranged T cell receptor (TCR) α variable region sequence comprising at least one human Vα gene segment and at least one human Jα gene segment that rearrange in a T cell to form a rearranged human Vα / Jα sequence, operably linked to a murine TCR α constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one human Vβ gene segment that rearranges in a T cell to form a rearranged human Vβ / Dβ / Jβ sequence, at least one human Dβ gene segment, and at least one human Jβ gene segment, operably linked to a murine TCRβ constant region sequence; the rearranged human Vα / Jα sequence operably linked to the murine TCRα constant region sequence encodes a humanized TCRα chain comprising a human TCRα variable domain operably linked to a murine TCRα constant domain; the rearranged human Vβ / Dβ / Jβ sequence operably linked to the murine TCRβ constant region sequence encodes a humanized TCRβ chain comprising a human TCRβ variable domain operably linked to a murine TCRβ constant domain; the mouse expresses a T cell receptor on the surface of a T cell, the T cell receptor comprising the humanized TCR alpha chain and the humanized TCR beta chain; Genetically modified mice.

2. In its germline genome, (a) the first nucleotide sequence encoding the chimeric CD8α polypeptide and the second nucleotide sequence encoding the chimeric CD8β polypeptide; (b) said third nucleotide sequence encoding said human or humanized MHC class I polypeptide; and (c) said unrearranged TCRα variable region sequence comprising at least one human Vα gene segment and at least one human Jα gene segment, and said unrearranged TCRβ variable region sequence comprising at least one human Vβ gene segment, at least one human Dβ gene segment, and at least one human Jβ gene segment; The genetically modified mouse of claim 1 .

3. (a) the first nucleotide sequence is present in the endogenous CD8α locus and the second nucleotide sequence is present in the endogenous CD8β locus; (b) the third nucleotide sequence is present in an endogenous MHC class I locus; and / or (c) the unrearranged TCR alpha variable region sequence is present at an endogenous TCR alpha variable region locus and the unrearranged TCR beta variable region sequence is present at an endogenous TCR beta variable region locus. The genetically modified mouse of claim 1.

4. (a) the first nucleotide sequence is present in the endogenous CD8α locus and is operably linked to a mouse CD8α polypeptide promoter and regulatory elements, and the second nucleotide sequence is present in the endogenous CD8β locus and is operably linked to a mouse CD8β polypeptide promoter and regulatory elements; (b) the third nucleotide sequence is present in the endogenous MHC class I locus and is operably linked to a mouse MHC class I promoter and regulatory elements; and / or (c) both (a) and (b); The genetically modified mouse of claim 3.

5. (a) the chimeric CD8α polypeptide comprises the IgV-like domain of the human CD8α polypeptide operably linked to the transmembrane and cytoplasmic domains of the murine CD8α polypeptide, and the chimeric CD8β polypeptide comprises the IgV-like domain of the human CD8β polypeptide operably linked to the transmembrane and cytoplasmic domains of the murine CD8β polypeptide; (b) the human or humanized MHC class I polypeptide comprises the α1, α2, and α3 domains of an HLA class I polypeptide operably linked to the transmembrane and cytoplasmic domains of a murine MHC class I polypeptide; or (c) both (a) and (b); The genetically modified mouse of claim 1.

6. The genetically modified mouse of claim 5, wherein the HLA class I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C.

7. The genetically modified mouse of claim 6, wherein the HLA class I polypeptide is HLA-A.

8. The genetically modified mouse of claim 1, wherein the unrearranged TCRα variable region sequence comprises a repertoire of human Vα gene segments and a repertoire of human Jα gene segments, and / or the unrearranged TCRβ variable region sequence comprises a repertoire of human Vβ gene segments, a repertoire of human Dβ gene segments, and a repertoire of human Jβ gene segments.

9. the endogenous TCRα variable region locus lacks all functional endogenous Vα gene segments and / or lacks all functional endogenous Jα gene segments, and / or The genetically modified mouse of claim 1, wherein the endogenous TCRβ variable region locus (a) lacks all functional endogenous Vβ gene segments, (b) lacks all functional endogenous Dβ gene segments, (c) lacks all functional endogenous Jβ gene segments, or (d) any combination of (a), (b), and (c).

10. (a) the first nucleotide sequence comprises a sequence encoding the extracellular portion of the human CD8α polypeptide, (i) replacing a sequence encoding the extracellular portion of the murine CD8α polypeptide, and (ii) operably linked to a murine CD8α transmembrane and cytoplasmic domain-encoding sequence at the endogenous CD8α locus; and the second nucleotide sequence (i) replaces a sequence encoding the extracellular portion of the mouse CD8β polypeptide at the endogenous CD8β locus, and (ii) comprises a sequence encoding the extracellular portion of the human CD8β polypeptide operably linked to a mouse CD8β transmembrane and cytoplasmic domain encoding sequence. (b) the third nucleotide sequence comprises a sequence encoding the extracellular portion of an HLA class I polypeptide that (i) replaces a sequence encoding the extracellular portion of a murine MHC class I polypeptide and (ii) is operably linked to a sequence encoding the transmembrane and cytoplasmic domains of a murine MHC class I polypeptide at the endogenous MHC class I locus; and / or (c) the unrearranged TCRα variable region sequence replaces one or more endogenous Vα and / or Jα gene segments at the endogenous TCRα variable region locus, and the unrearranged TCRβ variable region sequence replaces one or more endogenous Vβ, Dβ and / or Jβ gene segments at the endogenous TCRβ variable region locus. The genetically modified mouse of claim 1.

11. The mouse (a) a functional mouse CD8 polypeptide from the endogenous CD8 locus; (b) a murine TCRα variable domain from the endogenous TCRα locus; (c) a mouse TCRβ variable domain from the endogenous TCRβ locus, and / or (e) the extracellular domain of a classical mouse MHC class I polypeptide from the endogenous MHC class I locus. The genetically modified mouse of claim 1, which does not express 12. The method of claim 1, further comprising: providing a β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide; The genetically modified mouse of claim 1 , wherein the genetically modified mouse expresses the human or humanized β2 microglobulin polypeptide.

13. The genetically modified mouse of claim 12, wherein the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide is operably linked to a mouse β2 microglobulin regulatory element.

14. The genetically modified mouse of claim 12, wherein the β2 microglobulin locus comprises the nucleotide sequence shown in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.

15. The genetically modified mouse of claim 14, wherein the β2 microglobulin locus further comprises a nucleotide sequence shown in exon 1 of the mouse β2 microglobulin gene.

16. The genetically modified mouse of claim 1, wherein the genetically modified mouse expresses at least 50% of all functional human TCRV alpha gene segments and / or at least 50% of all functional human TCRV beta gene segments. (a) the chimeric CD8α polypeptide comprises an IgV-like domain of the human CD8α polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of the mouse CD8α polypeptide, and the chimeric CD8β polypeptide comprises an IgV-like domain of the human CD8β polypeptide operably linked to a transmembrane domain and a cytoplasmic domain of the CD8β polypeptide; (b) the human or humanized MHC class I polypeptide comprises the α1, α2, and α3 domains of an HLA class I polypeptide operably linked to the transmembrane and cytoplasmic domains of a murine MHC class I polypeptide; and / or (c) the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence set forth in exon 1 of the mouse β2 microglobulin gene operably linked to a nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene; The genetically modified mouse of claim 12.

18. A method for producing the genetically modified mouse according to claim 1, comprising: (a) the first and second nucleotide sequences encoding the chimeric CD8α polypeptide and the chimeric CD8β polypeptide, respectively; (b) said third nucleotide sequence encoding said human or humanized MHC class I polypeptide; and (c) said unrearranged TCRα variable region sequence comprising at least one human Vα gene segment and at least one human Jα gene segment, and said unrearranged TCRβ variable region sequence comprising at least one human Vβ gene segment, at least one human Dβ gene segment, and at least one human Jβ gene segment; modifying the genome of the mouse to comprise:

19. 19. The method of claim 18, wherein modifying the genome comprises homologous recombination in one or more mouse embryonic stem cells, thereby adding, in any order, the first, second, and third nucleotide sequences, and the unrearranged TCR alpha variable region sequence and the unrearranged TCR beta variable region sequence into the genome of the mouse embryonic stem cell.

20. the first, second, and third nucleotide sequences, the unrearranged TCR alpha variable region sequence, and the unrearranged TCR beta variable region sequence.

20. The method of claim 19, further comprising generating a mouse from the mouse embryonic stem cells comprising:

21. 1. A method for obtaining a cell that expresses a TCR protein that is specific for an antigen and comprises a human TCR variable domain, comprising: The method comprises: A step of isolating T cells that are specific to an antigen and express a TCR protein containing both a human TCR alpha variable domain and a human TCR beta variable domain from the genetically modified mouse of claim 1. A method comprising:

22. 1. A method for obtaining a TCR protein specific for an antigen and comprising a human TCR variable domain, comprising: The method comprises: Obtaining T cells that are specific to an antigen and express a TCR protein containing both a human TCR alpha variable domain and a human TCR beta variable domain from the genetically modified mouse of claim 1; isolating the TCR protein. A method comprising:

23. 1. A method for obtaining a human TCR variable domain of a TCR protein specific for an antigen, comprising: The method comprises: Obtaining a TCR protein specific to an antigen and comprising both a human TCR alpha variable domain and a human TCR beta variable domain from the genetically modified mouse of claim 1; isolating the human TCR alpha variable domain and / or the human TCR beta variable domain. A method comprising:

24. 1. A method for obtaining a nucleic acid sequence encoding a human TCR variable domain of a TCR protein specific for an antigen, comprising: The method comprises: Obtaining a TCR protein specific to an antigen and comprising both a human TCR alpha variable domain and a human TCR beta variable domain from the genetically modified mouse of claim 1; isolating a nucleic acid sequence encoding the human TCR alpha variable domain and / or a nucleic acid sequence encoding the human TCR beta variable domain. A method comprising:

25. introducing into a host cell one or more expression vectors comprising (i) a nucleic acid sequence encoding said human TCR alpha variable domain operably linked to a human TCR alpha constant region and / or (ii) a nucleic acid sequence encoding said human TCR beta variable domain operably linked to a human TCR beta constant region; culturing said host cells under conditions sufficient to express (i) and / or (ii). Further comprising: the nucleic acid sequences encoding the human TCR alpha variable domain and the human TCR beta variable domain are on the same or different expression vectors; The method according to any one of claims 21 to 24.

26. 26. The method of claim 25, wherein the one or more expression vectors comprise the nucleic acid sequence encoding the human TCR alpha variable domain operably linked to a sequence encoding a human TCR alpha constant region, and the nucleic acid sequence encoding the human TCR beta variable domain operably linked to a sequence encoding a human TCR beta constant region.

27. The method of any one of claims 21 to 24, wherein the antigen is a tumor antigen, a viral antigen, or a bacterial antigen.

28. A T cell that expresses a TCR protein comprising a human TCR variable domain specific for an antigen, the T cell comprising in its genome: (a) comprising a first nucleotide sequence encoding a chimeric CD8α polypeptide and a second nucleotide sequence encoding a chimeric CD8β polypeptide; the chimeric CD8α polypeptide comprises the extracellular portion of a human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of a murine CD8α polypeptide; the chimeric CD8β polypeptide comprises an extracellular portion of a human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of a murine CD8β polypeptide; (b) a third nucleotide sequence encoding a human or humanized MHC class I polypeptide; and (c) a rearranged human Vα / Jα sequence operably linked to a murine TCRα constant region sequence, encoding a humanized TCRα chain comprising a human TCRα variable domain operably linked to a murine TCRα constant domain, and a rearranged human Vβ / Dβ / Jβ sequence operably linked to a murine TCRβ constant region sequence, encoding a humanized TCRβ chain comprising a human TCRβ variable domain operably linked to a murine TCRβ constant domain; and the TCR protein comprises the humanized TCR alpha chain and the humanized TCR beta chain; T cells.

29. A hybridoma expressing a TCR protein comprising a human TCR variable domain specific to an antigen, wherein the hybridoma is produced from a T cell expressing a TCR protein comprising a human TCR variable domain specific to the antigen; The hybridoma (a) comprising a first nucleotide sequence encoding a chimeric CD8α polypeptide and a second nucleotide sequence encoding a chimeric CD8β polypeptide; the chimeric CD8α polypeptide comprises the extracellular portion of a human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of a murine CD8α polypeptide; the chimeric CD8β polypeptide comprises the extracellular portion of a human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of a mouse CD8β polypeptide; and (b) a rearranged human Vα / Jα sequence operably linked to a murine TCRα constant region sequence, encoding a humanized TCRα chain comprising a human TCRα variable domain operably linked to a murine TCRα constant domain, and a rearranged human Vβ / Dβ / Jβ sequence operably linked to a murine TCRβ constant region sequence, encoding a humanized TCRβ chain comprising a human TCRβ variable domain operably linked to a murine TCRβ constant domain; and the TCR protein comprises the humanized TCR alpha chain and the humanized TCR beta chain; Hybridoma.

30. A genetically modified mouse embryonic stem cell, wherein the mouse embryonic stem cell has in its genome: (a) comprising a first nucleotide sequence encoding a chimeric CD8α polypeptide and a second nucleotide sequence encoding a chimeric CD8β polypeptide; the chimeric CD8α polypeptide comprises the extracellular portion of a human CD8α polypeptide and at least the transmembrane and cytoplasmic domains of a mouse CD8α polypeptide; the chimeric CD8β polypeptide comprises an extracellular portion of a human CD8β polypeptide and at least the transmembrane and cytoplasmic domains of a mouse CD8β polypeptide; (b) a third nucleotide sequence encoding a human or humanized MHC class I polypeptide; and (c) an unrearranged TCRα variable region sequence comprising at least one human Vα gene segment and at least one human Jα gene segment, which are capable of rearranging in a T cell to form a rearranged human Vα / Jα sequence, operably linked to a murine TCR) α constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one human Vβ gene segment, at least one human Dβ gene segment, and at least one human Jβ gene segment, which are capable of rearrangement in a T cell to form a rearranged human Vβ / Dβ / Jβ sequence, operably linked to a murine TCRβ constant region sequence. Genetically modified mouse embryonic stem cells.

31. (a) the first nucleotide sequence is present in the endogenous CD8α locus and the second nucleotide sequence is present in the endogenous CD8β locus; (b) the third nucleotide sequence is present in an endogenous MHC class I locus; and / or (c) the unrearranged TCR alpha variable region sequence is present at an endogenous TCR alpha variable region locus and the unrearranged TCR beta variable region sequence is present at an endogenous TCR beta variable region locus. The genetically modified mouse embryonic stem cell of claim 30.

32. (a) the first nucleotide sequence is present in the endogenous CD8α locus and is operably linked to a mouse CD8α polypeptide promoter and regulatory elements, and the second nucleotide sequence is present in the endogenous CD8β locus and is operably linked to a mouse CD8β polypeptide promoter and regulatory elements; (b) the third nucleotide sequence is present in the endogenous MHC class I locus and is operably linked to a mouse MHC class I promoter and regulatory elements; or (c) both (a) and (b); The genetically modified mouse embryonic stem cell of claim 31.

33. (a) the chimeric CD8α polypeptide comprises the IgV-like domain of the human CD8α polypeptide operably linked to the transmembrane and cytoplasmic domains of the murine CD8α polypeptide, and the chimeric CD8β polypeptide comprises the IgV-like domain of the human CD8β polypeptide operably linked to the transmembrane and cytoplasmic domains of the murine CD8β polypeptide; (b) the human or humanized MHC class I polypeptide comprises the α1, α2, and α3 domains of an HLA class I polypeptide operably linked to the transmembrane and cytoplasmic domains of a mouse MHC class I polypeptide; The genetically modified mouse embryonic stem cell of claim 30.

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