Genetically engineered T cell receptor mice

Abstract

Provided is a non-human animal (e.g., a rodent) that comprises unrearranged human T-cell variable region gene segments that can be rearranged to form a gene encoding a human T-cell receptor variable domain, the gene comprising domains that are cognate to each other and that specifically bind to an antigen of interest. The present invention provides a non-human animal (e.g., a rodent) comprising non-human cells that express humanized molecules that function in a cellular immune response. The present invention also provides a non-human animal comprising an unrearranged TCR variable locus.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 61 / 552,582, filed October 28, 2011; U.S. Provisional Application No. 61 / 621,198, filed April 6, 2012; and U.S. Provisional Application No. 61 / 700,908, filed September 14, 2012, all of which are incorporated by reference in their entireties.

[0002] The present invention relates to genetically modified non-human animals, e.g., rodents (e.g., mice or rats), that comprise human or humanized T cell receptor (TCR) variable loci (e.g., TCR alpha and TCR beta variable loci and / or TCR delta and TCR gamma variable loci) in their genome and express human or humanized TCR polypeptides (e.g., TCR alpha and TCR beta polypeptides and / or TCR delta and TCR gamma polypeptides) from the human or humanized TCR variable loci. Non-human animals having human or humanized TCR variable loci of the invention comprise unrearranged human TCR variable region gene segments (e.g., V, D and / or J segments) at the endogenous non-human TCR locus. The present invention also relates to embryos, tissues and cells (e.g., T cells) that comprise human or humanized TCR variable loci and express human or humanized TCR polypeptides. Methods for making genetically modified non-human animals that contain human or humanized TCR variable loci; and methods of using the non-human animals, embryos, tissues and cells that contain human or humanized TCR variable loci and express human or humanized TCR polypeptides from these loci are also provided. [Background technology]

[0003] In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptors or TCRs). Pathogens in the blood and extracellular space are recognized by antibodies during the humoral immune response, whereas destruction of pathogens inside cells is mediated by T cells during the cellular immune response.

[0004] T cells recognize and attack antigens presented to them in the context of major histocompatibility complexes (MHC) on the cell surface. Antigen recognition is mediated by the TCR expressed on the surface of the T cell. Two main types of T cells perform this function: cytotoxic T cells, which express the cell surface protein CD8, and helper T cells, which express the cell surface protein CD4. Cytotoxic T cells activate a signaling cascade that leads to the direct destruction of cells presenting the antigen (in the context of MHC I), whereas helper T cells differentiate into several types, and their activation (primed by recognition of antigens presented in the context of MHC II) leads to macrophage-mediated destruction of pathogens and stimulates antibody production by B cells.

[0005] Antibodies, due to their antigen specificity, are currently being widely studied for their therapeutic potential for a number of human disorders. To create antibodies that can neutralize human targets while avoiding the activation of immune responses against such antibodies, scientists are focusing their efforts on generating human or humanized immunoglobulins. One method of generating humanized antibodies in vivo is by using VELOCIMMUNE® mice, a humanized mouse that (1) contains a repertoire of unrearranged human immunoglobulin V, D, and J segments operably linked to each other and to mouse constant regions at the endogenous mouse immunoglobulin heavy chain locus, and (2) contains a repertoire of unrearranged human Vκ and Jκ segments operably linked to each other and to mouse constant κ regions at the endogenous mouse immunoglobulin κ light chain locus. Thus, VELOCIMMUNE® mice provide a rich source of diverse rearranged antibody variable domains for use in generating human antibodies.

[0006] Like antibodies, T cell receptors contain variable regions encoded by unrearranged loci (alpha and beta loci or delta and gamma loci) that contain V(D)J variable region segments, which confer antigen-binding specificity to T cells. Also like antibodies, TCR specificity for antigens can be exploited to develop novel therapies. Thus, there is a need in the art for non-human animals (e.g., rodents, e.g., rats or mice) that contain unrearranged human T cell variable region gene segments that can be rearranged to form genes encoding human T cell receptor variable domains that contain domains that are cognate to each other and that specifically bind to an antigen of interest. There is also a need for non-human animals that contain T cell variable region loci that contain conservative humanization, e.g., non-human animals that contain unrearranged human gene segments that can be rearranged to form T cell receptor variable region genes linked to non-human (endogenous) T cell receptor constant gene sequences. There is also a need for non-human animals that can produce a diverse repertoire of human T cell receptor variable sequences. There is a need for non-human animals that are capable of rearranging most or all functional T cell receptor variable region segments in response to an antigen of interest to form a T cell receptor polypeptide that contains a completely human variable domain. Summary of the Invention [Means for solving the problem]

[0007] Non-human animals (e.g., rodents) are provided that include non-human cells that express humanized molecules that function in a cellular immune response. Also provided are non-human animals that include unrearranged TCR variable loci. In vivo and in vitro systems are provided that include humanized rodent cells, where the rodent cells express one or more humanized immune system molecules. Also provided are unrearranged humanized TCR rodent loci that encode humanized TCR proteins.

[0008] In one embodiment, provided herein is a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) that comprises in its genome: (a) an unrearranged TCR alpha variable locus comprising at least one human Valpha segment and at least one human Jalpha segment operably linked to a non-human (e.g., a rodent, e.g., a mouse or rat) TCR alpha constant gene sequence; and / or (b) an unrearranged TCR beta variable locus comprising at least one human Vbeta segment, at least one human Dbeta segment, and at least one human Jbeta segment operably linked to a non-human (e.g., a rodent, e.g., a mouse or rat) TCR beta constant gene sequence.

[0009] In one embodiment, the unrearranged TCR alpha variable locus replaces an endogenous non-human (e.g., rodent) TCR alpha variable locus at the endogenous TCR alpha variable locus. In one embodiment, the unrearranged TCR beta variable locus replaces an endogenous non-human (e.g., rodent) TCR beta variable locus at the endogenous TCR beta variable locus. In one embodiment, the endogenous non-human (e.g., rodent) V alpha and J alpha segments are incapable of rearrangement to form a rearranged V alpha / J alpha sequence. In one embodiment, the endogenous non-human (e.g., rodent) V beta, D beta and J beta segments are incapable of rearrangement to form a rearranged V beta / D beta / J beta sequence. In one embodiment, the non-human animal comprises a deletion such that the genome of the animal does not comprise a functional V alpha and a functional J alpha segment. In one embodiment, the non-human animal comprises a deletion such that the genome of the animal does not comprise a functional endogenous Vβ, a functional endogenous Dβ and a functional endogenous Jβ segment. In one embodiment, the animal comprises a deletion of all functional endogenous Vα and Jα segments. In one embodiment, the rodent comprises a deletion of all functional endogenous Vβ, Dβ and Jβ segments. In some embodiments, the human Vα segments and the human Jα segments are rearranged to form a rearranged Vα / Jα sequence. In some embodiments, the human Vβ segments, the human Dβ segments and the human Jβ segments are rearranged to form a rearranged Vβ / Dβ / Jβ sequence. Thus, in various embodiments, the non-human animal (e.g., rodent) expresses a T cell receptor comprising a human variable region and a non-human (e.g., rodent) constant region on the surface of the T cell.

[0010] In some embodiments, the T cells of the non-human animal undergo T cell development in the thymus to generate CD4 single positive T cells and CD8 single positive T cells. In some embodiments, the non-human animal comprises a normal ratio of splenic CD3+ T cells to total splenocytes. In various embodiments, the non-human animal produces a population of central memory T cells and effector memory T cells in the periphery.

[0011] In one embodiment, the unrearranged TCR alpha variable locus in the non-human animal described herein comprises 61 human Jalpha segments and 8 human Valpha segments, hi another embodiment, the unrearranged TCR alpha variable locus in the non-human animal comprises a complete repertoire of human Jalpha segments and a complete repertoire of human Valpha segments.

[0012] In one embodiment, the unrearranged TCR beta variable locus in the non-human animal described herein comprises 14 human J beta segments, 2 human D beta segments and 14 human V beta segments. In another embodiment, the unrearranged TCR beta variable locus in the non-human animal comprises a complete repertoire of human J beta segments, a complete repertoire of human D beta segments and a complete repertoire of human V beta segments.

[0013] In a further embodiment, the non-human animal (e.g., rodent) described herein further comprises a nucleotide sequence of a human TCR delta variable segment at the humanized TCR alpha locus. In one embodiment, the non-human animal (e.g., rodent) further comprises at least one human V delta, D delta and J delta segment at the humanized TCR alpha locus, e.g., a complete repertoire of human V delta, D delta and J delta segments.

[0014] In one embodiment, the non-human animal retains an endogenous non-human TCR α and / or TCR β locus, and the locus is a non-functional locus.

[0015] In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the rodent is a mouse.

[0016] In one aspect, the invention provides a genetically modified mouse comprising in its genome: (a) an unrearranged TCR α variable locus comprising a repertoire of human Jα segments and a repertoire of human Vα segments operably linked to a non-human (e.g., mouse or rat) TCR α constant gene sequence; and / or (b) an unrearranged TCR β variable locus comprising a repertoire of human Jβ segments, a repertoire of human Dβ segments, and a repertoire of human Vβ segments operably linked to a non-human (e.g., mouse or rat) TCR β constant gene sequence. In one embodiment, the mouse comprises a complete repertoire of human Vα segments. In one embodiment, the mouse comprises a complete repertoire of human Vβ segments. In one embodiment, the mouse comprises a complete repertoire of human Vα segments and human Jα segments. In one embodiment, the mouse comprises a complete repertoire of human Vα segments and human Vβ segments. In one embodiment, the mouse comprises a complete repertoire of human Vα, human Jα, human Vβ, human Dβ, and human Jβ segments.

[0017] In one embodiment, the mouse comprises at least one endogenous mouse Vα segment and at least one endogenous mouse Jα segment, the endogenous segments being incapable of rearrangement to form a rearranged Vα / Jα sequence, and also comprises at least one endogenous mouse Vβ segment, at least one endogenous mouse Dβ segment, and at least one endogenous mouse Jβ segment, the endogenous segments being incapable of rearrangement to form a rearranged Vβ / Dβ / Jβ sequence.

[0018] In one embodiment, an unrearranged TCR alpha variable locus comprising a human TCR alpha variable region segment replaces the mouse TCR alpha variable genes at the endogenous mouse TCR alpha variable locus, and an unrearranged TCR beta variable locus comprising a human TCR beta variable region segment replaces the mouse TCR beta variable genes at the endogenous mouse TCR beta variable locus.

[0019] In one embodiment, the human Vα and Jα segments are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ, Dβ and Jβ segments are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. In one embodiment, the rearranged human Vα / Jα sequence is operably linked to a mouse TCR α constant region sequence. In one embodiment, the rearranged human Vβ / Dβ / Jβ sequence is operably linked to a mouse TCR β constant region sequence. Thus, in various embodiments, the mouse expresses a T cell receptor on the surface of the T cell, the T cell receptor comprising a human variable region and a mouse constant region.

[0020] In one embodiment, the mouse further comprises a repertoire of human TCR delta variable region segments (e.g., human V delta, J delta and D delta segments) at the humanized TCR alpha locus. In one embodiment, the repertoire of human TCR delta variable region segments is a complete human TCR delta variable region segment repertoire. In one embodiment, the human TCR delta variable region segments are at the endogenous TCR alpha locus. In one embodiment, the human TCR delta variable region segments replace endogenous mouse TCR delta variable region segments.

[0021] In one embodiment, the genetically modified mouse expresses a T cell receptor comprising a human variable region and a mouse constant region on the surface of T cells.In one embodiment, the T cells of the mouse undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells.In one embodiment, the mouse contains a normal ratio of splenic CD3+ T cells to total splenocytes, and in one embodiment, the mouse produces a population of central memory T cells and effector memory T cells against an antigen of interest.

[0022] Methods for producing the genetically modified non-human animals described herein (eg, rodents, such as mice or rats) are also provided.

[0023] In one embodiment, a method for making a humanized rodent (e.g., a mouse or rat) is provided that includes replacing rodent TCR alpha and TCR beta variable region segments, rather than rodent constant genes, with human unrearranged TCR alpha and TCR beta variable region segments at the endogenous rodent TCR locus. In one embodiment, the method comprises replacing rodent TCR alpha variable region segments (Valpha and / or Jalpha) with human TCR alpha variable region segments (Valpha and / or Jalpha), where the TCR alpha variable region segments are operably linked to a non-human TCR constant region gene to form a humanized TCR alpha locus, and replacing rodent TCR beta variable region segments (Vbeta and / or Dbeta and / or Jbeta) with human TCR beta variable region segments (Vbeta and / or Dbeta and / or Jbeta), where the TCR beta variable region segments are operably linked to a non-human TCR constant region gene to form a humanized TCR beta locus. In one embodiment, the humanized rodent is a mouse, and the germline of the mouse comprises a human TCR alpha variable region segment operably linked to an endogenous mouse TCR alpha constant sequence at the endogenous TCR alpha locus, and the germline of the mouse comprises a human TCR beta variable region segment operably linked to an endogenous mouse TCR beta constant sequence at the endogenous TCR beta locus.

[0024] In one embodiment, a method for generating a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a T cell receptor comprising a human or humanized variable region and a non-human (e.g., rodent) constant region on the surface of a T cell, comprising: replacing in a first 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, wherein the humanized TCR α variable locus is operably linked to the endogenous non-human TCR α constant region. replacing in a second non-human animal an endogenous non-human TCR β variable locus 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, wherein said humanized TCR β variable locus is operably linked to an endogenous TCR β constant region; and mating said first and said second non-human animals to obtain a non-human animal expressing a T cell receptor comprising a human or humanized variable region and a non-human constant region.

[0025] In one embodiment of this method, endogenous non-human (e.g., rodent) Vα and Jα segments cannot rearrange to form rearranged Vα / Jα sequences, and endogenous non-human (e.g., rodent) Vβ, Dβ and Jβ segments cannot rearrange to form rearranged Vβ / Dβ / Jβ sequences. In one embodiment of this method, human Vα segments and human Jα segments are rearranged to form rearranged Vα / Jα sequences, and human Vβ segments, human Dβ segments and human Jβ segments are rearranged to form rearranged Vβ / Dβ / Jβ sequences. In one embodiment of this method, the unrearranged humanized TCR α variable locus comprises 61 human Jα segments and 8 human Vα segments, and the unrearranged humanized TCR β variable locus comprises 14 human Vβ segments, 2 human Dβ segments and 14 human Jβ segments. In another embodiment of this method, the unrearranged humanized TCR alpha variable locus comprises a complete repertoire of human Jalpha segments and a complete repertoire of human Valpha segments, and the unrearranged humanized TCR beta variable locus comprises a complete repertoire of human Vbeta segments, a complete repertoire of human Dbeta segments, and a complete repertoire of human Jbeta segments.

[0026] In one embodiment of this method, the T cells of the non-human animal (e.g., rodent) undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells. In one embodiment, the non-human animal (e.g., rodent) comprises a normal ratio of splenic CD3+ T cells to total splenocytes. In one embodiment, the non-human animal (e.g., rodent) produces a population of central memory T cells and effector memory T cells against the antigen of interest.

[0027] In some embodiments, the replacement of the endogenous non-human TCR alpha variable locus described herein is performed in a single ES cell and the single ES cell is introduced into a non-human (e.g., rodent, e.g., mouse or rat) embryo to generate a genetically modified non-human animal (i.e., a first non-human animal, e.g., a first rodent); the replacement of the endogenous non-human TCR beta variable locus described herein is performed in a single ES cell and the single ES cell is introduced into a non-human (e.g., rodent, e.g., mouse or rat) embryo to generate a genetically modified non-human animal (i.e., a second non-human animal, e.g., a second rodent). In one embodiment, the first rodent and the second rodent are mated to form progeny, which comprise a humanized TCR alpha variable locus and a humanized TCR beta variable locus in their germline.

[0028] In one embodiment of this method, the non-human animal is a rodent, such as a mouse. Thus, the present invention also provides a method for producing a genetically modified mouse.

[0029] Also provided herein are cells, e.g., isolated T cells (e.g., cytotoxic T cells, helper T cells, memory T cells, etc.) derived from the non-human animals described herein (e.g., rodents, e.g., mice or rats). Tissues and embryos derived from the non-human animals described herein are also provided.

[0030] In one aspect, a method is provided for producing human TCR variable domains comprising genetically modifying a rodent as described herein to comprise a humanized TCR alpha locus and / or a humanized TCR beta locus, and maintaining the rodent under conditions sufficient to generate T cells, wherein the T cells express human TCR alpha and / or human TCR beta variable domains.

[0031] In one aspect, there is provided a method for generating a nucleic acid sequence encoding a human TCR variable domain that binds an epitope of interest, comprising exposing a non-human animal as described herein to the epitope of interest, maintaining said non-human animal under conditions sufficient for said animal to present the epitope of interest to its humanized TCR, and identifying a nucleic acid from said animal that encodes a human TCR variable domain polypeptide that binds the epitope of interest.

[0032] In one embodiment, there is provided the use of the non-human animal described herein for producing a humanized TCR receptor.In one embodiment, there is provided the use of the non-human animal described herein for producing a human TCR variable domain.In one embodiment, there is provided the use of the non-human animal described herein for producing a nucleic acid sequence encoding a human TCR variable domain.

[0033] In one aspect, there is provided the use of a nucleic acid sequence encoding a human TCR variable domain or a fragment thereof to generate an antigen binding protein. In one embodiment, the antigen binding protein comprises a TCR variable domain comprising a human TCR alpha and / or a human TCR beta variable domain that binds to an antigen of interest.

[0034] In one embodiment, there is provided the use of a non-human cell as described herein to generate a non-human cell which expresses a humanized T cell receptor on its surface.

[0035] In one embodiment, there is provided a humanized T cell receptor derived from a non-human animal as described herein.

[0036] In one aspect there is provided a nucleic acid sequence encoding a human TCR variable domain or a fragment thereof, produced in a non-human animal as described herein.

[0037] All of the embodiments and aspects described herein can be used in conjunction with one another unless otherwise indicated or clear from the context. Other embodiments will become apparent to those skilled in the art upon review of the detailed description set forth below. The following detailed description is illustrative of various embodiments of the invention and does not limit the invention as claimed. The accompanying drawings constitute a part of this specification and, together with the description, serve solely to illustrate embodiments and not to limit the invention. In certain embodiments, for example, the following are provided: (Item 1) A genetically modified non-human animal comprising in its genome an unrearranged T cell receptor (TCR) alpha variable locus comprising at least one human V alpha segment and at least one human J alpha segment operably linked to a non-human TCR alpha constant gene sequence. (Item 2) 2. The animal of item 1, wherein the unrearranged TCR alpha variable locus replaces an endogenous non-human TCR alpha variable locus. (Item 3) The animal of item 1, wherein the endogenous non-human Vα segment and the endogenous non-human Jα segment are incapable of rearrangement to form a rearranged Vα / Jα sequence. (Item 4) 2. The animal of item 1, which lacks a functional endogenous non-human TCR alpha variable locus. (Item 5) 5. The animal of item 4, wherein the lack of a functional endogenous non-human TCR alpha variable locus comprises a deletion selected from the group consisting of: (a) a deletion of all endogenous V alpha gene segments, (b) a deletion of all endogenous J alpha gene segments, and (c) a combination thereof. (Item 6) The animal of item 1, wherein the human Vα segment and the human Jα segment are rearranged to form a rearranged human Vα / Jα sequence. (Item 7) The animal of item 6, which expresses a T cell receptor comprising a human TCR alpha variable region on the surface of the T cell. (Item 8) The animal of item 1, wherein the T cells of the animal undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells. (Item 9) The animal described in item 1, comprising a normal ratio of splenic CD3+ T cells to total splenocytes. (Item 10) The animal of item 1, which produces a population of central memory T cells and effector memory T cells against an antigen of interest. (Item 11) 2. The animal of item 1, wherein the unrearranged TCR alpha variable locus comprises a complete repertoire of human J alpha segments and a complete repertoire of human V alpha segments. (Item 12) 2. The animal of item 1, wherein the animal possesses an endogenous non-human TCR alpha variable locus, and the locus is a non-functional locus. (Item 13) 2. The animal according to item 1, which is a rodent. (Item 14) 14. The animal of item 13, wherein the rodent is a mouse. (Item 15) A genetically modified non-human animal comprising in its genome an unrearranged 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 operably linked to a non-human TCR beta constant gene sequence. (Item 16) 16. The animal of paragraph 15, wherein the unrearranged TCR β variable locus replaces an endogenous non-human TCR β variable locus. (Item 17) The animal of item 15, wherein the endogenous rodent Vβ segments, endogenous rodent Dβ segments and endogenous rodent Jβ segments are incapable of rearrangement to form a rearranged Vβ / Dβ / Jβ sequence. (Item 18) The animal of item 15, which lacks a functional endogenous non-human TCR β variable locus. (Item 19) 19. The animal of item 18, wherein the lack of a functional endogenous rodent TCR β variable locus comprises a deletion selected from the group consisting of: (a) a deletion of all endogenous V β gene segments, (b) a deletion of all endogenous D β gene segments, (c) a deletion of all endogenous J β gene segments, and (d) a combination thereof. (Item 20) 16. The animal of item 15, wherein the human Vβ segments, the human Dβ segments and the human Jβ segments are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. (Item 21) 21. The animal of item 20, which expresses a T cell receptor comprising a human TCRβ variable region on the surface of the T cell. (Item 22) 16. The animal of item 15, wherein the T cells of the animal undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells. (Item 23) The animal of item 15, comprising a normal ratio of splenic CD3+ T cells to total splenocytes. (Item 24) The animal of item 15, which produces a population of central memory T cells and effector memory T cells against an antigen of interest. (Item 25) 16. The animal of item 15, wherein the unrearranged TCR β variable locus comprises a complete repertoire of human J β segments, a complete repertoire of human D β segments, and a complete repertoire of human V β segments. (Item 26) 16. The animal of item 15, wherein the animal possesses an endogenous non-human TCR β variable locus, and the locus is a non-functional locus. (Item 27) 16. The animal according to item 15, which is a rodent. (Item 28) 28. The animal of item 27, wherein the rodent is a mouse. (Item 29) an unrearranged T cell receptor (TCR) alpha variable locus comprising at least one human V alpha segment and at least one human J alpha segment operably linked to a non-human TCR alpha constant gene sequence; an unrearranged 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 operably linked to a non-human TCR beta constant gene sequence; A genetically modified non-human animal comprising in its genome. (Item 30) 30. The animal of item 29, wherein the unrearranged TCR alpha variable locus replaces an endogenous non-human TCR alpha variable locus and the unrearranged TCR beta variable locus replaces an endogenous non-human TCR beta variable locus. (Item 31) The animal of item 29, wherein endogenous non-human Vα segments and endogenous non-human Jα segments are incapable of rearrangement to form rearranged Vα / Jα sequences, and endogenous non-human Vβ segments, endogenous non-human Dβ segments and endogenous non-human Jβ segments are incapable of rearrangement to form rearranged Vβ / Dβ / Jβ sequences. (Item 32) 30. The animal of item 29, wherein the animal lacks a functional endogenous non-human TCR alpha variable locus and lacks a functional endogenous non-human TCR beta variable locus. (Item 33) the lack of a functional endogenous non-human TCR α variable locus comprises a deletion selected from the group consisting of: (a) a deletion of all endogenous Vα gene segments, (b) a deletion of all endogenous Jα gene segments, and (c) a combination thereof; and 33. The animal of item 32, wherein the lack of a functional endogenous non-human TCR β variable locus comprises a deletion selected from the group consisting of: (a) a deletion of all endogenous V β gene segments, (b) a deletion of all endogenous D β gene segments, (c) a deletion of all endogenous J β gene segments, and (d) a combination thereof. (Item 34) The animal of item 29, wherein the human Vα segment and the human Jα segment are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ segment, the human Dβ segment and the human Jβ segment are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. (Item 35) 35. The animal of item 34, wherein the animal expresses a T cell receptor comprising a human variable region and a non-human constant region on the surface of the T cell. (Item 36) 30. The animal of item 29, wherein the T cells of the animal undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells. (Item 37) 30. The animal of item 29, comprising a normal ratio of splenic CD3+ T cells to total splenocytes. (Item 38) 30. The animal of item 29, which produces a population of central memory T cells and effector memory T cells against an antigen of interest. (Item 39) 30. The animal of item 29, wherein the unrearranged TCR alpha variable locus comprises 61 human Jalpha segments and 8 human Valpha segments, and the unrearranged TCR beta variable locus comprises 14 human Jbeta segments, 2 human Dbeta segments, and 14 human Vbeta segments. (Item 40) 30. The animal of item 29, wherein the unrearranged TCR alpha variable locus comprises a complete repertoire of human Jalpha segments and a complete repertoire of human Valpha segments, and the unrearranged TCR beta variable locus comprises a complete repertoire of human Jbeta segments, a complete repertoire of human Dbeta segments, and a complete repertoire of human Vbeta segments. (Item 41) 30. The animal of item 29, wherein the animal retains an endogenous non-human TCR alpha variable locus and an endogenous non-human TCR beta variable locus, and the loci are non-functional loci. (Item 42) 30. The animal of item 29, further comprising a nucleotide sequence of a human Vδ segment at the humanized TCR alpha locus. (Item 43) 43. The animal of item 42, further comprising a complete repertoire of human Vδ segments, a complete repertoire of human Dδ segments and a complete repertoire of human Jδ segments at the humanized TCR alpha locus. (Item 44) 30. The animal according to item 29, which is a rodent. (Item 45) 45. The animal of item 44, wherein the rodent is a mouse. (Item 46) 1. A method for generating a genetically modified non-human animal that expresses a T cell receptor comprising a human variable region and a non-human constant region on the surface of the T cell, the method comprising: replacing an endogenous non-human TCR α variable locus in a first non-human animal with an unrearranged humanized TCR α variable locus comprising at least one human Vα segment and at least one human Jα segment to create 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; replacing an endogenous non-human TCR β variable locus in a second non-human animal 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 create 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; breeding the first animal and the second animal to obtain a non-human animal expressing a T cell receptor comprising a human variable region and a non-human constant region; A method comprising: (Item 47) 47. The method of claim 46, wherein endogenous non-human Vα segments and endogenous non-human Jα segments are incapable of rearrangement to form rearranged Vα / Jα sequences, and endogenous non-human Vβ segments, endogenous non-human Dβ segments and endogenous non-human Jβ segments are incapable of rearrangement to form rearranged Vβ / Dβ / Jβ sequences. (Item 48) 47. The method of claim 46, wherein the genetically modified animal lacks a functional endogenous non-human TCR alpha variable locus and lacks a functional endogenous non-human TCR beta variable locus. (Item 49) the lack of a functional endogenous non-human TCR α variable locus comprises a deletion selected from the group consisting of: (a) a deletion of all endogenous Vα gene segments, (b) a deletion of all endogenous Jα gene segments, and (c) a combination thereof; and 49. The method of claim 48, wherein the lack of a functional endogenous non-human TCR β variable locus comprises a deletion selected from the group consisting of: (a) a deletion of all endogenous Vβ gene segments, (b) a deletion of all endogenous Dβ gene segments, (c) a deletion of all endogenous Jβ gene segments, and (d) a combination thereof. (Item 50) 47. The method of claim 46, wherein the human Vα segment and the human Jα segment are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ segment, the human Dβ segment and the human Jβ segment are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. (Item 51) 47. The method of claim 46, wherein the rodent T cells undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells. (Item 52) 47. The method of claim 46, wherein the animal comprises a normal ratio of splenic CD3+ T cells to total splenocytes. (Item 53) 47. The method of claim 46, wherein the animal produces a population of central memory T cells and effector memory T cells against the antigen of interest. (Item 54) 47. The method of claim 46, wherein the unrearranged humanized TCR alpha variable locus comprises 61 human Jalpha segments and 8 human Valpha segments, and the unrearranged humanized TCR beta variable locus comprises 14 human Jbeta segments, 2 human Dbeta segments, and 14 human Vbeta segments. (Item 55) 47. The method of claim 46, wherein the unrearranged humanized TCR alpha variable locus comprises a complete repertoire of human Jalpha segments and a complete repertoire of human Valpha segments, and the unrearranged humanized TCR beta variable locus comprises a complete repertoire of human Jbeta segments, a complete repertoire of human Dbeta segments, and a complete repertoire of human Vbeta segments. (Item 56) 47. The method of claim 46, wherein the non-human animal is a rodent. (Item 57) 57. The method of claim 56, wherein the rodent is a mouse. (Item 58) an unrearranged T cell receptor (TCR) alpha variable locus comprising a complete repertoire of human J alpha segments and a complete repertoire of human V alpha segments operably linked to a murine TCR alpha constant gene sequence; an unrearranged TCR β variable locus comprising a complete repertoire of human J β segments, a complete repertoire of human D β segments, and a complete repertoire of human V β segments operably linked to a murine TCR β constant gene sequence; A genetically modified mouse that contains the above gene in its genome. (Item 59) 59. The mouse of paragraph 58, wherein the unrearranged TCR alpha variable locus replaces an endogenous mouse TCR alpha variable locus and the unrearranged TCR beta variable locus replaces an endogenous mouse TCR beta variable locus. (Item 60) 59. The mouse of item 58, wherein endogenous mouse Vα segments and endogenous mouse Jα segments are incapable of rearrangement to form rearranged Vα / Jα sequences, and endogenous mouse Vβ segments, endogenous mouse Dβ segments and endogenous mouse Jβ segments are incapable of rearrangement to form rearranged Vβ / Dβ / Jβ sequences. (Item 61) The mouse of item 58, wherein the human Vα segment and the human Jα segment are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ segment, the human Dβ segment and the human Jβ segment are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. (Item 62) 62. The mouse of item 61, wherein the mouse expresses a T cell receptor comprising a human variable region and a mouse constant region on the surface of the T cell. (Item 63) 59. The mouse of item 58, wherein T cells of the mouse undergo thymic T cell development to generate CD4 single positive T cells and CD8 single positive T cells. (Item 64) The mouse of paragraph 58, comprising a normal ratio of splenic CD3+ T cells to total splenocytes. (Item 65) The mouse of item 58, which produces a population of central memory T cells and effector memory T cells against an antigen of interest. (Item 66) 59. The mouse of item 58, further comprising a complete repertoire of Vδ segments, a complete repertoire of Dδ segments, and a complete repertoire of Jδ segments at the humanized TCR alpha locus. (Item 67) The mouse of item 58, wherein the mouse retains an endogenous mouse TCR alpha variable locus and an endogenous mouse TCR beta variable locus, and the loci are non-functional loci. (Item 68) 1. A method for generating a human T cell receptor against an antigen of interest, the method comprising: Immunizing the non-human animal according to item 1 with the antigen of interest; Initiating an immune response in the animal; isolating T cells from the animal that are reactive to the antigen of interest; determining the nucleic acid sequence of the human TCR variable region expressed by the T cell; cloning a human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region, wherein the human TCR variable region is operably linked to the human TCR constant region; expressing a human T cell receptor in the cell; A method comprising: [Brief description of the drawings]

[0038] [Figure 1] Figure 1 depicts the interaction of TCR and MHC molecules in mice. The left panel shows mouse T cells (top) from a humanized TCR mouse containing a T cell receptor with human variable TCR domains and mouse constant TCR domains that recognizes antigen (grey ball) presented via MHC class I by an antigen presenting cell (bottom), and the right panel shows the same for MHC class II. The MHCI and MHCII complexes are shown together with their respective co-receptors CD8 and CD4. Mouse regions are in black and human regions are in white. [Diagram 2] 2 is a diagram (not to scale) depicting the overall organization of the mouse TCR alpha locus (top panel, first locus) and the human TCR alpha locus (top panel, second locus). The bottom panel illustrates a strategy for replacing mouse TCR alpha variable region segments (filled symbols) with human TCR alpha variable region segments (open symbols) at the endogenous mouse locus on chromosome 14, showing a humanized TCR alpha locus with human Valpha and Jalpha segments along with a mouse constant region and mouse enhancer, and in the embodiment shown, deleting the TCR delta locus during the humanization process. [Diagram 3] Figure 3 depicts (not to scale) a progressive strategy for humanization of the mouse TCR alpha locus, where TCR alpha variable region gene segments are sequentially added upstream of an initial humanization of the deleted mouse locus (MAID1540). Mouse sequences are indicated by filled symbols and human sequences by open symbols. MAID refers to the ID number of the modified allele. TRAV = TCR V alpha segment, TRAJ = TCR J alpha segment (hTRAJ = human TRAJ), TRAC = TCR C alpha domain, TCRD = TCR delta. [Figure 4A] FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Figure 4B]FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Figure 4C]FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Figure 4D]FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Figure 4E]FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Figure 4F]FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Figure 4G]FIG. 4 shows a detailed representation (not to scale) of the progressive humanization strategy at the TCR α locus. 4A depicts the deletion of mouse TCR α V and J segments, FIG. 4B depicts a strategy for inserting two human V segments and 61 human J segments into a deleted mouse TCR α locus, FIG. 4C depicts a strategy for inserting additional human V segments resulting in a total of 8 human V segments and 61 human J segments, FIG. 4D depicts a strategy for inserting additional human V segments resulting in a total of 23 human V segments and 61 human J segments, FIG. 4E depicts a strategy for inserting additional human V segments resulting in 35 human V segments and 61 human J segments, FIG. 4F depicts a strategy for inserting additional human segments resulting in 48 human V segments and 61 human J segments, and FIG. 4G depicts a strategy for inserting additional human segments resulting in 54 human V segments and 61 human J segments. MAID refers to the ID number of the altered allele. [Diagram 5] Figure 5 depicts (not to scale) one embodiment of a humanization strategy for the mouse TCR alpha locus, where human TCR delta (TCR delta V, TCR delta D, TCR delta J, TCR delta enh (enhancer), and TCR delta constant (C)) sequences are also placed into the humanized TCR alpha locus. Mouse sequences are indicated by filled symbols and human sequences are indicated by open symbols. LTVEC refers to large targeting vector. hTRD = human TCR delta. [Figure 6]6 depicts (not to scale) the overall organization of the mouse TCR β locus (top panel, first locus; on mouse chromosome 6) and the human TCR β locus (top panel, second locus; on human chromosome 7). The bottom panel illustrates a strategy to replace mouse TCR β variable region segments (filled symbols) with human TCR β variable region segments (open symbols) at the endogenous mouse locus on mouse chromosome 6. A humanized TCR β locus with human Vβ, Dβ, and Jβ segments is shown with mouse constant regions and mouse enhancers, and in the embodiment shown, the humanized locus retains the mouse trypsinogen gene (filled box), and in the specific embodiment shown, a single mouse V segment is retained upstream of the 5' mouse trypsinogen gene. [Figure 7] Figure 7 is a diagram (not to scale) depicting a stepwise strategy for humanization of the mouse TCRβ locus, in which TCRβ variable region gene segments are sequentially added to a deleted mouse TCRβ variable locus. Mouse sequences are shown by filled symbols and human sequences are shown by open symbols. MAID refers to the ID number of the modified allele. TRBV or TCRBV = TCRβ V segment. [Figure 8A]Figure 8 is a detailed depiction of the progressive humanization strategy at the TCR β locus: Figure 8A depicts a strategy to delete mouse TCR β V segments, Figure 8B depicts a strategy to insert 14 V segments into the deleted TCR β locus, Figure 8C depicts a strategy to insert 2 D segments and 14 J segments into the TCR β locus (i) followed by deletion of loxP sites (ii) resulting in 14 human V segments, 2 human D segments, and 14 human J segments, and Figure 8D depicts a strategy to insert 40 human V segments, 2 human D segments, and 14 human J segments into the deleted TCR β locus (i). 8A depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8B depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8C depicts replacement of a mouse V segment downstream of the mouse enhancer resulting in 67 human V segments, 2 human D segments, and 14 human J segments. In this particular embodiment, one mouse V segment is retained 5' of the mouse trypsinogen gene. [Figure 8B]Figure 8 is a detailed depiction of the progressive humanization strategy at the TCR β locus: Figure 8A depicts a strategy to delete mouse TCR β V segments, Figure 8B depicts a strategy to insert 14 V segments into the deleted TCR β locus, Figure 8C depicts a strategy to insert 2 D segments and 14 J segments into the TCR β locus (i) followed by deletion of loxP sites (ii) resulting in 14 human V segments, 2 human D segments, and 14 human J segments, and Figure 8D depicts a strategy to insert 40 human V segments, 2 human D segments, and 14 human J segments into the deleted TCR β locus (i). 8A depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8B depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8C depicts replacement of a mouse V segment downstream of the mouse enhancer resulting in 67 human V segments, 2 human D segments, and 14 human J segments. In this particular embodiment, one mouse V segment is retained 5' of the mouse trypsinogen gene. [Figure 8C]Figure 8 is a detailed depiction of the progressive humanization strategy at the TCR β locus: Figure 8A depicts a strategy to delete mouse TCR β V segments, Figure 8B depicts a strategy to insert 14 V segments into the deleted TCR β locus, Figure 8C depicts a strategy to insert 2 D segments and 14 J segments into the TCR β locus (i) followed by deletion of loxP sites (ii) resulting in 14 human V segments, 2 human D segments, and 14 human J segments, and Figure 8D depicts a strategy to insert 40 human V segments, 2 human D segments, and 14 human J segments into the deleted TCR β locus (i). 8A depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8B depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8C depicts replacement of a mouse V segment downstream of the mouse enhancer resulting in 67 human V segments, 2 human D segments, and 14 human J segments. In this particular embodiment, one mouse V segment is retained 5' of the mouse trypsinogen gene. [Figure 8D]Figure 8 is a detailed depiction of the progressive humanization strategy at the TCR β locus: Figure 8A depicts a strategy to delete mouse TCR β V segments, Figure 8B depicts a strategy to insert 14 V segments into the deleted TCR β locus, Figure 8C depicts a strategy to insert 2 D segments and 14 J segments into the TCR β locus (i) followed by deletion of loxP sites (ii) resulting in 14 human V segments, 2 human D segments, and 14 human J segments, and Figure 8D depicts a strategy to insert 40 human V segments, 2 human D segments, and 14 human J segments into the deleted TCR β locus (i). 8A depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8B depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8C depicts replacement of a mouse V segment downstream of the mouse enhancer resulting in 67 human V segments, 2 human D segments, and 14 human J segments. In this particular embodiment, one mouse V segment is retained 5' of the mouse trypsinogen gene. [Figure 8E]Figure 8 is a detailed depiction of the progressive humanization strategy at the TCR β locus: Figure 8A depicts a strategy to delete mouse TCR β V segments, Figure 8B depicts a strategy to insert 14 V segments into the deleted TCR β locus, Figure 8C depicts a strategy to insert 2 D segments and 14 J segments into the TCR β locus (i) followed by deletion of loxP sites (ii) resulting in 14 human V segments, 2 human D segments, and 14 human J segments, and Figure 8D depicts a strategy to insert 40 human V segments, 2 human D segments, and 14 human J segments into the deleted TCR β locus (i). 8A depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8B depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8C depicts replacement of a mouse V segment downstream of the mouse enhancer resulting in 67 human V segments, 2 human D segments, and 14 human J segments. In this particular embodiment, one mouse V segment is retained 5' of the mouse trypsinogen gene. [Figure 8F]Figure 8 is a detailed depiction of the progressive humanization strategy at the TCR β locus: Figure 8A depicts a strategy to delete mouse TCR β V segments, Figure 8B depicts a strategy to insert 14 V segments into the deleted TCR β locus, Figure 8C depicts a strategy to insert 2 D segments and 14 J segments into the TCR β locus (i) followed by deletion of loxP sites (ii) resulting in 14 human V segments, 2 human D segments, and 14 human J segments, and Figure 8D depicts a strategy to insert 40 human V segments, 2 human D segments, and 14 human J segments into the deleted TCR β locus (i). 8A depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8B depicts a strategy for inserting an additional human V segment resulting in 66 human V segments, 2 human D segments, and 14 human J segments, and FIG. 8C depicts replacement of a mouse V segment downstream of the mouse enhancer resulting in 67 human V segments, 2 human D segments, and 14 human J segments. In this particular embodiment, one mouse V segment is retained 5' of the mouse trypsinogen gene. [Figure 9]FIG. 9 shows a wild type (WT) mouse; a mouse homozygous for a deleted TCR α locus (first top panel; MAID1540 in FIG. 3); a mouse homozygous for a deleted TCR α locus and containing 8 human Vα and 61 human Jα segments (second top panel; MAID1767 in FIG. 3 or humanized TCR α mouse); a mouse homozygous for a deleted TCR β locus except for one upstream mouse Vβ segment and one downstream mouse Vβ segment (first bottom panel; MAID1545 in FIG. 7); a mouse homozygous for a deleted TCR β locus and containing 14 human Vβ, 2 human Dβ, and 14 human Jβ segments, with one upstream mouse Vβ segment and one downstream mouse Vβ segment (second top panel; MAID1767 in FIG. 3 or humanized TCR α mouse). Representative FACS histograms (Y-axis is cell number, X-axis is mean fluorescence intensity, and gates indicate frequency of CD3+ T cells within a single lymphocyte population) of spleen cells stained with anti-CD3 antibody in a mouse homozygous for deletion of both the TCRα and TCRβ loci (except for the two mouse Vβ segments) that contains 8 human Vα and 61 human Jα segments at the endogenous TCRα locus, as well as 14 human Vβ, 2 human Dβ, and 14 human Jβ segments at the endogenous TCRβ locus (MAID1767 / 1716 or humanized TCRα / β mouse). [Figure 10]Figure 10 shows representative FACS contour plots of mouse thymocytes from WT mice, homozygous humanized TCR alpha mice (1767HO; hTCR alpha); homozygous humanized TCR beta mice (1716HO; hTCR beta); and homozygous humanized TCR alpha / beta mice (1716HO1767HO; hTCR alpha / beta) stained with anti-CD4 (Y-axis) and anti-CD8 (X-axis) antibodies (upper panel) and anti-CD44 (Y-axis) and anti-CD25 (X-axis) antibodies (lower panel). The FACS plots in the upper panel make it possible to distinguish double negative (DN) T cells, double positive (DP) T cells, CD4 single positive (CD4 SP) T cells, and CD8 single positive (SP CD8) T cells. The FACS plots in the lower panel make it possible to distinguish the various stages (DN1, DN2, DN3, and DN4) of T cell development of double negative T cells. 1716 and 1767 refer to the MAID numbers identified in Figures 3 and 7. [Figure 11] FIG. 11 demonstrates the frequency (upper panels) or absolute numbers (lower panels) of DN T cells, DP T cells, CD4 SP T cells, and CD8 SP T cells in the thymus of WT mice, hTCRα mice (1767HO), hTCRβ mice (1716HO), or hTCRα / β mice (1716HO1767HO) (n=4). [Figure 12]FIG. 12 shows representative FACS analyses of spleen cells from WT mice, hTCRα mice (1767HO), hTCRβ mice (1716HO), or hTCRα / β mice (1716HO1767HO). The left panel shows the analysis of single cell-based anti-CD19 antibody staining (Y-axis; staining for B lymphocytes) or anti-CD3 antibody staining (X-axis; staining for T lymphocytes), the middle panel shows the analysis of CD3+ cells based on anti-CD4 antibody staining (Y-axis) or anti-CD8 antibody staining (X-axis), and the right panel shows the analysis of CD4+ or CD8+ cells based on anti-CD44 antibody staining (Y-axis) or anti-CD62L antibody staining (X-axis), showing that the staining makes it possible to distinguish different types of T cells in the periphery (naive T cells vs. central memory T cells (Tcm) vs. effector T cells or effector memory T cells (Teff / Tem)). [Figure 13] FIG. 13 demonstrates the number of CD4+ T cells (left panel) or CD8+ T cells (right panel) per spleen (Y axis) from WT, hTCRα (1767HO), hTCRβ (1716HO), or hTCRα / β mice (1716HO1767HO) (n=4). [Figure 14] FIG. 14 demonstrates the numbers of naive T cells, Tcm cells, and Teff / Tem cells per spleen (Y-axis) among CD4+ T cells (upper panel) or CD8+ T cells (lower panel) from WT, hTCRα (1767HO), hTCRβ (1716HO), or hTCRα / β mice (1716HO1767HO) (n=4). [Figure 15] Figure 15 is a table summarizing the expression of various human TCRβ V segments (as determined by FACS analysis using variable segment-specific antibodies) on splenic CD8+ T cells (Figure 15A) or CD4+ T cells (Figure 15B) of WT, hTCRβ (1716HO), or hTCRα / β (1716HO1767HO) mice. Data are presented as mean ± SD (n=4 mice per group). [Figure 16A]Figure 16 depicts the mRNA expression (Y axis) in thymic or splenic T cells of various human TCRβ V segments present in WT mice, hTCRα mice (1767HO), hTCRβ mice (1716HO), or hTCRα / β mice (1716HO1767HO), where Figure 16A represents an analysis of the mRNA expression of human TCRβ variable segments (hTRBV) 18, 19, 20, and 24, and Figure 16B represents an analysis of the mRNA expression of hTRBV25, 27, 28, and 29. [Figure 16B] Figure 16 depicts the mRNA expression (Y axis) in thymic or splenic T cells of various human TCRβ V segments present in WT mice, hTCRα mice (1767HO), hTCRβ mice (1716HO), or hTCRα / β mice (1716HO1767HO), where Figure 16A represents an analysis of the mRNA expression of human TCRβ variable segments (hTRBV) 18, 19, 20, and 24, and Figure 16B represents an analysis of the mRNA expression of hTRBV25, 27, 28, and 29. [Figure 17] FIG. 17 shows representative FACS histograms of spleen cells stained with anti-CD3 antibody in WT mice, mice homozygous for a deleted TCR α locus (TCRAΔV), mice homozygous for a deleted TCR α locus with two human V segments and 61 human J segments (TCRA2hV; MAID1626 in FIG. 3), mice homozygous for a deleted TCR α locus with eight human V segments and 61 human J segments (TCRA8hV; MAID1767 in FIG. 3), and mice homozygous for a deleted TCR α locus with 23 human V segments and 61 human J segments (TCRA23hV; MAID1979 in FIG. 3). (Y-axis is cell number, X-axis is mean fluorescence intensity, and gates indicate frequency of CD3+ T cells within a single lymphocyte population.) [Figure 18]FIG. 18 shows, in the upper left panel, representative FACS analysis of thymic CD3+ T cells from WT mice or homozygous hTCRα mice with 23 human V segments and 61 human J segments (1979HO) stained with anti-CD4 antibody (Y-axis) or anti-CD8 antibody (X-axis); in the lower left panel, FACS analysis of DN T cells from WT or 1979 mice stained with anti-CD44 (Y-axis) or anti-CD25 (X-axis); and in the right panel, graphs of the percentage of thymocytes that are DN, DP, CD4 SP, or CD8 SP (Y-axis) in WT or 1979HO mice (n=4). [Figure 19] FIG. 19 shows, in the left panel, representative FACS analysis of splenic lymphocytes from WT or 1979HO mice stained with anti-CD19 or anti-CD3 antibodies, and in the right panel, a graph of the percent of splenocytes that are CD3+ (Y axis) from WT and 1979HO mice (n=4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] definition The present invention provides genetically modified non-human animals, such as rodents, such as mice or rats, that express humanized T cell receptors.The present invention also relates to genetically modified non-human animals that contain unrearranged T cell receptor variable loci in their germline.The present invention also provides embryos, cells and tissues that contain them; their method of making; and their method of using.Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have acquired in the art, unless otherwise clearly indicated or clearly understood from the context in which the terms or phrases are used.

[0040] The term "conservative" when used to describe a conservative amino acid substitution includes replacing an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). A conservative amino acid substitution can be achieved by modifying a nucleotide sequence to introduce a nucleotide change that codes for a conservative substitution. In general, a conservative amino acid substitution does not substantially alter the desired functional property of a protein, e.g., the ability of T cells to recognize peptides presented by MHC molecules. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains (e.g., glycine, alanine, valine, leucine, and isoleucine); aliphatic-hydroxyl side chains (e.g., serine and threonine); amide-containing side chains (e.g., asparagine and glutamine); aromatic side chains (e.g., phenylalanine, tyrosine, and tryptophan); basic side chains (e.g., lysine, arginine, and histidine); acidic side chains (e.g., aspartic acid and glutamic acid); and sulfur-containing side chains (e.g., cysteine ​​and methionine). Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamate / aspartate, and asparagine / glutamine. In some embodiments, conservative amino acid substitutions can be substitutions of any native residue in a protein with alanine, for example as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), incorporated herein by reference. In some embodiments, the substitutions are moderately conservative, and have a non-negative value in the PAM250 log-likelihood matrix.

[0041] Thus, genetically modified non-human animals that express humanized TCR alpha and beta polypeptides (and / or humanized TCR delta and gamma polypeptides) that contain conservative amino acid substitutions in the amino acid sequences described herein are encompassed by the present invention.

[0042] One skilled in the art will appreciate that, in addition to the nucleic acid residues encoding the humanized TCR alpha and beta polypeptides described herein, due to the degeneracy of the genetic code, other nucleic acids may also encode the polypeptides of the invention. Thus, in addition to genetically modified non-human animals that contain in their genome nucleotide sequences encoding the humanized TCR polypeptides described herein, also provided are non-human animals that, due to the degeneracy of the genetic code, contain in their genome nucleotide sequences that differ from those described herein.

[0043] The term "identity" when used in relation to sequences includes identity determined by a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments described herein, identity is determined using ClustalW v.1.83 (slow) alignment with an open gap penalty of 10.0 and an extend gap penalty of 0.1, using the Gonnet similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of sequences compared for sequence identity varies depending on the individual sequence. In various embodiments, identity is determined by comparing the sequence of the mature protein from its N-terminus to its C-terminus. In various embodiments, when comparing a chimeric human / non-human sequence to a human sequence, the human portion of the chimeric human / non-human sequence (rather than the non-human portion) is used to compare 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 with the human ectodomain of a human protein).

[0044] The term "homology" or "homologous" with respect to a sequence, e.g., a nucleotide or amino acid sequence, refers to two sequences that, upon optimal alignment and comparison, are identical in at least about 75% of the nucleotides or amino acids, at least about 80% of the nucleotides or amino acids, at least about 90-95% of the nucleotides or amino acids, e.g., more than 97% of the nucleotides or amino acids. Those skilled in the art will appreciate that for optimal gene targeting, the targeting construct should contain arms that are homologous to the endogenous DNA sequence (i.e., "homology arms"), so that homologous recombination can occur between the targeting construct and the endogenous sequence being targeted.

[0045] The term "operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in the intended manner. Thus, the nucleic acid sequence encoding the protein can be operably linked to a control sequence (e.g., a promoter, enhancer, silencer sequence, etc.) to maintain proper transcriptional regulation. In addition, the various portions of the humanized protein of the present invention can be operably linked to maintain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless otherwise specified, the various domains of the humanized protein of the present invention are operably linked to each other.

[0046] The term "replacement" with respect to gene replacement refers to placing foreign genetic material at an endogenous locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. In one example, an endogenous non-human gene or a fragment thereof is replaced with a corresponding human gene or a fragment thereof. The corresponding human gene or a fragment thereof is an ortholog, a homologue, or a human gene or fragment that is substantially identical or the same in structure and / or function as the endogenous non-human gene or fragment thereof that is replaced. As demonstrated in the following examples, the nucleotide sequences of the endogenous non-human TCR alpha and beta variable loci were replaced with nucleotide sequences corresponding to the human TCR alpha and beta variable loci.

[0047] As used herein, for example with respect to a functional protein, "functional" refers to a protein that retains 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 at an endogenous non-human TCR alpha, beta, TCR delta and / or TCR gamma variable locus) results in the locus being unable to express a functional endogenous protein.

[0048] As used herein, a TCR locus or TCR locus (e.g., a TCR alpha (gene) locus or a TCR beta (gene) locus) refers to genomic DNA that includes the TCR coding region, including the entire TCR coding region, including unrearranged V(D)J sequences, enhancer sequences, constant sequence(s) and any upstream or downstream (UTRs, control regions, etc.) or intervening DNA sequences (introns, etc.). A TCR variable locus or TCR variable locus (e.g., a TCR alpha (gene) locus or a TCR beta (gene) locus) refers to genomic DNA that includes a region that includes a TCR variable region segment (the V(D)J region), but does not include the TCR constant sequences and, in various embodiments, enhancer sequences. Other sequences (e.g., selection cassettes, restriction sites, etc.) can also be included in the TCR variable locus for purposes of genetic engineering and are encompassed by the present invention.

[0049] Genetically modified TCR animals In various embodiments, the invention generally provides genetically modified non-human animals that comprise unrearranged, humanized TCR variable loci in their genome.

[0050] T cells bind epitopes on 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 these epitopes through the T cell receptor (TCR) complex on the surface of the T cell. 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 chains are encoded by nucleic acid sequences located within the α locus (on human or mouse chromosome 14), which also encompasses the entire δ locus, and the β chains are encoded by nucleic acid sequences located within the β locus (on mouse chromosome 6 or human chromosome 7). The majority of T cells have αβ TCRs, and a minority have γδ TCRs. The interaction of the TCR with MHC class I (presented to CD8+ T cells) and MHC class II (presented to CD4+ T cells) molecules is shown in FIG. 1 (closed symbols represent non-human sequences; open symbols represent human sequences, showing one particular embodiment of the TCR protein of the invention).

[0051] T cell receptor α and β polypeptides (and similarly γ and δ polypeptides) are linked to each other by disulfide bonds. Each of the two polypeptides constituting the TCR contains an extracellular domain including a constant region and a variable region, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytoplasmic tail are also part of the constant region). The variable region of the TCR determines its antigen specificity and, like immunoglobulins, contains three complementarity determining regions (CDRs). Also, like immunoglobulin genes, the T cell receptor variable loci (e.g., TCRα and TCRβ loci) contain multiple unrearranged V(D)J segments (variable (V) segments, joining (J) segments, and in TCRβ and δ, diversity (D) segments). During T cell development in the thymus, the TCR alpha variable locus rearranges such that the resulting TCR alpha chain is encoded by a specific combination of V-J segments (the Valpha / Jalpha sequence); the TCR beta variable locus rearranges such that the resulting TCR beta chain is encoded by a specific combination of V-D-J segments (the Vbeta / Dbeta / Jbeta sequence).

[0052] Interaction with the thymic stroma triggers thymocytes to undergo several developmental stages characterized by the expression of various cell surface markers. A summary of characteristic cell surface markers at different developmental stages in the thymus is given in Table 1. Rearrangement of the TCRβ variable locus begins at the DN2 stage and is completed during the DN4 stage, whereas rearrangement of the TCRα variable locus occurs at the DP stage. After completion of rearrangement of the TCRβ locus, the cells express the TCRβ chain at the cell surface together with an alternative α chain, pTα. See Janeway's Immunobiology, Chapter 7 (above).

[0053] [Table 1]

[0054] Naive CD4+ and CD8+ T cells leave the thymus and enter peripheral lymphoid organs (e.g., the spleen), where they are exposed to antigen, become activated, undergo clonally expansion and produce multiple effector T cells (Teff), e.g., cytotoxic T cells, T REG cell, T H 17 cells, T H 1 cell, T H After infection, several T cells remain as memory T cells. These are classified as either central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708-12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401:659-60. Sallusto and colleagues proposed that after initial infection, Tem cells represent a readily accessible pool of primed memory T cells with effector function in peripheral tissues, and Tcm cells represent 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), whereas Tcm are characterized by expression of L-selectin (also known as CD62L) and CCR7+, which are important for binding to and signaling in peripheral lymphoid organs and lymph nodes. Id. Thus, all T cells found in peripheral lymphoid organs (e.g., naive T cells, Tcm cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express several 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).

[0055] While the TCR variable domains function primarily in antigen recognition, the extracellular portion of the constant domain of the TCR as well as the transmembrane and cytoplasmic domains also play important roles. A complete TCR receptor complex does not only require α and β or γ and δ polypeptides; additional molecules required include CD3γ, CD3δ and CD3ε and 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 which recruits the CD3γε heterodimer and the other recruits ζζ through their respective acidic residues. TCRβ has an additional basic residue in its transmembrane domain that is believed to recruit the CD3δε heterodimer. See, e.g., Kuhns et al. (2006) Deconstructing the Form and Function of the TCR / CD3 Complex, Immunity 24:133-39; Wucherpfennig et al. (2009) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2:a005140. An assembled complex containing the TCRαβ heterodimer, CD3γε, CD3δε, and ζζ is expressed on the T cell surface. Polar residues in the transmembrane domain have been suggested to serve as quality controls for the exiting endoplasmic reticulum; it has been demonstrated that in the absence of the CD3 subunit, the TCR chains are retained in the ER and targeted for degradation.See, e.g., Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Function, Annu. Rev. Immunol. 23:101-25.

[0056] Since the TCRαβ heterodimer (or TCRγδ heterodimer) alone has no signaling activity, the CD3 and ζ chains of the assembled complex provide the components for TCR signaling. Each CD3 chain has one Immune-Receptor-Tyrosine-based-Activation-Motif (ITAM), whereas 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 by the T-Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. vol. 2:e002485. Following engagement of the TCR, ITAM motifs are phosphorylated by the Src family tyrosine kinases, Lck and Fyn, thereby initiating a signaling cascade that results in Ras activation, calcium mobilization, rearrangement of the actin cytoskeleton and activation of transcription factors, all of which ultimately lead to T cell differentiation, proliferation and effector functions. See id., and also Janeway's Immunobiology, 7th Edition, edited by Murphy et al., Garland Science, 2008 (both of which are incorporated herein by reference).

[0057] In addition, the TCRβ transmembrane and cytoplasmic domains are believed to be involved in mitochondrial targeting and induction of apoptosis; indeed, naturally occurring N-terminally truncated TCRβ molecules exist 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 carried out by the TCR constant region (which in various embodiments includes the extracellular as well as parts of the transmembrane and cytoplasmic domains); in various embodiments, the structure of this region should be taken into consideration when designing humanized TCRs or genetically modified non-human animals expressing them.

[0058] Mice transgenic 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, such as 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 can produce a diverse repertoire of human T cell receptor variable region sequences; thus, the present invention provides non-human animals that express TCRs with fully human variable domains that bind epitopes of the antigen of interest in response to the antigen of interest. In some embodiments, non-human animals are provided that produce a diverse repertoire of T cell receptors that can react with a variety of antigens, including but not limited to antigens presented by APCs.

[0059] 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 an unrearranged human TCR variable region segment (V(D)J segment), where the unrearranged human TCR variable region segment replaces an endogenous non-human TCR variable region segment at an endogenous non-human (e.g., rodent) TCR variable locus (e.g., a TCR alpha, beta, delta and / or gamma variable locus). In one embodiment, the unrearranged human TCR variable locus replaces an endogenous non-human TCR variable locus.

[0060] In another embodiment, the invention provides a genetically modified non-human animal (e.g., a rodent, e.g., a rat, a mouse) that comprises an unrearranged human TCR variable region segment (V(D)J segment) in its genome, where the unrearranged human TCR variable region segment is operably linked to a non-human TCR constant region gene sequence, thereby resulting in a humanized TCR locus, where the humanized TCR locus is at a site in the genome 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 an unrearranged human TCR variable region segment operably linked to a non-human TCR constant region sequence.

[0061] In one aspect, the genetically modified non-human animal of the present invention comprises a human TCR variable region segment in its genome, and at the same time retains a non-human (e.g., rodent, e.g., mouse, rat) TCR constant gene segment. In various embodiments, the constant region 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, such as a non-human non-endogenous TCR transmembrane domain and cytoplasmic tail. As indicated above, the constant region of the TCR is involved in the signaling cascade that is initiated during the activation of primed T cells; thus, the endogenous TCR constant region interacts with various non-human anchors and signaling proteins in T cells. Thus, in one embodiment, the genetically modified non-human animals of the 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 that are recruited to the TCR complex is described in Janeway's Immunobiology (supra). In addition, similar to VELOCIMMUNE® mice, which exhibit normal B cell developmental processes and normal clonal selection processes that are believed to be at least in part due to the placement of variable regions at the endogenous mouse locus and the maintenance of mouse constant domains, in one embodiment, the non-human animals of the invention exhibit normal T cell developmental and T cell differentiation processes.

[0062] In some embodiments, a non-human animal is provided that comprises an unrearranged human TCR alpha variable region segment in its genome, the unrearranged human TCR alpha variable region segment operably linked to a non-human TCR alpha constant region gene sequence to produce a humanized TCR alpha locus. In one embodiment, the humanized TCR alpha locus is at a site in the genome other than the endogenous non-human TCR alpha locus. In another embodiment, the unrearranged human TCR alpha variable region segment replaces the endogenous non-human TCR alpha variable region segment while retaining the endogenous non-human TCR alpha constant region. In one embodiment, the unrearranged human TCR alpha variable locus replaces the endogenous non-human TCR alpha variable locus. In some embodiments, the animal retains the endogenous non-human TCR beta variable and constant region gene sequences. Thus, the animal expresses a TCR that comprises a chimeric human / non-human (i.e., humanized) TCR alpha chain and a non-human TCR beta chain.

[0063] In other embodiments, a non-human animal is provided that comprises an unrearranged human TCR β variable region segment in its genome, the unrearranged human TCR β variable region segment operably linked to a non-human TCR β constant region gene sequence to produce a humanized TCR β locus. In one embodiment, the humanized TCR β locus is at a site in the genome other than the endogenous non-human TCR β locus. In another embodiment, the unrearranged human TCR β variable region segment replaces the endogenous non-human TCR β variable region segment while retaining the endogenous non-human TCR β constant region. 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 region and constant region gene sequences. Thus, the animal expresses a TCR that includes a chimeric human / non-human (i.e., humanized) TCR β chain and a non-human TCR α chain.

[0064] In certain embodiments, the present invention provides genetically modified non-human animals (e.g., rodents, e.g., mice or rats) that comprise in their genome: (a) an unrearranged T cell receptor (TCR) alpha variable locus comprising at least one human Valpha segment and at least one human Jalpha segment operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR alpha constant gene sequence; and / or (b) an unrearranged TCR beta variable locus comprising at least one human Vbeta segment, at least one human Dbeta segment, and at least one human Jbeta segment operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR beta constant gene sequence.

[0065] In various embodiments of the invention, unrearranged human or humanized TCR variable loci (e.g., TCR alpha and / or TCR beta 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, replacement of TCR V(D)J segments with unrearranged human TCR V(D)J segments (e.g., Valpha and Jalpha and / or Vbeta and Dbeta and Jbeta segments) is at an endogenous non-human TCR variable locus (or multiple loci), and the unrearranged human V and J and / or V and D and J segments are operably linked to the non-human TCR constant region genes.

[0066] In some embodiments of the invention, the non-human animal comprises two copies of unrearranged human or humanized TCR alpha variable loci and / or two copies of unrearranged human or humanized TCR beta variable loci. Thus, the non-human animal is homozygous for one or both unrearranged human or humanized TCR alpha and TCR beta variable loci. In some embodiments of the invention, the non-human animal comprises one copy of unrearranged human or humanized TCR alpha variable loci and / or one copy of unrearranged human or humanized TCR beta variable loci. Thus, the non-human animal is heterozygous for one or both unrearranged human or humanized TCR alpha and TCR beta variable loci.

[0067] In one embodiment, the unrearranged TCR alpha variable locus comprising human variable region segments (e.g., human Vα and Jα segments) is positioned in the non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, the unrearranged TCR alpha variable locus comprising human variable region segments replaces the endogenous TCR alpha variable locus. In one aspect, the endogenous non-human Vα segment and the endogenous non-human Jα segment cannot rearrange to form a rearranged Vα / Jα sequence. Thus, in one aspect, the human Vα and Jα segments in the unrearranged TCR alpha variable locus can rearrange to form a rearranged human Vα / Jα sequence.

[0068] Similarly, in one embodiment, an unrearranged TCR beta variable locus comprising a human variable region segment (e.g., a human Vβ, Dβ and Jβ segment) is positioned in a non-human genome such that the human variable region segment replaces the corresponding non-human variable region segment. In one embodiment, the unrearranged TCR beta variable locus comprising a human variable region segment replaces an endogenous TCR beta variable locus. In one aspect, the endogenous non-human Vβ segment, endogenous non-human Dβ segment and endogenous non-human Jβ segment cannot rearrange to form a rearranged Vβ / Dβ / Jβ sequence. Thus, in one aspect, the human Vβ, Dβ and Jβ segments in an unrearranged TCR beta variable locus can rearrange to form a rearranged human Vαβ / Dβ / Jβ sequence.

[0069] In yet another embodiment, both unrearranged TCR alpha and beta variable loci containing human variable region segments replace the respective endogenous TCR alpha and beta variable loci. In one embodiment, the endogenous non-human Valpha and Jalpha segments cannot rearrange to form rearranged Valpha / Jalpha sequences, and the endogenous non-human Vbeta, Dbeta and Jbeta segments cannot rearrange to form rearranged Vbeta / Dbeta / Jbeta sequences. Thus, in one embodiment, the human Valpha and Jalpha segments in the unrearranged TCR alpha variable loci can rearrange to form rearranged human Valpha / Jalpha sequences, and the human Vbeta, Dbeta and Jbeta segments in the unrearranged TCR beta variable loci can rearrange to form rearranged human Valpha / Dbeta / Jbeta sequences.

[0070] In some aspects of the invention, the non-human animal comprising a humanized TCR alpha and / or TCR beta locus (including an unrearranged TCR alpha and / or TCR beta variable locus) retains an endogenous non-human TCR alpha and / or TCR beta variable locus. In one embodiment, the endogenous non-human TCR alpha and / or TCR beta 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 with respect to the constant region sequence such that rearrangement cannot be successful using a variable region segment from an inverted locus). In one embodiment, the humanized TCR alpha and / or TCR beta variable locus is located between the endogenous non-human TCR alpha and / or TCR beta variable locus and the endogenous non-human TCR alpha and / or TCR beta constant locus.

[0071] The number, nomenclature, location and other aspects of the V and J and / or V, D and J segments of the human and mouse TCR locus can be ascertained using the IMGT database available at www.imgt.org. The mouse TCR alpha variable locus is about 1.5 megabases and contains a total of 110 Vα and 60 Jα segments (FIG. 2). The human TCR alpha variable locus is about 1 megabase and contains a total of 54 Vα and 61 Jα segments, with 45 Vα and 50 Jα believed to be functional. Unless otherwise stated, 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, the genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) contains at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal comprises a humanized TCR alpha locus that comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50 or 54 human Vα segments. In some embodiments, the humanized TCR alpha locus comprises 2, 8, 23, 35, 48 or 54 human Vα segments. Thus, in some embodiments, the humanized TCR alpha locus of a non-human animal can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% human Va; in some embodiments, about 2%, about 3%, about 15%, about 65%, about 90% or 100% human Va.

[0072] 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 (the Vα segment is also referred to as "TRAV" or "TCRAV") and a DNA fragment comprising the contiguous human sequence of 61 human Jα segments (the Jα segment is 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 alpha locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV6-TRAV41 and 61 human TRAJ. In one embodiment, the non-human animal comprises a humanized TCR alpha locus comprising a DNA fragment comprising the contiguous human sequence of human TRAV1-1-TRAV41 and 61 human TRAJ. In various embodiments, the DNA fragment comprising the contiguous human sequence of the human TCR alpha variable region segment also comprises restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection in the locus humanization process. In various embodiments, these additional sites do not interfere with the proper function (e.g., rearrangement, splicing, etc.) of the various genes in the TCR alpha locus.

[0073] In one embodiment, the humanized TCR α locus comprises 61 human Jα segments, i.e., 100% human Jα segments. In a specific embodiment, the humanized TCR α locus comprises 8 human Vα segments and 61 human Jα segments; in another specific embodiment, the humanized TCR α locus comprises 23 human Vα segments and 61 human Jα segments. In another specific embodiment, the humanized TCR α locus comprises a complete repertoire of human Vα and Jα segments, i.e., all human variable α region gene segments encoded by the α locus, i.e., 54 human Vα segments and 61 human Jα segments. In various embodiments, the non-human animal does not comprise an endogenous non-human Vα segment or an endogenous non-human Jα segment at the TCR α locus.

[0074] The mouse TCR beta variable locus is about 0.6 megabases and contains a total of 33 V beta segments, 2 D beta segments, and 14 J beta segments (Figure 6). The human TCR beta variable locus is about 0.6 megabases and contains a total of 67 V beta segments, 2 D beta segments, and 14 J beta 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 V beta, at least one human D beta, and at least one human J alpha segment. In one embodiment, the non-human animal contains a humanized TCR beta locus that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or 67 or less human V beta segments. In some embodiments, the humanized TCR β locus comprises 8, 14, 40, 66 or 67 human Vβ segments. Thus, in some embodiments, the humanized TCR β locus of a non-human animal can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% human Vβ; in some embodiments, about 20%, about 60%, about 15%, about 98% or 100% human Vβ.

[0075] In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising the contiguous human sequence of human Vβ18 to Vβ29-1 (the Vβ segments are also referred to as "TRBV" or "TCRBV"). In one embodiment, the non-human animal comprises a humanized TCR β locus comprising a DNA fragment comprising the contiguous human sequence of human TRBV18 to TRBV29-1, another DNA fragment comprising the contiguous human sequence of human Dβ1-Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 segment), and another DNA fragment comprising the 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 the contiguous human sequence of human TRBV6-5 to TRBV29-1, another DNA fragment comprising the contiguous human sequence of human Dβ1-Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 segment), and another DNA fragment comprising the 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 the contiguous human sequence of human TRBV1 to TRBV29-1, another DNA fragment comprising the contiguous human sequence of human Dβ1-Jβ1, and another DNA fragment comprising the 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 the contiguous human sequence of human TRBV1-TRBV29-1, another DNA fragment comprising the contiguous human sequence of human Dβ1-Jβ1, another DNA fragment comprising the contiguous human sequence of human Dβ2-Jβ2, and another DNA fragment comprising the 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 in the locus humanization process. In various embodiments, these additional sites do not interfere with the proper function (e.g., rearrangement, splicing, etc.) of the various genes at the TCR β locus.

[0076] In one embodiment, the humanized TCR beta locus comprises 14 human J beta segments, i.e., 100% human J beta segments, and 2 human J beta segments, i.e., 100% human J beta segments. In another embodiment, the humanized TCR beta locus comprises at least one human V beta segment, e.g., 14 human V beta segments and all mouse D beta and J beta segments. In a specific embodiment, the humanized TCR beta locus comprises 14 human V beta segments, 2 human D beta segments, and 14 human J beta segments. In another specific embodiment, the humanized TCR beta locus comprises a complete repertoire of human V beta, D beta, and J beta segments, i.e., all human variable beta region gene segments encoded by the beta locus, or 67 human V beta segments, 2 human D beta segments, and 14 human J beta segments. In one embodiment, the non-human animal comprises one (e.g., 5') non-human V beta segment in the humanized TCR beta locus. In various embodiments, the non-human animal does not comprise an endogenous non-human Vβ segment, an endogenous non-human Dβ segment, or an endogenous non-human Jβ segment at the TCR β locus.

[0077] 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 produce a diverse repertoire of TCR molecules against different antigens.

[0078] In various embodiments, the non-human animal comprises a contiguous portion of a human genomic TCR variable locus, including V, D and J, or D and J, or V and J, or V segments organized as in an unrearranged human genomic variable locus, e.g., including promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc. organized as in an unrearranged non-human genomic TCR variable locus. In other embodiments, the various segments are organized as in an unrearranged non-human genomic TCR variable locus. In various embodiments of a humanized TCR alpha and / or beta locus, the humanized locus can include two or more human genomic segments not found juxtaposed in the human genome, e.g., a fragment of a V segment of a human V locus located adjacent to a constant region in the human genome juxtaposed with a fragment of a V segment of a human V locus located at the upstream end of the human V locus in the human genome.

[0079] In both mice and humans, the TCR delta gene segments are located with the TCR alpha locus (see Figures 2 and 5). The TCR delta J and D segments are located between the V alpha and J alpha segments, whereas the TCR delta V segments are scattered throughout the TCR alpha locus, mostly located between the various V alpha segments. The number and location of the various TCR delta segments can be determined from the IMGT database. Due to the genomic organization of the TCR delta gene segments within the TCR alpha locus, successful rearrangement of the TCR alpha locus generally results in the deletion of the TCR delta gene segments.

[0080] In some embodiments of the invention, the non-human animal comprising an unrearranged human TCR alpha variable locus also comprises at least one human V delta segment, for example, up to a complete repertoire of human V delta segments. Thus, in some embodiments, the replacement of the endogenous TCR alpha variable locus results in the replacement of at least one non-human V delta segment with a human V delta segment. In other embodiments, the non-human animal of the invention comprises a complete repertoire of human V delta, D delta and J delta segments at the unrearranged humanized TCR alpha locus; in yet other embodiments, the non-human animal comprises a complete unrearranged human TCR delta locus (i.e., a TCR delta locus comprising human variable region segments and human enhancers and constant regions) at the unrearranged humanized TCR alpha locus. An exemplary embodiment for constructing an unrearranged humanized TCR alpha locus comprising a complete unrearranged TCR delta locus is shown in FIG. 5.

[0081] In yet another embodiment, the non-human animal of the invention further comprises an unrearranged humanized TCR gamma locus, e.g., a TCR gamma locus comprising at least one human V gamma and at least one human J gamma segment (e.g., a complete repertoire of human V gamma and human J gamma variable region segments). The human TCR gamma locus is located on human chromosome 7, and the mouse TCR gamma locus is located on mouse chromosome 13. For more detailed information regarding the TCR gamma locus, see the IMGT database.

[0082] 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, a humanized TCR delta / gamma variable loci) described herein expresses a humanized T cell receptor comprising a human variable region and a non-human (e.g., a rodent, e.g., a mouse or rat) constant region on the surface of the T cell. In some embodiments, the non-human animal can express a diverse repertoire of humanized T cell receptors that recognize a variety of presented antigens.

[0083] In various embodiments of the invention, the humanized T cell receptor polypeptides described herein comprise a human leader sequence. In alternative embodiments, the humanized TCR receptor nucleic acid sequence is engineered such that the humanized TCR polypeptide comprises a non-human leader sequence.

[0084] 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. Alternatively, the humanized TCR polypeptides described herein may be expressed under the control of human regulatory elements. In various embodiments, the non-human animals described herein further comprise all of the regulatory and other sequences normally found in situ in the human genome.

[0085] 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., 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 protein. In one aspect, the second cell, e.g., an APC, is an endogenous non-human cell that expresses a human or humanized MHC molecule. In a different embodiment, the second cell is a human cell that expresses a human MHC molecule.

[0086] In one embodiment, the non-human animal expresses a humanized T cell receptor with a non-human constant region on the surface of the T cell, said receptor being capable of interacting with a non-human molecule, e.g., an anchor or signaling molecule expressed in the T cell (e.g., a CD3 molecule, a zeta chain, or other protein that is tethered to the TCR via the CD3 molecule or the zeta chain).

[0087] Thus, in one aspect, a cell complex is provided that comprises a non-human T cell expressing a TCR comprising a humanized TCR alpha chain as described herein and a humanized TCR beta chain as described herein, and a non-human antigen-presenting cell comprising an antigen bound to MHC I or MHC II. In one embodiment, the non-human constant TCR alpha and beta 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.

[0088] The genetically modified non-human animal 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, rhesus monkeys). For non-human animals for which suitable genetically modified ES cells are not readily available, other methods are used to generate non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of a non-ES cell (e.g., fibroblasts or induced pluripotent cells) and using nuclear transfer to implant the modified genome into a suitable cell, e.g., an oocyte, and gestation of the modified cell (e.g., modified oocyte) in the non-human animal under conditions suitable for the formation of an embryo.

[0089] In one embodiment, the non-human animal is a mammal. In one embodiment, the non-human animal is a small mammal, for example, the superfamily Dipodidae (Dipodoidea) or the superfamily Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mouse, rat and hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from the Calomyscidae family (e.g., mouse-like hamsters), the Cricetidae family (e.g., hamsters, New World rats and mice, and voles), the Muridae family (Old World mice and rats, gerbils, spiny mice, and crested rats), the Nesomyidae family (tree mice, rock mice, with-tailed rats, and long-legged mice), the Platacanthomyidae family (e.g., spiny dormice), and the Spalacidae family (e.g., naked mole rats, bamboo rats, and common mole rats). In a particular embodiment, the genetically modified rodent is selected from Old World mice and rats (family Muridae), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0090] In certain embodiments, the non-human animal is a rodent that is a mouse of the C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836; see also Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In a particular embodiment, the genetically modified mouse is a cross between said 129 strain and said C57BL / 6 strain. In another particular embodiment, the mouse is a cross between said 129 strain or a cross between said BL / 6 strain. In a particular embodiment, the 129 strain of the cross is a 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 cross between a BALB strain and another of the aforementioned strains.

[0091] 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 cross between two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

[0092] Thus, in one embodiment, the present invention provides a genetically modified mouse comprising an unrearranged human or humanized TCR variable locus in its genome, such as a TCR alpha, TCR beta, TCR delta and / or TCR gamma variable locus. In some embodiments, the unrearranged human or humanized TCR variable locus replaces an endogenous mouse TCR variable locus. In other embodiments, the unrearranged human and humanized TCR variable locus is at a site in the genome other than the corresponding endogenous mouse TCR locus. In some embodiments, the unrearranged human or humanized TCR variable locus is operably linked to a mouse TCR constant region.

[0093] In one embodiment, a genetically modified mouse is provided, the genetically modified mouse comprising in its genome an unrearranged TCR alpha variable locus comprising at least one human Jalpha segment and at least one human Valpha segment operably linked to a mouse TCR alpha constant gene sequence, and an unrearranged TCR beta variable locus comprising at least one human Jbeta segment, at least one human Dbeta segment, and at least one human Vbeta segment operably linked to a mouse TCR beta constant gene sequence. In a specific embodiment, the mouse comprises in its genome an unrearranged TCR alpha variable locus comprising a full repertoire of human Jalpha segments and a full repertoire of human Valpha segments operably linked to a mouse TCR alpha constant gene sequence, and an unrearranged TCR beta variable locus comprising a full repertoire of human Jbeta segments, a full repertoire of human Dbeta segments, and a full repertoire of human Vbeta segments operably linked to a mouse TCR beta constant gene sequence.

[0094] In some embodiments, an unrearranged TCR alpha variable locus comprising a human TCR alpha variable region segment replaces an endogenous mouse TCR alpha variable locus, and an unrearranged TCR beta variable locus comprising a human TCR beta variable region segment replaces an endogenous mouse TCR beta variable locus. In some embodiments, the endogenous mouse V alpha and J alpha segments are unable to rearrange to form a rearranged V alpha / J alpha sequence, and the endogenous mouse V beta, D beta and J beta segments are unable to rearrange to form a rearranged V beta / D beta / J beta sequence. In some embodiments, the human V alpha and human J alpha segments are rearranged to form a rearranged human V alpha / J alpha sequence, and the human V beta, human D beta and human J beta segments are rearranged to form a rearranged human V beta / D beta / J beta sequence.

[0095] In various embodiments, the non-human animals described herein (e.g., rodents, e.g., mice or rats) generate T cells that can go through thymic development and proceed from DN1→DN2→DN3→DN4→DP and 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.) that are typically produced by T cells during certain stages of thymic development. Thus, the non-human animals described herein express pTα complexed with TCRβ at the DN3 stage of thymic development. The non-human animals described herein express T cells that can go through thymic development and generate CD4+ and CD8+ T cells. Normally, the physiological ratio of CD4+ T cells to CD8+ T cells in the thymus is about 2:1 to 3:1. See, e.g., Ge and Stanley (2008) The O-fucose glycan in the ligand-binding domain of Notch 1 regulates embryogenesis and T cell development, Proc. Natl. Acad. Sci. USA 105:1539-44. Thus, in one embodiment, the non-human animals described herein generate CD4+ and CD8+ T cells in the thymus at a ratio of about 2:1 to 3:1 (CD4+:CD8+).

[0096] In various embodiments, the non-human animals described herein generate T cells that are capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non-human animals described herein generate a normal repertoire of effector T cells, e.g., CTLs (cytotoxic T lymphocytes), T H 1. T H 2. T REG , T H17, etc. Thus, in these embodiments, the non-human animals described herein produce effector T cells that perform different functions typical of individual T cell types, e.g., recognize, bind, and respond to foreign antigens. In various embodiments, the non-human animals described herein produce effector T cells that kill cells displaying peptide fragments of cytosolic pathogens expressed in association with MHC I molecules; recognize peptides derived from antigens degraded in intracellular vesicles and presented by MHC II molecules on the surface of macrophages to induce macrophages to kill microorganisms; produce cytokines that drive B cell differentiation; activate B cells to produce opsonizing antibodies; induce epithelial cells to produce chemokines that recruit neutrophils to the site of infection, etc.

[0097] In further embodiments, the non-human animals described herein comprise a normal number of CD3+ T cells in the periphery, e.g., in the spleen. In some embodiments, the percentage of peripheral CD3+ T cells in the non-human animals described herein is comparable to that in wild-type animals (i.e., animals that comprise all endogenous TCR variable region segments). In one embodiment, the non-human animals described herein comprise a normal ratio of splenic CD3+ T cells to total splenocytes.

[0098] In other embodiments, the non-human animals described herein are capable of producing a population of memory T cells in response to an antigen of interest, for example, the non-human animals produce 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).

[0099] 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 normal numbers of B cells, myeloid cells (e.g., dendritic cells), mast cells, and NK cells. In some embodiments, the non-human animals described herein develop normal dendritic cell populations in the thymus.

[0100] The predominant type 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 present invention, the T cells of a non-human animal comprising a humanized TCRα and / or β locus exhibit normal utilization of the TCRα / β and TCRδ / γ loci, e.g., utilization of the TCRα / β and TCRδ / γ loci similar to that of a wild-type animal (e.g., the T cells of the non-human animals described herein express TCRα / β and TCRδ / γ proteins in a ratio comparable to that 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 normal utilization of all loci.

[0101] In addition to the genetically engineered non-human animals described herein, there is also provided a non-human embryo (e.g., a rodent embryo, e.g., a mouse or rat embryo) that comprises a donor ES cell derived from a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein. In one embodiment, the embryo comprises an ES donor cell that comprises an unrearranged humanized TCR locus and a host embryo cell.

[0102] Also provided are tissues derived from the non-human animals (e.g., mice or rats) described herein and expressing humanized TCR polypeptides (e.g., TCR alpha and / or TCR beta, or TCR delta and / or TCR gamma polypeptides).

[0103] Additionally, non-human cells isolated from the non-human animals described herein are also provided. In one embodiment, the cells are ES cells. In one embodiment, the cells are T cells. In one embodiment, the T cells are CD4+ T cells. In another embodiment, the T cells are CD8+ T cells.

[0104] Also provided is a non-human cell comprising a chromosome or fragment thereof of a non-human animal described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal described herein. In one embodiment, the non-human cell comprises a chromosome or fragment thereof as a result of nuclear transfer.

[0105] Also provided is a non-human cell expressing a TCR protein comprising a human variable region and a non-human constant region. The TCR protein may comprise TCR alpha, TCR beta, or a combination thereof. In one embodiment, the cell is a T cell, e.g., a CD4+ or CD8+ T cell.

[0106] In one aspect, there is provided a non-human induced pluripotent cell comprising an unrearranged humanized TCR locus encoding a humanized TCR polypeptide as described herein. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.

[0107] In one aspect, there is provided a hybridoma or quadroma derived from a cell of a non-human animal described herein. In one embodiment, the non-human animal is a rodent, such as a mouse or a rat.

[0108] Also provided are methods for making the genetically modified non-human animals (e.g., rodents, e.g., mice or rats) described herein. The methods for making genetically modified non-human animals result in animals whose genomes comprise unrearranged humanized TCR loci (e.g., unrearranged humanized TCR alpha, TCR beta, TCR delta and / or TCR gamma loci). In one embodiment, a method for making a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) that expresses a T cell receptor on the surface of a T cell comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region comprises replacing in a first non-human animal an endogenous non-human TCR alpha variable locus with an unrearranged humanized TCR alpha variable locus comprising at least one human V alpha segment and at least one human J alpha segment, wherein the humanized TCR alpha variable locus operably binds to the endogenous TCR alpha constant region. replacing in a second non-human animal an 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, wherein the humanized TCR beta variable locus is operably linked to an endogenous TCR beta constant region; and mating the first and second non-human animals to obtain a non-human animal expressing a T cell receptor comprising a human variable region and a non-human constant region. In other embodiments, the invention provides a genetically modified non-human animal whose genome comprises an unrearranged humanized TCR alpha locus created according to the methods described herein, or a method for producing a non-human animal whose genome comprises an unrearranged humanized TCR beta locus created according to the methods described herein. In various embodiments, the replacement is at the endogenous locus.In some embodiments, the method utilizes one or more targeting constructs generated using VELOCIGENE® technology, as described in the Examples, to introduce the construct(s) into ES cells, and introduce the targeted ES cell clones into mouse embryos using VELOCIMOUSE® technology. In some embodiments, the ES cells are derived from a cross between 129 and C57BL / 6 mice. In various embodiments, the method involves a progressive humanization strategy in which constructs containing additional variable region segments are introduced into the ES cells at each subsequent step of humanization, ultimately resulting in mice containing a full repertoire of human variable region segments (see, e.g., Figures 3 and 7).

[0109] Thus, also provided is a nucleotide construct for use in making the genetically engineered non-human animals described herein. In one aspect, the nucleotide construct comprises 5' and 3' homology arms, a human DNA fragment comprising a human TCR variable region gene segment(s), and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a TCR alpha gene fragment and comprises at least one human TCR alpha variable region segment. In another embodiment, the human DNA fragment is a TCR beta fragment and comprises at least one human TCR beta variable region gene segment. In one aspect, at least one homology arm is a non-human homology arm and is homologous to a non-human TCR locus (e.g., a non-human TCR alpha or TCR beta locus).

[0110] Selection cassettes are nucleotide sequences inserted into targeting constructs to facilitate the selection of cells (e.g., ES cells) that have integrated the construct of interest. Many suitable selection cassettes are known in the art. In general, selection cassettes allow for positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, Spec, etc.). In addition, selection cassettes can be flanked by recombination sites, allowing for the deletion of the selection cassette upon treatment with recombinase enzymes. Commonly used recombination sites are loxP and Frt, which are recognized by Cre and Flp enzymes, respectively, but others are known in the art.

[0111] 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, e.g., within a human intron. In another embodiment, the selection cassette is located at the junction of a human and mouse DNA fragment.

[0112] Various exemplary embodiments of targeting strategies for generating genetically engineered non-human animals, constructs, and targeting vectors for use therewith are shown in FIGS.

[0113] After completion of gene targeting, the ES cells or genetically modified non-human animals are screened to confirm successful incorporation of the desired exogenous nucleotide sequence or expression of the exogenous polypeptide (e.g., human TCR variable region segment). Numerous techniques are known to those of skill in the art, including, but not limited to, Southern blotting, long PCR, quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescent in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, and the like. In one example, non-human animals (e.g., mice) carrying the desired genetic modification can be identified by screening for loss of mouse alleles and / or gain of human alleles using a modification of the allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659. Other assays for identifying specific nucleotide or amino acid sequences in genetically modified animals are known to those of skill in the art.

[0114] The present disclosure also provides a method of modifying a TCR variable locus (e.g., a TCR alpha, TCR beta, TCR delta and / or TCR gamma locus) of a non-human animal to express a humanized TCR protein as described herein. In one embodiment, the invention provides a method of modifying a TCR variable locus to express a humanized TCR protein on the surface of a T cell, comprising replacing an endogenous non-human TCR variable locus in a non-human animal with an unrearranged humanized TCR variable locus. In one embodiment, the TCR variable locus is a TCR alpha variable locus, the unrearranged humanized TCR variable locus comprises at least one human Valpha segment and at least one human Jalpha segment. In one embodiment, the TCR variable locus is a TCR beta variable locus, the unrearranged humanized TCR variable locus comprises at least one human Vbeta segment, at least one human Dbeta segment and at least one human Jbeta segment. In various embodiments, the unrearranged humanized TCR variable loci are operably linked to corresponding endogenous non-human TCR constant regions.

[0115] Also provided is a humanized TCR protein made by a non-human animal (e.g., a rodent, e.g., a mouse or rat) as described herein, comprising a human variable region and a non-human constant region. Thus, the humanized TCR protein comprises human complementarity determining regions of its variable domain (i.e., human CDR1, 2 and 3) and a non-human constant region.

[0116] Although the following examples describe genetically engineered non-human animals whose genomes contain humanized TCR alpha and / or TCR beta variable loci, one of skill in the art will appreciate that similar strategies may be used to generate genetically engineered animals whose genomes contain humanized TCR delta and / or TCR gamma variable loci. Also provided are genetically engineered non-human animals in which all four TCR variable loci are humanized.

[0117] Use of genetically modified TCR animals In various embodiments, the genetically modified non-human animals of the invention generate T cells with humanized TCR molecules on their surface, and as a result will recognize peptides presented to them by MHC complexes in a manner similar to humans. The genetically modified non-human animals described herein can be used to study human T cell development and function and the process of immune tolerance; to test human vaccine candidates; to generate TCRs with particular specificities for TCR gene therapy; to generate TCR libraries against disease-associated antigens (e.g., tumor-associated antigens (TAA)); and the like.

[0118] T cell therapy has been of increasing interest in the art, since T cells (e.g., cytotoxic T cells) can be directed to attack and destroy antigens of interest, such as viral antigens, bacterial antigens, tumor antigens, etc., or cells that present them. Early studies of cancer T cell therapy aimed to isolate tumor-infiltrating lymphocytes (TILs; a population of lymphocytes in tumor masses that are believed to contain T cells reactive to tumor antigens) from tumor cell masses, expand them in vitro with T cell growth factors, and return them to patients in a process called adoptive T cell transfer. See, for example, Restifo et al. (2012) Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews 12:269-81; Linnermann et al. (2011) T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success, J. Invest. Dermatol. 131:1806-16. However, the success of these therapies has so far been limited to melanoma and renal cell carcinoma; TIL adoptive transfer is not specifically directed against defined tumor-associated antigens (TAA). Linnermann et al. (supra).

[0119] Attempts have been made to initiate TCR gene therapy, in which T cells are selected or programmed to target an antigen of interest, e.g., a TAA. Current TCR gene therapy relies on the identification of sequences of TCRs that target specific antigens, e.g., tumor-associated antigens. For example, Rosenberg and colleagues have published several studies in which genes encoding TCR α and β chains specific for the melanoma-associated antigen MART-1 epitope were transduced into peripheral blood lymphocytes derived from melanoma patients, and the resulting expanded lymphocytes were used for adoptive T cell therapy. Johnson et al. (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen, Blood 114:535-46; Morgan et al. (2006) Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes, Science 314:126-29. MART-1-specific TCRs have been isolated from patients who experienced tumor regression following TIL therapy. However, the identification of such TCRs, especially high avidity TCRs (most likely to be therapeutically useful), is complicated by the fact that most tumor antigens are self-antigens, and TCRs targeting these antigens are often missing or have suboptimal affinity, primarily due to immune tolerance.

[0120] In various embodiments, the present invention solves this problem by providing genetically engineered non-human animals that contain unrearranged human TCR variable loci in their genome. The non-human animals described herein can produce T cells with a diverse repertoire of humanized T cell receptors. Thus, the non-human animals described herein can be a source of a diverse repertoire of humanized T cell receptors, e.g., high avidity humanized T cell receptors for use in adoptive T cell transfer.

[0121] Thus, in one embodiment, the invention provides a method of generating a T cell receptor against a human antigen, the method comprising the steps of immunizing a non-human animal as described herein (e.g., a rodent, e.g., a mouse or rat) with the antigen of interest, mounting an immune response in the animal, isolating activated T cells from the animal having specificity for the antigen of interest, and determining the nucleic acid sequence of the T cell receptor expressed by the antigen-specific T cells.

[0122] In one embodiment, the invention provides a method of generating a human T cell receptor specific for an antigen of interest (e.g., a disease-associated antigen), comprising immunizing a non-human animal as described herein with the antigen of interest, mounting an immune response in the animal, isolating T cells from the animal reactive to the antigen of interest, determining the nucleic acid sequence of the human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of the human TCR constant region such that the human TCR variable region is operably linked to the human TCR constant region, and expressing a human T cell receptor specific for the antigen of interest from the construct. In one embodiment, the steps of isolating the T cell, determining the nucleic acid sequence of the human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of the human TCR constant region, and expressing the human T cell receptor are performed using standard techniques known to those of skill in the art.

[0123] In one embodiment, a nucleotide sequence encoding a T cell receptor specific for an antigen of interest is expressed in a cell. In one embodiment, the cell expressing the TCR is selected from CHO, COS, 293, HeLa, PERC.6™ cells, and the like.

[0124] The antigen of interest can be any antigen known to cause or be associated with a disease or condition, such as a tumor-associated antigen; an antigen from a virus, bacteria, or other pathogen, etc. Many tumor-associated antigens are known in the art. A selection of tumor-associated antigens is shown in Cancer Immunity (A Journal of the Cancer Research Institute) Peptide Database (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the present invention, the antigen of interest is a human antigen, such as a human tumor-associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and uses a T cell receptor to kill cells expressing the antigen.

[0125] In one embodiment, provided herein is a method of identifying T cells having specificity for an antigen of interest, e.g., a tumor associated antigen, comprising immunizing a non-human animal as described herein with the antigen of interest, mounting an immune response in the animal, and isolating T cells from the non-human animal having specificity for the antigen.

[0126] The present invention provides a new method for adoptive T cell therapy. Thus, provided herein is a method for treating or ameliorating a disease or condition (e.g., cancer) in a subject (e.g., a mammalian subject, e.g., a human subject), comprising immunizing a non-human animal as described herein with an antigen associated with the disease or condition, initiating an immune response in the animal, isolating a population of antigen-specific T cells from the animal, and injecting the isolated antigen-specific T cells into the subject. In one embodiment, the present invention provides a method of treating or ameliorating a disease or condition in a human subject, comprising immunizing a non-human animal as described herein with an antigen of interest (e.g., a disease- or condition-associated antigen, e.g., a tumor-associated antigen), initiating an immune response in the animal, isolating a population of antigen-specific T cells from the animal, determining the nucleic acid sequence of a T cell receptor expressed by the antigen-specific T cells, cloning the nucleic acid sequence of the T cell receptor into an expression vector (e.g., a retroviral vector), introducing the vector into T cells derived from the subject such that the T cells express the antigen-specific T cell receptor, and injecting the T cells into the subject. In one embodiment, the T cell receptor nucleic acid sequence is further humanized before being introduced into T cells derived from the subject. For example, the sequence encoding the non-human constant region is modified to more resemble a human TCR constant region (e.g., replacing the non-human constant region with a human constant region). In some embodiments, the disease or condition is cancer. In some embodiments, the antigen-specific T cell population is expanded before infusion into the subject. In some embodiments, the immune cell population of the subject is immunodepleted before infusion of the antigen-specific T cell. In some embodiments, the antigen-specific TCR is a high avidity TCR, for example, a TCR with high avidity for tumor-associated antigen. In some embodiments, the T cell is a cytotoxic T cell. In other embodiments, the disease or condition is caused by a virus or bacteria.

[0127] In another embodiment, the disease or condition is an autoimmune disease. REG These cells are a subpopulation of T cells that maintain tolerance to self-antigens and prevent pathological self-reactivity. Thus, antigen-specific T cells in the non-human animals of the invention described herein REG Methods for treating autoimmune diseases that rely on the production of cells are also provided herein.

[0128] Also provided herein is a method of treating or ameliorating a disease or condition (e.g., cancer) in a subject, the method comprising introducing cells affected by the disease or condition (e.g., cancer cells) from the subject into a non-human animal, causing the animal to mount an immune response against the cells, isolating from the animal a population of T cells reactive to the cells, determining the nucleic acid sequence of a T cell receptor expressed by the T cells, cloning the T cell receptor sequence into a vector, introducing the vector into T cells derived from the subject, and injecting the subject's T cells bearing the T cell receptor into the subject.

[0129] Also provided herein is the use of a non-human animal as described herein to generate a nucleic acid sequence encoding a human TCR variable domain (e.g., a TCR alpha and / or beta variable domain). In one embodiment, also provided is a method for generating a nucleic acid sequence encoding a human TCR variable domain, comprising immunizing a non-human animal as described herein with an antigen of interest, causing said non-human animal to mount an immune response against the antigen of interest, and obtaining therefrom a nucleic acid sequence encoding a human TCR variable domain that binds to the antigen of interest. In one embodiment, the method further comprises the step of generating a nucleic acid sequence encoding a human TCR variable domain operably linked to a non-human TCR constant region, comprising isolating T cells from the non-human animal as described herein, and obtaining therefrom a nucleic acid sequence encoding a TCR variable domain linked to a TCR constant region.

[0130] Also provided herein is the use of a non-human animal as described herein to generate a human therapeutic, comprising the steps of immunizing the non-human animal with an antigen of interest (e.g., a tumor-associated antigen), mounting an immune response in the non-human animal, obtaining T cells from the animal that are reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized TCR protein that binds the antigen of interest, and using the nucleic acid sequence(s) encoding the humanized TCR protein in a human therapeutic.

[0131] Accordingly, there is also provided a method for making a human therapeutic comprising the steps of immunizing a non-human animal as described herein with an antigen of interest, mounting an immune response in the non-human animal, obtaining T cells from the animal that are reactive to said antigen of interest, obtaining from said T cells a nucleic acid sequence(s) encoding a humanized T cell receptor that binds said antigen of interest, and using said humanized T cell receptor for a human therapeutic.

[0132] In one embodiment, the human therapeutic is a T cell (e.g., a human T cell, e.g., a T cell derived from a human subject) that harbors a nucleic acid sequence of interest (e.g., transfected, transduced or otherwise introduced with a nucleic acid of interest) and that expresses a humanized TCR protein having affinity for an antigen of interest. In one embodiment, the subject in which the therapeutic is used is in need of therapy for a particular disease or condition, and the antigen is associated with the disease or condition. In one embodiment, the T cell is a cytotoxic T cell, the antigen is a tumor-associated antigen, and the disease or condition is cancer. In one embodiment, the T cell is derived from the subject.

[0133] In another embodiment, the human therapeutic is a T cell receptor. In one embodiment, the therapeutic receptor is a soluble T cell receptor. Great efforts have been developed to create soluble T cell receptors or TCR variable regions for use in therapeutics. Creating soluble T cell receptors relies on obtaining rearranged TCR variable regions. One approach is to design single-chain TCRs containing TCRα and TCRβ and fuse them by a linker, similar to the format of scFv immunoglobulins (see, for example, International Application WO2011 / 044186). The resulting scTv, if similar to scFv, will provide a thermostable and soluble form of TCRα / β binding protein. Alternative approaches have included engineering soluble TCRs with TCR β constant domains (see, e.g., Chung et al. (1994) Functional three-domain single-chain T-cell receptors, Proc. Natl. Acad. Sci. USA. 91:12654-58); and engineering non-native disulfide bonds at the interface between the TCR constant domains (reviewed in Boulter and Jakobsen (2005) Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens, Clinical and Experimental Immunology 142:454-60; see also U.S. Pat. No. 7,569,664). Other formats of soluble T cell receptors have also been described. The non-human animals described herein can be used to determine the sequence of a T cell receptor that binds an antigen of interest with high affinity and then design a soluble T cell receptor based on that sequence.

[0134] Soluble T cell receptors derived from TCR receptor sequences expressed by non-human animals can be used to block the function of proteins of interest, e.g., viral, bacterial or tumor-associated proteins. Alternatively, soluble T cell receptors can be fused to moieties capable of killing infected or cancerous cells, e.g., cytotoxic molecules (e.g., chemotherapeutic drugs), toxins, radionuclides, prodrugs, antibodies, etc. Soluble T cell receptors can also be fused to immunomodulatory molecules, e.g., cytokines, chemokines, etc. Soluble T cell receptors can also be fused to immunosuppressive molecules, e.g., molecules that inhibit T cells from killing other cells that carry the antigen recognized by the T cell. Such soluble T cell receptors fused to immunosuppressive molecules can be used, for example, to block autoimmunity. Various exemplary immune suppressive molecules that can be fused to soluble T cell receptors are reviewed in Ravetch and Lanier (2000) Immune Inhibitory Receptors, Science 290:84-89, which is incorporated herein by reference.

[0135] The present invention also provides methods for studying immune responses associated with human TCRs, including human TCR rearrangements, T cell development, T cell activation, immune tolerance, and the like.

[0136] Methods for testing vaccine candidates are also provided. In one embodiment, methods are provided herein for determining whether a vaccine activates an immune response (e.g., T cell proliferation, cytokine release, etc.) and results in the production of effector and memory T cells (e.g., central memory T cells and effector memory T cells). EXAMPLES

[0137] The present invention is further illustrated by the following non-limiting examples. These examples are presented for the purpose of aiding in the understanding of the present invention, and are not intended, and should not be viewed as limiting its scope in any way. The examples do not include detailed descriptions of conventional methods well known to those skilled in the art, such as molecular cloning methods. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are given in degrees Celsius, and pressures are at or near atmospheric pressure.

[0138] Example 1. Generation of mice with humanized TCR variable loci Mice containing a deletion of an endogenous TCR (α or β) variable locus and a replacement of an endogenous V segment and an endogenous J segment, or a replacement of an endogenous V segment, an endogenous D segment, and an endogenous J segment, are generated using VELOCIGENE® genetic engineering methods (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela, DM et al. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6):652-659), in which human sequences derived from a BAC library using bacterial homologous recombination are used to generate large targeting vectors (LTVECs) containing genomic fragments of the human TCR variable locus flanked by targeting arms for targeting the LTVECs to the endogenous mouse TCR variable locus in mouse ES cells. The LTVECs are linearized and electroporated into mouse ES cell lines according to Valenzuela et al. ES cells are selected for hygromycin or neomycin resistance and screened for loss of the mouse allele or gain of the human allele.

[0139] Targeted ES cell clones are introduced into 8-cell stage (or earlier) mouse embryos by the VELOCIMOUSE® method (Poueymirou, WT et al. (2007). F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat. Biotech. 25:91-99). VELOCIMICE® (F0 mice derived entirely from donor ES cells) carrying humanized TCR loci are identified by screening for loss of endogenous TCR variable alleles and gain of human alleles using a modified allelic assay (Valenzuela et al.). F0 pups are genotyped and bred to homozygosity. Mice homozygous for humanized TCR alpha and / or beta variable loci (e.g., comprising a subset of human TCR alpha and / or beta variable segments) are generated and phenotyped as described herein.

[0140] All mice were housed and bred in specialized pathogen-free facilities at Regeneron Pharmaceuticals, and all animal studies were approved by the IACUC and Regeneron Pharmaceuticals.

[0141] Example 2 Progressive humanization of the TCR alpha variable locus 1.5 megabases of DNA in the mouse TCR alpha locus corresponding to 110 mouse V segments and 60 mouse J segments were replaced with 1 megabase of DNA corresponding to 54 V segments and 61 J segments of human TCR alpha using the progressive humanization strategy summarized in Figures 2 and 3. The nucleic acid sequences of the junctions of the various targeting vectors used for the progressive humanization strategy of the TCR alpha locus are summarized in Table 2 and incorporated in the Sequence Listing.

[0142] [Table 2]

[0143] In particular, as demonstrated in Figure 4A, DNA from mouse BAC clone RP23-6A14 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1539) to replace the TCRAJ1-TCRAJ28 region of the endogenous mouse TCRα locus with a Ub-hygromycin cassette followed by loxP sites. DNA from mouse BAC clone RP23-117i19 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1535) to replace TCRAV1 of the endogenous mouse TCRα locus and an approximately 15 kb region surrounding (and including) the endogenous mouse TCRδ locus with a PGK-neomycin cassette followed by loxP sites. ES cells carrying a double-targeted chromosome (i.e., a single endogenous mouse TCR α locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods known in the art (e.g., TAQMAN™). The modified ES cells were treated with CRE recombinase, which mediates the deletion of the region between the two loxP sites (i.e., the region consisting of the endogenous mouse TCR α locus spanning TCRAV1-TCRAJ1), leaving behind only the single loxP site, the neomycin cassette, and the mouse constant region and mouse enhancer region. This strategy resulted in the creation of a deleted mouse TCR α / δ locus (MAID1540).

[0144] The first human targeting vector for TCRα had 191,660 bp of human DNA derived from the CTD2216p1 and CTD2285m07 BAC clones (Invitrogen) containing the first two consecutive human TCRαV gene segments (TRAV40 and 41) and 61 TCRαJ (50 functional) gene segments. This BAC was modified to contain a Not1 site 403 bp downstream (3') of the TCRαJ1 gene segment for ligation of the 3' mouse homology arm by homologous recombination, and a 5' AsiSI site for ligation of the 5' mouse homology arm. Two different homology arms were used for ligation to this human fragment: the 3' homology arm contained the endogenous mouse TCRα sequence from the RP23-6A14 BAC clone, and the 5' homology arm contained the 5' endogenous TCRα sequence of mouse TCRαV from mouse BAC clone RP23-117i19. This mouse-human chimeric BAC was used as a targeting vector (MAID1626) for the initial insertion of the human TCRα gene segment into the mouse TCRα locus, plus an upstream loxP-Ub-hygromycin-loxP cassette (Figure 4B). The nucleic acid sequences of the junctions for the targeting vector MAID1626 (SEQ ID NOs: 1-3) are listed in Table 2.

[0145] A series of human targeting vectors were then generated that used the same mouse 5' arm containing endogenous TCRα sequences 5' to mouse TCRαV derived from RP23-117i19, a mouse BAC clone alternating with loxP-neomycin-loxP and loxP-hygromycin-loxP (or frt-hygromycin-frt for MAID1979) selection cassettes.

[0146] To create a human TCRα minilocus containing a total of eight human TCRαV (seven functional) gene segments and 61 human TCRαJ (50 functional) gene segments, DNA from the human BAC clone RP11-349p11 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1767) (Figure 4C). This added 104,846 bp of human DNA containing the next six (five functional) consecutive human TCRαV gene segments (TRAV35-TRAV39) and a 5' loxP-Ub-neomycin-loxP cassette. The resulting TCRα locus contained a total of eight human TCRαV (seven functional) gene segments and 61 human TCRαJ gene segments operably linked to the mouse TCRα constant gene and mouse enhancer in addition to the 5' loxP-Ub-neomycin-loxP cassette. The nucleic acid sequences of the junctions for the MAID1767 targeting vector (SEQ ID NOs: 4 and 5) are set forth in Table 2.

[0147] To generate a human TCRα minilocus containing a total of 23 human TCRαV (17 functional) and 61 human TCRαJ gene segments, DNA from a mouse BAC clone containing, from 5' to 3', a unique I-CeuI site, a 20 kb mouse TCRA arm 5' of the mouse TCRA locus used for homologous recombination into ES cells, and a reverse loxP-Ub-Hyg-loxP cassette was modified by bacterial homologous recombination to contain, from 5' to 3', a unique I-CeuI site, a 20 kb mouse TCRA arm 5' of the mouse TCRA locus, a frt-pgk-Hyg-frt cassette, and a unique AsiSI site. DNA from RP11-622o20 (Invitrogen), a human BAC clone carrying human TCRαV22-V34, was modified by homologous recombination to contain a Spec cassette flanked by unique I-CeuI and AsiSI sites. The Spec cassette in the modified human BAC clone was then replaced by sequences contained between the I-CeuI and AsiSI sites in the modified mouse BAC clone by standard restriction digestion / ligation methods. The resulting targeting vector (MAID1979; Figure 4D) added 136,557 bp of human DNA containing the following 15 (10 functional) contiguous human TCRαJ gene segments (TRAV22-TRAV34) and the 5' frt-pgk-Hyg-frt cassette. The resulting TCRα locus contained a total of 23 human TCRαV (17 functional) and 61 human TCRαV gene segments operably linked to the mouse TCRα constant gene and mouse enhancer in addition to the 5' frt-pgk-Hyg-frt cassette. The nucleic acid sequences of the junctions for the MAID1979 targeting vector (SEQ ID NOs: 6 and 7) are listed in Table 2.

[0148] To create a human TCRα minilocus containing a total of 35 human TCRαV (28 functional) gene segments and 61 human TCRαJ gene segments, DNA from human BAC clone CTD2501-k5 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1769) (Figure 4E). This added 124,118 bp of human DNA containing the following 12 (11 functional) contiguous human TCRαV gene segments (TRAV13-2 to TRAV21) and a 5' loxP-Ub-neomycin-loxP cassette. The resulting TCRα locus contained a total of 35 human TCRαV (28 functional) gene segments and 61 human TCRαJ gene segments operably linked to the mouse TCRα constant gene and mouse enhancer in addition to the 5' loxP-Ub-neomycin-loxP cassette. The nucleic acid sequences of the junctions for the MAID1769 targeting vector (SEQ ID NOs: 8 and 9) are set forth in Table 2.

[0149] To create a human TCRα minilocus containing a total of 48 human TCRαV (39 functional) gene segments and 61 human TCRαJ gene segments, DNA from human BAC clone RP11-92F11 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1770) (Figure 4F). This added 145,505 bp of human DNA containing the following 13 (11 functional) contiguous human TCRαJ gene segments (TRAV6 to TRAV8.5) and a 5' loxP-Ub-hygromycin-loxP cassette. The resulting TCRα locus contains a total of 48 human TCRαV (39 functional) gene segments and 61 human TCRαJ gene segments operably linked to the mouse TCRα constant gene and mouse enhancer in addition to the 5' loxP-Ub-hygromycin-loxP cassette. The nucleic acid sequences of the junctions for the MAID1770 targeting vector (SEQ ID NOs: 10 and 11) are set forth in Table 2.

[0150] To create a human TCRα minilocus containing a total of 54 human TCRαV (45 functional) gene segments and 61 human TCRαJ gene segments, DNA from human BAC clone RP11-780M2 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1771) (Figure 4G). This added 148,496 bp of human DNA containing the following six (six functional) contiguous human TCRαV gene segments (TRAV1-1 to TRAV5) and a 5' loxP-Ub-neomycin-loxP cassette. The resulting TCRα locus contains a total of 54 human TCRαV (45 functional) gene segments and 61 human TCRαJ gene segments operably linked to the mouse TCRα constant gene and mouse enhancer in addition to the 5' loxP-Ub-neomycin-loxP cassette. The nucleic acid sequences of the junctions for the MAID1771 targeting vector (SEQ ID NOs: 12 and 13) are set forth in Table 2.

[0151] In any of the above steps, the selection cassette is removed via deletion with Cre or Flp recombinase. In addition, the human TCR delta locus can be introduced as depicted in Figure 5.

[0152] Example 3 Progressive humanization of TCRβ variable loci Using the progressive humanization strategy summarized in Figures 6 and 7, 0.6 megabases of DNA in the mouse TCR beta locus corresponding to 33 mouse V segments, 2 mouse D segments, and 14 mouse J segments were replaced with 0.6 megabases of DNA corresponding to 67 V segments, 2 D segments, and 14 J segments of human TCR beta. The nucleic acid sequences of the junctions of the various targeting vectors used for the progressive humanization strategy of the TCR beta locus are summarized in Table 3 and incorporated in the Sequence Listing.

[0153] [Table 3]

[0154] In particular, DNA from mouse BAC clone RP23-153p19 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1544) to replace a 17 kb region immediately upstream of the 3' trypsinogen gene cluster in the endogenous mouse TCRβ locus (including TCRBV30) with a PGK-neo cassette followed by a loxP site (Figure 8A). DNA from mouse BAC clone RP23-461h15 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1542) to replace a 8355 bp region downstream of the 5' trypsinogen gene cluster in the endogenous mouse TCRβ locus (including TCRBV2 and TCRBV3) with a Ub-hygromycin cassette followed by a loxP site. ES cells carrying a double-targeted chromosome (i.e., a single endogenous mouse TCRβ locus targeted by both targeting vectors) were confirmed by karyotyping and screening methods known in the art (e.g., TAQMAN™). The modified ES cells were treated with CRE recombinase to mediate deletion of the region between the 5′ and 3′ loxP sites (comprising the endogenous mouse TCRβ locus spanning TCRBV2-TCRBV30), leaving behind only a single loxP site, a hygromycin cassette, and mouse TCRBD, mouse TCRBJ, mouse constant, and mouse enhancer sequences. As noted in FIG. 8A, one mouse TCRVβ was left upstream of the 5′ cluster of trypsinogen gene, and one mouse TCRBβ was left downstream of mouse Eβ.

[0155] The first human targeting vector for TCRβ had 125,781 bp of human DNA from BAC clone CTD2559j2 (Invitrogen), which contains the first 14 consecutive human TCRβ V gene segments (TRBV18-TRBV29-1). This BAC was modified to contain 5′ AsiSI and 3′ AscI sites for ligating 5′ and 3′ mouse homology arms by homologous recombination. Two different homology arms were used to ligate into the human fragment: one set of homology arms contained endogenous TCRβ sequences around the downstream mouse trypsinogen gene from BAC clone RP23-153p19, and another set contained endogenous TCRβ sequences around the upstream mouse trypsinogen gene from mouse BAC clone RP23-461h15. This mouse-human chimeric BAC was used as a targeting vector (MAID1625) to perform the initial insertion of human TCRβ gene segments, plus an upstream frt-Ub-neomycin-frt cassette, in the mouse TCRβ locus, resulting in a human TCRβ minilocus containing 14 (8 functional) human TCRβVs (Figure 8B). The nucleic acid sequences of the junctions for the MAID1625 targeting vector (SEQ ID NOs: 14-16) are listed in Table 3.

[0156] To replace the mouse TCRβD segment and the mouse TCRβJ segment with the human TCRβD segment and the human TCRβJ segment, DNA from the mouse BAC clone RP23-302p18 (Invitrogen) and the human BAC clone RP11-701D14 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1715) for ES cells containing the TCRβV minilocus described above (i.e., MAID1625). This modification replaced an approximately 18,540 bp region in the endogenous mouse TCRβ locus (100 bp downstream of the polyA of the 3' trypsinogen gene to 100 bp downstream from the J segment in the D2 cluster encompassing mouse TCRBD1-J1, mouse constant1, and mouse TCRBD2-J2) with an approximately 25,425 bp sequence containing human TCRBD1-J1, a loxP-Ub-hygromycin-loxP cassette, mouse constant1, and human TCRBD2-J2 (Figure 8C(i)). ES cells carrying a double-targeted chromosome (i.e., a single endogenous mouse TCRβ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods known in the art (e.g., TAQMAN™). The modified ES cells were treated with CRE recombinase, which mediated the deletion of the hygromycin cassette, leaving behind only a single loxP site downstream from the human J segment within the D1J cluster (Figure 8C(ii)). The nucleic acid sequences of the junctions for the MAID1715 targeting vector (SEQ ID NOs: 17-21) are listed in Table 3.

[0157] A series of human targeting vectors were then generated using the same mouse 5' arms containing the endogenous TCRβ sequence surrounding the upstream mouse trypsinogen gene derived from RP23-461h15, a mouse BAC clone with alternating selection cassettes.

[0158] To generate a human TCRβ minilocus containing a total of 40 human TCRβV (30 functional) segments as well as human TCRβD and J segments, DNA from human BAC clones RP11-134h14 and RP11-785k24 (Invitrogen) was modified by homologous recombination and combined into a targeting vector (MAID1791) using standard bacterial homologous recombination, restriction digestion / ligation, and other cloning methods. Introduction of the MAID1791 targeting vector resulted in the addition of 198,172 bp of human DNA containing the following 26 (22 functional) contiguous human TCRβV gene segments (TRBV6-5 to TRBV17) and a 5′ frt-Ub-hygromycin-frt cassette. The resulting TCRβ locus contained a total of 40 human TCRβV (30 functional) gene segments, as well as human TCRβD and J gene segments, operably linked to the mouse TCRβ constant gene and mouse enhancer, in addition to the 5' frt-Ub-hygromycin-frt cassette (FIG. 8D). The nucleic acid sequences of the junctions for the MAID1791 targeting vector (SEQ ID NOs: 22 and 23) are listed in Table 3.

[0159] To generate a human TCRβ minilocus containing a total of 66 human TCRβV (47 functional) segments as well as human TCRβD and J segments, DNA from the human BAC clone RP11-902B7 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID1792), resulting in the addition of 159,742 bp of human DNA containing the following 26 (17 functional) contiguous human TCRβV gene segments (TRBV1 to TRBV12-2) and a 5′ frt-Ub-neomycin-frt cassette. The resulting TCRβ locus contained a total of 66 human TCRβV (47 functional) gene segments, as well as human TCRβD and J gene segments, operably linked to the mouse TCRβ constant gene and mouse enhancer, in addition to the 5' frt-Ub-neomycin-frt cassette (FIG. 8E). The nucleic acid sequences of the junctions for the MAID1792 targeting vector (SEQ ID NOs: 24 and 25) are listed in Table 3.

[0160] In any of the above steps, the selection cassette is removed via deletion with Cre or Flp recombinase. For example, as depicted in Figure 7, MAID1716 corresponds to MAID1715 with the hygromycin cassette deleted.

[0161] Finally, a human TCRβ minilocus was created that contains a total of 67 human TCRβV (48 functional) segments as well as human TCRβD and J segments. Mouse TCRBV31 is located approximately 9.4 kb 3' to TCRBC2 (the second TCRB constant region sequence) and in the reverse orientation relative to the other TCRBV segments. The equivalent human V segment is TCRBV30, which is also located in a similar location within the human TCRB locus.

[0162] To humanize TCRBV31, a mouse BAC clone containing mouse TCRBV31 was modified by bacterial homologous recombination to generate the LTVEC MAID6192 (Figure 8F). The entire coding region starting at the start codon in exon 1 of TCRBV31, the intron, the 3' UTR, and the recombination signal sequence (RSS) were replaced with the homologous human TCRBV30 sequence. The 5' UTR was left as the mouse sequence. For selection, a self-deletion cassette (lox2372-ubiquitin promoter-Hyg-PGKpolyA-protamine promoter-Cre-SV40polyA-lox2372) was inserted within the intron (72 bp 3' of exon 1 and 289 bp 5' of exon 2). For convenience, Figures 7 and 8 depict the selection cassette 3' of hTCRBV30 when engineered to be located in the intron between exons 1 and 2 of the hTCRBV30 gene. The protamine promoter that drives Cre expression is transcribed exclusively in post-meiotic spermatids, so the cassette "self-deletes" in the F1 generation of mice.

[0163] The nucleic acid sequences of the junctions for the MAID6192 targeting vector (SEQ ID NOs:26 and 27) are set forth in Table 3. MAID6192 DNA is electroporated into MAID1792 ES cells. ES cell clones are selected for hygromycin resistance and screened for loss of the mouse TCRB31 allele and gain of the human TCRB30 allele.

[0164] Optionally, the remaining 5' mouse TCR β V segment is also deleted using a similar engineering strategy.

[0165] Example 4: Generation of TCRα / TCRβ mice At each step of the incremental humanization of the TCR alpha and beta loci, mice homozygous for the humanized TCR alpha and beta variable loci can be bred with mice homozygous for the humanized TCR beta variable loci to form progeny that contain the humanized TCR alpha and beta variable loci. The progeny are bred to be homozygous for the humanized TCR alpha and beta loci.

[0166] In one embodiment, a mouse homozygous for a humanized TCR alpha variable locus containing 8 human Va and 61 human Ja (MAID1767; "1767HO") was bred to a mouse homozygous for a humanized TCR beta variable locus containing 14 human Vβ, 2 human Dβ, and 14 human Jβ (MAID1716; "1716HO"). The offspring were bred to be homozygous for both humanized loci.

[0167] Example 5 Generation of splenic T cells in mice homozygous for a humanized TCR α locus and / or a humanized TCR β locus Spleens from wild-type (WT) mice; mice with a deleted mouse TCR alpha locus ("MAID1540", see FIG. 3); mice homozygous for the human TCR alpha locus ("MAID1767", see FIG. 3); mice with a deleted TCR beta V segment except for the remaining two mouse V segments ("MAID1545", see FIG. 7); mice homozygous for the human TCR beta locus and also containing the remaining two mouse V segments ("MAID1716", see FIG. 7); and mice homozygous for both the human TCR alpha locus and the human TCR beta locus and also containing the remaining two mouse V segments ("MAID1767 1716") were perfused with collagenase D (Roche Bioscience) and red blood cells were lysed with ACK lysis buffer, followed by washing in RPMI medium.

[0168] Splenocytes from a single representative animal of WT, MAID1540, 1767, 1545, 1716, and 1716 1767 were evaluated by flow cytometry. Briefly, cell suspensions were generated using standard methods. 1 × 10 6 Cells were incubated with anti-mouse CD16 / CD32 (2.4G2, BD) for 10 min on ice and stained with the appropriate antibody cocktail for 30 min on ice. After staining, cells were washed and then fixed in 2% formaldehyde. Data collection was performed on a LSRII / CantoII / LSRFortessa flow cytometer and analyzed with FlowJo.

[0169] Anti-mouse FITC-CD3 (17A2, BD) was used to stain splenocytes. As demonstrated in Figure 9, mice with human TCR segments were able to generate significant numbers of CD3+ T cells, whereas mice with deleted TCRα mouse locus were unable to do so. Mice with deleted TCRβ locus also generated CD3+ T cells, likely due to usage of the remaining 3' mouse V segments (see below).

[0170] Example 6. Thymic T cell development in mice homozygous for a humanized TCR α locus and / or a humanized TCR β locus. To determine whether mice homozygous for the humanized TCR α and / or β loci exhibit normal T cell development in the thymus, splenocytes from four of each of WT, 1767HO, 1716HO, and 1716HO1767HO age-matched animals (7-10 weeks old) were used in flow cytometry to assess the generation of T cells at various developmental stages, as well as the frequency and absolute numbers of DN, DP, CD4, and CD8 SP T cells.

[0171] Cell type determination was made based on the presence of CD4, CD8, CD44, and CD25 cell surface markers, summarized in Table 1. Correlation between cell type designation and expression of cell surface markers in the thymus is as follows: double negative (DN) cells (CD4- CD8-), double positive (DP) cells (CD4+ CD8+), CD4 single positive cells (CD4+ CD8-), CD8 single positive cells (CD4- CD8+), double negative 1 / DN1 cells (CD4- CD8-, CD25- CD44+), double negative 2 / DN2 cells (CD4- CD8-, CD25+ CD44+), double negative 3 / DN3 cells (CD4- CD8-, CD25+ CD44-), double negative 4 / DN4 cells (CD4- CD8-, CD25- CD44-).

[0172] Thymocytes were evaluated by flow cytometry. Briefly, cell suspension was prepared using standard methods. Flow cytometry was performed as described in Example 5. The antibodies used were anti-mouse PE-CD44 (IM7, BioLegend), PeCy7-CD25 (PC61, BioLegend), APC-H7-CD8a (53-6.7, BD), and APC-CD4 (GK1.5, eBioscience).

[0173] As shown in Figures 10 and 11, mice homozygous for humanized TCR alpha, humanized TCR beta, and both humanized TCR alpha and humanized TCR beta were able to generate DN1 T cells, DN2 T cells, DN3 T cells, DN4 T cells, DP T cells, CD4 SP T cells, and CD8 SP T cells, indicating that T cells generated from the humanized loci are also capable of undergoing T cell development in the thymus.

[0174] Example 7 Differentiation of Splenic T Cells in Mice Homozygous for a Humanized TCR α Locus and / or a Humanized TCR β Locus To determine whether mice homozygous for the humanized TCRα and / or TCRβ loci exhibit normal T cell differentiation in the periphery (e.g., spleen), four of each of WT, 1767HO, 1716HO, and 1716HO1767HO age-matched animals (7-10 weeks old) were used in flow cytometry to assess the generation of diverse T cell types in the spleen (CD3+, CD4+, CD8+, naive T, Tcm, and Teff / Tem), as well as the absolute numbers of each T cell type in the spleen.

[0175] Cell type determination was based on the presence of CD19 (B cell marker), CD3 (T cell marker), CD4, CD8, CD44, and CD62L (L-selectin) cell surface markers. Correlation between cell type designation and expression of cell surface markers in the spleen is as follows: T cells (CD3+), CD4 T cells (CD3+ CD4+ CD8-), CD8 T cells (CD3+ CD4- CD8+), CD4 effector / effector memory T cells (CD3+ CD4+ CD8- CD62L- CD44+), CD4 central memory T cells (CD3+ CD4+ CD8- CD62L+ CD44+), CD4 naive T cells (CD3+ CD4+ CD8- CD62L+ CD44-), CD8 effector / effector memory T cells (CD3+ CD4- CD8+ CD62L- CD44+), CD8 central memory T cells (CD3+ CD4- CD8+ CD62L+ CD44+), CD8 naive T cells (CD3+ CD4- CD8+ CD62L+ CD44-).

[0176] Splenocytes were evaluated by flow cytometry. Briefly, cell suspension was prepared using standard methods. Flow cytometry was performed as described in Example 5. The antibodies used were anti-mouse FITC-CD3 (17A2, BD), PE-CD44 (IM7, BioLegend), PerCP-Cy5.5-CD62L (Mel-14, BioLegend), APC-H7-CD8a (53-6.7, BD), APC-CD4 (GK1.5, eBioscience), and V450-CD19 (1D3, BD).

[0177] As shown in Figures 12-14, T cells in the spleens of mice homozygous for humanized TCRα, humanized TCRβ, and both humanized TCRα and humanized TCRβ were able to undergo T cell differentiation, and both CD4+ and CD8+ T cells were present. In addition, memory T cells were also detected in the spleens of the test mice.

[0178] Example 8 Use of human V segments in humanized TCR mice Expression of the human TCRβ V-segment was assessed at the protein and RNA levels using flow cytometry and TAQMAN™ real-time PCR, respectively, in mice homozygous for the humanized TCRβ locus (1716HO) and in mice homozygous for both the humanized TCRβ locus and the humanized TCRα locus (1716HO1767HO).

[0179] For flow cytometry, splenic T cells were prepared and analyzed as described in Example 5. For flow cytometry, a TCRβ repertoire kit (IOTEST® Beta Mark, Beckman Coulter) was used. The kit contains anti-human antibodies specific to multiple human TCRBVs, such as hTRBV-18, hTRBV-19, hTRBV-20, hTRBV-25, hTRBV-27, hTRBV-28, and hTRBV-29.

[0180] The results are summarized in Figure 15. The tables shown in Figure 15A (CD8 T cell overlay) and Figure 15B (CD4 T cell overlay) confirm that splenic T cells in both 1716HO and 1716HO1767HO mice used large numbers of human TCRβV segments. Wild-type mice were used as negative controls.

[0181] For real-time PCR, total RNA was purified from spleen and thymus using MAGMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies) according to the manufacturer's specifications. Genomic DNA was removed using MAGMAX™ TURBO™ DNase Buffer and TURBO DNase from the MAGMAX kit listed above (Ambion by Life Technologies). mRNA (up to 2.5ug) was reverse transcribed into cDNA using SUPERSCRIPT® VILO™ Master Mix (Invitrogen by Life Technologies). cDNA was diluted to 2-5ng / μL and 10-25ng of cDNA was amplified with TAQMAN® Gene Expression Master Mix (Applied Biosystems by Life Technologies) using an ABI 7900HT Sequence Detection System (Applied Biosystems) with the primers depicted in Table 4 and TAQMAN MGB probe (Applied Biosystems) or BHQ1 / BHQ-PLUS probe (Biosearch Technologies) according to the manufacturer's instructions. Relative expression of each gene was normalized to the mouse TCR beta constant 1 (TRBC1) control.

[0182] [Table 4]

[0183] As shown in Figures 16A-B, mice homozygous for the humanized TCRβ locus (1716HO) and mice homozygous for both the humanized TCRβ and TCRα loci (1716HO1767HO) displayed RNA expression of various human TCRβ segments in both the thymus and spleen. The mice also displayed RNA expression of mouse TRBV-1 and TRBV-31 segments (data not shown), but mouse TRBV-1 protein was not detected by flow cytometry (data not shown).

[0184] As shown in Figure 8F, mouse TRBV-31 segment is replaced with human TRBV-30 segment, and mice are generated from MAID6192 ES cells as described herein. The spleen and thymus of the resulting homozygous animals are examined for the use of human Vβ segments, including TRBV-30, by flow cytometry and / or real-time PCR as described herein. Also, mTRBV-1 segment can be deleted.

[0185] Example 9. Development of T cells in mice homozygous for 23 human TCR Vα segments. The homozygous humanized TCRα mice characterized in the previous examples (1767HO, see FIG. 3) contained 8 human Vα segments and 61 human Jα segments. Homozygous humanized TCRα mice (1979HO, see FIG. 3) containing 23 human Vα segments and 61 human Jα segments were examined for their ability to generate splenic CD3+ T cells and demonstrate T cell development in the thymus.

[0186] Experimental data were obtained using flow cytometry with the appropriate antibodies as described in the previous examples. As depicted in Figure 17, mice homozygous for 23 human Vα segments and 61 human Jα segments generated significant numbers of splenic CD3+ T cells, and the percentage of peripheral CD3+ T cells was similar to that of wild-type animals (Figure 19).

[0187] Furthermore, thymocytes in 1979HO mice were capable of undergoing T cell development and contained DN1, DN2, DN3, DN4, DP, CD4 SP, and CD8 SP stage T cells (Figure 18).

[0188] Example 10. T cell development and differentiation in mice homozygous for a complete repertoire of both human TCR alpha and beta variable region segments. Mice "1771HO6192HO" (see Figures 3 and 7), homozygous for a complete repertoire of human TCR alpha variable region segments (i.e., 54 human Va and 61 human Jα) and homozygous for a complete repertoire of human TCR beta variable region segments (67 human Vβ, 2 human Dβ, and 14 human Jβ), are examined for their ability to generate thymocytes that undergo normal T cell development and generate T cells that undergo normal T cell differentiation in the periphery and use their complete repertoire of human Va and Vβ segments.

[0189] Flow cytometry is performed using anti-mouse CD4, anti-mouse CD8, anti-mouse CD25, and CD44 antibodies described above in Examples 5 and 6 to determine the presence of DN1 T cells, DN2 T cells, DN3 T cells, DN4 T cells, DP T cells, CD4 SP T cells, and CD8 SP T cells in the thymus. Flow cytometry is also performed to determine the number of CD3+ T cells in the periphery, as well as to evaluate T cell differentiation in the periphery (e.g., the presence of effector and memory T cells in the periphery). Experiments are performed using anti-mouse CD3, anti-mouse CD19, anti-mouse CD4, anti-mouse CD8, anti-mouse CD44, and anti-mouse CD62L antibodies described above in Examples 5 and 7.

[0190] Finally, flow cytometry and / or real-time PCR are performed to determine whether T cells in 1771HO6192HO mice use the complete repertoire of TCRB V segments and TCRA V segments. For protein expression using flow cytometry, use the TCR beta repertoire kit (IOTEST® Beta Mark, Beckman Coulter) containing anti-human hTCRBV specific antibody (see Example 8). For RNA expression using real-time PCR, amplify cDNA from spleen or thymus using human TCR-V primers and TAQMAN probe according to the manufacturer's instructions and as described in Example 8 above.

[0191] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the scope of the following claims.

[0192] The entire contents of all non-patent literature, patent applications, and patents cited throughout this application are hereby incorporated by reference in their entirety.

Claims

1. 1. A targeting vector comprising an unrearranged human TCRα variable region sequence, the unrearranged human TCR α variable region sequence comprises a functional unrearranged human TCR Vα gene segment operably linked to a functional unrearranged human TCR Jα gene segment; a targeting vector, wherein the unrearranged human TCR α variable region sequence is flanked by targeting arms for targeting the targeting vector to an endogenous TCR α locus in a mouse embryonic stem (ES) cell, whereby an endogenous mouse TCR Vα gene segment is replaced by the functional unrearranged human TCR Vα gene segment, and an endogenous mouse TCR Jα gene segment is replaced by the functional unrearranged human TCR Jα gene segment, and the functional unrearranged human TCR Vα gene segment and the functional unrearranged human TCR Jα gene segment are operably linked to an endogenous TCR Cα gene sequence.

2. Upon homologous recombination between the targeting vector and the endogenous TCRα locus, (I) all endogenous TCR Vα gene segments are replaced with said functional, unrearranged human TCR Vα gene segments; (II) The targeting vector of claim 1, wherein all endogenous TCR Jα gene segments are replaced with said functional, unrearranged human TCR Jα gene segments.

3. (I) the functional, unrearranged human TCR Vα gene segment comprises an unrearranged human TRAV40 gene segment, an unrearranged human TRAV41 gene segment, an unrearranged human TRAV39 gene segment, an unrearranged human TRAV35 gene segment, an unrearranged human TRAV34 gene segment, an unrearranged human TRAV22 gene segment, an unrearranged human TRAV21 gene segment, an unrearranged human TRAV13-2 gene segment, an unrearranged human TRAV8.5 gene segment, an unrearranged human TRAV6 gene segment, an unrearranged human TRAV5 gene segment, and / or an unrearranged human TRAV1-1 gene segment; (II) The targeting vector of claim 1, wherein the functional, unrearranged human TCR Jα gene segments comprise all functional, unrearranged human TCR Jα gene segments.

4. The targeting vector of claim 1 , comprising a recombination site downstream of the functional, unrearranged human TCR Vα gene segment.

5. 5. The targeting vector of claim 4, wherein the recombination site downstream of the functional, unrearranged human TCR Vα gene segment is upstream of the functional, unrearranged human TCR Jα gene segment.

6. The following: (i) an engineered AsiSI restriction site; (ii) an engineered AscI restriction site; (iii) a nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and / or SEQ ID NO:13, and (iv) Selection Cassette The targeting vector of claim 1 , comprising at least one of:

7. 1. A targeting vector comprising an unrearranged human TCRβ variable region sequence, The unrearranged human TCRβ variable region sequence comprises: (i) a functional, unrearranged human TCR Vβ gene segment, or (ii) a functional, unrearranged, human TCR Dβ gene segment and a functional, unrearranged, human TCR Jβ gene segment. Including, the unrearranged human TCR beta variable region sequence is flanked by targeting arms for targeting the targeting vector to an endogenous mouse TCR beta variable locus in a mouse ES cell, thereby (i) an endogenous mouse TCR Vβ gene segment is replaced by said functional unrearranged human TCR Vβ gene segment, and said functional unrearranged human TCR Vβ gene segment is inserted upstream of one or more trypsinogen genes and operably linked to a functional unrearranged human TCR Dβ gene segment and a functional unrearranged human TCR Jβ gene segment and to the endogenous TCR Cβ gene sequence; or (ii) a targeting vector in which an endogenous mouse TCR Dβ gene segment is replaced with said functional, unrearranged human TCR Dβ gene segment, an endogenous mouse TCR Jβ gene segment is replaced with said functional, unrearranged human TCR Jβ gene segment, and said functional, unrearranged human TCR Dβ gene segment and functional, unrearranged human TCR Jβ gene segment are inserted downstream of one or more trypsinogen genes and operably linked to a functional, unrearranged human TCR Vβ gene segment and an endogenous TCR Cβ gene sequence.

8. (a) upon homologous recombination between the targeting vector comprising the functional, unrearranged human TCR Vβ gene segments and (b) the endogenous mouse TCR β variable locus, all endogenous TCR Vβ gene segments located between the 5′ trypsinogen cluster and the 3′ trypsinogen cluster are replaced by the functional, unrearranged human TCR Vβ gene segments; or 8. The targeting vector of claim 7, wherein upon homologous recombination between (a) the targeting vector comprising the functional, unrearranged human TCR Dβ gene segments and functional, unrearranged human TCR Jβ gene segments, and (b) the endogenous mouse TCR β variable locus, all endogenous TCR Dβ gene segments are replaced by the functional, unrearranged human TCR Dβ gene segments and all endogenous TCR Jβ gene segments are replaced by the functional, unrearranged human TCR Jβ gene segments.

9. (I) the functional, unrearranged human TCR Vβ gene segment comprises an unrearranged human TRBV18 gene segment, an unrearranged human TRBV19 gene segment, an unrearranged human TRBV20 gene segment, an unrearranged human TRBV24 gene segment, an unrearranged human TRBV25 gene segment, an unrearranged human TRBV27 gene segment, an unrearranged human TRBV28 gene segment, and / or an unrearranged human TRBV29 gene segment; (II) the functional, unrearranged, human TCR Dβ gene segment comprises all functional, unrearranged, human TCR Dβ gene segments; or (III) The targeting vector of claim 7, wherein the functional, unrearranged, human TCR Jβ gene segments comprise all functional, unrearranged, human TCR Jβ gene segments.

10. The following: (i) an engineered AsiSI restriction site; (ii) an engineered AscI restriction site; (iii) the nucleotide sequence set forth in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and / or SEQ ID NO:27, and (iv) Selection Cassette The targeting vector of claim 7, comprising at least one of the following:

11. A method of modifying mouse embryonic stem (ES) cells, comprising contacting the ES cells with a targeting vector according to any one of claims 1 to 10.

12. A mouse ES cell comprising the targeting vector of any one of claims 1 to 10.

13. A mouse comprising the targeting vector of any one of claims 1 to 10.

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