T-cell receptor gene-modified mice

Abstract

This invention provides a genetically modified non-human animal whose genome contains an unrearranged T-cell receptor variable gene locus, and provides a non-human embryo, cell, and tissue containing the unrearranged T-cell receptor variable gene locus. It also provides a construct for preparing the genetically modified non-human animal and a method for preparing the genetically modified non-human animal. This invention further provides various methods for using the genetically modified non-human animal.

Description

[0001] Cross-references to related applications

[0002] This application is a divisional application of Chinese Patent Application No. 201710930556.4, filed on October 26, 2012, entitled "T-cell receptor gene-modified mouse". This Chinese patent application is a divisional application of Chinese Patent Application No. 201280064216.3, filed on October 26, 2012, entitled "T-cell receptor gene-modified mouse". The original application was a national phase application with international application No. PCT / US2012 / 062065, which claimed priority to U.S. Provisional Application Nos. 61 / 552,582, filed on October 28, 2011; U.S. Provisional Application Nos. 61 / 621,198, filed on April 6, 2012; and U.S. Provisional Application Nos. 61 / 700,908, filed on September 14, 2012, the entire contents of which are incorporated herein by reference. Invention Field

[0003] This invention relates to genetically modified non-human animals, such as rodents (e.g., mice or rats), whose genomes contain human or humanized T-cell receptor (TCR) variable gene loci (e.g., TCRα and TCRβ variable gene loci and / or TCRδ and TCRγ variable gene loci), and express human or humanized TCR polypeptides (e.g., TCRα and TCRβ polypeptides and / or TCRδ and TCRγ polypeptides) derived from human or humanized TCR variable gene loci. Non-human animals possessing the human or humanized TCR variable gene loci of this invention contain unrearranged human TCR variable region gene segments (e.g., V, D, and / or J segments) at the endogenous non-human TCR loci. This invention also relates to embryos, tissues, and cells (e.g., T cells) containing human or humanized TCR variable gene loci and expressing human or humanized TCR polypeptides. This application also provides methods for preparing genetically modified nonhuman animals containing human or humanized TCR variable gene loci; and methods for using nonhuman animals, embryos, tissues, and cells containing human or humanized TCR variable gene loci and expressing human or humanized TCR polypeptides from these loci. Background of the Invention

[0004] In adaptive immune responses, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptors or TCRs). Pathogens in the blood and extracellular space are recognized by antibodies during humoral immune responses, while the destruction of pathogens within cells is mediated by T cells during cellular immune responses.

[0005] T cells recognize and attack antigens presented on the cell surface in the context of the major histocompatibility complex (MHC). Antigen recognition is mediated by TCRs expressed on the T cell surface. There are two main types of T cells with this function: cytotoxic T cells, which express the cell surface protein CD8, and helper T cells, which express the cell surface protein CD4. The activation cascade of cytotoxic T cells leads to the direct destruction of the cell presenting the antigen (in the context of MHC I), while helper T cells differentiate into several types, and their activation (in the context of MHC II, triggered by recognition of the presented antigen) leads to macrophage-mediated pathogen destruction and stimulates B cells to produce antibodies.

[0006] Due to their antigen specificity, antibodies have been extensively studied for their therapeutic potential against a variety of human diseases. To generate antibodies capable of neutralizing human targets while avoiding simultaneous activation of the immune response against such antibodies, scientists have focused on producing human or humanized immunoglobulins. One method for producing humanized antibodies in vivo is to use... The mouse, this humanized mouse comprising (1) a library of unrearranged human immunoglobulin V, D, and J segments operably linked to each other, the segments and the mouse constant region located at the endogenous mouse immunoglobulin heavy chain locus, and (2) a library of unrearranged human Vκ and Jκ segments operably linked to each other, the segments and the mouse constant κ region located at the endogenous mouse immunoglobulin κ light chain locus. Thus, Mice provide a rich source of highly diverse rearranged antibody variable domains for the engineered production of human antibodies.

[0007] Similar to antibodies, T-cell receptors contain a variable region encoded by unrearranged loci (α and β loci, or δ and γ loci) containing a V(D)J variable region segment, and this variable region confers T-cell antigen-binding specificity. Also similar to antibodies, the antigen specificity of TCRs can be used to develop novel therapies. Therefore, there is a need in the art for non-human animals (e.g., rodents, such as rats or mice) containing unrearranged human T-cell variable region gene segments capable of rearranging to form genes encoding human T-cell receptor variable domains, including homologous domains and domains that specifically bind to the target antigen. There is also a need for a non-human animal containing a T-cell variable region locus containing conserved humanization, comprising a non-human animal containing unrearranged human gene segments capable of rearranging to form T-cell receptor variable region genes linked to a non-human (endogenous) T-cell receptor constant gene sequence. There is also a need for non-human animals capable of generating diverse libraries of human T-cell receptor variable sequences. It is necessary to be able to rearrange most or all of the functional T cell receptor variable regions in response to the target antigen to form a non-human animal containing a T cell receptor polypeptide with a fully human variable domain. Invention Overview

[0008] This application provides non-human animals, such as rodents, comprising non-human cells expressing humanized molecules that function in cellular immune responses. This application also provides non-human animals comprising an unrearranged TCR variable gene locus. This application provides in vivo and in vitro systems comprising humanized rodent cells expressing one or more humanized immune system molecules. This application also provides an unrearranged humanized TCR rodent locus encoding a humanized TCR protein.

[0009] In one aspect, this application provides a genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) whose genome contains (a) an unrearranged TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment operatively linked to a non-human (e.g., rodent, such as a mouse or rat) TCRα constant gene sequence, and / or (b) an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operatively linked to a non-human (e.g., rodent, such as a mouse or rat) TCRβ constant gene sequence.

[0010] In one embodiment, the unrearranged TCRα variable gene locus replaces the endogenous non-human (e.g., rodent) TCRα variable gene locus at the endogenous TCRα variable gene locus. In one embodiment, the unrearranged TCRβ variable gene locus replaces the endogenous non-human (e.g., rodent) TCRβ variable gene locus at the endogenous TCRβ variable gene locus. In one embodiment, the endogenous non-human (e.g., rodent) Vα and Jα regions cannot be rearranged to form a rearranged Vα / Jα sequence. In one embodiment, the endogenous non-human (e.g., rodent) Vβ, Dβ, and Jβ regions cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence. In one embodiment, the non-human animal contains deletions such that the animal's genome does not contain functional Vα and functional Jα regions. In one embodiment, the non-human animal contains deletions such that its genome does not contain functional endogenous Vβ, Dβ, and Jβ regions. In one embodiment, the animal contains deletions of all functional endogenous Vα and Jα regions. In one embodiment, the rodent contains deletions of all functional endogenous Vβ, Dβ, and Jβ regions. In some embodiments, the human Vα and Jα regions are rearranged to form rearranged Vα / Jα sequences. In some embodiments, the human Vβ, Dβ, and Jβ regions are rearranged to form rearranged Vβ / Dβ / Jβ sequences. Thus, in various embodiments, the non-human animal (e.g., rodent) expresses T cell receptors on the surface of T cells containing human variable regions and non-human (e.g., rodent) constant regions.

[0011] In some aspects, the T cells of the non-human animal undergo T cell development in the thymus to produce CD4 and CD8 single-positive T cells. In some aspects, the non-human animal contains a normal ratio of splenic CD3+ T cells to total splenic cells. In various embodiments, the non-human animal generates peripheral centers and effector memory T cell populations.

[0012] In one embodiment, the unrearranged TCRα variable gene locus in the non-human animal described in this application comprises 61 human Jα segments and 8 human Vα segments. In another embodiment, the unrearranged TCRα variable gene locus in the non-human animal comprises a complete library of human Jα segments and a complete library of human Vα segments.

[0013] In one embodiment, the unrearranged TCRβ variable gene locus in the non-human animal described in this application comprises 14 human Jβ segments, 2 human Dβ segments, and 14 human Vβ segments. In another embodiment, the unrearranged TCRβ variable gene locus in the non-human animal comprises a complete human Jβ segment library, a complete human Dβ segment library, and a complete human Vβ segment library.

[0014] In an additional embodiment, the non-human animal (e.g., rodent) described in this application further includes the nucleotide sequence of a human TCRδ variable segment at the humanized TCRα locus. In one embodiment, the non-human animal (e.g., rodent) further includes at least one human Vδ, Dδ, and Jδ segment, for example, a complete library of human Vδ, Dδ, and Jδ segments at the humanized TCRα locus.

[0015] In one embodiment, the non-human animal retains endogenous non-human TCRα and / or TCRβ loci, wherein the loci are non-functional loci.

[0016] In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from mice and rats. In one embodiment, the rodent is a mouse.

[0017] In one aspect, the present invention provides a genetically modified mouse whose genome includes (a) an unrearranged TCRα variable gene locus containing a human Jα segment library and a human Vα segment library operatively linked to a non-human (e.g., mouse or rat) TCRα constant gene sequence, and / or (b) an unrearranged TCRβ variable gene locus containing a human Jβ segment library, a human Dβ segment library, and a human Vβ segment library operatively linked to a non-human (e.g., mouse or rat) TCRβ constant gene sequence. In one embodiment, the mouse contains a complete human Vα segment library. In one embodiment, the mouse contains a complete human Vβ segment library. In one embodiment, the mouse contains a complete human Vα and human Jα segment library. In one embodiment, the mouse contains a complete human Vα and human Vβ segment library. In one embodiment, the mouse contains a complete human Vα, human Jα, human Vβ, human Dβ, and human Jβ segment library.

[0018] In one embodiment, the mouse comprises at least one endogenous mouse Vα and at least one endogenous mouse Jα segment, wherein the endogenous segments cannot be rearranged to form a rearranged Vα / Jα segment, and further comprises at least one endogenous mouse Vβ, at least one endogenous mouse Dβ, and at least one endogenous mouse Jβ segment, wherein the endogenous segments cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence.

[0019] In one implementation, an unrearranged TCRα variable gene locus containing a human TCRα variable region segment replaces the mouse TCRα variable gene at the endogenous mouse TCRα variable gene locus, and an unrearranged TCRβ variable gene locus containing a human TCRβ variable region segment replaces the mouse TCRβ variable gene at the endogenous mouse TCRβ variable gene locus.

[0020] In one embodiment, the human Vα and Jα regions are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ, Dβ, and Jβ regions are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence. In one embodiment, the rearranged human Vα / Jα sequence is operatively linked to a mouse TCRα constant region sequence. In one embodiment, the rearranged human Vβ / Dβ / Jβ sequence is operatively linked to a mouse TCRβ constant region sequence. Thus, in various embodiments, the mouse expresses a T cell receptor on the surface of T cells, wherein the T cell receptor comprises a human variable region and a mouse constant region.

[0021] In one embodiment, the mouse at the humanized TCRα locus further includes a library of human TCRδ variable region segments (e.g., human Vδ, Jδ, and Dδ segments). In one embodiment, the human TCRδ variable region segment library is a complete human TCRδ variable region segment library. In one embodiment, the human TCRδ variable region segments are located at the endogenous TCRα locus. In one embodiment, the human TCRδ variable region segments replace the endogenous mouse TCRδ variable region segments.

[0022] 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 its T cells. In one aspect, the mouse's T cells undergo thymic T-cell development to produce CD4 and CD8 single-positive T cells. In one aspect, the mouse comprises a normal ratio of splenic CD3+ T cells to total splenic cells; in one aspect, the mouse generates central and effector memory T-cell populations in response to a target antigen.

[0023] This application also provides a method for preparing the genetically modified non-human animals (e.g., rodents, such as mice or rats) described in this application.

[0024] In one aspect, this application provides a method for preparing humanized rodents (e.g., mice or rats), the method comprising replacing rodent TCRα and TCRβ variable regions, rather than rodent constant regions, with human, unrearranged TCRα and TCRβ variable regions at an endogenous rodent TCR locus. In one embodiment, the method comprises replacing rodent TCRα variable regions (Vα and / or Jα) with human TCRα variable regions (Vα and / or Jα), wherein the TCRα variable regions are operatively linked to non-human TCR constant regions to form a humanized TCRα locus, and replacing rodent TCRβ variable regions (Vβ and / or Dβ and / or Jβ) with human TCRβ variable regions (Vβ and / or Dβ and / or Jβ), wherein the TCRβ variable regions are operatively linked to non-human TCR constant regions to form a humanized TCRβ locus. In one embodiment, the humanized rodent is a mouse and the mouse germline includes a human TCRα variable region segment operatively linked to an endogenous mouse TCRα constant sequence at an endogenous TCRα locus; and the mouse germline includes a human TCRβ variable region segment operatively linked to an endogenous mouse TCRβ constant sequence at an endogenous TCRβ locus.

[0025] In one embodiment, this application provides a method for preparing genetically modified non-human animals (e.g., rodents, such as mice or rats), said animals expressing T cell receptors containing human or humanized variable regions and non-human (e.g., rodent) constant regions on the surface of T cells, said method comprising: replacing an endogenous non-human TCRα variable gene locus in a first non-human animal with an unrearranged humanized TCRα variable gene locus, said humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein said humanized TCRα variable gene locus can... The process involves operatively linking the endogenous non-human TCRα constant region; replacing the endogenous non-human TCRβ variable gene locus in a second non-human animal with an unrearranged humanized TCRβ variable gene locus segment comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the humanized TCRβ variable gene locus is operatively linked to the endogenous TCRβ constant region; and mating the first and second non-human animals to obtain a non-human animal expressing a T cell receptor containing a human or humanized variable region and a non-human constant region.

[0026] In one embodiment of the method, the endogenous non-human (e.g., rodent) Vα and Jα regions cannot be rearranged to form a rearranged Vα / Jα sequence, and the endogenous non-human (e.g., rodent) Vβ, Dβ, and Jβ regions cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence. In one embodiment of the method, the human Vα and Jα regions are rearranged to form a rearranged Vα / Jα sequence, and the human Vβ, Dβ, and Jβ regions are rearranged to form a rearranged Vβ / Dβ / Jβ sequence. In one embodiment of the method, the unrearranged humanized TCRα variable gene locus comprises 61 human Jα regions and 8 human Vα regions, and the unrearranged humanized TCRβ variable gene locus comprises 14 human Vβ regions, 2 human Dβ regions, and 14 human Jβ regions. In another embodiment of the method, the unrearranged humanized TCRα variable gene locus contains a complete human Jα segment library and a complete human Vα segment library, and the unrearranged humanized TCRβ variable gene locus contains a complete human Vβ segment library, a complete human Dβ segment library and a complete human Jβ segment library.

[0027] In one aspect of the method, the T cells of the non-human animal (e.g., rodent) undergo thymic T cell development to produce CD4 and CD8 single-positive T cells. In another aspect, the non-human animal (e.g., rodent) comprises a normal ratio of splenic CD3+ T cells to total splenic cells. In yet another aspect, the non-human animal (e.g., rodent) targets a target antigen-generating center and an effector memory T cell population.

[0028] In some embodiments, the substitution of the endogenous non-human TCRα variable gene locus described in this application is performed in a single ES cell, and the single ES cell is introduced into a non-human (e.g., rodent, such as a mouse or rat) embryo to prepare a genetically modified non-human animal (i.e., a first non-human animal, such as a first rodent); and the substitution of the endogenous non-human TCRβ variable gene locus described in this application is performed in a single ES cell, and the single ES cell is introduced into a non-human (e.g., rodent, such as a mouse or rat) embryo to prepare a genetically modified non-human animal (i.e., a second non-human animal, such as a second rodent). In one embodiment, the first rodent and the second rodent are mated to form offspring, wherein the offspring contain humanized TCRα variable gene loci and humanized TCRβ variable gene loci in their lineage.

[0029] In one embodiment of the method, the non-human animal is a rodent, such as a mouse. Therefore, the present invention also provides a method for preparing genetically modified mice.

[0030] This application also provides cells derived from the non-human animals described in this application (e.g., rodents, such as mice or rats), such as isolated T cells (e.g., cytotoxic T cells, helper T cells, memory T cells, etc.). This application also provides tissues and embryos derived from the non-human animals described in this application.

[0031] In one aspect, this application provides a method for preparing human TCR variable domains, the method comprising genetically modifying a rodent to include a humanized TCRα locus and / or a humanized TCRβ locus as described in this application, maintaining the rodent under conditions sufficient for the formation of T cells, wherein the T cells express humanized TCRα and / or humanized TCRβ variable domains.

[0032] In one aspect, this application provides a method for preparing a nucleic acid sequence encoding a human TCR variable domain that binds to a target epitope, the method comprising contacting a non-human animal as described in this application with the target epitope, maintaining the non-human animal under conditions sufficient to allow the animal to present the target epitope to the animal's humanized TCR, and identifying the nucleic acid encoded by the animal as a polypeptide of the human TCR variable domain that binds to the target epitope.

[0033] In one aspect, this application provides the use of non-human animals as described in this application for preparing humanized TCR receptors. In another aspect, this application provides the use of non-human animals as described in this application for preparing human TCR variable domains. In yet another aspect, this application provides the use of non-human animals as described in this application for preparing nucleic acid sequences encoding human TCR variable domains.

[0034] In one aspect, this application provides the use of nucleic acid sequences encoding a human TCR variable domain or fragments thereof for preparing antigen-binding proteins. In one embodiment, the antigen-binding protein comprises a TCR variable domain including human TCRα and / or human TCRβ variable domains that bind to a target antigen.

[0035] In one aspect, this application provides the use of non-human animals as described in this application for the preparation of non-human cells expressing humanized T-cell receptors on their surface.

[0036] In one aspect, this application provides humanized T-cell receptors derived from non-human animals as described in this application.

[0037] In one aspect, this application provides nucleic acid sequences encoding human TCR variable domains or fragments thereof prepared in non-human animals as described in this application.

[0038] Any embodiments and aspects described in this application may be used in combination with each other unless otherwise stated or apparent in the context. Other embodiments will be apparent to those skilled in the art from the summary of the following detailed description. The following detailed description includes exemplary descriptions of different embodiments of the invention, which are not restrictive of the claimed invention. The accompanying drawings form part of this specification and, together with the specification, are used only to illustrate embodiments and not to limit the invention.

[0039] Brief description of the attached figures

[0040] Figure 1 The interaction between TCR and MHC molecules in mice is described: The left panel shows mouse T cells (top) from humanized TCR mice, containing T cell receptors with human variable TCR domains and mouse constant TCR domains, recognizing antigens (gray spheres) presented by antigen-presenting cells (bottom) via MHC class I molecules; the right panel shows the same process presented via MHC class II molecules. The MHC I and MHC II complexes are shown together with their respective co-receptors CD8 and CD4. Mouse regions are shown in black and human regions in white.

[0041] Figure 2 The general organization of mouse (top, first locus) and human (top, second locus) TCRα loci (not to scale) is described. The figure below shows the strategy of replacing the mouse (solid symbol) TCRα variable region segment with a human TCRα variable region segment (hollow symbol) at the endogenous mouse locus on chromosome 14; the humanized TCRα locus with human Vα and Jα segments is shown alongside the mouse constant region and mouse enhancer in the figure; in the illustrated embodiment, the TCRδ locus is omitted during humanization.

[0042] Figure 3 A strategy for the stepwise humanization of the mouse TCRα locus (not to scale) is described, in which the TCRα variable region gene segment was sequentially added upstream of the initially humanized, missing mouse locus (MAID1540). Mouse sequences are represented by solid symbols; human sequences are represented by hollow symbols. MAID refers to the modified allele ID number. TRAV = TCR Vα region, TRAJ = TCR Jα region (hTRAJ = human TRAJ), TRAC = TCR Cα domain, TCRD = TCRδ.

[0043] Figure 4 is a detailed description of the stepwise humanization strategy at the TCRα locus (not to scale). Figure 4A The deletion process of the TCRαV and J regions in mice was described; Figure 4BA strategy for inserting humanized 2V and 61J segments into a deleted mouse TCRα locus is described. Figure 4C The strategy for inserting additional human-source V segments is described, resulting in a total of 8 V and 61 J human-source segments; Figure 4D The strategy for inserting additional human-source V segments is described, resulting in a total of 23 V and 61 J human-source segments; Figure 4E The strategy for inserting additional human V segments that results in 35 V and 61 J human-derived segments is described. Figure 4F The strategy for inserting additional human-source segments, resulting in 48 V and 61 J human-source segments, is described; and Figure 4G The strategy for inserting additional human-derived segments, resulting in 54 V and 61 J human-derived segments, is described. MAID refers to the modified allele ID number.

[0044] Figure 5 One implementation of a humanization strategy for the mouse TCRα locus (not to scale) is described, wherein human TCRδ sequences (TCRδVs, TCRδDs, TCRδJs, TCRδenh (enhancer), and TCRδ constant (C)) are also located at the humanized TCRα locus. Mouse sequences are represented by solid symbols; human sequences are represented by hollow symbols. LTVEC denotes a larger targeting vector; hTRD = human TCRδ.

[0045] Figure 6 The general organization of the mouse (top, first locus; on mouse chromosome 6) and human (top, second locus; on human chromosome 7) TCRβ loci (not to scale) is described. The figure below shows the strategy of replacing the mouse (solid symbol) TCRβ variable region segment with a human TCRβ variable region segment (hollow symbol) at the endogenous mouse locus on mouse chromosome 6. The humanized TCRβ locus with human Vβ, Dβ, and Jβ segments is shown in the figure along with the mouse constant region and mouse enhancer; in the illustrated embodiment, the humanized locus retains the mouse trypsinogen gene (solid rectangle); and in the specific illustrated embodiment, a single mouse V segment is retained upstream of the 5' mouse trypsinogen gene.

[0046] Figure 7 A strategy for the stepwise humanization of the mouse TCRβ locus (not to scale) is described, in which TCRβ variable region gene segments are sequentially added to deleted mouse TCRβ variable loci. Mouse sequences are represented by solid symbols; human sequences are represented by hollow symbols. MAID refers to the modified allele ID number. TRBV or TCRBV = TCRβV segment.

[0047] Figure 8 is a detailed description of the stepwise humanization strategy at the TCRβ locus. Figure 8A The strategy for TCRβV segment deletion in mice is described; Figure 8B The strategy for inserting 14 V segments into the deleted TCRβ locus is described; Figure 8C The strategy of inserting 2 D and 14 J segments into the TCRβ locus (i) followed by loxP deletion (ii) is described, resulting in 14 V, 2 D and 14 J human segments; Figure 8D The strategy described is the insertion of additional human-derived V segments that results in 40 V, 2 D, and 14 J human-derived segments; and Figure 8E The strategy for inserting additional human V segments that results in 66 V, 2 D, and 14 J human-source segments is described. Figure 8F The substitution of the mouse V region downstream of the mouse enhancer is described, resulting in 67 V, 2 D, and 14 J human-derived regions. In this particular embodiment, one mouse V region is the 5' end of the preserved mouse trypsinogen gene.

[0048] Figure 9 A typical FACS analysis histogram depicting the percentage of spleen cells stained with anti-CD3 antibody is shown (where the Y-axis represents cell number, the X-axis represents mean fluorescence intensity, and a molecular grid indicates the frequency of CD3+ T cells in a single lymphocyte population). The mouse species included are: wild-type (WT) mice and homozygous mice lacking the TCRα locus (first image above). Figure 3 MAID 1540); homozygous mice lacking the TCRα locus and containing 8 human Vα and 61 human Jα segments (second image above); Figure 3 MAID 1767 or humanized TCRα mice); homozygous mice lacking the TCRβ locus except for one upstream and one downstream mouse Vβ segment (first image below). Figure 7 MAID 1545); a homozygous mouse that lacks the TCRβ locus except for one upstream and one downstream mouse Vβ segment and contains 14 human Vβ, 2 human Dβ and 14 human Jβ segments (second image below). Figure 7 The MAID 1716 or humanized TCRβ mice and homozygous mice (MAID 1767 / 1716 or humanized TCRα / β mice) with deletions of the TCRα and TCRβ loci (except for the two mouse Vβ segments mentioned above) and containing 8 human Vα and 61 human Jα segments at the endogenous TCRα locus and 14 human Vβ, 2 human Dβ and 14 human Jβ segments at the endogenous TCRβ locus.

[0049] Figure 10These are WT mice, humanized TCRα homozygous (1767 HO; hTCRα), humanized TCRβ homozygous (1716 HO; hTCRβ), and humanized TCRα / β homozygous mice stained with anti-CD4 (Y-axis) and anti-CD8 (X-axis) antibodies (top image) and anti-CD44 (Y-axis) and anti-CD25 (X-axis) antibodies (bottom image) (bottom image).

[0050] Typical FACS contour plot of mouse thymocytes (hTCRα / β). The FACS curves in the upper figure can distinguish between double-negative (DN), double-positive (DP), CD4 single-positive (CD4 SP), and CD8 single-positive (SP CD8) T cells. The FACS curves in the lower figure can distinguish between double-negative T cells (DN1, DN2, DN3, and DN4) at different stages of T cell development. 1716 and 1767 refer to... Figure 3 and Figure 7 The MAID number specified in the code.

[0051] Figure 11 The table shows the frequency (top) or absolute number (bottom) of DN, DP, CD4 SP, and CDSP T cells in the thymus of WT, hTCRα (1767 HO), hTCRβ (1716 HO), or hTCRα / β (1716 HO + 1767 HO) mice (n=4).

[0052] Figure 12 Typical FACS analysis of mouse spleen cells in WT, hTCRα (1767 HO), hTCRβ (1716 HO), or hTCRα / β (1716 HO + 1767 HO) mice: The left panel shows the analysis of single cells stained with anti-CD19 antibody (Y-axis; staining for B lymphocytes) or anti-CD3 antibody (X-axis; staining for T lymphocytes); the middle panel shows the analysis of CD3+ cells stained with anti-CD4 (Y-axis) or anti-CD8 (X-axis) antibody; and the right panel shows the analysis of CD4+ or CD8+ cells stained with anti-CD44 (Y-axis) or anti-CD62L (X-axis) antibody, which can distinguish different types of peripheral T cells (naive T cells vs. central memory T cells (Tcm) vs. effector or effector memory T cells (Teff / Tem)).

[0053] Figure 13 The figures represent the number of CD4+ (left) or CD8+ (right) T cells in each spleen (Y-axis) of WT, hTCRα (1767 HO), hTCRβ (1716 HO), or hTCRα / β (1716 HO + 1767 HO) mice (n=4).

[0054] Figure 14 The figures represent the number of T initial, Tcm, and Teff / em cells in each spleen (Y-axis) of WT, hTCRα (1767 HO), hTCRβ (1716 HO), or hTCRα / β (1716 HO + 1767 HO) mice (n=4).

[0055] Figure 15 This study summarizes the splenic CD8+ T cells in WT, hTCRβ (1716 HO), or hTCRα / β (1716 HO 1767 HO) mice. Figure 15 A) or CD4+ T cells ( Figure 15 Table B) shows the expression of different human TCRβV segments (determined by FACS analysis using variable segment-specific antibodies). Data are expressed as mean ± SD (n = 4 mice per group).

[0056] Figure 16 illustrates the expression (Y-axis) of different human TCRβV segment mRNAs present in thymic or splenic T cells of WT, hTCRα(1767 HO); hTCRβ(1716 HO); or hTCRα / β(1716 HO 1767 HO) mice. Figure 16A Analysis of the expression of human TCRβ variable region (hTRBV) mRNAs at 18, 19, 20, and 24. Figure 16B This represents an analysis of the expression of hTRBV25, 27, 28, and 29 mRNA.

[0057] Figure 17 Typical FACS histograms of spleen cells stained with anti-CD3 antibody are described (where the Y-axis represents cell number, the X-axis represents mean fluorescence intensity, and a molecular grating shows the frequency of CD3+ T cells in a single lymphocyte population). The mice included are: WT mice, homozygous mice lacking the TCRα locus (TCRAΔV), and homozygous mice lacking the TCRα locus but possessing 2 human V segments and 61 human J segments (TCRA 2hV). Figure 3 MAID 1626), a homozygous mouse lacking the TCRα locus and possessing 8 human V segments and 61 human J segments (TCRA 8hV); Figure 3 MAID 1767) and a homozygous mouse (TCRA 23hV) that lacks the TCRα locus and has 23 human V segments and 61 human J segments; Figure 3 (MAID1979).

[0058] Figure 18The top left panel shows a typical FACS analysis of CD3+ T cells from the thymus obtained from WT or homozygous hTCRα mice (1979 HO) with 23 human V segments and 61 human J segments, stained with anti-CD4 (Y-axis) or anti-CD8 (X-axis) antibodies; the bottom left panel shows a FACS analysis of DN T cells from WT or 1979 mice stained with anti-CD44 (Y-axis) or anti-CD25 (X-axis); the right panel shows the percentage (Y-axis) of thymocytes of DN, DP, CD4 SP, or CD8 SP in WT or 1979 HO mice (n=4).

[0059] Figure 19 The left panel shows a typical FACS analysis of spleen lymphocytes from WT or 1979HO mice stained with anti-CD19 or anti-CD3 antibodies; the right panel shows the percentage of CD3+ spleen cells (Y-axis) obtained from WT and 1979 HO mice (n=4). Invention Details

[0060] definition

[0061] This invention provides genetically modified non-human animals, such as rodents like mice or rats, that express humanized T-cell receptors. The invention also relates to genetically modified non-human animals containing, in their lineage, an unrearranged T-cell receptor variable gene locus. This application also provides embryos, cells, and tissues comprising the thereof; methods for preparing the thereof; and methods for using the thereof. Unless otherwise defined, all terms and phrases used in this application include their meanings as they have in the art, unless expressly stated to the contrary or obvious from the context of their use.

[0062] When used to describe conserved amino acid substitutions, the term "conservative" includes the substitution of an amino acid residue by another amino acid residue with a side chain R group having similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying the nucleotide sequence to introduce nucleotide changes that will encode the conserved substitution. Generally, conserved amino acid substitutions will not substantially alter the functional properties of the target protein, such as the ability of T cells to recognize peptides presented by MHC molecules. Examples of amino acid groups with side chains having similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxy side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Conserved amino acid substituents include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine. In some embodiments, the conserved amino acid substitution may be the substitution of any native residue in the protein with alanine, for example, for alanine scanning mutagenesis. In some embodiments, conserved substitutions with positive values ​​in the PAM250 log-likelihood matrix are prepared, as disclosed by Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), which is incorporated herein by reference. In some embodiments, the substitution is a moderately conserved substitution, wherein the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0063] Therefore, the present invention also includes a genetically modified non-human animal expressing humanized TCRα and β polypeptides (and / or humanized TCRδ and TCRγ polypeptides), wherein the polypeptides contain conserved amino acid substitutions of the amino acid sequences described in this application.

[0064] Those skilled in the art will understand that, in addition to the nucleic acid residues encoding the humanized TCRα and β polypeptides described in this application, other nucleic acids can also encode the polypeptides of this invention due to the degeneracy of the genetic code. Therefore, besides genetically modified non-human animals whose genomes contain nucleotide sequences encoding humanized TCR polypeptides, this application also provides non-human animals whose genomes contain nucleic acid sequences different from those described in this application due to the degeneracy of the genetic code.

[0065] The term "identity," when used in conjunction with sequence, includes identity determined by a variety of different algorithms known in the art capable of determining the identity of nucleotide and / or amino acid sequences. In some embodiments described in this application, the ClustalW v.1.83 (slow) algorithm with an open gap penalty of 10.0 and an extended gap penalty of 0.1 is used, along with the Gonnet similarity matrix (MacVector). TM 10.0.2, MacVector Inc., 2008) Determining Identity. The length of the sequence used for sequence identity comparison depends on the specific sequence. In various embodiments, identity is determined by comparing the sequence of a mature protein from its N-terminus to its C-terminus. In various embodiments, when comparing a human / non-human chimeric sequence with a human sequence, the human portion (not the non-human portion) of the human / non-human chimeric sequence is used for comparison to determine the level of identity between the human sequence and the human portion of the human / non-human chimeric sequence (e.g., comparing the human extracellular domain of a human / mouse chimeric protein with the human extracellular domain of a human protein).

[0066] The term "homology" or "homogeneous" in relation to sequences, such as nucleotide or amino acid sequences, refers to two sequences that, when optimally aligned and compared, have at least about 75% of their nucleotides or amino acids, at least about 80% of their nucleotides or amino acids, or at least about 90-95% of their nucleotides or amino acids, for example, more than 97% of their nucleotides or amino acids being identical. Those skilled in the art will understand that, for optimal gene targeting, the targeting construct should contain an arm homologous to the endogenous DNA sequence (i.e., a "homologous arm"); thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

[0067] The term "operably linked" refers to a juxtaposition, where the components described in this way are in a relationship that allows them to function in the intended manner. Thus, the nucleic acid sequence encoding a protein can be operably linked to regulatory sequences (e.g., promoters, enhancers, silencer sequences, etc.) to preserve appropriate transcriptional regulation. Furthermore, different portions of the humanized protein of the present invention can be operably linked to proteins in the cell that retain appropriate folding, processing, targeting, expression, and other functional properties. Unless otherwise expressly stated, the different domains of the humanized protein of the present invention are operably linked to each other.

[0068] The term "substitution" in relation to gene substitution refers to the insertion of exogenous genetic material into an endogenous genetic locus to replace all or part 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 by a corresponding human gene or a fragment thereof. The corresponding human gene or fragment thereof is an ortholog, homolog, or a human gene or fragment thereof that is substantially identical or the same in structure and / or function as the replaced endogenous non-human gene or fragment thereof. As illustrated in the examples below, the nucleotide sequences of the endogenous non-human TCRα and β variable gene loci are replaced with nucleotide sequences corresponding to the human TCRα and β variable gene loci.

[0069] As used herein, “functional”, for example, a functional protein, refers to a protein that retains at least one biological activity typically associated with natural proteins. For example, in some embodiments of the invention, substitutions at endogenous loci (e.g., substitutions at endogenous nonhuman TCRα, TCRβ, TCRδ, and / or TCRγ variable gene loci) render the loci unable to express functional endogenous proteins.

[0070] As used in this application, a TCR locus or TCR gene locus (e.g., TCRα or TCRβ locus) refers to genomic DNA containing the TCR coding region, which includes the entire TCR coding region, including the unrearranged V(D)J sequence, enhancer sequence, constant sequence, and any upstream or downstream (UTR, regulatory region, etc.) or intermediate DNA sequence (intron, etc.). A TCR variable locus or TCR variable gene locus (e.g., TCRα or TCRβ variable gene locus) refers to genomic DNA containing a region including the TCR variable region segment (V(D)J region) but not the TCR constant sequence, and in different embodiments, does not contain enhancer sequence. Other sequences may be included in the TCR variable gene locus for gene manipulation purposes (e.g., selective expression cassettes, restriction sites, etc.), and these are included in this application.

[0071] Genetically modified TCR animals

[0072] In various embodiments, the present invention typically provides genetically modified non-human animals, wherein the non-human animals contain unrearranged humanized TCR variable gene loci in their genome.

[0073] T cells bind to epitopes on small antigenic determinants on the surface of antigen-presenting cells that bind to the major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complex. T cells bind to these epitopes via the T cell receptor (TCR) complex on their surface. The T cell receptor is a heterodimeric structure composed of two types of chains: α (alpha) and β (beta) chains, or γ (gamma) and δ (delta) chains. The α chain is encoded by a nucleic acid sequence located at the α locus (on human or mouse chromosome 14), which also includes the entire δ locus, and the β chain is encoded by a nucleic acid sequence located at the β locus (on mouse chromosome 6 or human chromosome 7). Most T cells possess αβTCRs; a minority possess γδTCRs. Interactions between the TCR and MHC class I (presented to CD8+ T cells) and MHC class II (presented to CD4+ T cells) molecules are described in [link to relevant documentation]. Figure 1 (Solid symbols indicate non-human sequences; hollow symbols indicate human sequences, showing a specific implementation of the TCR protein of the present invention).

[0074] 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 that make up the TCR contains an extracellular domain comprising constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable region of the TCR determines its antigen specificity and, similar to immunoglobulins, contains three complementarity-determining regions (CDRs). Also similar to immunoglobulin genes, T cell receptor variable gene loci (e.g., TCRα and TCRβ loci) contain multiple unrearranged V(D)J segments (variable (V), linked (J), and diversity (D) segments in TCRβ and δ). During T cell development in the thymus, the TCRα variable gene locus undergoes rearrangement so that the resulting TCRα chain is encoded by a specific combination of VJ segments (Vα / Jα sequence); and the TCRβ variable gene locus undergoes rearrangement so that the resulting TCRβ chain is encoded by a specific combination of VDJ segments (Vβ / Dβ / Jβ sequence).

[0075] Interactions with the thymic stroma trigger several developmental stages in thymocytes, characterized by the expression of different cell surface markers. A summary of characteristic cell surface markers at different developmental stages in the thymus is shown in Table 1. Rearrangement at the TCRβ variable locus begins at stage DN2 and ends at stage DN4, while rearrangement at the TCRα variable locus occurs at stage DP. After TCRβ locus rearrangement is complete, the cells express the TCRβ chain and the alternative α chain pTα on the cell surface. See Janeway's Immunobiology, Chapter 7, as described above.

[0076] Table 1: Developmental stages of T cells in the thymus

[0077]

[0078] Naïve CD4+ and CD8+ T cells leave the thymus and enter peripheral lymphoid organs (such as the spleen) where they come into contact with antigens and are activated to clone and differentiate into multiple effector T cells (Teffs), such as cytotoxic T cells, T cells, etc. REG Cells, T H 17 cells, T H 1 cell, T H 2. Cells, etc. Following infection, multiple T cells persist as memory T cells and are classified as central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708-12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401:659-60. Sallusto and colleagues proposed that after initial infection, Tem cells are a mixture of antigen-stimulated memory T cells with effector functions readily available in peripheral tissues, while Tcm cells are antigen-stimulated memory T cells in peripheral lymphoid organs that can form new effector T cells upon secondary stimulation. Although all memory T cells express the CD45RO isoform of CD45 (naive T cells express the CD45RA isoform), Tcm cells are characterized by the expression of L-selectin (also known as CD62L) and CCR7+, which are crucial for binding to peripheral lymphoid organs and lymph nodes and for signal transduction, as described above. Therefore, all T cells in peripheral lymphoid organs (e.g., naive T cells, Tcm cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express a variety of different cell surface markers, such as CD44. For an overview of the different cell surface markers on T cells, see Janeway's Immunobiology, Chapter 10, as described above.

[0079] Although the primary function of the variable domain of the TCR is antigen recognition, the extracellular portion of the constant domain, as well as the transmembrane and cytoplasmic domains, also play important roles. The complete TCR receptor complex requires not only α and β or γ and δ peptides, but also other molecules including CD3γ, CD3δ, and CD3ε, as well as the ζ-chain homodimer (ζζ). After the TCRβ rearrangement, this pre-TCR complex, along with CD3, is present on the cell surface when the cell expresses TCRβ / pTα. On the cell surface, TCRα (or pTα) has two basic residues in its transmembrane domain, one recruiting the CD3γε heterodimer, and the other recruiting ζζ through its respective acidic residues. TCRβ has additional basic residues in its transmembrane domain, believed to recruit the CD3δε heterodimer. See, for example, Kuhns et al., (2006) Deconstructing the Form and Function of the TCR / CD3 Complex, Immunity 24:133-39; Wucherpfennig et al., (2009) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2:a005140. The assembled complex, containing TCRαβ heterodimers, CD3γε, CD3δε, and ζζ, is expressed on the surface of T cells. Polar residues in the transmembrane domain have been proposed to control the quality of the TCR chain leaving the endoplasmic reticulum; it has been demonstrated that in the absence of the CD3 subunit, the TCR chain is retained in the ER and undergoes targeted degradation. See, for example, 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.

[0080] Since the TCRαβ heterodimer (or TCRγδ heterodimer) itself lacks signal transduction activity, the CD3 and ζ chains of the assembled complex provide components for TCR signaling. Each CD3 chain has an immunoreceptor tyrosine activation motif (ITAM), while the ζ chain contains three tandem ITAMs. Each ITAM contains tyrosine residues that can be phosphorylated by linked kinases. Therefore, 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. 2:e002485. After TCR binding, the ITAM motifs are phosphorylated by Src family tyrosine kinases Lck and Fyn, initiating a signaling cascade that leads to Ras activation, calcium mobilization, actin cytoskeleton rearrangement, and transcription factor activation, all of which ultimately result in T cell differentiation, proliferation, and effector function. As noted above, see also Janeway's Immunobiology, 7th edition, edited by Murphy et al., Garland Science, 2008, all of which are incorporated herein by reference.

[0081] Furthermore, the transmembrane and cytoplasmic domains of TCRβ are believed to play a role in mitochondrial targeting and apoptosis induction; in fact, naturally occurring N-terminally truncated TCRβ molecules are present in thymocytes. (Shani et al., (2009) Incomplete T-cell receptor--βpeptides target the mitochondrion and induce apoptosis, Blood 113:3530-41). Therefore, the TCR constant region has several important functions (which in different embodiments includes part of the extracellular, transmembrane, and cytoplasmic domains); and in different embodiments, the structure of this region should be considered when designing humanized TCRs or gene-modified non-human animals expressing them.

[0082] Transgenic mice with rearranged T-cell receptor sequences are well known in the art. This invention relates to genetically modified non-human animals (e.g., rodents, such as rats and mice) containing unrearranged human or humanized T-cell variable gene loci capable of rearranging to form nucleic acid sequences encoding variable domains of human T-cell receptors, including animals containing T cells containing rearranged human variable domains and non-human (e.g., mouse or rat) constant regions. The invention also provides non-human animals (e.g., rodents, such as rats and mice) capable of generating diverse libraries of human T-cell receptor variable region sequences; thus, the invention provides non-human animals expressing TCRs with fully human variable domains that respond to and bind to epitopes of target antigens. In some embodiments, this application provides non-human animals that generate diverse libraries of T-cell receptors capable of responding to different antigens, including but not limited to antigens presented by antigen-presenting cells (APCs).

[0083] In one embodiment, the present invention provides genetically modified non-human animals (e.g., rodents, such as rats and mice) whose genomes contain unrearranged human TCR variable regions (V(D)J regions), wherein the unrearranged human TCR variable regions replace endogenous non-human TCR variable regions at endogenous non-human (e.g., rodent) TCR variable gene loci (e.g., TCRα, β, δ, and / or γ variable gene loci). In one embodiment, the unrearranged human TCR variable gene loci replace endogenous non-human TCR variable gene loci.

[0084] In another embodiment, the present invention provides genetically modified non-human animals (e.g., rodents, such as rats and mice) containing an unrearranged human TCR variable region segment (V(D)J segment) in their genome, wherein the unrearranged human TCR variable region segment is operatively linked to a non-human TCR constant region gene sequence, resulting in a humanized TCR locus, wherein the humanized TCR locus is located at a different site in the genome than an endogenous non-human TCR locus. Therefore, in one embodiment, the present application also provides a transgenic non-human animal (e.g., rodents, such as mice and rats) containing an unrearranged human TCR variable region segment operatively linked to a non-human TCR constant region sequence.

[0085] In one aspect, the genetically modified non-human animal of the present invention includes a human TCR variable region segment in its genome while retaining a non-human (e.g., rodent, such as mouse or rat) TCR constant gene segment. In various embodiments, the constant region includes 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 includes a non-human, non-endogenous TCR constant gene sequence, such as the non-human, non-endogenous TCR transmembrane domain and cytoplasmic tail. As described above, the constant region of the TCR participates in the signaling cascade initiated in antigen-triggered T cell activation; therefore, the endogenous TCR constant region interacts with various non-human anchoring and signaling proteins in T cells. Therefore, in one aspect, the genetically modified non-human animal of the present invention expresses a humanized T-cell receptor that retains the ability to recruit a variety of endogenous non-human anchoring or signaling molecules, such as CD3 molecules (e.g., CD3γ, CD3δ, CD3ε), ζ chain, Lck, Fyn, ZAP-70, etc. A non-limiting list of molecules recruited to the TCR complex is provided in Janeway's Immunobiology, as described above. Furthermore, with Similar to mice, which exhibit normal B cell development and normal clonal selection processes, believed to be at least in part due to the location of the variable region at an endogenous mouse locus and the preservation of the mouse constant domain, the non-human animals of the present invention exhibit normal T cell development and T cell differentiation processes.

[0086] In some embodiments, this application provides a non-human animal whose genome contains an unrearranged human TCRα variable region segment, wherein the unrearranged human TCRα variable region segment is operatively linked to a non-human TCRα constant region gene sequence to produce a humanized TCRα locus. In one embodiment, the humanized TCRα locus is located at a different site in the genome than the endogenous non-human TCRα locus. In another embodiment, the unrearranged human TCRα variable region segment replaces the endogenous non-human TCRα variable region segment while retaining the endogenous non-human TCRα constant region. In one embodiment, the unrearranged human TCRα variable gene locus replaces the endogenous non-human TCRα variable gene locus. In some embodiments, the animal retains the endogenous non-human TCRβ variable region and constant region gene sequences. Therefore, the animal expresses a TCR comprising a human / non-human chimeric (i.e., humanized) TCRα chain and a non-human TCRβ chain.

[0087] In other embodiments, this application provides a non-human animal whose genome contains an unrearranged human TCRβ variable region segment, wherein the unrearranged human TCRβ variable region segment is operatively linked to a non-human TCRβ constant region gene sequence to produce a humanized TCRβ locus. In one embodiment, the humanized TCRβ locus is located at a different site in the genome than the endogenous non-human TCRβ locus. In another embodiment, the unrearranged human TCRβ variable region segment replaces the endogenous non-human TCRβ variable region segment while retaining the endogenous non-human TCRβ constant region. In one embodiment, the unrearranged human TCRβ variable gene locus replaces the endogenous non-human TCRβ variable gene locus. In some embodiments, the animal retains the endogenous non-human TCRα variable region and constant region gene sequences. Therefore, the animal expresses a TCR comprising a human / non-human chimeric (i.e., humanized) TCRβ chain and a non-human TCRα chain.

[0088] In some specific embodiments, the present invention provides a genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) whose genome includes (a) an unrearranged T-cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment operably linked to an endogenous non-human (e.g., rodent, such as a mouse or rat) TCRα constant gene sequence, and / or (b) an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment operably linked to an endogenous non-human (e.g., rodent, such as a mouse or rat) TCRβ constant gene sequence.

[0089] In various embodiments of the invention, unrearranged human or humanized TCR variable gene loci (e.g., TCRα and / or TCRβ variable gene loci) are included in non-human animal (e.g., rodent, such as mouse or rat) lineages. In various embodiments, the TCR V(D)J fragment is replaced by unrearranged human TCR V(D)J fragments (e.g., Vα and Jα, and / or Vβ and Dβ and Jβ fragments) at an endogenous non-human TCR variable gene locus (or locus), wherein the unrearranged human V and J and / or V and D and J segments are operatively linked to non-human TCR constant region genes.

[0090] In some embodiments of the present invention, the non-human animal comprises two copies of the unrearranged human or humanized TCRα variable gene locus and / or two copies of the unrearranged human or humanized TCRβ variable gene locus. Therefore, the non-human animal is homozygous for one or both of the unrearranged human or humanized TCRα and TCRβ variable gene loci. In some embodiments of the present invention, the non-human animal comprises one copy of the unrearranged human or humanized TCRα variable gene locus and / or one copy of the unrearranged human or humanized TCRβ variable gene locus. Therefore, the non-human animal is heterozygous for one or both of the unrearranged human or humanized TCRα and TCRβ variable gene loci.

[0091] In one embodiment, an unrearranged TCRα variable gene locus containing human variable regions (e.g., human Vα and Jα regions) is located in a non-human genome such that the human variable region replaces the corresponding non-human variable region. In another embodiment, an unrearranged TCRα variable gene locus containing a human variable region replaces an endogenous TCRα variable gene locus. In one aspect, the endogenous non-human Vα and Jα regions cannot be rearranged to form rearranged Vα / Jα sequences. Therefore, in one aspect, the human Vα and Jα regions at the unrearranged TCRα variable gene locus can be rearranged to form rearranged human Vα / Jα sequences.

[0092] Similarly, in one embodiment, an unrearranged TCRβ variable gene locus containing human variable region segments (e.g., human Vβ, Dβ, and Jβ segments) is located in a non-human genome such that the human variable region segment replaces the corresponding non-human variable region segment. In one embodiment, an unrearranged TCRβ variable gene locus containing a human variable region segment replaces an endogenous TCRβ variable gene locus. In one aspect, endogenous non-human Vβ, Dβ, and Jβ segments cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence. Therefore, in one aspect, human Vβ, Dβ, and Jβ segments in an unrearranged TCRβ variable gene locus can be rearranged to form a rearranged human Vβ / Dβ / Jβ sequence.

[0093] In yet another embodiment, unrearranged TCRα and β variable gene loci comprising human variable region segments replace endogenous TCRα and β variable gene loci, respectively. In one aspect, endogenous non-human Vα and Jα segments cannot be rearranged to form rearranged Vα / Jα sequences, and endogenous non-human Vβ, Dβ, and Jβ segments cannot be rearranged to form rearranged Vβ / Dβ / Jβ sequences. Therefore, in one aspect, human Vα and Jα segments at unrearranged TCRα variable gene loci can be rearranged to form rearranged human Vα / Jα sequences, and human Vβ, Dβ, and Jβ segments at unrearranged TCRβ variable gene loci can be rearranged to form rearranged human Vαβ / Dβ / Jβ sequences.

[0094] In some aspects of the invention, the non-human animal containing the humanized TCRα and / or TCRβ loci (containing unrearranged TCRα and / or TCRβ variable gene loci) retains the endogenous non-human TCRα and / or TCRβ variable gene loci. In one embodiment, the endogenous non-human TCRα and / or TCRβ variable gene loci are non-functional loci. In one embodiment, the non-functional loci are inactive loci, such as inverted loci (e.g., the nucleic acid sequence encoding the variable gene locus is oriented opposite to the constant region sequence, making successful rearrangement using the variable region segment of the inverted locus impossible). In one embodiment, the humanized TCRα and / or TCRβ variable gene loci are located between the endogenous non-human TCRα and / or TCRβ variable gene loci and the endogenous non-human TCRα and / or TCRβ constant gene loci.

[0095] The IMGT database at www.imgt.org can be used to determine the numbering, name, location, and other information of human and mouse TCR loci V and J and / or V, D, and J regions. The mouse TCRα variable locus is approximately 1.5 million bases long and contains a total of 110 Vα and 60 Jα regions. Figure 2The human TCRα variable locus is approximately one million base pairs and contains a total of 54 Vα and 61 Jα segments, of which 45 Vα and 50 Jα are believed to be functional. Unless otherwise expressly stated, the number of human V(D)J segments throughout the specification refers to the total number of V(D)J segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) contains at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal contains a humanized TCRα locus containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Vα segments. In some embodiments, the humanized TCRα locus contains 2, 8, 23, 35, 48, or 54 human Vα segments. Therefore, in some embodiments, the humanized TCRα locus in non-human animals may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% human Vα; in some embodiments, it may comprise about 2%, about 3%, about 15%, about 65%, about 90%, or 100% human Vα.

[0096] In one embodiment, the non-human animal comprises a humanized TCRα locus containing a DNA fragment with consecutive human Vα40 to Vα41 (the Vα region is also referred to as "TRAV" or "TCRAV") human sequences and a DNA fragment with 61 consecutive human Jα region (the Jα region is also referred to as "TRAJ" or "TCRAJ") human sequences. In one embodiment, the non-human animal comprises a humanized TCRα locus containing a DNA fragment with consecutive human TRAV35 to TRAV41 human sequences and a DNA fragment with 61 consecutive human TRAJ human sequences. In one embodiment, the non-human animal comprises a humanized TCRα locus containing a DNA fragment with consecutive human TRAV22 to TRAV41 human sequences and a DNA fragment with 61 consecutive human TRAJ human sequences. In one embodiment, the non-human animal comprises a humanized TCRα locus containing a DNA fragment containing consecutive human TRAV13-2 to TRAV41 sequences and a DNA fragment containing 61 consecutive human TRAJ sequences. In another embodiment, the non-human animal comprises a humanized TCRα locus containing a DNA fragment containing consecutive human TRAV6 to TRAV41 sequences and 61 human TRAJ sequences. In yet another embodiment, the non-human animal comprises a humanized TCRα locus containing a DNA fragment containing consecutive human TRAV1-1 to TRAV41 sequences and 61 human TRAJ sequences. In various embodiments, the DNA fragment containing consecutive human sequences of the human TCRα variable region further includes restriction endonuclease sites, selective expression cassettes, nuclease sites, or other inserted sites to facilitate cloning and selection during locus humanization. In various embodiments, these additional sites do not interfere with the normal function (e.g., rearrangement, splicing, etc.) of the different genes within the TCRα locus.

[0097] In one embodiment, the humanized TCRα locus comprises 61 human Jα segments, or 100% human Jα segments. In one specific embodiment, the humanized TCRα locus comprises 8 human Vα segments and 61 human Jα segments; in another specific embodiment, the humanized TCRα locus comprises 23 human Vα segments and 61 human Jα segments. In yet another specific embodiment, the humanized TCRα locus comprises a complete library of human Vα and Jα segments, i.e., all human variable α region gene segments encoded by the α locus, or 54 human Vα and 61 human Jα segments. In various embodiments, the non-human animal does not contain any endogenous non-human Vα or Jα segments at the TCRα locus.

[0098] The mouse TCRβ variable locus is approximately 0.6 million bases long and contains a total of 33 Vβ, 2 Dβ, and 14 Jβ segments. Figure 6 The human TCRβ variable locus is approximately 0.6 million bases long and contains a total of 67 Vβ, 2 Dβ, and 14 Jβ segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) contains at least one human Vβ, at least one human Dβ, and at least one human Jβ segment. In one embodiment, the non-human animal contains a humanized TCRβ locus containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or up to 67 human Vβ segments. In some embodiments, the humanized TCRβ locus contains 8, 14, 40, 66, or 67 human Vβ segments. Therefore, in some embodiments, the humanized TCRβ locus in non-human animals may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% human Vβ; in some embodiments, it may comprise about 20%, about 60%, about 15%, about 98%, or 100% human Vβ.

[0099] In one embodiment, the non-human animal comprises a humanized TCRβ locus containing a DNA fragment with consecutive human sequences from Vβ18 to Vβ29-1 (the Vβ region is also referred to as "TRBV" or "TCRBV"). In another embodiment, the non-human animal comprises a humanized TCRβ locus containing a DNA fragment with consecutive human sequences from Vβ18 to Vβ29-1, a separated DNA fragment containing consecutive human sequences from Dβ1 to Jβ1 (i.e., the human Dβ1-Jβ1-1-Jβ1-6 region), and a separated DNA fragment containing consecutive human sequences from Dβ2 to Jβ2 (i.e., the human Dβ2-Jβ2-1-Jβ2-7 region). In one embodiment, the non-human animal comprises a humanized TCRβ locus, the locus comprising a DNA fragment containing consecutive human TRBV6-5 to TRBV29-1 sequences, a separated DNA fragment containing consecutive human Dβ1-Jβ1 sequences (i.e., the human Dβ1-Jβ1-1-Jβ1-6 region), and a separated DNA fragment containing consecutive human Dβ2-Jβ2 sequences (i.e., the human Dβ2-Jβ2-1-Jβ2-7 region). In another embodiment, the non-human animal comprises a humanized TCRβ locus, the locus comprising a DNA fragment containing consecutive human TRBV1 to TRBV29-1 sequences, a separated DNA fragment containing consecutive human Dβ1-Jβ1 sequences, and a separated DNA fragment containing consecutive human Dβ2-Jβ2 sequences. In one embodiment, the non-human animal comprises a humanized TCRβ locus containing a DNA fragment with consecutive human TRBV1 to TRBV29-1 sequences, a separated DNA fragment with consecutive human Dβ1-Jβ1 sequences, a separated DNA fragment with consecutive human Dβ2-Jβ2 sequences, and a separated DNA fragment with a human TRBV30 sequence. In other embodiments, the DNA fragment containing consecutive human sequences of the human TCRβ variable region further includes restriction endonuclease sites, selective expression cassettes, nuclease sites, or other inserted sites to facilitate cloning and selection during the locus humanization process. In other embodiments, these additional sites do not interfere with the normal function (e.g., rearrangement, splicing, etc.) of the different genes within the TCRβ locus.

[0100] In one embodiment, the humanized TCRβ locus comprises 14 human Jβ segments or 100% human Jβ segments, and 2 human Dβ segments or 100% human Jβ segments. In another embodiment, the humanized TCRβ locus comprises at least one human Vβ segment, such as 14 human Vβ segments, and all mouse Dβ and Jβ segments. In a particular embodiment, the humanized TCRβ locus comprises 14 human Vβ segments, 2 human Dβ segments, and 14 human Jβ segments. In another particular embodiment, the humanized TCRβ locus comprises a complete library of human Vβ, Dβ, and Jβ segments, i.e., all human variable β region gene segments encoded by the β locus or 67 human Vβ, 2 human Dβ, and 14 human Jβ segments. In one embodiment, the non-human animal at the humanized TCRβ locus comprises one (e.g., 5') non-human Vβ segment. In different implementations, the non-human animal does not contain any endogenous non-human Vβ, Dβ, or Jβ segments at the TCRβ locus.

[0101] In various embodiments, the non-human animal (e.g., rodent) contains a library of human TCRα and TCRβ (and optionally human TCRδ and TCRγ) variable region segments (e.g., a complete library of variable region segments), and the animal uses the library of different segments (e.g., a complete library of different segments) to generate a diverse library of TCR molecules for different antigens.

[0102] In various aspects, the non-human animal comprises a continuous human genome TCR variable locus portion containing V, D, and J, or D and J, or V and J, or V segments arranged in an unrearranged human genome variable locus, such as promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., arranged in a human genome TCR variable locus. In other aspects, the different segments are arranged in an unrearranged non-human genome TCR variable locus. In different embodiments of the humanized TCRα and / or β locus, the humanized locus may contain two or more human genome segments that are not parallel to each other in the human genome, for example, a V segment fragment of a human V locus located in a human genome adjacent to a constant region is parallel to a V segment fragment of a human V locus located upstream of a human V locus in the human genome.

[0103] In mice and humans, the TCRδ gene segment is located at the TCRα gene locus (see [link to TCRα gene locus]). Figure 2 and 5The TCRδJ and D regions are located between the Vα and Jα regions, while the TCRδV region is distributed throughout the TCRα locus, with most of it situated within different Vα regions. The numbering and location of different TCRδ regions can be determined from the IMGT database. Due to the genomic distribution of TCRδ gene regions within the TCRα locus, successful rearrangements within the TCRα locus typically involve the deletion of a TCRδ gene region.

[0104] In some embodiments of the invention, the non-human animal containing the unrearranged human TCRα variable gene locus also contains at least one human Vδ segment, such as a complete library of human Vδ segments. Therefore, in some embodiments, substitution of the endogenous TCRα variable gene locus results in the replacement of at least one non-human Vδ segment with a human Vδ segment. In other embodiments, the non-human animal of the invention contains a complete library of human Vδ, Dδ, and Jδ segments at the unrearranged humanized TCRα locus; in yet another embodiment, the non-human animal contains a complete unrearranged human TCRδ locus (i.e., a TCRδ locus including human variable regions as well as human enhancers and constant regions) at the unrearranged humanized TCRα locus. Figure 5 An exemplary implementation of constructing an unrearranged humanized TCRα locus containing the complete unrearranged TCRδ locus is described.

[0105] In yet another embodiment, the non-human animal of the present invention further comprises an unrearranged humanized TCRγ locus, such as a TCRγ locus comprising at least one human Vγ and at least one human Jγ segment (e.g., a complete library of human Vγ and human Jγ variable region segments). The human TCRγ locus is located on human chromosome 7, while the mouse TCRγ locus is located on mouse chromosome 13. For details on the TCRγ locus, please refer to the IMGT database.

[0106] In one aspect, non-human animals (e.g., rodents, such as mice or rats) containing the humanized TCRα and β variable gene loci (and optionally humanized TCRδ / γ variable gene loci) described in this application express humanized T cell receptors on the surface of T cells, said receptors comprising humanized variable regions and non-human (e.g., rodent, such as mice or rats) constant regions. In some aspects, said non-human animals are capable of expressing a diverse repertoire of humanized T cell receptors recognizing a variety of presented antigens.

[0107] In different embodiments of the present invention, the humanized T-cell receptor polypeptide described herein comprises a human leader sequence. In an alternative embodiment, the humanized TCR receptor nucleic acid sequence is modified so that the humanized TCR polypeptide comprises a non-human leader sequence.

[0108] The humanized TCR peptide described in this application can be expressed under the regulation of endogenous non-human regulatory elements (e.g., rodent regulatory elements), such as promoters, silencers, enhancers, etc. Alternatively, the humanized TCR peptide described in this application can be expressed under the regulation of human regulatory elements. In different embodiments, the non-human animals described in this application further include all regulatory sequences and other sequences commonly found in situ in the human genome.

[0109] In various embodiments, the humanized variable region of the humanized TCR protein is capable of interacting with different proteins on the surface of the same cell or other cells. In one embodiment, the humanized 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 another cell, such as an antigen-presenting cell (APC). In some embodiments, the MHC class I or II protein is a non-human protein (e.g., rodent, such as mouse or rat). In other embodiments, the MHC class I or II protein is a human protein. In one aspect, the other cell, such as an APC, is an endogenous non-human cell expressing human or humanized MHC molecules. In various embodiments, the other cell is a human cell expressing human MHC molecules.

[0110] In one aspect, the non-human animal expresses a humanized T-cell receptor with a non-human constant region on the surface of its T cells, wherein the receptor is capable of interacting with non-human molecules such as anchoring or signaling molecules expressed on the T cells (e.g., CD3 molecules, ζ chains, or other proteins anchored to the TCR via CD3 molecules or ζ chains).

[0111] Therefore, in one aspect, this application provides a cell complex comprising nonhuman T cells expressing a TCR containing the humanized TCRα chain and the humanized TCRβ chain described in this application, and a nonhuman antigen-presenting cell containing an antigen that binds to MHC I or MHC II. In one embodiment, the nonhuman constant TCRα and TCRβ chains are complexed with a nonhuman (ζ) 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.

[0112] Genetically modified non-human animals can be selected from the following group: mice, rats, rabbits, pigs, cattle (e.g., dairy cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, macaques). For non-human animals from which suitable genetically modified ES cells are not readily available, other methods can be used to prepare genetically modified non-human animals. These methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and applying nuclear transfer to transfer the modified genome to suitable cells such as oocytes, and then, under suitable conditions, inducing pregnancy in the non-human animal using the modified cells (e.g., modified oocytes) to form an embryo.

[0113] In one aspect, the non-human animal is a mammal. In another aspect, the non-human animal is a small mammal, such as a jerboa or a superfamily of murines. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from a superfamily of murines. In one embodiment, the genetically modified animal is selected from families such as Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny rats, crested rats), Madagascar Muridae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), spiny dwarfs (e.g., spiny dormice), and mole-like mice (e.g., mole rats, bamboo rats, and mole rats). In a particular embodiment, the genetically modified rodent is selected from true mice or rats (muridae), gerbils, spiny rats, and yellow-crowned rats. In one embodiment, the genetically modified mouse is a member of the murine family. In one embodiment, the animal is a rodent. In one particular embodiment, the rodent is selected from mice and rats. In another embodiment, the non-human animal is a mouse.

[0114] In one particular embodiment, the non-human animal is a rodent, specifically 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 mice are selected from the following 129 strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, for example, Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, and also Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In one specific embodiment, the genetically modified mouse is a mixture of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another specific embodiment, the mouse is a mixture of the aforementioned 129 strain or a mixture of the aforementioned BL / 6 strain. In one specific embodiment, the mixed 129 strain is the 129S6 (129 / SvEvTac) strain. In another embodiment, the mouse is a BALB strain, such as the BALB / c strain. In yet another embodiment, the mouse is a mixture of the BALB strain and another of the aforementioned strains.

[0115] In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from Wistar rats, the LEA strain, the Sprague Dawley strain, the Fischer strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0116] Therefore, in one embodiment, the present invention provides a genetically modified mouse containing, in its genome, an unrearranged human or humanized TCR variable gene locus, such as TCRα, TCRβ, TCRδ, and / or TCRγ variable gene locus. In some embodiments, the unrearranged human or humanized TCR variable gene locus replaces an endogenous mouse TCR variable gene locus. In other embodiments, the unrearranged human or humanized TCR variable gene locus is located at a different site in the genome than the corresponding endogenous mouse TCR genome. In some embodiments, the human or humanized unrearranged TCR variable gene locus is operatively linked to a mouse TCR constant region.

[0117] In one embodiment, this application provides a genetically modified mouse, wherein the mouse contains an unrearranged T-cell receptor (TCR) α variable gene locus in its genome, the locus comprising at least one human Jα segment and at least one human Vα segment operably linked to a mouse TCRα constant gene sequence, and an unrearranged TCRβ variable gene locus, the locus comprising at least one human Jβ segment, at least one human Dβ segment, and at least one human Vβ segment operably linked to a mouse TCRβ constant gene sequence. In a particular embodiment, the mouse contains an unrearranged TCRα variable gene locus in its genome, the locus comprising a complete human Jα segment library and a complete human Vα segment library operably linked to a mouse TCRα constant gene sequence, and an unrearranged TCRβ variable gene locus comprising a complete human Jβ segment library, a complete human Dβ segment library, and a complete human Vβ segment library operably linked to a mouse TCRβ constant gene sequence.

[0118] In some embodiments, the unrearranged TCRα variable gene locus comprises a human TCRα variable region replacing the endogenous mouse TCRα variable gene locus, and the unrearranged TCRβ variable gene locus comprises a human TCRβ variable region replacing the endogenous mouse TCRβ variable gene locus. In some embodiments, the endogenous mouse Vα and Jα regions cannot be rearranged to form a rearranged Vα / Jα sequence, and the endogenous mouse Vβ, Dβ, and Jβ regions cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence. In some embodiments, the human Vα and Jα regions are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ, Dβ, and Jβ regions are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence.

[0119] In various embodiments, the T cells produced by the non-human animals (e.g., rodents, such as mice or rats) described in this application can undergo thymic development, which proceeds from DN1 to DN2 to DN3 to DN4 to DP and then to CD4 or CD8SP T cells. The cell surface molecules expressed by these T cells in the non-human animals of this invention are typically produced by T cells at specific stages of thymic development (e.g., CD25, CD44, kit, CD3, pTα, etc.). Therefore, the pTα expressed by the non-human animals described in this application complexes with TCRβ at the DN3 stage of thymic development. The T cells expressed by the non-human animals described in this application can undergo thymic development to produce CD4+ and CD8+ T cells. Under normal circumstances, the physiological ratio of CD4+ to CD8+ T cells in the thymus is between approximately 2:1 and 3:1. See, for example, 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. Therefore, in one embodiment, the ratio of CD4+ to CD8+ T cells produced in the thymus of the non-human animal described in this application is between approximately 2:1 and 3:1 (CD4+:CD8+).

[0120] In various embodiments, the T cells generated by the non-human animals described in this application are capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non-human animals described in this application are capable of generating a normal pool of effector T cells, such as CTLs (cytotoxic T lymphocytes) and T cells. H 1. T H 2. T REG T H 17. Therefore, in these embodiments, the effector T cells generated by the non-human animals described in this application perform typical functions of different T cell types, such as recognition, binding, and response to foreign antigens. In different embodiments, the effector T cells generated by the non-human animals described in this application, expressed in the context of MHC I molecules, kill cells displaying cytoplasmic pathogen peptide fragments; recognize peptides derived from antigens degraded in intracellular vesicles and presented to the surface of macrophages via MHC II molecules and induce macrophages to kill microorganisms; produce cytokines that drive B cell differentiation; activate B cells to produce antibodies that promote opsonization; induce epithelial cells to produce chemokines that recruit neutrophils to the site of infection, etc.

[0121] In additional embodiments, the non-human animals described in this application contain a normal number of CD3+ T cells in the periphery, such as the spleen. In some embodiments, the percentage of peripheral CD3+ T cells in the non-human animals described in this application is comparable to that of wild-type animals (i.e., animals containing all endogenous TCR variable region segments). In one embodiment, the non-human animals described in this application have a normal ratio of splenic CD3+ T cells to total spleen cells.

[0122] In other respects, the non-human animals described in this application are capable of generating a population of memory T cells in response to a target antigen. For example, the non-human animals generate central memory T cells (Tcm) and effector memory T cells (Tem) in response to antigens such as target antigens (e.g., antigens detected for vaccine development).

[0123] 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, for example, 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 in this application 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 in this application develop a normal population of dendritic cells in the thymus.

[0124] The primary 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 invention, T cells from non-human animals containing humanized TCRα and / or β loci show normal utilization of the TCRα / β and TCRδ / γ loci, for example, utilization of the TCRα / β and TCRδ / γ loci is similar to that of wild-type animals (e.g., the TCRα / β and TCRδ / γ proteins expressed by the T cells of the non-human animals described in this application have similar proportions to those expressed in wild-type animals). Therefore, in some embodiments, non-human animals containing humanized TCRα / β and endogenous non-human TCRδ / γ loci show normal utilization of all loci.

[0125] In addition to genetically modified non-human animals, this application also provides non-human embryos (e.g., rodent embryos, such as mouse or rat embryos), wherein the embryo contains donor ES cells derived from non-human animals (e.g., rodents, such as mice or rats) as described in this application. In one aspect, the embryo comprises ES donor cells including unrearranged humanized TCR loci and host embryonic cells.

[0126] This application also provides an tissue derived from a non-human animal (e.g., mouse or rat) as described in this application, and expressing humanized TCR peptides (e.g., TCRα and / or TCRβ, or TCRδ and / or TCRγ peptides).

[0127] Furthermore, this application provides non-human cells isolated from the non-human animals described in this application. 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.

[0128] This application also provides a non-human cell comprising chromosomes or fragments thereof from a non-human animal as described in this application. In one embodiment, the non-human cell comprises a nucleus from a non-human animal as described in this application. In another embodiment, the non-human cell comprises chromosomes or fragments thereof obtained through nuclear transfer.

[0129] This application also provides a non-human cell expressing a TCR protein, wherein the TCR protein comprises a human variable region and a non-human constant region. The TCR protein may comprise TCRα, TCRβ, or a combination thereof. In one embodiment, the cell is a T cell, such as a CD4+ or CD8+ T cell.

[0130] In one aspect, this application provides a non-human induced pluripotent stem cell containing an unrearranged humanized TCR locus encoding the humanized TCR polypeptide as described in this application. In one embodiment, the induced pluripotent stem cell is derived from a non-human animal as described in this application.

[0131] In one aspect, this application provides a hybridoma or tetravalent tumor derived from non-human animal cells as described in this application. In one embodiment, the non-human animal is a rodent, such as a mouse or rat.

[0132] This application also provides a method for preparing a genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) as described in this application. The method for preparing a genetically modified non-human animal includes, in the genome of the animal, a humanized, unrearranged TCR locus (e.g., a humanized, unrearranged TCRα, TCRβ, TCRδ, and / or TCRγ locus). In one embodiment, this application provides a method for preparing a genetically modified non-human animal (e.g., a rodent, such as a mouse or rat) expressing a T cell receptor on the surface of T cells, the receptor comprising a humanized variable region and a non-human (e.g., rodent, such as a mouse or rat) constant region, wherein the method comprises replacing an endogenous non-human TCRα variable gene locus in a first non-human animal with an unrearranged humanized TCRα variable gene locus comprising at least one humanized Vα segment and at least one humanized Jα segment, wherein the humanized TCRα variable gene locus... The Rα variable gene locus is operatively linked to an endogenous TCRα constant region; in a second non-human animal, the endogenous non-human TCRβ variable gene locus is replaced with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, one human Dβ segment, and one human Jβ segment, wherein the humanized TCRβ variable gene locus is operatively linked to the endogenous TCRβ constant region; and the first and second non-human animals are mated 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, this application provides a method for preparing a genetically modified non-human animal whose genome comprises a humanized unrearranged TCRα locus, or a non-human animal whose genome comprises a humanized unrearranged TCRβ locus, generated according to the method described in this application. In various embodiments, the substitution is performed at an endogenous locus. In some embodiments, as described in the examples, the method utilizes... One or more targeted constructs prepared by the technology are introduced into ES cells and used. The technique involves introducing targeted ES cell clones into mouse embryos. In some embodiments, the ES cells are derived from mice of a hybrid strain of 129 and C57BL / 6. In various embodiments, the method comprises a stepwise humanization strategy, wherein in each subsequent humanization step, a construct containing additional variable region segments is introduced into the ES cells, ultimately resulting in the inclusion of a complete human variable region segment library in the mouse (see, for example...). Figure 3 and Figure 7 ).

[0133] Therefore, this application also provides nucleotide constructs for producing the genetically modified non-human animals described in this application. In one aspect, the nucleotide construct comprises: 5' and 3' homologous arms, a human DNA fragment containing a human TCR variable region gene segment, and a selective expression cassette between recombination sites. In one embodiment, the human DNA fragment is a TCRα gene fragment and contains at least one human TCRα variable region segment. In another embodiment, the human DNA fragment is a TCRβ fragment and contains at least one human TCRβ variable region gene segment. In one aspect, at least one homologous arm is a non-human homologous arm and is homologous to a non-human TCR locus (e.g., a non-human TCRα or TCRβ locus).

[0134] Selective expression cassettes are nucleotide sequences inserted into a target construct that facilitate the selection of cells (e.g., ES cells) that have integrated into the target construct. A variety of suitable selective expression cassettes are well known in the art. Typically, selective expression cassettes result in positive selection in the presence of specific antibiotics (e.g., Neo, Hyg, Pur, CM, Spec, etc.). Furthermore, selective expression cassettes can be located between recombination sites, allowing for removal after treatment with recombinases. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, respectively, but others are also well known in the art.

[0135] In one embodiment, the selective expression cassette is located at the 5' end of the human DNA fragment. In another embodiment, the selective expression cassette is located at the 3' end of the human DNA fragment. In yet another embodiment, the selective expression cassette is located within the human DNA fragment, for example, within a human intron. In yet another embodiment, the selective expression cassette is located at the junction of human and mouse DNA fragments.

[0136] Various exemplary implementations of targeting strategies for generating genetically modified non-human animals, constructs, and targeting vectors for use are described in [link to relevant documentation]. Figure 3 4, 5, 7 and 8.

[0137] After gene targeting is completed, ES cells or genetically modified non-human animals are screened to confirm successful incorporation of the target exogenous nucleotide sequence or expression of exogenous peptides (e.g., human TCR variable region segments). Various techniques are well known to those skilled in the art, including (but not limited to) Southern blotting, long PCR, and quantitative PCT (e.g., using...). Real-time PCR, fluorescence in situ hybridization, Northern blotting, flow cytometry, Western spectroscopy, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (e.g., mice) carrying the target gene modification can be identified by screening for the deletion of mouse alleles and / or the availability of human alleles, using the revised allele detection 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 detection methods for identifying specific nucleotide or amino acid sequences in genetically modified animals are well known to those skilled in the art.

[0138] This disclosure also provides a method for modifying a non-human animal TCR variable gene locus (e.g., TCRα, TCRβ, TCRδ, and / or TCRγ gene locus) to express the humanized TCR protein described in this application. In one embodiment, the present invention provides a method for modifying a TCR variable gene locus to express a humanized TCR protein on the surface of T cells, wherein the method comprises replacing an endogenous non-human TCR variable gene locus in a non-human animal with an unrearranged humanized TCR variable gene locus. In one embodiment, the TCR variable gene locus is a TCRα variable gene locus, and the unrearranged humanized TCR variable gene locus comprises at least one human Vα segment and at least one human Jα segment. In one embodiment, the TCR variable gene locus is a TCRβ variable gene locus, and the unrearranged humanized TCR variable gene locus comprises at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment. In different respects, the unrearranged humanized TCR variable gene locus is operatively linked to the corresponding endogenous non-human TCR constant region.

[0139] This application also provides a humanized TCR protein prepared from the non-human animal (e.g., rodent, such as mouse or rat) described in this application, wherein the humanized TCR protein comprises a human variable region and a non-human constant region. Therefore, the humanized TCR protein includes human complementarity-determining regions (i.e., human CDR1, 2, and 3) and a non-human constant region in its variable domain.

[0140] Although the following embodiments describe a genetically modified animal whose genome contains humanized TCRα and / or humanized TCRβ variable gene loci, those skilled in the art will understand that similar strategies can be used to produce genetically modified animals whose genomes contain humanized TCRδ and / or TCRγ variable gene loci. This application also provides genetically modified non-human animals in which all four TCR variable gene loci are humanized.

[0141] Uses of genetically modified TCR animals

[0142] In various embodiments, the genetically modified non-human animals of the present invention imbue T cells with humanized TCR molecules on their surface, and as a result, they recognize peptides presented to them by the MHC complex in a human-like manner. The genetically modified non-human animals described in this application can be used to study the development and function of human T cells and the process of immune tolerance; to detect human vaccine candidates; to generate TCRs with certain specificities for TCR gene therapy; and to generate TCR libraries of disease-associated antigens (e.g., tumor-associated antigens (TAAs)).

[0143] There is growing interest in T-cell therapy in this field because T cells (e.g., cytotoxic T cells) can be directed to attack and cause the destruction of target antigens such as viral antigens, bacterial antigens, tumor antigens, etc., or the cells that present them. Early research into cancer T-cell therapy aimed to isolate tumor-infiltrating lymphocytes (TILs; a population of lymphocytes in a tumor mass that may contain T cells responding to tumor antigens) from tumor cell masses, expand them in vitro using T-cell growth factors, and transfer them back into the patient in a process known as adoptive T-cell therapy. 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 to date has been limited to melanoma and renal cell carcinoma; and adoptive metastasis of TILs is not specific to defined tumor-associated antigens (TAAs). (Linnermann et al., as stated above.)

[0144] There have been some attempts to initiate TCR gene therapy, in which T cells are selected or programmed to target a specific antigen, such as TAA. Currently, TCR gene therapy relies on the identification of TCR sequences targeting specific antigens, such as tumor-associated antigens (TAAs). For example, Rosenberg and colleagues have published results showing that they transduced peripheral blood lymphocytes from melanoma patients with genes encoding TCRα and β chains specific to the MART-1 epitope and used the expanded lymphocytes for adoptive T-cell therapy. (Johnson et al., (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen, Blood 114:535-46; Morgan et al., (2006) Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes, Science 314:126-29.) MART-1 specific TCRs are isolated from patients whose tumors regressed after TIL treatment. However, the identification of such TCRs, especially high-affinity TCRs (which are most likely to be useful in treatment), is complicated because most tumor antigens are autoantigens, and TCRs targeting these antigens are often absent or have suboptimal affinity, mainly due to immune tolerance.

[0145] In various embodiments, the present invention addresses this problem by providing genetically modified non-human animals whose genomes contain unrearranged human TCR variable gene loci. The non-human animals described in this application are capable of generating T cells with a diverse repertoire of humanized T cell receptors. Therefore, the non-human animals described in this application can serve as a source of diverse humanized T cell receptor repertoires, such as high-affinity humanized T cell receptors for adoptive T cell therapy.

[0146] Therefore, in one embodiment, the present invention provides a method for generating a T-cell receptor against a human antigen, the method comprising immunizing a non-human animal (e.g., a rodent, such as a mouse or rat) described in this application with a target antigen to induce an immune response in the animal, isolating activated T cells specific to the target antigen from the animal, and determining the nucleic acid sequence of the T-cell receptor expressed by the antigen-specific T cells.

[0147] In one embodiment, the present invention provides a method for generating a human T-cell receptor specific to a target antigen (e.g., a disease-associated antigen), the method comprising immunizing a non-human animal as described in this application with the target antigen; inducing an immune response in the animal; isolating T cells reactive to the target antigen from the animal; determining the nucleic acid sequence of a human TCR variable region expressed by the T cells; cloning the human TCR variable region into a nucleotide construct containing a nucleic acid sequence of a human TCR constant region, such that the human TCR variable region is operatively linked to the human TCR constant region; and expressing a human T-cell receptor specific to the target antigen from the construct. In one embodiment, the steps of isolating T cells, determining the nucleic acid sequence of a human TCR variable region expressed by the T cells, cloning the human TCR variable region into a nucleotide construct containing a nucleic acid sequence of a human TCR constant region, and expressing the human T-cell receptor are performed using standard techniques known to those skilled in the art.

[0148] In one embodiment, the nucleotide sequence encoding a T-cell receptor specific to the target antigen is expressed in cells. In one embodiment, the cells expressing the TCR are selected from CHO, COS, 293, HeLa, and PERC.6. TM Cells, etc.

[0149] The target antigen can be any antigen known to cause or be associated with a disease or condition, such as tumor-associated antigens; antigens of viruses, bacteria, or other pathogens. Many tumor-associated antigens are well known in the art. For selection of tumor-associated antigens, refer to the Cancer Immunity (Journal of the Cancer Institute) peptide database (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the invention, the target antigen is a human antigen, such as a human tumor-associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and T cell receptors are used to kill cells expressing the antigen.

[0150] In one embodiment, this application provides a method for identifying T cells specific to a target antigen, such as a tumor-associated antigen, the method comprising immunizing a non-human animal described in this application with the target antigen to induce an immune response in the animal, and isolating T cells specific to the target antigen from the non-human animal.

[0151] This invention provides a novel method for adoptive T-cell therapy. Therefore, this application provides a method for treating or alleviating a disease or condition (e.g., cancer) in a subject (e.g., a mammalian subject, such as a human subject), the method comprising immunizing a non-human animal described in this application with an antigen associated with the disease or condition to induce an immune response in the animal, isolating an antigen-specific T-cell population from the animal, and introducing the isolated antigen-specific T-cells into the subject. In one embodiment, the invention provides a method for treating or alleviating a disease or condition in a human subject, the method comprising immunizing a non-human animal described in this application with a target antigen (e.g., a disease or condition-related antigen, such as a tumor-associated antigen) to induce an immune response in the animal, isolating an antigen-specific T-cell population 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 to cause the T cells to express the antigen-specific T-cell receptor, and introducing the T cells into the subject. In one embodiment, the nucleic acid sequence of the T cell receptor is further humanized before the introduction of T cells derived from the subject, for example, by modifying the sequence encoding a non-human constant region to make it more similar to 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, an antigen-specific T cell population is expanded before infusion into the subject. In some embodiments, the subject's immune cell population is immune-depleted before infusion of antigen-specific T cells. In some embodiments, the antigen-specific TCR is a high-affinity TCR, such as a TCR with high affinity for tumor-associated antigens. In some embodiments, the T cells are cytotoxic T cells. In other embodiments, the disease or condition is caused by a virus or bacteria.

[0152] In another embodiment, the disease or condition is an autoimmune disease. REG T cells are a subset of cells that maintain tolerance to self-antigens and prevent pathological autoimmune reactions. Therefore, this application also provides a method for treating autoimmune diseases, the method relying on the generation of antigen-specific T cells in the non-human animals of the present invention. REG cell.

[0153] This application also provides a method for treating or alleviating a disease or condition (e.g., cancer) in a subject, the method comprising introducing disease- or condition-affected cells (e.g., cancer cells) of the subject into a non-human animal, causing the animal to generate an immune response against the cells, isolating a population of T cells that are responsive to the cells from the animal, 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 introducing T cells carrying the T cell receptor from the subject into the subject.

[0154] This application also provides the use of non-human animals for preparing nucleic acid sequences encoding human TCR variable domains (e.g., TCRα and / or β variable domains) as described in this application. In one embodiment, this application provides a method for preparing a nucleic acid sequence encoding a human TCR variable domain, the method comprising immunizing a non-human animal as described in this application with an antigen of interest, causing the non-human animal to generate 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 preparing a nucleic acid sequence encoding a human TCR variable domain operatively linked to a non-human TCR constant region, which comprises isolating T cells from the non-human animal described in this application and obtaining therefrom a nucleic acid sequence encoding a TCR variable domain operatively linked to a TCR constant region.

[0155] This application also provides a use of non-human animals as described in this application to prepare human therapeutic agents, the use comprising immunizing the non-human animal with an antigen of interest (e.g., a tumor-associated antigen) to induce an immune response in the non-human animal, obtaining animal T cells that are reactive to the antigen of interest, obtaining a nucleic acid sequence encoding a humanized TCR protein that binds to the antigen of interest, and using the nucleic acid sequence encoding the humanized TCR protein in the human therapeutic agent.

[0156] Therefore, this application also provides a method for preparing a human therapeutic agent, the method comprising immunizing a non-human animal as described in this application with an antigen of interest, causing the non-human animal to produce an immune response, obtaining animal T cells that are reactive to the antigen of interest, obtaining a nucleic acid sequence encoding a humanized T cell receptor that binds to the antigen of interest, and using the humanized T cell receptor in the human therapeutic agent.

[0157] In one embodiment, the human therapeutic agent is a T cell (e.g., a human T cell, such as a T cell derived from a human subject) carrying a sequence of interest (e.g., transfected, transduced, or otherwise introduced into the nucleic acid of interest) to express a humanized TCR protein with affinity for the antigen of interest. In one aspect, the subject using the therapeutic agent requires treatment for a specific disease or condition, and the antigen is associated with the disease or condition. In one aspect, the T cell is a cytotoxic T cell, the antigen is a tumor-associated antigen, and the disease or condition is cancer. In one aspect, the T cell is derived from a human subject.

[0158] In another embodiment, the human therapeutic agent is a T-cell receptor. In one embodiment, the therapeutic receptor is a soluble T-cell receptor. Significant effort has been devoted to generating soluble T-cell receptors or TCR variable regions for therapeutic agents. The generation of soluble T-cell receptors depends on obtaining rearranged TCR variable regions. One approach is to engineer single-chain TCRs containing TCRα and TCRβ, and fuse them together via linkers, similar to the scFv immunoglobulin form (see, for example, International Application No. WO 2011 / 044186). The resulting scTv, if similar to scFv, will provide a thermostable and soluble form of the TCRα / β binding protein. Alternative approaches include designing soluble TCRs with a constant TCRβ domain (see, for example, Chung et al., (1994) Functional three-domain single-chain T-cell receptors, Proc. Natl. Acad. Sci. USA. 91:12654-58); and designing non-naturally occurring disulfide bonds at the junctions between constant TCR domains (Boulter and Jakobsen review (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. Patent No. 7,569,664). Other forms of soluble T-cell receptors have also been described. The non-human animals described in this application can be used to determine the sequence of a T-cell receptor that binds with high affinity to the antigen of interest, and subsequently to design soluble T-cell receptors based on said sequence.

[0159] Soluble T-cell receptors derived from TCR receptor sequences expressed in non-human animals can be used to block the function of proteins of interest, such as viral, bacterial, or tumor-associated proteins. Alternatively, soluble T-cell receptors can be fused with portions capable of killing infected or cancerous cells, such as cytotoxic molecules (e.g., chemotherapeutic agents), toxins, radionuclides, prodrugs, antibodies, etc. Soluble T-cell receptors can also be fused with immunomodulatory molecules, such as cytokines, chemokines, etc. Soluble T-cell receptors can also be fused with immunosuppressive molecules, such as molecules that prevent T cells from killing other cells carrying antigens recognized by T cells. Such soluble T-cell receptors fused with immunosuppressive molecules can be used, for example, to block autoimmunity. For different exemplary immunosuppressive molecules that can be fused with soluble T-cell receptors, see Ravetch and Lanier (2000) Immune Inhibitory Receptors, Science 290:84-89, which is incorporated herein by reference.

[0160] This invention also provides methods for studying immune responses in the context of human TCR, including human TCR rearrangement, T cell development, T cell activation, and immune tolerance.

[0161] This application also provides a method for testing vaccine candidates. In one embodiment, this application provides a method for determining whether a vaccine will activate an immune response (e.g., T cell proliferation, cytokine release, etc.) and lead to the generation of effector and memory T cells (e.g., central and effector memory T cells).

[0162] This application also includes the following implementation methods:

[0163] Implementation Method 1. A genetically modified non-human animal, whose genome contains:

[0164] An unrearranged T-cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment operatively linked to a non-human TCRα constant gene sequence.

[0165] Implementation Method 2. The animal according to Implementation Method 1, wherein the unrearranged TCRα variable gene locus replaces the endogenous non-human TCRα variable gene locus.

[0166] Implementation Method 3. The animal according to Implementation Method 1, wherein the endogenous non-human Vα and Jα segments cannot be rearranged to form rearranged Vα / Jα sequences.

[0167] Implementation Method 4. The animal according to Implementation Method 1, wherein the animal lacks a functional endogenous non-human TCRα variable locus.

[0168] Implementation 5. The animal according to Implementation 4, wherein the lack of functional endogenous non-human TCRα variable locus comprises deletions selected from the group consisting of: (a) deletions of all endogenous Vα gene segments, (b) deletions of all endogenous Jα gene segments, and (c) combinations thereof.

[0169] Implementation 6. The animal according to Implementation 1, wherein the human Vα and Jα segments are rearranged to form a rearranged human Vα / Jα sequence.

[0170] Embodiment 7. The animal according to Embodiment 6, wherein the animal expresses a T cell receptor containing a human TCRα variable region on the surface of its T cells.

[0171] Implementation Method 8. The animal according to Implementation Method 1, wherein the T cells of the animal undergo thymic T cell development to produce CD4 and CD8 single-positive T cells.

[0172] Implementation Method 9. The animal according to Implementation Method 1, wherein the animal comprises a normal ratio of splenic CD3+ T cells to total splenic cells.

[0173] Implementation Method 10. An animal according to Implementation Method 1, wherein the animal produces a population of central and effector memory T cells against a target antigen.

[0174] Implementation 11. The animal according to Implementation 1, wherein the unrearranged TCRα variable gene locus contains a complete human Jα segment library and a complete human Vα segment library.

[0175] Implementation 12. The animal according to Implementation 1, wherein the animal retains an endogenous non-human TCRα variable gene locus, and wherein the locus is a non-functional locus.

[0176] Embodiment 13. The animal according to Embodiment 1, wherein the animal is a rodent.

[0177] Embodiment 14. The animal according to Embodiment 13, wherein the rodent is a mouse.

[0178] Implementation Method 15. A genetically modified non-human animal, whose genome contains:

[0179] An unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operatively linked to a non-human TCRβ constant gene sequence.

[0180] Embodiment 16. The animal according to Embodiment 15, wherein the unrearranged TCRβ variable gene locus replaces the endogenous non-human TCRβ variable gene locus.

[0181] Implementation Method 17. The animal according to Implementation Method 15, wherein the endogenous rodent Vβ, Dβ and Jβ segments cannot be rearranged to form the rearranged Vβ / Dβ / Jβ sequence.

[0182] Implementation Method 18. The animal according to Implementation Method 15, wherein the animal lacks a functional endogenous non-human TCRβ variable locus.

[0183] Implementation 19. The animal according to Implementation 18, wherein the deficiency of the functional endogenous rodent TCRβ 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 of the above deletions.

[0184] Implementation 20. The animal according to Implementation 15, wherein the human Vβ, Dβ and Jβ segments are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence.

[0185] Embodiment 21. The animal according to Embodiment 20, wherein the animal expresses a T cell receptor containing a human TCRβ variable region on the surface of its T cells.

[0186] Implementation 22. The animal according to Implementation 15, wherein the animal’s T cells undergo thymic T cell development to produce CD4 and CD8 single-positive T cells.

[0187] Implementation 23. The animal according to Implementation 15, wherein the animal comprises a normal ratio of splenic CD3+ T cells to total splenic cells.

[0188] Implementation Method 24. The animal according to Implementation Method 15, wherein the animal generates a population of central and effector memory T cells against the target antigen.

[0189] Implementation 25. The animal according to Implementation 15, wherein the unrearranged TCRβ variable gene locus comprises a complete human Jβ segment library, a complete human Dβ segment library, and a complete human Vβ segment library.

[0190] Implementation 26. The animal according to Implementation 15, wherein the animal retains an endogenous non-human TCRβ variable gene locus, and wherein the locus is a non-functional locus.

[0191] Embodiment 27. The animal according to Embodiment 15, wherein the animal is a rodent.

[0192] Embodiment 28. The animal according to Embodiment 27, wherein the rodent is a mouse.

[0193] Implementation Method 29. A genetically modified non-human animal, whose genome contains:

[0194] An unrearranged T-cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment operatively linked to a non-human TCRα constant gene sequence; and,

[0195] An unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operatively linked to a non-human TCRβ constant gene sequence.

[0196] Implementation 30. The animal according to Implementation 29, wherein the unrearranged TCRα variable gene locus replaces the endogenous non-human TCRα variable gene locus, and wherein the unrearranged TCRβ variable gene locus replaces the endogenous non-human TCRβ variable gene locus.

[0197] Implementation Method 31. The animal according to Implementation Method 29, wherein the endogenous non-human Vα and Jα segments cannot be rearranged to form the rearranged Vα / Jα sequence, and wherein the endogenous non-human Vβ, Dβ, and Jβ segments cannot be rearranged to form the rearranged Vβ / Dβ / Jβ sequence.

[0198] Implementation 32. The animal according to Implementation 29, wherein the animal lacks a functional endogenous non-human TCRα variable locus and a functional endogenous non-human TCRβ variable locus.

[0199] Implementation 33. The animal according to Implementation 32, wherein the deficiency of the functional endogenous non-human TCRα 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 of the above deletions.

[0200] Furthermore, the absence of the functional endogenous non-human TCRβ variable locus includes deletions selected from the following groups: (a) deletion of all endogenous Vβ gene segments, (b) deletion of all endogenous Dβ gene segments, (c) deletion of all endogenous Jβ gene segments, and (d) a combination of the above deletions.

[0201] Implementation 34. The animal according to Implementation 29, wherein 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.

[0202] Embodiment 35. The animal according to Embodiment 34, wherein the animal expresses a T cell receptor on the surface of its T cells comprising a human variable region and a non-human animal constant region.

[0203] Implementation 36. The animal according to Implementation 29, wherein the animal’s T cells undergo thymic T cell development to produce CD4 and CD8 single-positive T cells.

[0204] Implementation 37. The animal according to Implementation 29, wherein the animal comprises a normal ratio of splenic CD3+ T cells to total splenic cells.

[0205] Implementation Method 38. An animal according to Implementation Method 29, wherein the animal is targeted at a target antigen-generating center and an effector memory T cell population.

[0206] Implementation 39. The animal according to Implementation 29, wherein the unrearranged TCRα variable gene locus comprises 61 human Jα segments and 8 human Vα segments, and wherein the unrearranged TCRβ variable gene locus comprises 14 human Jβ segments, 2 human Dβ segments and 14 human Vβ segments.

[0207] Implementation 40. The animal according to Implementation 29, wherein the unrearranged TCRα variable gene locus contains a complete human Jα segment library and a complete human Vα segment library, and wherein the unrearranged TCRβ variable gene locus contains a complete human Jβ segment library, a complete human Dβ segment library and a complete human Vβ segment library.

[0208] Implementation 41. The animal according to Implementation 29, wherein the animal retains endogenous non-human TCRα and TCRβ variable gene loci, and wherein the loci are non-functional loci.

[0209] Implementation 42. The animal according to Implementation 29, wherein the animal further comprises a nucleotide sequence of the humanized Vδ region at the humanized TCRα locus.

[0210] Implementation 43. The animal according to Implementation 42, wherein the animal further comprises a complete human Vδ segment library, a complete human Dδ segment library and a complete human Jδ segment library at the humanized TCRα locus.

[0211] Embodiment 44. The animal according to Embodiment 29, wherein the animal is a rodent.

[0212] Embodiment 45. The animal according to Embodiment 44, wherein the rodent is a mouse.

[0213] Implementation Method 46. A method for preparing a genetically modified non-human animal, wherein the animal expresses a T-cell receptor comprising a human variable region and a non-human constant region on the surface of T cells, the method comprising:

[0214] In a first non-human animal, an endogenous non-human TCRα variable gene locus is replaced with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operatively linked to an endogenous non-human TCRα constant region.

[0215] In a second non-human animal, an endogenous non-human TCRβ variable gene locus is replaced with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operatively linked to an endogenous non-human TCRβ constant region; and

[0216] The first and second animals were mated to obtain a non-human animal expressing a T-cell receptor containing a human variable region and a non-human constant region.

[0217] Implementation 47. The method according to Implementation 46, wherein the endogenous non-human Vα and Jα segments cannot be rearranged to form a rearranged Vα / Jα sequence, and wherein the endogenous non-human Vβ, Dβ, and Jβ segments cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence.

[0218] Implementation 48. The method according to Implementation 46, wherein the genetically modified animal lacks a functional endogenous non-human TCRα variable locus and lacks a functional endogenous non-human TCRβ variable locus.

[0219] Implementation 49. The method according to implementation 48, wherein the deficiency of the functional endogenous non-human TCRα 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 of the above deletions.

[0220] Furthermore, the absence of the functional endogenous non-human TCRβ variable locus includes deletions selected from the following group: (a) deletion of all endogenous Vβ gene segments, (b) deletion of all endogenous Dβ gene segments, (c) deletion of all endogenous Jβ gene segments, and (d) a combination of the above deletions.

[0221] Implementation 50. The method according to Implementation 46, wherein 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.

[0222] Implementation 51. The method according to Implementation 46, wherein the rodent's T cells undergo thymic T cell development to produce CD4 and CD8 single-positive T cells.

[0223] Implementation 52. The method according to implementation 46, wherein the animal comprises a normal ratio of splenic CD3+ T cells to total splenic cells.

[0224] Implementation 53. The method according to implementation 46, wherein the animal generates a population of central and effector memory T cells against the target antigen.

[0225] Implementation 54. The method according to implementation 46, wherein the unrearranged humanized TCRα variable gene locus comprises 61 human Jα segments and 8 human Vα segments, and wherein the unrearranged humanized TCRβ variable gene locus comprises 14 human Jβ segments, 2 human Dβ segments and 14 human Vβ segments.

[0226] Implementation 55. The method according to Implementation 46, wherein the unrearranged humanized TCRα variable gene locus comprises a complete human Jα segment library and a complete human Vα segment library, and wherein the unrearranged humanized TCRβ variable gene locus comprises a complete human Jβ segment library, a complete human Dβ segment library and a complete human Vβ segment library.

[0227] Implementation 56. The method according to implementation 46, wherein the non-human animal is a rodent.

[0228] Implementation 57. The method according to Implementation 56, wherein the rodent is a mouse.

[0229] Implementation Method 58. A genetically modified mouse, comprising in its genome:

[0230] The unrearranged T-cell receptor (TCR) α variable gene locus contains a complete human Jα segment library and a complete human Vα segment library operatively linked to the mouse TCRα constant gene sequence, and

[0231] The unrearranged TCRβ variable gene locus contains a complete human Jβ segment library, a complete human Dβ segment library, and a complete human Vβ segment library that are operatively linked to the mouse TCRβ constant gene sequence.

[0232] Implementation 59. The mouse according to Implementation 58, wherein the unrearranged TCRα variable gene locus replaces the endogenous mouse TCRα variable gene locus, and wherein the unrearranged TCRβ variable gene locus replaces the endogenous mouse TCRβ variable gene locus.

[0233] Implementation 60. The mouse according to Implementation 58, wherein the endogenous mouse Vα and Jα regions cannot be rearranged to form a rearranged Vα / Jα sequence, and wherein the endogenous mouse Vβ, Dβ, and Jβ regions cannot be rearranged to form a rearranged Vβ / Dβ / Jβ sequence.

[0234] Implementation 61. The mouse according to Implementation 58, wherein 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.

[0235] Embodiment 62. The mouse according to Embodiment 61, wherein the mouse expresses a T cell receptor comprising a human variable region and a mouse constant region on the surface of its T cells.

[0236] Implementation 63. The mouse according to Implementation 58, wherein the mouse's T cells undergo thymic T cell development to produce CD4 and CD8 single-positive T cells.

[0237] Implementation 64. The mouse according to Implementation 58, wherein the mouse comprises a normal ratio of splenic CD3+ T cells to total splenic cells.

[0238] Implementation Method 65. The mouse according to Implementation Method 58, wherein the mouse generates a population of central and effector memory T cells against the target antigen.

[0239] Implementation 66. The mouse according to Implementation 58, wherein the mouse further comprises a complete human Vδ segment library, a complete human Dδ segment library and a complete human Jδ segment library at the humanized TCRα locus.

[0240] Implementation 67. The mouse according to Implementation 58, wherein the mouse retains the endogenous mouse TCRα and TCRβ variable gene loci, and wherein the loci are non-functional loci.

[0241] Implementation Method 68. A method for generating a human T-cell receptor against a target antigen, comprising:

[0242] Immunize the non-human animals described in Implementation 1 with the target antigen;

[0243] Inducing an immune response in the animals;

[0244] Isolate T cells that respond to the target antigen from the animals;

[0245] Determine the nucleic acid sequence of the variable region of the human TCR expressed by the T cells;

[0246] Cloning the human TCR variable region into a nucleotide construct containing a nucleic acid sequence of a human TCR constant region, wherein the human TCR variable region is operatively linked to the human TCR constant region; and

[0247] It expresses human T-cell receptors in cells. Example

[0248] The invention will be further illustrated by the following non-limiting embodiments. These embodiments are listed to aid in understanding the invention, but are not intended and should not be construed as limiting its scope in any way. The embodiments do not include descriptions of conventional methods (molecular cloning techniques, etc.) well known to those skilled in the art. Unless otherwise stated, parts are parts by weight, molecular weight is average molecular weight, temperature is expressed in degrees Celsius, and pressure is at or near atmospheric pressure.

[0249] Example 1: Generation of mice with humanized TCR variable gene loci

[0250] use Genetic engineering techniques (see, for example, US Patent 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) were used to prepare mice containing deletions of endogenous TCR (α or β) variable loci and substitutions of endogenous V and J or V, D, and J segments, wherein human sequences derived from a BAC library obtained using bacterial homologous recombination were used to prepare a large targeting vector (LTVEC) comprising a genomic fragment of the human TCR variable locus positioned between the targeting arms to target the LVEC to the endogenous mouse TCR variable locus in mouse ES cells. The LVEC was linearized and electrotransfected into mouse ES cell lines according to Valenzuela et al. Hygromycin- or neomycin-resistant ES cells were selected, and screening was performed for deletion of mouse alleles and acquisition of human alleles.

[0251] Targeting ES cell clones through The 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.) was introduced into mouse embryos at the 8-cell stage (or earlier). A modified allele detection method (Valenzuela et al.) was used to screen for the deletion of endogenous TCR variable alleles and the acquisition of human alleles to detect mice carrying humanized TCR loci. Identification was performed on F0 mice (derived entirely from donor ES cells). F0 fetuses were genotyped and bred to homozygote status. Mice homozygous for humanized TCRα and / or TCRβ variable loci (e.g., including human TCRα and / or TCRβ variable segment subclasses) were prepared as described in this application and phenotypically classified.

[0252] All mice were bred and housed in Regeneron Pharmaceuticals’ specific pathogen-free laboratory. All animal studies were approved by IACUC and Regeneron Pharmaceuticals.

[0253] Example 2: Stepwise humanization of the TCRα variable locus

[0254] Figure 2 and 3 This paper outlines the use of a stepwise humanization strategy to replace 1.5 million bases of DNA corresponding to the 110 V and 60 J mouse regions of the mouse TCRα locus with 1 million bases of DNA corresponding to the 54 V and 61 J regions of the human TCRα locus. The junctional nucleic acid sequences of different targeting vectors used for the stepwise humanization strategy of the TCRα locus are summarized in Table 2 and are included in the sequence listing.

[0255] Table 2: Nucleic acid sequences at the junction of different TCRα locus targeting vectors

[0256]

[0257]

[0258] The variable regions of human TCRα were numbered according to the IMGT database. Each junction had at least 100 bp (approximately 50 bp at each end) included in the sequence list.

[0259] In particular, such as Figure 4A As shown, DNA from mouse BAC clone RP23-6A14 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1539) to replace the endogenous mouse TCRα locus TCRJ1-TCRAJ28 region with a Ub-hygromycin expression cassette following the loxP site. DNA from mouse BAC clone RP23-117i19 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1535) to replace the region surrounding (including) the endogenous mouse TCRα and δ locus TCRAV1 within a 15kb range with a PGK-neomycin expression cassette following the loxP site. Chromosome karyotype analysis and screening methods known in the art (e.g., TAQMAN) were used to determine the target vector. TM ES cells carrying dual-targeting chromosomes (i.e., a single endogenous mouse TCRα locus targeted by two targeting vectors) were validated. Modified ES cells were treated with CRE recombinase to mediate the deletion of the region between the two loxP sites (i.e., the region consisting of endogenous mouse TCRα loci from TCRAV1 to TCRAJ1) while retaining only a single loxP site, neomycin expression cassette, and mouse constant and enhancer regions. This strategy resulted in the generation of the deleted mouse TCRα / δ locus (MAID 1540).

[0260] The first human targeting vector for TCRα has BAC clones from CTD2216p1 and CTD2285m07.

[0261] The 191,660 bp human DNA from Invitrogen contained the first two consecutive human TCRαV gene segments (TRAV40 & 41) and 61 TCRαJ (50 functional) gene segments. This BAC was modified via homologous recombination to include a Not1 site 403 bp downstream of the TCRαJ1 gene segment used to link the 3' mouse homologous arm and a 5' AsiSI site used to link the 5' mouse homologous arm. Two distinct homologous arms were used to link this human fragment: the 3' homologous arm contained an endogenous mouse TCRα sequence from the RP23-6A14 BAC clone, and the 5' homologous arm contained a 5' endogenous TCRα sequence from the mouse TCRα of the mouse BAC clone RP23-117i19. This mouse-human chimeric BAC was used as a targeting vector for the initial insertion of the human TCRα gene segment at the mouse TCRα locus and the upstream loxp-ub-hygromycin-loxp expression cassette (MAID 1626). Figure 4B The junctional nucleic acid sequences (SEQ ID NO: 1-3) of the MAID 1626 targeting vector are shown in Table 2.

[0262] Subsequently, a series of human-derived targeting vectors were prepared using the same mouse 5' arm, which contained an endogenous 5' TCRα sequence from mouse BAC clone RP23-117i19 mouse TCRα and alternative loxP-neomycin-loxP and loxP-hygromycin-loxP (or frt-hygromycin-frt for MAID 1979) selective expression cassettes.

[0263] To generate a human TCRα small locus containing a total of 8 human TCRαV (7 functional) and 61 human TCRαJ (50 functional) gene segments, DNA from the human BAC clone RP11-349p11 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1767). Figure 4C The added 104,846 bp human DNA contained the next six (five functional) consecutive human TCRαV gene segments (TRAV35 to TRAV39) and a 5' loxP-ub-neomycin-loxP expression cassette. The resulting TCRα locus, containing the 5' loxP-ub-neomycin-loxP expression cassette and a total of eight human TCRαV (seven functional) and 61 human TCRαJ gene segments, was operatively linked to the mouse TCRα constant gene and enhancer. The junctional nucleic acid sequences (SEQ ID NO: 4 and 5) of the MAID 1767 targeting vector are shown in Table 2.

[0264] To generate a small human TCRα locus containing a total of 23 human TCRαV (17 functional) and 61 human TCRαJ gene segments, DNA from a mouse BAC clone containing a unique I-CeuI site from 5' to 3', a 20kb mouse TCRA arm at the 5' end of the mouse TCRA locus for homologous recombination into ES cells, and an inverted loxP-Ub-Hyg-loxP expression cassette was modified via bacterial homologous recombination to include, from 5' to 3', a unique I-CeuI site, a 20kb mouse TCRA arm at the 5' end of the mouse TCRA locus, a frt-pgk-Hyg-frt expression cassette, and a unique AsiSI site. DNA from the human BAC clone RP11-622o20 (Invitrogen) carrying human TCRαV22-V34 was modified via homologous recombination to include a Spec expression cassette between the unique I-CeuI and AsiSI sites. Subsequently, the Spec expression cassette in the modified human BAC clone was replaced with the sequence between the I-Ceul and AsiSI sites in the modified mouse BAC clone using standard restriction enzyme digestion / ligation techniques. The resulting targeting vector (MAID 1979; Figure 4D A 136,557 bp human DNA sequence was introduced, containing the next 15 (10 functional) consecutive human TCRαJ gene segments (TRAV22 to TRAV34) and a 5'frt-pgk-Hyg-frt expression cassette. The resulting TCRα locus, containing the 5'frt-pgk-Hyg-frt expression cassette and a total of 23 human TCRαV (17 functional) and 61 human TCRαV gene segments, was operatively linked to the mouse TCRα constant gene and enhancer. The junctional nucleic acid sequences (SEQ ID NO: 6 and 7) of the MAID 1779 targeting vector are shown in Table 2.

[0265] To generate a human TCRα small locus containing a total of 35 human TCRαV (28 functional) and 61 human TCRαJ gene segments, DNA from the human BAC clone CTD2501-k5 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1769). Figure 4EThe added 124,118 bp human DNA contained the next 12 (11 functional) consecutive human TCRαV gene segments (TRAV13-2 to TRAV21) and a 5' loxP-ub-neomycin-loxP expression cassette. The resulting TCRα locus, containing the 5' loxP-ub-neomycin-loxP expression cassette and a total of 35 human TCRαV (28 functional) and 61 human TCRαJ gene segments, was operatively linked to the mouse TCRα constant gene and enhancer. The junctional nucleic acid sequences (SEQ ID NO: 8 and 9) of the MAID 1769 targeting vector are shown in Table 2.

[0266] To generate a human TCRα small locus containing a total of 48 human TCRαV (39 functional) and 61 human TCRαJ gene segments, DNA from the human BAC clone RP11-92F11 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1770). Figure 4F The 145,505 bp human DNA added contained the following 13 (11 functional) consecutive human TCRαJ gene segments (TRAV6 to TRAV8.5) and a 5' loxP-ub-hygromycin-loxP expression cassette. The resulting TCRα locus, containing the 5' loxp-ub-hygromycin-loxP expression cassette and a total of 48 human TCRαV (39 functional) and 61 human TCRαJ gene segments, was operatively linked to the mouse TCRα constant gene and enhancer. The junctional nucleic acid sequences (SEQ ID NO: 10 and 11) of the MAID 1770 targeting vector are shown in Table 2.

[0267] To generate a human TCRα small locus containing a total of 54 human TCRαV (45 functional) and 61 human TCRαJ gene segments, DNA from the human BAC clone RP11-780M2 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1777). Figure 4GThe 148,496 bp human DNA added contained the next six (six functional) consecutive human TCRαV gene segments (TRAV1-1 to TRAV5) and a 5' loxP-ub-neomycin-loxP expression cassette. The resulting TCRα locus, containing the 5' loxP-ub-neomycin-loxP expression cassette and a total of 54 human TCRαV (45 functional) and 61 human TCRαJ gene segments, was operatively linked to the mouse TCRα constant gene and enhancer. The junctional nucleic acid sequences (SEQ ID NO: 12 and 13) of the MAID 1771 targeting vector are shown in Table 2.

[0268] In any of the above steps, the selective expression cassette deletion is removed using Cre or Flp recombinase. Additionally, it can be done as follows: Figure 5 The human TCRδ locus is introduced as shown.

[0269] Example 3: Stepwise humanization of the TCRβ variable locus

[0270] Figure 6 and 7 This paper outlines the use of a stepwise humanization strategy to replace 0.6 million bases of DNA corresponding to the mouse TCRβ locus (33 V, 2 D, and 14 J) with 0.6 million bases of DNA corresponding to the 67 V, 2 D, and 14 J regions of the human TCRβ locus. The junctional nucleic acid sequences of different targeting vectors used for the stepwise humanization strategy of the TCRβ locus are summarized in Table 3 and are included in the sequence listing.

[0271] Table 3: Nucleic acid sequences at the junction of different TCRβ locus targeting vectors

[0272]

[0273]

[0274] The variable regions of human TCRβ were numbered according to the IMGT database. Each junction had at least 100 bp (approximately 50 bp at each end) included in the sequence list.

[0275] Specifically, DNA from mouse BAC clone RP23-153p19 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1544) to replace the 17kb region (including TCRBV30) immediately upstream of the 3' trypsinogen gene cluster at the endogenous mouse TCRβ locus with a PGK-neo expression cassette following the loxP site. Figure 8ADNA from mouse BAC clone RP23-461h15 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1542) to replace the 8355 bp region downstream of the 5' trypsinogen gene cluster at the endogenous mouse TCRβ locus (including TCRBV2 and TCRBV3) with a Ub-hygromycin expression cassette following the loxP site. Chromosome karyotype analysis and screening methods known in the art (e.g., TAQMAN) were then used. TM ES cells carrying dual-targeting chromosomes (i.e., a single endogenous mouse TCRβ locus targeted by two targeting vectors) were validated. Modified ES cells were treated with CRE recombinase to mediate the deletion of the region between the 5' and 3' loxP sites (composed of endogenous mouse TCRβ loci from TCRBV2 to TCRBV30) while retaining only a single loxP site, hygromycin expression cassette, and mouse TCRBD, TCRBJ, constant, and enhancer sequences. A mouse TCRβ locus was retained upstream of the 5' end of the trypsinogen cluster, and a mouse TCRBβ locus was retained downstream of the mouse Eβ locus. Figure 8A As shown.

[0276] The first human targeting vector for TCRβ contains 125,781 bp of human DNA from the CTD2559j2 BAC clone (Invitrogen), which contains the first 14 consecutive human TCRβV gene segments (TRBV18-TRBV29-1). This BAC was modified via homologous recombination to include a 5' AsiSI site and a 3' AscI site for linking the 5' and 3' mouse homologous arms. Two different homologous arms were used to link this human fragment: one containing an endogenous TCRβ sequence surrounding the downstream of the mouse trypsinogen gene from the RP23-153p19 BAC clone, and the other containing an endogenous TCRβ sequence surrounding the upstream of the mouse trypsinogen gene from the RP23-461h15 mouse BAC clone. The mouse-human chimeric BAC was used as the targeting vector (MAID 1625) for the initial insertion of the human TCRβ gene segment and the upstream frt-ub-neomycin-frt expression cassette at the mouse TCRβ locus. The resulting human TCRβ minilocus contained 14 human (8 functional) TCRβVs. Figure 8B The junctional nucleic acid sequences (SEQ ID NO: 14-16) of the MAID 1625 targeting vector are shown in Table 3.

[0277] To replace the mouse TCRβD and J regions with human TCRβD and J regions, DNA from mouse BAC clone RP23-302p18 (Invitrogen) and human BAC clone RP11-701D14 (Invitrogen) was modified via homologous recombination and introduced as a targeting vector (MAID 1715) into ES cells containing the aforementioned TCRβV small locus (i.e., MAID 1625). This modification involved replacing a ~25425 bp sequence containing human TCRBD1-J1, loxP Ub-hygromycin-loxP expression cassette, mouse constant region 1, and human TCRBD2-J2 in a ~18540 bp region (from 100 bp downstream of the polyA region of the 3' trypsinogen gene to 100 bp downstream of the J region in the D2 cluster, including mouse TCRBD1-J1, mouse constant region 1, and mouse TCRBD2-J2) at the endogenous mouse TCRβ locus. Figure 8C (i)). Chromosomal karyotype analysis and screening methods known in the art (e.g., TAQMAN) TM ES cells carrying a dual-target chromosome (i.e., a single endogenous mouse TCRβ locus targeted by two targeting vectors) were validated. Modified ES cells were treated with CRE recombinase to mediate hygromycin expression cassette deletion, thus retaining only a single loxP site downstream of the human J region of the D1J cluster. Figure 8C (ii)). The junctional nucleic acid sequences (SEQ ID NO:17-21) of the MAID 1715 targeting vector are shown in Table 3.

[0278] Subsequently, a series of human targeting vectors were prepared using the same mouse 5' arm, which contained an endogenous TCRβ sequence from mouse BAC clone RP23-461h15 surrounding the upstream of the mouse trypsinogen gene, along with alternative selective expression cassettes.

[0279] To generate a human TCRβ minilocus containing a total of 40 human TCRβV (30 functional) and human TCRβD and J gene segments, DNA from human BAC clones RP11-134h14 and RP11-785k24 (Invitrogen) was modified and incorporated into the targeting vector (MAID 1791) using standard bacterial homologous recombination, restriction enzyme digestion / ligation, and other cloning techniques. The introduction of the MAID 1791 targeting vector added 198,172 bp of human DNA containing the next 26 (22 functional) consecutive human TCRβV gene segments (TRBV6-5 to TRBV17) and the 5'frt-ub-hygromycin-frt expression cassette. The obtained TCRβ loci containing the 5'frt-ub-hygromycin-frt expression cassette and a total of 40 human TCRβV (30 functional) and human TCRβD and J gene segments were operatively linked to the mouse TCRβ constant gene and enhancer. Figure 8D The junctional nucleic acid sequences (SEQ ID NO: 22 and 23) of the MAID 1791 targeting vector are shown in Table 3.

[0280] To generate a human TCRβ small locus containing a total of 66 human TCRβV (47 functional) and human TCRβD and J gene segments, the DNA of human BAC clone RP11-902B7 (Invitrogen) was modified via homologous recombination and used as a targeting vector (MAID 1792). The added 159,742 bp of human DNA contained the next 26 (17 functional) consecutive human TCRβV gene segments (TRBV1 to TRBV12-2) and a 5'frt-ub-neomycin-frt expression cassette. The resulting TCRβ locus containing the 5'frt-ub-neomycin-frt expression cassette and a total of 66 human TCRβV (47 functional) and human TCRβD and J gene segments was operatively linked to the mouse TCRβ constant gene and enhancer. Figure 8E The junctional nucleic acid sequences (SEQ ID NO: 24 and 25) of the MAID 1792 targeting vector are shown in Table 3.

[0281] In any of the above steps, the selective expression cassette deletion is removed using Cre or Flp recombinase. For example, as... Figure 7 As shown, MAID 1716 corresponds to MAID 1715, which lacks the hygromycin expression cassette.

[0282] Finally, a small human TCRβ locus containing a total of 67 human TCRβVs (48 functional) and human TCRβD and J segments was generated. Mouse TCRBV31 is located at the 3' end of TCRBC2, approximately 9.4 kb (second TCRB constant region sequence), and its orientation is opposite to that of other TCRBV segments. The equivalent human V segment is TCRBV30, which is located at a similar position within the human TCRB locus.

[0283] To humanize TCRBV31, a mouse BAC clone containing mouse TCRBV31 was modified via bacterial homologous recombination to prepare LTVEC MAID 6192. Figure 8F The entire coding region was replaced with a homologous human TCRBV30 sequence, starting from the start codon of exon 1, introns, the 3' UTR, and the recombination signal sequence (RSS) of TCRBV31. The 5' UTR was preserved as a mouse sequence. A self-deletion expression cassette (lox2372-ubiquitin promoter-Hyg-PGKpolyA-protamine promoter-Cre-SV40polyA-lox2372) was inserted into the introns (exon 1 3'72bp, exon 2 5'1, 289bp) for selection. For simplicity, Figure 7 Figure 8 illustrates the selective expression cassette of hTCRBV30 3', which is designed to be located in an intron between exon 1 and exon 2 of the hTCRBV30 gene. In post-meiotic sperm cells, the protamine promoter drives Cre-specific transcriptional expression, thus the expression cassette is "self-deleted" in F1 generation mice.

[0284] The junctional nucleic acid sequences (SEQ ID NO: 26 and 27) of the MAID 6192 targeting vector are shown in Table 3. MAID6192 DNA was electrotransfected into MAID1792 ES cells. ES cell clones were selected for hygromycin resistance and further screened for deletion of the mouse TCRB31 allele and acquisition of the human TCRB30 allele.

[0285] The remaining 5' mouse TCRβV segment can be selectively deleted using a similar modification strategy.

[0286] Example 4: Generation of TCRα / TCRβ mice

[0287] In the progressive humanization steps of the TCRα and TCRβ loci, homozygous mice targeting the humanized TCRα variable locus can be mated with homozygous mice targeting the humanized TCRβ variable locus to produce offspring containing both the humanized TCRα and TCRβ variable loci. The offspring targeting the humanized TCRα and humanized TCRβ loci are then mated to obtain homozygotes.

[0288] In one implementation, homozygous mice targeting the humanized TCRα variable locus (MAID 1767; "1767 HO") containing 8 human Vα and 61 human Jα were mated with homozygous mice targeting the humanized TCRβ variable locus (MAID 1716; "1716 HO") containing 14 human Vβ, 2 human Dβ, and 14 human Jβ. Offspring were mated targeting the two humanized loci to obtain homozygotes.

[0289] Example 5: The generation of splenic T cells in homozygous mice targeting humanized TCRα and / or TCRβ loci will be derived from wild-type (WT) mice; mice lacking the mouse TCRα locus (“MAID1540”, see Figure 3 ); Homozygous mice targeting the human TCRα locus (“MAID 1767”, see Figure 3 Mice lacking the TCRβV region except for the two remaining mouse V regions (“MAID1545”, see…) Figure 7 ); for homozygous mice containing two remaining mouse V regions of the human TCRβ locus and (“MAID 1716”, see Figure 7 Spleens from homozygous mice (“MAID 1767 1716”) that also contain two remaining mouse V segments of the TCRβ locus were perfused with collagenase D (Roche Bioscience) and erythrocytes were lysed with ACK lysis buffer, followed by washing in RPMI medium.

[0290] Spleen cells from single WT, MAID 1540, 1767, 1545, 1716, and 1716-1767 representative animals were evaluated using flow cytometry. Briefly, cell suspensions were prepared using standard methods. 1 x 102 6 Cells were incubated with anti-mouse CD16 / CD32 (2.4G2, BD) on ice for 10 min and stained on ice for 30 min with a suitable antibody mixture. After staining, cells were washed and subsequently fixed in 2% formaldehyde. Data were acquired on an LSRII / CantoII / LSRFortessa flow cytometer and analyzed using FlowJo.

[0291] Spleen cells were stained using anti-mouse FITC-CD3 (17A2, BD). For example... Figure 9As shown, mice with the human TCR locus can produce a significant number of CD3+ T cells, while mice lacking the TCRα locus do not. Mice lacking the TCRβ locus also produce CD3+ T cells, possibly due to the utilization of the remaining 3' mouse V locus (see below).

[0292] Example 6: Development of thymic T cells in homozygous mice targeting humanized TCRα and / or TCRβ loci

[0293] To determine whether homozygous mice targeting humanized TCRα and / or TCRβ loci exhibit normal T cell development in the thymus, spleen cells from four age-matched animals (7-10 weeks old) at each of the WT, 1767 HO, 1716 HO, and 1716 HO 1767 HO loci were used for flow cytometry to assess T cell production at different developmental stages, as well as the frequency and absolute number of T cells at each of the DN, DP, CD4SP, and CD8SP loci.

[0294] Cell types were determined based on the presence of CD4, CD8, CD44, and CD25 cell surface markers summarized in Table 1. The relationship between cell types in the thymus and cell surface marker expression 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-), and double-negative 4 / DN4 cells (CD4-CD8-, CD25-CD44-).

[0295] Thymocytes were evaluated using flow cytometry. Briefly, cell suspensions were 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).

[0296] like Figure 10 and Figure 11As shown, homozygous mice with humanized TCRα, TCRβ, and both TCRα and TCRβ can generate DN1, DN2, DN3, DN4, DP, CD4 SP, and CD8 SP T cells, indicating that T cells generated by humanized loci can undergo T cell development in the thymus.

[0297] Example 7: Differentiation of spleen T cells in homozygous mice targeting humanized TCRα and / or TCRβ loci

[0298] To determine whether homozygous mice targeting humanized TCRα and / or TCRβ loci exhibit normal T cell differentiation in the periphery (e.g., spleen), four age-matched WT, 1767 HO, 1716 HO, and 1716 HO 1767 HO animals (7-10 weeks old) were used for flow cytometry to assess the production of different T cell types (CD3+, CD4+, CD8+, T naïve, Tcm, and Teff / em) in the spleen, as well as to assess the absolute number of each T cell type in the spleen.

[0299] Cell types are determined based on the presence of cell surface markers such as CD19 (B cell marker), CD3 (T cell marker), CD4, CD8, CD44, and CD62L (L-selectin). The relationship between cell type selection and cell surface marker expression in the spleen is as follows: T cells (CD3+), CD4 T cells (CD3+CD4+CD8-), CD8 T cells (CD3+CD4-CD8+), CD4 effector / effect 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 / effect memory T cells (CD3+CD4-CD8+CD62L-CD44+), CD8 central memory T cells (CD3+CD4-CD8+CD62L+CD44+), and CD8 naive T cells (CD3+CD4-CD8+CD62L+CD44-).

[0300] Splenic cells were evaluated using flow cytometry. Briefly, cell suspensions were 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).

[0301] like Figure 12-14 As shown, T cells underwent T cell differentiation in the spleens of homozygous mice with humanized TCRα, TCRβ, and both TCRα and TCRβ, and CD4+ and CD8+ T cells were present. Furthermore, memory T cells were detected in the spleens of the mice examined.

[0302] Example 8: Utilization of the human V region in humanized TCR mice

[0303] In homozygous mice targeting the humanized TCRβ locus (1716 HO) and homozygous mice targeting the humanized TCRβ and TCRα loci (1716 HO and 1767 HO), flow cytometry and TAQMAN assays were used, respectively. TM Real-time PCR was used to assess the expression of the human TCRβV region at both the protein and RNA levels.

[0304] For flow cytometry, spleen T cells were prepared and analyzed according to Example 5. For flow cytometry, a TCRβ library kit was used (…). Beta Mark (Beckman Coulter). The kit contains anti-human antibodies specific to various human TCRVBs, such as hTRBV-18, -19, -20, -25, -27, -28, and -29.

[0305] Results are summarized in Figure 15 . Figure 15 A (CD8 T cell overlap) and Figure 15 The table in B(CD4 T cell overlap) shows splenic T cells in 1716 HO and 1716 HO 1767 HO mice utilizing multiple human TCRβV segments. Wild-type mice were used as negative controls.

[0306] For real-time PCR, MAGMAX is used. TMThe -96 Microarray Total RNA Isolation Kit (Ambion by LifeTechnologies) purified total RNA from the spleen and thymus according to the manufacturer's instructions. MAGMAX was used. TM TURBO TM DNase buffer and TURBO DNase from the MAGMAX kit (Ambion by Life Technologies) listed above were used to remove genomic DNA. VILO TM Master Mix (Invitrogen by Life Technologies) reverse transcribes mRNA (up to 2.5 μg) into cDNA. The cDNA is diluted to 2–5 ng / μL and sequenced using an ABI 7900HT sequencing system (Applied Biosystems). Gene ExpressionMaster Mix (Applied Biosystems by Life Technologies) amplifies 10-25 ng of cDNA, using the primers and Taqman MGB probe (Applied Biosystems) or BHQ1 / BHQ-Plus probe (Biosearch Technologies) as shown in Table 4, according to the manufacturer's instructions. The relative expression of each gene is normalized to the mouse TCRβ constant 1 (TRBC1) control.

[0307] Table 4: Using real-time PCR (TAQMAN) TM Primers and probes used to detect RNA expression in the TCR βV region and homeostasis region in humanized TCR mice.

[0308]

[0309]

[0310] like Figure 16A As shown in -B, homozygous mice targeting the humanized TCRβ locus (1716 HO) and homozygous mice targeting the humanized TCRβ and TCRα loci (1716 HO and 1767 HO) both showed expression of different humanized TCRβ RNA segments in the thymus and spleen. The mice also showed expression of mouse TRBV-1 and TRBV-31 RNA segments (data not shown), but mouse TRBV-1 protein was not detected by flow cytometry (data not shown).

[0311] like Figure 8FAs shown, the mouse TRBV-31 region was replaced by the human TRBV-30 region, and the mice were generated from MAID 6192ES cells as described in this application. The utilization of the human Vβ region including TRBV-30 in the spleen and thymus of homozygous animals was detected by flow cytometry and / or real-time PCR as described in this application. The mTRBV-1 region can also be deleted.

[0312] Example 9: T cell development in homozygous mice targeting the Vα region of 23 human TCRs

[0313] In the previous examples, homozygous humanized TCRα mice were characterized, which contained 8 human Vα segments and 61 human Jα segments (1767 HO, see...). Figure 3 The ability to generate splenic CD3+ T cells and the development of T cells observed in the spleen were tested in homozygous humanized TCRα mice containing 23 human Vα segments and 61 human Jα segments (1979 HO, see [reference]). Figure 3 ).

[0314] As described in the foregoing embodiments, experimental data were obtained using flow cytometry with suitable antibodies. Figure 17 As shown, homozygous mice targeting 23 human Vα regions and 61 human Jα regions produced a significant number of splenic CD3+ T cells, and the percentage of peripheral CD3+ T cells was comparable to that of wild-type animals. Figure 19 ).

[0315] Furthermore, thymocytes in 1979 HO mice were able to undergo T cell development and contained T cells at the DN1, DN2, DN3, DN4, DP, CD4 SP, and CD8 SP stages. Figure 18 ).

[0316] Example 10: T cell development and differentiation in homozygous mice targeting the complete human TCRα and TCRβ variable region repertoire

[0317] The study investigated the ability of the mice to produce thymocytes undergoing normal T cell development, to produce T cells undergoing normal T cell differentiation in the periphery, and to utilize its complete human Vα and Vβ segment libraries to detect the "1771 HO 6192 HO" in homozygous mice (i.e., 54 human Vα and 61 human Jα) and homozygous mice (67 human Vβ, 2 human Dβ, and 14 human Jβ) targeting the complete human TCRα variable region segment library (see [link to study]). Figure 3 and Figure 7 ).

[0318] As described in Examples 5 and 6 above, flow cytometry was used to determine the presence of DN1, DN2, DN3, DN4, DP, CD4 SP, and CD8 SP T cells in the thymus. Flow cytometry was also used to determine the number of peripheral CD3+ T cells and to assess the differentiation of peripheral T cells (e.g., the presence of effector and memory T cells in the periphery). Experiments were performed using anti-mouse CD3, CD19, CD4, CD8, CD44, and CD61L antibodies as described in Examples 5 and 7 above.

[0319] Finally, flow cytometry and / or real-time PCR were used to determine whether T cells in 1771 HO 6192 HO mice utilized the complete TCRB and TCRA V segment library. For protein expression assays performed by flow cytometry, a TCRβ library kit containing an anti-human hTCRBV-specific antibody was used. (See Example 8). For RNA expression detection using real-time PCR, human TCR-V primers and Taqman probes were used to amplify spleen or thymus cDNA according to the manufacturer's instructions and as described in Example 8.

[0320] equivalent

[0321] Those skilled in the art will recognize, or be able to determine, various equivalents of specific embodiments of the invention described in this application using experiments not exceeding those of conventional methods. Such equivalents are intended to be included in the following claims.

[0322] All non-patent documents, patent applications and patents cited throughout this application are incorporated herein by reference in their entirety. sequence list <110> Rizeen Pharmaceuticals <120> T-cell receptor gene-modified mice <130> 070860-8013CN04 <140> Pending allocation <141> Submitted with letter <150> 61 / 552,582 <151> 2011-10-28 <150> 61 / 621,198 <151> 2012-04-06 <150> 61 / 700,908 <151> 2012-09-14 <160> 54 <170> FastSEQ Windows version 4.0 <210> 1 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 1 atggagtagt cagaacacac tcttcagaag ggactcctga tttcaaaggg ggtaccgggc 60 cccccctcga ggtcgacata acttcgtata gcatacatta 100 <210> 2 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 2 ggccatgcat ataacttcgt atagcataca ttatacgaag ttataccggt gcgatcgcgc 60 gcttccctct tctaaccact aattcaaaaa ggattgtaag taatgttt 108 <210> 3 <211> 145 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 3 agacagaccc ctaaacacct ccaaattaaa agcggcaaag agataaggtt ggagctccac 60 cgcggtggcg gccgccaccg cggtggagct cgaggtttcc ggtacttaac aacagagcac 120 agatttagtg gtgagggactctctc 145 <210> 4 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 4 atggagtagt cagaacacac tcttcagaag ggactcctga tttcaaaggg ggtaccgggc 60 cccccctcga ggtcgacata acttcgtata gcatacatta 100 <210> 5 <211> 109 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 5 ggccatgcat ataacttcgt atagcataca ttatacgaag ttataccggt gcgatcgctc 60 aagcatgcaa gggtaacata tgttatgaga ttatattttc tttatctca 109 <210> 6 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 6 atggagtagt cagaacacac tcttcagaag ggactcctga tttcaaaggg gggtaccggg 60 ccccccctcg agaagttcct attccgaagt tcctattctc 100 <210> 7 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 gttcctattc cgaagttcct attctctaga aagtatagga acttcctagg gcgatcgctc 60 ctctccaggc tcgaattagt attacagttg aggcacgttg tcctcccg 108 <210> 8 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 8 atggagtagt cagaacacac tcttcagaag ggactcctga tttcaaaggg ggtaccgggc 60 cccccctcga ggtcgacata acttcgtata gcatacatta 100 <210> 9 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 9 ggccatgcat ataacttcgt atagcataca ttatacgaag ttataccggt gcgatcgccg 60 cctccatttc cttcatagga aacatgaagt gaatggggct gtgtgtgt 108 <210> 10 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 10 atggagtagt cagaacacac tcttcagaag ggactcctga tttcaaaggg ggtaccgggc 60 cccccctcga ggtcgacata acttcgtata gcatacatta 100 <210> 11 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 11 ggccatgcat ataacttcgt atagcataca ttatacgaag ttataccggt gcgatcgctg 60 ggagcacgtt ccattattat aacaactttc tgaacacaag agggcagt 108 <210> 12 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 12 atggagtagt cagaacacac tcttcagaag ggactcctga tttcaaaggg ggtaccgggc 60 cccccctcga ggtcgacata acttcgtata gcatacatta 100 <210> 13 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 13 ggccatgcat ataacttcgt atagcataca ttatacgaag ttataccggt gcgatcgctt 60 [[ID=5,4]]taaggtgagg aggcaggcaa taccccctct ccaccgcatt ctcaatcc 108 <210> 14 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 14 gggggggtgg ggtggaggag gagggtacag catctcctct ccttcctctc tggtaccgaa 60 gttcctattc cgaagttcct attctctaga aagtatagga 100 <210> 15 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 15 gaagttccta ttctctagaa agtataggaa cttcctaggg tttcaccggt gcgatcgcgt 60 gaatatacta aaaaccactt aattatatat ttgaaagggt ggatgtta 108 <210> 16 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 16 ctctctccta cccagctcct ctcacacgag cctgaaggcc ctgccaaggt ggcgcgcctt 60 tcaaattgtt gttgagttca aagtgggcaa cagaaaaggg ggtgtgag 1o8 <210> 17 <211> 130 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 17 aataaatagt aaatttctgt agaatcataa tgaggtctag acccccgggc tcgataacta 60 taacggtcct aaggtagcga aatggcgcgt aatcaagccc agctcttcat gctgcatttt 120 tatcttcttt 130 <210> 18 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 18 ttgactcggg ggtgcctggg tttgactgca atgatcagtt gctgggaagg accggtataa 60 cttcgtataa tgtatgctat acgaagttat atgcatggcc <210> 19 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 19 ccggcgcgcc ataacttcgt ataatgtatg ctatacgaag ttatgtcgac ataaggtaag 60 acagagtcgt cccttcccat ctggaaccct ctacctttct 100 <210> 20 <211> <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 20 gttgatgaat cataaaagaa gagatattca agaaaaggat ggccacactg cggccgcaga 60 ggtattcaag gaaaatgcag actcttcacg taagagggat gaggggc 107 <210> 21 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 21 tccccggagt cggagggtgg accggagctg gaggagctgc cgcggtggcg gccgatgcca 60 tttcattacc tctttctccg cacccgacat agataaagct 100 <210> 22<000099​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <213> Artificial sequence <220> <223> Synthetic <400> twenty four gggggggtgg ggtggaggag gagggtacag catctcctct ccttcctctc tggtaccgaa 60 gttcctattc cgaagttcct attctctaga aagtatagga 100 <210> 25 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 25 gaagttccta ttctctagaa agtataggaa cttcctaggg tttcaccggt gcgatcgcgt 60 tatctagtag acttaattaa ggatcgatcc ggcgcgccaa tagtcatg 108 <210> 26 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 26 gttttccaga cttcaacttg actatcagcc agaaattcag tggcaaaccc ccacccagtc 60 cctaagtgaa ggcccctggg gagtatggtt agggctcagg 100 <210> 27 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 27 cacccacccaa agaaagtgcc caggagaagg gcaaggagag agcagagcat agttcaagat 60 ggtctttgtc taggcttgtc tactctgcac ttgtacttcc 100 <210> 28 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 28 ccggcgtcat gcagaa 16 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 29 gggctgcatc tcagtcttgc 20 <210> 30 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 30 cacctggtca ggaggagg 18 <210> 31 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 31 ggaatcactc agtccccaaa g 21 <210> 32 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 32 attctgttca caactcaggg tca 23 <210> 33 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 33 tcagaaagga aggacagaat 20 <210> 34 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 34 cgagcaaggc gtcgagaa 18 <210> 35 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 35 ggacaaggtc aggcttgca 19 <210> 36 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 36 acaagtttct catcaacc 18 <210> 37 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 37 tgttacccag accccaagga 20 <210> 38 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 38 tctgagaaca ttccagcata atcct 25 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 39 taggatcaca aagacaggaa 20 <210> 40 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 40 tcccctgacc ctggagtct 19 <210> 41 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 41 tgctggcaca gaggtactga ga 22 <210> 42 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 42 caggccctca catac 15 <210> 43 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 43 aagcccaagt gacccagaa 19 <210> 44 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 44 attctgagaa caagtcactg ttaacttc 28 <210> 45 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 45 ctcatcacag tgactggaa 19 <210> 46 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 46 gtgaaagtaa cccagagctc gag 23 <210> 47 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 47 atcctggaca cattccagaa aaac 24 <210> 48 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 48 atatctagtc aaaaggacgg ga 22 <210> 49 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 49 tgtcattgac aagtttccca tcag 24 <210> 50 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 50 tgctgtcttc agggctcatg 20 <210> 51 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 51 tcaactctga ctgtgagca 19 <210> 52 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 52 agccgcctga gggtctct 18 <210> 53 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 53 gccacttgtc ctcctctgaa ag 22 <210> 54 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 54 taccttctgg cacaatcctc gca 23

Claims

1. A method for generating a human T-cell receptor (TCR) and / or A method for sequencing nucleic acid sequences with variable domains, the method comprising: Immunizing genetically modified mice with the target antigen The mice were made to develop an immune response to the target antigen, and From therein, human TCRs encoding a TCR that binds to the target antigen are obtained. Variable structural domains and / or human TCR Nucleic acid sequences with variable structural domains The genetically modified mice described above contain the following in their genome: Unrearranged TCR α variable gene loci, comprising replacing an endogenous TCR Vα segment with at least one functionally unrearranged human TCR Vα segment and replacing an endogenous TCR Jα segment with at least one functionally unrearranged human TCR Jα segment. in, The at least one functionally unrearranged human TCR Vα segment and the at least one functionally unrearranged human TCR Jα segment are operatively linked to the mouse TCR α constant gene sequence, and / or Unrearranged TCR β variable gene loci, comprising replacing an endogenous TCR Vβ region with at least one functionally unrearranged human TCR Vβ region, replacing an endogenous TCR Dβ region with at least one functionally unrearranged human TCR Dβ region, and replacing an endogenous TCR Jβ region with at least one functionally unrearranged human TCR Jβ region. Wherein, the at least one functionally unrearranged human TCR Vβ segment, the at least one functionally unrearranged human TCR Dβ segment, and the at least one functionally unrearranged human TCR Jβ segment are operatively linked to the mouse TCR β constant gene sequence. in (i) the at least one functional unrearranged human TCR V The segment and the at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, which encodes a TCR that specifically binds to the target antigen. Variable structural domains, and / or (ii) the at least one functional unrearranged human TCR V The segment, the at least one functionally unrearranged human TCR D The segment and the at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, which encodes a TCR that specifically binds to the target antigen. Variable structural domain.

2. A method for generating encoded human-derived TCRs A method for sequencing the nucleic acid sequence of a polypeptide, the method comprising: Immunizing genetically modified mice with the target antigen The mice were made to develop an immune response to the target antigen. From therein, human TCRs encoding a TCR that binds to the target antigen are obtained. Nucleic acid sequences with variable structural domains, and Human TCR encoding a TCR that binds to the target antigen Nucleic acid sequences containing variable domains were cloned into human TCRs. Nucleic acid constructs in the constant region, thereby enabling the human TCR Variable structural domain and the human TCR The constant region is operably connected. The genetically modified mice described herein contain an unrearranged TCR α variable gene locus in their genome, which includes The endogenous TCR Vα segment was replaced with at least one functional, unrearranged human TCR Vα segment, and the endogenous TCR Jα segment was replaced with at least one functional, unrearranged human TCR Jα segment. The at least one functionally unrearranged human TCR Vα region and the at least one functionally unrearranged human TCR Jα region are operatively linked to the mouse TCR α constant gene sequence, and in, The at least one functionally unrearranged human TCR V Segment and at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, which encodes a TCR that specifically binds to the target antigen. Variable structural domain.

3. A method for generating a nucleic acid sequence encoding a human TCR β polypeptide, the method comprising: Immunizing genetically modified mice with the target antigen The mice were made to develop an immune response to the target antigen. From therein, a nucleic acid sequence encoding the human TCR β-variable domain that binds to the target antigen is obtained, and The nucleic acid sequence encoding the human TCR β-variable domain that binds to the target antigen was cloned into a TCR containing the human TCR. A nucleic acid construct with a constant region, thereby enabling the human TCR β-variable domain to interact with the human TCR. The constant region is operably connected. The genetically modified mice contain unrearranged TCRs in their genome. Variable gene loci, comprising replacing an endogenous TCR Vβ segment with at least one functionally unrearranged human TCR V The segment replaces the endogenous TCRDβ segment with at least one functional, unrearranged human TCRD. The segment and replacing the endogenous TCR Jβ segment with at least one functional, unrearranged human TCR J Section, The at least one functionally unrearranged human TCR Vβ segment, the at least one functionally unrearranged human TCR Dβ segment, and the at least one functionally unrearranged human TCR Jβ segment are related to the mouse TCR Constant gene sequences can be operatively linked, and in, The at least one functionally unrearranged human TCR V The segment, the at least one functionally unrearranged human TCR D The segment and the at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, which encodes a TCR that specifically binds to the target antigen. Variable structural domain.

4. A method for generating human TCR Methods involving polypeptides, the methods comprising Immunizing genetically modified mice with the target antigen The mice were made to develop an immune response to the target antigen. From therein, human TCRs encoding a TCR that binds to the target antigen are obtained. Nucleic acid sequences with variable structural domains Human TCR encoding a TCR that binds to the target antigen Nucleic acid sequences containing variable domains were cloned into human TCRs. In the nucleotide construct of the constant region, thereby enabling the human TCR Variable structural domain and the human TCR The constant region is operatively connected, and Human TCR expressed from the nucleotide construct polypeptide, The genetically modified mice contain unrearranged TCRs in their genome. Variable gene loci, comprising replacing an endogenous TCR Vα segment with at least one functionally unrearranged human TCR V. The segment and replacing the endogenous TCR Jα segment with at least one functional, unrearranged human TCR J Section, The at least one functionally unrearranged human TCR Vα segment and the at least one functionally unrearranged human TCR Jα segment are related to the mouse TCR Constant gene sequences can be operatively linked, and in, The at least one functionally unrearranged human TCR V The segment and the at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, which encodes a TCR that specifically binds to the target antigen. Variable structural domain.

5. A method for generating a human TCRβ polypeptide, the method comprising: Immunizing genetically modified mice with the target antigen The mice were made to develop an immune response to the target antigen. From this, a nucleic acid sequence encoding the human TCR β-variable domain that binds to the target antigen is obtained. The nucleic acid sequence encoding the human TCR β variable domain that binds to the target antigen is cloned into a nucleotide construct containing the human TCR β constant region, thereby operatively linking the human TCR β variable domain to the human TCR β constant region; and Human TCR β peptide was expressed from the nucleotide construct. The genetically modified mouse contains an unrearranged TCR β variable gene locus in its genome, comprising replacing the endogenous TCR Vβ region with at least one functionally unrearranged human TCR Vβ region, replacing the endogenous TCRDβ region with at least one functionally unrearranged human TCR Dβ region, and replacing the endogenous TCR Jβ region with at least one human TCR Jβ region. in, The at least one functionally unrearranged human TCR Vβ segment, the at least one functionally unrearranged human TCR Dβ segment, and the at least one functionally unrearranged human TCR Jβ segment are operatively linked to the mouse TCR β constant gene sequence. The at least one functionally unrearranged human TCR Vβ segment, the at least one functionally unrearranged human TCR Dβ segment, and the at least one functionally unrearranged human TCR Jβ segment can be rearranged to form a nucleic acid sequence, wherein the nucleic acid sequence encodes the TCR β variable domain of a TCR that specifically binds to the target antigen.

6. A method for preparing a therapeutic agent for human use, the method comprising: Immunizing genetically modified mice with the target antigen Inducing an immune response in the mice, T cells that respond to the target antigen were obtained from the mice. Obtain from the T cells a T cell receptor that binds to the target antigen and / or a nucleic acid sequence encoding the T cell receptor, wherein the T cell receptor includes a human TCR variable region, and The human TCR variable region is used in human therapeutic agents. The genetically modified mice described above contain the following in their genome: Unrearranged TCR α variable gene loci, comprising replacing an endogenous TCR Vα segment with at least one functionally unrearranged human TCR Vα segment and replacing an endogenous TCR Jα segment with at least one functionally unrearranged human TCR Jα segment. The at least one functionally unrearranged human TCR Vα segment and the at least one functionally unrearranged human TCR Jα segment are operatively linked to the mouse TCRα constant gene sequence, and / or Unrearranged TCR β variable gene loci, comprising replacing an endogenous TCR Vβ region with at least one functionally unrearranged human TCR Vβ region, replacing an endogenous TCR Jβ region with at least one functionally unrearranged human TCR Dβ region, and replacing an endogenous TCR Jβ region with at least one functionally unrearranged human TCR Jβ region. in, The at least one functionally unrearranged human TCR Vβ segment, the at least one functionally unrearranged human TCR Dβ segment, and the at least one functionally unrearranged human TCR Jβ segment are operatively linked to the mouse TCR β constant gene sequence. in (i) the at least one functional unrearranged human TCR V The segment and the at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, the nucleic acid sequence encoding a TCR that specifically binds to the target antigen. Variable structural domains, and / or (ii) the at least one functional unrearranged human TCR V The segment, the at least one functionally unrearranged human TCR D The segment and the at least one functionally unrearranged human TCR J The segments can be rearranged to form a nucleic acid sequence, the nucleic acid sequence encoding a TCR that specifically binds to the target antigen. Variable structural domain.

7. The method of claim 6, wherein the human therapeutic agent is: (i) Soluble T-cell receptors, (ii) Single-chain TCR, (iii) scTv or three-domain single-chain T-cell receptor, or Any combination of (iv)(i)-(iii).

8. The method of claim 7, wherein the soluble T-cell receptor (i) It fuses with parts that can kill infected or cancerous cells. (ii) fusion with immunomodulatory molecules, and / or (iii) Fusion with immunosuppressive molecules.

9. The method of claim 8, wherein the portion capable of killing infected cells or cancer cells is a cytotoxic molecule, a toxin, a radionuclide, a prodrug, an antibody, or any combination thereof.

10. The method according to any one of claims 1-9, wherein the mouse further comprises an unrearranged human TCR Variable area segment library.

11. The method of claim 10, wherein the mouse further comprises an intact human TCR V. Segment library, complete human TCR D Segment library and complete human TCR J Section warehouse.

12. The method according to any one of claims 1-9, wherein the mouse comprises an endogenous TCR Vβ segment.

13. The method according to any one of claims 1-9, wherein the mouse expression (A) TCR α variable structural domain, derived from: Human TRAV40 gene fragment, Human TRAV41 gene fragment, Human TRAV39 gene fragment, Human TRAV35 gene fragment, Human TRAV34 gene fragment, Human TRAV22 gene fragment, Human TRAV21 gene fragment, Human TRAV13-2 gene fragment, Human TRAV8.5 gene fragment, Human TRAV6 gene fragment, Human TRAV5 gene fragment, or Human TRAV1-1 gene fragment; (B) TCRβ variable structural domain, derived from Human TRBV-18 gene fragment, Human TRBV-19 gene fragment, Human TRBV-20 gene fragment, Human TRBV-24 gene fragment, Human TRBV-25 gene fragment, Human TRBV-27 gene fragment, Human TRBV-28 gene fragment, or Human TRBV-29 gene fragment, or (C) A combination of (A) and (B).

14. The method according to any one of claims 1-9, wherein the mouse comprises a spleen cell population, at least 20% of which is CD3. + .

15. The method according to any one of claims 1-9, wherein the mouse comprises (A) At least one of the following at the endogenous TCRα locus: (i) Recombinant AsiSI restriction site, (ii) Recombinant AscI restriction enzyme site, (iii) The nucleic acid sequences shown 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. (B) At least one of the following at the endogenous TCRβ locus: (i) Recombinant AsiSI restriction site, (ii) Recombinant AscI restriction site, and (iii) The nucleic acid sequences shown 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, or (C) A combination of (A) and (B).

16. A method for producing in vitro host cells, comprising: The method according to any one of claims 1-3 yields: (i) A nucleic acid sequence encoding the human TCR α-variable domain that specifically binds to the target antigen, and / or (ii) The nucleic acid sequence encoding the human TCR β-variable domain that specifically binds to the target antigen, and Inserting the nucleic acid sequence described in (i) and / or (ii) into the host cell, and The host cells are maintained in vitro.

17. The method of claim 16, wherein (i) The nucleic acid sequence encoding the human TCR α variable domain that specifically binds to the target antigen is cloned into a nucleic acid construct containing the human TCR α constant region, such that the human TCR α variable domain is operatively linked to the human TCR α constant region, and / or, (ii) The nucleic acid sequence encoding the human TCR β variable domain that specifically binds to the target antigen is cloned into a nucleic acid construct containing the human TCR β constant region, such that the human TCR β variable domain is operatively linked to the human TCR β constant domain.

18. The method of claim 17, wherein the host cell expresses a human therapeutic agent, the human therapeutic agent being: (i) Soluble T-cell receptors, (ii) Single-chain TCR, (iii) scTv or three-domain single-chain T-cell receptor, or Any combination of (iv)(i)-(iii).

19. The method of claim 16, wherein the host cell is selected from CHO cells, COS cells, 293 cells, HeLa cells, and PERC6. TM cell.

20. A method for obtaining a nucleic acid sequence encoding a variable domain of a human TCR, comprising: Determine the nucleic acid sequence of a rearranged human TCR Vα / Jα sequence and / or a rearranged human TCR Vβ / Dβ / Jβ sequence, said nucleic acid sequence being expressed by isolated mouse T cells, hybridomas derived from said isolated mouse T cells, or tetravalent tumors derived from said isolated mouse T cells. The isolated mouse T cells, the hybridoma, or the tetravalent tumor include: A rearranged human TCR Vα / Jα sequence located at the endogenous TCRα locus and operatively linked to the endogenous TCR Cα gene sequence, and / or A rearranged human TCR Vβ / Dβ / Jβ sequence located at the endogenous TCRβ locus and operatively linked to the endogenous TCR Cβ gene sequence. The isolated mouse T cells, the hybridoma, or the tetravalent tumor express a T cell receptor on their cell surface, the T cell receptor comprising: A functional chimeric TCRα polypeptide encoded by a rearranged human TCR Vα / Jα sequence operatively linked to an endogenous TCR Cα gene sequence; wherein the functional chimeric TCRα polypeptide comprises a human TCR α variable domain operatively linked to an endogenous TCR Cα domain; and / or A functional chimeric TCRβ polypeptide encoded by a rearranged human TCR Vβ / Dβ / Jβ sequence operatively linked to an endogenous TCR Cβ gene sequence; wherein the functional chimeric TCRβ polypeptide comprises a human TCRβ variable domain operatively linked to an endogenous TCR Cβ domain.

21. A nucleic acid obtained by the method of claim 20, comprising a mouse TCR Cα gene sequence operably linked to a rearranged human TCR Vα / Jα sequence, and / or a mouse TCR Cβ gene sequence operably linked to a rearranged human TCR Vβ / Dβ / Jβ sequence.

22. A method for preparing a genetically modified mouse, comprising modifying the mouse genome to include: (A) In endogenous T cell receptor (TCR) The locus contains the following substitutions: (I) Using unrearranged human TCR variable (V) Gene segment substitution for endogenous TCR V Gene segments, and (II) Ligation with unrearranged human TCR (J) Gene segment substitution of endogenous TCR J Gene segments, The unrearranged human TCR V Gene segments and the aforementioned unrearranged human TCR J Gene segments: (i) Relative to each other and constant with endogenous TCR (C) Gene sequences can be operatively linked, and (ii) Rearrangement within T cells to make the endogenous TCR The locus encodes a functional chimeric TCR The polypeptide, the functional chimeric TCR Peptides contain endogenous TCR C The structural domain can be operatively connected to human or humanized TCRs. Variable structural domain; (B) In endogenous TCR The locus contains the following substitutions: (I) Using unrearranged human TCR variable (V) Gene segment substitution for endogenous TCR V Gene segments, (II) Using unrearranged human TCR diversity (D) Gene segment substitution of endogenous TCR D Gene segments, and (III) Ligation with unrearranged human TCR (J) Gene segment substitution of endogenous TCR J Gene segments, The unrearranged human TCR V Gene segment, the aforementioned unrearranged human TCR D Gene segments and the aforementioned unrearranged human TCR J Gene segments: (i) Relative to each other and constant with endogenous TCR (C) Gene sequences can be operatively linked, and (ii) Rearrangement within T cells to make the endogenous TCR The locus encodes a functional chimeric TCR The polypeptide, the functional chimeric TCR Peptides contain endogenous TCR C The structural domain can be operatively connected to human or humanized TCRs. Variable structural domain, or (C) Both (A) and (B) The genetically modified mouse strain has at least 20% of its spleen cell population expressing CD3.

Images