NZ623147B2 - Genetically modified t cell receptor mice - Google Patents

Abstract

Disclosed is a genetically modified non-human animal, comprising in its genome: -an unrearranged T cell receptor (TCR) ? variable gene locus comprising at least one human V? segment and at least one human J? segment, wherein the TCR? variable gene locus is operably linked to a non-human TCR? constant gene sequence, wherein the unrearranged TCR? variable gene locus replaces an endogenous non-human TCR? variable gene locus, and/or -an unrearranged TCR? variable gene locus comprising at least one human V? segment, at least one human D? segment, and at least one human J? segment, wherein the unrearranged TCR? variable gene locus is operably linked to a non-human TCR? constant gene sequence, wherein the unrearranged TCR? variable gene locus replaces an endogenous non-human TCR? variable gene locus. nt gene sequence, wherein the unrearranged TCR? variable gene locus replaces an endogenous non-human TCR? variable gene locus, and/or -an unrearranged TCR? variable gene locus comprising at least one human V? segment, at least one human D? segment, and at least one human J? segment, wherein the unrearranged TCR? variable gene locus is operably linked to a non-human TCR? constant gene sequence, wherein the unrearranged TCR? variable gene locus replaces an endogenous non-human TCR? variable gene locus.

Description

CALLY MODIFIED T CELL RECEPTOR MICE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to US. Provisional Application No. 61/552,582, filed October 28, 2011; US. Provisional Application No. 61/621,198, filed April 6, 2012; and US. Provisional Application No. 61/700,908, filed September 14, 2012, all of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION Present invention relates to a genetically ed man animal, e.g., a rodent (e.g., a mouse or a rat), that comprises in its genome human or humanized T Cell Receptor (TCR) variable gene loci (e.g., TCR): and TCRfi le gene loci and/or TCR) and TCRy variable gene loci), and expresses human or humanized TCR polypeptides (e.g., TCRoc and TCRS polypeptides and/or TCR?) and TCRy polypeptides) from the human or humanized TCR variable gene loci. A non-human animal with human or humanized TCR variable gene loci of the invention comprises unrearranged human TCR variable region gene segments (e.g., V, D, and/or J segments) at endogenous non-human TCR gene loci. The invention also relates to embryos, tissues, and cells (e.g., T cells) that comprise human or zed TCR variable gene loci and express human or humanized TCR polypeptides.

Also provided are methods for making the genetically modified non—human animal comprising human or humanized TCR le gene loci; and methods of using man animals, embryos, tissues, and cells that se human or humanized TCR variable gene loci and express human or humanized TCR polypeptides from those loci.

BACKGROUND OF THE INVENTION in the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunogiobulins) and T lymphocytes (e.g., T cell receptors or TCRs). While pathogens in the blood and extracellular space are recognized by antibodies in the course of humoral immune response, destruction of pathogens inside cells is mediated in the course of cellular immune response by T cells.

T cells ize and attack antigens presented to them in the context of a Major Histocompatibility Complex (MHC) on the cell surface. The antigen ition is mediated by TCRs sed on the surface of the T cells. Two main ciasses of T ceiis serve this function: cytotoxic T cells, which express a cell-surface protein CD8, and helper T cells, which express a ceii—surface protein CD4. Cytotoxic T celis activate signaling cascades that result in direct destruction of the cell presenting the n (in the context of MHC i), while helper T cells differentiate into several classes, and their activation (primed by recognition of antigen presented in the context of MHC ii) resuits in macrophage-mediated pathogen destruction and stimulation of antibody production by B cells.

Because of their antigen specificity, antibodies are presently widely studied for their therapeutic potential against numerous human disorders. To te antibodies capable of neutralizing human targets, whiie simultaneously avoiding activation of immune responses against such antibodies, scientists have concentrated their s on producing human or humanized immunogiobulins. One way of producing humanized antibodies in vivo is by using VELOCIMMUNE® mouse, a humanized mouse comprising (1) ranged human immunogiobuiin V, D, and J segment oire operably linked to each other and a mouse constant region at the endogenous mouse immunogiobuiin heavy chain locus and (2) ranged human VK and JK segment repertoire operably linked to each other and a mouse constant K region at the endogenous mouse giobulin K light chain locus. As such, VELOCIMMUNE® mice provide a rich source of highly diverse rearranged antibody variable domains for use in engineering human antibodies.

Similar to an antibody, a T cell receptor comprises a variable region, d by unrearranged loci (a and [5 loci, or a and y loci) sing V(D)J variable region segments, and this variable region confers upon the T cell its antigen binding specificity. Also r to an antibody, the TCR specificity for its antigen can be utilized for development of novel therapeutics. Thus, there is a need in the art for man animals (e.g., rodents, e.g., rats or mice) that comprise unrearranged human T cell variable region gene segments capable of rearranging to form genes that encode human T cell receptor variable domains, ing domains that are cognate with one another, and including domains that specifically bind an antigen of interest. There is also a need for non-human animals that comprise T cell le region loci that comprise conservative humanizations, including non-human animals that comprise ranged human gene segments that can nge to form T cell receptor variable region genes that are linked to non-human (endogenous) T cell receptor constant gene sequences. There remains a need for non-human animals that are capable of generating a diverse repertoire of human T cell receptor variable sequences. There is a need for non-human animals that are capable of rearranging most or all functional T cell receptor variable region segments, in response to an antigen of interest, to form T cell receptor polypeptides that comprise fully human variable domains.

SUMMARY OF THE ION Non-human animals, e.g., rodents, comprising non-human cells that express humanized les that function in the cellular immune response are provided.

Nonhuman animals that comprise unrearranged TCR variable gene loci are also provided. In vivo and in vitro systems are provided that comprise humanized rodent cells, wherein the rodent cells express one or more humanized immune system molecules. ranged humanized TCR rodent loci that encode humanized TCR proteins are also provided.

In one aspect, provided herein is a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or a rat) that comprises in its genome (a) an unrearranged TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human (e.g., a rodent, e.g., a mouse or a 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 a non-human (e.g., a rodent, e.g., a mouse or a rat) TCRβ constant gene sequence. [0008a] In another aspect, provided herein is a cally modified non-human animal, comprising in its genome: an unrearranged T cell receptor (TCR)  variable gene locus comprising at least one human V t and at least one human J segment, wherein the TCR variable gene locus is operably linked to a man TCR constant gene sequence, wherein the unrearranged TCR le gene locus replaces an endogenous man TCR variable gene locus, and/or an unrearranged TCR variable gene locus comprising at least one human V segment, at least one human D segment, and at least one human J segment, wherein the unrearranged TCR variable gene locus is operably linked to a non-human TCR constant gene sequence, wherein the unrearranged TCR variable gene locus replaces an endogenous non-human TCR variable gene locus.

In one embodiment, the unrearranged TCRα variable gene locus replaces endogenous non-human (e.g., rodent) TCRα variable gene locus at an endogenous TCRα variable gene locus. In one ment, the unrearranged TCRβ le gene locus replaces the endogenous non-human (e.g., rodent) TCRβ variable gene locus at an endogenous TCRβ variable gene locus. In one embodiment, the nous non-human (e.g., rodent) Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence. In one embodiment, the endogenous non-human (e.g., rodent) Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Jβ ce. In one embodiment, the non-human animal comprises a deletion such that the genome of the animal does not comprise a functional Vα and functional Jα segment. In one embodiment, the non—human animal comprises a deletion such that the genome of the animal does not comprise a functional endogenous VB, a functional endogenous DB, and a functional endogenous Jfi t. in one embodiment, the animal comprises a deletion of all functional endogenous Va and Ja segments. in one embodiment, the rodent comprises a deletion of all functional endogenous VB, D6, and J0 segments. in some embodiments, the human V0: and Joe segments rearrange to form a nged VOL/Jet sequence. in some ments, the human VB, DB, and J13 segments rearrange to form a nged VB/DB/Jfi sequence. Thus, in various embodiments, the non—human animal (e.g., rodent) ses a T cell receptor comprising a human le region and a non—human (e.g., rodent) constant region on a surface of a T cell. ln some aspects, 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 comprises a normai ratio of splenic CD3+ T cells to total splenocytes. In various embodiments, the non—human animal generates a population of central and effector memory T cells in the periphery.

In one embodiment, the unrearranged TCRa le gene locus in the non- human animal described herein ses 61 human Ja segments and 8 human Von segments. in another embodiment, the unrearranged TCRoc variable gene locus in the non— human animal comprises a complete repertoire of human Jo: segments and a complete repertoire of human Va segments.

In one embodiment, the unrearranged TCRB variable gene locus in the non- human animal described herein comprises 14 human JB segments, 2 human DB segments, and 14 human st segments. in another embodiment, the unrearranged TCRfi le gene locus in the non-human animal ses a complete repertoire of human JB segments, a complete repertoire of human DB ts, and a complete repertoire of human Vfi segments. in an additional embodiment, the non-human animal described herein (e.g., a rodent) further comprises nucleotide sequences of human TCRo variable segments at a humanized TCRoc locus. in one embodiment, the non-human animal (e.g., rodent) r comprises at least one human V5, D6, and J6 segments, e.g., a complete repertoire of human V6, D5, and J6 segments at the humanized TCRor locus. in one embodiment, the non-human animal retains an endogenous non-human TCRor and/or TCRfi locus, wherein the locus is a non—functional locus. ln one embodiment, the non—human animal is a rodent. In one embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the rodent is a mouse.

In one aspect, the invention provides a genetically modified mouse comprising in its genome (a) an unrearranged TCRa variable gene locus comprising a repertoire of human Joc segments and a repertoire of human Va segments, operably linked to a non—human (e.g., mouse or rat) TCRoc constant gene ce, and/or (b) an ranged TCRfi variable gene locus comprising a repertoire of human JB segments, a repertoire of human DB segments, and a repertoire of human V13 segments, operably linked to a non-human (e.g., a mouse or rat) TCRB constant gene sequence. In one ment, the mouse comprises a complete repertoire of human Va segments. In one embodiment, the mouse comprises a complete repertoire of human Vfi segments. ln one ment, the mouse comprises a te repertoire of human Va segments and human Jon segments. In one embodiment, the mouse comprises a complete repertoire of human Voc segments and human VB segments. In one embodiment, the mouse comprises a complete repertoire of human Voc, human Jon, human V6, human DB, and human JB segments.

In one embodiment, the mouse comprises at least one endogenous mouse Va and at least one endogenous mouse Jo: segment, wherein the endogenous segments are incapable of nging to form a rearranged t sequence, and also comprises at least one endogenous mouse Vfi, at least one endogenous mouse Dfs, and at least one nous mouse J13 segment, wherein the endogenous segments are incapable of rearranging to form a rearranged VB/DB/Jfi sequence.

In one embodiment, the unrearranged TCRa variable gene locus that comprises human TCRa variable region segments replaces mouse TCRoc variable genes at the endogenous mouse TCRoc variable locus, and the unrearranged TCRB variable gene locus that ses human TCRB variable region ts replaces mouse TCRfi variable genes at the endogenous mouse TCRB variable locus.

In one embodiment, the human Va and Ja ts rearrange to form a rearranged human Von/Jon sequence, and the human VB, D0, and JB ts rearrange to form a rearranged human Vp/DB/Jfi sequence. in one embodiment, the rearranged human Von/Jo: sequence is operably linked to a mouse TCRoc constant region sequence. in one embodiment, the rearranged human Vfi/DB/JB sequence is operably linked to a mouse TCR[3 constant region ce. Thus, in various embodiments, the mouse expresses a T cell receptor on the surface of a T cell, n the T cell or comprises a human variable region and a mouse constant region.

In one embodiment, the mouse further comprises a repertoire of human TCRé variable region segments (e.g., human V6, J6, and D6 segments) at a humanized TCRa locus. In one embodiment, the repertoire of human TCR?) variable region segments is a complete human TCRé variable region segment repertoire. In one embodiment, the human TCR6 variable region segments are at the nous TCRoc locus. In one embodiment, the human TCRo variable region segments replace endogenous mouse TCRé variable region segments.

In one embodiment, the genetically modified mouse ses a T cell receptor comprising a human variable region and a mouse constant region on a surface of a T cell.

In one aspect, the T cells of the mouse undergo thymic T cell development to produce CD4 and CD8 single ve T cells. In one aspect, the mouse comprises a normal ratio of splenic CD3+ T cells to total splenocytes; in one aspect, the mouse generates a population of central and effector memory T cells to an antigen of interest.

Also provided are methods for making genetically modified non-human animals (e.g., rodents, e.g., mice or rats) described herein.

In one aspect, a method for making a humanized rodent (e.g., a mouse or rat) is provided, comprising replacing rodent TCRor and TCRiS le region segments, but not rodent constant genes, with human unrearranged TCRor and TCRB variable region segments, at endogenous rodent TCR loci. In one embodiment, the method comprises replacing rodent TCRa variable region ts (Va and/or Jor) with human TCRoc variable region segments (Von and/or Jor), wherein the TCRor variable region segments are operably linked to a non—human TCR constant region gene to form a zed TCRoc locus; and replacing rodent TCR|3 variable region ts (VB and/or DB and/or JB) with human TCRfi variable region segments (VB and/or D6 and/or J6), wherein the TCRB variable region segments are operably linked to a non-human TCR constant region gene to form a humanized TCRiS locus. In one embodiment, the humanized rodent is a mouse and the germline of the mouse comprises the human TCRoc variable region segments operably linked to an endogenous mouse TCRoc constant ce at an endogenous TCRor locus; and the germline of the mouse ses the human TCRB le region segments operably linked to an endogenous mouse TCRB constant sequence at an endogenous TCRB locus. in one embodiment, provided herein is a method for making a genetically modified non—human animal (e.g., rodent, e.g., mouse or rat) that expresses a T cell receptor comprising a human or humanized le region and a non-human (e.g., rodent) constant region on a surface of a T cell comprising: replacing in a first non-human animal an endogenous non-human TCRoc variable gene locus with an unrearranged humanized TCRa variable gene locus sing at least one human Va segment and at least one human Ja segment, wherein the zed TCRoc le gene locus is operably linked to endogenous non-human TCRoc constant region; replacing in a second man animal an endogenous non-human TCRB variable gene locus with an unrearranged humanized TCRfi variable gene locus comprising at least one human V6 segment, at least one human DE» t, and at least one human JB t, wherein the humanized TCRfi variable gene locus is operably linked to endogenous TCRB constant ; and breeding the first and the second man animal to obtain a non-human animal that expresses a T cell receptor comprising a human or humanized variable region and a non—human constant . in one embodiment of the method, the endogenous non—human (e.g., rodent) V0: and Jon segments are incapable of rearranging to form a rearranged Von/Joe sequence and the endogenous non-human (e.g., rodent) V6, DB, and J6 segments are incapable of rearranging to form a rearranged Vfi/Dfi/JB sequence. in one embodiment of the method, the human Va and Joe segments rearrange to form a rearranged Va/Ja sequence and the human W5, DB, and J6 segments rearrange to form a rearranged Vp/Dfi/Jfi ce. In one embodiment of the method, the unrearranged zed TCRoc variable gene locus comprises 61 human Jor segments and 8 human Va segments, and the unrearranged humanized TCRfi variable gene locus comprises 14 human VB segments, 2 human DB segments, and 14 human Jfi segments. in another embodiment of the method, the ranged humanized TCRa variable gene locus comprises a complete repertoire of human Ja segments and a complete oire of human Va ts, and the unrearranged humanized TCRB le gene locus comprises a complete repertoire of human V5 segments, a complete repertoire of human DB segments, and a complete repertoire of human Jfi segments. 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 one aspect, the non—human animal (e.g., the rodent) comprises a normal ratio of splenic CD3+ T cells to total splenocytes. In one aspect, the non—human animal (e.g., rodent) generates a population of central and effector memory T cells to an antigen of interest.

In some embodiments, the replacement of the endogenous non-human TCRoc variable gene locus described herein is made in a single ES cell, and the single ES cell is introduced into a non-human (e.g., a rodent, e.g., a mouse or rat) embryo to make a genetically modified non—human animal (i.e., the first non-human , e.g., the first rodent); and the replacement of the endogenous non-human TCRfi variable gene locus described herein is made in a single ES cell, and the single ES cell is introduced into a non- human (e.g., a rodent, e.g., a mouse or rat) embryo to make a genetically modified non- human animal (i.e., the second non-human animal, e.g., the second rodent). in one embodiment, the first rodent and the second rodent are bred to form a progeny, wherein the progeny comprises in its germline a humanized TCRa variable locus and a humanized TCRfi variable locus. in one embodiment of the method, the non-human animal is a rodent, e.g., a mouse. Thus, the t invention also provides a method for making a genetically modified mouse.

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

In one aspect, a method for making a human TCR variable domain is provided, comprising cally modifying a rodent as described herein to comprise a humanized TCRa locus and/or a humanized TCRB locus, maintaining the rodent under conditions sufficient to form a T cell, wherein the T cell expresses a human TCRa and/or a human TCRB variable domain. in one aspect, a method for making a nucleic acid sequence encoding a human TCR variable domain that binds an epitope of interest is provided, comprising exposing a non-human animal as described herein to an epitope of st, ining the non-human animal under conditions ient for the animal to present the epitope of interest to a humanized TCR of the animal, and identifying a nucleic acid of the animal that encodes a human TCR variable domain ptide that binds the epitope of interest. in one , use of a non-human animal as bed herein is provided for making a humanized TCR receptor. in one aspect, use of a non-human animal as described herein is provided for making a human TCR variable domain. in one aspect, use of a non— human animal as described herein is provided for making a c acid sequence encoding a human TCR le domain.

In one aspect, use of nucleic acid sequence encoding a human TCR variable domain or fragment thereof to make an antigen-binding protein is provided. In one embodiment, the antigen-binding protein comprises a TCR variable domain comprising a human TCRa and/or human TCRL’» variable domain that binds an n of interest.

In one , use of a non-human as described herein is provided for making a non—human cell that expresses on its surface a zed T cell receptor.

In one aspect, a humanized T cell receptor from a non-human animal as described herein is provided.

In one , a nucleic acid sequence encoding a human TCR variable domain or fragment thereof, made in a non-human animal as described herein, is provided.

Any of the embodiments and aspects described herein can be used in conjunction with one another, unless otherwise indicated or apparent from the context.

Other embodiments will become nt to those skilled in the art from a review of the ensuing detailed description. The following detailed description es exemplary entations of various embodiments of the invention, which are not restrictive of the invention as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate embodiments and not to limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS depicts ction in a mouse between a TCR molecule and an MHC molecule: the left panel shows a mouse T cell (top) from a humanized TCR mouse comprising a T cell receptor with human variable TCR domains and mouse constant TCR domains, which recognizes an antigen (grey ball) presented through an MHC class I by an antigen presenting cell (bottom); the right panel shows the same for an MHC class II. The MHC I and MHC II complexes are shown together with their respective co—receptors, CD8 and CD4. Mouse regions are in black and human s are in white. depicts (not to scale) the l organization of a mouse (top panel, first locus) and human (top panel, second locus) TCRa locus. The bottom panel illustrates a strategy for ing TCRoc variable region segments in a mouse (closed s) with human TCRoc variable region segments (open symbols) at the endogenous mouse locus on chromosome 14; a humanized TCRoc locus having human Va and Jo: segments is shown with a mouse constant region and a mouse enhancer; in the embodiment shown, the TCRE) locus is d in the course of humanization. depicts (not to scale) a progressive strategy for humanization of the mouse TCRoc locus, wherein TCRa variable region gene segments are sequentially added upstream of an initial humanization of a deleted mouse locus (MAID1540). Mouse sequence is indicated by closed symbols; human sequence is indicated by open symbols. MAID refers to modified allele ID number. TRAV=TCR Voc segment, TRAJ=TCR Jet segment (hTRAJ=human TRAJ), TRAC=TCR Cor domain, TCRD=TCR6. is a detailed depiction (not to scale) of progressive humanization strategy at the TCRd locus. depicts on of the mouse TCRd V and J segments; depicts strategy for insertion of 2V and 61J human segments into the deleted mouse TCRa locus; depicts strategy for insertion of additional human V ts, resulting in a total of 8V and SH human segments; s strategy for insertion of additional human V segments, resulting in a total of 23V and GM human segments; depicts strategy for insertion of additional human V ts resulting in 35V and (SH human segments; depicts strategy for insertion of additional human segments resulting in 48V and SH human segments; and depicts strategy for ion of additional human segments ing in 54V and 61J human segments. MAID refers to modified allele ID number. depicts (not to scale) one embodiment of mouse TCRa locus humanization gy, in which human TCR 6 sequences (TCRo Vs, TCRo Ds, TCRo Js, TCR8 enh (enhancer), and TCRo constant (0)) are also placed at the humanized TCRor locus. Mouse ce is indicated by closed symbols; human sequence is indicated by open symbols. LTVEC refers to a large targeting ; hTRD=human TCRé. depicts (not to scale) the general organization of a mouse (top panel, first locus; on mouse some 6) and human (top panel, second locus; on human chromosome 7) TCRB loci. The bottom panel illustrates a strategy for replacing TCRfi variable region segments in the mouse (closed symbols) with human TCRE variable region segments (open symbols) at the endogenous mouse locus on mouse chromosome 6. The humanized TCRB locus having human Vfi, DB, and JB segments is shown with mouse constant s and a mouse enhancer; in the embodiment shown, the zed locus retains mouse trypsinogen genes (solid rectangles); and in the particular embodiment shown, a single mouse V segment is retained upstream of the 5’ mouse trypsinogen genes. depicts (not to scale) a progressive strategy for zation of the mouse TCRB locus, wherein TCRfi variable region gene segments are sequentially added to a deleted mouse TCRfi variable locus. Mouse sequence is ted by closed symbols; human sequence is indicated by open symbols. MAID refers to modified allele ID number.

TRBV or TCRBV= TCRB V segment. is a detailed depiction of progressive humanization strategy at the TCRB locus. depicts the strategy for deletion of the mouse TCRB V segments; FIG. BB depicts the strategy for insertion of 14V segments into the deleted TCRfS locus; depicts the strategy for insertion of 2D and 14J segments into TCRB locus (i), followed by deletion of the onP site (ii), resulting in 14V, 2D and 14J human ts; depicts the strategy for inserting additional human V ts resulting in 40V, 2D and 14J human segments; and depicts the strategy for insertion of additional human V ts resulting in the 66V, 2D and 14J human segments; depicts replacement of the mouse V segment downstream of a mouse enhancer, resulting in 67V, 2D and 14J human segments. In this particular embodiment, one mouse V segment is retained 5’ of the mouse trypsinogen genes. depicts representative FACS analysis histograms for percent spleen cells (where Y axis is number of cells, X axis is mean scence intensity, and the gate shows frequency of CD3+ T cells within the single lymphocyte population) d with anti—CD3 antibody in a wild type (WT) mouse; a mouse homozygous for a deleted TCRoc locus (first top panel; MAID 1540 of ; a mouse homozygous for a deleted TCRoc locus and comprising 8 human Va and 61 human Joc segments (second top panel; MAID 1767 of or a zed TCRa mouse); a mouse homozygous for a deleted TCRB locus with the exception of one upstream and one downstream mouse V6 segments (first bottom panel; MAID 1545 of ; a mouse homozygous for a deleted TCRB locus with one upstream and one downstream mouse V13 segments and comprising 14 human VB, 2 human D6, and 14 human JB segments (second bottom panel; MAID 1716 of or a humanized TCRfi mouse); and a mouse homozygous for both TCRa and TCRB loci ons (with the exception of said two mouse V13 segments) and comprising 8 human Va and 61 human Ja ts at the endogenous TCRa locus as well as 14 human VB, 2 human D6, and 14 human J6 ts at the endogenous TCRB loci (MAID 1767/1716 or a zed TCRa/B mouse). is a representative FACS contour plot of mouse thymus cells from a WT, homozygous zed TCch (1767 H0; hTCRoc); homozygous humanized TCRB (1716 H0; hTCsz’o); and gous humanized TCRoc/B mouse (1716 H0 1767 H0; hTCRoc/fi) stained with anti—CD4 (Y axis) and anti—CD8 (X axis) antibodies (top panel), and anti—CD44 (Y axis) and anti-C025 (X axis) antibodies (bottom panel). The FACS plot in the top panel allows to distinguish double negative (DN), double positive (DP), CD4 single positive (CD4 SP), and CD8 single positive (SP CD8) T cells. The FACS plot in the bottom panel allows to distinguish various stages of double negative T cells during T cell development (DN1, DN2, DN3, and DN4). 1716 and 1767 referto MAID numbers as identified in FIGs. 3 and 7. trates either frequency (top panel) or absolute number (bottom panel) of DN, DP, CD4 SP, and CD SP T cells in the thymus of either WT, hTCRa (1767 H0); hTCRB (1716 H0); or hTCRoc/ffi (1716 H0 1767 H0) mice (n=4). is a representative FACS analysis of spleen cells of a WT, hTCRa (1767 H0); hTCRfi (1716 H0); or hTCRoc/B (1716 H0 1767 H0) mouse: left panel represents is of singlet cells based anti—CD19 antibody (Y axis; stain for B lymphocytes) or anti- CD3 dy (X axis; stain for T lymphocytes) staining; middle panel represents analysis of CD3+ cells based on anti-CD4 (Y axis) or D8 (X axis) antibody staining; and right panel represents analysis of either CD4+ or CD8+ cells based on anti—CD44 (Y axis) or anti- CD62L (X axis) antibody staining, the stains allow to distinguish various types of T cells in the periphery (naive T cells vs. l memory T cells (Tcm) vs. effector or effector memory T cells (Teff/Tem)). demonstrates the number of CD4+ (left panel) or CD8+ (right panel) T cells per spleen (Y axes) of WT, hTCRa (1767 H0); hTCRfi (1716 H0); or hTCRoc/fi (1716 .H0 1767 H0) mice (n=4). demonstrates the number of T na'ive, Tcm, and Teff/em cells per spleen (Y axes) of CD4+ (top panel) or CD8+ (bottom panel) T cells of WT, hTCRa (1767 H0); hTCRB (1716 H0); or hTCRon/[B (1716 H0 1767 H0) mice (n=4). are tables izing expression (determined by FACS analysis using variable segment-specific antibodies) of various human TCRB V segments in the splenic CD8+ T cells (A) or CD4+ T cells (B) of WT, hTCRB (1716 H0) or hTCRa/B (1716 H0 1767 H0) mice. Data presented as Mean:SD (n=4 mice per group) depicts mRNA expression (Y axes) of various human TCRB V ts present in WT, hTCRa (1767 H0); hTCRB (1716 H0); or hTCRa/B (1716 HO1767 HO) mice in thymic or splenic T cells; A represents analysis of mRNA sion of human TCRB variable segment (hTRBV) 18, 19, 20, and 24; and B ents analysis of mRNA expression of hTRBV 25, 27, 28, and 29. depicts representative FACS histograms of spleen cells (where Y axis is number of cells, X axis is mean fluorescence intensity, and the gate shows frequency of CD3+ T cells within the single lymphocyte population) stained with anti-CD3 antibody in a WT mouse, a mouse homozygous for a deleted TCRor locus (TCRA AV), a mouse homozygous for deleted TCRoc locus With 2 human V segments and 61 human J segments (TCRA 2 hV; MAID 1626 of , a mouse homozygous for deleted TCRoc locus with 8 human V segments and 61 human J segments (TCRA 8 hV; MAID 1767 of , and a mouse homozygous for deleted TCRoc locus with 23 human V segments and 61 human J segments (TCRA 23 hV; MAID 1979 of . , at left top panel, is a representative FACS analysis of CD3+ T cells of the thymus obtained from either a WT or homozygous hTCRoc mouse with 23 human V segments and 61 human J segments (1979 H0) stained with either anti-CD4 (Y axis) or anti— CD8 (X axis) antibody; at left bottom panel, is a FACS analysis of DN T cells from either a WT or 1979 mouse stained with either anti—CD44 (Y axis) or anti—CD25 (X axis); at right panel are graphs of percent of ytes (Y axis) that are DN, DP, CD4 SP, or CD8 SP in either WT or 1979 H0 mice (n=4). , at left panel, is a representative FACS is of splenic lymphocytes from a WT or 1979 H0 mouse d either with anti—CD19 or anti—CDB antibodies; at right panel, are graphs of percent splenocytes (Y axis) obtained from WT and 1979 H0 mice (n=4) that are CD3+.

DETAILED DESCRIPTION OF THE INVENTION Definitions The present invention provides genetically modified non-human s, e.g., rodents, e.g., mice or rats, that express zed T cell receptors. The present invention also relates to genetically modified non-human animals that comprise in their germline unrearranged T cell receptor variable gene loci. Also provided are embryos, cells, and tissues comprising the same; methods of making the same; as well as methods of using the same. Unless defined othenNise, all terms and s used herein include the meanings that the terms and s have attained in the art, unless the contrary is clearly ted or clearly apparent from the context in which the term or phrase is used.

The term "conservative,” when used to describe a conservative amino acid substitution, includes substitution of an amino acid e by another amino acid e having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). vative amino acid substitutions may be achieved by modifying a tide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a n, for e, the ability of a T cell to recognize a peptide presented by an MHC molecule. Examples ofgroups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide— containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids tution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution can be a tution of any native residue in a protein with alanine, as used in, for e, alanine ng mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443—45), hereby incorporated by reference. ln some embodiments, the substitution is a moderately conservative tution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.

Thus, encompassed by the invention is a genetically modified man animal expressing humanized TCR oz and {3 polypeptides (and/or humanized TCRB and TCRy polypeptides) comprising conservative amino acid substitutions in the amino acid sequence described herein.

One skilled in the art would understand that in addition to the nucleic acid residues encoding humanized TCR a and [3 polypeptides described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the ptides of the invention. ore, in addition to a genetically modified non-human animal that comprises in its genome nucleotide sequences encoding humanized TCR polypeptides described herein, a non-human animal that comprises in its genome nucleotide sequences that differ from those described herein due to the racy of the genetic code are also provided.

The term “identity” when used in connection with sequence includes identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid ce identity. In some embodiments described herein, identities are ined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet rity matrix (MacVectorTM 10.0.2, MacVector Inc, 2008). The length of the ces compared with respect to identity of sequences will depend upon the particular ces. In various 2012/062065 embodiments, identity is determined by comparing the sequence of a mature protein from its inal to its C-terminal. In various embodiments, when comparing a chimeric human/non-human sequence to a human sequence, the human portion of the chimeric human/non-human sequence (but not the non-human portion) is used in making a ison for the purpose of ascertaining a level of identity between a human sequence and a human portion of a chimeric human/non—human sequence (e.g., comparing a human main of a chimeric mouse protein to a human ectodomain of a human protein).

The terms “homology” or “homologous” in reference to sequences, e.g., nucleotide or amino acid sequences, means two sequences which, upon optimal ent and comparison, are identical in at least about 75% of nucleotides or amino acids, at least about 80% of nucleotides or amino acids, at least about 90-95% nucleotides or amino acids, e.g,, greater than 97% nucleotides or amino acids. One skilled in the art would understand that, for optimal gene targeting, the targeting construct should contain arms homologous to endogenous DNA sequences (i.e., “homology arms”); thus, homologous recombination can occur between the ing construct and the targeted endogenous sequence.

The term "operably linked" refers to a juxtaposition wherein the components so described are in a onship permitting them to function in their intended . As such, a nucleic acid sequence ng a protein may be operably linked to regulatory ces (e.g., promoter, er, silencer sequence, etc.) so as to retain proper transcriptional regulation. in addition, various portions of the humanized protein of the ion may be operably linked to retain proper folding, processing, targeting, sion, and other functional properties of the protein in the cell. Unless stated othenrvise, various domains of the humanized protein of the invention are operably linked to each other.

The term “replacement” in reference to gene replacement refers to placing exogenous genetic material at an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. in one instance, an endogenous non-human gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. A corresponding human gene or fragment f is a human gene or fragment that is an ortholog of, a homolog of, or is substantially identical or the same in ure and/or function, as the endogenous non—human gene or nt thereof that is replaced. As demonstrated in the Examples below, nucleotide sequences of endogenous non—human TCR 0L and 5 variable gene loci were replaced by nucleotide sequences corresponding to human TCR a and [3 variable gene loci.

“Functional” as used herein, e.g., in reference to a functional protein, refers to a protein that retains at least one biological activity normally ated with the native protein.

For example, in some embodiments of the invention, a replacement at an nous locus (e.g., replacement at endogenous non—human TCRor, TCRB, TCRo and/or TCRy le gene loci) results in a locus that fails to express a functional endogenous protein.

TCR locus or TCR gene locus (e.g., TCRoc locus or TCRB locus), as used herein, refer to the genomic DNA comprising the TCR coding region, including the entire TCR coding , including unrearranged V(D)J sequences, enhancer, sequence, constant sequence(s), and any upstream or downstream (UTR, regulatory regions, etc), or intervening DNA sequence (introns, etc). TCR variable locus or TCR variable gene locus (e.g., TCRor le gene locus or TCRB variable gene locus), refers to genomic DNA comprising the region that includes TCR variable region segments (V(D)J region) but excludes TCR constant sequences and, in various embodiments, enhancer sequences.

Other sequences may be included in the TCR le gene locus for the purposes of genetic manipulation (e.g., ion cassettes, restriction sites, etc), and these are encompassed herein. cally Modified TCR Animals in s embodiments, the invention generally provides genetically modified non—human animals wherein the non—human animals comprise in the genome unrearranged humanized TCR variable gene loci.

T cells bind epitopes on small antigenic determinants on the surface of antigen- presenting cells that are associated with a major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complex. T cells bind these epitopes through a T cell receptor (TCR) complex on the surface of the T cell. T cell receptors are heterodimeric structures composed of two types of chains: an a ) and (3 (beta) chain, or a y (gamma) and r3 (delta) chain. The or chain is encoded by the nucleic acid sequence located within the on locus (on human or mouse chromosome 14), which also encompasses the entire 6 locus, and the (3 chain is encoded by the c acid sequence located within the [3 locus (on mouse chromosome 6 or human chromosome 7). The majority of T cells have an afz’) TCR; while a ty of T cells bear a yo TCR. lnteractions of TCRs with MHC class l (presenting to CD8+ T cells) and MHC class ll (presenting to CD4+ T cells) molecules are shown in (closed symbols represent non-human sequences; open s represent human ces, showing one particular embodiment of the TCR protein of the present invention).

T cell receptor or and [3 polypeptides (and similarly y and 6 polypeptides) are linked to each other via a disulfide bond. Each of the two polypeptides that make up the TCR contains an extracellular domain sing nt and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytopiasmic tail also being a part of the constant ). The variable region of the TCR determines its antigen specificity, and similar to immunoglobulins, comprises 3 complementary determining regions (CDRs). Also similar to immunoglobulin genes, T cell receptor variable gene loci (e.g., TCRoc and TCRB loci) contain a number of unrearranged V(D)J segments (variable (V), joining (J), and in TCRB and 6, diversity (D) segments).

During T cell development in the thymus, TCRa le gene locus undergoes rearrangement, such that the resultant TCR a chain is encoded by a specific combination of VJ segments (Va/Jon ce); and TCRB variable gene locus undergoes rearrangement, such that the resuitant TCR {3 chain is encoded by a specific combination of VDJ segments (Vfi/DB/st sequence). interactions with thymic stroma trigger thymocytes to undergo several developmental stages, terized by expression of various cell surface markers. A summary of characteristic cell surface markers at various developmental stages in the thymus is presented in Table 1. Rearrangement at the TCRB variable gene locus begins at the DN2 stage and ends during the DN4 stage, while rearrangement of the TCRa variable gene locus occurs at the DP stage. After the completion of TCRB iocus ngement, the cells express TCRB chain at the cell surface together with the ate 0t chain, qu. See, Janeway’s bioiogy, Chapter 7, supra.

Table 1: Developmental Stages of T cells in the Thymus “"rWhiDevelopmental CD44+ICD25~ CD44+ICD25+ CD44 °wIC025+ CD44-l CD4+/008+ CD4+ CD25- or CD8+ Naive CD4+ and CD8+ T cells exit the thymus and enter the peripheral lymphoid organs (e.g., spleen) where they are exposed to antigens and are activated to ly expand and entiate into a number of effector T cells (Teff), e.g., xic T celis, TREG cells, TH17 cells, TH1 celis, TH2 cells, etc. Subsequent to infection, a number ofT cells persist as memory T cells, and are classified as either central memory T cells (Tcm) or effector memory T ceils (Tern). Sallusto et ai. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708—12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401:659-60. Sallusto and colleagues proposed that, after initial infection, Tem cells represent a readily available pool of antigen—primed memory T cells in the peripheral tissues with effector functions, while Tcm cells represent antigen-primed memory T cells in the peripheral lymphoid organs that upon secondary challenge can become new effector T cells. While all memory T cells express CD45RO m of CD45 (naive T cells express CD45RA m), Tcm are characterized by expression of L-selectin (also known as CD62L) and CCR7+, which are important for binding to and signaling in the peripheral lymphoid organs and lymph nodes. Id. Thus, all T cells found in the peripheral lymphoid organs (e.g., naive T cells, Tcm cells, etc.) express CD62L. in on to CD45RO, all memory T cells are known to express a number of ent cell surface markers, e.g., CD44. For y of various cell surface markers on T cells, see Janeway’s lmmunobiology, Chapter 10, supra.

While TCR variable domain functions primarily in antigen recognition, the extracellular portion of the constant , as well as transmembrane, and asmic domains of the TCR also serve important functions. A complete TCR receptor complex requires more than the a and [3 or y and 5 polypeptides; additional molecules required include CD3], CD36, and CD35, as well as the C chain homodimer (it). At the completion of TCRB rearrangement, when the cells express TCRB/pTa, this pre-TCR complex exists together with CD3 on the cell surface. TCRoc (or pToc) on the cell surface has two basic residues in its transmembrane , one of which recruits a CD3ys heterodimer, and another recruits t]; via their respective acidic residues. TCRB has an additional basic residue in its transmembrane domain that is believed to recruit CD365 dimer. See, e.g., Kuhns et al. (2006) tructing the Form and Function of the TCR/CD3 Complex, immunity 24:133-39; pfennig et al. (2009) Structural y of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and tion of Signaling, Cold Spring Harb. Perspect. Biol. 2:a005140. The assembled complex, comprising TCRocfs heterodimer, CD3ys, CD352, and 2;; is expressed on the T cell e. The polar residues in the transmembrane domain have been suggested to serve as quality l for exiting endoplasmic reticulum; it has been demonstrated that in the absence of CD3 subunits, TCR chains are retained in the ER and targeted for degradation. See, e.g., Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Function, Annu. Rev. lmmunol. 23:101—25.

CD3 and C chains of the assembled complex provide components for TCR signaling as TCRocB heterodimer (or TCRyt‘) heterodimer) by itself lacks signal transducing activity. The CD3 chains possess one lmmune-Receptor-Tyrosine-based-Activation-Motif (lTAM) each, while the 2; chain contains three tandem lTAMs. ITAMs contain tyrosine residues capable of being phosphorylated by associated kinases, Thus, the led 3 complex ns 10 lTAM . See, e.g., Love and Hayes (2010) lTAM- Mediated Signaling by the T—Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. 21e002485. Following TCR engagement, lTAM motifs are orylated by Src family tyrosine kinases, Lck and Fyn, which tes a signaling cascade, resulting in Ras activation, calcium mobilization, actin cytoskeleton rearrangements, and activation of transcription factors, all tely leading to T cell differentiation, proliferation, and effector actions. Id., see also, Janeway’s lmmunobiology, 7th Ed., Murphy et al. eds., Garland Science, 2008; both incorporated herein by reference. onally, TCRB transmembrane and cytoplasmic domains are thought to have a role in mitochondrial targeting and induction of apoptosis; in fact, naturally occurring N- terminally truncated TCRB molecules exist in thymocytes. Shani et al. (2009) lncompiete T— cell receptor-—B peptides target the mitochondrion and induce sis, Blood 113:3530-41.

Thus, several ant functions are served by the TCR constant region (which, in various embodiments, comprises a portion of extracellular as well as transmembrane and cytoplasmic domains); and in various embodiments the structure of this region should be taken into consideration when designing humanized TCRs or genetically modified non- human animals expressing the same.

Mice transgenic for rearranged T cell receptor sequences are known in the art.

The present invention relates to genetically modified non—human animals (e.g., rodents, e.g., rats, mice) that comprise unrearranged human or zed T cell variable gene loci that are capable of rearranging to form nucleic acid sequences that encode human T cell receptor variable domains, including animals that comprise T cells that comprise rearranged human le domains and non-human (e.g., mouse or rat) constant s. The present ion also provides non-human animals (e.g., rodents, e.g., rats, mice) that are e of generating a diverse repertoire of human T cell receptor variable region sequences; thus, the present invention provides non-human animals that express TCRs with fully human le domains in response to an antigen of interest and that bind an e of the antigen of interest. In some embodiments, provided are non-human animals that generate a diverse T cell receptor repertoire capable of reacting with various antigens, including but not limited to antigens presented by APCs. ln one embodiment, the invention provides cally modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise in their genome unrearranged human TCR le region segments (V(D)J segments), wherein the unrearranged human TCR variable region segments replace, at an endogenous non-human (e.g., rodent) TCR variable gene locus (e.g., TCRa, f3, 5, and/or y variable gene locus), endogenous non—human TCR variable region segments. in one embodiment, unrearranged human TCR variable gene locus replaces endogenous non-human TCR variable gene locus. in another embodiment, the invention provides cally modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise in their genome unrearranged human TCR variable region segments (V(D)J segments), n the unrearranged human TCR variable region segments are operably linked to a non-human TCR constant region gene sequence resulting in a humanized TCR locus, wherein the humanized TCR locus is at a site in the genome other than the endogenous non-human TCR locus. Thus, in one ment, a non—human animal (e.g., rodent, e.g., mouse, rat) comprising a transgene that comprises unrearranged human TCR variable region segments ly linked to non- human TCR constant region sequence is also ed. in one aspect, the genetically modified non-human animals of the invention comprise in their genome human TCR variable region ts, while ing non—human (e.g., , e.g., mouse, rat) TCR constant gene segments. in various embodiments, constant s include transmembrane domain and the cytoplasmic tail of the TCR. Thus, in various ments of the present invention, the genetically modified non-human animals retain endogenous non-human TCR transmembrane domain and asmic tail.

In other embodiments, non—human animals comprise non—human non—endogenous TCR constant gene ces, e.g., non-human non-endogenous TCR transmembrane domain and cytoplasmic tail. As indicated above, the constant region of the TCR participates in a signaling e initiated during antigen-primed T cell activation; thus, endogenous TCR constant region cts with a variety of non-human anchor and signaling proteins in the T cell. Thus, in one aspect, the genetically modified non-human animals of the invention express zed T cell receptors that retain the ability to recruit a variety of endogenous non—human anchor or signaling molecules, e.g., CD3 molecules (e.g., CD3y, C036, CD32), the C chain, Lck, Fyn, ZAP-70, etc. A nonlimiting list of molecules that are recruited to the TCR complex is described in Janeway’s lmmunobiology, supra. In addition, similar to VELOClMMUNE® mice, which exhibit normal B cell development and normal clonal selection processes believed to be due at least in part to the placement of le regions at the endogenous mouse loci and the maintenance of mouse constant domains, in one aspect, the non-human animals of the present invention exhibit normal T cell development and T cell differentiation processes.

In some embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRa variable region segments, wherein the unrearranged human TCRoc variable region segments are operably linked to a non-human TCRoc constant region gene sequence resulting in a humanized TCRoc locus. in one embodiment, the humanized TCRa locus is at a site in the genome other than the endogenous non—human TCRa locus. in another embodiment, the unrearranged human TCRoc variable region segments e endogenous non-human TCRoc variable region segments while ing endogenous non-human TCRoc constant region. ln one embodiment, the unrearranged human TCRor le gene locus replaces endogenous non-human TCRa variable gene locus. in some embodiments, the animal retains endogenous non-human TCRfi le region and constant region gene sequences. Thus, the animal expresses a TCR that comprises a chimeric non-human (i.e., humanized) TCRa chain and a non-human TCRB chain. in other embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRB variable region segments, wherein the unrearranged human TCRfi le region segments are operably linked to a non-human TCRB constant region gene ce resulting in a humanized TCRB locus. In one embodiment, the humanized TCRE locus is at a site in the genome other than the nous non-human TCRB locus. ln another ment, the unrearranged human TCRB variable region segments replace endogenous non-human TCRB variable region segments while ing endogenous non-human TCRB constant region. in one embodiment, the unrearranged human TCRB le gene locus replaces endogenous non-human TCRB variable gene locus. In some embodiments, the animal retains endogenous non—human TCRa variable region and constant region gene sequences. Thus, the animal expresses a TCR that comprises a chimeric human/non—human (i.e., humanized) TCRB chain and a non-human TCRoc chain. in some specific embodiments, the invention provides a genetically modified non— human animal (e.g., rodent, e.g., mouse or rat) that comprises in its genome (a) an unrearranged T cell receptor (TCR) on variable gene locus comprising at least one human Va t and at least one human Jor segment, operably linked to an endogenous non- human (e.g., rodent, e.g., mouse or rat) TCRa constant gene sequences, and/or (b) an unrearranged TCRB variable gene locus sing at least one human VB segment, at least one human D5 segment, and at least one human JB t, operably linked to an endogenous non—human (e.g., rodent, e.g., mouse or rat) TCRffi constant gene sequence.

In various embodiments of the invention, the unrearranged human or humanized TCR variable gene locus (e.g., TCRa and/or TCRfi variable gene locus gene locus) is comprised in the germline of the non—human animal (e.g., rodent, e.g., mouse or rat). in various ments, the replacements of TCR V(D)J segments by unrearranged human TCR V(D)J segments (e.g., V0: and Jet, and/or VB and DB and J6 segments) are at an nous non-human TCR variable locus (or loci), wherein the unrearranged human V and J and/or V and D and J segments are operably linked to non-human TCR constant region genes. ln some embodiments of the invention, the non-human animal comprises two copies of the unrearranged human or humanized TCRoc variable gene locus and/or two copies of the unrearranged human or humanized TCRB variable gene locus. Thus, the non- human animal is homozygous for one or both unrearranged human or humanized TCRor and TCRIB variable gene locus. in some embodiments of the invention, the non—human animal comprises one copy of the unrearranged human or humanized TCRor variable gene locus and/or one copy of the unrearranged human or humanized TCRiS variable gene locus.

Thus, the non-human animal is heterozygous for one or both unrearranged human or humanized TCRor and TCRfi variable gene locus.

In one embodiment, the unrearranged TCRa variable gene locus sing human variable region segments (e.g., human Va and Jet ts) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human le region segments. In one embodiment, the unrearranged TCRa variable gene locus comprising human variable region segments replaces endogenous TCRa variable gene locus. In one aspect, endogenous non-human Va and Jon ts are incapable of rearranging to form a rearranged VOL/Jot ce. Thus, in one aspect, the human Va and Ja segments in the ranged TCRor variable gene locus are capable of rearranging to form a rearranged human Vor/Jor ce.

Similarly, in one embodiment, the unrearranged TCRB le gene locus comprising human variable region ts (e.g., human VB, DB, and JB segments) is positioned in the non—human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRfS variable gene locus comprising human variable region segments replaces endogenous TCRfi variable gene locus. in one aspect, endogenous non-human V13, DB, and JB segments are incapable of rearranging to form a rearranged VB/DB/JB sequence.

Thus, in one aspect, the human V13, DB, and Jfi segments in the unrearranged TCRB variable gene locus are capable of rearranging to form a rearranged human VqB/DB/Jfi sequence.

In yet another embodiment, both the unrearranged TCRa and B variable gene loci sing human variable region segments replace respective endogenous TCRa and [3 variable gene loci. In one aspect, endogenous non—human Va and Jet segments are incapable of rearranging to form a rearranged Voc/Joc sequence, and endogenous non— human VB, DB, and J13 segments are incapable of rearranging to form a rearranged Vfi/DB/Jfi sequence. Thus, in one aspect, the human Vor and Ja segments in the unrearranged TCRa variable gene locus are capable of rearranging to form a rearranged human Von/Jon sequence and the human V6, DE», and JB ts in the unrearranged TCRfi le gene locus are capable of rearranging to form a rearranged human Vafi/Dfi/JB sequence.

In some aspects of the invention, the non—human animal comprising a humanized TCRq and/or TCRfi gene locus (comprising an unrearranged TCRa and/or TCRfi variable gene locus) retains an endogenous non-human TCRa and/or TCRB variable gene locus. In one embodiment, the endogenous non—human TCRa and/or TCRfS variable gene locus is a non—functional locus. In one embodiment, the non-functional locus is an vated locus, e.g., an ed locus (e.g., the coding nucleic acid sequence of the variable gene locus is in inverted orientation with t to the constant region sequence, such that no successful rearrangements are possible utilizing variable region segments from the inverted locus). In one embodiment, the zed TCRa and/or TCRB variable gene locus is positioned between the endogenous non-human TCRoc and/or TCRB variable gene locus and the endogenous man TCRa and/or TCRfi constant gene locus.

The number, nomenclature, position, as well as other aspects of V and J and/or V, D, and J segments of the human and mouse TCR loci may be ascertained using the lMGT database, available at www.imgt.org. The mouse TCRq variable locus is approximately 1.5 megabases and ses a total of 110Va and 60 Jet segments (.

The human TCRor variable locus is approximately 1 megabase and comprises a total of 54Va and 61Ja segments, with 45Va and 50Ja believed to be functional. Unless stated otherwise, the numbers; of human V(D)J segments referred to hout the ication refers to the total number of V(D)J segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) ses at least one human Va and at least one human Jo: segment. In one ment, the non-human animal comprises a humanized TCRoc locus that comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 2012/062065 , 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Va segments. In some embodiments, the zed TCRa locus comprises 2, 8, 23, 35, 48, or 54 human Va segments. Thus, in some embodiments, the humanized TCRa locus in the non—human animal may comprise %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% of human Va; in some embodiments, it may comprise about 2%, about 3%, about 15%, about 65%, about 90%, or 100% of human Va.

In one embodiment, the non-human animal comprises a humanized TCRa locus that comprises a DNA fragment sing a contiguous human sequence of human Va40 to Va41 (Va segment is also referred to as “TRAV” or “TCRAV”) and a DNA fragment comprising a contiguous human sequence of 61 human Ja segments (Ja segment is also referred to as “TRAJ” or “TCRAJ”). in one embodiment, the non—human animal comprises a humanized TCRa locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV35 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. in one embodiment, the non-human animal comprises a humanized TCRa locus that comprises a DNA nt comprising a contiguous human ce of human TRAV22 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. in one embodiment, the non-human animal comprises a humanized TCRa locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV13-2 to TRAV41 and a DNA fragment comprising a contiguous human sequence of61 human TRAJs. in one embodiment, the non—human animal comprises a humanized TCRa locus that ses a DNA fragment comprising a contiguous human sequence of human TRAV6 to TRAV41 and 61 human TRAJs. in one embodiment, the non-human animal comprises a humanized TCRa locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV1-1 to TRAV 41 and 61 human TRAJs. In s embodiments, the DNA fragments comprising contiguous human sequences of human TCRa variable region segments also comprise ction enzyme sites, selection cassettes, endonucleases sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with proper functioning (e.g., ngement, splicing, etc.) of various genes at the TCRa locus. in one embodiment, the humanized TCRa locus comprises 61 human Ja segments, or 100% of human Ja ts. in a particular embodiment, humanized TCRa locus ses 8 human Va segments and 61 human Ja segments; in another particular embodiment, humanized TCRa locus ses 23 human Va segments and 61 human Ja segments. in another particular embodiment, the humanized TCRa locus comprises a te repertoire of human Va and Jon segments, i.e., all human variable on region gene segments d by the oz locus, or 54 human Von and 61 human Ja segments. in various embodiments, the non-human animal does not comprise any endogenous non-human Va or Joc ts at the TCRq locus.

The mouse TCRffi variable locus is approximately 0.6 megabases and comprises a total of 33 V6, 2 DB, and 14 J6 segments (. The human TCRB variable locus is approximately 0.6 megabases and comprises a total of 67 VB, 2 DB, and 14 J6 segments.

In one embodiment of the invention, the genetically modified non—human animal (e.g., rodent, e.g., mouse or rat) comprises at least one human V6, at least one human D8, and at least one human Ja segment. In one embodiment, the non-human animal ses a humanized TCRfi locus that comprises 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 human 67 V6 segments. In some embodiments, the humanized TCRfS locus comprises 8, 14, 40, 66, or human 67 V6 ts. Thus, in some embodiments, the humanized TCRfi locus in the non-human animal may comprise 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% of human V6; in some embodiments, it may comprise about 20%, about 60%, about 15%, about 98%, or 100% of human V6. in one embodiment, the non-human animal comprises a humanized TCRfi locus that comprises a DNA fragment comprising a uous human sequence of human V618 to V6294 (VB segment is also referred to as “TRBV” or “TCRBV”). in one embodiment, the man animal comprises a humanized TCRB locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV18 to -1, a separate DNA fragment comprising a contiguous human sequence of human Dfi1-Ji31 (i.e., human D91- Jf31Jf51-6 segments), and a separate DNA fragment sing a contiguous human sequence of human z (i.e., human DBZ-J62-1—J62—7 segments). in one embodiment, the non—human animal comprises a humanized TCRfi locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV6-5 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human D61-JB1 (i.e., human DB1-Jl31Jf31-6 segments), and a separate DNA fragment comprising a uous human sequence of human DBZ—JfiZ (i.e., human D62-JBZJ62-7 ts). in one embodiment, the non-human animal comprises a humanized TCRB locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV1 to TRBV29—1, a separate DNA fragment comprising a uous human sequence of human DB1—Jf31, and a separate DNA fragment comprising a contiguous human sequence of human DB2—JB2. In one ment, the man animal comprises a humanized TCRB locus that comprises a DNA nt comprising a contiguous human sequence of human TRBV1 to TRBV29—1, a separate DNA fragment comprising a contiguous human ce of human DB1-JB1, a separate DNA fragment comprising a contiguous human sequence of human DB2—JB2, and a separate DNA fragment comprising the sequence of human TRBV30. In various embodiments, the DNA fragments sing contiguous human sequences of human TCRB variable region segments also se restriction enzyme sites, selection cassettes, endonucleases sites, or other sites inserted to facilitate cloning and selection during the locus humanization s. In s embodiments, these additional sites do not interfere with proper functioning (e.g., rearrangement, splicing, etc.) of various genes at the TCRB locus.

In one embodiment, the humanized TCRB locus comprises 14 human JB segments, or 100% of human JB segments, and 2 human DB ts or 100% of human JB segments. In another embodiment, the humanized TCRB locus ses at least one human VB segment, e.g., 14 human VB segments, and all mouse DB and JB segments. In a particular embodiment, humanized TCRB locus ses 14 human VB segments, 2 human DB segments, and 14 human JB segments. In another particular embodiment, the humanized TCRB locus comprises a complete repertoire of human VB, DB, and JB segments, i.e., all human variable B region gene segments encoded by the B locus or 67 human VB, 2 human DB, and 14 human JB segments. In one embodiment, the non-human animal ses one (e.g., 5’) non-human VB segment at the humanized TCRB locus. In various embodiments, the non—human animal does not comprise any endogenous non- human VB, DB, or JB segments at the TCRB locus.

In various embodiments, wherein the non—human animal (e.g., rodent) comprises a repertoire of human TCRa and TCRB (and optionally human TCRB and TCRy) variable region segments (e.g., a complete repertoire of variable region segments), the repertoire of s segments (e.g., the complete oire of various segments) is utilized by the animal to generate a diverse repertoire of TCR molecules to various antigens.

In various aspects, the non-human animals se contiguous portions of the human genomic TCR variable loci that comprise V, D, and J, or D and J, or V and J, or V segments arranged as in an unrearranged human genomic variable locus, e.g., comprising promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., ed as in a human genomic TCR variable locus. In other aspects, the various segments are arranged as in an unrearranged non-human genomic TCR variable locus. in various ments of the zed TCRa and/or [3 locus, the humanized locus can comprise two or more human genomic segments that do not appear in a human genome juxtaposed, e.g., a fragment ofV segments of the human V locus located in a human genome proximal to the constant region, juxtaposed with a nt of V segments of the human V locus located in a human genome at the upstream end of the human V locus. in both mouse and human, the TCRé gene segments are located with the TCRa locus (see Fle. 2 and 5). TCRo J and D segments are located between VOL and Ja segments, while TCRB V ts are interspersed throughout the TCRa locus, with the majority d among various Von segments. The number and locations of various TCRE) segments can be determined from the lMGT database. Due to the genomic arrangement of TCRE) gene segments within the TCRoc locus, successful rearrangement at the TCRa locus generally deletes the TCRB gene ts. in some embodiments of the invention, a non—human animal comprising an unrearranged human TCRa variable gene locus also comprises at least one human V5 segment, e.g., up to complete repertoire of human V6 segments. Thus, in some embodiments, the replacement of endogenous TCRa le gene iocus results in a replacement of at least one non-human V6 segment with a human V5 segment. in other embodiments, the non—human animal of the ion comprises a complete repertoire of human V6, D6, and J6 segments at the unrearranged humanized TCRoc locus; in yet other embodiments, the non—human animal comprises a complete unrearranged human TCRé locus at the ranged humanized TCRor locus (Le, a TCRo locus including human variable region segments, as well as human enhancer and constant region). An exemplary embodiment for ucting an unrearranged humanized TCRoc locus comprising te unrearranged TCRo locus is depicted in In yet another embodiment, the non—human animal of the invention further comprises an unrearranged humanized TCRy locus, e.g., a TCRy locus comprising at least one human Vy and at least one human Jy segments (e.g., a complete repertoire of human Vy and human Jy variable region segments). The human TCRy locus is on human chromosome 7, while the mouse TCRy locus is on mouse chromosome 13. See the lMGT database for more detail on the TCRy locus. in one aspect, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising humanized TCRoc and [3 variable gene loci (and, optionally humanized TCRB/y variable gene loci) described herein ses a humanized T cell receptor comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region on a surface of a T cell. ln some aspects, the non-human animal is e or expressing a e repertoire of zed T cell receptors that recognize a y of presented antigens.

In various embodiments of the invention, the humanized T cell receptor polypeptides described herein se human leader sequences. in alternative embodiments, the humanized TCR receptor nucleic acid sequences are engineered such that the humanized TCR ptides comprise non—human leader sequences.

The zed TCR polypeptides bed herein may be expressed under control of endogenous non-human regulatory elements (e.g., rodent regulatory elements), e.g., promoter, silencer, enhancer, etc. The humanized TCR polypeptides described herein may alternatively be expressed under control of human regulatory elements. In various embodiments, the non—human animals described herein further comprise all tory and other ces normally found in situ in the human genome. in various embodiments, the human variable region of the humanized TCR protein is capable of interacting with various proteins on the surface of the same cell or another cell. in one embodiment, the human variable region of the humanized TCR interacts with MHC proteins (e.g., MHC class I or ll proteins) ting antigens on the surface of the second cell, e.g., an antigen presenting cell (APC). in some embodiments, the MHC l or ll n is a non-human (e.g., rodent, e.g., mouse or rat) protein. In other embodiments, the MHC l or ll protein is a human protein. In one aspect, the second cell, e.g., the APC, is an endogenous non-human cell expressing a human or humanized MHC molecule. ln a different embodiment, the second cell is a human cell expressing a human MHC molecule.

In one aspect, the non—human animal expresses a humanized T cell receptor with a non—human constant region on the surface of a T cell, wherein the receptor is capable of interacting with non-human les, e.g., anchor or signaling molecules sed in the T cell (e.g., CD3 molecules, the C, chain, or other proteins ed to the TCR through the CD3 molecules or the C .

Thus, in one aspect, a cellular complex is provided, comprising a non—human T- cell that expresses a TCR that comprises a humanized TCRa chain as described herein and humanized TCR[5 chain as described herein, and a non-human antigen-presenting cell comprising an antigen bound to an MHC l or MHC ll. in one embodiment, the non-human nt TCch and TCRB chains are complexed with a non-human zeta (C) chain homodimer and CD3 heterodimers. in one embodiment, the cellular complex is an in vivo cellular complex. in one embodiment, the cellular complex is an in vitro cellular complex.

] The genetically modified non—human animal may be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). Forthe non—human animals where suitable genetically modifiable ES cells are not y available, other methods are employed to make a non—human animal sing the genetic modification.

Such methods include, e.g., modifying a non—ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.

In one aspect, the non-human animal is a mammal. ln one aspect, the nonhuman animal is a small , e.g., of the superfamily Dipodoidea or Muroidea. in one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one ment, the rodent is selected from the superfamily Muroidea. ln one embodiment, the genetically ed animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). ln a specific embodiment, the cally modified rodent is selected from a true mouse or rat (family e), a gerbil, a spiny mouse, and a crested rat. ln one ment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. ln one embodiment, the non—human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is a mouse of a CS7BL strain selected from C57BL/A, CS7BL/An, GrFa, C57BL/KaLwN, CS7BL/6, C57BL/6J, CS7BL/6ByJ, CS7BL/6NJ, C57BL/10, CS7BL/108c8n, C57BL/1OCr, and Ola. in another embodiment, the mouse is a 129 strain selected from the group consisting ofa strain that is 129P1, 129P2, 129P3, 129X1, 12981 (e.g., 12981/8V, 12981/8vlm), 12982, 12984, 12985, SvaH, 12986 (129/SvaTac), 12987, 12988, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al (2000) Establishment and a is of 129/8va- and C57BL/6—Derived Mouse Embryonic Stem Cell Lines). ln a specific embodiment, the genetically modified mouse is a mix of an entioned 129 strain and an aforementioned CS7BL/6 strain. In another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 s. In a specific embodiment, the 129 strain of the mix is a 12986 (129/8vaTac) strain. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. in yet another embodiment, the mouse is a mix of a BALB strain and another aforementioned strain. in one ment, the non-human animal is a rat. in one embodiment, the rat is selected from a Wistar rat, an LEA , a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. in one embodiment, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark .

Thus, in one embodiment, the ion provides a genetically modified mouse sing in its genome an unrearranged human or humanized TCR variable gene locus, e.g., TCRa, TCRB, TCRB, and/or TCRy variable gene locus. in some embodiments, the unrearranged human or humanized TCR variable gene locus replaces endogenous mouse TCR variable gene locus. in other embodiments, unrearranged human or humanized TCR variable gene locus is at a site in the genome other than the corresponding endogenous mouse TCR locus. in some embodiments, human or humanized unrearranged TCR variable gene locus is operably linked to mouse TCR constant region. in one embodiment, a genetically modified mouse is provided, wherein the mouse comprises in its genome an unrearranged T cell receptor (TCR) 0: le gene locus comprising at least one human Joc segment and at least one human Va segment, operably linked to a mouse TCRa constant gene sequence, and an unrearranged TCRB variable gene locus comprising at least one human JB segment, at least one human DB segment, and at least one human VB segment, operably linked to a mouse TCRB constant gene sequence. In one ic embodiment, the mouse ses in its genome an unrearranged TCRa le gene locus comprising a complete oire of human Ja segments and a complete repertoire of human Von segments, operably linked to a mouse TCRoc constant gene sequence, and an unrearranged TCRB variable gene locus comprising a complete repertoire of human JB ts, a complete repertoire of human DB segments, and a complete repertoire of human VB segments, operably linked to a mouse TCRB constant gene sequence. in some ments, the unrearranged TCRa variable gene locus comprising human TCRa variable region segments replaces endogenous mouse TCRa variable gene locus, and the unrearranged TCRB variable gene locus comprising human TCRB variable region segments replaces the endogenous mouse TCRB le gene locus. In some embodiments, the endogenous mouse Va and Jo: segments are incapable of rearranging to form a rearranged VOL/Jet sequence, and the endogenous mouse VB, DB, and JB segments are incapable of rearranging to form a rearranged /JB sequence. in some embodiments, the human V0: and Jet segments rearrange to form a rearranged human Von/Jo: sequence, and the human VB, DB, and J0 segments rearrange to form a rearranged human VB/DB/JB sequence.

In various embodiments, the non-human animals (e.g., s, e.g., mice or rats) described herein produce T cells that are capable of undergoing thymic development, ssing from DN1 to DN2 to DN3 to DN4 to DP and to CD4 or CD8 SP T cells. Such T cells of the non-human animal of the invention express cell e molecules typically produced by a T cell during a particular stage of thymic development (e.g., CD25, CD44, Kit, CD3, pToc, etc.). Thus, the non-human animals described herein express pToc complexed with TCRB at the DN3 stage of thymic development. The non—human animals described herein express T cells capable of undergoing thymic development to produce CD4+ and CD8+ T cells. Normally, in the thymus the physiological ratio of CD4+ to CD8+ T cells is between about 2:1 and 321. See, e.g., Ge and Stanley (2008) The O-fucose glycan in the ligand-binding domain of Notch 1 regulates embryogenesis and T cell development, Proc.

Natl. Acad. Sci. USA 105:1539-44. Thus, in one embodiment, the man s bed herein produce CD4+ and CD8+ T cells in the thymus at a ratio of between about 2:1 and 3:1 (CD4+:CD8+). in various embodiments, the non—human animals described herein produce T cells that are capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non—human animals described herein are capable of producing a normal repertoire of effector T cells, e.g., CTL (cytotoxic T lymphocytes), TH1, TH2, TREg, TH17, etc.

Thus, in these embodiments, the non—human animals described herein te effector T cells that fulfill different ons l of the particularT cell type, e.g., recognize, bind, and respond to foreign antigens. In various embodiments, the non-human animals described herein produce effector T cells that kill cells displaying peptide nts of cytosolic pathogens expressed in the context of MHC l molecules; recognize peptides derived from antigens degraded in intracellular vesicles and presented by MHC ll molecules on the surface of macrophages and induce macrophages to kill microorganisms; e cytokines that drive B cell differentiation; activate B cells to produce opsonizing antibodies; induce lial cells to produce chemokines that t neutrophils to infection sites; etc. ln onal embodiments, the non-human animals described herein comprise a normal number of CD3+ T cells in the periphery, e.g., in the spleen. In some embodiments, the percent of peripheral CD3+ T cells in the non—human animals described herein is the comparable to that of the wild type animals (i.e., animals comprising all endogenous TCR 2012/062065 variable region segments). In one embodiment, the non—human animals described herein comprise a normal ratio of splenic CD3+ T cells to total splenocytes. in other aspects, the non-human animals described herein are capable of ting a population of memory T cells in response an antigen of st. For example, the non—human animals generate both central memory T cells (Tcm) and effector memory T cells (Tern) to an antigen, e.g., antigen of interest (e.g., antigen being tested for vaccine development, etc.).

DN1 and DN2 cells that do not receive sufficient signals (e.g., Notch signals) may develop into B cells, myeloid cells (e.g., dendritic cells), mast cells and NK cells. See, e.g., Yashiro-Ohtani et al. (2010) Notch regulation of early thymocyte pment, Seminars in immunology 222261—69. in some embodiments, the non-human animals described herein develop normal numbers of B cells, myeloid cells (e.g., dendritic cells), mast cells and NK cells. In some embodiments, the man s described herein develop normal dendritic cell population in the thymus.

The predominant type of T cell receptors expressed on the surface of T cells is TCRa/B, with the minority of the cells expressing TCRé/y. In some embodiments of the invention, the T cells of the non—human animals comprising humanized TCRoc and/or [3 loci exhibit normal utilization of TCRoc/B and TCRa/y loci, e.g., utilization of TCRoc/{S and TCRé/y loci that is r to the wild type animal (e.g., the T cells of the non—human animals described herein express TCRa/B and TCRtS/y ns in comparable proportions to that expressed by wild type animals). Thus, in some embodiments, the non-human animals comprising humanized TCRoc/B and endogenous non—human TCRé/y loci exhibit normal utilization of all loci.

In addition to genetically engineered non-human animals described herein, a non-human embryo (e.g., a rodent embryo, e.g., mouse or a rat embryo) is also ed, wherein the embryo comprises a donor ES cell that is d from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein. in one aspect, the embryo comprises an E8 donor cell that comprises an unrearranged humanized TCR locus, and host embryo cells.

Also ed is a tissue, wherein the tissue is derived from a man animal (e.g., a mouse or a rat) as described , and expresses a humanized TCR polypeptide (e.g., TCRoc and/or TCRB, or TCRé, and/or TCRy polypeptide).

In addition, a non—human cell isolated from a non—human animal as described herein is provided. in one embodiment, the cell is an ES cell. in one embodiment, the cell is a T cell. In one embodiment, the T cell is a CD4+ T cell. in r embodiment, the T cell is a CD8+ T cell.

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

Also provided is a man cell that expresses a TCR protein comprising a human variable region and a non-human nt region. The TCR protein may comprise TCRa, TCRB, or a combination thereof. In one embodiment, the cell is a T cell, e.g., a CD4+ or a CD8+ T cell.

In one aspect, a man induced pluripotent cell comprising an unrearranged humanized TCR locus encoding a humanized TCR polypeptide as described herein is provided. In one ment, the d pluripotent cell is derived from a man animal as described herein. in one aspect, a hybridoma or quadroma is provided, derived from a cell of a non-human animal as described herein. in one embodiment, the non-human animal is a rodent, e.g., a mouse or rat.

Also provided is a method for making a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) described . The method for making a genetically modified man animal results in the animal whose genome comprises a zed unrearranged TCR locus (e.g., a humanized unrearranged TCRa, TCRfi, TCRé, and/or TCRy locus). in one embodiment, a method for making a genetically modified non—human animal (e.g., rodent, e.g., mouse or rat) that expresses a T cell receptor comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region on a surface of a T cell is ed, wherein the method comprises replacing in a first non—human animal an endogenous non—human TCRa variable gene locus with an unrearranged humanized TCRoc variable gene locus comprising at least one human Va t and at least one human Jot segment, wherein the zed TCRa variable gene locus is ly linked to endogenous TCRa constant region; replacing in a second non-human animal an endogenous non-human TCRfi variable gene locus with an unrearranged humanized TCRB variable gene locus comprising at least one human VB segment, one human DB segment, and one human JB segment, wherein the humanized TCRB variable gene locus is operably linked to endogenous TCRB constant region; and breeding the first and the second non- WO 63361 human animal to obtain a non—human animal that expresses a T cell receptor comprising a human variable region and a non—human constant region. In other embodiments, the invention provides methods of making a genetically modified non—human animal whose genome comprises a humanized unrearranged TCRa locus, or a non-human animal whose genome comprises a humanized unrearranged TCRB locus, generated according to the methods described herein. In various embodiments, the replacements are made at the endogenous loci. In some embodiments, the method utilizes one or more targeting constructs made using VELOCIGENE® technology, introducing the construct(s) into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples. In some embodiments, the ES cells are derived from a mouse that is a mix of 129 and C57BL/6 strains. In various embodiments, the method comprises ssive humanization strategy, wherein a construct comprising additional variable region segments is introduced into ES cells at each subsequent step of zation, ultimately resulting in a mouse sing a complete repertoire of human variable region segments (see, e.g., Fle. 3 and 7).

Thus, nucleotide constructs used for generating genetically engineered non- human animals described herein are also provided. In one , the nucleotide construct comprises: 5’ and 3’ homology arms, a human DNA fragment comprising human TCR variable region gene t(s), and a selection cassette flanked by recombination sites.

In one embodiment, the human DNA fragment is a TCRa gene fragment and it comprises at least one human TCRoc variable region segment. In another embodiment, the human DNA fragment is a TCRB fragment and it comprises at least one human TCRfi variable region gene segment. In one , at least one gy arm is a non-human homology arm and it is homologous to non-human TCR locus (e.g., non—human TCRa or TCRB locus).

A selection cassette is a tide sequence ed into a targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. A number of suitable ion tes are known in the art. Commonly, a selection cassette enables positive selection in the ce of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, Spec, etc.) In addition, a selection cassette may be flanked by recombination sites, which allow deletion of the selection cassette upon treatment with recombinase enzymes.

Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, tively, but others are known in the art.

In one embodiment, the selection cassette is located at the 5’ end the human DNA fragment. In another embodiment, the selection te is located at the 3’ end of the human DNA nt. In another embodiment, the selection cassette is located within the human DNA fragment, e.g., within the human . In another embodiment, the selection cassette is located at the on of the human and mouse DNA fragment.

] Various exemplary embodiments of the targeting strategy for generating genetically engineered non-human animals, the constructs, and the ing vectors used for the same are ted in Fle. 3, 4, 5, 7, and 8.

Upon completion of gene targeting, ES cells or genetically modified non-human animals are screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide (e.g., human TCR variable region ts). Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCT (e.g., real-time PCR using ®), fluorescence in situ hybridization, Northern blotting, flow try, Western is, immunocytochemlstry, immunohistochemistry, etc. in one e, non-human animals (e.g., mice) bearing the genetic modification of interest can be identified by screening for loss of mouse allele and/or gain of human allele using a modification of allele assay described in Valenzuela et al. (2003) High—throughput engineering of the mouse genome coupled with high-resolution sion analysis, Nature Biotech. 21(6):652—659.

Other assays that identify a specific nucleotide or amino acid sequence in the genetically modified animals are known to those skilled in the art.

The disclosure also provides a method of modifying a TCR variable gene locus (e.g., TCRoc, TCRB, TCRo, and/or TCRy gene locus) of a non—human animal to express a humanized TCR protein bed herein. In one embodiment, the invention provides a method of ing a TCR variable gene locus to express a humanized TCR protein on a surface of a T cell wherein the method comprises replacing in a non-human animal an endogenous non-human TCR variable gene locus with an ranged humanized TCR variable gene locus. In one embodiment wherein the TCR variable gene locus is a TCRa variable gene locus, the unrearranged humanized TCR variable gene locus comprises at least one human Voc segment and at least one human Jon segment. in one embodiment wherein the TCR variable gene locus is a TCRfi variable gene locus, the unrearranged humanized TCR variable gene locus comprises at least one human Vl3 segment, at least one human DB segment, and at least one human JB segment. ln various aspects, the unrearranged humanized TCR variable gene locus is operably linked to the corresponding endogenous non-human TCR constant region.

A humanized TCR protein made by a non-human animal (e.g., rodent, e.g., mouse or rat) as described herein is also provided, wherein the zed TCR n comprises a human variable region and a non—human constant region. Thus, the humanized TCR protein ses human complementary determining regions (i.e., human CDR1, 2, and 3) in its variable domain and a non—human constant region.

Although the Examples that follow describe a genetically engineered non—human animal whose genome comprises humanized TCRa and/or humanized TCRB variable gene locus, one skilled in the art would understand that a similar strategy may be used to produce genetically engineered animals whose genome comprises zed TCRB and/or TCRy variable gene locus. A genetically engineered non—human animal with humanization of all four TCR le gene loci is also provided.

Use of Genetically Modified TCR Animals In various embodiments, the cally modified non—human animals of the invention make T cells with humanized TCR molecules on their surface, and as a result, would recognize peptides presented to them by MHC complexes in a human-like manner.

The genetically modified non—human animals described herein may be used to study the development and function of human T cells and the ses of immunological tolerance; to test human vaccine ates; to generate TCRs with certain specificities for TCR gene therapy; to generate TCR libraries to e associated antigens (e.g., tumor associated antigens (TAAs); etc.

There is a growing st in T cell therapy in the art, as T cells (e.g., xic T cells) can be directed to attack and lead to destruction of antigen of interest, e.g., viral antigen, bacterial antigen, tumor antigen, etc, or cells that present it. Initial studies in cancer T cell therapy aimed at isolation of tumor infiltrating lymphocytes (TlLs; lymphocyte populations in the tumor mass that presumably comprise T cells reactive against tumor antigens) from tumor cell mass, expanding them in vitro using T cell growth factors, and transferring them back to the patient in a process called adoptive T cell transfer. See, e.g., Restifo et al. (2012) ve immunotherapy for cancer: harnessing the T cell response, Nature Reviews 122269-81; Linnermann et al. (2011) T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success, J. Invest. Dermatol. 131:1806—16. However, success of these therapies have thus far been limited to melanoma and renal cell carcinoma; and the TlL adoptive transfer is not specifically directed to defined tumor associated antigens . Linnermann et al., supra.

Attempts have been made to initiate TCR gene therapy where T cells are either selected or programmed to target an n of interest, e.g., a TAA. t TCR gene therapy relies on identification of sequences of TCRs that are ed to specific antigens, e.g., tumor ated ns. For example, Rosenberg and colleagues have published several studies in which they uced peripheral blood lymphocytes derived from a melanoma patient with genes encoding TCR and  chains specific for melanomaassociated antigen MART-1 epitopes, and used resulting expanded lymphocytes for adoptive T cell therapy. Johnson et al. (2009) Gene therapy with human and mouse T-cell ors mediates cancer regression and targets normal s 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. The MART-1 specific TCRs were isolated from patients that experienced tumor sion following TIL therapy. However, identification of such TCRs, particularly high-avidity TCRs (which are most likely to be therapeutically useful), is complicated by the fact that most tumor antigens are self antigens, and TCRs ing these antigens are often either deleted or s suboptimal affinity, due primarily to immunological tolerance.

In various embodiments, the present invention addresses this problem by providing genetically ered non-human animals comprising in their genome an unrearranged human TCR variable gene locus. The non-human animal described herein is capable of generating T cells with a diverse repertoire of humanized T cell receptors. Thus, the non-human s bed herein may be a source of a diverse repertoire of humanized T cell receptors, e.g., high-avidity humanized T cell receptors for use in adoptive T cell transfer.

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

In one ment, the invention provides a method of producing a human T cell receptor specific for an antigen of interest (e.g., a disease-associated antigen) comprising immunizing a non-human animal described herein with the antigen of interest; allowing the animal to mount an immune response; isolating from the animal a T cell ve to the n of interest; determining a nucleic acid sequence of a human TCR variable region expressed by the T cell; g the human TCR variable region into a nucleotide uct comprising a nucleic acid sequence of a human TCR constant region such that the human TCR variable region is operably linked to the human TCR nt region; and expressing from the uct a human T cell receptor specific for the antigen of interest. In one embodiment, the steps of isolating a T cell, determining a nucleic acid sequence of a human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide uct comprising a nucleic acid sequence of a human TCR constant region, and expressing a human T cell receptor are performed using standard techniques known to those of skill the art.

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

The antigen of interest may be any antigen that is known to cause or be associated with a disease or condition, e.g., a tumor associated antigen; an n of viral, bacterial or other pathogenic origin; etc. Many tumor associated antigens are known in the art. A selection of tumor associated ns is presented in Cancer Immunity (A Journal of the Cancer Research Institute) Peptide se (archive.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm). In some embodiments of the invention, the n of interest is a human antigen, e.g., a human tumor associated antigen. In some embodiments, the n is a cell type-specific intracellular antigen, and a T cell receptor is used to kill a cell expressing the antigen.

In one embodiment, provided herein is a method of identifying a T cell with specificity against an antigen of interest, e.g., a tumor associated antigen, sing immunizing a man animal described herein with the antigen of interest, allowing the animal to mount an immune response, and isolating from the non-human animal a T cell with icity for the n.

The t invention provides new methods for adoptive T cell therapy. Thus, provided herein is a method of treating or ameliorating a disease or condition (e.g., a cancer) in a subject (e.g., a mammalian subject, e.g., a human subject) comprising immunizing a non—human animal described herein with an antigen ated with the disease or condition, allowing the animal to mount an immune response, isolating from the animal a population of antigen-specific T cells, and infusing isolated n-specific T cells into the t. ln one embodiment, the invention provides a method of treating or ameliorating a disease or condition in a human subject, comprising immunizing the non—human animal described herein with an antigen of interest (e.g., a disease- or condition-associated antigen, e.g., a tumor associated antigen), allowing the animal to mount an immune response, isolating from the animal a population of antigen—specific T cells, determining the nucleic acid ce of a T cell receptor expressed by the antigen-specific T cells, g the nucleic acid sequence of the T cell receptor into an expression vector (e.g., a retroviral vector), introducing the vector into T cells derived from the subject such that the T cells express the antigen-specific T cell receptor, and infusing the T cells into the subject. In one embodiment, the T cell receptor nucleic acid sequence is further humanized prior to introduction into T cells derived from the subject, e.g., the sequence encoding the non- human nt region is modified to further resemble a human TCR constant region (e.g., the non-human constant region is replaced with a human constant ). in some embodiments, the disease or condition is cancer. in some embodiments, an antigen-specific T cell population is expanded prior to ng into the subject. ln some embodiments, the subject’s immune cell population is immunodepleted prior to the infusion. of antigen-specific T cells. In some embodiments, the antigen—specific TCR is a high avidity TCR, e.g., a high avidity TCR to a tumor associated n. In some embodiments, the T cell is a xic T cell. In other embodiments, the disease or condition is caused by a virus or a bacterium. in another embodiment, a disease or condition is an autoimmune disease. TREG cells are a subpopulation of T cells that maintain tolerance to self-antigens and prevent pathological self—reactivity. Thus, also provided herein are methods of treating autoimmune disease that rely on generation of antigen—specific TREG cells in the non-human animal of the invention described herein.

Also provided herein is a method of treating or ameliorating a disease or condition (e.g., a cancer) in a subject comprising ucing the cells affected by the disease or condition (e.g., cancer cells) from the subject into a non-human animal, allowing the animal to mount an immune se to the cells, isolating from the animal a population of T cells reactive to the cells, determining the nucleic acid sequence of a T cell receptor expressed by the T cells, cloning the T cell or sequence into a vector, introducing the vector into T cells derived from the subject, and ng the subject’s T cells harboring the T cell receptor into the subject.

Also provided herein is the use of a non-human animal as described herein to make nucleic acid sequences encoding human TCR variable domains (e.g., TCR 0t and/or {3 variable s). in one embodiment, a method is provided for making a nucleic acid sequence encoding a human TCR le domain, comprising immunizing a non-human animal as described herein with an antigen of interest, ng the man animal to mount an immune response to the antigen of interest, and obtaining therefrom a nucleic acid ce encoding a human TCR variable domain that binds the antigen of interest. in one embodiment, the method further comprises making a nucleic acid sequence ng a human TCR variable domain that is operably linked to a non-human TCR constant , comprising isolating a T cell from a non-human animal described herein and obtaining rom the nucleic acid sequence encoding TCR variable domain linked to TCR constant region.

Also provided herein is the use of a non-human animal as described herein to make a human therapeutic, comprising immunizing the non—human animal with an antigen of interest (e.g., a tumor associated antigen), allowing the non-human animal to mount an immune response, obtaining from the animal T cells reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized TCR protein that binds the n of interest, and employing the nucleic acid sequence(s) encoding a humanized TCR protein in a human therapeutic.

Thus, also provided is a method for making a human therapeutic, comprising immunizing a non—human animal as described herein with an antigen of interest, allowing the non-human animal to mount an immune response, ing from the animal T cells ve to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized T cell receptor that binds the antigen of interest, and employing the humanized T cell receptor in a human therapeutic.

In one embodiment, the human therapeutic is a T cell (e.g., a human T cell, e.g., a T cell derived from a human subject) harboring a nucleic acid ce of interest (e.g., ected or transduced or otherwise introduced with the nucleic acid of interest) such that the T cell expresses the humanized TCR protein with ty for an antigen of interest. In one aspect, a t in whom the therapeutic is ed is in need of therapy for a particular disease or ion, and the antigen is associated with the disease or ion.

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 the subject.

In another embodiment, the human therapeutic is a T cell receptor. In one ment, the therapeutic receptor is a soluble T cell receptor. Much effort has been expanded to generate soluble T cell receptors or TCR le regions for use therapeutic agents. Generation of soluble T cell receptors depends on obtaining rearranged TCR variable regions. One approach is to design single chain TCRs comprising TCRoc and TCRB, and, similarly to scFv immunoglobulin format, fuse them together via a linker (see, e.g., International Application No. ). The resulting sch, if analogous to scFv, would provide a thermally stable and soluble form of TCRa/B binding protein.

Alternative approaches included designing a soluble TCR having TCRfi constant domains (see, 6.9., Chung et al., (1994) onal domain single-chain T-ceII receptors, Proc.

Natl. Acad. Sci. USA. 91 :12654-58); as well as engineering a non—native disuifide bond into the ace between TCR constant domains (reviewed in Boulter and Jakobsen (2005) Stable, soluble, high-affinity, engineered T cell receptors: novel dy-like proteins for specific targeting of peptide antigens, CIinical and Experimental Immunoiogy 142:454—60; see also, US. Patent No. 7,569,664). Other formats of soluble T cell receptors have been described. The non-human animals described herein may be used to ine a ce of a T cell or that binds with high affinity to an antigen of interest, and uently design a soluble T cell receptor based on the sequence.

] A soluble T cell receptor derived from the TCR receptor sequence expressed by the non—human animal can be used to block the function of a protein of interest, e.g., a viral, bacterial, or tumor associated protein. Alternatively, a‘s‘bluble T cell receptor may be fused to a moiety that can kill an infected or cancer cell, e.g., a cytotoxic les (e.g., a chemotherapeutic), toxin, radionuclide, prodrug, antibody, etc. A soluble T cell receptor may also be fused to an immunomodulatory molecule, e.g., a cytokine, chemokine, etc. A soluble T cell receptor may also be fused to an immune inhibitory molecule, e.g., a molecule that inhibits a T cell from killing other cells harboring an antigen recognized by the T cell.

Such soluble T cell receptors fused to immune inhibitory molecules can be used, e.g., in blocking autoimmunity. Various exemplary immune inhibitory molecules that may be fused to a soluble T cell receptor are reviewed in Ravetch and Lanier (2000) immune inhibitory Receptors, Science 290284-89, incorporated herein by reference.

The present invention also provides s for studying immunological response in the context of human TCR, including human TCR rearrangement, T cell development, T cell activation, immunological tolerance, etc.

Also provided are methods of testing vaccine candidates. in one embodiment, provided herein is a method of determining whether a vaccine will activate an immunological response (e.g., T cell proliferation, cytokine e, etc), and lead to tion of effector, as well as memory T cells (e.g., central and or memory T cells).

The invention will be r illustrated by the ing noniimiting examples.

These Examples are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to, limit its scope in any way. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless indicated othen/vise, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Celsius, and pressure is at or near atmospheric.

Example 1. Generation of Mice with Humanized TCR le Gene Loci Mice comprising a deletion of nous TCR (a or {3) variable loci and replacement of nous V and J or V, D, and J segments are made using 2012/062065 VELOClGENE® genetic engineering technology (see, 9.9., US Pat. No. 6,586,251 and Valenzuela, D.M., et al. (2003) High-throughput engineering of the mouse genome coupled with esolution expression analysis. Nat. Biotech. 21(6): 652-659), wherein human sequences derived from BAC ies using bacterial homologous recombination are used to make large targeting vectors (LTVECs) comprising genomic fragments of human TCR variable loci flanked by targeting arms to target the LTVECs to endogenous mouse TCR variable loci in mouse ES cells. LTVECs re linearized and oporated into a mouse ES cell line according to Valenzuela et al. ES cells are selected for hygromycin or neomycin resistance, and screened for loss of mouse allele or gain of human allele.

Targeted ES cell clones are introduced into 8-cell stage (or earlier) mouse embryos by the VELOCIMOUSE® method (Poueymirou, W.T. et al. (2007). F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat. Biotech. 25: ). VELOClMlCE® (F0 mice fully derived from the donor ES cell) bearing humanized TCR loci are identified by screening for loss of endogenous TCR le allele and gain of human allele using a modification of allele assay (Valenzuela et al.). F0 pups are genotyped and bred to homozygosity. Mice homozygous for humanized TCRa and/or TCRB variable loci (e.g., comprising a subset of human TCRoc and/or TCRB variable segments) are made and phenotyped as described herein.

] All mice were housed and bred in the specific pathogen-free facility at Regeneron ceuticals. All animal experiments were ed by lACUC and Regeneron ceuticals.

Example 2: Progressive Humanization of TCRa Variable Locus 1.5 megabases of DNA at mouse TCRa locus ponding to 110 V and 60 J mouse segments was replaced with 1 megabase of DNA corresponding to 54V and 61 J segments of human TCRoc using a progressive humanization strategy summarized in Fle. 2 and 3. Junctional nucleic acid sequences of various targeting vectors used for progressive humanization strategy of TCRoc locus are summarized in Table 2, and included in the Sequence Listing.

Table 2: Junctional Nucleic Acid Sequences for Various TCRa Locus Targeting Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRoc variable locus and the 5’ end of loxP-Ub—Hyg-onP cassette.

Description Junctional nucleic acid sequence between the 3’ end of loxP-Ub- Hyg-onP cassette and the 5’ end of human 40-TCRVoc41— TCRJa1 insertion, including AsiSl site.

Junctional nucleic acid ce between the 3’ end of human TCRVa40-TCRVoc41—TCRJoc1 insertion and the 5’ end of the mouse sequence downstream of the human TCRa variable locus, including Notl site.

Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRa variable locus and the 5’ end of loxP-Ub—Neo—onP cassette.

Junctional c acid sequence between the 3’ end of onP-Ub— Neo-loxP cassette and the 5’ end of human TCRch35—TCRVa39 insertion, including AsiSl site.

Junctionai nucleic acid sequence between the 3’ end of mouse sequence am of the TCRoc variable locus and the 5’ end of frt—ng-Hyg—frt cassette.

Junctional nucleic acid sequence n the 3’ end of frt—ng— Hyg-frt te and the 5’ end of human TCRVocZZ—TCRVa34 insertion, including AsiSl site.

Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRa variable locus and the 5’ end of onP-Ub-Neo—IOXP cassette. onal nucleic acid ce between the 3’ end of loxP-Ub- Neo-onP cassette and the 5’ end of human TCRVa13—2- TCRch21 insertion, including AsiSl site.

Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRa variable locus and the 5’ end of onP—Ub—Hyg—IOXP cassette. onal c acid sequence between the 3’ end of loxP—Ub— Hyg-loxP cassette and the 5’ end of human TCRVaG—TCRVa8-5 insertion, including AsiSl site.

Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream and the TCRa variable locus to the 5’ end of loxP-Ub-Neo-onP cassette.

Junctional nucleic acid sequence between the 3’ end of onP-Ub- Neo-onP cassette and the 5’ end of human TCRVai-t-TCRVa5 insertion, including AsiSl site.

Human TCRa le region segments are numbered as in lMGT database. At least 100 bp at each junction (about 50 bp from each end) are included in the Sequence Listing.

Specifically, as trated in , DNA from mouse BAC clone RP23-6A14 (Invitrogen) was modified by gous recombination and used as a targeting vector (MAlD 1539) to replace TCRAJ1-TCRAJ28 region of the endogenous mouse TCRa locus with a Ub-hygromycin cassette followed by a onP site. DNA from mouse BAC clone RP23—1 17i19 (lnvitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1535) to replace ~15kb region surrounding (and including) TCRAV1 of the endogenous mouse TCRoc and 6 locus with a PGK—neomycin cassette followed by a onP site. ES cells g a double-targeted chromosome (i.e., a single endogenous mouse TCRoc locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMANTM) known in the art. Modified ES cells were treated with CRE recombinase, thereby mediating the deletion of the region between the two onP sites (i.e., the region ting of the endogenous mouse TCRoc locus from TCRAV1 to TCRAJ1) and leaving behind only a single onP site, neomycin te and the mouse constant and enhancer regions. This gy resulted in generation of a deleted mouse TCR alts locus (MAID 1540).

The first human targeting vector for TCRa had 191,660 bp of human DNA from the CTD2216p1 and CTD2285mO7 BAC clones (lnvitrogen) that contained the first two consecutive human TCRocV gene segments (TRAV4O & 41) and 61 TCRaJ (50 functional) gene ts. This BAC was modified by homologous recombination to contain a Not1 site 403 bp downstream (3’) of the TCRocJ1 gene segment for ligation of a 3’ mouse homology arm and a 5’ AsiSI site for ligation of a 5’ mouse homology arm. Two different homology arms were used for ligation to this human fragment: the 3’ homology arm contained endogenous mouse TCRa sequences from the RP23-6A14 BAC clone and the 5’ homology arm contained endogenous TCRa ce 5’ of mouse TCchV from mouse BAC clone RP23-117i19. This mouse-human chimeric BAC was used as a targeting vector (MAID 1626) for making an initial insertion of human TCRoc gene segments plus an upstream onp-ub—hygromycin-loxp cassette at the mouse TCRoc loci (). The junctional c acid sequences (SEQ ID NOs: 1-3) for the MAID 1626 targeting vector are described in Table 2.

Subsequently, a series of human targeting vectors were made that utilized the same mouse 5’ arm that contained endogenous TCRa sequence 5’ of mouse TCRaV from mouse BAC clone RP23-117i19 with ating onP-neomycin-onP and loxP-hygromycin- loxP (or gromycin—f/t for MAID 1979) ion tes.

To generate a human TCRa mini—locus containing a total 8 human TCRaV (7 functional) and 61 human TCRdJ (50 functional) gene segments, DNA from human BAC clone RP11-349p11 (lnvitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1767) (). This added 104,846 bp of human DNA containing the next 6 (5 functional) utive human TCRaV gene segments (TRAV35 to TRAV39) and a 5’ loxP—ub-neomycin—loxP cassette. Resulting TCRq locus contained a 5’ loxp-ub-neomycin-JOXP cassette plus a total of 8 human TCRqV (7 functional) and 61 human TCRaJ gene segments operabiy linked to mouse TCRq nt genes and enhancers. The junctional nucleic acid ces (SEQ ID NOs: 4 and 5) for the MAID 1767 targeting vector are described in Table 2.

] To generate a human TCRq mini-locus ning total of 23 human TCRocV (17 onal) and 61 human TCRqJ gene segments, DNA from mouse BAC clone ning from 5’ to 3’: a unique I—Ceul site, a 20 kb mouse TCRA arm 5’ of the mouse TCRA locus to be used for homologous recombination into ES cells, and a onP—Ub—Hyg~/oxP te in reverse orientation, was modified by bacterial homologous recombination to contain from 5’ to 3’: a unique I—Ceul site, a 20 kb mouse TCRA arm 5’ of the mouse TCRA locus, an frt— pgk-Hyg-frt cassette, and a unique AsiSI site. DNA from human BAC clone RP11-622020 (Invitrogen), harboring human TCRaV22-V34 was modified by homoiogous recombination to contain a Spec te flanked by unique I-CeuI and AsiSI sites. Subsequently, the Spec cassette in the ed human BAC clone was replaced by the sequence comprised between the l-CeuI and AsiSI sites in the modified mouse BAC clone by standard restriction digestion/ligation techniques. The resulting targeting vector (MAID 1979; ) added 136,557 bp of human DNA that contained the next 15 (1O functional) consecutive human TCRqJ gene ts (TRAV22 to TRAV34) and a 5’ frt—pgk—Hyg-fn‘ cassette. Resuiting TCch locus contained a 5’ frt-pgk-Hyg-frt cassette plus a total of 23 human TCRocV (17 functional) and 61 human TCRocV gene segments operably linked to mouse TCRoc constant genes and enhancers. The onaI nucIeic acid sequences (SEQ ID NOs: 6 and 7) for the MAID 1979 targeting vector are described in Tabie 2.

To generate human TCRa mini-locus containing a totaI of 35 human TCRaV (28 functional) and 61 human TCRaJ gene segments, DNA from human BAC cIone CTD2501— k5 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1769) (). This added 124,118 bp of human DNA that contained the next 12 (11 functionaI) consecutive human TCRqV gene segments (TRAV13-2 to ) and a 5’ loxp-ub-neomycin-loxP cassette. Resulting TCRa locus contained a 5’ loxp-ub—neomycin- loxP cassette plus a total of 35 human TCRocV (28 functional) and 61 human TCRqJ gene segments operably linked to mouse TCRoc constant genes and enhancers. The junctional nucIeic acid sequences (SEQ ID NOs: 8 and 9) for the MAID 1769 targeting vector are described in Table 2.

WO 63361 To generate a human TCRa mini-locus containing total of 48 human TCRaV (39 functional) and 61 human TCRaJ gene segments, DNA from human BAC clone RP11- 92F11 (lnvitrogen) was modified by homologous recombination and used as a targeting vector (MAlD 1770) (HS. 4F). This added 145,505 bp of human DNA that contained the next 13 (11 functional) consecutive human TCRaJ gene segments (TRAV6 to TRAV8.5) and a 5’ onp-ub—hygromycin-loxP cassette. ing TCRa locus contains a 5’ loxp—ub— hygromycin-onP cassette plus a total of 48 human TCRaV (39 functional) and 61 human TCRaJ gene segments operably linked to mouse TCRa constant genes and enhancers.

Thejunctional nucleic acid ces (SEQ ID N05: 10 and 11) for the MAID 1770 targeting vector are described in Table 2.

To generate a human TCRa mini-locus containing total of 54 human TCRorV (45 functional) and 61 human TCRaJ gene segments, DNA from human BAC clone RP11- 780M2 (lnvitrogen) was modified by homologous ination and used as a ing vector (MAlD 1771) (). This added 148,496 bp of human DNA that contained the next 6 (6 functional) consecutive human TCRaV gene ts (TRAV1—1 to TRAV5) and a ’ onp-ub—neomycin-onP cassette. Resulting TCRa locus contains a 5’ onp-ub—neomycin- onP cassette plus a total of 54 human TCRaV (45 functional) and 61 human TCRaJ gene segment operably linked to mouse TCRa constant genes and enhancers. The junctional nucleic acid sequences (SEQ ID N05: 12 and 13) for the MAlD 1771 targeting vector are described in Table 2.

In any of the above steps, the selection cassettes are removed by deletion with Cre or Flp recombinase. In addition, human TCRa locus may be introduced as depicted in Example 3: Progressive Humanization of TCRfi Variable Locus 0.6 megabases of DNA at mouse TCRfS locus corresponding to 33 V, 2 D, and 14 J mouse ts were ed with 0.6 megabases of DNA corresponding to 67 V, ZD, and 14 J segments of human TCRfi using a progressive humanization strategy summarized in Fle 6 and 7. Junctional nucleic acid sequences of various targeting s used for progressive humanization strategy of TCRB locus are summarized in Table 3, and included in the Sequence Listing.

Table 3: Junctional Nucleic Acid Sequences for s TCRB Locus Targeting Vectors MAID SEQ lD Description NO. NO 14 Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRB le locus y the am mouse trypsinogen genes) and the 5’ end of frt-Ub-Neo— fn‘ cassette.

Junctional nucleic acid sequence between the 3’ end of frt-Ub- Neo-frt cassette and the 5’ end of human TCRVBtB—TCRVBZQ-t insertion.

Junctional c acid sequence between the 3’ end of human TCRVB18-TCRV529-1 insertion and the 5’ end of the mouse sequence downstream of the mouse TCRVlS segments (nearby downstream mouse trypsinogen genes).

Junctional nucleic acid sequence between 3’ of the downstream mouse trypsinogen genes and the 5’ end of human TCRD(31— TCRJB1TCRJBt—6 ion, ing lceul site. onal nucleic acid sequence between the 3’ end of human TCRDBt-TCRJB1TCRJBt-6 insertion and the 5’ end of loxP— Ub—Hyg-loxP cassette.

Junctional nucleic acid sequence between the 3’ end of - Hyg-loxP cassette and the 5’ end of mouse ce nearby the mouse CB1 gene.

Junctional nucleic acid sequence between the 3’ end of the mouse sequence nearby the mouse C(31 gene and the 5’ end of human TCRDBZ—TCRJBZ-t—TCRJBZJ insertion, including Notl site. 21 Junctional nucleic acid sequence between the 3’ end of human TCRDfiZ-TCRJBZTCRJBZ-7 insertion and the 5’ end of the mouse sequence downstream of the TCRfi‘» variable locus y the 032 mouse sequence). 22 Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRB variable locus (nearby the upstream mouse trypsinogen genes) and the 5’ end of frt-Ub-Hyg- 1791 frt cassette. 3 Junctional nucleic acid sequence between the 3’ end of frt-Ub— Hyg-frt cassette and the 5’ end of human TCRVfiB—5-TCRV517 insertion. 24 Junctional nucleic acid sequence between the 3’ end of mouse sequence upstream of the TCRfs variable locus (nearby the upstream mouse nogen genes) and the 5’ end of frt-Ub-Neo- 1792 frt cassette.

Junctional nucleic acid sequence between the 3’ end of frt-Ub- H o-frt cassette and the 5’ end of human TCRV 1—TCRV 12—2 SEQ ID Description NO. NO insertion.

Junctional nucleic acid sequence between the 3’ end of mouse sequence nearby the mouse CB2 gene and the 5’ end of the human TCRBV30 exon 2 sequence. 6192 Junctional nucleic acid sequence between the 3’ end human TCRBV30 exon 1 sequence and the 5’ end of mouse sequence downstream of TCR|3 locus.

Human TCRB variable region ts are numbered as in lMGT database. At least 100 bp at each junction (about 50 bp from each end) are included in the Sequence Listing.

Specifically, DNA from mouse BAC clone RP23—153p19 (lnvitrogen) was modified by homologous recombination and used as a targeting vector (MAlD 1544) to replace 17kb region (including TCRBV30) just upstream of the 3’ trypsinogen gene cluster in the endogenous mouse TCRB locus with a PGK-neo cassette followed by a onP site (FlG. 8A). DNA from mouse BAC clone RP23—461h15 (lnvitrogen) was modified by gous recombination and used as a targeting vector (MAlD 1542) to replace 8355 bp region (including TCRBV2 and TCRBV3) downstream of 5’ trypsinogen gene cluster in the endogenous mouse TCRB locus with a Ub-hygromycin cassette ed by a onP site. ES cells bearing a double~targeted chromosome (Le, a single endogenous mouse TCRB locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (9.9., TAQMANTM) known in the art. Modified ES cells were treated with CRE recombinase, mediating the on of the region n the 5’ and 3’ loxP sites (consisting of the endogenous mouse TCRB locus from TCRBV2 to 0) and leaving behind only a single onP site, hygromycin cassette and the mouse TCRBDs, TCRBJs, constant, and enhancer sequences. One mouse TCRVfi was left am of the 5’ cluster of nogen genes, and one mouse TCRBfS was left downstream of the mouse Eff», as noted in Fig 8A.

The first human targeting vector for TCRB had 125,781 bp of human DNA from the CTD2559j2 BAC clone (lnvitrogen) that ned the first 14 consecutive human TCRfiV gene segments (TRBV18—TRBV29—1). This BAC was modified by gous recombination to contain a 5’ AsiSl site and a 3’ Ascl site for ligation of a 5’ and 3’ mouse homology arms. Two different homology arms were used for on to this human fragment: one set of homology arms contained endogenous TCRf3 sequence surrounding the downstream mouse trypsinogen genes from the RP23—1 53p19 BAC clone and another set ned endogenous TCRfi ce surrounding the upstream mouse trypsinogen genes from mouse BAC clone RP23-461h15. This mouse-human chimeric BAC was used as a targeting vector (MAID 1625) for making an initial insertion of human TCRfi gene ts plus an upstream frt-ub-neomycin-frt te at the mouse TCRp locus, and resulted in a human TCRB mini-locus containing 14 human (8 functional) TCRBV (FIG. BB).

Thejunctional nucleic acid sequences (SEQ ID NOs: 14-16) for the MAID 1625 targeting vector are described in Table 3.

In order to replace mouse TCRfi D and J segments with human TCRB D and J segments, DNA from mouse BAC clone RP23—302p18 (lnvitrogen) and from human BAC clone RP11-701D14 (lnvitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1715) into the ES cells that contained the TCRBV mini-locus described above (i.e., MAID 1625). This modification replaced ~18540 bp region (from 100 bp downstream of the polyA of the 3’ trypsinogen genes to 100bp downstream from the J segments in the D2 cluster which included mouse TCRBD1-J1, mouse constant 1, and mouse TCRBD2-J2) in the endogenous mouse TCRB locus with ~25425 bp of sequence containing human TCRBD1-J1, loxP Ub-hygromycin-onP cassette, mouse constant 1, human TCRBD2—J2 ((i)). ES cells bearing a double-targeted chromosome (Le, a single endogenous mouse TCRfi locus targeted with both ing vectors) were confirmed by karyotyping and screening methods (e.g., TAQMANTM) known in the art. Modified ES cells were d with CRE recombinase thereby mediating the on the hygromycin cassette leaving behind only a single onP site downstream from human J segments in D1J cluster ((ii)). Thejunctional nucleic acid sequences (SEQ ID NOs: 17-21) for the MAID 1715 targeting vector are described in Table 3.

] Subsequently, a series of human targeting vectors were made that utilized the same mouse 5’ arm that contained endogenous TCRB sequence surrounding the upstream mouse trypsinogen genes from mouse BAC clone RP23-461h15 with alternating selection cassette.

] To generate a human TCRB mini-locus containing a total 40 human TCRfiV (30 functional) and the human TCRB D and J segments, DNA from human BAC clones RP11—134h14 and RP11-785k24 (lnvitrogen) was modified by homologous recombination and combined into a targeting vector (MAID 1791) using standard bacterial gous recombination, restriction digestion/ligation, and other cloning techniques.

Introduction of the MAID 1791 targeting vector ed in addition of 198,172 bp of human DNA that ned the next 26 (22 onal) consecutive human TCRBV gene segments (TRBV6—5 to TRBV17) and a 5’ frt—ub—hygromycin-frt cassette. Resulting TCRp locus contained a 5’ frt—ub-hygromycin—frt cassette plus a total of 40 human TCR|3V (30 onal) and human TCRB D and J gene segments ly linked to mouse TCRB constant genes and enhancers (). The junctional nucleic acid sequences (SEQ ID NOs: 22 and 23) for the MAID 1791 targeting vector are described in Table 3.

To generate a human TCRB mini—locus containing a total 66 human TCRBV (47 functional) and the human TCRB D and J segments, DNA from human BAC clone RP11-902B7 (lnvitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1792). This resulted in addition of 2 bp of human DNA that contained the next 26 (17 functional) consecutive human TCRBV gene segments (TRBV1 to TRBV12-2) and a 5’ —neomycin—fn‘ cassette. Resulting TCRB locus contained a 5’ fri— ub—neomycin-fn‘ cassette plus a total of 66 human TCRBV (47 onal) and human TCRB D and J gene segments ly linked to mouse TCRB constant genes and enhancers. (). The junctional nucleic acid sequences (SEQ ID N05: 24 and 25) for the MAID 1792 targeting vector are described in Table 3.

In any of the above steps, the selection cassettes are removed by deletion with Cre or Flp recombinase. For example, as depicted in MAID 1716 corresponds to MAID 1715 with the hygromycin cassette deletion.

Finally, a human TCRB mini-locus containing a total 67 human TCRBV (48 functional) and the human TCRfi D and J segments was generated. Mouse TCRBV31 is located ~94 kb 3’ of TCRBCZ (second TCRB constant region ce) and is in the opposite orientation to the other TCRBV ts. The equivalent human V segment is TCRBV30, which is located in a similar position in the human TCRB locus.

To humanize TCRBV31, the mouse BAC clone containing mouse TCRBV31, was modified by bacterial homologous recombination to make LTVEC MAID 6192 (). The entire coding , beginning at the start codon in exon 1, the intron, the 3’ UTR, and the recombination signal sequences (RSS) of TCRBV31 were replaced with the homologous human TCRBV30 sequences. The 5’ UTR was kept as mouse sequence. For selection, a self-deleting cassette (lox2372-Ubiquitin promoter-Hyg-PGKpolyA—Protamine promoter-Cre- SV40polyA-on2372) was inserted in the intron (72 bp 3’ of exon 1,289 bp 5’ of exon 2). For city, FIGS. 7 and 8 depict the selection cassette 3’ of the hTCRBV30, while it was engineered to be located in the intron n exon 1 and exon 2 of the 3O gene.

The protamine promoter g Cre expression is transcribed ively in post—meiotic spermatids, so the cassette is “self-deleted” in the F1 generation of mice.

The junctional nucleic acid sequences (SEQ ID N03: 26 and 27) for the MAID 6192 targeting vector are described in Table 3. MAID 6192 DNA is electroporated into MAID1792 ES cells. ES cell clones are selected for hygromycin-resistance and screened for loss of mouse TCRB31 allele and gain of human TCRB30 allele.

Similar engineering strategy is used to optionally delete the remaining 5’ mouse TCRB V segment.

Example 4: Generation of TCRa/TCRfi Mice At each step of progressive humanization of TCRa and TCRfi loci, mice homozygous for humanized TCRa variable locus may be bred with mice homozygous for humanized TCRB variable locus to form progeny sing humanized TCRa and TCRB variable loci. Progeny are bred to homozygosity with respect to humanized TCRa and humanized TCRfi loci.

In one embodiment, mice gous for humanized TCRoc variable locus sing 8 human Von and 61 human Jet (MAID 1767; “1767 H0”) were bred with mice homozygous for humanized TCRB variable locus comprising 14 human VB, 2 human DB, and 14 human J8 (MAID 1716; “1716 H0”). Progeny were bred to homozygosity with respect to both humanized loci.

Example 5: Splenic T cell tion in Mice gous for zed TCRot and/or TCRfl Locus Spleens from wild type (WT) mice; mice with d mouse TCRa locus (“MAID1540”, see ; mice gous for human TCRa locus (“MAID 1767”, see ; mice with deleted TCRfi V segments with the exception of two remaining mouse V segments (“MAID1545”, see ; mice homozygous for human TCR{3 locus, also comprising the two remaining mouse V segments (“MAID 1716”, see ; and mice homozygous for both human TCRoc and TCRB loci, with TCRB locus also comprising the two remaining mouse V segments (“MAID 1767 1716”) were perfused with Collagenase D (Roche Bioscience) and erythrocytes were lysed with ACK lysis buffer, followed by washing in RPMI medium.

Splenocytes from a single WT, MAID 1540, 1767, 1545, 1716, and 1716 1767 representative animal were evaluated by flow try. Briefly, cell suspensions were made using standard methods. 1x106 cells were ted with anti—mouse CD16/CD32 (2.4G2, BD) on ice for 10 minutes stained with the appropriate cocktail of antibodies for 30 minutes on ice. Following staining, cells were washed and then fixed in 2% formaldehyde.

Data acquisition was performed on an LSRll/Cantoll/LSRFortessa flow ter and analyzed with FlowJo.

WO 63361 For staining of splenocytes, anti—mouse FlTC-CD3 (17A2, BD) was used. As demonstrated in mice with human TCR segments were able to produce significant numbers of CD3+ T cells, while mice with TCRd mouse locus deletion did not. Mice with TCRB locus deletion also produced CD3+ T cells, presumably due to utilization of the ing 3’ mouse V segment (see below).

Example 6: Thymic T Cell Development in Mice Homozygous for Humanized TCRa and/or TCRfi Locus To determine whether mice homozygous for humanized TCRoc and/or TCRB locus exhibited normal T cell development in the thymus, cytes from four of each WT, 1767 H0, 1716 H0, and 1716 H0 1767 H0 age matched animals (7-10 weeks old) were used in flow cytometry to te production of T cells at various developmental stages, as well as to evaluate frequency and absolute number of each of DN, DP, CD4 SP, and CD8 SP T cells.

Cell type determinations were made based on the presence of CD4, CD8, CD44, and CD25 cell surface markers as summarized in Table 1. Correlation n cell type designation and expression of cell surface markers in the thymus is as follows: double negative (DN) cells (CD4— CD8-), double positive (DP) cells (CD4+ CD8+), CD4 single positive cells (CD4+ CDS-), CD8 singie positive cells (CD4— CD8+), double ve 1/DN1 cells (CD4- CD8-, CD25- CD44+), double negative 2/DN2 cells (CD4- CD8—, CD25+ CD44+), double negative 3/DN3 cells (CD4- CD8—, CD25+ CD44-), double negative 4/DN4 cells (CD4- CD8-, CD25- CD44-).

Thymocytes were evaluated by flow cytometry. Briefly, cell suspensions were made using standard methods. Flow cytometry was conducted as described in Example 5.

Antibodies used were: anti—mouse PE-CD44 (lM7, BioLegend), PeCy7-CD25 (PC61, BioLegend), APC-H7-CD8a (53-67, BD), and APC-CD4 (GK1.5, eBioscience).

As shown in Fle 10 and 11, mice homozygous for humanized TCRoc, TCRB, and both TCRa and TCRp were able to produce a DN1, DN2, DN3, DN4, DP, CD4 SP, and CDS SP T cells, indicating that the T cells produced from the humanized loci are capable of undergoing T cell development in the thymus.

Example 7: c T Cell entiation in Mice Homozygous for Humanized TCRa and/or TCRB Locus ] To determine whether mice homozygous for humanized TCRa and/or TCRfi locus exhibited normal T cell differentiation in the periphery (e.g., ), four of each WT, 1767 H0, 1716 H0, and 1716 H0 1767 H0 age matched animals (7-10 weeks old) were WO 63361 used in flow cytometry to evaluate production of various T cell types in the spleen (CD3+, CD4+, CD8+, T naive, Tom, and Teff/em), as well as to evaluate the absolute number of each T cell type in the spleen.

Cell type determinations were made based on the presence of CD19 (8 cell marker), CD3 (T cell marker), CD4, CD8, CD44, and CD62L (L-selectin) cell surface markers. Correlation between cell type designation and expression of cell surface s in the spleen is as follows: T cells (CD3+), CD4 T cells (CD3+ CD4+ CD8—), CD8 T cells (CD3+ CD4- CD8+), CD4 effector/effector memory T cells (CD3+ CD4+ CD8— CD62L- , CD4 central memory T cells (CD3+ CD4+ CD8- CD62L+ CD44+), CD4 naive T cells (CD3+ CD4+ CD8- CD62L+ CD44-), CD8 effector/effector memory T cells (CD3+ CD4- CD8+ CD62L- CD44+), CD8 central memory T cells (CD3+ CD4— CD8+ CD62L+ , CD8 naive T cells (CD3+ CD4- CD8+ CD62L+ CD44-).

Splenocytes were evaluated by flow cytometry. y, cell sions were made using standard methods. Flow cytometry was conducted as described in Example 5.

Antibodies used were: ouse FITC-CD3 (17A2, BD), PE-CD44 (IM7, BioLegend), Cy5.5—CD62L (Mel-14, end), APC-H7-CD8a (53-67, BD), APC—CD4 (GK1.5, ience), and V450-CD19 (1 D3, BD).

As shown in FIGS. 12-14, T cells in the spleen of mice homozygous for humanized TCRd, TCRB, and both TCRoc and TCRB were able to undergo T cell differentiation, and both CD4+ and CD8+ T cells were present. In addition, memory T cells were detected in the spleens of the mice tested.

Example 8: Utilization of Human V Segments in Humanized TCR Mice Expression of human TCR[3 V segments was evaluated on protein and RNA level using flow cytometry and TAQMANTM real-time PCR, respectively, in mice homozygous for humanized TCRB locus (1716 H0) and mice homozygous for both humanized TCRB and TCRa locus (1716 H0 1767 H0).

For flow cytometry, c T cell were prepared and analysis conducted as described in Example 5. For flow cytometry, TCRB repertoire kit (lOTEST® Beta Mark, Beckman Coulter) was used. The kit contains anti-human antibodies specific for a number of human TCRBVs, e.g., hTRBV—18, -19, -20, -25, -27, -28, and —29.

Results are summarized in . The tables presented in A (CD8 T cell overlay) and B (CD4 T cell y) demonstrate that splenic T cells in both 1716 H0 and 1716 H0 1767 H0 mice utilized a number of human TCRB V segments. The wild type mice were used as a negative control.

] For real—time PCR, total RNA was purified from spleen and thymus using MAGMAXTM-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies) according to manufacturer's ications. Genomic DNA was d using MAGMAXTMTURBOTMDNase Buffer and TURBO DNase from the MAGMAX kit listed above (Ambion by Life Technologies). mRNA (up to 2.5ug) was reverse—transcribed into cDNA using SUPERSCRIPT® VILOTM Master Mix (Invitrogen by Life Technologies). cDNA was diluted to nL, and 10-25ng cDNA was ed with the TAQMAN® Gene Expression Master Mix (Applied Biosystems by Life Technologies) using the ABI 79OOHT Sequence Detection System (Applied Biosystems), using the primers and Taqman MGB probes ed Biosystems) or BHQ1/BHQ-Plus probes (Biosearch Technologies) depicted in Table 4 according to manufacturer’s instructions. The relative expression of each gene was normalized to the murine TCR beta constant 1 (TRBCl) l.

Table 4: Primers and Probes Used for Detecting RNA Expression of TCRB V Segments and Constant Region in Humanized TCR Mice by Real-Time PCR (TAQMANW) TRBV Sense Primer (5’-3’) Antisense Primer (5’-3’) Probe (5’-3’) E-E-E hTRBV CCGGCGTCATGC 28 GGGCTGCATCTCAGT 29 18 AGAA CTTGC CACCTGGTCAGG AGGAGG —MGB hTRBV GGAATCACTCAG ATTCTGTTCACAACTC 32 19 TCCCCAAAG A TCAGAAAGGAAG GACAGAAT-MGB hTRBV CGAGCAAGGCGT 34 GGACAAGGTCAGGCT 35 FAM- CGAGAA TGCA ACAAGTTTCTCAT CAACC- MGB TGTTACCCAGAC 37 TCTGAGAACATTCCA FAM- CCCAAGGA GCATAATCCT TAGGATCACAAAG ACAGGAA-MGB GACCCT 4O TGCTGGCACAGAGGT FAM- GGAGTCT ACTGAGA CAGGCCCTCACAT AC- MGB AAGCCCAAGTGA ATTCTGAGAACAAGT FAM— CCCAGAA CACTGTTAACTTC CTCATCACAGTGA CTGGAA- MGB 2012/062065 TRBV Sense Primer (5’-3’) Antisense Primer (5’-3’) Probe (5’-3’) Sequence SED Sequence SED ce 559 ID NO ID NO ID No hTRBV GTGAAAGTAACC 46 ATCCTGGACACATTC 47 FAM- 48 28 CAGAGCTCGAG CAGAAAAAC ATATCTAGTCAAA AGGACGGGA- TGTCATTGACAA TGCTGTCTTCAGGGC FAM- 51 GTTTCCCATCAG TCATG TCAACTCTGACTG TGAGCA— MGB AGCCGCCTGAGG GCCACTTGTCCTCCT FAM— 54 1 GTCTCT CTGAAAG TACCTTCTGGCAC AATCCTCGCA — As demonstrated in FIGs. 16A—B, mice homozygous for humanized TCRfi locus (1716 H0) and mice homozygous for both humanized TCRB and TCRa locus (1716 H0 1767 H0) exhibited RNA expression of various human TCRB segments in both the thymus and the spleen. Mice also exhibited RNA expression of mouse TRBV—1 and TRBV—31 segments (data not shown), but no mouse TRBV-1 protein was detected by flow cytometry (data not shown).

Mouse TRBV-31 segment is replaced with human TRBV—30 segment as demonstrated in , and mice are generated from MAID 6192 ES cells as bed . The spleens and thymi of ing homozygous animals are tested for utilization of human VB segments, including TRBV-30, by flow cytometry and/or real—time PCR as described herein. mTRBV—1 segment may also be deleted.

Example 9: T Cell Development in Mice Homozygous for 23 Human TCR Va Segments Homozygous humanized TCRoc mice characterized in the previous examples ned 8 human Va segments and 61 human Joe segments (1767 H0, see .

Homozygous humanized TCRoc mice comprising 23 human Va segments and 61 human Ja segments (1979 H0, see were tested for their y to te splenic CD3+ T cells and exhibit T cell development in the thymus. mental data was obtained using flow cytometry using appropriate antibodies as described in the preceding es. As depicted in , a mouse homozygous for 23 human Von segments and 61 human Jo: segments produced a significant number of splenic CD3+ T cells, and the percent of eral CD3+ T cells was comparable to that of the wild type animals ().

Moreover, thymocytes in 1979 H0 mice were able to undergo T cell development and contained T cells at DN1, DN2, DN3, DN4, DP, CD4 SP, and CD8 SP stages (FlG. 18).

Example 10: T Cell pment and Differentiation in Mice Homozygous for a te Repertoire of Both Human TCR: and TCR [3 Variable Region Segments Mice homozygous for a te repertoire of human TCRa variable region segments (Le, 54 human Von and 61 human Jon) and gous for a complete repertoire of human TCRB variable region segments (67 human VB, 2 human DB, and 14 human J3), “1771 H0 6192 H0” (see Fle. 3 and 7), are tested for their ability to produce thymocytes that undergo normal T cell development, produce T cells that undergo normal T cell differentiation in the periphery, and utilize the complete repertoire of their human Va and VB segments.

Flow cytometry is conducted to determine the presence of DN1, DN2, DN3, DN4, DP, CD4 SP and CD8 SP T cells in the thymus using anti—mouse CD4, CD8, CD25, and CD44 antibodies as described above in Examples 5 and 6. Flow cytometry is also conducted to determine the number of CD3+ T cells in the periphery, as well as to evaluate T cell differentiation in the ery (e.g., presence of effector and memory T cells in the periphery). The experiment is conducted using anti-mouse CD3, CD19, CD4, CD8, CD44, and CD62L antibodies as bed above in Examples 5 and 7, Finally, flow cytometry and/or ime PCR are conducted to determine whether T cells in 1771 H0 6192 H0 mice utilize a complete repertoire of TCRB and TCRA V segments. For protein expression using flow cytometry, TCRB repertoire kit (lOTEST® Beta Mark, Beckman Coulter), containing anti—human hTCRBV—specific antibodies, is utilized (see Example 8). For RNA expression using ime PCR, cDNAs from spleens or thymi are amplified using human TCR-V primers and Taqman , according to manufacturers instructions and as described above in Example 8.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many lents of the specific embodiments of the invention described . Such equivalents are intended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications and patents cited throughout this application are incorporated by reference herein in their entirety.

Where the terms “comprise”, ises”, “comprised” or “comprising” areused in this specification (including the claims) they are to be reted as specifying the ce of the stated features, integers, steps or ents, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Claims (21)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A genetically modified non-human animal, comprising in its genome: an unrearranged T cell receptor (TCR)  variable gene locus comprising at least one human V t and at least one human J segment, wherein the TCR variable gene locus is operably linked to a man TCR nt gene sequence, wherein the unrearranged TCR variable gene locus replaces an endogenous non-human TCR variable gene locus, and/or an unrearranged TCR variable gene locus sing at least one human V segment, at least one human D segment, and at least one human J segment, wherein the unrearranged TCR variable gene locus is operably linked to a non-human TCR constant gene sequence, wherein the unrearranged TCR variable gene locus replaces an endogenous man TCR variable gene locus.

2. The animal of claim 1, wherein endogenous man V and J segments are incapable of rearranging to form a rearranged V/J sequence and/or endogenous rodent V, D, and J segments are incapable of rearranging to form a rearranged V/D/J sequence.

3. The animal of claim 1, wherein the animal lacks a onal endogenous non-human TCR and/or TCR variable locus.

4. The animal of claim 3, wherein the lack of the functional endogenous non-human TCR variable gene locus comprises a on selected from the group consisting of (a) a deletion of all endogenous V gene segments, (b) a deletion of all endogenous J gene segments, and (c) a combination thereof and/or the lack of the functional endogenous rodent TCR variable gene locus comprises a deletion selected from the group ting of (a) a deletion of all endogenous V gene segments, (b) a on of all endogenous D gene segments, (c) a deletion of all endogenous J gene segments, and (d) a combination thereof.

5. The animal of claim 1, wherein the human V and J segments rearrange to form a rearranged human V/J sequence and/or the human V, D, and J segments rearrange to form a rearranged human V/D/J sequence.

6. The animal of any one of claims 1 to 5, wherein the animal expresses a T cell or comprising a human TCR and/or TCR variable region on the surface of a T cell.

7. The animal of any one of claims 1 to 6, wherein T cells of the animal undergo thymic T cell development to produce CD4 and CD8 single positive T cells.

8. The animal of any one of claims 1 to 7, wherein the animal comprises a normal ratio of splenic CD3+ T cells to total splenocytes.

9. The animal of any one of claims 1 to 8, wherein the animal generates a tion of central and effector memory T cells to an antigen of interest.

10. The animal of any one of claims 1 to 9, wherein the unrearranged TCR variable gene locus comprises a complete repertoire of human J segments and a complete repertoire of human V segments and/or the ranged TCR variable gene locus comprises a complete repertoire of human J segments, a complete repertoire of human D segments, and a te repertoire of human V segments.

11. The animal of claim 1, wherein the animal retains an endogenous non-human TCR variable gene locus and/or an endogenous non-human TCR variable gene locus, and n the endogenous non-human TCR variable gene locus and/or the endogenous nonhuman TCR variable gene locus is a non-functional locus.

12. The animal of any one of claims 1 to 11, wherein the animal is a rodent.

13. The animal of claim 12, n the rodent is a mouse.

14. A genetically modified non-human animal according to any one of claims 1 to 13, comprising in its : an unrearranged T cell receptor (TCR)  variable gene locus comprising at least one human V t and at least one human J segment, operably linked to a non-human TCR constant gene sequence; and, an unrearranged TCR variable gene locus comprising at least one human V segment, at least one human D segment, and at least one human J segment, operably linked to a non-human TCR constant gene ce.

15. The animal of claim 14, wherein the animal expresses a T cell receptor comprising a human variable region and a non-human constant region on a surface of a T cell.

16. The animal of claim 14, wherein the unrearranged TCR variable gene locus comprises 61 human J segments and 8 human V segments, and n the unrearranged TCR variable gene locus comprises 14 human J segments, 2 human D segments, and 14 human V segments.

17. The animal of claim 14, wherein the animal r comprises an unrearranged TCR variable region locus comprising human V segments.

18. The animal of claim 17, wherein the animal further comprises a complete repertoire of human V segments, a complete repertoire of human D segments, and a complete repertoire of human J segments at the humanized TCR locus.

19. A genetically modified non-human animal according to any one of claims 1 to 18, wherein the non-human animal is a mouse comprising in its genome: an unrearranged T cell receptor (TCR)  variable gene locus sing a complete repertoire of human J segments and a te repertoire of human V segments, operably linked to a mouse TCR constant gene sequence, and an unrearranged TCR variable gene locus comprising a complete repertoire of human J segments, a complete repertoire of human D segments, and a complete repertoire of human V segments, operably linked to a mouse TCR constant gene sequence.

20. A method of producing a human T cell receptor to an antigen of interest comprising: immunizing a non-human animal of any one of claims 1 to 19 with the antigen of interest; allowing the animal to mount an immune response; ing from the animal a T cell reactive to the antigen of interest; determining a nucleic acid sequence of a human TCR variable region expressed by the T cell; cloning the nucleic acid sequence of the human TCR variable region into a nucleotide uct comprising a c acid sequence of a human TCR constant region, wherein the nucleic acid sequence of the human TCR variable region is operably linked to the nucleic acid sequence of the human TCR nt region; and expressing a human T cell receptor in a cell.

21. The genetically modified non-human animal of claim 1, substantially as hereinbefore described with reference to the es and/or