NZ719373B2 - Genetically modified major histocompatibility complex mice - Google Patents

Abstract

Disclosed is a chimeric human/non-human animal MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises ?1, ?2, and ?3 domains of a human HLA class I polypeptide and a non-human portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a non-human animal MHC I polypeptide. Further discloses are nucleic acids encoding said polypeptide. animal MHC I polypeptide. Further discloses are nucleic acids encoding said polypeptide.

Description

CALLY MODIFIED MAJOR HISTOCOMPATIBILITY COMPLEX MICE CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a divisional application from New Zealand patent application number 623456, the entire disclosure of which is incorporated herein by reference. [0001a] This application claims benefit of priority to U.S. Provisional Patent Application Nos. 61/552,582 and 61/552,587, both filed October 28, 2011, and U.S. Provisional Patent Application No. 61/700,908, filed September 14, 2012, all of which are hereby incorporated by reference herein in their entireties.

FIELD OF THE INVENTION Present ion relates to a genetically ed non-human animal, e.g., a rodent (e.g., a mouse or a rat), that expresses a human or humanized Major Histocompatibility Complex (MHC) class I molecule. The invention also relates to a genetically modified non-human animal, e.g., a mouse or a rat, that expresses a human or humanized MHC I protein (e.g., MHC I  chain) and/or a human or humanized 2 microglobulin; as well as embryos, tissues, and cells expressing the same. The ion further provides methods for making a genetically modified man animal that expresses human or humanized MHC class I protein (e.g., MHC I  chain) and/or 2 microglobulin. Also provided are methods for identifying and evaluating es in the context of a humanized cellular immune system in vitro or in a genetically modified nonhuman animal, and methods of modifying an MHC I and/or a 2 lobulin locus of a non-human animal, e.g., a mouse or a rat, to express a human or humanized MHC I and/or 2 lobulin.

OUND OF THE INVENTION In the ve immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell 2012/062042 receptor or TCR). These foreign antigens are presented on the surface of cells as e fragments by specialized ns, generically referred to as major histocompatibility complex (MHC) molecules. MHC les are encoded by multiple loci that are found as a linked cluster of genes that spans about 4 Mb. ln mice, the MHC genes are found on chromosome 17, and for historical reasons are referred to as the histocompatibility 2 (H-2) genes. in humans, the genes are found on chromosome 6 and are called human leukocyte antigen (HLA) genes. The loci in mice and humans are polygenic; they include three highly rphic classes of MHC genes (class I, ll and Ill) that exhibit similar organization in human and murine genomes (see and respectively).

MHC loci exhibit the highest polymorphism in the genome; some genes are represented by >300 alleles (e.g., human HLA—DRfi and human HLA-B). All class l and II MHC genes can present peptide fragments, but each gene expresses a protein with different binding characteristics, reflecting polymorphisms and allelic variants. Any given individual has a unique range of peptide fragments that can be presented on the cell surface to B and T cells in the course of an immune response.

Both humans and mice have class i MHC genes (see HS. 2 and . In , the classical class l genes are termed HLA-A, HLA-B and HLA—C, s in mice they are H-2K, H-ZD and H—2L. Class I molecules consist of two : a polymorphic 0c— chain (sometimes referred to as heavy chain) and a smaller chain called BZ-microglobulin (also known as light chain), which is generally not polymorphic (HQ 1). These two chains form a non-covalent dimer on the cell surface. The a-chain contains three domains (a1, a2 and 0L3). Exon 1 of the n gene encodes the leader sequence, exons 2 and 3 encode the (11 and a2 domains, exon 4 encodes the 0:3 domain, exon 5 encodes the transmembrane , and exons 6 and 7 encode the cytoplasmic tail. The a-chain forms a peptide-binding cleft involving the (x1 and a2 domains (which resemble lg-like domains) followed by the 0:3 domain, which is similar to BZ-microglobulin. (52 microglobulin is a non-glycosyiated 12 kDa protein; one of its functions is to stabilize the MHC class l a~chain. Unlike the a-chain, the [32 lobulin does not span the membrane. The human (32 microglobulin locus is on chromosome 15, while the mouse locus is on chromosome 2. [32 microglobulin gene consists of 4 exons and 3 introns. ating forms of [32 microglobulin are present in the serum, urine, and other body fluids; thus, the non-covalently MHC l—associated (32 microglobulin can be exchanged with circulating [32 microglobulin under physiological conditions.

Class I MHC molecules are expressed on all nucleated cells, including tumor cells. They are expressed specifically on T and B lymphocytes, macrophages, dendritic cells and neutrophils, among other cells, and function to display e fragments (typically 8-10 amino acids in length) on the surface to CD8+ cytotoxic T lymphocytes (CTLs). CTLs are specialized to kill any cell that bears an MHC I-bound peptide recognized by its own membrane-bound TCR. When a cell displays peptides derived from ar proteins not normally present (e.g., of viral, tumor, or other non-self origin), such peptides are recognized by CTLs, which become ted and kill the cell ying the e.

Typically, presentation of normal (i.e., self) proteins in the context of MHC I molecules does not elicit CTL activation due to the tolerance mechanisms. r, in some diseases (e.g., cancer, mune diseases) peptides derived from self-proteins become a target of the ar component of the immune system, which results in destruction of cells presenting such peptides. Although there has been advancement in recognizing some self-derived antigens that elicit cellular immune response (e.g., antigens associated with various cancers), in order to improve identification of peptides recognized by human CTLs through MHC class I molecules there remains a need for both in vivo and in vitro systems that mimic aspects of the human cellular immune system. s that mimic the human cellular immune system can be used in identifying disease-associated antigens in order to develop human therapeutics, e.g., es and other biologics. Systems for assessing antigen recognition in the context of the human immune system can assist in identifying therapeutically useful CTL populations (e.g., useful for ng and combatting human disease). Such systems can also assist in enhancing the ty of human CTL populations to more effectively combat infections and n antigen-bearing entities. Thus, there is a need for biological systems (e.g., genetically engineered animals) that can generate an immune system that displays components that mimic the function of human immune .

SUMMARY OF THE INVENTION A biological system for generating or identifying peptides that associate with human MHC class I proteins and chimeras thereof, and bind to CD8+ T cells, is provided. man animals comprising non-human cells that express human or humanized molecules that function in the cellular immune response are provided. Humanized rodent loci that encode human or human or humanized MHC I and 2 microglobulin proteins are also provided. Humanized rodent cells that express human or humanized MHC and 2 microglobulin molecules are also provided. In vivo and in vitro systems are provided that comprise humanized rodent cells, wherein the rodent cells express one or more human or humanized immune system molecules.

Provided herein is a nucleic acid comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein the nucleotide sequence comprises a first nucleic acid sequence that encodes α1, α2 and α3 domains of a human HLA class I polypeptide operably linked to a second nucleic acid sequence that encodes transmembrane and cytoplasmic domains of the non human MHC I polypeptide. [0010a] Further provided herein is a chimeric human/non-human animal MHC I polypeptide, wherein a human portion of the chimeric polypeptide ses α1, α2, and α3 domains of a human HLA class I polypeptide and a non-human portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a non-human animal MHC I polypeptide. [0010b] r provided herein is a non-human animal, e.g., a rodent (e.g., a mouse or a rat), comprising in its genome a nucleotide sequence encoding a chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an ellular domain of a human MHC I polypeptide. Specifically, provided herein is a non-human animal comprising at an endogenous MHC I locus a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide, and n the animal expresses the chimeric human/non-human MHC I polypeptide. In one aspect, the animal does not express an extracellular domain of an endogenous non-human MHC I polypeptide from an endogenous man MHC I locus. In one aspect of the invention, the non-human animal (e.g., a rodent, e.g., a mouse or a rat) comprises two copies of the MHC I locus comprising a nucleotide sequence encoding chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide. In another aspect of the invention, the animal comprises one copy of the MHC I locus comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide. Thus, the animal may be gous or heterozygous for the MHC I locus comprising a tide sequence encoding chimeric human/non-human MHC I ptide. In various embodiments, the tide ce ng a chimeric human/non-human MHC I polypeptide is comprised in the germline of the non-human animal (e.g., rodent, e.g., rat or mouse).

In one aspect, the nucleotide ce encoding the chimeric human/non-human MHC I is ly linked to endogenous man regulatory elements, e.g., promoter, enhancer, silencer, etc. In one embodiment, a human portion of the chimeric polypeptide comprises a human leader sequence. In an additional embodiment, the human portion of the chimeric polypeptide comprises 1, 2, and 3 domains of the human MHC I polypeptide.

The human MHC I ptide may be selected from a group consisting of HLA-A, HLA-B, and HLA-C. In one embodiment, the human MHC I polypeptide is an HLA-A2 ptide, e.g., an HLA-A2.1 polypeptide.

In one aspect, the genetically engineered non-human animal is a rodent. In one embodiment, the rodent is a mouse. Thus, in one embodiment, the endogenous non-human locus is a mouse locus, e.g., a mouse H-2K, H-2D or H-2L locus. In one embodiment, the non-human portion of the chimeric non-human MHC I polypeptide comprises embrane and cytoplasmic domains of the endogenous non-human MHC I polypeptide.

Thus, in an embodiment wherein the non-human animal is a mouse, the endogenous nonhuman MHC I locus may be an H-2K locus (e.g., H-2Kb locus) and the endogenous nonhuman MHC I polypeptide may be an H-2K polypeptide; therefore, the chimeric human/non-human MHC I polypeptide may comprise transmembrane and cytoplasmic domains of H-2K polypeptide. In another embodiment wherein the non-human animal is a mouse, the endogenous non-human MHC I locus may be an H-2D locus and the endogenous non-human MHC I polypeptide may be an H-2D polypeptide; therefore, the chimeric human/non-human MHC I ptide may comprise transmembrane and cytoplasmic domains of H-2D polypeptide. Similarly, in another embodiment, the endogenous non-MHC I locus may be an H-2L locus and the endogenous non-human MHC I polypeptide may be an H-2L ptide; therefore, the chimeric human/non-human MHC I polypeptide may comprise transmembrane and cytoplasmic domains of H-2L ptide.

Also provided herein is a mouse comprising at an endogenous H-2K locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A (e.g., HLA-A2) polypeptide and a mouse n comprises embrane and cytoplasmic domains of a mouse H-2K ptide, and wherein the mouse expresses the chimeric human/mouse MHC I polypeptide. In some embodiments, the mouse does not express an ellular domain of the mouse H-2K polypeptide from an endogenous H-2K locus. In one aspect, the nucleotide sequence encoding a ic human/mouse MHC I polypeptide is operably linked to endogenous mouse regulatory elements. The human portion of the chimeric polypeptide may comprise a human leader sequence. It may also comprise 1, 2, and 3 domains of the human MHC I ptide. The human MHC I polypeptide may be HLA-A polypeptide, e.g., HLA-A2.1 polypeptide. In one aspect, the mouse H-2K locus is an H-2Kb locus.

Another aspect of the invention s to a non-human animal, e.g., a rodent (e.g., a mouse or a rat), comprising in its genome a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide. Thus, provided herein is a non-human animal comprising at an endogenous non-human 2 microglobulin locus a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide, wherein the animal expresses the human or humanized 2 lobulin polypeptide. In one , the animal does not express a onal endogenous non-human 2 microglobulin polypeptide from an endogenous non-human 2 microglobulin locus. In one aspect, the animal comprises two copies of the 2 lobulin locus encoding the human or humanized 2 microglobulin polypeptide; in another ment, the animal comprises one copy of the 2 microglobulin locus encoding the human or humanized 2 microglobulin polypeptide. Thus, the animal may be homozygous or heterozygous for the 2 microglobulin locus encoding the human or humanized 2 microglobulin polypeptide. In various embodiments, the nucleotide sequence encoding the human or humanized 2 microglobulin polypeptide is comprised in the germline of the non-human animal (e.g., rodent, e.g., rat or mouse). In one embodiment, a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide comprises a nucleotide sequence encoding a ptide comprising a human 2 microglobulin amino acid ce. In one embodiment, the polypeptide is capable of binding to an MHC I protein.

In some embodiments, the nucleotide ce encoding the human or humanized 2 microglobulin polypeptide is operably linked to endogenous man 2 lobulin regulatory elements. In one aspect, the nucleotide ce encoding the human or humanized 2 microglobulin polypeptide comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human 2 microglobulin gene. In another aspect, the nucleotide ce encoding the human or zed 2 microglobulin ptide ses nucleotide sequences set forth in exons 2, 3, and 4 of a human 2 microglobulin gene. In a further aspect, the nucleotide sequence also comprises a nucleotide sequence set forth in exon 1 of a non-human 2 microglobulin gene. In some embodiments, the nonhuman animal is a rodent (e.g., mouse or a rat); thus, the non-human 2 microglobulin locus is a rodent (e.g., a mouse or a rat) 2 microglobulin locus.

Also provided is a mouse comprising at an endogenous 2 microglobulin locus a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide, wherein the mouse expresses the human or humanized 2 microglobulin polypeptide. In some embodiments, the mouse does not express a functional endogenous mouse 2 microglobulin from an endogenous 2 microglobulin locus. The nucleotide sequence may be linked to nous mouse regulatory elements. In one aspect, the nucleotide sequence ses a nucleotide sequence set forth in exon 2 to exon 4 of a human 2 microglobulin gene. Alternatively, the nucleotide sequence encoding the human or humanized 2 microglobulin polypeptide may comprise nucleotide sequences set forth in exons 2, 3, and 4 of a human 2 microglobulin gene. The nucleotide sequence encoding the human or humanized 2 microglobulin polypeptide may further comprise a nucleotide sequence of exon 1 of a mouse 2 microglobulin gene. In one embodiment, a nucleotide sequence ng a human or humanized 2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide sing a human 2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to an MHC I protein.

The invention further provides a man animal (e.g., a rodent, e.g., a mouse or a rat) comprising in its genome a nucleotide sequence encoding a chimeric human/nonhuman MHC I polypeptide and a tide sequence encoding a human or humanized 2 microglobulin polypeptide. In one ment, the invention provides a non-human animal comprising in its genome a first nucleotide sequence encoding a chimeric human/nonhuman MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide; and a second nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous non-human MHC I locus, and the second nucleotide sequence is located at an endogenous non-human 2 microglobulin locus, and wherein the animal expresses the ic human/non-human MHC I polypeptide and the human or humanized 2 microglobulin ptide. In one aspect, the animal is a mouse. Thus, the endogenous MHC I locus may be selected from a group consisting of H-2K, H-2D, and H-2L locus. In one embodiment, the endogenous mouse locus is an H-2K locus (e.g., H-2Kb locus). In one embodiment, t he human MHC I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C polypeptide. In one aspect, the human MHC I polypeptide is HLA-A, e.g., HLA-A2 (e.g., HLA-A2.1). In various embodiments, the first and the second nucleotide sequences are comprised in the germline of the non-human animal (e.g., rodent, e.g., mouse or rat).

Therefore, the invention provides a mouse comprising in its genome a first nucleotide sequence ng a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A (e.g., ) and a mouse portion comprises embrane and asmic domains of a mouse H-2K; and a second nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide, wherein the first nucleotide ce is located at an endogenous H-2K locus and the second nucleotide sequence is located at an endogenous mouse 2 microglobulin locus, and n the mouse expresses the chimeric mouse MHC I polypeptide and the human or humanized 2 microglobulin polypeptide. In one embodiment, the non-human animal (e.g., the mouse) comprising both the chimeric MHC I ptide and human or humanized 2 lobulin polypeptide does not express an extracellular domain of an endogenous non-human MHC I polypeptide (e.g., the mouse H-2K polypeptide) and/or a functional endogenous non-human (e.g., the mouse) 2 lobulin polypeptides from their respective endogenous loci. In one aspect, the animal (e.g., the mouse) comprises two copies of each of the first and the second nucleotide sequence. In another aspect, the animal (e.g., the mouse) comprises one copy of the first and one copy of the second nucleotide sequences. Thus, the animal may be homozygous or heterozygous for both the first and the second nucleotide sequences.

In one aspect, the first tide sequence is operably linked to endogenous non-human (e.g., mouse) MHC I tory elements, and the second nucleotide ce is operably linked to endogenous non-human (e.g., mouse) 2 microglobulin elements. The human portion of the chimeric polypeptide may comprise 1, 2 and 3 domains of the human MHC I polypeptide. The second nucleotide sequence may comprise a nucleotide sequence set forth in exon 2 to exon 4 of a human 2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences set forth in exons 2, 3, and 4 of a human 2 lobulin gene. In one aspect, the mouse comprising both the chimeric MHC I polypeptide and human or humanized 2 microglobulin polypeptide may be such that the expression of human or humanized 2 microglobulin increases the expression of the chimeric human/mouse MHC I polypeptide as compared to the expression of the ic human/mouse MHC I polypeptide in the absence of expression of human or zed 2 microglobulin polypeptide.

Also provided are methods of making genetically engineered non-human animals (e.g., rodents, e.g., mice or rats) described herein. Thus, in one embodiment, provided is a method of modifying an MHC I locus of a rodent (e.g., a mouse or a rat) to express a chimeric human/rodent (e.g., human/mouse or human/rat) MHC I polypeptide, wherein the method comprises ing at the endogenous MHC I locus a nucleotide sequence ng an extracellular domain of a rodent MHC I polypeptide with a nucleotide sequence encoding an extracellular domain of a human MHC I ptide. In another embodiment, provided is a method of modifying a 2 microglobulin locus of a rodent (e.g., a mouse or a rat) to express a human or humanized 2 microglobulin polypeptide, wherein the method comprises replacing at the endogenous rodent (e.g., mouse or rat) 2 microglobulin locus a tide sequence encoding a rodent (e.g., a mouse or a rat) 2 microglobulin ptide with a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide.

In such methods, the replacement may be made in a single ES cell, and the single ES cell may be introduced into a rodent (e.g., a mouse or a rat) to make an embryo. The resultant rodent (e.g., a mouse or a rat) can be bred to generate a double humanized animal.

Thus, the invention also provides a method of making double zed animals, e.g., rodents (e.g., mice or rats). In one embodiment, p rovided is a method of making a genetically ed mouse comprising (a) modifying an MHC I locus of a first mouse to express a chimeric human/mouse MHC I polypeptide sing ing at the endogenous mouse MHC I locus a nucleotide sequence encoding an extracellular domain of a mouse MHC I polypeptide with a nucleotide sequence encoding an extracellular domain of a human MHC I ptide, (b) modifying a 2 lobulin locus of a second mouse to express a human or humanized 2 microglobulin polypeptide comprising replacing at the endogenous mouse 2 microglobulin locus a nucleotide sequence encoding a mouse 2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized 2 lobulin polypeptide; and (c) breeding the first and the second mouse to generate a genetically modified mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide and a second nucleotide sequence encoding a human or zed 2 lobulin polypeptide, wherein the genetically modified mouse ses the chimeric human/mouse MHC I polypeptide and the human or zed 2 microglobulin polypeptide. In some embodiments, the MHC I locus is selected from H-2K, H-2D, and H-2L; in some embodiments, the human MHC I polypeptide is selected from HLAA , HLA-B, and HLA-C. In one embodiment, the MHC I locus is an H-2K locus, the human MHC I polypeptide is HLA-A (e.g., HLA-A2), and the mouse expresses a ic HLA-A/H- 2K polypeptide (e.g., /H-2K polypeptide). In one aspect, the chimeric HLA -A2/H-2K polypeptide comprises an extracellular domain of the HLA-A2 polypeptide and cytoplasmic and transmembrane domains of H-2K polypeptide. In one aspect, the second nucleotide ce comprises nucleotide sequences set forth in exons 2, 3, and 4 (e.g., exon 2 to exon 4) of a human 2 microglobulin gene, and a tide sequence set forth in exon 1 of a mouse 2 microglobulin gene.

Also provided herein are cells, e.g., isolated antigen-presenting cells, derived from the non-human animals (e.g., rodents, e.g., mice or rats) described herein. Tissues and s derived from the non-human animals described herein are also provided.

In yet another embodiment, the invention provides methods for identification of antigens or antigen epitopes that elicit immune response, methods for evaluating a vaccine candidate, methods for identification of high affinity T cells to human pathogens or cancer antigens.

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 apparent to those skilled in the art from a review of the g detailed ption. The following detailed description includes exemplary representations 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 is a schematic drawing of the four domains of a class I MHC molecule: - chain containing the 1, 2 and 3 domains and the non-covalently associated fourth domain, roglobulin (2m). The gray circle represents a peptide bound in the peptidebinding cleft. is a schematic representation (not to scale) of the relative genomic structure of the human HLA, showing class I, II and III genes. is a schematic representation (not to scale) of the ve c structure of the mouse MHC, showing class I, II and III genes. illustrates a viral vector construct containing a cDNA ng a chimeric HLA-A/H-2K polypeptide with an IRES-GFP reporter (A); and histograms comparing expression of human HLA-A2 in MG87 cells transduced with HLA-A2 (dashed line), HLAA2 /H-2K (dotted line), or no transduction (solid line) either alone (left) or co-transduced with humanized 2 microglobulin (right) (B). Data from ntal gates presented graphically in (B) is illustrated as percent of cells expressing the construct in the table in (C). is a schematic m (not to scale) of the targeting strategy used for making a chimeric H-2K locus that expresses an extracellular region of a human HLA-A2 protein. Mouse sequences are represented in black and human sequences are represented in white. L=leader, UTR=untranslated region, TM=transmembrane domain, CYT=cytoplasmic domain, HYG=hygromycin. demonstrates sion (% total cells) of HLA-A2 (left) and H-2K ) in cells isolated from either a wild-type (WT) mouse or a heterozygous mouse carrying the chimeric HLA-A2/H-2K locus (HLA-A/H-2K HET). is a dot plot of in vivo expression of the ic HLA-A2/H-2K n in a heterozygous mouse harboring a chimeric HLA-A2/H-2K locus. shows a targeting strategy (not to scale) for humanization of a 2 microglobulin gene at a mouse 2 microglobulin locus. Mouse sequences are in black and human sequences are in white. NEO=neomycin. shows a representative dot plot of HLA class I and human 2 microglobulin expression on cells isolated from the blood of wild-type (WT) mice, mice heterozygous for chimeric HLA-A2/H-2K, and mice heterozygous for chimeric HLA-A2/H-2K and heterozygous for humanized 2 lobulin (double heterozygous; class I/2m HET). shows a representative histogram of human HLA class I sion (X axis) on cells isolated from the blood of ype (WT), chimeric HLA-A2/H-2K heterozygous (class I HET), and chimeric HLA-A2/H2K/humanized 2 microglobulin double heterozygous (class I/ 2m HET) mice. shows the results of IFN Elispot assays for human T cells d to antigen-presenting cells (APCs) from wild-type mice (WT APCs) or mice heterozygous for both ic HLA-A2/H-2K and humanized 2 microglobulin e HET APCs) in the presence of flu (left) or EBV (right) peptides. Statistical analysis was performed using one way ANOVA with a Tukey’s Multiple Comparison Post Test.

DETAILED PTION OF THE INVENTION Definitions The present invention provides genetically modified non-human animals (e.g., mice, rats, rabbits, etc.) that express human or zed MHC I and/or 2 microglobulin polypeptides; embryos, cells, and s comprising the same; methods of making the same; as well as methods of using the same. Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated 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 residue by r amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity).

Conservative amino acid substitutions may be achieved by modifying a nucleotide ce so as to introduce a nucleotide change that will encode the conservative tution. In general, a conservative amino acid tution will not substantially change the functional properties of interest of a protein, for example, the ability of MHC I to present a peptide of interest. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, e, 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 substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and gine/glutamine. In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning 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. In some ments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.

Thus, also encompassed by the invention is a genetically modified non-human animal whose genome comprises a nucleotide sequence encoding a human or humanized MHC I polypeptide and/or 2 microglobulin polypeptide, wherein the polypeptide(s) ses vative amino acid substitutions of the amino acid sequence(s) described herein.

One skilled in the art would understand that in addition to the nucleic acid residues encoding a human or zed MHC I polypeptide and/or 2 microglobulin described , due to the degeneracy of the genetic code, other nucleic acids may encode the ptide(s) of the invention. Therefore, in addition to a cally ed non-human animal that comprises in its genome a nucleotide sequence encoding MHC I and/or 2 microglobulin polypeptide(s) with conservative amino acid substitutions, a nonhuman animal whose genome comprises a nucleotide sequence(s) that differs from that described herein due to the degeneracy of the genetic code is also ed.

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 tide and/or amino acid sequence identity. In some embodiments described herein, ties are determined 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 similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to identity of sequences will depend upon the particular ces. In various embodiments, identity is determined by comparing the sequence of a mature protein from its N-terminal 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 comparison for the e of ascertaining a level of identity between a human sequence and a human portion of a ic human/non-human ce (e.g., comparing a human ectodomain of a chimeric mouse protein to a human ectodomain of a human protein).

The terms “homology” or ogous” in reference to sequences, e.g., nucleotide or amino acid sequences, means two sequences which, upon optimal alignment 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 nous DNA ces (i.e., “homology ; thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

The term "operably linked" refers to a juxtaposition n the components so described are in a relationship permitting them to function in their intended manner. As such, a nucleic acid sequence encoding a protein may be operably linked to tory sequences (e.g., promoter, enhancer, silencer ce, etc.) so as to retain proper riptional regulation. In addition, various portions of the chimeric or humanized protein of the invention may be operably linked to retain proper folding, processing, ing, expression, and other functional properties of the protein in the cell. Unless stated ise, various domains of the chimeric or humanized proteins of the invention are operably linked to each other.

The term “MHC I complex” or the like, as used herein, includes the complex between the MHC I  chain polypeptide and the roglobulin polypeptide. The term “MHC I polypeptide” or the like, as used herein, includes the MHC I  chain polypeptide alone. Typically, the terms “human MHC” and “HLA” can be used interchangeably.

The term “replacement” in nce 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.

As demonstrated in the Examples below, c acid sequences of endogenous loci encoding portions of mouse MHC I and 2 microglobulin polypeptides were replaced by nucleotide sequences encoding portions of human MHC I and 2 microglobulin polypeptides, respectively.

“Functional” as used herein, e.g., in reference to a functional ptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some ments of the invention, a replacement at an endogenous locus (e.g., replacement at an endogenous non-human MHC I and/or 2 microglobulin locus) results in a locus that fails to express a functional endogenous polypeptide.

Several aspects described herein below for the cally modified MHC I an animals, e.g., animal type; animal s; cell types; ing, detection and other methods; methods of use; etc., will be applicable to the genetically engineered 2 microglobulin and MHC I/2 microglobulin animals. cally Modified MHC I Animals In various embodiments, the invention generally provides cally modified non-human animals that comprise in their genome a nucleotide ce encoding a human or humanized MHC I ptide; thus, the animals express a human or humanized MHC I ptide.

MHC genes are categorized into three classes: class I, class II, and class III, all of which are encoded either on human chromosome 6 or mouse chromosome 17. A schematic of the relative organization of the human and mouse MHC classes is ted in FIGs. 2 and 3, respectively. The MHC genes are among the most polymorphic genes of the mouse and human genomes. MHC polymorphisms are presumed to be important in providing evolutionary advantage; changes in sequence can result in differences in peptide binding that allow for better presentation of pathogens to cytotoxic T cells.

MHC class I protein comprises an extracellular domain (which comprises three domains: 1, 2, and 3), a transmembrane domain, and a cytoplasmic tail. The 1 and 2 domains form the e-binding cleft, while the 3 interacts with 2-microglobulin.

In addition to its ction with 2-microglobulin, the 3 domain interacts with the TCR co-receptor CD8, facilitating antigen-specific activation. Although binding of MHC class I to CD8 is about 100-fold weaker than binding of TCR to MHC class I, CD8 binding enhances the affinity of TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding ties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366. Interestingly, increasing MHC class I binding to CD8 abrogated antigen specificity in CTL activation. Id.

CD8 binding to MHC class I molecules is species-specific; the mouse homolog of CD8, Lyt-2, was shown to bind H-2Dd molecules at the 3 domain, but it did not bind HLA-A molecules. Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class I 3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168:325-341.

Differential binding was presumably due to CDR-like determinants (CDR1- and CDR2-like) on CD8 that was not conserved between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J.

Exp. Med. 174:371-379; Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice ng a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. 173:1007-1015; and, Gao et al. (1997) Crystal structure of the complex between human CD8 and HLA-A2, Nature 387:630-634. It has been reported that CD8 binds HLA -A2 in a conserved region of the 3 domain (at on 9). A single substitution (V245A) in HLA -A reduced binding of CD8 to HLA-A, with a concomitant large ion in T cell-mediated lysis. Salter et al. (1989), Polymorphism in the 3 domain of HLA-A molecules affects binding to CD8, Nature 338:345-348. In general, polymorphism in the 3 domain of HLA-A molecules also affected binding to CD8. Id. In mice, amino acid substitution at e 227 in H-2Dd affected the binding of mouse Lyt-2 to H-2Dd, and cells transfected with a mutant H-2Dd were not lysed by CD8+ T cells. Potter et al. (1989) tution at e 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73-75.

Therefore, due to species specificity of interaction between the MHC class I 3 domain and CD8, an MHC I complex comprising a replacement of an H-2K 3 domain with a human HLA-A2 3 domain was nonfunctional in a mouse (i.e., in vivo) in the absence of a human CD8. In animals transgenic for HLA-A2, substitution of human 3 domain for the mouse 3 domain resulted in restoration of T cell response. Irwin et al. (1989) srestricted interactions between CD8 and the 3 domain of class I influence the magnitude of the xenogeneic se, J. Exp. Med. 170:1091-1101; Vitiello et al. (1991), supra.

The transmembrane domain and cytoplasmic tail of mouse MHC class I proteins also have important functions. One function of MHC I transmembrane domain is to facilitate modulation by HLA-A2 of homotypic cell adhesion (to enhance or inhibit adhesion), ably as the result of cross-linking (or ligation) of surface MHC molecules. Wagner et al. (1994) Ligation of MHC Class I and Class II Molecules Can Lead to Heterologous Desensitization of Signal Transduction Pathways That Regulate pic Adhesion in Human Lymphocytes, J. Immunol. 152:5275-5287. Cell adhesion can be ed by mAbs that bind at diverse epitopes of the HLA-A2 molecule, suggesting that there are multiple sites on HLA-A2 implicated in modulating homotypic cell adhesion; depending on the epitope bound, the affect can be to enhance or to inhibit HLA-A2-dependent adhesion. Id.

The cytoplasmic tail, encoded by exons 6 and 7 of the MHC I gene, is reportedly necessary for proper expression on the cell surface and for LIR1-mediated inhibition of NK cell cytotoxicity. Gruda et al. (2007) Intracellular ne es in the Tail of MHC Class I Proteins Are Crucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1, J.

Immunol. 179:3655-3661. A cytoplasmic tail is required for multimerizaton of at least some MHC I molecules through formation of ide bonds on its cysteine residues, and thus may play a role in clustering and in recognition by NK cells. Lynch et al. (2009) Novel MHC Class I Structures on Exosomes, J. Immunol. 183:1884-1891.

The cytoplasmic domain of HLA-A2 contains a constitutively phosphorylated serine residue and a phosphorylatable tyrosine, although—in Jurkat mutant HLA-A2 molecules lacking a cytoplasmic domain appear normal with respect to expression, cytoskeletal association, aggregation, and endocytic internalization. Gur et al. (1997) Structural Analysis of Class I MHC Molecules: The Cytoplasmic Domain Is Not ed for Cytoskeletal Association, ation, and Internalization, Mol. Immunol. 125-132.

Truncated HLA-A2 molecules lacking the cytoplasmic domain are apparently normally expressed and ate with 2 microglobulin. Id.

However, several studies have trated that the cytoplasmic tail is critical in intracellular trafficking, dendritic cell (DC)-mediated antigen presentation, and CTL priming.

A tyrosine residue encoded by exon 6 was shown to be required for MHC I trafficking h endosomal compartments, presentation of exogenous antigens, and CTL priming; while deletion of exon 7 caused enhancement of anti-viral CTL responses. Lizee et al. (2003) Control of Dendritic Cross-Presentation by the Major Histocompatibility x Class I asmic Domain, Nature l. 4:1065-73; Basha et al. (2008) MHC Class I Endosomal and Lysosomal Trafficking Coincides with ous Antigen Loading in Dendritic Cells, PLoS ONE 3: e3247; and Rodriguez-Cruz et al. (2011) Natural Splice Variant of MHC Class I Cytoplasmic Tail Enhances Dendritic Cell-Induced CD8+ T-Cell Responses and Boosts Anti-Tumor Immunity, PLoS ONE 6:e22939.

In various embodiments, the invention provides a genetically modified nonhuman animal (e.g., mouse, rat, rabbit, etc.) that comprises in its genome a nucleotide sequence encoding a human or humanized MHC class I polypeptide. The non-human animal may comprise in its genome a tide sequence that encodes an MHC I polypeptide that is partially human and partially non-human, e.g., a non-human animal that expresses a chimeric human/non-human MHC I polypeptide. In one aspect, the non-human animal only expresses the human or zed MHC I polypeptide, e.g., chimeric human/non-human MHC I polypeptide, and does not express an endogenous non-human MHC I protein from an endogenous MHC I locus.

In one embodiment, the chimeric human/non-human MHC I polypeptide comprises in its human portion a peptide binding domain of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I. In this ment, the human portion of the chimeric polypeptide comprises an extracellular domain of an  chain of a human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises 1 and 2 domains of a human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises 1, 2, and 3 domains of a human MHC I.

The human or humanized MHC I polypeptide may be derived from a functional human HLA molecule encoded by any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci. A list of commonly used HLA antigens is described in Shankarkumar et al. ((2004) The Human Leukocyte n (HLA) System, Int. J. Hum. Genet. 4(2):91-103), orated herein by reference. Shankarkumar et al. also present a brief explanation of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and –DQ antigens, Tissue Antigens 73:95-170, and a recent update by Marsh et al. (2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens 75:291-455, both incorporated herein by reference. Thus, the human or humanized MHC I polypeptide may be derived from any functional human HLA class I les described therein.

In one specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., and .1 polypeptide). In one embodiment, the HLA -A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., 02:01:01:01 allele. The HLAA *0201 allele is ly used amongst the North American population. Although the present Examples describe this particular HLA sequence, any suitable HLA-A sequence is assed herein, e.g., polymorphic variants of HLA-A2 exhibited in human population, sequences with one or more conservative or non-conservative amino acid cations, nucleic acid sequences differing from the ce described herein due to the degeneracy of genetic code, etc.

In one aspect, a non-human animal that expresses a human HLA-A2 sequence is provided, wherein the human HLA-A2 sequence comprises one or more conservative or non-conservative cations.

In one aspect, a non-human animal that expresses a human HLA-A2 sequence is ed, wherein the human HLA-A2 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human HLA-A2 sequence. In a specific embodiment, the human HLA-A2 ce is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human HLA-A2 ce described in the Examples. In one embodiment, the human HLA-A2 sequence comprises one or more conservative substitutions. In one embodiment, the human HLA-A2 sequence comprises one or more non-conservative substitutions.

In another specific aspect, the human or humanized MHC I polypeptide is derived from human MHC I selected from HLA-B and HLA-C. In one aspect, the human or humanized MHC I is derived from HLA-B, e.g., HLA-B27.

In one aspect, the non-human portion of the chimeric human/non-human MHC I polypeptide comprises transmembrane and/or cytoplasmic domains of the non-human MHC I polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human MHC I polypeptide is selected from H-2K, H-2D, and H-2L. In one embodiment, the nonhuman MHC I polypeptide is H-2K, e.g., H-2Kb. gh specific H-2K sequences are described in the Examples, any suitable H-2K sequences, e.g., polymorphic variants, conservative/non-conservative amino acid substitutions, etc., are encompassed herein.

The non-human animal described herein may comprise in its genome a nucleotide sequence encoding a human or humanized MHC I polypeptide, e.g., ic non-human MHC I polypeptide, wherein the nucleotide sequence encoding such polypeptide is d at an nous non-human MHC I locus (e.g., H-2K locus). In one aspect, this results in a replacement of an endogenous MHC I gene or a portion thereof with a nucleotide ce encoding a human or humanized MHC I ptide, e.g., a chimeric gene encoding a chimeric human/non-human MHC I polypeptide described herein. In one embodiment, the replacement comprises a replacement of an endogenous nucleotide sequence encoding a non-human MHC I peptide binding domain or a non-human MHC I extracellular domain with a human nucleotide sequence (e.g., HLA-A2 nucleotide sequence) ng the same. In this embodiment, the replacement does not comprise a replacement of an MHC I sequence encoding transmembrane and/or cytoplasmic domains of a nonhuman MHC I polypeptide (e.g., H-2K polypeptide). Thus, the non-human animal contains chimeric non-human tide sequence at an nous non-human MHC I locus, and expresses chimeric human/non-human MHC polypeptide from the endogenous non-human MHC I locus.

A chimeric human/non-human ptide may be such that it comprises a human or a non-human leader (signal) sequence. In one embodiment, the chimeric polypeptide comprises a non-human leader sequence of an nous MHC I protein. In r embodiment, the chimeric polypeptide ses a leader sequence of a human MHC I protein, e.g., HLA-A2 protein (for instance, HLA-A2.1 leader sequence). Thus, the nucleotide sequence encoding the chimeric MHC I ptide may be operably linked to a nucleotide ce encoding a human MHC I leader sequence.

A chimeric human/non-human MHC I polypeptide may comprise in its human portion a te or substantially complete extracellular domain of a human MHC I polypeptide. Thus, the human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g., 95% or more of the amino acids ng an extracellular domain of a human MHC I polypeptide (e.g., HLA-A2 polypeptide). In one e, ntially complete extracellular domain of the human MHC I ptide lacks a human MHC I leader sequence. In another example, the chimeric human/non-human MHC I polypeptide comprises a human MHC I leader sequence. er, the chimeric MHC I polypeptide may be expressed under the control of endogenous non-human regulatory elements, e.g., rodent MHC I regulatory animals. Such arrangement will facilitate proper expression of the chimeric MHC I polypeptide in the nonhuman animal, e.g., during immune se in the non-human animal.

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, , primate (e.g., marmoset, rhesus monkey). For the non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification.

Such methods e, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an d pluripotent cell) and employing nuclear transfer to er the modified genome to a suitable cell, e.g., an oocyte, and ing 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. In one aspect, the nonhuman animal is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one ment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family ed 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), idae (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). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment, 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. In 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 C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, 6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) d nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al (2000) Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse nic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In a specific embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac) . In r embodiment, the mouse is a BALB , e.g., BALB/c strain. In yet another embodiment, the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from a Wistar rat, an LEA strain, 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 Agouti.

Thus, in one embodiment, the invention relates to a genetically modified mouse that comprises in its genome a nucleotide sequence encoding a ic human/mouse MHC I polypeptide, wherein a human n of the chimeric polypeptide comprises a peptide g domain or an ellular domain of a human MHC I (e.g., human HLA-A, e.g., human HLA-A2, e.g., human HLA-A2.1). In some embodiments, the mouse does not express a peptide binding or an extracellular domain of an endogenous mouse polypeptide from its endogenous mouse locus. The e binding domain of the human MHC I may comprise 1 and 2 domains. Alternatively, the peptide binding domain of the human MHC I may comprise 1, 2, and 3 domains. In one aspect, the extracellular domain of the human MHC I comprises an extracellular domain of a human MHC I  chain. In one embodiment, the endogenous mouse locus is an H-2K (e.g., H-2Kb) locus, and the mouse portion of the chimeric polypeptide ses transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide.

Thus, in one ment, a genetically modified mouse is provided, wherein the mouse comprises at an endogenous H-2K (e.g., H-2Kb) locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide ses an extracellular domain of a human HLA-A2 (e.g., HLAA2.1 ) polypeptide and a mouse portion comprises embrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. In one aspect, the mouse does not express an extracellular domain of the mouse H-2K (e.g., H-2Kb) polypeptide from an endogenous MHC I locus. In one embodiment, the mouse expresses a chimeric HLA-A2/H-2K (e.g., a chimeric HLA-A2.1/H-2Kb) polypeptide from an endogenous H-2K (e.g., H-2Kb) locus. In various embodiments, expression of the chimeric gene is under control of nous mouse MHC class I tory elements. In some aspects, the mouse comprises two copies of the chimeric MHC I locus containing a nucleotide sequence encoding a chimeric HLA-A2/H-2K polypeptide; while in other aspects, the mouse comprises one copy of the chimeric MHC I locus ning a nucleotide ce encoding a chimeric HLA-A2/H-2K polypeptide.

Thus, the mouse may be gous or heterozygous for the nucleotide sequence encoding the chimeric /H-2K polypeptide.

In some embodiments described herein, a mouse is provided that comprises a chimeric MHC I locus located at an endogenous mouse H-2K locus. The chimeric locus comprises a nucleotide sequence that encodes an extracellular domain of a human HLA-A2 protein, e.g., 1, 2, and 3 domains of a human HLA-A2 gene. The chimeric locus lacks a nucleotide sequence that encodes an extracellular domain of a mouse H-2K protein (e.g., 1, 2, and 3 domains of the mouse H-2K). In one aspect, the chimeric locus lacks a nucleotide sequence that encodes a leader peptide, 1, 2, and 3 domains of a mouse H- 2K; and comprises a leader peptide, 1, 2, and 3 domains of a human HLA-A2, and transmembrane and cytoplasmic domains of a mouse H-2K. The various domains of the chimeric locus are operably linked to each other such that the chimeric locus expresses a functional chimeric human/mouse MHC I n.

In various embodiments, a non-human animal, e.g., a rodent (e.g., a mouse or a rat), that expresses a functional chimeric MHC I protein from a ic MHC I locus as described herein displays the chimeric protein on a cell surface. In one embodiment, the non-human animal expressed the ic MHC I protein on a cell surface in a cellular distribution that is the same as observed in a human. In one aspect, the cell displays a peptide fragment (an antigen fragment) bound to an extracellular n (e.g., human HLAA2 extracellular portion) of the ic MHC I protein. In an embodiment, the extracellular portion of such chimeric n cts with other proteins on the surface of said cell, e.g., 2-microglobulin.

In various embodiments, a cell displaying the chimeric MHC I protein, e.g., HLAA2 /H-2K protein, is a nucleated cell. In various aspects, the cell is an antigen-presenting cell (APC). Although most cells in the body can t an antigen in the context of MHC I, some nonlimiting examples of antigen presenting cells include hages, dendritic cells, and B cells. Other antigen presenting cells, including professional and nonprofessional APCs, are known in the art, and are encompassed herein. In some embodiments, the cell displaying the chimeric MHC I protein is a tumor cell, and a peptide fragment presented by the chimeric protein is derived from a tumor. In other em nts, the peptide fragment presented by the chimeric MHC I protein is derived from a pathogen, e.g., a bacterium or a virus.

The chimeric MHC I protein described herein may interact with other ns on the surface of the same cell or a second cell. In some embodiments, the chimeric MHC I protein interacts with endogenous non-human proteins on the e of said cell. The chimeric MHC I protein may also interact with human or humanized proteins on the surface of the same cell or a second cell.

On the same cell, HLA class I molecules may functionally interact with both nonhuman (e.g., rodent, e.g., mouse or rat) and human 2-microglobulin. Thus, in one embodiment, the chimeric MHC I protein, e.g., HLA-A2/H-2K protein, interacts with a mouse 2-microglobulin. gh interaction between some human HLA class I molecules and mouse 2-microglobulin is possible, it nevertheless may be greatly reduced in comparison to interaction between human HLA class I and human 2-microglobulin. Thus, in the absence of human 2-microglobulin, expression of human MHC I on the cell surface may be reduced.

Perarnau et al. (1988) Human 2-microglobulin ically Enhances Cell-Surface Expression of HLA Class I Molecules in Transfected Murine Cells, J. Immunol. 141:1383-89.

Other HLA molecules, e.g., HLA-B27, do not ct with mouse 2-microglobulin; see, e.g., Tishon et al. (2000) Transgenic Mice Expressing Human HLA and CD8 les Generate HLA-Restricted s Virus Cytotoxic T Lymphocytes of the Same Specificity as Humans with Natural Measles Virus Infection, Virology 275:286-293, which reports that HLA-B27 function in transgenic mice es both human 2-microglobulin and human CD8.

Therefore, in another embodiment, the chimeric MHC I protein interacts with a human or humanized roglobulin. In some such embodiments, as described herein below, the non-human animal, e.g., a rodent (e.g., a mouse or a rat), comprises in its genome a human or humanized 2-microglobulin gene, and the animal expresses a functional human or humanized 2-microglobulin ptide; ore, the chimeric MHC I protein interacts with a human or humanized roglobulin polypeptide.

In various aspects, the chimeric protein (e.g., HLA-A2/H-2K protein) also interacts with proteins on the surface of a second cell (through its extracellular portion). The second cell may be a cell of a non-human, e.g., a mouse, or a human origin. The second cell may be derived from the same non-human animal or the same non-human animal specie as the cell expressing the chimeric MHC I ptide. Nonlimiting examples of proteins with which the extracellular portion of a chimeric protein (e.g., HLA-A2/H-2K) may interact include T cell receptor (TCR) and its co-receptor CD8. Thus, a second cell may be a T cell. In addition, the extracellular portion of the chimeric MHC I protein may bind a protein on the surface of Natural Killer (NK) cells, e.g., killer globulin receptors (KIRs) on the surface of NK cells.

A T cell or NK cell may bind a complex formed between the chimeric MHC I ptide and its displayed peptide fragment. Such binding may result in T cell activation or inhibition of NK-mediated cell killing, respectively. One hypothesis is that NK cells have evolved to kill either infected or tumor cells that have evaded T cell mediated cytotoxicity by downregulating their MHC I complex. However, when MHC I complex is expressed on cell surface, NK cell receptors recognize it, and NK-mediated cell g is inhibited. Thus, in some aspects, when an NK cell binds a complex formed between the chimeric MHC I polypeptide (e.g., HLA-A2/H-2K polypeptide) and a displayed peptide fragment on the surface of ed or tumor cell, the NK-mediated cell g is inhibited.

In one example, the chimeric MHC I ptide described , e.g., a chimeric HLA-A2/H-2K polypeptide, interacts with CD8 protein on the surface of a second cell. In one embodiment, the chimeric /H-2K polypeptide interacts with endogenous rodent (e.g., mouse or rat) CD8 protein on the surface of a second cell. In one embodiment, the second cell is a T cell. In another embodiment, the second cell is ered to express CD8. In certain s, the chimeric HLA-A2/H-2K polypeptide interacts with a human CD8 on the surface of the second cell (e.g., a human cell or a rodent cell). In some such embodiments, the non-human animal, e.g., a mouse or a rat, comprises a human CD8 transgene, and the mouse or the rat expresses a functional human CD8 protein.

The ic MHC I polypeptide bed herein may also interact with a nonhuman (e.g., a mouse or a rat) TCR, a human TCR, or a humanized TCR on a second cell.

The chimeric MHC I polypeptide may interact with an endogenous TCR (e.g., mouse or rat TCR) on the surface of a second cell. The chimeric MHC I polypeptide may also interact with a human or humanized TCR expressed on the surface of a second cell, wherein the cell is derived from the same animal or animal specie (e.g., mouse or rat) as the cell expressing the chimeric MHC I polypeptide. The chimeric MHC I polypeptide may interact with a human TCR sed on the surface of a human cell.

In addition to genetically engineered non-human animals, a non-human embryo (e.g., a rodent embryo, e.g., mouse or a rat ) is also provided, wherein the embryo comprises a donor ES cell that is derived 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 ES donor cell that comprises the chimeric MHC I gene, and host embryo cells.

Also provided is a tissue, wherein the tissue is derived from a non-human animal (e.g., a mouse or a rat) as described herein, and expresses the ic MHC I polypeptide (e.g., HLA-A2/H-2K polypeptide).

In addition, a non-human cell isolated from a non-human animal as described herein is ed. In one embodiment, the cell is an ES cell. In one embodiment, the cell is an antigen-presenting cell, e.g., dendritic cell, macrophage, B cell. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is a lymphocyte.

Also provided is a man 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 r transfer.

In one aspect, a non-human induced otent cell comprising gene encoding a chimeric MHC I polypeptide (e.g., HLA-A2/H-2K polypeptide) as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.

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

Also provided is a method for making a cally engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or a rat) described . The method for making a cally ered non-human animal s in the animal whose genome comprises a nucleotide sequence encoding a chimeric MHC I polypeptide. In one embodiment, the method results in a genetically engineered mouse, whose genome comprises at an endogenous MHC I locus, e.g., H-2K locus, a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human n of the ic MHC I polypeptide comprises an extracellular domain of a human HLA-A2 and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K. In some embodiments, the method utilizes a targeting construct made using VELOCIGENE technology, ucing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE technology, as described in the es. In one embodiment, the ES cells are a mix of 129 and C57BL/6 mouse strains; in another embodiment, the ES cells are a mix of BALB/c and 129 mouse strains.

Thus, a nucleotide construct used for generating cally engineered nonhuman animals described herein is also provided. In one aspect, the nucleotide construct comprises: 5’ and 3’ non-human homology arms, a human DNA nt comprising human HLA-A gene sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human HLA-A gene. In one embodiment, the non-human homology arms are homologous to a non-human MHC class I locus (e.g., a mouse H-2K locus).

In one embodiment, the genomic fragment comprises a human HLA-A leader, an 1 domain, an 2 domain and an 3 domain coding sequence. In one ment, the human DNA fragment comprises, from 5’ to 3’: an HLA-A leader sequence, an HLA-A leader/1 intron, an HLA-A 1 exon, an HLA-A 1-2 intron, an HLA-A 2 exon, an HLA-A 2-3 intron, and an HLA-A 3 exon.

A selection te is a nucleotide sequence inserted into a targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. ly, a ion 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, respectively, but others are known in the art.

In one embodiment, the ion cassette is located at the 5’ end the human DNA fragment. In another embodiment, the selection cassette is located at the 3’ end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA nt. In another embodiment, the selection te is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located within the 2-3 intron.

In one embodiment, the 5’ and 3’ non-human homology arms comprise genomic sequence at 5’ and 3’ locations of an endogenous non-human (e.g., murine) MHC class I gene locus, respectively (e.g., 5’ of the first leader sequence and 3’ of the 3 exon of the non-human MHC I gene). In one ment, the nous MHC class I locus is selected from mouse H-2K, H-2D and H-2L. In a specific embodiment, the endogenous MHC class I locus is mouse H-2K.

In one aspect, a nucleotide construct is provided, comprising, from 5’ to 3’: a 5’ homology arm containing mouse genomic sequence 5’ of the endogenous mouse H-2K locus, a first human DNA fragment comprising a first genomic ce of an HLA-A gene, a 5’ recombination sequence site (e.g., loxP), a selection cassette, a 3’ recombination ce site (e.g., loxP), a second human DNA fragment comprising a second genomic sequence of an HLA-A gene and a 3’ homology arm containing mouse genomic sequence 3’ of an endogenous H-2K 3 exon. In one embodiment, the nucleotide construct comprises, from 5’ to 3’: a 5’ homology arm containing mouse genomic sequence 5’ of the endogenous mouse H-2K locus, a human genomic sequence including an HLA-A leader, an HLA-A leader/1 intron sequence, an HLA-A 1 exon, an HLA-A 1-2 intron, an HLA-A 2 exon, a first 5’ portion of an 2-3 intron, a selection cassette flanked by recombination sites, a second 3’ portion of an 2-3 intron, an HLA-A 3 exon, and a 3’ homology arm containing non-mouse genomic sequence 3’ of the endogenous mouse H-2K 3 exon. In one embodiment, a 5’ homology arm sequence is set forth in SEQ ID NO:1, and a 3’ homology arm sequence is set forth in SEQ ID NO:2.

Upon completion of gene targeting, ES cells or genetically modified non-human animals are screened to confirm successful oration of exogenous tide sequence of interest or expression of exogenous polypeptide. Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, tative PCT (e.g., real-time PCR using TAQMAN), fluorescence in situ hybridization, Northern blotting, flow try, Western is, immunocytochemistry, immunohistochemistry, etc. In one example, man animals (e.g., mice) bearing the c 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 expression analysis, Nature Biotech. 21(6):652-659. Other assays that identify a specific nucleotide or amino acid sequence in the genetically modified s are known to those skilled in the art.

The disclosure also provides a method of modifying an MHC I locus of a nonhuman animal to express a ic human/non-human MHC I polypeptide bed herein. In one embodiment, the invention provides a method of modifying an MHC I locus of a mouse to express a chimeric human/mouse MHC I polypeptide n the method comprises replacing at an endogenous MHC I locus a nucleotide sequence encoding a peptide binding domain of a mouse MHC ptide with a tide sequence encoding a peptide binding domain of a human MHC I polypeptide. In some aspects, a nucleotide sequence of an extracellular domain of a mouse MHC I is replaced by a nucleotide sequence of an extracellular domain of a human MHC I. The mouse may fail to express the peptide binding or the extracellular domain of the mouse MHC I from an endogenous MHC I locus. In some embodiments, a nucleotide ce of an extracellular domain of a mouse H-2K is replaced by a nucleotide sequence of an extracellular domain of a human HLA-A2, such that the modified mouse MHC I locus ses a chimeric HLA-A2/H-2K polypeptide.

In one aspect, a method for making a chimeric human HLA class I/non-human MHC class I molecule is provided, comprising expressing in a single cell a chimeric HLAA /H-2K protein from a nucleotide construct, wherein the nucleotide construct comprises a cDNA ce that encodes an 1, 2, and 3 domain of an HLA-A protein and a transmembrane and asmic domain of a non-human H-2K protein, e.g., mouse H-2K protein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a iral . In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a cell that expresses a chimeric human HLA Class I/non-human MHC I protein (e.g., HLA-A/H-2K protein) is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric MHC class I gene, wherein the chimeric MHC class I gene comprises a sequence of a human HLA-A gene fused in operable linkage with a sequence of a non-human H-2K gene, e.g., mouse H-2K gene. In one embodiment, the sequence of the human HLA-A gene comprises the exons that encode 1, 2 and 3 domains of an HLA-A protein. In one embodiment, the sequence of the non- human H-2K gene comprises the exons that encode transmembrane and asmic domains of an H-2K protein. In one embodiment, the cell is ed from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a ™ cell).

A chimeric MHC class I molecule made by a non-human animal as described herein is also provided, wherein the chimeric MHC class I molecule comprises 1, 2, and 3 domains from a human HLA-A protein and embrane and cytoplasmic s from a non-human, e.g., mouse, H-2K protein. The chimeric MHC I polypeptide described herein maybe detected by anti-HLA-A antibodies. Thus, a cell displaying chimeric human/non-human MHC I polypeptide may be detected and/or ed using anti-HLA-A antibody. In some instances, the ic MHC I polypeptide described herein maybe detected by an anti-HLA-A2 antibody. gh the following Examples describe a genetically engineered animal whose genome comprises a replacement of a nucleotide sequence ng an extracellular domain of mouse H-2K polypeptide with the sequence encoding an extracellular domain of a human HLA-A at the endogenous mouse H-2K locus, one skilled in the art would understand that a similar strategy may be used to replace other mouse MHC I loci (H-2D, H-2L, etc.) with their corresponding human HLA loci (HLA-B, HLA-C, etc.). Thus, a non-human animal comprising in its genome a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide wherein a human portion of the polypeptide is derived from another HLA class I protein is also provided. The replacement of multiple MHC I loci is also contemplated.

Genetically Modified 2 Microglobulin Animals The invention lly provides genetically modified non-human animals that comprise in their genome a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide; thus, the animals express a human or humanized 2 microglobulin polypeptide. 2 microglobulin or the light chain of the MHC class I complex (also abbreviated “2M”) is a small (12 kDa) non-glycosylated protein, that ons primarily to ize the MHC I  chain. The human 2 microglobulin gene encodes a n of 119 amino acids, with 20 N-terminal amino acids encoding a leader sequence. The mature protein comprises 99 amino acids. The gene contains 4 exons, with the first exon containing the 5’ untranslated region, the entire leader sequence and the first two amino acids of the mature polypeptide; the second exon ng the ty of the mature protein; the third exon encoding the last four amino acids of the mature protein and a stop codon; and the fourth exon containing the 3’ non-translated . Gussow et al. (1987) The 2-Microglobulin Gene. Primary Structure and Definition of the Transcriptional Unit, J. Immunol. 139:3131-38. 2 lobulin is non-covalently associated with MHC I. Unbound 2 microglobulin is found in body , such as plasma, and is carried to the kidney for excretion. Kidney dysfunction causes accumulation of 2 microglobulin, which can be pathogenic (e.g., Dialysis Related Amyloidosis); the accumulated protein forms ntous fibrils resembling amyloid plaques in joints and tive tissues.

In addition to Dialysis Related Amyloidosis, 2 microglobulin has been implicated in a number of other disorders. Elevated levels of 2 microglobulin were detected in lymphocytic malignancies, e.g., non-Hodgkin’s lymphoma and multiple myeloma. See, e.g., Shi et al. (2009) 2 Microglobulin: Emerging as a Promising Cancer Therapeutic Target, Drug Discovery Today 14:25-30. Some other malignancies with elevated levels of 2 microglobulin e breast cancer, prostate , lung cancer, renal cancer, gastrointestinal and nasopharyngeal s. Overexpression of 2 microglobulin has been suggested to have tumor growth promoting effects. Id. It has also been recently shown that 2 microglobulin drives epithelial to mesenchymal transition, promoting cancer bone and soft tissue metastasis in breast, te, lung and renal cancers. Josson et al. (2011) 2 microglobulin Induces Epitelial to Mesenchymal Transition and Confers Cancer ity and Bone Metastasis in Human Cancer Cells. Cancer Res. 71(7): 1-11. 2 microglobulin interacts with a non-classical MHC I member, hemochromatosis (HFE) protein, and with the transferrin receptor, and modulates iron homeostasis. Id. Involvement of 2 microglobulin in other hallmarks of ancy (self-renewal, angiogenesis enhancement, resistance to treatment) is widely documented in the art.

Mice deficient in 2 microglobulin have been reported. S ee, Koller et al. (1990) Normal development of mice deficient in 2m, MHC class I proteins, and CD8+ T cells, e 248: 1227-1230. As reported in Koller et al., these mice appeared y, however, MHC class I expression was not detected. Further, most T cell populations appeared normal in some tissues, while a marked decrease of CD8+ T cells was ed in others. This purported lack of MHC I expression ees with previous results obtained by Allen et al. ((1986) 2 microglobulin Is Not Required for Cell Surface Expression of the Murine Class I Histocompatibility Antigen H-2Db or of a Truncated H-2Db, Proc. Natl. Acad.

Sci. USA 83:7447-7451). Allen et al. reported that 2 microglobulin was not absolutely required for cell surface expression of all MHC I xes, because cells lacking 2 lobulin were able to express H-2Db. However, the on of H -2Db. in these cells was presumably mised, and conformation of was different from the native protein, which explains the ity of Koller and colleagues to detect this protein using dies against native H-2Db. r, cells lacking 2 microglobulin can reportedly present endogenous antigen to CD8+ T cells (including exogenous CD8+ T cells from normal mice), and 2 lobulin is reportedly not required in order to develop high levels of H-2d MHC class I-restricted CD8+ CTLs in response to antigen challenge in mice, although it is required in order to sustain an effective immune response. Quinn et al. (1997) Virus-Specific, CD8+ Major Histocompatibility Complex Class I-Restricted Cytotoxic T Lymphocytes in Lymphocytic Choriomeningitis Virus-Infected 2-Microglobulin-Deficient Mice, J. Virol. 71:8392-8396. It is of note that the ability to generate high levels of such T cells in the absence of 2 microglobulin is reportedly limited to an H-2d MHC class I- restricted response. 2 microglobulin deficient mice have been reported to have a host of dramatic characteristics, such as, for example, an increased susceptibility to some parasitic diseases, an increased susceptibility to hepatitis infections, a deficiency in iron metabolism, and an impaired breeding phenotype. Cooper et al. (2007) An impaired breeding phenotype in mice with a c deletion of Beta-2 microglobulin and diminished MHC class I sion: Role in reproductive fitness, Biol. Reprod. 77:274-279.

Mice that express human 2 microglobulin as well as human HLA class I molecules (i.e., HLA-B7) on a randomly inserted transgene have been reported.

Chamberlain et al. (1988) Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (2-microglobulin) chain genes in transgenic mice, Proc. Natl. Acad. Sci. USA 85:7690-7694. The expression of human HLA class I was consistent with that of endogenous class I with a marked decrease in the liver. Id. The expression of human 2 microglobulin was also consistent with the endogenous 2 microglobulin, while sion of the human HLA class I molecule was increased 10- to 17-fold in double transgenic mice. Id. However, the authors did not t a replacement of a mouse endogenous 2 microglobulin locus with a human 2 microglobulin locus.

] Therefore, disclosed herein is a genetically engineered non-human animal (e.g., a rodent, e.g., a mouse or a rat) whose genome comprises a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide. In one aspect, the animal does not express an endogenous non-human 2 microglobulin from an endogenous non-human 2 microglobulin locus. In some embodiments, the nucleotide sequence encodes a 2 microglobulin polypeptide that is partially human and partially non-human, e.g., it contains some amino acids that correspond to human and some amino acids that correspond to nonhuman 2 microglobulin. In one aspect, the non-human animal does not express an endogenous non-human 2 microglobulin polypeptide from an endogenous non-human locus, and only expresses the human or humanized 2 microglobulin polypeptide. In one example, the non-human animal does not s a complete endogenous non-human 2 lobulin polypeptide but only expresses a portion of a non-human endogenous 2 microglobulin polypeptide from an endogenous 2 microglobulin locus. Thus, in s embodiments, the animal does not express a functional man 2 microglobulin polypeptide from an endogenous non-human 2 microglobulin locus. In a specific aspect, the nucleotide ce encoding the human or humanized 2 microglobulin is located at an endogenous man 2 microglobulin locus. In one , the animal comprises two copies of 2 microglobulin locus comprising a nucleotide sequence encoding a human or zed 2 microglobulin polypeptide. In another aspect, the animal comprises one copy of 2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide. Thus, the animal may be homozygous or heterozygous for 2 microglobulin locus comprising a nucleotide sequence that encodes a human or humanized 2 microglobulin polypeptide. The nucleotide sequence of the human or humanized 2 microglobulin may be derived from a collection of 2 microglobulin sequences that are naturally found in human populations. In s embodiments, the genetically engineered non-human animal of the invention ses in its germline a nucleotide sequence encoding a human or humanized 2 microglobulin. In one embodiment, a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide comprising a human 2 microglobulin amino acid ce. In one embodiment, the polypeptide is capable of binding to an MHC I protein.

The nucleotide sequence encoding the human or humanized 2 microglobulin polypeptide may se nucleic acid residues corresponding to the entire human 2 lobulin gene. Alternatively, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 21-119 of a human 2 microglobulin protein (i.e., amino acid residues corresponding to the mature human 2 microglobulin). In an alternative embodiment, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 23-115 of a human 2 microglobulin protein, for example, amino acid sequence set forth in amino acids 23-119 of a human 2 microglobulin protein. The nucleic and amino acid sequences of human 2 microglobulin are described in Gussow et al., supra, incorporated herein by nce.

Thus, the human or humanized 2 microglobulin polypeptide may comprise amino acid sequence set forth in amino acids 23-115 of a human 2 microglobulin ptide, e.g., amino acid sequence set forth in amino acids 23-119 of a human 2 microglobulin ptide, e.g., amino acid sequence set forth in amino acids 21-119 of a human 2 microglobulin polypeptide. Alternatively, the human 2 microglobulin may comprise amino acids 1-119 of a human 2 microglobulin ptide.

In some embodiments, the nucleotide sequence encoding a human or humanized 2 microglobulin ses a nucleotide sequence set forth in exon 2 to exon 4 of a human 2 microglobulin gene. Alternatively, the tide sequence ses nucleotide sequences set forth in exons 2, 3, and 4 of a human 2 microglobulin gene. In this embodiment, the nucleotide sequences set forth in exons 2, 3, and 4 are operably linked to allow for normal transcription and ation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to exon 4 of a human 2 lobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human 2 microglobulin gene. In a specific embodiment, the human sequence comprises about 2.8 kb of a human 2 microglobulin gene.

Thus, the human or humanized 2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequence set forth in exon 2 to exon 4 of a human 2 microglobulin, e.g., tide sequence corresponding to exon 2 to exon 4 of a human 2 lobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequences set forth in exons 2, 3, and 4 of a human 2 microglobulin gene. In a ic embodiment, the human or humanized 2 microglobulin polypeptide is d by a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human 2 lobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide ce comprising about 2.8 kb of a human 2 microglobulin gene. As exon 4 of the 2 microglobulin gene contains the 5’ untranslated region, the human or humanized polypeptide may be encoded by a nucleotide sequence comprising exons 2 and 3 of the 2 microglobulin gene.

It would be understood by those of ordinary skill in the art that although specific nucleic acid and amino acid sequences to te genetically ered animals are described in the t examples, sequences of one or more conservative or nonconservative amino acid substitutions, or sequences differing from those bed herein due to the degeneracy of the genetic code, are also provided. ore, a non-human animal that expresses a human 2 microglobulin sequence is provided, wherein the 2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human 2 microglobulin sequence. In a specific embodiment, the 2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% cal to the human 2 microglobulin ce described in the Examples. In one embodiment, the human 2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human 2 microglobulin sequence comprises one or more non-conservative substitutions.

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

Similarly to a non-human animal comprising a nucleotide ce encoding a chimeric human/non-human MHC I polypeptide, the man animal comprising a nucleotide sequence encoding a human or zed 2 microglobulin 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, , primate (e.g., marmoset, rhesus monkey). In some embodiments of the invention, the non-human animal is a mammal. In a specific embodiment, the non-human animal is a murine, e.g., a rodent (e.g., a mouse or a rat). In one embodiment, the animal is a mouse.

Thus, in some aspects, a genetically engineered mouse is provided, wherein the mouse comprises a nucleotide sequence ng a human or a humanized 2 microglobulin polypeptide as bed herein. A genetically engineered mouse is provided, wherein the mouse comprises at its nous 2 microglobulin locus a nucleotide sequence ng a human or humanized 2 microglobulin polypeptide (e.g., a human or substantially human 2 microglobulin polypeptide). In some embodiments, the mouse does not express an nous 2 microglobulin polypeptide (e.g., a functional endogenous 2 microglobulin polypeptide) from an endogenous 2 lobulin locus. In some embodiments, the genetically engineered mouse comprises a nucleotide sequence comprising exon 1 of a mouse 2 lobulin gene and exons 2, 3, and 4 of a human 2 microglobulin gene. In some embodiments, the mouse expresses the human or humanized 2 microglobulin polypeptide.

In one aspect, a modified non-human 2 microglobulin locus is provided that comprises a heterologous 2 microglobulin sequence. In one embodiment, the heterologous 2 microglobulin ce is a human or a humanized sequence.

In one embodiment, the modified locus is a rodent locus. In a specific embodiment, the rodent locus is selected from a mouse or rat locus. In one embodiment, the non-human locus is modified with at least one human 2 microglobulin coding sequence.

In one embodiment, the logous 2 microglobulin sequence is operably linked to endogenous regulatory elements, e.g., endogenous promoter and/or expression control sequence. In a specific embodiment, the heterologous 2 microglobulin sequence is a human sequence and the human sequence is operably linked to an endogenous er and/or expression control sequence.

In one aspect, a modified non-human 2 microglobulin locus is provided that comprises a human sequence operably linked to an nous er and/or expression control sequence.

In various aspects, the human or humanized 2 microglobulin expressed by a genetically modified non-human animal, or cells, embryos, or tissues derived from a nonhuman animal, ves all the functional s of the endogenous and/or human 2 microglobulin. For example, it is preferred that the human or humanized 2 microglobulin binds the  chain of MHC I polypeptide (e.g., endogenous non-human or human MHC I polypeptide). The human or humanized 2 microglobulin polypeptide may bind, recruit or otherwise ate with any other molecules, e.g., receptor, anchor or signaling molecules that associate with endogenous man and/or human 2 microglobulin (e.g., HFE, etc.).

In addition to genetically modified animals (e.g., rodents, e.g., mice or rats), also provided is a tissue or cell, wherein the tissue or cell is derived from a non-human animal as described , and comprises a heterologous 2 microglobulin gene or 2 microglobulin sequence, i.e., nucleotide and/or amino acid sequence. In one ment, the heterologous 2 microglobulin gene or 2 microglobulin sequence is a human or humanized 2 microglobulin gene or human or zed 2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express MHC I complex, e.g., an antigen presenting cell. The human or humanized 2 microglobulin polypeptide expressed by said cell may interact with endogenous non-human MHC I (e.g., rodent MHC I), to form a functional MHC I complex. The resultant MHC I complex may be capable of interacting with a T cell, e.g., a cytotoxic T cell. Thus, also provided is an in vitro complex of a cell from a non-human animal as described herein and a T cell.

Also provided are non-human cells that se human or humanized 2 lobulin gene or sequence, and an additional human or humanized sequence, e.g., chimeric MHC I polypeptide presently disclosed. In such an instance, the human or humanized 2 microglobulin polypeptide may interact with, e.g., a chimeric human/nonhuman MHC I ptide, and a functional MHC I complex may be . In some aspects, such complex is capable of interacting with a TCR on a T cell, e.g., a human or a non-human T cell. Thus, also provided in an in vitro x of a cell from a non-human animal as described herein and a human or a non-human T cell.

Another aspect of the disclosure is a rodent embryo (e.g., a mouse or a rat embryo) comprising a heterologous 2 microglobulin gene or 2 lobulin sequence as described herein. In one embodiment, the embryo comprises an ES donor cell that comprises the heterologous 2 microglobulin gene or 2 microglobulin sequence, and host embryo cells. The heterologous 2 microglobulin gene or 2 microglobulin sequence is a human or humanized 2 microglobulin gene or 2 microglobulin sequence.

This invention also encompasses a non-human cell sing a chromosome or fragment thereof of a non-human animal as described herein (e.g., wherein the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide). The non-human cell may comprise a nucleus of a non-human animal as described . In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear er.

] In one , a non-human induced otent cell comprising a heterologous 2 microglobulin gene or 2 microglobulin sequence is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein. In one embodiment, the heterologous 2 microglobulin gene or 2 microglobulin sequence is a human or humanized gene or sequence.

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

The disclosure also provides methods for making a genetically engineered nonhuman animal (e.g., a cally engineered rodent, e.g., a mouse or a rat) described . The s result in an animal whose genome comprises a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide. In one , the s result in a cally engineered mouse, whose genome comprises at an endogenous 2 microglobulin locus a nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide. In some instances, the mouse does not s a functional mouse 2 microglobulin from an endogenous mouse 2 microglobulin locus. In some aspects, the methods utilize a targeting construct made using VELOCIGENE technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE technology, as described in the Examples. In one embodiment, the ES cells are mix of 129 and C57BL/6 mouse strains; in another embodiment, the ES cells are a mix of BALB/c and 129 mouse strains.

Also provided is a nucleotide construct used for generating cally engineered non-human animals. The nucleotide construct may comprise: 5’ and 3’ non- human homology arms, a human DNA fragment comprising human 2 microglobulin sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human 2 microglobulin gene. In one embodiment, the non-human homology arms are homologous to a man 2 microglobulin locus. The genomic fragment may comprise exons 2, 3, and 4 of the human 2 microglobulin gene. In one instance, the genomic fragment comprises, from 5’ to 3’: exon 2, , exon 3, intron, and exon 4, all of human 2 microglobulin sequence. The selection cassette may be located anywhere in the construct outside the 2 microglobulin coding region, e.g., it may be located 3’ of exon 4 of the human 2 lobulin. The 5’ and 3’ non-human homology arms may se genomic sequence 5’ and 3’ of endogenous non-human 2 microglobulin gene, respectively. In another embodiment, the 5’ and 3’ man homology arms comprise genomic sequence ’ of exon 2 and 3’ of exon 4 of nous non-human gene, respectively.

Another aspect of the invention relates to a method of modifying a 2 microglobulin locus of a non-human animal (e.g., a rodent, e.g., a mouse or a rat) to s a human or humanized 2 microglobulin polypeptide described . One method of modifying a 2 microglobulin locus of a mouse to express a human or humanized 2 microglobulin polypeptide comprises replacing at an endogenous 2 microglobulin locus a nucleotide sequence encoding a mouse 2 microglobulin with a nucleotide sequence encoding the human or humanized 2 microglobulin polypeptide. In one ment of such method, the mouse does not express a functional 2 microglobulin polypeptide from an endogenous mouse 2 microglobulin locus. In some specific embodiments, the nucleotide ce encoding the human or humanized 2 microglobulin polypeptide comprises nucleotide sequence set forth in exons 2 to 4 of the human 2 microglobulin gene. In other embodiments, the tide sequence encoding the human or humanized 2 lobulin polypeptide comprises nucleotide sequences set forth in exons 2, 3, and 4 of the human 2 microglobulin gene. cally Modified MHC I / 2 Microglobulin Animals In various embodiments, the invention lly provides genetically modified man animals that se in their genome nucleotide sequences encoding both human or humanized MHC I and 2 microglobulin polypeptides; thus, the animals express both human or humanized MHC I and 2 microglobulin polypeptides.

Functional differences arise in the use of mixed human/non-human system components. HLA class I binds 2 microglobulin tighter than mouse class I. Bernabeu (1984) 2-microgobulin from serum associates with MHC class I antigens on the surface of cultured cells, Nature 308:642-645. Attempts to abrogate onal differences are reflected in the construction of particular humanized MHC mice. H-2 class I and class 2 knockout mice (in a mouse 2 microglobulin KO background) that s a randomly integrated human HLA-A2.1/HLA-DR1 chimeric transgene having an 1 and 2 of human HLA-A2.1, and 3 of mouse H-2Db, attached at its inal via a linker to the C-terminus of human 2-microglobulin have been developed. See, e.g., Pajot et al. (2004) A mouse model of human ve immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class ss II- knockout mice, Eur. J. Immunol. 34:3060-3069. These mice reportedly generate antigenspecific antibody and CTL responses against hepatitis B virus, whereas mice merely transgenic for HLA-A2.1 or H-2 class I/class II knockout mice do not. The deficiency of mice that are merely transgenic for the genes ably stems from the ability of such mice to employ nous class I and/or class II genes to circumvent any transgene, an option not available to MHC ut mice. However, the mice may s at least H-2Db, presumably due to breedings into the mouse 2 microglobulin knockout mouse background (see, Pajot et al., supra; which apparently comprised an intact endogenous class I and class II locus).

Cell surface expression of the chimeric fusion with human 2 microglobulin is reportedly lower than endogenous MHC expression, but survivability/rate of NK killing is not reported, nor is the rate of NK self-killing. Pajot et al., supra. Some improvement in CD8+ T cell numbers was ed over MHC class I-deficient 2-microglobulin knockout mice (2- 3% of total splenocytes, vs. 0.6-1% in the 2 KO mice). However, T cell variable region usage exhibited altered profiles for BV 5.1, BV 5.2, and BV 11 gene segments. Both CD8+ and CD4+ T cell responses were reportedly restricted to the appropriate hepatitis B antigen used to immunize the mice, although at least two mice killed cells bearing either of the antigens, where the mice were immunized with only one antigen, which might be due to a lack of NK cell tion or lack of NK cell selectivity.

As mentioned above, mice transgenic for both human MHC I and human 2 microglobulin comprise a tide ce encoding a chimeric MHC I/2 microglobulin protein, wherein the MHC I and 2 microglobulin portions are contained within a single polypeptide chain, resulting in MHC I  chain and 2 microglobulin being covalently linked to each other and thereby tethered at the cell surface. A mouse which comprises in its genome two independent nucleotide sequences, one encoding a human or humanized MHC I polypeptide and the other encoding a human or humanized 2 microglobulin polypeptide is provided. The mouse provided herein would s an MHC I complex that more closely resembles an MHC I complex present in nature, wherein MHC I  chain and 2 microglobulin are provided on two separate ptide chains with 2 microglobulin noncovalently associating with the MHC I  chain.

] Thus, the present sure provides a non-human animal comprising in its genome: a first tide sequence encoding a human or humanized MHC I polypeptide, and a second nucleotide sequence encoding a human or zed 2 microglobulin polypeptide. In one aspect, provided is a non-human animal comprising in its genome: (a) a first nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises a peptide binding domain or an ellular domain of a human MHC I (e.g., HLA-A, HLA-B, or HLA-C; e.g., HLA-A2), and (b) a second nucleotide sequence encoding a human or humanized 2 microglobulin polypeptide.

The first nucleotide sequence may be located at an endogenous non-human MHC I locus such that the animal comprises in its genome a replacement at the MHC I locus of all or a portion of endogenous MHC I gene (e.g., a portion encoding a peptide binding domain or an extracellular domain) with the corresponding human MHC I sequence. Thus, the animal may comprise at an endogenous MHC I locus a nucleotide sequence encoding an extracellular domain of a human MHC I (e.g., HLA-A, HLA-B, or HLA-C; e.g., HLA-A2) and transmembrane and asmic domains of endogenous non-human MHC I (e.g., H- 2K, H-2D, etc., e.g., H-2Kb). In one aspect, the animal is a mouse, and the first nucleotide sequence ses a nucleotide sequence encoding an ellular domain of a human HLA-A2 (e.g., HLA-A2.1) and embrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb).

] The second nucleotide sequence may be located at an endogenous non-human 2 microglobulin locus such that the animal comprises in its genome a replacement at the 2 microglobulin locus of all or a n of endogenous 2 lobulin gene with the corresponding human 2 microglobulin sequence. The second nucleotide sequence may comprise a nucleotide sequence set forth in exon 2 to exon 4 of a human 2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences set forth in exons 2, 3, and 4 of a human 2 microglobulin gene. In this embodiment, nucleotide sequences are operably linked to each other. The second nucleotide sequence may further comprise the sequence of exon 1 of a non-human 2 microglobulin gene.

] In one aspect, the animal does not express a functional MHC I from an endogenous non-human MHC I locus (e.g., does not express either a peptide binding domain or an extracellular domain of the endogenous MHC I), and the animal does not express a functional 2 microglobulin polypeptide from an endogenous non-human 2 microglobulin locus. In some aspects, the animal is homozygous for both an MHC I locus comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide and a 2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized 2 microglobulin. In other aspects, the animal is heterozygous for both an MHC I locus comprising a nucleotide sequence encoding a chimeric human/nonhuman MHC I polypeptide and a 2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized 2 microglobulin. ably, the first and the second tide sequences are operably linked to endogenous expression control elements (e.g., promoters, enhancers, silencers, etc.).

Various other embodiments of the first and second nucleotide sequences (and the polypeptides they encode) encompassed herein may be readily understood from the embodiments described hout the ication, e.g., those described in the sections related to genetically engineered MHC I animals and genetically engineered 2 microglobulin animals.

In one aspect, the disclosure provides a mouse sing in its genome (a) a first nucleotide sequence encoding a chimeric human/mouse MHC I ptide fically, HLA-A2/H-2K polypeptide), wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 and the mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K, and (b) a second tide sequence encoding a human or humanized 2 microglobulin polypeptide (e.g., wherein the nucleotide sequence comprises a nucleotide ce set forth in exon 2 to exon 4 of the human 2 microglobulin gene or nucleotide ces set forth in exon 2, 3, and 4 of the human 2 microglobulin gene), wherein the first nucleotide sequence is d at an endogenous H-2K locus, and the second sequence is located at an nous 2 microglobulin locus. In one embodiment, the mouse does not express functional H-2K and mouse 2 lobulin polypeptides from their respective endogenous loci. In one embodiment, the mouse expresses both the chimeric human/mouse MHC I ptide and the human or humanized 2 microglobulin polypeptide.

As shown in the following Examples, animals genetically ered to coexpress both the human or humanized MHC I and 2 microglobulin displayed increased expression of chimeric MHC class I on cell surface in comparison to animals humanized for MHC I alone. In some embodiments, co-expression of human or humanized MHC I and 2 microglobulin increases cell surface expression of human or humanized MHC I by more than about 10%, e.g., more than about 20%, e.g., about 50% or more, e.g., about 70%, over the expression of human or humanized MHC I in the absence of human or humanized 2 microglobulin.

The disclosure also provides a method of making genetically engineered nonhuman animals (e.g., s, e.g., rats or mice) whose genome comprises a first and a second nucleotide ce as described herein. The method generally comprises generating a first genetically engineered non-human animal whose genome comprises a first nucleotide sequence described herein (i.e., a human or humanized MHC I sequence), generating a second genetically engineered non-human animal whose genome comprises a second nucleotide sequence described herein (i.e., a human or humanized 2 microglobulin sequence), and breeding the first and the second animal to obtain progeny whose genome contains both nucleotide sequences. In one embodiment, the first and the second animal are heterozygous for the first and the second nucleotide sequence, respectively. In one embodiment, the first and the second animal are homozygous for the first and the second nucleotide sequence, tively. In one embodiment, the first and second animals are generated through replacement of endogenous non-human loci with the first and the second nucleotide sequences, respectively. In one aspect, the first and the second animals are generated through utilization of constructs generated via VELOCIGENE technology, and introducing targeted ES cell clones g such constructs into an embryo (e.g., a rodent embryo, e.g., a mouse or a rat embryo) via the MOUSE method.

Use of Genetically Modified Animals In various ments, the genetically modified non-human animals described herein make APCs with human or humanized MHC I and/or 2 microglobulin on the cell surface and, as a result, t peptides derived from cytosolic proteins as epitopes for CTLs in a like manner, because substantially all of the components of the complex are human or humanized. The genetically ed non-human animals of the invention can be used to study the function of a human immune system in the zed animal; for identification of antigens and antigen es that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer es), e.g., for use in vaccine development; for identification of high affinity T cells to human pathogens or cancer antigens (i.e., T cells that bind to antigen in the context of human MHC I complex with high avidity), e.g., for use in adaptive T cell therapy; for evaluation of vaccine candidates and other e strategies; for studying human autoimmunity; for ng human infectious diseases; and otherwise for devising better therapeutic strategies based on human MHC expression.

The MHC I complex binds es and presents them on cell surface. Once presented on the surface in the context of such a complex, the peptides are recognizable by T cells. For example, when the peptide is derived from a pathogen or other antigen of interest (e.g., a tumor n), T cell recognition can result in T cell activation, macrophage killing of cells bearing the presented peptide sequence, and B cell activation of antibodies that bind the presented sequence.

T cells ct with cells expressing MHC I complex through the peptide-bound MHC class I ectodomain and the T cell's CD8 ectodomain. CD8+ T cells that encounter APC's that have suitable antigens bound to the MHC class I molecule will become cytotoxic T cells. Thus, antigens that in the context of MHC class I bind with high avidity to a T cell receptor are potentially important in the development of treatments for human pathologies.

However, presentation of ns in the context of mouse MHC I is only somewhat relevant to human disease, since human and mouse MHC complexes recognize antigens ently, e.g., a mouse MHC I may not recognize the same antigens or may present ent epitopes than a human MHC I. Thus, the most relevant data for human pathologies is obtained through studying the presentation of antigen es by human MHC I.

Thus, in various embodiments, the genetically engineered animals of the present invention are useful, among other things, for evaluating the capacity of an antigen to initiate an immune response in a human, and for generating a diversity of antigens and identifying a specific antigen that may be used in human vaccine development.

In one , a method for determining antigenicity in a human of a peptide sequence is provided, comprising ng a genetically modified non-human animal as described herein to a molecule comprising the peptide sequence, allowing the non-human animal to mount an immune response, and detecting in the non-human animal a cell that binds a sequence of the e ted by a chimeric human/non-human MHC I, or a humanized MHC I complex (comprising a ic human/non-human MHC I and a human or zed 2 microglobulin) as described herein.

] In one aspect, a method for determining whether a peptide will provoke a cellular immune response in a human is provided, comprising exposing a genetically modified nonhuman animal as bed herein to the peptide, allowing the non-human animal to mount an immune se, and detecting in the non-human animal a cell that binds a sequence of the peptide by a chimeric human/non-human MHC class I molecule as described herein. In one embodiment, the non-human animal following exposure ses an MHC class I- restricted CD8+ cytotoxic T lymphocyte (CTL) that binds the peptide. In one embodiment, the CTL kills a cell bearing the peptide.

In one aspect, a method for identifying a human CTL epitope is provided, comprising exposing a non-human animal as described herein to an antigen comprising a ve CTL epitope, allowing the non-human animal to mount an immune response, isolating from the non-human animal an MHC class I-restricted CD8+ CTL that binds the e, and identifying the epitope bound by the MHC class I-restricted CD8+ CTL.

In one aspect, a method is provided for identifying an HLA class I-restricted peptide whose presentation by a human cell and binding by a human lymphocyte (e.g., human T cell) will result in cytotoxicity of the peptide-bearing cell, comprising ng a non-human animal (or MHC class I-expressing cell f) as described herein to a molecule sing a peptide of st, isolating a cell of the non-human animal that ses a chimeric human/non-human class I molecule that binds the peptide of interest, exposing the cell to a human lymphocyte that is capable of conducting HLA class I-restricted cytotoxicity, and measuring peptide-induced cytotoxicity.

In one aspect, a method is provided for identifying an antigen that generates a cytotoxic T cell response in a human, comprising exposing a ve antigen to a mouse as described herein, allowing the mouse to generate an immune response, and identifying the antigen bound by the HLA-A-restricted molecule.

] In one embodiment, the antigen comprises a bacterial or viral surface or envelope protein. In one embodiment, the antigen comprises an antigen on the surface of a human tumor cell. In one embodiment, the n comprises a putative vaccine for use in a human. In one ment, the antigen ses a human epitope that generates antibodies in a human. In another embodiment, the antigen comprises a human epitope that generates high affinity CTLs that target the epitope/MHC I complex.

In one aspect, a method is provided for determining whether a putative antigen contains an epitope that upon exposure to a human immune system will generate an HLA-A- restricted immune response (e.g., HLA-A2-restricted response), comprising exposing a mouse as described herein to the putative antigen and measuring an antigen-specific HLAA-restricted (e.g., HLA-A2-restricted) immune response in the mouse.

In one embodiment, the putative antigen is ed from a biopharmaceutical or fragment f, a non-self protein, a surface antigen of a non-self cell, a surface antigen of a tumor cell, a surface antigen of a bacterial or yeast or fungal cell, a surface n or envelope protein of a virus.

In addition, the genetically engineered non-human animals described herein may be useful for identification of T cell ors, e.g., high-avidity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. The method may se: exposing the non-human animal described herein to an antigen, allowing the nonhuman animal to mount an immune response to the antigen, isolating from the non-human animal a T cell sing a T cell receptor that binds the antigen presented by a human or humanized MHC I, and determining the sequence of said T cell receptor.

In one , a method for identifying a T cell receptor variable domain having high affinity for a human tumor antigen is ed, comprising ng a mouse comprising humanized MHC I 1, 2, and 3 domains (e.g., HLA-A2 1, 2, and 3 domains) to a human tumor antigen; allowing the mouse to generate an immune response; and, isolating from the mouse a nucleic acid sequence encoding a T cell receptor variable domain, wherein the T cell receptor variable domain binds the human tumor antigen with a KD of no higher than about 1 nanomolar.

] In one embodiment, the mouse further comprises a replacement at the endogenous mouse T cell receptor variable region gene locus with a plurality of unrearranged human T cell or variable region gene segments, wherein the unrearranged human T cell receptor variable region gene segments recombine to encode a chimeric human-mouse T cell receptor gene comprising a human variable region and a mouse nt region. In one embodiment, the mouse comprises a human CD8 ene, and the mouse expresses a functional human CD8 protein.

T cell receptors having high y to tumor antigens are useful in cell-based therapeutics. T cell populations with high avidity to human tumor antigens have been prepared by exposing human T cells to HLA-A2 that has been d to minimize CD8 binding to the 3 subunit, in order to select only those T cells with extremely high avidity to the tumor antigen (i.e., T cell clones that recognize the antigen in spite of the inability of CD8 to bind 3). See, Pittet et al. (2003) 3 Domain s of e/MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells, J. Immunol. 171:1844-1849. The non-human animals, and cells of the non-human animals, are useful for identifying es that will form a complex with human HLA class I that will bind with high avidity to a T cell receptor, or activate a lymphocyte bearing a T cell receptor.

Antigen/HLA class I binding to a T cell, or activation of a T cell, can be measured by any suitable method known in the art. Peptide-specific APC-T cell binding and activation are measurable. For example, T cell engagement of antigen-presenting cells that express HLA-A2 reportedly causes PIP2 to accumulate at the immunosynapse, s crosslinking MHC class I molecules does not. See, Fooksman et al. (2009) Cutting Edge: Phosphatidylinositol 4,5-Bisphosphate Concentration at the APC Side of the Immunological Synapse Is Required for Effector T Cell Function, J. Immunol. 182:5179-5182.

Functional consequences of the interaction of a lymphocyte bearing a TCR, and a class I-expressing APC, are also measurable and include cell killing by the lymphocyte.

For e, contact points on the 2 subunit of HLA-A2 by CD8+ CTLs reportedly generate a signal for Fas-independent killing. HLA-A2-expressing Jurkat cells apoptose when contacted (by antibodies) at epitopes on the HLA-A2 molecule known (from crystallographic studies) to contact CD8, without any apparent reliance on the asmic domain. See, Pettersen et al. (1998) The TCR-Binding Region of the HLA Class I 2 Domain Signals Rapid Fas-Independent Cell Death: A Direct y for T Cell-Mediated Killing of Target Cells? J. Immunol. 160:4343-4352. It has been postulated that the rapid killing induced by HLA-A2 2 contact with a CD8 of a CD8+ CTL may primarily be due to this Fas-independent HLA-A2-mediated pathway (id.), as distinguished from TCR- ndent 3 domain-mediated killing—which by itself can induce apoptosis (see, Woodle et al. (1997) Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas n-mediated pathway, J. Immunol. 158:2156-2164).

The consequence of interaction between a T cell and an APC displaying a peptide in the context of MHC I can also be measured by a T cell eration assay.

Alternatively, it can be determined by measuring cytokine e commonly associated with activation of immune se. In one embodiment, IFN ELISPOT can be used to monitor and quantify CD8+ T cell activation.

As described herein, CD8+ T cell activation can be hampered in the genetically modified man animals bed herein due to species-specific binding of CD8 to MHC I. For embodiments where a species-specific CD8 interaction is desired, a cell of a genetically modified animal as described herein (e.g., a rodent, e.g., a mouse or a rat) is exposed (e.g., in vitro) to a human cell, e.g., a human CD8-bearing cell, e.g., a human T cell.

In one embodiment, an MHC class essing cell of a mouse as bed herein is exposed in vitro to a T cell that comprises a human CD8 and a T cell receptor. In a specific embodiment, the T cell is a human T cell. In one embodiment, the MHC class I-expressing cell of the mouse ses a e bound to a chimeric human/mouse MHC I or a humanized MHC I complex (which includes human 2 microglobulin), the T cell is a human T cell, and the ability of the T cell to bind the peptide-displaying mouse cell is determined. In one embodiment, activation of the human T cell by the peptide-displaying mouse cell is determined. In one embodiment, an in vitro method for measuring activation of a human T cell by the peptide-displaying cell is provided, comprising exposing a mouse or a mouse cell as described herein to an antigen of interest, ng a cell from said mouse or said mouse cell (presumably bearing a peptide derived from the antigen in complex with human or humanized MHC I) to a human T cell, and measuring activation of the human T cell. In one embodiment, the method is used to identify a T cell epitope of a human pathogen or a human sm. In one embodiment, the method is used to identify an epitope for a vaccine.

In one embodiment, a method is provided for determining T cell activation by a putative human therapeutic, comprising exposing a genetically modified animal as described herein to a putative human therapeutic (or e.g., exposing a human or humanized MHC I- expressing cell of such an animal to a peptide sequence of the putative therapeutic), exposing a cell of the cally modified animal that displays a human or humanized MHC I/peptide x to a T cell comprising a human T cell receptor and a CD8 capable of binding the cell of the genetically modified animal, and measuring activation of the human T cell that is induced by the peptide-displaying cell of the genetically modified animal.

In various embodiments, a x formed n a human or humanized MHC class I-expressing cell from an animal as described herein is made with a T cell that comprises a human CD8 sequence, e.g., a human T cell, or a T cell of a non-human animal that comprises a transgene that encodes human CD8. Mice transgenic for human CD8 are known in the art. Tishon et al. (2000) Trangenic Mice sing Human HLA and CD8 Molecules Generate HLA-Restricted Measles Virus xic T cytes of the Same Specificity as Humans with Natural Measles Virus ion, Virology 275(2):286-293; also, LaFace et al. (1995) Human CD8 Transgene Regulation of HLA Recognition by Murine T Cells, J. Exp. Med. 15-1325.

In addition to the ability to identify antigens and antigen epitopes from human pathogens or neoplasms, the genetically modified animals of the invention can be used to identify autoantigens of relevance to human autoimmune diseases, e.g., type I diabetes, multiple sclerosis, etc. For example, Takaki et al. ((2006) HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes, J. Immunol. 176:3257-65) describe the utility of NOD mice bearing HLA/2 microglobulin monochain in identifying type 1 es autoantigens. Also, the genetically modified animals of the invention can be used to study s s of human autoimmune disease. As some rphic alleles of human MHC I are known to be associated with development of certain diseases, e.g., autoimmune diseases (e.g., Graves’ disease, enia gravis, psoriasis, etc.; see Bakker et al. (2006) A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC, Nature Genetics 38:1166-72 and Supplementary Information and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated es, Proc. Natl. Acad. Sci. USA 106:18680-85, both incorporated herein by nce), a genetically modified animal of the invention sing a humanized MHC I locus including such an allele may be useful as an autoimmune disease model. In one embodiment, the disease allele is HLA-B27, and the disease is ankylosing spondylitis or reactive arthritis; thus, in one ment, the animal used for the study of these diseases comprises a human or humanized HLA-B27.

Other aspects of cellular immunity that involve MHC I complexes are known in the art; therefore, genetically engineered non-human animals described herein can be used to study these aspects of immune biology. For instance, binding of TCR to MHC class I is modulated in vivo by additional factors. Leukocyte immunoglobulin-like receptor subfamily B member (LILRB1, or LIR-1) is expressed on MHC Class I-restricted CTLs and downregulates T cell stimulation by binding a specific determinant on the 3 subunit of MHC class I molecules on APCs. Structural studies show that the binding site for LIR-1 and CD8 overlap, suggesting that inhibitory LIR-1 competes with stimulatory CD8 for binding with MHC class I les. Willcox et al. (2003) Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor, Nature Immunology 4(9):913-919; also, Shirioshi et al. (2003) Human inhibitory ors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G, Proc. Natl.

Acad. Sci. USA ):8856-8861. LIR-1 transduces inhibitory signals h its (intracellular) immunoreceptor tyrosine-based inhibitory motif (ITIM). In NK cells, studies have shown that KIRs itory killer cell e receptors) lacking ITIMs (normally incapable of inhibition) can inhibit in the presence of LIR-1 (presumably h the LIR-1 ITIM) bound to the 3 domain of an MHC class I molecule (see, Kirwin et al. (2005) Killer Cell Ig-Like or-Dependent Signaling by Ig-Like Transcript 2 (ILT2/CD85j/LILRB1/LIR- 1) J. Immunol. 06-5015), suggesting cooperation between LIR-1 bound to MHC class I and KIRs and thus a role for HLA 3 domain binding in modulating NK cell inhibition.

As described above, MHC molecules interact with cells that do not express a TCR. Among these cell s are NK cells. NK cells are cytotoxic lymphocytes (distinguished from CTLs, or cytotoxic T lymphocytes) that play a central role in the cellular immune response, and in particular innate immunity. NK cells are the first line of defense against ng microorganisms, viruses, and other non-self (e.g., tumor) entities. NK cells are ted or inhibited through surface receptors, and they s CD8 but do not express TCRs. NK cells can interact with cells that express MHC class I, but interaction is through the CD8-binding 3 domain rather than the TCR-binding, peptide-bearing 1 and 2 domains. A primary function of NK cells is to destroy cells that lack sufficient MHC class I surface protein.

Cross-linking MHC class I molecules on the surface of human natural killer (NK) cells results in ellular tyrosine phosphorylation, migration of the MHC class I molecule from the immunosynapse, and down-regulation of tumor cell killing. Rubio et al. (2004) Cross-linking of MHC class I molecules on human NK cells inhibits NK cell function, segregates MHC I from the NK cell synapse, and s intracellular phosphotyrosines, J. yte Biol. 76:116-124.

Another function of MHC class I in NK cells is apparently to prevent self-killing.

NK cells bear both activating receptor 2B4 and the 2B4 ligand CD48; MHC class I appears to bind 2B4 and prevent its activation by CD48. Betser-Cohen (2010) The Association of MHC Class I ns with the 2B4 Receptor Inhibits Self-Killing of Human NK Cells, J.

Immunol. 184:2761-2768.

Thus, the genetically engineered non-human animals described herein can be used to study these non-TCR or non-CTL ed processes and to design approaches for their modulation.

EXAMPLES The invention will be further illustrated by the following nonlimiting examples.

These Examples are set forth to aid in the tanding 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 tional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless indicated ise, parts are parts by weight, molecular weight is e molecular weight, temperature is ted in Celsius, and pressure is at or near atmospheric.

Example 1. Construction and Characterization of Genetically Modified HLA-A2 Mice Example 1.1: Expression of HLA-A2/H-2K in MG87 Cells.

] A viral construct containing a ic HLA-A2/H-2K gene sequence () was made using standard molecular cloning techniques known to a skilled artisan in order to analyze chimeric human/mouse MHC I expression in transfected cells.

Briefly, a chimeric human HLA-A/mouse H-2K viral construct was made using the exon sequences encoding the 1, 2 and 3 domains of the  chain and cloning them in frame with the mouse coding sequences for the transmembrane and cytoplasmic domains from the H-2K gene (, pMIG-HLA-A2/H2K). As illustrated in the construct contained an IRES-GFP reporter sequence, which allowed for determining if the construct was able to express in cells upon transfection.

Viruses containing the chimeric construct described above were made and propagated in human embryonic kidney 293 (293T) cells. 293T cells were plated on 10 cm dishes and allowed to grow to 95% confluency. A DNA transfection mixture was prepared with 25 µg of pMIG-HLA-A2/H2K, pMIG-human HLA-A2, or pMIG-humanized 2 microglobulin, and 5 µg of pMDG ope d), 15 µg of pCL-Eco (packaging construct without packaging signal ), 1 mL of Opti-MEM (Invitrogen). Added to this 1 mL DNA mixture was 80 µL of Lipofectamine-2000 (Invitrogen) in 1 mL of Opti-MEM, which was previously mixed together and allowed to incubate at room temperature for 5 minutes. The Lipofectamine/DNA e was allowed to incubate for an additional 20 minutes at room temperature, and then was added to 10 cm dishes, and the plates were incubated at 37C.

Media from the cells was collected after 24 hours and a fresh 10 mL of R10 (RPMI 1640 + % FBS) media was added to the cells. This media exchange was repeated twice. After a total of four days, the collected media was pooled, centrifuged and passed through a sterile filter to remove cellular debris.

The propagated viruses made above were used to transduce MG87 (mouse fibroblast) cells. MG87 cells from a single T-75 flask were washed once with PBS. 3 mL of 0.25% Trypsin + EDTA was added to the cells and allowed to incubate at room temperature for three minutes. 7 mL of D10 (high glucose DMEM; 10% Fetal Bovine Serum) was added to the cells/trypsin mixture and transferred to a 15 mL tube to centrifuge at 1300 rpm for five minutes. After centrifuging the cells, the media was aspirated and the cells resuspended in mL D10. Cells were counted and 05 cells were placed per well in a 6-well plate. pMIG-human HLA-A2 or pMIG-HLA-A2/H-2K either alone or with pMIG-humanized 2 microglobulin virus were added to the wells, with non-transduced cells as a control. Cells were incubated at 37C with 5% CO2 for 2 days. Cells were ed for FACS is (using anti-HLA-A2 antibody, clone BB7.2) for HLA-A2 expression with or without 2 microglobulin.

] The graphs (), as well as the table summarizing the data obtained from the graphs () demonstrate that nsduction with humanized 2 lobulin ses the expression of human HLA-A2 or chimeric human/non-human HLA-A2/H-2K, as demonstrated by the shift of curves to the right. e 1.2. Engineering a Chimeric HLA-A2/H-2K Locus.

The mouse H-2K gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., US Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution sion analysis. Nat. Biotech. 21(6): 652-659). DNA from mouse BAC clone RP23- 173k21 (Invitrogen) was modified by homologous recombination to e the genomic DNA encoding the 1, 2 and 3 domains of the mouse H-2K gene with human genomic DNA encoding the 1, 2 and 3 subunits of the human HLA-A gene (.

Briefly, the genomic sequence ng the mouse the 1, 2 and 3 subunits of the H-2K gene is replaced with the human genomic DNA encoding the 1, 2 and 3 domains of the human HLA-A*0201 gene in a single targeting event using a targeting vector comprising a hygromycin cassette flanked by loxP sites with a 5’ mouse homology arm containing sequence 5’ of the mouse H-2K locus including the 5’ untranslated region (UTR; ’ homology arm is set forth in SEQ ID NO: 1) and a 3’ mouse homology arm containing genomic sequence 3’ of the mouse H-2K 3 coding sequence (3’ homology arm is set forth in SEQ ID NO: 2).

The final construct for targeting the endogenous H-2K gene locus from 5’ to 3’ included (1) a 5’ homology arm ning ~200 bp of mouse genomic sequence 5’ of the endogenous H-2K gene including the 5’UTR, (2) ~1339 bp of human genomic sequence including the HLA-A*0201 leader sequence, the HLA-A*0201 leader/1 intron, the HLAA *0201 1 exon, the HLA-A*0201 1-2 intron, the 0201 2 exon, ~316 bp of the 5’ end of the 2-3 intron, (3) a 5’ loxP site, (4) a hygromycin cassette, (5) a 3’ loxP site, (6) ~580 bp of human genomic sequence including ~304 bp of the 3’ end of the 2-3 intron, the HLA-A*0201 3 exon, and (7) a 3’ gy arm containing ~200 bp of mouse genomic sequence ing the intron between the mouse H-2K 3 and transmembrane coding sequences (see for schematic representation of the H-2K targeting vector). The sequence of 149 nucleotides at the junction of the human sequences at the 5’ of the targeting vector is set forth in SEQ ID NO: 3, and the sequence of 159 nucleotides at the junction of the human/mouse sequences at the 3’ of the targeting vector is set forth in SEQ ID NO:4. Homologous recombination with this targeting vector created a modified mouse H- 2K locus containing human genomic DNA ng the 1, 2 and 3 domains of the HLAA *0201 gene operably linked to the endogenous mouse H-2K transmembrane and cytoplasmic domain coding sequences which, upon translation, leads to the formation of a ic human/mouse MHC class I protein.

The targeted BAC DNA was used to electroporate mouse F1H4 ES cells to create modified ES cells for generating mice that express a chimeric MHC class I protein on the surface of nucleated cells (e.g., T and B lymphocytes, macrophages, phils). ES cells ning an insertion of human HLA ces were identified by a quantitative TAQMAN™ assay. Specific primer sets and probes were designed for detecting insertion of human HLA sequences and associated selection tes (gain of allele, GOA) and loss of endogenous mouse sequences (loss of allele, LOA). Table 1 identifies the names and locations detected for each of the probes used in the quantitative PCR .

Table 1: Probes Used For Genotyping Region Detected by Probe Assay Sequence ID Probe ACGAGCGGGT TCGGCCCATT HYG GOA Hygromycin cassette 5 Human HLA-A2 2- AGTCCTTCAG CCTCCACTCA 1665H1 GOA 6 3 intron GGTCAGG Human HLA-A2 2 TACCACCAGT ACGCCTACGA 1665H2 GOA 7 exon CGGCA Human HLA-A22- 5112H2 GOA ATCCTGTACC TG 8 3 intron The selection cassette may be removed by methods known by the skilled artisan.

For example, ES cells bearing the chimeric human/mouse MHC class I locus may be transfected with a construct that expresses Cre in order to remove the “loxed” hygromycin cassette uced by the insertion of the targeting construct containing human HLAA *0201 gene sequences (See . The hygromycin cassette may optionally be removed by breeding to mice that express Cre recombinase. Optionally, the ycin cassette is retained in the mice.

] Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., US Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully d from the donor gene-targeted ES cells allowing immediate phenotypic es Nature Biotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human 0201 gene ces.

Example 1.3. In V ivo Expression of Chimeric HLA-A/H-2K in Genetically Modified Mice.

A heterozygous mouse carrying a genetically modified H-2K locus as described in Example 1.2 was analyzed for sion of the chimeric HLA-A/H-2K protein in the cells of the animal.

Blood was obtained tely from a wild-type and a HLA-A/H-2K chimeric heterozygote (A2/H2K) mouse. Cells were stained for human HLA-A2 with a phycoerythrinconjugated (PE) anti-HLA-A antibody, and exposed to an allophycocyanin-conjugated anti- H-2Kb antibody for one hour at 4oC. Cells were analyzed for expression by flow cytometry using antibodies specific for HLA-A and H-2Kb. shows the expression of H-2Kb and HLA-A2 in the wild-type and chimeric heterozygote, with chimeric heterozygote expressing both proteins. F IG. 6B shows expression of both the H-2Kb and the chimeric HLA-A2/H2K in the heterozygous mouse.

Example 2: Construction and Characterization of Genetically Modified 2 Microglobulin Mice Example 2.1: ering a Humanized 2 Microglobulin Locus The mouse 2 microglobulin (2m) gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial some (BAC) DNA using VELOCIGENE® technology (see, e.g., US Pat. No. 6,586,251 and Valenzuela et al., supra).

] Briefly, a targeting vector was generated by bacterial homologous ination ning mouse 2m upstream and downstream homology arms from BAC clone 89C24 from the RPCI-23 library (Invitrogen). The mouse homology arms were engineered to flank a 2.8 kb human 2m DNA fragment extending from exon 2 to about 267 nucleotides downstream of non-coding exon 4 (. A drug selection cassette (neomycin) flanked by recombinase recognition sites (e.g., loxP sites) was engineered into the targeting vector to allow for subsequent selection. The final targeting vector was linearized and oporated into a F1H4 mouse ES cell line zuela et al., supra).

Targeted ES cell clones with drug cassette removed (by uction of Cre recombinase) were introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., US Pat. No. 7,294,754 and Poueymirou et al., supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing the humanized 2m gene were fied by screening for loss of mouse allele and gain of human allele using a modification of allele assay zuela et al., supra).

Example 2.2: Characterization of Humanized 2 Microglobulin Mice Mice heterozygous for a humanized 2 microglobulin (2m) gene were evaluated for expression using flow cytometry (FIGs 8. and 9).

] Briefly, blood was isolated from groups (n=4 per group) of wild type, humanized 2m, humanized MHC (i.e., human HLA) class I, and double humanized 2m and MHC class I mice using ques known in art. The blood from each of the mice in each group was treated with ACK lysis buffer (Lonza sville) to ate red blood cells.

Remaining cells were stained using fluorochrome conjugated D3 (17A2), anti-CD19 (1D3), anti-CD11b (M1/70), anti-human HLA class I, and anti-human 2 microglobulin (2M2) antibodies. Flow cytometry was performed using BD-FACSCANTO (BD Biosciences).

Expression of human HLA class I was detected on cells from single zed and double humanized s, while expression of 2 microglobulin was only detected on cells from double humanized mice (. Co -expression of human 2m and human HLA class I resulted in an increase of detectable amount of human HLA class I on the cell surface compared to human HLA class I expression in the absence of human 2m ( mean fluorescent intensity of 2370 versus 1387).

Example 3. Immune Response to Flu an Epstein-Barr Virus (EBV) es Presented by APCs from Genetically Modified Mice Expressing HLA-A2/H-2K and Humanized 2 Microglobulin.

] PBMCs from several human donors were screened for both HLA-A2 expression and their ability to mount a response to flu and EBV es. A single donor was selected for subsequent experiments.

Human T cells are isolated from PBMCs of the selected donor using negative selection. c non-T cells were isolated from a mouse heterozygous for a chimeric HLAA2 /H-2K and heterozygous for a humanized 2-microglobulin gene, and a wild-type mouse.

About 50,000 splenic non-T cells from the mice were added to an Elispot plate coated with anti-human IFN antibody. Flu peptide (10 micromolar) or a pool of EBV peptides (5 micromolar each) was added. Poly IC was added at 25 micrograms/well, and the wells were incubated for three hours at 37ºC at 5% CO2. Human T cells (50,000) and anti -human CD28 were added to the splenic non T cells and the peptides, and the wells were incubated for 40 hours at 37ºC at 5% CO2, after which an IFN Elispot assay was performed.

As shown in , human T cells were able to mount a response to flu and EBV peptides when presented by mouse APCs that expressed the chimeric HLA-A2/H-2K and humanized 2 microglobulin on their surface.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention bed herein. 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 orated by reference herein in their entirety.

Throughout the description and claims of the specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission or a suggestion that that document was, known or that the ation it ns was part of the common general knowledge as at the priority date of any of the claims.

Claims (12)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A nucleic acid comprising a tide sequence encoding a chimeric human/nonhuman MHC I polypeptide, wherein the nucleotide sequence comprises a first nucleic acid sequence that encodes α1, α2 and α3 domains of a human HLA class I polypeptide operably linked to a second nucleic acid sequence that encodes transmembrane and cytoplasmic domains of the man MHC I polypeptide.
2. 2. The nucleic acid of claim 1, wherein the non-human MHC I polypeptide is a rodent MHC I polypeptide, and wherein the second nucleic acid sequence encodes transmembrane and cytoplasmic s of the rodent MHC I polypeptide.
3. 3. The nucleic acid of claim 1 or claim 2, wherein the second nucleic acid ce encodes transmembrane and cytoplasmic domains of the mouse MHC I polypeptide.
4. 4. The nucleic acid of claim 3, n the mouse MHC I polypeptide is selected from the group consisting of H-2K, H-2D and H-2L.
5. 5. The nucleic acid of any of the preceding claims, wherein the human HLA class I polypeptide is selected from the group ting of HLA-A, HLA-B, and HLA-C.
6. 6. The nucleic acid of any of the preceding claims, n the first nucleic acid ce encodes α1, α2 and α3 domains of a human HLA-A polypeptide and the second nucleic acid sequence encodes transmembrane and cytoplasmic domains of mouse H-2K polypeptide.
7. 7. The nucleic acid of claim 6, n the HLA-A polypeptide is a HLA-A2 polypeptide.
8. 8. The nucleic acid of claim 7, wherein the HLA-A2 polypeptide is a HLA-A2.1 polypeptide.
9. 9. The nucleic acid of any one of claims 6 to 8, wherein the H-2K polypeptide is a H-2Kb polypeptide.
10. 10. The nucleic acid of any one of the preceding claims, further comprising a regulatory element and a leader sequence operably linked to the nucleotide sequence.
11. 11. The nucleic acid of claim 10, wherein the regulatory element is a non-human regulatory element and the leader sequence is ed from a human HLA class I polypeptide leader ce, a non-human MHC I polypeptide leader sequence, and a combination thereof.
12. 12. The nucleic acid of claim 11, wherein the regulatory element is a rodent regulatory 13. The nucleic acid of claim 12, wherein the regulatory element is a mouse regulatory element. 14. An isolated cell comprising the nucleotide sequence of any of the preceding claims. 15. The isolated cell of claim 14, n the cell expresses the ic human/nonhuman MHC I polypeptide on its surface. 16. The ed cell of claim 14 or claim 15, further comprising a nucleotide sequence encoding a human or humanized β2 lobulin polypeptide operably linked to endogenous non-human β2 microglobulin regulatory elements. 17. The isolated cell of any one of claims 14 to 16, wherein the cell is an embryonic stem cell. 20. A ic human/non-human animal MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises α1, α2, and α3 domains of a human HLA class I polypeptide and a non-human portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a non-human animal MHC I polypeptide. 21. The chimeric human/mouse MHC I polypeptide of claim 20, wherein the MHC I polypeptide is non-covalently bound to a human or humanized β2 microglobulin. 22. The chimeric human/mouse MHC I polypeptide of claim 20 or 21, wherein the nonhuman portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a rodent MHC I polypeptide. 23. The chimeric human/mouse MHC I polypeptide of any of claims 20-22, wherein the non-human portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a mouse MHC I polypeptide. 24. The ic human/mouse MHC I polypeptide of claim 23, wherein the mouse MHC I polypeptide is selected from the group consisting of H-2K, H-2D and H-2L. 25. The chimeric human/mouse MHC I ptide of any one of claims 20 to 24, n the human HLA class I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C. 26. The chimeric human/mouse MHC I ptide of any one of claims 20 to 25, wherein the human HLA class I polypeptide is a human HLA-A polypeptide and the non-human animal MHC I polypeptide is a mouse H-2K polypeptide. 27. The chimeric mouse MHC I polypeptide of claim 26, wherein the HLA-A polypeptide is a HLA-A2 polypeptide. 28. The chimeric mouse MHC I polypeptide of claim 27, wherein the HLA-A2 polypeptide is a HLA-A2.1 ptide. 29. The chimeric human/mouse MHC I polypeptide of any one of claims 26-28, wherein the H-2K polypeptide is a H-2Kb polypeptide. 30. A nucleic acid of claim 1, substantially as hereinbefore described with reference to the