NZ623146B2 - Transgenic mice expressing chimeric major histocompatibility complex (mhc) class ii molecules - Google Patents

Abstract

Disclosed is a non-human animal comprising at an endogenous Major Histocompatibility Complex II (MHC II) ? gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II ? polypeptide and/or comprising at an endogenous MHC II ? gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II ? polypeptide, wherein a human portion of the chimeric human/non-human MHC II ? polypeptide comprises a human MHC II ?2 extracellular domain, and/or a human portion of the chimeric human/non-human MHC II ? polypeptide comprises human MHC II ?2 domains, and wherein the animal expresses a functional MHC II complex on a surface of a cell of the animal. non-human MHC II ? polypeptide, wherein a human portion of the chimeric human/non-human MHC II ? polypeptide comprises a human MHC II ?2 extracellular domain, and/or a human portion of the chimeric human/non-human MHC II ? polypeptide comprises human MHC II ?2 domains, and wherein the animal expresses a functional MHC II complex on a surface of a cell of the animal.

Description

TRANSGENIC MICE SING CHIMERIC MAJOR HISTOCOMPATIBILITY X (MHC) CLASS II MOLECULES CROSS-REFERENCE TO RELATED APPLICATION This application claims benefit of priority to US. Provisional Application No. 61/552,584, filed October 28, 2011, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION Present invention relates to a non-human animal, e.g., a rodent (e.g., a mouse or a rat) that is genetically engineered to s a zed Major Histocompatibiiity Complex (MHC) class II protein, as well as embryos, tissues, and cells expressing the same.

The invention r relates to methods for making a genetically modified non—human animal that ses a humanized MHC II protein. Also provided are methods for using non-human s, cells, and tissues that express a humanized MHC class II protein for identifying peptides that activate lymphocytes and engage T cells, and for developing human es and other therapeutics.

BACKGROUND OF THE INVENTION In the adaptive immune response, foreign antigens are recognized by receptor molecules on B cytes (e.g., immunoglobulins) and T cytes (e.g., T cell receptor or TCR). These foreign antigens are presented on the surface of cells as peptide fragments by specialized proteins, generically referred to as major histocompatibility complex (MHC) molecules. MHC molecules are encoded by multiple loci that are found as a linked cluster of genes that spans about 4 Mb. In 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 n (HLA) genes. The loci in mice and humans are nic; they include three highly polymorphic classes of MHC genes (class l, 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-DRB and human HLA—B). All class I and ll MHC genes can present peptide fragments, but each gene ses a protein with different g characteristics, reflecting polymorphisms and allelic variants. Any given individual has a unique range of peptide fragments that can be presented on the cell e to B and T cells in the course of an immune response.

Both humans and mice have class ll MHC genes (see Fle. 2 and 3). In humans, the classical MHC ll genes are termed HLA-DP, , and HLA-DR, whereas in mice they are H-2A and H-2E (often iated as l-A and l—E, respectively). Additional proteins encoded by genes in the MHC ll locus, HLA—DM and HLA-DO in humans, and H-2M and H-20 in mice, are not found on the cell surface, but reside in the endocytic compartment and ensure proper loading of MHC ll molecules with peptides. Class ll molecules consist of two polypeptide chains: on chain and [3 chain. The extracellular portion of the on chain contains two extracellular domains, at and (x2; and the extracellular portion of the [3 chain also contains two extracellular domains, [31 and [32 (see . The on and the [3 chains are non-covalently associated with each other.

MHC class ll molecules are expressed on antigen—presenting cells (APCs), e.g., B cells, macrophages, dendritic cells, endothelial cells during a course of inflammation, etc.

MHC ll molecules sed on the surface of APCs typically present antigens ted in intracellular vesicles to CD4+ T cells. In order to participate in CD4+ T cell engagement, the MHC class ll complex with the antigen of interest must be sufficiently stable to survive long enough to engage a CD4+ T cell. When a CD4+ T helper cell is engaged by a foreign e/MHC |l complex on the surface of APC, the T cell is activated to release cytokines that assist in immune response to the r.

Not all antigens will provoke T cell activation due to tolerance mechanisms. r, in some diseases (e.g., cancer, autoimmune diseases) peptides derived from self- proteins become the target of the cellular component of the immune system, which s in ction of cells presenting such peptides. There has been significant advancement in recognizing antigens that are clinically significant (e.g., antigens associated with various types of cancer). However, in order to improve identification and selection of peptides that will provoke a suitable response in a human T cell, in particular for peptides of clinically significant antigens, there remains a need for in vivo and in vitro systems that mimic aspects of human immune system. Thus, there is a need for biological systems (e.g., cally modified non-human animals and cells) that can display components of a human immune system.

SUMMARY OF THE INVENTION A biological system for generating or identifying peptides that associate with human MHC class II ns and chimeras thereof, and bind to CD4+ T cells, is provided.

Non-human animals comprising non-human cells that s humanized les that function in the cellular immune response are provided. zed rodent loci that encode humanized MHC II proteins are also provided. Humanized rodent cells that express humanized MHC 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 humanized immune system molecules.

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 ng a zed MHC II x, wherein a human portion of the zed MHC II complex comprises an extracellular domain of a human MHC II complex, e.g., a zed MHC II  extracellular domain and a humanized MHC II  extracellular domain.

In one aspect, provided herein is a non-human animal comprising at an endogenous Major Histocompatibility Complex II (MHC II)  gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II  polypeptide and/or comprising at an endogenous MHC II β gene locus a nucleotide sequence encoding a ic human/nonhuman MHC II β ptide, wherein a human portion of the chimeric human/non-human MHC II  polypeptide comprises a human MHC II 2 extracellular domain, and/or a human portion of the chimeric human/non-human MHC II β polypeptide comprises human MHC II β2 domains, and wherein the animal expresses a functional MHC II complex on a surface of a cell of the animal.

] In a further aspect, provided herein is a method of modifying an MHC II locus of a mouse to express a ic human/mouse MHC II complex comprising replacing at the endogenous mouse MHC II α locus a nucleotide sequence encoding a mouse MHC II α protein with a first nucleotide sequence encoding a chimeric human/mouse MHC II α protein and/or at the endogenous mouse MHC II β locus a nucleotide sequence encoding a mouse MHC II β protein with a second nucleotide sequence encoding a chimeric human/mouse MHC II β protein, wherein the chimeric human/mouse MHC II α protein comprises an α2 domain of a human MHC II  polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II  polypeptide and the chimeric human/mouse MHC II β protein ses a β2 domain of a human MHC II β polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide. [0010b] In a further aspect, ed herein is a non-human animal comprising at an endogenous MHC II  gene locus a nucleotide sequence encoding a chimeric human/nonhuman MHC II  polypeptide. In one embodiment, a human portion of such chimeric human/non-human MHC II  polypeptide comprises a human MHC II  extracellular domain.

In one embodiment, the non-human animal expresses a onal MHC II complex on a surface of a cell of the animal. In one embodiment, the human MHC II  extracellular domain in the animal comprises human MHC II 1 and 2 domains; in one embodiment, a nonhuman portion of the ic human/non-human MHC II  polypeptide comprises transmembrane and cytoplasmic domains of an endogenous man MHC II  polypeptide. In one embodiment, the nucleotide ce encoding a chimeric nonhuman MHC II  polypeptide is expressed under regulatory control of endogenous nonhuman MHC II  promoter and regulatory elements. In one embodiment, the human portion of the chimeric polypeptide is derived from a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g., the human portion is derived from HLA-DR4 protein. The non-human animal may be a rodent, e.g., a mouse. In one , the non-human animal comprising at an nous MHC II  gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II  polypeptide further comprises at an endogenous MHC II [5 gene locus a tide sequence encoding a chimeric human/non-human MHC II [3 polypeptide. Also provided herein is a method of making a genetically modified non—human animal comprising at an endogenous MHC II a gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II on polypeptide. Such method may comprise replacing at an endogenous MHC II on gene locus a nucleotide sequence encoding an endogenous non-human MHC II a ptide with a nucleotide sequence encoding a chimeric human/non-human MHC II on polypeptide.

Also provided herein is a non—human animal comprising at an endogenous MHC II (3 gene locus a nucleotide ce encoding a chimeric human/non-human MHC II [3 ptide. In one embodiment, a human portion of such chimeric human/non-human MHC II [3 polypeptide comprises a human MHC II p extracellular domain. In one embodiment, the non—human animal ses a functional MHC II complex on a surface of a cell of the animal. In one embodiment, the human MHC ll B extracellular domain in the animal comprises human MHC || [31 and [52 domains; in one embodiment, a non-human portion of the chimeric human/non-human MHC II [3 polypeptide ses transmembrane and cytoplasmic s of an endogenous non-human MHC II p polypeptide. In one embodiment, the nucleotide sequence encoding a chimeric human/non-human MHC II [3 polypeptide is expressed under regulatory control of endogenous man MHC II [3 promoter and regulatory elements. In one embodiment, the human portion of the ic polypeptide is derived from a human HLA class II n selected from the group consisting of HLA-DR, , and HLA-DP, e.g., the human n is derived from HLA-DR4 protein. The non-human animal may be a rodent, e.g., a mouse. In one aspect, the non— human animal comprising at an endogenous MHC II [3 gene locus a tide sequence encoding a chimeric human/non-human MHC II [3 polypeptide further comprises at an endogenous MHC II on gene locus a nucleotide sequence encoding a chimeric human/non- human MHC II a ptide. Also ed herein is a method of making a genetically modified non-human animal comprising at an endogenous MHC II [3 gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II [3 polypeptide. Such method may comprise replacing at an endogenous MHC II B gene locus a tide sequence encoding an endogenous non—human MHC ll {3 polypeptide with a nucleotide sequence encoding a chimeric human/non-human MHC II [5 polypeptide.

In one aspect, a non-human animal is provided sing at an endogenous MHC II gene locus a first nucleotide sequence encoding a chimeric human/non-human MHC II 0t polypeptide and a second nucleotide sequence encoding a chimeric human/non—human MHC II [3 polypeptide, wherein a human portion of the ic human/non-human MHC II or polypeptide comprises a human MHC II or extracellular domain and a human portion of the chimeric human/non-human MHC II [3 polypeptide comprises a human MHC II B extracellular domain. In one embodiment, the chimeric human/non-human MHC II or and B ptides form a functional chimeric MHC II complex (e.g., non-human MHC II complex) on a surface of a cell. In one embodiment, the human MHC II or extracellular domain comprises human a1 and a2 domains of human MHC II. In one embodiment, the human MHC II B extracellular domain comprises human [31 and [32 domains of human MHC II. In various aspects, the first nucleotide sequence is sed under regulatory control of endogenous non-human MHC II or promoter and regulatory elements. In s aspects, the second nucleotide sequence is expressed under regulatory control of endogenous non-human MHC II [5 promoter and regulatory elements. In some embodiments, a non—human portion of the chimeric human/non-human MHC II or polypeptide ses transmembrane and cytoplasmic domains of an endogenous non-human MHC II or ptide. In some embodiments, a non-human portion of the chimeric human/non-human MHC II [5 polypeptide comprises embrane and asmic domains of an endogenous non-human MHC II [5 polypeptide.

In various embodiments, the non-human animal is a , and the human portions of the chimeric human/rodent MHC II or and fi polypeptides comprise human sequences derived from HLA class M protein ed from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments of the invention, the human portions of the chimeric human/rodent MHC II or and [3 sequences are derived from a human 4 sequence; thus, the nucleotide sequence encoding the MHC II or extracellular domain is derived from a sequence of an HLA-DRa*01 gene, and the nucleotide ce encoding the MHC II [3 extracellular domain is derived from a sequence encoding an HLA-DR[31*O4 gene.

In various embodiments of the invention, the first and the second nucleotide sequences are located on the same chromosome. In some aspects, the animal comprises two copies of the MHC II locus containing the first and the second nucleotide sequences, while in other aspects, the animal comprises one copy of the MHC II locus containing the first and the second nucleotide sequences. Thus, the animal may be homozygous or zygous for the MHC II locus containing the first and the second tide sequences.

In some aspects, the chimeric MHC II or polypeptide and/or the chimeric MHC II {5 polypeptide is operably linked to a non-human leader sequence.

WO 63340 In one aspect, the cally engineered non-human animal is a rodent. In one embodiment, the rodent is selected from the group consisting of a mouse and a rat. Thus, in some embodiments, non-human sequences of the chimeric MHC ll or and [3 genes are derived from nucleotide sequences encoding mouse MHC ll protein, e.g., a mouse H-2E n. in one embodiment, the rodent (e.g., the mouse or the rat) of the invention does not express functional endogenous MHC II polypeptides from their endogenous loci. In one embodiment, wherein the rodent is a mouse, the mouse does not express functional endogenous H-2E and H-2A polypeptides from their endogenous loci.

Thus, in some embodiments, a mouse is provided comprising at an endogenous mouse MHC ll locus a first nucleotide sequence encoding a chimeric human/mouse MHC II 0L polypeptide and a second nucleotide ce encoding a chimeric human/mouse MHC ll [3 polypeptide, n a human portion of the chimeric MHC II or polypeptide comprises an extracellular domain derived from an on polypeptide of a human HLA-DR4 protein and a human portion of the ic human/mouse MHC ll 6 polypeptide comprises an extracellular domain derived from a B polypeptide of a human HLA-DR4 protein, wherein a mouse portion of the chimeric MHC ll or ptide comprises transmembrane and asmic domains of a mouse H-2E a chain and a mouse portion of the chimeric MHC ll [3 polypeptide comprises transmembrane and cytoplasmic domains of a mouse H-2E [3 chain, and wherein the mouse expresses a functional chimeric HLA-DR4/H-2E MHC ll complex. In some aspects, the extracellular domain of the ic MHC ll 0L polypeptide comprises human 0:1 and a2 domains; in some aspects, the extracellular domain of the ic MHC II [3 polypeptide ses human B1 and {52 domains. In some embodiments, the first nucleotide sequence is sed under regulatory l of endogenous mouse MHC ll 0t er and regulatory elements, and the second nucleotide sequence is expressed under regulatory control of endogenous mouse MHC II [3 promoter and regulatory elements. In various ments, the mouse does not express functional endogenous MHC ll polypeptides, e.g., H-2E and H-2A polypeptides, from their endogenous loci. In some aspects, the mouse comprises two copies of the MHC II locus containing the first and the second nucleotide sequences, while in other aspects, the mouse comprises one copy of the MHC II locus containing the first and the second nucleotide sequences.

Methods of making genetically engineered non-human animals (e.g., rodents, e.g., mice or rats) as described herein are also provided. In various embodiments, non- human animals (e.g., rodents, e.g., mice or rats) of the invention are made by replacing endogenous MHC II sequences with nucleotide sequences encoding chimeric human/non- human (e.g., human/mouse) MHC II or and [3 ptides. In one embodiment, the invention provides a method of ing an MHC II locus of a rodent (e.g., a mouse or a rat) to s a chimeric rodent MHC II complex comprising replacing at the endogenous mouse MHC II locus a nucleotide sequence ng a rodent MHC II complex with a nucleotide sequence encoding a chimeric human/rodent MHC II complex. In one aspect of the method, the nucleotide sequence encoding the chimeric human/rodent MHC II complex comprises a first nucleotide sequence encoding an extracellular domain of a human MHC II at chain and transmembrane and cytoplasmic domains of a rodent MHC II 0t chain and a second nucleotide sequence encoding an extracellular domain of a human MHC II [3 chain and transmembrane and cytoplasmic domains of a rodent MHC II B chain. In some aspects, a rodent portion of the chimeric MHC II complex is derived from a mouse H-2E n, and a human portion is derived from a human HLA—DR4 n. In some embodiments, the replacement of the endogenous MHC II loci described herein is made in a single ES cell, and the single ES cell is introduced into a rodent (e.g., mouse or rat) embryo to make a genetically modified rodent (e.g., mouse or rat).

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

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

Other embodiments will become nt to those skilled in the art from a review of the ensuing detailed description. The following detailed description 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 invenfion.

BRIEF DESCRIPTION OF THE DRAWINGS is a schematic drawing of the MHC II class molecule expressed on the surface of an antigen presenting cell (APC), containing four domains: (11, a2, [31, and $2.

The gray circle ents a peptide bound in the peptide-binding cleft. is a tic representation (not to scale) of the relative genomic structure of the human HLA, showing class I, II and Ill genes. is a schematic representation (not to scale) of the relative genomic ure of the mouse MHC, showing class I, II and Ill genes.

(A-D) is a schematic ration (not to scale) of the strategy for generating a targeting vector comprising humanized l-E [3 and l-E 0L (i.e., H-2EB/HLA—DRfi1*O4 and H- 2Ea/HLA—DRa*O1 chimera, respectively). In , the final zed MHC II sequence from is ligated between l and I-Ceul restriction sites of the final construct from , to generate a construct comprising humanized MHC II and exon 1 of I-Eoc from . Pg=pseudogene; BHR= bacterial homologous recombination; CM=chloramphenicol; spec=spectinomycin; hyg=hygromycin; neo=neomycin; EP=electroporation. Triangles represent exons, filled les represent mouse exons from 057BL/6 mouse (with the exception of hashed triangles, which represent exon 1 of l-Ea from BALB/c mouse) and open triangles represent human exons. shows a schematic illustration, not to scale, of MHC class II l—E and l-A genes, showing knockout of the mouse locus using a ycin cassette, ed by introduction of a vector comprising a humanized I-E [3 and l-E or (i.e., H-2EB/HLA—DR61*04 and /HLA-DRor*01 chimera, respectively). Open triangles represent human exons; filled triangles represent mouse exons. Probes used for genotyping are encircled. shows a schematic illustration, not to scale, of Ore—mediated removal of the neomycin cassette of Open triangles represent human exons; filled triangles represent mouse exons. Top two strands represent MHC II loci in humanized MHC II heterozygous mouse ing a neomycin selection cassette, and bottom two strands represent MHC II loci in humanized MHC II heterozygous mouse with neomycin cassette removed. ’ shows a schematic ative ration, not to scale, of mouse and human class II loci. Class II genes are represented by boxes, and empty boxes represent pseudogenes. Relative sizes (kb) of various nucleic acid fragments are included. at left panel, is a schematic illustration (not to scale) of humanization strategy for the MHC II or chain; in particular, the figure shows a replacement of a1 and 012 domains, encoded by exons 2 and 3 of MHC II or gene, while retaining mouse transmembrane and cytoplasmic tail sequences. In the humanized locus, the MHC II a leader sequence is derived from the mouse BALB/c strain. The right panel illustrates humanization of the MHC II [5 chain; in particular, the figure shows a replacement of {31 and [32 domains, encoded by exons 2 and 3 of MHC II [3 gene, while retaining the mouse leader and mouse transmembrane and asmic tail sequences. Top row are all human sequences; middle row are all mouse sequences; bottom row are all humanized sequences, with exons 2 and 3 derived from human HLA-DR genes. shows FACS analysis with anti-HLA-DR antibody of B cells from a mouse heterozygous for a chimeric HLA-DR4 (neo cassette d) in the presence (1681 HET + po|y(l:C) or absence (1681 HET) of poly(I:C), and a wild-type mouse (WT mouse).

DETAILED DESCRIPTION OF THE INVENTION Definitions The present invention provides genetically modified non-human animals (e.g., mice, rats, rabbits, etc.) that express human or humanized MHC ll polypeptide; embryos, cells, and tissues comprising the same; s of making the same; as well as methods of using the same. Unless defined ise, 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 vative amino acid substitution, includes substitution of an amino acid e by another amino acid e having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity).

Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of MHC II to t 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, alanine, , 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 alanine, ne, and tryptophan; basic side chains such as , ne, 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 asparagine/glutamine. In some embodiments, a conservative amino acid tution can be a substitution of any native residue in a n 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) tive ng of the Entire Protein Sequence Database, Science 256:1443—45), hereby incorporated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix. 2012/062029 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 II polypeptide, wherein the polypeptide comprises conservative amino acid substitutions in the amino acid sequence described herein.

One skilled in the art would understand that in addition to the nucleic acid residues encoding a human or humanized MHC ll ptide bed herein, due to the degeneracy of the c code, other c acids may encode the polypeptide of the invention. Therefore, in addition to a genetically modified non-human animal that ses in its genome a nucleotide sequence encoding MHC II polypeptide with conservative amino acid substitutions, a non-human animal whose genome comprises a nucleotide ce that s from that described herein due to the degeneracy of the genetic code is also The term “identity” when used in connection with ce includes identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are ined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MacVectorTM 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to identity of sequences will depend upon the particular sequences. In various embodiments, identity is determined by comparing the sequence of a mature protein from its inal to its C-terminal. In various embodiments when comparing a chimeric human/non-human sequence to a human sequence, the human portion of the chimeric human/non—human sequence (but not the man portion) is used in making a comparison for the purpose of ascertaining a level of ty between a human sequence and a human portion of a chimeric human/non-human ce (e.g., comparing a human ectodomain of a chimeric human/mouse protein to a human ectodomain of a human protein).

The terms ogy” or “homologous” 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 endogenous DNA sequences (i.e., “homology arms”); thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

The term bly 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 regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. In addition, various portions of the chimeric or humanized protein of the invention may be operably linked to retain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless stated otherwise, various domains of the chimeric or humanized protein of the invention are operably linked to each other.

The terms “MHC ll complex,” “MHC ll protein,” or the like, as used herein, e the complex between an MHC II a polypeptide and an MHC ll [3 polypeptide. The term “MHC II 0L ptide” or “MHC II [3 polypeptide” (or the like), as used herein, includes the MHC I OL polypeptide alone or MHC ll [3 polypeptide alone, respectively. rly, the terms “HLA—DR4 complex”, “HLA—DR4 n,” “H—2E x,” “H-2E” protein,” or the like, refer to complex between on and [3 polypeptides. Typically, the terms “human MHC” and ”HLA” are used interchangeably.

The term “replacement” in nce to gene replacement refers to g 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, nucleic acid sequence of endogenous MHC ll locus was replaced by a tide sequence comprising sequences encoding portions of human MHC ll 0L and [3 polypeptides; specifically, ng the extracellular portions of the MHC ll 0t and [3 polypeptides.

“Functional” as used herein, e.g., in nce to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a replacement at an endogenous locus (e.g., replacement at an endogenous non—human MHC II locus) results in a locus that fails to express a functional endogenous polypeptide.

Genetically ed MHC ll Animals In various s, the invention generally provides genetically modified non- human animals that comprise in their genome a nucleotide sequence encoding a human or humanized MHC ll x; thus, the animals express a human or humanized MHC II complex (e.g., MHC II 0t and [3 polypeptides).

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 presented in FIGs. 2 and 3, respectively. The majority of MHC genes are polymorphic, in fact they are the most polymorphic genes of the mouse and human genomes. MHC polymorphisms are ed to be important in providing evolutionary advantage; changes in sequence can result in differences in peptide binding that allow for better n presentation. One exception is the human a chain and its mouse homolog, Ea (i.e., H-2Ea), which are monomorphic.

MHC class II complex comprises two valently associated domains: an on chain and a 0 chain, also referred herein as an 0L ptide and a [3 polypeptide (.

The protein spans the plasma membrane; thus it contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular n of the a chain includes (11 and (12 domains, and the extracellular portion of the [3 chain includes [31 and [32 domains. The (11 and [31 domains form a peptide—binding cleft on the cell surface. Due to the three-dimensional confirmation of the peptide-binding cleft of the MHC II complex, there is theoretically no upper limit on the length of the bound antigen, but typically peptides presented by MHC II are between 13 and 17 amino acids in length.

In addition to its interaction with the antigenic peptides, the peptide-binding cleft of the MHC II molecule interacts with invariant chain (Ii) during the ses of MHC II complex formation and peptide acquisition. The (x/B MHC II dimers assemble in the asmic lum and associate with Ii chain, which is responsible for control of peptide binding and targeting of the MHC II into endocytic pathway. In the endosome, Ii undergoes lysis, and a small fragment of Ii, Class II-associated invariant chain peptide (CLIP), remains at the peptide-binding cleft. In the endosome, under control of HLA-DM (in humans), CLIP is exchanged for antigenic peptides.

MHC II interacts with T cell co—receptor CD4 at the hydrophobic crevice at the junction between a2 and [32 domains. Wang and Reinherz (2002) Structural Basis of T Cell Recognition of Peptides Bound to MHC Molecules, lar Immunology, 38:1039-49.

When CD4 and T cell receptor bind the same MHC II moIecule complexed with a peptide, the sensitivity of a T cell to antigen is increased, and it es 100-fold Iess antigen for activation. See, Janeway’s Immunobiology, 7th Ed., Murphy et al. eds., Garland Science, 2008, incorporated herein by reference.

Numerous functions have been proposed for transmembrane and asmic domains of MHC II. In the case of asmic domain, it has been shown to be important for intracellular signaling, cking to the plasma membrane, and tely, antigen presentation. For example, it was shown that T cell hybridomas respond poorly to antigen- presenting cells (APCs) transfected with MHC II [:3 chains truncated at the cytoplasmic domain, and induction of B cell differentiation is hampered. See, e.g., Smiley et al. (1996) Truncation of the class II B-chain cytoplasmic domain influences the level of class II/invariant chain-derived peptide complexes, Proc. Natl. Acad. Sci. USA, 93:241-44. Truncation of Class II molecules seems to impair CAMP production. It has been postulated that deletion of the cytoplasmic tail of MHC II affects intracellular trafficking, thus preventing the complex from coming across relevant antigens in the endocytic pathway. Smiley et al. (supra) demonstrated that truncation of class II molecules at the cytoplasmic domain reduces the number of CLIP/class II complexes, postulating that this affects the ability of CLIP to effectively regulate n presentation.

It has been hypothesized that, since MHC II clustering is important for T cell receptor (TCR) ring, if MHC II molecules truncated at the asmic domain were prevented from binding cytoskeleton and thus aggregating, antigen presentation to T cells would be affected. Ostrand—Rosenberg et al. (1991) Abrogation of Tumorigenicity by MHC Class II Antigen Expression Requires the Cytoplasmic Domain of the Class II Molecule, J.

Immunol. 147:2419-22. In fact, it was ly shown that HLA—DR truncated at the cytoplasmic domain failed to associate with the cytoskeleton following oligomerization. El Fakhy et al. (2004) Delineation of the HLA—DR Region and the Residues Involved in the ation with the Cytoskeleton, J. Biol. Chem. 279:18472-80. Importantly, actin eleton is a site of localized signal transduction activity, which can effect antigen presentation. In on to ation with cytoskeleton, recent studies have also shown that up to 20% of all HLA-DR molecules constitutively reside in the lipid rafts of APCs, which are microdomains rich in cholesterol and glycosphingolipids, and that such localization is important for antigen presentation, immune synapse formation, and MHC II-mediated signaling. See, e.g., Dolan et al. (2004) Invariant Chain and the MHC II Cytoplasmic Domains Regulate Localization of MHC Class II Molecules to Lipid Rafts in Tumor Cell- Based Vaccines, J. Immunol. 7~14. Dolan et al. ted that truncation of cytoplasmic domain of MHC II reduces constitutive localization of MHC II to lipid rafts.

In addition, the asmic domain of MHC II, in particular the [3 chain, ns a leucine residue that is subject to ubiquitination by ubiquitin ligase, membrane-associated RING-CH | (MARCH I), which controls endocytic trafficking, alization, and degradation of MHC II; and it has been shown that MARCH-mediated ubiquitination ceases upon dendritic cell maturation resulting in increased levels of MHC II at the plasma membrane.

Shin et al. (2006) Surface expression of MHC class II in tic cells is controlled by regulated ubiquitination, Nature 444:115-18; De Gassart et al. (2008) MHC class ll stabilization at the surface of human dendritic cells is the result of maturation-dependent MARCH l down-regulation, Proc. Natl. Acad. Sci. USA 105:3491—96.

Transmembrane domains of 0c and [5 chains of MHC ll ct with each other and this interaction is important for proper assembly of class ll MHC complex. Cosson and Bonifacino (1992) Role of Transmembrane Domain interactions in the Assembly of Class ll MHC Molecules, Nature 258:659-62. In fact, MHC ll molecules in which the transmembrane domains of the 0L and [3 chains were ed by the OL chain of lL-2 receptor were retained in the ER and were barely detectable at the cell surface. Id. Through mutagenesis studies, conserved Gly residues at the on and [5 transmembrane domains were found to be responsible for MHC ll assembly at the cell surface. Id. Thus, both transmembrane and cytoplasmic domains are crucial for the proper function of the MHC ll complex.

In various embodiments, the invention provides a cally modified non- human animal (e.g., mouse, rat, rabbit, etc.) that comprises in its genome a nucleotide sequence encoding a human or humanized MHC ll complex, e.g., a human or humanized MHC ll oz and/or B polypeptide(s). The non—human animal may comprise in its genome a nucleotide sequence that s an MHC II complex that is partially human and lly man, e.g., a non-human animal that expresses a chimeric human/non—human MHC ll complex (e.g., a non-human animal that ses chimeric human/non-human MHC ll cc and B ptides). in one aspect, the non-human animal only ses the human or humanized MHC II complex, e.g., a chimeric human/non-human MHC ll complex, and does not express an endogenous non-human MHC II complex from an endogenous MHC ll locus. in some embodiments, the animal is incapable of expressing any endogenous non—human MHC ll complex from an endogenous MHC ll locus, but only ses the human or humanized MHC ll complex. In various embodiments, the genetically modified non-human animal (e.g., mouse, rat, rabbit, etc.) ses in its germline a nucleotide sequence encoding a human or humanized MHC l| complex, e.g., a human or humanized MHC ll 0t and/or [3 polypeptide(s). in one aspect, a chimeric human/non-human MHC ll complex is provided. in one ment, the chimeric human/non-human MHC ll complex comprises a chimeric human/non—human MHC II a polypeptide and a ic human/non-human MHC II [3 polypeptide. in one aspect, a human portion of the chimeric MHC ll a polypeptide and/or a human portion of the ic MHC ll [3 polypeptide comprises a peptide-binding domain of a human MHC II 0t polypeptide and/or human MHC ll [3 polypeptide, respectively. In one aspect, a human portion of the chimeric MHC II (1 and/or [5 polypeptide comprises an extracellular domain of a human MHC II on and/or [3 polypeptide, tively. In one embodiment, a human portion of the chimeric MHC ll 0L polypeptide comprises a1 domain of a human MHC II on polypeptide; in another embodiment, a human portion of the chimeric MHC ll 0L polypeptide comprises a1 and a2 domains of a human MHC II 0t ptide. In an additional embodiment, a human portion of the chimeric MHC ll (5 polypeptide comprises [31 domain of a human MHC ll {3 polypeptide; in r embodiment, a human portion of the chimeric MHC ll [3 polypeptide comprises [31 and [32 domains of a human MHC ll [3 polypeptide.

The human portion of the MHC II on and [3 polypeptides described herein may be encoded by any of HLA-DP, -DQ, and —DR loci. A list of commonly used HLA antigens and alleles is described in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, lnt. J. Hum. Genet. 4(2):91-103), incorporated herein by nce. rkumar 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 —455, both incorporated herein by reference. Thus, the human or humanized MHC II polypeptide may be derived from any functional human HLA molecules described therein.

In one specific aspect, the human portions of the humanized MHC ll complex described herein are derived from human HLA-DR, e.g., HLA-DR4. Typically, HLA-DR 0L chains are monomorphic, e.g., the on chain of HLA-DR complex is encoded by A gene (e.g., HLA-DRa*01 gene). On the other hand, the HLA-DR B chain is polymorphic.

Thus, 4 comprises an 0: chain encoded by A gene and a [3 chain encoded by HLA—DRB1 gene (e.g., HLA-DRB1*04 gene). As described herein below, HLA-DR4 is known to be associated with nce of a number of autoimmune diseases, e.g., rheumatoid arthritis, type I diabetes, multiple sis, etc. ln one embodiment of the invention, the HLA-DRA allele is HLA-DRa*01 allele, e.g., HLA-DRa*01:01:01:01. In another embodiment, the HLA-DRB allele is HLA—DRB1*04, e.g., HLA-DR[51*04:01 :01.

Although the present Examples describe these particular HLA sequences; any le HLA— DR sequences are encompassed herein, e.g., rphic variants ted in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequences described herein due to the degeneracy of genetic code, etc.

The human portions of the humanized MHC II complex may be encoded by nucleotide sequences of HLA alleles known to be associated with common human diseases.

Such HLA alleles e, but are not limited to, HLA-DRB1*O401, O301, — 501, -DQB1*0201,—DRB1*1501,-DRBt*1502, -DQB1*0602, —DQA1*O102, - DQA1*0201, -DQB1*0202, -DQA1*0501, and combinations thereof. For a summary of HLA allele/disease associations, see Bakker et al. (2006) A high—resolution HLA and SNP ype map for disease association studies in the extended human MHC, Nature Genetics 38:1166-72 and Supplementary Information, incorporated herein by nce.

In one , a non-human portion of the chimeric human/non-human MHC II x comprises transmembrane and/or asmic domains of an endogenous non— human (e.g., rodent, e.g., mouse, rat, etc.) MHC II complex. Thus, a man portion of the chimeric human/non-human MHC II or polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non—human MHC II on polypeptide. A man portion of the ic human/non-human MHC II [3 polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC II B polypeptide. In one , the animal is a mouse, and non-human portions of the chimeric 0t and [3 polypeptides are derived from a mouse H-2E protein. Thus, non—human portions of the ic a and t3 ptides may comprise transmembrane and asmic domains derived from a mouse H-2E protein. Although specific H-2E sequences are contemplated in the Examples, any suitable sequences, e.g., polymorphic variants, conservative/non- conservative amino acid tutions, etc., are encompassed herein.

In various aspects of the invention, the sequence(s) encoding a chimeric human/non-human MHC II complex are located at an endogenous non—human MHC II locus (e.g., mouse H-2A and/or H—2E locus). In one embodiment, this results in a replacement of an endogenous MHC II gene(s) or a portion thereof with a nucleotide sequence(s) encoding a human or humanized MHC II protein, e.g., a chimeric gene encoding a chimeric human/non-human MHC II protein described herein. Since the nucleotide sequences encoding MHC II on and [3 polypeptides are located in proximity to one r on the chromosome, a replacement can be designed to target the two genes either independently or together; both of these possibilities are encompassed herein. In one embodiment, the replacement comprises a replacement of an endogenous nucleotide sequence encoding an MHC II or and [3 polypeptides with a nucleotide sequence encoding a chimeric human/non- human MHC 0L polypeptide and a chimeric human/non-human MHC [3 polypeptide. In one aspect, the replacement comprises ing nucleotide sequences representing one or more (e.g., two) endogenous MHC II genes. Thus, the non-human animal contains a chimeric human/non-human nucleotide sequence at an endogenous MHC II locus, and expresses a chimeric human/non—human MHC ll protein from the endogenous non—human locus.

Thus, provided herein is a non-human animal comprising at an endogenous MHC ll gene locus a first nucleotide sequence encoding a chimeric non-human MHC ll 0. polypeptide and a second nucleotide sequence encoding a chimeric human/non-human MHC ll {5 polypeptide, n a human n of the chimeric human/non-human MHC II or polypeptide comprises a human MHC ll or extracellular domain and a human portion of the chimeric human/non-human MHC II [3 polypeptide comprises a human MHC ll 0 extracellular domain, and n the chimeric human/non-human MHC II or and MHC ll [3 polypeptides form a functional MHC ll x on a surface of a cell.

A chimeric human/non-human polypeptide may be such that it comprises a human or a non-human leader l) ce. in one embodiment, the chimeric MHC ll 0t polypeptide comprises a non—human leader sequence of an endogenous MHC II or polypeptide. In one embodiment, the chimeric MHC ll [5 polypeptide comprises a non- human leader sequence of an endogenous MHC ll B polypeptide. In an alternative embodiment, the chimeric MHC II on and/or MHC ll {3 polypeptide comprises a non-human leader sequence of MHC ll or and/or MHC ll [3 polypeptide, respectively, from another non- human animal, e.g., another rodent or another mouse strain. Thus, the nucleotide sequence encoding the chimeric MHC II or and/or MHC ll (3 ptide may be operably linked to a tide ce encoding a non-human MHC II 0. and/or MHC ll [3 leader sequence, respectively. in yet another embodiment, the chimeric MHC II or and/or MHC II 13 polypeptide comprises a human leader ce of human MHC ll 0L and/or human MHC ll [3 polypeptide, respectively (e.g., a leader sequence of human HLA-DRA and/or human HLA- DRB1*O4, respectively).

A chimeric human/non-human MHC ll 0: and/or MHC ll [3 polypeptide may comprise in its human portion a complete or substantially complete ellular domain of a human MHC II or and/or human MHC II [3 polypeptide, respectively. Thus, a 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 encoding an extracellular domain of a human MHC ll or and/or human MHC ll [3 polypeptide (e.g., human HLA-DRA and/or human HLA-DR[31*04). In one example, substantially complete extracellular domain of the human MHC II on and/or human MHC II [3 polypeptide lacks a human leader sequence. In r example, the chimeric 2012/062029 human/non-human MHC II 0t and/or the chimeric human/non-human MHC II [3 polypeptide comprises a human leader sequence.

Moreover, the ic MHC II on and/or MHC Ii (3 polypeptide may be expressed under the control of endogenous non-human promoter and regulatory elements, e.g., mouse MHC II 0t and/or MHC II [3 regulatory elements, respectively. Such arrangement will facilitate proper expression of the chimeric MHC II polypeptides in the non-human animal, e.g., during immune response in the non—human animal.

The genetically ed non-human animal may be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, e (e.g., marmoset, rhesus ). 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 cation.

Such methods include, e.g., modifying a non—ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable ions 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 ed animal is a rodent. In one embodiment, 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., like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, s, spiny mice, d rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and SpaIacidae (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 cally 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 ment, the non-human animal is a rodent that is a mouse of a C57BL strain ed from C57'BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, 057BL/6, C57BL/SJ, C57BL/GByJ, C57BL/6NJ, C57BL/10, C57BL/1OScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting ofa strain that is 129P1, 129P2, 129P3, 129X1, 12981 (e.g., 12981/SV, 12981/Svlm), 12982, 12984, 12985, 12989/8vaH, 12986 (129/8vaTac), 12987, 12988, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome , see also, Auerbach et al (2000) Establishment and a Analysis of 129/8va- and 6-Derived Mouse Embryonic 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 12986 (129/8vaTac) strain. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment, the mouse is a mix of a BALB strain and another entioned strain.

In one embodiment, the man 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 , 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 chimeric mouse MHC II complex, e.g., chimeric human/mouse MHC II on and [3 polypeptides. In one embodiment, a human portion of the chimeric human/mouse MHC II a polypeptide comprises a human MHC II a peptide binding or extracellular domain and a human portion of the chimeric human/mouse MHC II {5 polypeptide comprises a human MHC II [5 peptide g or extracellular . In some embodiments, the mouse does not express a peptide binding or an extracellular domain of endogenous mouse oz and/or[3 polypeptide from an endogenous mouse locus (e.g., H-2A and/or H-2E locus). In some embodiments, the mouse comprises a genome that lacks a gene that encodes a functional MHC class II molecule comprising an H-2Ab1, H-2Aa, H—2Eb1, H—2Eb2, H-2Ea, and a combination thereof. The e-binding domain of the human MHC II 0L polypeptide may se (11 domain and the e-binding domain of the human MHC II B polypeptide may comprise a [51 domain; thus, the peptide-binding domain of the chimeric MHC II complex may comprise human a1 and [31 domains. The extracellular domain of the human MHC II 0t ptide may comprise (11 and a2 domains and the extracellular domain of the human MHC II [3 polypeptide may comprise [51 and {32 domains; thus, the extracellular domain of the chimeric MHC II complex may comprise human a1, a2, [31 and [32 domains. In one embodiment, the mouse n of the chimeric MHC II x comprises transmembrane and cytosolic domains of mouse MHC II, e.g. mouse H-2E (e.g., transmembrane and cytosolic domains of mouse H-2E or and [3 chains). ore, in one embodiment, a genetically modified mouse is provided, wherein the mouse comprises at an endogenous mouse MHC II locus a first nucleotide sequence encoding a chimeric human/mouse MHC II 0L ptide and a second nucleotide sequence encoding a chimeric human/mouse MHC II [3 polypeptide, wherein a human portion of the chimeric MHC II a ptide ses an extracellular domain derived from an or polypeptide of a human HLA-DR4 protein and the human portion of the chimeric MHC II B polypeptide comprises an ellular domain derived from a [3 polypeptide of a human HLA—DR4 protein, wherein a mouse portion of the chimeric MHC II or polypeptide comprises transmembrane and cytoplasmic domains of a mouse H—2E or chain and a mouse portion of the chimeric MHC II {3 polypeptide comprises transmembrane and asmic domains of a mouse H—2E [3 chain, and wherein the mouse expresses a functional ic HLA—DR4/H- 2E MHC II complex. In one embodiment the chimeric HLA—DR4/H-2E MHC II x comprises an MHC II a chain that includes extracellular domains (e.g., at, and (12 domains) derived from HLA—DR4 protein RA a1, and (x2 domains) and transmembrane and cytoplasmic domains from a mouse H-2E 0c chain, as well as an MHC II [3 chain that includes extracellular domains (e.g., [31 and [32 domains) derived from HLA—DR4 (HLA—DR[31*O4 [31 and [52 domains) and embrane and cytoplasmic domains from mouse H-2E (3 chain.

In one aspect, the mouse does not express functional nous H-2A and H-2E polypeptides from their endogenous mouse loci (e.g., the mouse does not express H-2Ab1, H-2Aa, H—2Eb1, H-2Eb2, and H-2Ea polypeptides). In various embodiments, expression of the first and second nucleotide sequences is under the control of respective endogenous mouse promoters and regulatory elements. In various embodiments of the invention, the first and the second nucleotide ces are located on the same chromosome. In some aspects, the mouse comprises two copies of the chimeric MHC II locus containing the first and the second nucleotide sequences, while in other aspects, the mouse ses one copy of the MHC II locus containing the first and the second nucleotide sequences. Thus, the mouse may be homozygous or heterozygous for the chimeric MHC II locus containing the first and the second nucleotide sequences. In various embodiments, the first and the second nucleotide sequences are comprises in the ne of the mouse.

In some embodiments described herein, a mouse is ed that comprises a ic MHC II locus at an endogenous mouse MHC II locus, e.g., via replacement of endogenous mouse H-2A and H—2E genes. In some aspects, the chimeric locus comprises a nucleotide sequence that encodes an extracellular domain of a human HLA—DRA and transmembrane and cytoplasmic domains of a mouse H-2E a chain, as well as an extracellular domain of a human HLA-DRB1*O4 and transmembrane and cytoplasmic domains of a mouse H—2E [3 chain. The s domains of the chimeric locus are linked in such a fashion that the locus expresses a functional chimeric human/mouse MHC II complex.

In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a onal chimeric MHC M protein from a chimeric MHC II locus as described herein displays the chimeric protein on a cell surface. In one embodiment, the non-human animal expresses the chimeric MHC M 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 nt (antigen fragment) bound to an extracellular portion (e.g., human HLA-DR4 extracellular portion) of the ic MHC II n.

In various embodiments, a cell displaying the chimeric MHC II protein, e.g., HLA— DR4/H-2E protein, is an antigen-presenting cell (APC) e.g., a macrophage, a tic cell, or a B cell. In some embodiments, the e nt presented by the chimeric protein is derived from a tumor. In other embodiments, the peptide fragment presented by the chimeric MHC II protein is derived from a pathogen, e.g., a bacterium, a virus, or a parasite.

The chimeric MHC II protein bed herein may interact with other proteins on the surface of the same cell or a second cell. In some embodiments, the chimeric MHC II protein interacts with endogenous non—human proteins on the surface of said cell. The chimeric MHC M protein may also interact with human or humanized proteins on the surface of the same cell or a second cell. In some embodiments, the second cell is a T cell, and the chimeric MHC II protein interacts with T cell receptor (TCR) and its co—receptor CD4. In some embodiments, the T cell is an nous mouse T cell. In other embodiments, the T cell is a human T cell. In some embodiments, the TCR is a human or humanized TCR. In additional ments, the CD4 is a human or humanized CD4. In other embodiment, either one or both of TCR and CD4 are non-human, e.g., mouse or rat.

In one embodiment, a genetically modified non—human animal as described herein is provided that does not develop tumors at a higher rate than a wild-type animal that lacks a chimeric MHC II gene. In some embodiments, the animal does not develop hematological malignancies, e.g., various T and B cell mas, Ieukemias, composite lymphomas (e.g., n’s lymphoma), at a higher rate than the wild-type animal.

In addition to a genetically engineered non-human animal, a non-human embryo (e.g., a , e.g., a mouse or a rat embryo) 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 , the embryo comprises an ES donor cell that comprises the chimeric MHC II gene, and host embryo cells.

Also provided is a , wherein the tissue is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein, and expresses the chimeric MHC II protein (e.g., HLA—DR4/H-2E n).

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

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

In one aspect, a non-human induced pluripotent cell comprising gene encoding a chimeric MHC II n (e.g., HLA—DR4/H-2E n) as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.

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

In one aspect, an in vitro preparation is provided that comprises a first cell that bears a chimeric human/rodent MHC II surface protein that comprises a bound peptide to form a chimeric human/rodent MHC ll/peptide complex, and a second cell that binds the ic human/rodent MHC ll/peptide complex. In one ment, the second cell comprises a human or humanized T-cell receptor, and in one embodiment r comprises a human or humanized CD4. In one embodiment, the second cell is a rodent (e.g., mouse or rat) cell comprising a human or humanized T—cell receptor and a human or humanized CD4 protein. In one embodiment, the second cell is a human cell.

Also provided is a method for making a genetically engineered man animal (e.g., a genetically engineered rodent, e.g., a mouse or rat) described herein. The method for making a genetically engineered non—human animal results in the animal whose genome comprises a nucleotide sequence encoding a chimeric MHC II protein (e.g., chimeric MHC II on and [3 polypeptides). In one embodiment, the method results in a genetically engineered mouse, whose genome comprises at an endogenous MHC II locus a nucleotide sequence encoding a chimeric human/mouse MHC II protein, wherein a human portion of the ic MHC ll protein comprises an extracellular domain of a human HLA- DR4 and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2E. In some embodiments, the method utilizes a targeting uct made using VELOCIGENE® technology, introducing the uct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOClMOUSE® technology, as described in the Examples. in one embodiment, the ES cells are a mix of 129 and C57BL/6 mouse strains; in one embodiment, the ES cells are a mix of BALB/c and 129 mouse strains.

A nucleotide construct used for ting genetically engineered non—human animals described herein is also provided. in one aspect, the nucleotide uct comprises: 5' and 3’ man homology arms, a DNA fragment comprising human HLA— DR or and B chain sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human HLA—DR or and {5 chain sequences are genomic sequences that comprise introns and exons of human HLA-DR 0L and 6 chain genes. in one embodiment, the non-human homology arms are homologous to non-human MHC ll genomic sequence.

In one embodiment, the human HLA-DR a chain sequence comprises an (11 and a2 domain coding sequence. in a specific embodiment, it comprises, from 5’ to 3’: (x1 exon (exon 2), a1/oc2 intron (intron 2), and a2 exon (exon 3). in one embodiment, the human HLA-DR [3 chain sequence comprises a [31 and [32 domain coding sequence. In a specific embodiment, it comprises, from 5’ to 3’: [31 exon (exon 2), [31/62 intron (intron 2), and [32 exon (exon 3).

A selection Cassette is a nucleotide sequence inserted into a targeting uct to facilitate ion of cells (e.g., ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. Commonly, a selection cassette enables positive selection in the presence of a particular otic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). in addition, a selection cassette may be d by recombination sites, which allow deletion of the selection te upon treatment with recombinase enzymes.

Commonly used ination sites are loxP and Fit, recognized by Cre and Flp enzymes, respectively, but others are known in the art. A ion cassette may be located anywhere in the construct outside the coding region. In one embodiment, the selection cassette is located in the [3 chain intron, e.g., BZ/transmembrane domain intron (intron 3).

In one embodiment, 5’ and 3’ homology arms comprise genomic sequence at 5’ and 3’ locations of endogenous non-human MHC II locus. In one embodiment, the 5’ homology arm comprises genomic sequence upstream of mouse H-2Ab1 gene and the 3’ homology arm comprises genomic sequence downstream of mouse H—2Ea gene. In this embodiment, the construct allows replacement of both mouse H-2E and H-2A genes.

Thus, in one aspect, a nucleotide uct is provided comprising, from 5’ to 3’: a 5’ gy arm containing mouse genomic sequence upstream of mouse H-2Ab1 gene, a first nucleotide ce comprising a sequence encoding a ic human/mouse MHC ll [5 chain, a second nucleotide sequence comprising a sequence ng a chimeric human/mouse MHC II or chain, and a 3’ homology arm containing mouse genomic sequence downstream of mouse H-2Ea gene. In a specific embodiment, the first nucleotide sequence comprising a sequence encoding a chimeric human/mouse MHC II {5 chain comprises human [31 exon, [SI/[32 , [32 exon, an a selection cassette flanked by recombination sites inserted in the intronic region n the human [32 exon sequence and the sequence of a mouse transmembrane domain exon. In a specific embodiment, the second nucleotide sequence comprising a sequence encoding a chimeric human/mouse MHC II or chain comprises human a1 exon, (11/012 intron, and human (x2 exon. An exemplary construct of the invention is ed in (MAID 1680).

Upon completion of gene targeting, ES cells or cally modified non-human animals are screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide. us techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCT (e.g., ime PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, histochemistry, etc. In one example, non-human animals (e.g., mice) bearing the genetic modification of interest can be fied by screening for loss of mouse allele and/or gain of human allele using a cation of allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression is, Nature Biotech. 21 (6):652-659. Other assays that identify a specific nucleotide or amino acid sequence in the genetically modified animals are known to those skilled in the art.

The disclosure also provides a method of modifying an MHC II locus of a nonhuman animal to express a chimeric human/non—human MHC II x described herein.

In one embodiment, the invention provides a method of modifying an MHC II locus of a mouse to express a chimeric human/mouse MHC II complex comprising replacing at the WO 63340 endogenous mouse MHC II locus a nucleotide ce encoding a mouse MHC II complex with a nucleotide sequence encoding a chimeric human/mouse MHC II complex. In a specific aspect, the nucleotide sequence encoding the chimeric human/mouse MHC II complex comprises a first nucleotide sequences encoding an extracellular domain of a human MHC II on chain (e.g., HLA-DR4 or chain) and transmembrane and cytoplasmic domains of a mouse MHC II a chain (e.g., H-2E or chain) and a second nucleotide sequence encoding an extracellular domain of a human MHC II B chain (e.g., HLA-DR4 [3 chain) and transmembrane and asmic domains of a mouse MHC II [5 chain (e.g., H-2E [3 chain, e.g., H-2Eb1 chain). In some embodiments, the modified mouse MHC II Iocus expresses a chimeric HLA-DR4/H-2E n.

In one aspect, a method for making a ic human HLA class II/non—human MHC class II molecule is provided, comprising expressing in a single cell a chimeric HLA- ADR4/H-2E protein from a nucleotide construct as described herein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a l celI expressing a viral nucleic acid sequence (e.g., a PERC.6T"" cell).

In one aspect, a cell that expresses a chimeric HLA-DR4/H-2E protein is provided. In one embodiment, the cell comprises an sion vector comprising a chimeric MHC class II sequence as bed herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a l cell expressing a viral nucleic acid sequence (e.g., a PERC.6TM cell).

A chimeric MHC class II molecule made by a non—human animal as described herein is also provided, wherein the chimeric MHC class II molecule comprises (11, a2, [51, and [32 domains from a human MHC II protein, e.g., 4 protein, and transmembrane and cytoplasmic domains from a non-human MHC II protein, e.g., mouse H-2E protein. The chimeric MHC II complex comprising an extracellular domain of 4 described herein maybe detected by anti-HLA-DR antibodies. Thus, a cell displaying chimeric human/non- human MHC II ptide may be detected and/or selected using anti—HLA—DR antibody.

Although the Examples that follow describe a genetically engineered animal whose genome comprises a replacement of a nucleotide sequence encoding mouse H-2A and H-2E proteins with a nucleotide sequence encoding a chimeric human/mouse HLA- 2E protein, one skilled in the art would understand that a similar strategy may be used to introduce chimeras sing other human MHC II genes (HLA-DP and HLA-DQ).

Thus, an additional embodiment of the invention is ed to a genetically ered animal whose genome comprises a nucleotide sequence encoding a chimeric HLA—DQ/H—ZA protein. in one ment, the tide sequence encodes a chimeric HLA—DQ2.5/H-2A protein. in another ment, the nucleotide sequence encodes a chimeric HLA-DQS/H— 2A n. In on, introduction of multiple humanized MHC II molecules (e.g., chimeric HLA—DR/H—ZE and HLA-DQ/H-ZA) is also contemplated.

Use of Genetically Modified Animals In s ments, the genetically modified non-human animals bed herein make APCs with human or humanized MHC II on the cell surface and, as a result, present peptides derived from cytosolic proteins as epitopes for T cells in a human-like manner, because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), e.g., for use in e development; for evaluation of vaccine candidates and other vaccine strategies; for studying human autoimmunity; for studying human infectious diseases; and othenrvise for devising better therapeutic strategies based on human MHC expression.

MHC II complex binds peptides derived from extracellular proteins, e.g., extracellular bacterium, neighboring cells, or polypeptides bound by B cell receptors and internalized into a B cell. Once extracellular proteins enter endocytic pathway, they are degraded into peptides, and peptides are bound and presented by MHC ll. Once a e presented by MHC ii is recognized by CD4+ T cells, T cells are activated, proliferate, differentiate to various T helper es (e.g., TH1, TH2), and lead to a number of events including activation of macrophage-mediated pathogen killing, B cell proliferation, and antibody production. Because of MHC II role in immune response, understanding of MHC II peptide presentation is important in the development of treatment for human pathologies. r, presentation of antigens in the context of mouse MHC II is only somewhat relevant to human disease, since human and mouse MHC xes ize antigens differently, e.g., a mouse MHC II may not recognize the same antigens or may present different epitopes than a human MHC II. Thus, the most nt data for human pathologies is obtained through studying the presentation of antigen epitopes by human MHC II.

Thus, in various embodiments, the genetically engineered animals of the present invention are useful, among other things, for evaluating the capacity of an n to initiate an immune se 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 aspect, a method for determining antigenicity in a human of a peptide sequence is provided, sing exposing 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 peptide ted by a zed MHC II complex described herein.

In one aspect, a method for determining whether a peptide will provoke an immune response in a human is ed, comprising exposing a genetically modified non- human animal as described herein to the peptide, allowing the non—human animal to mount an immune response, and ing in the non-human animal a cell that binds a sequence of the peptide by a chimeric human/non-human MHC class II molecule as described herein. In one embodiment, the non-human animal following exposure comprises an MHC class II- restricted CD4+ T cell that binds the peptide.

In one aspect, a method for identifying a human CD4+ T cell epitope is provided, comprising ng a non—human animal as described herein to an antigen comprising a putative T cell epitope, allowing the non-human animal to mount an immune response, and identifying the epitope bound by the MHC class ll-restricted CD4+ T cell.

In one aspect, a method is provided for identifying an antigen that generates a CD4+ 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, detecting a CD4+ T cell response that is specific for the antigen in the context of a human MHC II molecule (e.g., an HLA-DR molecule), and identifying the antigen bound by the human MHC ll-restricted molecule (e.g., human HLA-DR restricted molecule).

In one embodiment, the antigen comprises a bacterial protein. In one embodiment, the antigen ses a human tumor cell antigen. In one embodiment, the n comprises a putative e for use in a human, or another biopharmaceutical. In one embodiment, the antigen comprises a human epitope that generates antibodies in a human. In yet another embodiment, an antigen comprises a yeast or fungal cell antigen. In yet another embodiment, an antigen is derived from a human parasite.

In one aspect, a method is ed for determining whether a ve n contains an epitope that upon exposure to a human immune system will generate an HLA— DR-restricted immune response (e.g., HLA—DR4-restricted se), comprising ng a mouse as described herein to the putative antigen and ing an antigen—specific HLA- DR-restricted (e.g., HLA-DR4-restricted) immune response in the mouse. In another aspect, a method is provided for determining wherein a ve antigen contains an epitope that upon exposure to a human immune system will generate an HLA—DQ-restricted response.

Also provided is a method of generating antibodies to an antigen, e.g., an antigen derived from bacterium, parasite, etc., ted in the context of a human MHC II complex, comprising exposing a mouse described herein to an antigen, allowing a mouse to mount an immune response, wherein the immune response comprises antibody tion, and isolating an antibody that recognizes the antigen presented in the t of human MHC II complex. In one ment, in order to generate antibodies to the peptide-MHC II, the MHC II zed mouse is immunized with a peptide—MHC II immunogen.

In one , a method for identifying a T cell receptor variable domain that recognizes an antigen ted in the context of MHC II (e.g., human tumor antigen, a vaccine, etc.) is provided, comprising exposing a mouse comprising a zed MHC II complex described herein to the antigen, allowing the mouse to generate an immune response, and isolating from the mouse a nucleic acid sequence encoding a variable domain of a T cell or that binds MHC lI-restricted antigen. In one embodiment, the n is presented in the context of a humanized MHC II (e.g., human HLA ll ectodomain/mouse MHC II transmembrane and/or cytoplasmic domain).

The uence of interaction between a T cell and an APC displaying a peptide in the context of MHC II (e.g., human HLA || ectodomain/mouse MHC II transmembrane and/or cytoplasmic domain) can be measured by a number of techniques known in the art, e.g., T cell proliferation assays, cytokine release assays, etc.

In on to the ability to fy antigens and their T cell epitopes from pathogens or neoplasms, the genetically modified animals of the invention can be used to identify autoantigens of relevance to human autoimmune disease, and othenlvise study human autoimmune disease progression. It is known that polymorphisms within the HLA loci play a role in predisposition to human autoimmune disease. In fact, specific polymorphisms in HLA-DR and HLA-DQ loci have been identified that correlate with development of rheumatoid arthritis, type I diabetes, Hashimoto’s thyroiditis, le sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn’s disease, ulcerative colitis, and other mune disorders. See, e.g., Wong and Wen (2004) What can the HLA transgenic mouse tell us about autoimmune diabetes?, Diabetologia 47:1476-87; Taneja and David (1998) HLA Transgenic Mice as Humanized Mouse Models of Disease and Immunity, J. Clin. Invest. 101:921—26; Bakker et al. (2006), supra; and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases, Proc. Natl. Acad. Sci. USA 106:18680-85.

Thus, the methods of making a zed MHC II complex animals described herein can be used to introduce MHC ll molecules thought to be associated with specific human mune diseases, and progression of human mune disease can be studied. In addition, non-human s described herein can be used to develop animal models of human autoimmune disease. Mice according to the invention ng humanized MHC II proteins described herein can be used to identify potential autoantigens, to map epitopes involved in disease progression, and to design strategies for autoimmune disease modulation.

In addition, the genetically modified animals described herein may be used in the study of human allergic response. As allergic responses appear to be associated with MHC II alleles, cally modified animals described herein may be used to determine HLA restriction of allergen specific T cell response and to develop strategies to combat ic response.

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

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

Example 1. Deletion of the Endogenous MHC class II H-2A and H-2E Loci The targeting vector for introducing a deletion of the nous MHC class ll H- 2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea genes was made using VELOCIGENE® genetic engineering technology (see, 9.9., US Pat. No. 6,586,251 and Valenzuela et al., supra).

Bacterial Artificial Chromosome (BAC) RP23—458i22 (lnvitrogen) DNA was modified to delete the endogenous MHC class II genes H—2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H—2Ea.

] Briefly, upstream and downstream homology arms were derived by PCR of mouse BAC DNA from locations 5’ of the H-2Ab1 gene and 3’ of the H—2Ea gene, respectively. As depicted in these homology arms were used to make a cassette that deleted ~79 kb of RP23-458i22 comprising genes H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea of the MHC class ll locus by bacterial homologous recombination (BHR). This region was replaced with a hygromycin cassette flanked by lox66 and lox71 sites. The final targeting vector from 5’ to 3’ included a 34 kb homology arm comprising mouse genomic sequence 5’ to the H-2Ab1 gene of the endogenous MHC class II locus, a 5’ on66 site, a hygromycin cassette, a 3’ lox71 site and a 63 kb homology arm comprising mouse genomic sequence 3’ to the H—2Ea gene of the endogenous MHC class II locus (MAID 5111, see FIG.

The BAC DNA targeting vector ibed above) was used to electroporate mouse ES cells to create modified ES cells comprising a deletion of the endogenous MHC class II locus. Positive ES cells containing a d endogenous MHC class II locus were identified by the tative PCR assay using TAQMANT'V' probes (Lie and Petropoulos (1998) Curr. Opin. Biotechnology 9:43-48). The upstream region of the deleted locus was confirmed by PCR using primers 5111U F CGCCAGGCTGTAAC; SEQ ID N0:1) and 5111U R (GGAGAGCAGGGTCAGTCAAC; SEQ ID N0:2) and probe 5111U P (CACCGCCACTCACAGCTCCTTACA; SEQ ID NO:3), whereas the downstream region of the deleted locus was confirmed using primers 5111D F (GTGGGCACCATCTTCATCATTC; SEQ ID NO:4) and 5111D R (CTTCCTTTCCAGGGTGTGACTC; SEQ ID NO:5) and probe 5111D P (AGGCCTGCGATCAGGTGGCACCT; SEQ ID N016). The presence of the ycin cassette from the targeting vector was confirmed using primers HYGF (TGCGGCCGATCTTAGCC; SEQ ID N027) and HYGR (TTGACCGATTCCTTGCGG; SEQ ID NO:8) and probe HYGP (ACGAGCGGGTTCGGCCCATTC; SEQ ID NO:9). The nucleotide sequence across the upstream deletion point (SEQ ID N0:10) included the following, which indicates endogenous mouse sequence upstream of the deletion point (contained within the parentheses below) linked uously to cassette sequence present at the on point: (TTTGTAAACA AAGTCTACCC AGAGACAGAT GACAGACTTC AGCTCCAATG CTGATTGGTT CCTCACTTGG GACCAACCCT) CTCGAGTACC GTTCGTATAA TGTATGCTAT TTAT ATGCATCCGG GAGG. The nucleotide sequence across the downstream deletion point (SEQ ID N0:11) included the following, which indicates cassette sequence contiguous with nous mouse sequence downstream of the deletion point ined within the heses below): CCTCGACCTG CAGCCCTAGG ATAACTTCGT ATAATGTATG CTATACGAAC GGTAGAGCTC (CACAGGCATT TGGGTGGGCA GGGATGGACG GTGACTGGGA CAATCGGGAT GCAT AGAATGGGAG TTAGGGAAGA). Positive ES cell clones were then used to implant female mice using the VELOCIMOUSE® method (described below) to generate a litter of pups containing a deletion of the endogenous MHC class II locus.

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, 9.9., US Pat. No.

WO 63340 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech. 25(1):91-99). Mice bearing a deletion of H-2Ab1, H—2Aa, H-2Eb1, H-2Eb2, and H- 2Ea genes in the endogenous MHC class II locus were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detected the presence of the hygromycin cassette and confirmed the absence of endogenous MHC class II sequences.

Mice bearing a deletion of H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea genes in the endogenous MHC class II locus can be bred to a Cre r mouse strain (see, e.g., ational Patent Application Publication No. ) in order to remove any loxed hygromycin te introduced by the targeting vector that is not d, e.g., at the ES cell stage or in the . Optionally, the hygromycin te is retained in the mice.

Example 2. Generation of Large Targeting Vector (LTVEC) Comprising Humanized H- 2Eb1 and H-2Ea Genes A targeting vector to introduce humanized MHC II sequences was designed as ed in Using VELOCIGENE® genetic engineering technology, Bacterial Artificial Chromosome (BAC) RP23-458i22 DNA was ed in various steps to: (1) create a vector comprising a functional l-E on exon 1 from BALB/c H-2Ea gene (); (2) create a vector comprising replacement of exons 2 and 3 of mouse l-E (3 gene with those of human DRfi1*O4 and replacement of exons 2 and 3 of mouse l-E or with those of human DRa1*01 (FIGS. 48); (3) create a vector carrying exons 2 and 3 of human DR[31*04 amongst remaining mouse I—E B exons, and exons 2 and 3 of human DRoc1*O1 amongst remaining mouse l—E 0L exons ing a functional l—E on exon 1 from BALB/c mouse (step (1) (); and (4) remove a cryptic splice site in the vector generated in (3) ().

Specifically, because in the C57Bl/6 mice, the l-E 0t gene is a pseudogene due to the presence of a non-functional exon 1, first, a vector comprising a functional l-E or exon 1 from BALB/c H—2Ea gene was created (). RP23-458i22 BAC was modified by bacterial homologous recombination (1 .BHR) to replace chloramphenicol resistance gene with that of spectromycin. The resultant vector was r ed by BHR to e the entire l—A and l-E coding region with a neomycin cassette flanked by recombination sites (2.BHR). Another round of BHR (3. BHR) with the construct comprising an exon encoding BALB/c l-Ea leader (exon 1) and chloramphenicol gene flanked by Pl-Scel and l—Ceul restriction sites resulted in a vector comprising a functional BALB/c H-2Ea exon 1.

Independently, in order to generate a vector comprising replacement of exons 2 and 3 of mouse l—E [3 gene with those of human DR|31*04 and replacement of exons 2 and 3 WO 63340 of mouse l-E or with those of human DRa1*O1, RP23—458i22 BAC was modified via several homologous ination steps, 4. BHR - 8. BHR (), The resultant nucleic acid sequence was flanked by Pl—Scel/I-Ceul restriction sites to allow ligation into the construct carrying BALB/c I-Eoc exon 1, mentioned above ().

The sequence of the final construct depicted in contained a c splice site at the 3’ end of the BALB/c intron. Several BHR steps (11. BHR — 12. BHR) followed by a deletion step were performed to obtain the final targeting vector (MAID 1680) that was used to electroporate into ES cells ().

In detail, the final ing vector (MAID 1680), from 5’ to 3’, was comprised of a ’ mouse homology arm consisting of ~26 kb of mouse genomic sequence ending just am of the H-2Ab1 gene of the endogenous MHC class II locus; an ~59 kb insert containing the humanized MHC II [3 chain gene (humanized H—2Eb1 gene) and humanized MHC II a chain gene (humanized H-2Ea gene) and a floxed neomycin cassette; and a 3’ mouse homology arm consisting of ~57 kb of mouse genomic sequence beginning just ream of the H—2Ea gene of the endogenous MHC class II locus. The nucleotide sequence across the junction between the 5’ arm and the insert (SEQ ID NO:12) included the following: (TGCTGATTGG TTCCTCACTT GGGACCAACC C) TAAGCTTTA TCTATGTCGG GTGCGGAGAA AGAGGTAATG AAATGGCACA AGGAGATCAC AAAC CAAACTCGCC, where the italicized sequence is a unique PI-Scel site, and mouse genomic sequence in the 5’ homology arm is in parentheses. The nucleotide sequence across the junction between the insert and the 3’ arm (SEQ ID NO:13) included the following: CACATCAGTG GAAT AAATTAAAAT CGCTAATATG AAAATGGGG (ATTTGTACCT GTGA AGGCTGGGAA GACTGCTTTC AAGGGAC), where the mouse genomic ce in the 3’ homology arm is in heses.

Within the ~59 kb insert, the H-2Eb1 gene was modified as s: a 5136 bp region of H-2Eb1, including the last 153 bp of intron1, exon 2, intron 2, exon 3, and the first 122 bp of intron 3, was replaced with the 3111 bp homologous region of human HLA— DRB1*04, including the last 148 bp of intron 1, exon 2, intron 2, exon 3, and the first 132 bp of intron 3. At the junction between the human and mouse sequences of intron 3, a cassette consisting of a 5’ lox2372 site, UbC promoter, neomycin resistance gene, and a 3’ lox2372 site, was inserted. The resulting gene encoded a chimeric HLA—DRB1*04/H-2Eb1 protein comprised of the mouse H-2Eb1 leader, the human [:31 and [32 s from DRB1*04, and the mouse transmembrane domain and cytoplasmic tail. The nucleotide sequence across the mouse/human junction in intron 1 (SEQ ID NO:14) included the following: (TCCATCACTT CACTGGGTAG CACAGCTGTA ACTGTCCAGC CTG) GGTACCGAGC 2012/062029 TCGGA TCCAC TAGTAACGGC CGCCAGTGTG CTGGAATTC GCCCTTGATC GAGCTCCCTG GGCTGCAGGT GGTGGGCGTT GCGGGTGGGG CCGGTTAA, where the italicized sequence is a multiple cloning site introduced during the g steps, and the mouse intron 1 sequences are in parentheses. The nucleotide sequence across the junction between the human intron 3 and neomycin cassette (SEQ ID NO:15) included the following: CATCA GAAGGGCACC GGT) ATAACTT CGTA ATCCTATACG AAGTTATATG CATGGCCTCC GCGCCGGGTT, where the 5’ |ox2372 site is italicized, and human intron 3 sequence is in parentheses. The nucleotide sequence across the junction between the neomycin cassette and mouse intron 3 (SEQ ID NO:16) included the following: ATAACTTCGTATAAGGTATC CTATACGAAG TTA G (TGGCTTACAG GTAGGTGCGT GAAGCTTCTA CAGT TGCCCCCTGG), where the 3’ lox2372 site is italicized, and the mouse intron 3 sequence is in parentheses.

Also within the ~59 kb insert, the H-2Ea gene was modified as follows: a 1185 bp region of H-2Ea, including the last 101 bp of intron1, exon 2, intron 2, exon 3, and the first 66 bp of intron 3, was replaced with the 1189 bp homologous region of human HLA—DRA1 *01, including the last 104 bp of intron 1, exon 2, intron 2, exon 3, and the first 66 bp of intron 3.

As described above, because exon 1 of the 057BL/6 allele of H-2Ea contains a deletion which s the gene nonfunctional, H-2Ea exon 1 and the remainder of intron 1 were replaced with the equivalent 2616 bp region from the BALB/c allele of H-2Ea, which is functional. The resulting gene encoded a chimeric H-2Ea/HLA-DRA1*O1 n comprised of the mouse H-2Ea leader from , the human a1 and a2 domains from DRA1*O1, and the mouse transmembrane domain and cytoplasmic tail. The nucleotide sequence across the mouse/human junction in intron 1 (SEQ lD NO:17) included the following: (CTGTTTCTTC CCTAACTCCC ATTCTATGCT CTTCCATCCC GA) CCGCGGCCCA ATCTCTCTCC ACTACTTCCT GCCTACATGT ATGTAGGT, where the ized sequence is a restriction enzyme site introduced during the cloning steps, and the BALB/c intron 1 sequences are in parentheses. The nucleotide sequence across the human/mouse junction in intron 3 (SEQ lD NO:18) included the ing: CAAGGTTTCC TCCTATGATG TGAA ACTCGGGGCC GGCC (AGCATTTAAC AGTACAGGGA TGGGAGCACA GCTCAC), where the italicized sequence is a ction enzyme site introduced during the cloning steps, and the mouse intron 3 sequences are in parentheses. The nucleotide sequence across the CS7BL/6-BALB/c junction 5’ of exon 1 (SEQ ID NO:19) included the following: (GAAAGCAGTC TTCCCAGCCT TCACACTCAG AGGTACAAAT) CCCCATTTTC ATATTAGCGA TTTTAATTTA TTCTAGCCTC, where the CS7BL/6-specific sequences are in parentheses. The nucleotide sequence across the BALB/c-C57BL/6 junction 3’ of exon 1 (SEQ ID NO:20) included the following: TCTTCCCTAA CTCCCATTCT ATGCTCTTCC ATCCCGA CCG CGG (CCCAATC TCTCTCCACT ACTTCCTGCC TACATGTATG), where Sacll restriction site is italicized, and C57BL/6 ces are in parenthesis.

Example 3. Generation of Humanized MHC II Mice ] Simplified diagrams of the strategy for generating humanized MHC ll mice using the vector of Example 2 are presented in Fle. 5 and 8.

Specifically, MAID1680 BAC DNA (described above) was used to oporate MAlD5111 ES cells to create modified ES cells comprising a replacement of the endogenous mouse l-A and l—E loci with a genomic nt comprising a ic human DR4/mouse l-E locus. Positive ES cells containing deleted endogenous l—A and l-E loci replaced by a c fragment comprising a chimeric human DR4/mouse l-E locus were identified by a quantitative PCR assay using TAQMANT'V' probes (Lie and Petropoulos, supra). The insertion of the human DRoc sequences was confirmed by PCR using primers hDRA1F (CTGGCGGCTTGAAGAATTTGG; SEQ ID NO:21), hDRA1R TTTCCAGGTTGGCTTTGTC; SEQ ID NO:22), and probe hDRA1P (CGATTTGCCAGCTTTGAGGCTCAAGG; SEQ ID NO:23). The insertion of the human DR[3 sequences was confirmed by PCR using primers hDRB1 F TGGGTGCTCCACTTG; SEQ ID NO:24), hDRB1R (GACCCTGGTGATGCTGGAAAC; SEQ ID N025), and probe hDRB1 P (CAGGTGTAAACCTCTCCACTCCGAGGA; SEQ lD NO:26).The loss of the hygromycin cassette from the targeting vector was confirmed with s HYGF (TGCGGCCGATCTTAGCC; SEQ ID NO:7) and HYGR (TTGACCGATTCCTTGCGG; SEQ ID NO:8) and probe HYGP (ACGAGCGGGTTCGGCCCATTC; SEQ ID NO:9).

Positive ES cell clones were then used to implant female mice using the MOUSE® method (supra) to generate a litter of pups containing a replacement of the endogenous l-A and l-E loci with a chimeric human DR4/mouse l—E locus. ed ES cells described above were used as donor ES cells and introduced into an 8—cell stage mouse embryo by the VELOCIMOUSE® method. Mice bearing a chimeric human DR4/mouse l-E locus were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detected the presence of a chimeric human DR4/mouse l-E locus.

Mice bearing a chimeric human DR4/mouse l-E locus can be bred to a Cre deletor mouse strain (see, e.g., International Patent Application Publication No. WO 2009/114400) in order to remove any loxed neomycin cassette introduced by the targeting vector that is not removed, e.g., at the ES cell stage or in the embryo (See .

Example 4. Expression of the ic HLA-DR4 in cally Modified Mice Spleens from WT or heterozygous humanized HLA-DR4 mice (“1681 HET”) were perfused with Collagenase D (Roche Bioscience) and erythrocytes were lysed with ACK lysis buffer. Splenocytes were cultured for two days with 25 micrograms/mL poly(I:C) to stimulate the expression of MHC-II genes. Cell surface expression of human HLA-DR4 was analyzed by FACS using fluorochrome-conjugated anti-CD3 (17A2), anti-CD19 (1D3), anti-CD11c (N418), anti-F480 (BM8), anti-I-A/I-E (M15) and anti-HLADR (L243). Flow cytometry was performed using BD-LSRII. Expression of human 4 was clearly detectable on the surface of CD19+ B cells and was significantly upregulated upon stimulation by toll-like receptor agonist poly(I:C) (see .

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 described herein. Such lents are ed to be encompassed by the following claims.

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

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

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

Claims (18)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A non-human animal comprising at an endogenous Major Histocompatibility Complex II (MHC II)  gene locus a nucleotide sequence encoding a ic human/non-human MHC II  polypeptide and/or comprising at an endogenous MHC II β gene locus a nucleotide sequence encoding a chimeric human/non-human MHC II β polypeptide, wherein a human portion of the chimeric non-human MHC II  polypeptide comprises a human MHC II 2 ellular domain, and/or a human portion of the chimeric human/non-human MHC II β polypeptide comprises human MHC II β2 domains, and wherein the animal expresses a functional MHC II complex on a surface of a cell of the animal.

2. The animal of claim 1, wherein the nucleotide sequence encoding a chimeric human/non-human MHC II  polypeptide is expressed under regulatory control of endogenous man MHC II  promoter and tory elements and/or the tide sequence encoding a chimeric human/non-human MHC II β polypeptide is expressed under regulatory control of endogenous non-human MHC II β er and regulatory ts.

3. The animal of claim 1, wherein a non-human portion of the chimeric human/nonhuman MHC II  polypeptide comprises embrane and cytoplasmic domains of an nous non-human MHC II  polypeptide and/or a non-human portion of the chimeric human/non-human MHC II β polypeptide ses transmembrane and cytoplasmic domains of an endogenous non-human MHC II β polypeptide.

4. The animal of claim 1, wherein the human portion of the chimeric human/non-human MHC II  polypeptide is encoded by a HLA class II gene selected from the group consisting of any α chain gene of HLA-DR, HLA-DQ, and HLA-DP and/or the human portion of the chimeric human/non-human MHC II β polypeptide is encoded by a human HLA class II gene selected from the group consisting of any β chain gene of HLA-DR, HLA-DQ, and HLA-DP.

5. The animal of claim 4, wherein the human portion of the chimeric human/non-human MHC II  polypeptide is encoded by a human HLA-DR4 α chain gene and/or the human portion of the ic human/non-human MHC II β polypeptide is encoded by a human HLADR4 β chain gene.

6. The animal of claim 1, n the animal is a rodent.

7. The rodent of claim 6, wherein the rodent is a mouse.

8. A non-human animal according to any one of claims 1 to 7, n the non-human animal is a rodent comprising at an endogenous MHC II gene locus a first nucleotide ce encoding a chimeric human/rodent MHC II  polypeptide and a second nucleotide sequence encoding a chimeric human/rodent MHC II  polypeptide, wherein a human portion of the chimeric human/rodent MHC II  polypeptide ses a human MHC II 2 domain and a human portion of the chimeric human/rodent MHC II  polypeptide comprises a human MHC II 2 domain, and wherein the chimeric human/rodent MHC II  and MHC II  ptides form a functional MHC II complex on a surface of a cell of the rodent.

9. The rodent of claim 8, wherein the rodent is a mouse.

10. The mouse of claim 9, wherein mouse portions of the chimeric MHC II  and  polypeptides are encoded by a mouse H-2E gene.

11. The rodent of claim 8, wherein the rodent does not express functional endogenous MHC II polypeptides from their endogenous rodent MHC II loci.

12. A method of modifying an MHC II locus of a mouse to express a chimeric human/mouse MHC II complex comprising replacing at the endogenous mouse MHC II α locus a nucleotide sequence encoding a mouse MHC II α protein with a first nucleotide sequence encoding a chimeric human/mouse MHC II α protein and/or at the endogenous mouse MHC II β locus a nucleotide sequence encoding a mouse MHC II β protein with a second nucleotide ce encoding a chimeric human/mouse MHC II β protein, wherein a chimeric human/mouse MHC II α protein comprises an α2 domain of a human MHC II  polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II  polypeptide and the chimeric human/mouse MHC II β protein comprises a β2 domain of a human MHC II β polypeptide and embrane and cytoplasmic domains of a mouse MHC II β polypeptide.

13. The method of claim 12, wherein a human portion of the chimeric MHC II complex is encoded by a human HLA class II  chain gene ed from the group consisting of any  chain gene of HLA-DR, HLA-DQ, and HLA-DP and/or by a human HLA class II β chain gene selected from the group ting of any β chain gene of HLA-DR, , and HLA-DP.

14. The method of claim 12, wherein a mouse portion of the chimeric MHC II complex is encoded by a mouse H-2E  gene and/or d by a mouse H-2E β gene, and a human portion of the chimeric MHC II complex is encoded by a human HLA-DR4  gene and/or encoded by a human HLA-DR4 β gene.

15. The method of claim 12, wherein the replacement is made in a single ES cell, and the single ES cell is introduced into a mouse embryo to make a mouse.

16. The non-human animal of claim 1, the rodent of claim 6, or the mouse of claim 7, wherein the extracellular domain of the α polypeptide further comprises a human α1 domain d by a human HLA-DR gene.

17. The non-human animal of claim 1, the rodent of claim 6, or the mouse of claim 7, wherein the extracellular domain of the β ptide further comprises a human β1 domain encoded by a human HLA-DR β gene.

18. The non-human animal according to claim 1, or the method according to claim 12, substantially as herein described with reference to any of the Examples and/or