Method for preparing major histocompatibility complex gene-modified mice

Abstract

The present invention provides a genetically modified non-human animal expressing a human / non-human chimeric MHC 1 polypeptide and / or a human or humanized β2 microglobulin polypeptide, as well as embryos, cells and tissues comprising the genetic modification. Also provided are a construct for preparing the genetically modified animal and a method for preparing the construct. The present invention also provides a method for using the genetically modified animal to study different aspects of the human immune system.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of a Chinese application with an application date of October 26, 2012 and an application number of 201710347715.8. The parent application of this application is a divisional application of a Chinese application with an application date of October 26, 2012 and an application number of 201280060540.8. The original application claims priority to U.S. Provisional Patent Application Nos. 61 / 552,582 and 61 / 552,587 with an application date of October 28, 2011 and U.S. Provisional Patent Application No. 61 / 700,908 with an application date of September 14, 2012, all of which are incorporated into this application by reference. Technical Field

[0003] The present invention relates to genetically modified non-human animals, such as rodents (e.g., mice or rats), which express human or humanized major histocompatibility complex (MHC) class I molecules. The present invention also relates to genetically modified non-human animals, such as mice or rats, which express human or humanized MHC I proteins (e.g., MHC Iα chains) and / or human or humanized β2 microglobulins; and embryos, tissues and cells expressing human or humanized MHC I proteins (e.g., MHC Iα chains) and / or human or humanized β2 microglobulins. The present invention further provides a method for preparing a genetically modified non-human animal, wherein the animal expresses human or humanized MHC class I proteins (e.g., MHC Iα chains) and / or β2 microglobulins. The present invention also provides a method for identifying and evaluating peptides in an in vitro humanized cellular immune system or a genetically modified non-human animal, and a method for modifying the MHC I and / or β2 microglobulin loci of a non-human animal, such as a mouse or rat, to express human or humanized MHC I and / or β2 microglobulins. Background Art

[0004] In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptors or TCRs). These foreign antigens are presented on the cell surface as peptide fragments of specific proteins, which are generally referred to as major histocompatibility complex (MHC) molecules. MHC molecules are encoded by multiple loci, which have been found to be linked gene clusters spanning approximately 4 Mb. In mice, the MHC genes are located on chromosome 17 and are called histocompatibility 2 (H-2) genes for historical reasons. In humans, the genes are located on chromosome 6 and are called human leukocyte antigen (HLA) genes. In mice and humans, the loci are polygenic, including three highly polymorphic types of MHC genes (class I, II, and III) with similar structures in the human and mouse genomes (see, respectively). Figure 2and Figure 3 ).

[0005] The MHC loci show the highest polymorphism in the genome; some genes are represented by >300 alleles (e.g., human HLA-DRβ and human HLA-B). All class I and class II MHC genes are capable of presentation as peptide fragments, but each gene expresses proteins with different binding properties, reflecting polymorphism and allelic variants. Any given individual has a unique range of peptide fragments that can be presented to the cell surface of B and T cells during the immune response.

[0006] Both humans and mice have class I MHC genes (see Figure 2 and Figure 3 In humans, the classical class I genes are called HLA-A, HLA-B, and HLA-C, and in mice they are called H-2K, H-2D, and H-2L. Class I molecules consist of two chains: the polymorphic α-chain (sometimes called the heavy chain) and a smaller chain called β2-microglobulin (also called the light chain), which is usually not polymorphic ( Figure 1 ). The two chains form non-covalent heterodimers on the cell surface. The α-chain contains three domains (α1, α2 and α3). Exon 1 of the α chain gene encodes the leader sequence, exons 2 and 3 encode the α1 and α2 domains, exon 4 encodes the α3 domain, exon 5 encodes the transmembrane domain, and exons 6 and 7 encode the cytoplasmic tail. The α-chain forms a peptide binding cleft containing the α1 and α2 domains (similar to the Ig-like domain) followed by the α3 domain similar to β2-microglobulin.

[0007] β2-microglobulin is a non-glycosylated 12 kDa protein; one of its functions is to stabilize the MHC class I α-chain. Unlike the α-chain, the β2-microglobulin does not cross membranes. The human β2-microglobulin gene locus is on chromosome 15, while the mouse locus is on chromosome 2. The β2-microglobulin gene consists of 4 exons and 3 introns. Circulating forms of β2-microglobulin are present in serum, urine, and other body fluids; therefore, non-covalently MHC I-bound β2-microglobulin can be exchanged with circulating β2-microglobulin under physiological conditions.

[0008] Class I MHC molecules are expressed in all nucleated cells, including tumor cells. They are specifically expressed on T and B lymphocytes, macrophages, dendritic cells, neutrophils and other cells and their function is to display peptide fragments (usually 8-10 amino acids in length) on the surface of CD8+ cytotoxic T lymphocytes (CTLs). CTLs specifically kill any cell carrying MHC I-bound peptides through their own membrane-bound TCRs. When cells display peptides from cell protein sources that are not normally present (e.g., viruses, tumors or other non-self sources), such peptides are recognized by CTLs, which are activated and kill cells that display the peptides.

[0009] Usually, due to the existence of tolerance mechanisms, the presentation of normal (i.e., self) proteins in the context of MHC I molecules will not trigger the activation of CTL. However, in some diseases (e.g., cancer, autoimmune diseases), peptides derived from self-proteins become target cell components of the immune system, resulting in the destruction of cells presenting such peptides. Although there has been progress in identifying some self-derived antigens (e.g., antigens associated with various cancers) that stimulate cellular immune responses, in order to improve the identification of peptides recognized by human CTLs through MHC I molecules, in vivo and in vitro systems are still needed to simulate various aspects of the human cellular immune system. Systems that simulate the human cellular immune system can be used to identify antigens associated with diseases and thus develop human therapeutic products such as vaccines and other biological products. Systems that assess antigen recognition in the human immune system can assist in identifying CTL groups that are useful for treatment (e.g., useful for research and prevention of human diseases). This system can also assist in enhancing the activity of human CTL groups so as to more effectively combat infections and entities carrying foreign antigens. Therefore, there is a need for a biological system (e.g., genetically modified animals) that can produce an immune system that displays components that simulate the functions of the human immune system. SUMMARY OF THE INVENTION

[0010] The present application provides a biological system for producing or identifying peptides, the peptides are associated with MHC class I proteins and chimeras thereof, and are bound to CD8+T cells. The present application provides a non-human animal comprising non-human cells expressing human or humanized molecules that function in cellular immune responses. The present application also provides humanized rodent loci encoding human or humanized MHC I and β2 microglobulin. The present application also provides humanized rodent cells expressing human or humanized MHC I and β2 microglobulin. The present application provides in vivo and in vitro systems comprising humanized rodent cells, wherein the rodent cells express one or more human or humanized immune system molecules.

[0011] The present application provides a non-human animal, such as a rodent (e.g., a mouse or a rat), comprising in its genome a nucleotide sequence encoding a human / non-human (e.g., human / rodent, such as human / mouse or human / rat) chimeric MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide. Specifically, the present invention provides a non-human animal, which comprises a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide at an endogenous MHC I locus, wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide, and wherein the animal expresses a human / non-human chimeric MHC I polypeptide. In one aspect, the animal does not express an extracellular domain of an endogenous non-human MHC I polypeptide at an endogenous non-human MHC I locus. In one aspect of the invention, the non-human animal (e.g., a rodent, such as a mouse or a rat) comprises two copies of an MHC I locus comprising a nucleotide sequence encoding a human / non-human (e.g., human / rodent, such as a human / mouse or human / rat) chimeric MHC I polypeptide. In another aspect of the invention, the animal comprises one copy of an MHC I locus comprising a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide. Thus, the animal may be homozygous or heterozygous for an MHC I locus comprising a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide. In various embodiments, a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide is contained in a germ cell of a non-human animal (e.g., a rodent, such as a rat or a mouse).

[0012] In one aspect, the nucleotide sequence encoding the human / non-human chimeric MHC I is operably linked to an endogenous non-human regulatory element, such as a promoter, enhancer, silencer, etc. In one embodiment, the human portion of the chimeric polypeptide comprises a human leader sequence. In another embodiment, the human portion of the chimeric polypeptide comprises the α1, α2, and α3 domains of a human MHC I polypeptide. The human MHC I polypeptide may be selected from the group consisting of HLA-A, HLA-B, and HLA-C. In one embodiment, the human MHC I polypeptide is an HLA-A2 polypeptide, such as an HLA-A2.1 polypeptide.

[0013] In one aspect, the genetically modified non-human animal is a rodent. In one embodiment, the rodent is a mouse. Therefore, in one embodiment, the endogenous non-human locus is a mouse locus, such as a mouse H-2K, H-2D or H-2L locus. In one embodiment, the non-human portion of the human / non-human chimeric MHC I polypeptide comprises the transmembrane and cytoplasmic domains of the endogenous non-human MHC I polypeptide. Therefore, in one embodiment, wherein the non-human animal is a mouse, the endogenous non-human MHC I locus can be an H-2K locus (e.g., an H-2kb locus) and the endogenous non-human HMC I polypeptide can be an H-2K polypeptide; therefore, the human / non-human chimeric MHC I polypeptide can comprise the transmembrane and cytoplasmic domains of the H-2K polypeptide. In another embodiment, wherein the non-human animal is a mouse, the endogenous non-human MHC I locus may be an H-2D locus and the endogenous non-human MHC I polypeptide may be an H-2D polypeptide; thus, the human / non-human chimeric MHC I polypeptide may comprise the transmembrane and cytoplasmic domains of the H-2D polypeptide. Similarly, in another embodiment, the endogenous non-MHC I locus may be an H-2L locus and the endogenous non-human MHC I polypeptide may be an H-2L polypeptide; thus, the human / non-human chimeric MHC I polypeptide may comprise the transmembrane and cytoplasmic domains of the H-2L polypeptide.

[0014] The present application also provides a mouse comprising a nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide at an endogenous H-2K locus, wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A (e.g., HLA-A2) polypeptide and the mouse portion comprises a transmembrane and cytoplasmic domain of a mouse H-2K polypeptide, and wherein the mouse expresses a human / mouse chimeric MHC I polypeptide. In some embodiments, the mouse does not express the extracellular domain of a mouse H-2K polypeptide from an endogenous H-2K locus. In one aspect, the nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide is operably linked to an endogenous mouse regulatory element. The human portion of the chimeric polypeptide may comprise a human leader sequence. It may also comprise the α1, α2, and α3 domains of a human MHC I polypeptide. The human MHC I polypeptide may be an HLA-A polypeptide, such as an HLA-A2.1 polypeptide. In one aspect, the mouse H-2K locus is an H-2Kb locus.

[0015] Another aspect of the present invention relates to a non-human animal, such as a rodent (e.g., a mouse or a rat), comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide in the genome of the animal. Therefore, the present application provides a non-human animal, the animal comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide in an endogenous non-human β2 microglobulin locus, wherein the animal expresses a human or humanized β2 microglobulin polypeptide. In one aspect, the animal does not express a functional endogenous non-human β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In one aspect, the animal comprises two copies of a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide; in another embodiment, the animal comprises one copy of a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. Therefore, the animal may be homozygous or heterozygous for a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In various embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide is contained in the germ cells of the non-human animal (e.g., a rodent, such as a rat or a mouse). In one embodiment, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of human β2 microglobulin. In one embodiment, the polypeptide is capable of binding to an MHC I protein.

[0016] In some embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide is operably linked to an endogenous non-human β2 microglobulin regulatory element. In one aspect, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene. In another aspect, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises the nucleotide sequence shown in exons 2, 3, and 4 of the human β2 microglobulin gene. In a further embodiment, the nucleotide sequence also includes the nucleotide sequence shown in exon 1 of the non-human β2 microglobulin gene. In some embodiments, the non-human animal is a rodent (e.g., a mouse or a rat); therefore, the non-human β2 immunoglobulin locus is a rodent (e.g., a mouse or a rat) β2 immunoglobulin locus.

[0017] The present application also provides a mouse, the mouse comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide at an endogenous β2 microglobulin locus, wherein the mouse expresses the human or humanized β2 microglobulin polypeptide. In some embodiments, the mouse does not express a functional endogenous mouse β2 microglobulin from an endogenous β2 microglobulin locus. The nucleotide sequence may be connected to an endogenous mouse regulatory element. In one aspect, the nucleotide sequence comprises the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may comprise the nucleotide sequence shown in exons 2, 3, and 4 of the human β2 microglobulin gene. The nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may further comprise the nucleotide sequence of exon 1 of the mouse β2 microglobulin gene. In one embodiment, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may comprise a nucleotide sequence encoding a polypeptide containing a human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to an MHC I protein.

[0018] The present invention further comprises a non-human animal (e.g., a rodent, such as a mouse or a rat), the animal comprising in its genome a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide and a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one embodiment, the present invention provides a non-human animal, the animal comprising in its genome a first nucleotide sequence encoding a human / non-human MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide; and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous non-human MHC I locus, and the second nucleotide sequence is located at an endogenous non-human β2 microglobulin locus, and wherein the animal expresses a human / non-human chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide. In one aspect, the animal is a mouse. Thus, the endogenous MHC I locus can be selected from the group consisting of H-2K, H-2D, and H-2L loci. In one embodiment, the endogenous mouse locus is an H-2K locus (e.g., an H-2Kb locus). In one embodiment, the human MHC I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C polypeptides. In one aspect, the human MHC I polypeptide is HLA-A, such as HLA-A2 (e.g., HLA-A2.1). In various embodiments, the first and second nucleotide sequences are contained in germ cells of the non-human animal (e.g., a rodent, such as a mouse or rat).

[0019] Therefore, the present invention provides a mouse, the mouse comprising in its genome a first nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises an extracellular domain of human HLA-A (e.g., HLA-A2) and the mouse portion comprises a transmembrane and cytoplasmic domain of mouse H-2K; and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous H-2K locus and the second nucleotide sequence is located at an endogenous mouse β2 immunoglobulin locus, and wherein the mouse expresses a human / mouse chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide. In one embodiment, the non-human animal (e.g., the mouse) comprising a chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide does not express an extracellular domain of an endogenous non-human MHC I polypeptide (e.g., a mouse H-2K polypeptide) and / or a functional endogenous non-human (e.g., mouse) β2 microglobulin polypeptide at its respective endogenous locus. In one aspect, the animal (e.g., mouse) comprises two copies of the first and second nucleotide sequences, respectively. In another aspect, the animal (e.g., mouse) comprises a copy of the first and a copy of the second nucleotide sequence. Therefore, the animal can be homozygous or heterozygous for the first and second nucleotide sequences.

[0020] In one aspect, the first nucleotide sequence is operably linked to an endogenous non-human (e.g., mouse) MHC I regulatory element, and the second nucleotide sequence is operably linked to an endogenous non-human (e.g., mouse) β2 microglobulin element. The human portion of the chimeric polypeptide may include a human MHC I polypeptide. and Domain. The second nucleotide sequence may comprise the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise the nucleotide sequence shown in exons 2, 3 and 4 of the human β2 microglobulin gene. In one aspect, the mouse comprising a chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide may be such that the expression of the human or humanized β2 microglobulin increases the expression of the human / mouse chimeric MHC I polypeptide compared to the expression of the human / mouse chimeric MHC I polypeptide when the human or humanized β2 microglobulin polypeptide is not expressed.

[0021] The present invention also provides a method for preparing a genetically modified non-human animal (e.g., a rodent, such as a mouse or a rat) as described herein. Therefore, in one embodiment, the present invention provides a method for modifying the MHC I locus of a rodent (e.g., a mouse or a rat) to express a human / rodent chimeric (e.g., a human / mouse or a human / rat) MHC I polypeptide, wherein the method comprises replacing a nucleotide sequence encoding an extracellular domain of a rodent MHC I polypeptide with a nucleotide sequence encoding an extracellular domain of a human MHC I polypeptide at the endogenous MHC I locus. In another embodiment, the present invention provides a method for modifying a β2 microglobulin locus of a rodent (e.g., a mouse or a rat) to express a human or humanized β2 microglobulin polypeptide, wherein the method comprises replacing a nucleotide sequence encoding a rodent (e.g., a mouse or a rat) β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide at the endogenous rodent (e.g., a mouse or a rat) β2 microglobulin locus. In this method, the replacement can be performed in a single ES cell, and the single ES cell can be introduced into a rodent (e.g., a mouse or a rat) to prepare an embryo. The resulting rodent (e.g., a mouse or a rat) can be mated to produce a double humanized animal.

[0022] Therefore, the present invention also provides a method for preparing a double humanized animal, such as a rodent (eg, a mouse or a rat). In one embodiment, the present application also provides a method for preparing a genetically modified mouse, the method comprising (a) modifying the MHC I locus of a first mouse to express a human / mouse chimeric MHC I polypeptide, wherein a nucleotide sequence encoding the extracellular domain of a mouse MHC I polypeptide contained in an endogenous mouse MHC I locus is replaced by a nucleotide sequence encoding the extracellular domain of a human MHC I polypeptide, (b) modifying the β2 microglobulin locus of a second mouse to express a human or humanized β2 microglobulin polypeptide, wherein a nucleotide sequence encoding a mouse β2 microglobulin polypeptide contained in an endogenous mouse β2 microglobulin locus is replaced by a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide; and (c) mating the first and second mice to generate a modified mouse, wherein the mouse comprises in its genome a first nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the genetically modified mouse expresses a human / mouse chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide. In some embodiments, the MHC I locus is selected from H-2K, H-2D and H-2L; In some embodiments, the human MHC I polypeptide is selected from HLA-A, HLA-B and HLA-C. In one embodiment, the MHC I locus is the H-2K locus, the human MHC I polypeptide is HLA-A (e.g., HLA-A2), and the mouse expresses a chimeric HLA-A / H-2K polypeptide (e.g., HLA-A2 / H-2K polypeptide). In one aspect, the chimeric HLA-A2 / H-2K polypeptide comprises an extracellular domain of an HLA-A2 polypeptide and a cytoplasmic and transmembrane domain of an H-2K polypeptide. In one aspect, the second nucleotide sequence comprises the nucleotide sequence shown in exons 2, 3 and 4 (e.g., exons 2 to exon 4) of a human β2 microglobulin gene, and the nucleotide sequence shown in exon 1 of a mouse β2 microglobulin gene.

[0023] The present invention also provides a cell derived from a non-human animal (eg, a rodent, such as a mouse or a rat) as described herein, such as an isolated antigen presenting cell. The present invention also provides tissues and embryos derived from a non-human animal as described herein.

[0024] In yet another embodiment, the present invention provides methods for identifying antigens or antigenic epitopes that stimulate an immune response, methods for evaluating candidate vaccines, and methods for identifying T cells with high affinity for human pathogens or cancer antigens.

[0025] Any embodiments and aspects of the present invention may be used in combination with each other, unless otherwise specified or apparent from the context. Other embodiments will be apparent to those skilled in the art through the subsequent detailed description. The following detailed description includes exemplary descriptions of different embodiments of the present invention, which are not limiting to the claimed invention. The accompanying drawings constitute a part of this specification, which together with the specification are only used for the description of the embodiments, and are not used to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic representation of the four domains of the class I MHC molecule: the α-chain containing the α1, α2, and α3 domains and the non-covalently bound fourth domain, β2-microglobulin (β2m). The gray circles represent peptides bound in the peptide-binding cleft.

[0027] Figure 2 is a schematic diagram of the relative genomic structure of human HLA (not to scale), showing class I, II, and III genes.

[0028] Figure 3 is a schematic diagram (not to scale) of the relative genomic organization of the mouse MHC, showing class I, II, and III genes.

[0029] Figure 4 Viral vector constructs containing cDNA encoding chimeric HLA-A / H-2K polypeptides and an IRES-GFP reporter gene are shown (A); and a histogram (B) comparing human HLA-A2 expression in MG87 cells transduced with HLA-A2 alone (dashed line), HLA-A2 / H-2K (dotted line), or not transduced (solid line), or co-transduced with humanized β2 microglobulin (right). The data corresponding to (B) are shown in Figure (C) as the percentage of cells expressing the construct.

[0030] Figure 5 Schematic representation of the mapping strategy used to generate a chimeric H-2K locus expressing the extracellular region of the human HLA-A2 protein (not to scale). Mouse sequences are shown in black and human sequences are shown in white. L = leader sequence, UTR = untranslated region, TM = transmembrane domain, CYT = cytoplasmic domain, HYG = hygromycin.

[0031] Fig. 6A Shown is the expression of HLA-A2 (left panel) and H-2K (right panel) (% of total cells) in cells isolated from wild-type (WT) mice or heterozygous mice carrying a chimeric HLA-A2 / H-2K locus (HLA-A / H-2K HET).

[0032] Figure 6BDot plots are shown for in vivo expression of chimeric HLA-A2 / H-2K proteins in heterozygous mice carrying the chimeric HLA-A2 / H-2K loci.

[0033] Figure 7 The directed strategy for humanizing the β2 microglobulin gene at the mouse β2 microglobulin locus is shown (not to scale). Mouse sequences are shown in black and human sequences are shown in white. NEO = neomycin.

[0034] Figure 8 Representative dot plots of the expression of HLA class I and human β2 microglobulin in cells isolated from the blood of wild-type (WT) mice, chimeric HLA-A2 / H-2K heterozygous mice, chimeric HLA-A2 / H-2K heterozygous and humanized β2 microglobulin heterozygous mice (double heterozygous; class I / β2m HET) are shown.

[0035] Fig. 9 Shown are representative histograms of human HLA class I expression (X-axis) in cells isolated from the blood of wild-type (WT), chimeric HLA-A2 / H-2K heterozygous (class I HET), and chimeric HLA-A2 / H2K / humanized β2 microglobulin double heterozygous (class I / β2m HET) mice.

[0036] Fig.10 Results of IFNγ Elispot assays of human T cells exposed to antigen presenting cells (APCs) derived from wild-type mice (WT APCs) or from mice heterozygous for chimeric HLA-A2 / H-2K and humanized β2 microglobulin (double HET APCs) in the presence of influenza (left panel) or EBV (right panel) peptides are shown. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc multiple comparison test. DETAILED DESCRIPTION OF THE INVENTION

[0037] definition

[0038] The present invention provides genetically modified non-human animals (e.g., mice, rats, rabbits, etc.) expressing human or humanized MHC I and / or β2 microglobulin; and embryos, cells and tissues comprising the genetic modification; methods for preparing the genetically modified animals and using the same. Unless otherwise defined, all terms and phrases used in this application include the meanings that the terms and phrases have in the art, unless there is a clear contrary description or it is obvious in the context of the use of the terms or phrases.

[0039] When used to describe conservative amino acid substitutions, the term "conservative" includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying the nucleotide sequence to introduce changes in the nucleotides encoding the conservative substitutions. In general, conservative amino acid substitutions will not substantially alter the functional properties of the target protein, such as the ability of MHC I to present the target peptide. Examples of amino acid groups having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxy side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid and asparagine / glutamine. In some embodiments, conservative amino acid substitutions can be any native residue in the protein replaced by alanine, for example, alanine scanning mutagenesis. In some embodiments, the conservative substitutions adopted have positive values ​​in the PAM250 logarithmic likelihood matrix, as disclosed in Gonnet et al., (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45, which is incorporated herein by reference. In some embodiments, the substitutions are appropriate conservative substitutions, wherein the substitutions have non-negative values ​​in the PAM250 logarithmic likelihood matrix.

[0040] Therefore, the present invention also includes a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding a human or humanized MHC I polypeptide and / or β2 microglobulin polypeptide, wherein the polypeptide comprises a conservative amino acid substitution of the amino acid sequence described in the present application.

[0041] Those skilled in the art will appreciate that, in addition to the nucleic acid residues encoding human or humanized MHC I polypeptides and / or β2 microglobulin described herein, other nucleic acids may also encode the polypeptides of the present invention due to the degeneracy of the genetic code. Therefore, in addition to genetically modified non-human animals containing nucleotide sequences encoding MHC I and / or β2 microglobulin polypeptides having conservative amino acid substitutions in their genomes, the present application also provides non-human animals containing nucleic acid sequences different from those described herein in their genomes due to the degeneracy of the genetic code.

[0042] When used with sequences, the term "identity" includes identity determined by a variety of different algorithms known in the art that can be used to determine nucleotide and / or amino acid sequence identity. In some embodiments described herein, the ClustalW v.1.83 (slow) algorithm with an open gap penalty of 10.0 and an extension gap penalty of 0.1 and a Gonnet similarity matrix (MacVector TM 10.0.2, MacVector Inc., 2008) to determine the identity. The length of the sequence used for sequence identity comparison depends on the specific sequence. In various embodiments, the identity is determined by comparing the sequence from the N-terminus to the C-terminus of the mature protein. In various embodiments, when a human / non-human chimeric sequence is compared with a human sequence, the human portion of the human / non-human chimeric sequence (rather than the non-human portion) is used for comparison to determine the level of identity between the human sequence and the human portion of the human / non-human chimeric sequence (e.g., comparing the human extracellular domain of a human / mouse chimeric protein with the human extracellular domain of a human protein).

[0043] The term "homology" or "homologous" with respect to sequences such as nucleotide or amino acid sequences refers to two sequences that are optimally aligned and compared with at least about 75% of the nucleotides or amino acids, at least about 80% of the nucleotides or amino acids, at least about 90-95% of the nucleotides or amino acids, such as more than 97% of the nucleotides or amino acids being identical. It will be appreciated by those skilled in the art that for optimal gene targeting, the targeting construct should contain arms that are homologous to the endogenous DNA sequence (i.e., "homology arms"); therefore, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

[0044] The term "operably connected" refers to juxtaposition, wherein the component described in this way is in a relationship that allows it to function in a predictable manner. Therefore, the nucleic acid sequence of the encoded protein can be operably connected to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc.) to keep suitable transcriptional regulation. In addition, the multiple parts of the chimeric or humanized protein of the present invention can be operably connected to keep its folding, processing, targeting, expression, and protein function in other cells in the cell. Unless otherwise expressly stated, the various domains of the chimeric or humanized protein of the present invention are operably connected to each other.

[0045] As used herein, the term "MHC I complex" or similar expressions include a complex between an MHC I α chain polypeptide and a β2-microglobulin polypeptide. As used herein, the term "MHC I polypeptide" or similar expressions include a single MHC I α chain polypeptide. Generally, the terms "human MHC" and "HLA" can be used interchangeably.

[0046] The term "replacement" in gene replacement refers to the addition of exogenous genetic material to an endogenous locus, thereby replacing all or part of an endogenous gene with an orthologous or homologous nucleic acid sequence. As shown in the following examples, nucleic acid sequences of endogenous loci encoding portions of mouse MHC I and β2 microglobulin polypeptides were replaced by nucleic acid sequences encoding portions of human MHC I and β2 microglobulin polypeptides, respectively.

[0047] As used herein, "functional", with respect to a functional polypeptide, refers to a polypeptide that retains at least one biological activity that is typically associated with a native protein. For example, in some embodiments of the invention, a substitution at an endogenous locus (e.g., a substitution at an endogenous non-human MHC I and / or β2 microglobulin locus) renders the locus unable to express a functional endogenous polypeptide.

[0048] Several aspects of the genetically modified MHC I non-human animals described below in this application, such as animal types, animal strains, cell types, screening, detection and other methods, methods of use, etc., will be applicable to animals genetically modified with β2 microglobulin and MHC I / β2 microglobulin.

[0049] Genetically modified MHC I animals

[0050] In various embodiments, the present invention generally provides a genetically modified non-human animal comprising in its genome a nucleotide sequence encoding a human or humanized MHC I polypeptide; thus, the animal expresses a human or humanized MHC I polypeptide.

[0051] MHC genes are divided into three classes: class I, class II, and class III, all of which are encoded on chromosome 6 in humans or chromosome 17 in mice. Schematic diagrams of the relative structures of the MHC types of humans and mice are shown in Figure 2 and Figure 3 The MHC genes are the most polymorphic genes in the mouse and human genomes. MHC polymorphism is presumed to be important in providing evolutionary advantage; changes in sequence can lead to differences in peptide binding for better presentation of pathogens to cytotoxic T cells.

[0052] MHC class I proteins contain an extracellular domain (which contains three domains: α 1 , α 2 and α 3 ), transmembrane domain and cytoplasmic tail. α 1 and α 2 domains form the peptide-binding cleft, while the α 3 Interacts with β2-microglobulin.

[0053] In addition to the interaction with β2-microglobulin, α 3The domain also interacts with the TCR co-receptor CD8 to promote antigen-specific activation. Although the binding of MHC class I to CD8 is about 100 times weaker than the binding of TCR to MHC class I, CD8 binding enhances the affinity of TCR binding. Wooldridge et al., (2010) MHC Class IMolecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184: 3357-3366. Interestingly, enhancing the binding of MHC class I to CD8 eliminates the antigen specificity in CTL activation. Same as above.

[0054] CD8 binds to MHC class I molecules in a species-specific manner; Lyt-2, a mouse homolog of CD8, has been found to bind to H-2D in the α3 domain. dMolecules bind to CD8, but not to HLA-A molecules. Connolly et al., (1988) The Lyt-2 Molecule Recognizes Residues in the Class I α3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168: 325-341. This differential binding may be caused by the fact that the CDR-like determinants (CDR1- and CDR2-like) on CD8 are not conserved between humans and mice. Sanders et al., (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174: 371-379; Vitiello et al., (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice Carrying a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. 173: 1007-1015; and Gao et al., (1997) Crystal structure of the complex between human CD8ααand HLA-A2, Nature 387: 630-634. CD8 has been reported to bind to HLA-A2 in a conserved region of the α3 domain (at positions 223-229). A single substitution in HLA-A (V245A) results in decreased binding of CD8 to HLA-A, accompanied by a significant reduction in T cell-mediated lysis. Salter et al., (1989), Polymorphism in the α 3 domainof HLA-A molecules affects binding to CD8,Nature 338:345-348. Under normal circumstances, polymorphisms in the α3 domain of HLA-A molecules also affect their binding to CD8. Same as above. In mice, in H-2D d Amino acid substitution at residue 227 affects the relationship between mouse Lyt-2 and H-2D d The combination of mutated H-2D dTransfected cells are not lysed by CD8+ T cells. Potter et al., (1989) Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73-75.

[0055] Thus, because the interaction between the MHC class I α3 domain and CD8 is species specific, an MHC I complex containing the H-2K α3 domain replaced by the human HLA-A2 α3 domain is nonfunctional in mice lacking human CD8 (i.e., in vivo). In HLA-A2 transgenic animals, the use of the human α3 domain to replace the mouse α3 domain restored T cell responses. Irwin et al., (1989) Species-restricted interactions between CD8 and the α3 domain of class I influence the magnitude of the xenogeneic response, J. Exp. Med. 170: 1091-1101; Vitiello et al., (1991), supra.

[0056] The transmembrane domain and cytoplasmic tail of mouse MHC class I proteins also have important functions. One function of the MHC I transmembrane domain is to promote the regulation of HLA-A2 homotypic cell adhesion (enhance or inhibit adhesion), which may be the result of cross-linking (or connection) of surface MHC molecules. Wagner et al., (1994) Ligation of MHC Class I and Class II Molecules Can Lead to Heterologous Desensitization of Signal Transduction Pathways That Regulate Homotypic Adhesion in Human Lymphocytes, J. Immunol. 152: 5275-5287. Cell adhesion can be affected by mAbs that bind to different epitopes of the HLA-A2 molecule, indicating that multiple sites on HLA-A2 are involved in regulating homotypic cell adhesion; depending on the different epitopes bound, this effect may enhance or inhibit HLA-A2-dependent adhesion. Same as above.

[0057] The cytoplasmic tail encoded by exons 6 and 7 of the MHC I gene is reported to be necessary for proper expression on the cell surface and inhibition of LIR1-mediated NK cell toxicity. Gruda et al., (2007) Intracellular Cysteine ​​Residues in the Tail of MHC Class I Proteins Are Crucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1, J. Immunol. 179: 3655-3661. The cytoplasmic tail is necessary for the multimerization of MHC I molecules at least by forming disulfide bonds on its cysteine ​​residues, so it may play an important role in clustering and being recognized by NK cells. Lynch et al., (2009) Novel MHC Class I Structures on Exosomes, J. Immunol. 183: 1884-1891.

[0058] Although the cytoplasmic domain of HLA-A2 contains a constitutively phosphorylated serine residue and a phosphorylated tyrosine, mutant HLA-A2 molecules lacking the cytoplasmic domain in Jurkat cells show normal expression, cytoskeleton binding, aggregation, and endocytosis. Gur et al., (1997) Structural Analysis of Class I MHC Molecules: The Cytoplasmic Domain Is Not Required for Cytoskeletal Association, Aggregation, and Internalization, Mol. Immunol. 34 (2): 125-132. Truncated HLA-A2 molecules lacking the cytoplasmic domain show normal expression and binding to β2 microglobulin, as above.

[0059] However, some studies have suggested that the cytoplasmic tail plays an important role in intracellular trafficking, dendritic cell (DC)-mediated antigen presentation, and CTL priming. It has been demonstrated that the tyrosine residue encoded by exon 6 is required for MHC I trafficking through endosomal compartments, presentation of exogenous antigens, and CTL priming; whereas the deletion of exon 7 leads to an enhancement of antiviral CTL responses. Lizee et al., (2003) Control of Dendritic Cross-Presentation by the MajorHistocompatibility Complex Class I Cytoplasmic Domain, Nature Immunol.4:1065-73; Basha et al., (2008) MHC Class I Endosomal and Lysosomal Trafficking Coincides with Exogenous Antigen Loading in Dendritic Cells, PLoS ONE 3:e3247; and Rodriguez-Cruz et al., (2011) Natural Splice Variant of MHC Class I Cytoplasmic TailEnhances Dendritic Cell-Induced CD8+T-Cell Responses and Boosts Anti-TumorImmunity, PLoS ONE 6:e22939.

[0060] In various embodiments, the present invention provides a genetically modified non-human animal (e.g., mouse, rat, rabbit, etc.), comprising in the genome of the animal a nucleotide sequence encoding a human or humanized MHC class I polypeptide. The non-human animal may comprise in its genome a nucleotide sequence encoding an MHC I polypeptide, wherein the MHC I polypeptide is partially human and partially non-human, such as a non-human animal expressing a human / non-human chimeric MHC I polypeptide. In one aspect, the non-human animal expresses only a human or humanized MHC I polypeptide, such as a human / non-human chimeric MHC I polypeptide, and does not express endogenous non-human MHC I proteins from an endogenous MHC I locus.

[0061] In one embodiment, the human / non-human chimeric MHC I polypeptide comprises a peptide binding domain of a human MHC I polypeptide in its human portion. In one aspect, the human portion of the chimeric polypeptide comprises the extracellular domain of human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises the extracellular domain of the α chain of human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises the α1 and α2 domains of human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises the α1, α2, and α3 domains of human MHC I.

[0062] The human or humanized MHC I polypeptide can be derived from a functional human HLA molecule encoded by any HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G locus. A list of commonly used HLA antigens can be found in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4 (2): 91-103), which is incorporated herein by reference. Shankarkumar et al. also provide a brief description of the HLA nomenclature used in the art. For further information on HLA nomenclature and different HLA alleles, see 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 the latest Marsh et al. (2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens 75: 291-455, all of which are incorporated herein by reference. Therefore, the human or humanized MHC I polypeptide can be derived from any functional human HLA class I molecule described in the present application.

[0063] In a specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., an HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele (e.g., an HLA-A*02:01:01:01 allele). The HLA-A*0201 allele is commonly used in the North American population. Although this particular HLA sequence is described in this example, any suitable HLA-A sequence is included in the present application, such as polymorphic variants of HLA-A2 in the population, sequences having one or more conservative or non-conservative amino acid modifications, nucleic acid sequences that are different from the sequences described in the present application due to the degeneracy of the genetic code, and the like.

[0064] In one aspect, the present application provides a non-human animal that expresses a human HLA-A2 sequence, wherein the human HLA-A2 sequence comprises one or more conservative or non-conservative modifications.

[0065] In one aspect, the present application provides a non-human animal expressing a human HLA-A2 sequence, wherein the human HLA-A2 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a human HLA-A2 sequence. In a specific embodiment, the human HLA-A2 sequence is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the human HLA-A2 sequence described in the Examples. In one embodiment, the human HLA-A2 sequence comprises one or more conservative substitutions. In one embodiment, the human HLA-A2 sequence comprises one or more non-conservative substitutions.

[0066] In another specific aspect, the human or humanized MHC I polypeptide is derived from a human MHC I selected from HLA-B and HLA-C. In one aspect, the human or humanized MHC I is derived from HLA-B, such as HLA-B27.

[0067] In one aspect, the non-human portion of the human / non-human chimeric MHC I polypeptide comprises a transmembrane and / or cytoplasmic domain of a non-human MHC I polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human MHC I polypeptide is selected from H-2K, H-2D, and H-2L. In one embodiment, the non-human MHC I polypeptide is H-2K, such as H-2Kb. Although specific H-2K sequences are described in the examples, any suitable H-2K sequence, such as polymorphic variants, conservative / non-conservative amino acid substitutions, etc., are included in the present application.

[0068] The non-human animal described in the present application may include a nucleotide sequence encoding a human or humanized MHC I polypeptide in its genome, such as a human / or non-human chimeric MHC I polypeptide, wherein the nucleotide sequence encoding such a polypeptide is located at an endogenous non-human MHC I locus (e.g., an H-2K locus). In one aspect, it is the result of an endogenous MHC I gene or a portion thereof being replaced by a nucleotide sequence encoding a human or humanized MHC I polypeptide, such as a chimeric gene encoding a human / non-human chimeric MHC I polypeptide described in the present application. In one embodiment, the replacement comprises an endogenous nucleotide sequence encoding a non-human MHC I polypeptide binding domain or a non-human MHC I extracellular domain being replaced by a human nucleotide sequence encoding it (e.g., an HLA-A2 nucleotide sequence). In this embodiment, the replacement does not include a replacement of an MHC I sequence encoding a transmembrane and / or cytoplasmic domain of a non-human MHC I polypeptide (e.g., an H-2K polypeptide). Thus, the non-human animal comprises a human / non-human chimeric sequence at an endogenous non-human MHC I locus and expresses a human / non-human chimeric MHC polypeptide from the endogenous non-human MHC I locus.

[0069] The human / non-human chimeric polypeptide may comprise a human or non-human leader (signal) sequence. In one embodiment, the chimeric polypeptide comprises a leader sequence of a non-human endogenous MHC I protein. In another embodiment, the chimeric polypeptide comprises a leader sequence of a human MHC I protein, such as an HLA-A2 protein (e.g., an HLA-A2.1 leader sequence). Thus, the nucleotide sequence encoding the chimeric MHC I polypeptide may be operably linked to a nucleotide sequence encoding a human MHC I leader sequence.

[0070] The human / or non-human chimeric MHC I polypeptide may comprise a complete or substantially complete extracellular domain of a human MHC I polypeptide in its human portion. Thus, the human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, for example 95% or more of the amino acids encoding the extracellular domain of a human MHC I polypeptide (e.g., an HLA-A2 polypeptide). In one example, the extracellular domain of a substantially complete human MHC I polypeptide lacks a human MHC I leader sequence. In another example, the human / non-human chimeric MHC I polypeptide comprises a human MHC I leader sequence.

[0071] Furthermore, the chimeric MHC I polypeptide can be expressed under the control of endogenous non-human regulatory elements, such as rodent MHC I regulatory elements. Such rearrangement will facilitate the appropriate expression of the chimeric MHC I polypeptide in a non-human animal, such as in an immune response of a non-human animal.

[0072] Genetically modified non-human animals can be selected from the following groups: mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques). For non-human animals that are not easy to obtain ES cells that are suitable for genetic modification, other methods can be used to prepare non-human animals comprising genetic modification. This method includes, for example, modifying non-ES cell genomes (e.g., fibroblasts or induced pluripotent stem cells) and introducing nuclear transfer so that the modified genome is transferred to suitable cells such as oocytes, and making the modified cells (e.g., modified oocytes) pregnant in non-human animals under suitable conditions to form embryos.

[0073] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as the superfamily of the family ... In a specific embodiment, the rodent is selected from the group consisting of a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0074] In a specific embodiment, the non-human animal is a rodent which is a mouse of the C57BL strain selected from the group consisting of C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. In another embodiment, the mouse is a 129 strain selected from the following group: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, also see Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mixed strain of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another specific embodiment, the mouse is a mixed strain of the aforementioned 129 strain or a mixed strain of the aforementioned BL / 6 strain. In a specific embodiment, the mixed strain of the 129 strain is the 129S6 (129 / SvEvTac) strain. In another embodiment, the mouse is a BALB strain, such as a BALB / c strain. In yet another embodiment, the mouse is a mixed strain of the BALB strain and another aforementioned strain.

[0075] In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mixed strain of two or more strains selected from the following groups: Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0076] Therefore, in one embodiment, the present invention relates to a genetically modified mouse, the mouse comprising a nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide in its genome, wherein the human portion of the chimeric polypeptide comprises a peptide binding domain or an extracellular domain of a human MHC I (e.g., human HLA-A, such as human HLA-A2, such as human HLA-A2.1). In some embodiments, the mouse does not express peptide binding or extracellular domains of endogenous mouse polypeptides from its endogenous mouse locus. The peptide binding domain of the human MHC I may comprise α1 and α2 domains. Alternatively, the peptide binding domain of the human MHC I may comprise α1, α2, and α3 domains. In one aspect, the extracellular domain of the human MHC I comprises the extracellular domain of the human MHC I α chain. In one embodiment, the endogenous mouse locus is an H-2K (e.g., H-2Kb) locus, and the mouse portion of the chimeric polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide.

[0077] Therefore, in one embodiment, a genetically modified mouse is provided, wherein the mouse comprises a nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide at an endogenous H-2K (e.g., H-2Kb) locus, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide and the mouse portion comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. In one aspect, the mouse does not express the extracellular domain of a mouse H-2K (e.g., H-2Kb) polypeptide from an endogenous MHC I locus. In one embodiment, the mouse expresses a chimeric HLA-A2 / H-2K (e.g., chimeric HLA-A2.1 / H-2Kb) polypeptide from an endogenous H-2K (e.g., H-2Kb) locus. In various embodiments, expression of the chimeric gene is performed under the regulation of an endogenous mouse MHC class I regulatory element. In some aspects, the mouse comprises two copies of a chimeric MHC I locus comprising a nucleotide sequence encoding a chimeric HLA-A2 / H-2K polypeptide, while in other aspects, the mouse comprises one copy of a chimeric MHC I locus comprising a nucleotide sequence encoding a chimeric HLA-A2 / H-2K polypeptide. Thus, the mouse can be homozygous or heterozygous for a nucleotide sequence encoding a chimeric HLA-A2 / H-2K polypeptide.

[0078] In some embodiments described herein, a mouse is provided, the mouse comprising a chimeric MHC I locus in an endogenous mouse H-2K locus. The chimeric locus comprises a nucleotide sequence encoding an extracellular domain of a human HLA-A2 protein, such as an α1, α2, and α3 domain of a human HLA-A2 gene. The chimeric locus lacks a nucleotide sequence encoding an extracellular domain of a mouse H-2K protein (e.g., an α1, α2, and α3 domain of mouse H-2K). In one aspect, the chimeric locus lacks a nucleotide sequence encoding a leader peptide, α1, α2, and α3 domain of mouse H-2K; and comprises a leader peptide, α1, α2, and α3 domain of human HLA-A2, and a transmembrane and cytoplasmic domain of mouse H-2K. The different domains of the chimeric locus are operably connected to each other so that the chimeric locus expresses a functional human / mouse chimeric MHC I protein.

[0079] In various embodiments, a non-human animal, such as a rodent (e.g., a mouse or rat), expressing a functional chimeric MHC I protein from a chimeric MHC I locus as described herein, displays the chimeric protein on the cell surface. In one embodiment, the distribution of the chimeric MHC I protein expressed on the cell surface by the non-human animal in the cell is the same as that observed in humans. In one aspect, the peptide fragments (antigenic fragments) displayed by the cell bind to the extracellular portion of the chimeric MHC I protein (e.g., the extracellular portion of human HLA-A2). In one embodiment, the extracellular portion of this chimeric protein interacts with other proteins on the cell surface, such as β2-microglobulin.

[0080] In various embodiments, the cell displaying the chimeric MHC I protein, such as the HLA-A2 / H-2K protein, is a nucleated cell. In various aspects, the cell is an antigen presenting cell (APC). However, most cells in the body can present antigens in the presence of MHC I, and some non-limiting examples of antigen presenting cells include macrophages, dendritic cells, and B cells. Other antigen presenting cells, including professional and non-professional APCs, are well known in the art and are all included in the present application. In some embodiments, the cell displaying the chimeric MHC I protein is a tumor cell, and the peptide fragments presented by the chimeric protein are from a tumor. In other embodiments, the peptide fragments presented by the chimeric MHC I protein are from a pathogen, such as a bacterium or a virus.

[0081] The chimeric MHC I protein described herein can interact with other proteins on the surface of the same cell or another cell. In some embodiments, the chimeric MHC I protein interacts with endogenous non-human proteins on the surface of the cell. The chimeric MHC I protein can also interact with human or humanized proteins on the surface of the same cell or another cell.

[0082] On the same cell, HLA class I molecules can functionally interact with non-human (e.g., rodent, such as mouse or rat) and human β2-microglobulin. Therefore, in one embodiment, the chimeric MHC I protein, such as HLA-A2 / H-2K protein, interacts with mouse β2-microglobulin. Although some interactions between human HLA class I molecules and mouse β2-microglobulin are possible, they are greatly reduced compared to the interactions between human HLA class I and human β2-microglobulin. Therefore, in the absence of human β2-microglobulin, the expression of human MHC I on the cell surface may be reduced. Perarnau et al., (1988) Human β2-microglobulin Specifically Enhances Cell-Surface Expression of HLA Class I Molecules in Transfected Murine Cells, J.Immunol.141:1383-89. Other HLA molecules such as HLA-B27 do not interact with mouse β2-microglobulin; see, for example, Tishon et al., (2000) Transgenic Mice Expressing Human HLA and CD8 Molecules Generate HLA-Restricted Measles Virus Cytotoxic T Lymphocytes of the Same Specificity as Humans with Natural Measles Virus Infection, Virology 275:286-293, which reported that the function of HLA-B27 in transgenic mice requires human β2-microglobulin and human CD8. Therefore, in another embodiment, the chimeric MHC I protein interacts with human or humanized β2-microglobulin. In some such embodiments, as described below in this application, the non-human animal, such as a rodent (e.g., a mouse or rat), comprises a human or humanized β2-microglobulin gene in its genome, and the animal expresses a functional human or humanized β2-microglobulin polypeptide; thus, the chimeric MHC I protein interacts with the human or humanized β2-microglobulin polypeptide.

[0083] In various aspects, the chimeric protein (e.g., HLA-A2 / H-2K protein) also interacts with a protein on the surface of another cell (through its extracellular portion). The other cell can be a non-human, such as a mouse or human cell. The other cell can be derived from the same non-human animal or the same non-human animal species as the cell expressing the chimeric MHC I polypeptide. Non-limiting examples of proteins that interact with the extracellular portion of the chimeric protein (e.g., HLA-A2 / H-2K) include T cell receptors (TCR) and their co-receptors CD8. Thus, the other cell can be a T cell. In addition, the extracellular portion of the chimeric MHC I protein can bind to a protein on the surface of a natural killer (NK) cell, such as a killer immunoglobulin receptor (KIR) on the surface of a NK cell.

[0084] T cells or NK cells can be combined with the complex formed between the chimeric MHC I polypeptide and the peptide fragment displayed thereon. This combination can lead to the inhibition of activation of T cells or NK-mediated cell killing, respectively. A hypothesis is that NK cells have evolved to kill infected or tumor cells that have escaped T cell-mediated cytotoxicity by lowering their MHC I complexes. However, when the MHC I complex is expressed on the cell surface, it can be recognized by the NK cell receptor and inhibit NK-mediated cell killing. Therefore, in some aspects, when NK cells are combined with the complex formed between the chimeric MHC I polypeptide (e.g., HLA-A2 / H-2K polypeptide) and the peptide fragment displayed on the infected or tumor cell surface, NK-mediated cell killing is inhibited.

[0085] In one example, the chimeric MHC I polypeptide described herein, such as a chimeric HLA-A2 / H-2K polypeptide, interacts with a CD8 protein on the surface of another cell. In one embodiment, the chimeric HLA-A2 / H-2K polypeptide interacts with an endogenous rodent (e.g., mouse or rat) CD8 protein on the surface of another cell. In one embodiment, the other cell is a T cell. In another embodiment, the other cell is modified to express CD8. In some aspects, the chimeric HLA-A2 / H-2K polypeptide interacts with human CD8 on the surface of another cell (e.g., a human cell or a rodent cell). In some such embodiments, the non-human animal, such as a mouse or a rat, comprises a human CD8 transgene, and the mouse or rat expresses a functional human CD8 protein.

[0086] The chimeric MHC I polypeptides described herein may also interact with a non-human (e.g., mouse or rat) TCR, a human TCR, or a humanized TCR on another cell. The chimeric MHC I polypeptides may interact with an endogenous TCR (e.g., a mouse or rat TCR) on the surface of another cell. The chimeric MHC I polypeptides may also interact with a human or humanized TCR expressed on the surface of another cell, wherein the cell is derived from the same animal or animal species (e.g., mouse or rat) as the cell expressing the chimeric MHC I polypeptide. The chimeric MHC I polypeptides may interact with a human TCR expressed on the surface of a human cell.

[0087] In addition to genetically modified non-human animals, the application also provides non-human embryos (e.g., rodent embryos, such as mouse or rat embryos), wherein the embryo comprises a donor ES cell derived from a non-human animal (e.g., rodent, such as mouse or rat) as described herein. In one aspect, the embryo comprises an ES donor cell and a host embryonic cell comprising the chimeric MHC I gene.

[0088] The present application also provides a tissue, wherein the tissue is derived from a non-human animal (eg, mouse or rat) as described in the present application, and expresses the chimeric MHC I polypeptide (eg, HLA-A2 / H-2K polypeptide).

[0089] In addition, the application provides non-human cells separated from non-human animals as described in the application. In one embodiment, the cell is an ES cell. In one embodiment, the cell is an antigen presenting cell, such as a dendritic cell, a macrophage, a B cell. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is a lymphocyte.

[0090] The present application also provides a non-human cell, the cell comprising a chromosome or a fragment thereof of a non-human animal as described in the present application. In one embodiment, the non-human cell comprises a cell nucleus of a non-human animal as described in the present application. In one embodiment, the non-human cell comprises a chromosome or a fragment thereof obtained by nuclear transfer.

[0091] In one aspect, the present application provides a non-human induced pluripotent stem cell, the cell comprising a gene encoding a chimeric MHC I polypeptide (e.g., HLA-A2 / H-2K polypeptide) as described herein. In one embodiment, the induced pluripotent stem cell is derived from a non-human animal as described herein.

[0092] In one aspect, the present application provides a hybridoma or quadrivalent tumor, which is derived from a non-human animal cell as described in the present application. In one embodiment, the non-human animal is a mouse or a rat.

[0093] The present application also provides a method for preparing a genetically modified non-human animal (e.g., a genetically modified rodent, such as a mouse or rat) as described herein. The method for preparing a genetically modified non-human animal is such that a nucleotide sequence encoding a chimeric MHC I polypeptide is included in the genome of the animal. In one embodiment, the genetically modified mouse generated by the method has a genome comprising a nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide at an endogenous MHC I locus, such as an H-2K locus, wherein the human portion of the chimeric MHC I polypeptide comprises a human HLA-A2 extracellular domain and the mouse portion comprises a transmembrane and cytoplasmic domain of mouse H-2K. In some embodiments, as described in the Examples, the method utilizes the use of The targeting constructs were prepared using the technique, introduced into ES cells and used The target ES cell clone is introduced into a mouse embryo by the technique. In one embodiment, the ES cell is a mixture of 129 and C57BL / 6 mouse strains; in another embodiment, the ES cell is a mixture of BALB / c and 129 mouse strains.

[0094] Therefore, the present application also provides a nucleotide construct for producing the genetically modified non-human animals described in the present application. In one aspect, the nucleotide construct comprises: 5' and 3' non-human homology arms, a human DNA fragment comprising a human HLA-A gene sequence, and recombination sites on both sides of the screening gene expression cassette. In one embodiment, the human DNA fragment is a genomic fragment comprising introns and exons of the human HLA-A gene. In one embodiment, the non-human homology arms have homology to a non-human MHC class I locus (e.g., a mouse H-2K locus).

[0095] In one embodiment, the genomic fragment comprises the coding sequence of the human HLA-A leader, α1 domain, α2 domain and α3 domain. In one embodiment, the human DNA fragment comprises, from 5' to 3': the HLA-A leader sequence, the HLA-A leader / α1 intron, the HLA-A α1 exon, the HLA-A α1-α2 intron, the HLA-A α2 exon, the HLA-A α2-α3 intron and the HLA-A α3 exon.

[0096] The screening gene expression cassette is a nucleotide sequence that inserts the targeting construct, which helps to screen the cells (e.g., ES cells) that have been inserted into the target construct. Some suitable screening gene expression cassettes are well known in the art. Usually, the screening gene expression cassette can obtain positive screening under the condition of having specific antibiotics (e.g., Neo, Hyg, Pur, CM, Spec, etc.). In addition, the screening gene expression cassette may have recombination sites on both sides, which makes it possible to remove the screening gene expression cassette after using recombinase treatment. Commonly used recombination sites are loxP and Frt, which are respectively recognized by Cre and Flp enzymes, but other are also well known in the art.

[0097] In one embodiment, the screening gene expression cassette is located at the 5' end of the human DNA fragment. In another embodiment, the screening gene expression cassette is located at the 3' end of the human DNA fragment. In another embodiment, the screening gene expression cassette is located in the human DNA fragment. In another embodiment, the screening gene expression cassette is located in the intron of the human DNA fragment. In another embodiment, the screening gene expression cassette is located in the α2-α3 intron.

[0098] In one embodiment, the 5' and 3' non-human homology arms comprise genomic sequences located at the 5' and 3' positions of an endogenous non-human (e.g., mouse) MHC class I locus, respectively (e.g., the 5' of the first leader sequence of a non-human MHC I gene and the 3' end of the α3 exon). In one embodiment, the endogenous MHC class I locus is selected from mouse H-2K, H-2D, and H-2L. In a specific embodiment, the endogenous MHC class I locus is mouse H-2K.

[0099] In one aspect, the present application provides a nucleotide construct, which comprises, from 5' to 3', a 5' homology arm at the 5' end of a mouse genomic sequence containing an endogenous mouse H-2K locus, a first human DNA fragment containing a first genomic sequence of an HLA-A gene, a 5' recombination sequence site (e.g., loxP), a selection gene expression cassette, a 3' recombination sequence site (e.g., loxP), a second human DNA fragment containing a second genomic sequence of an HLA-A gene, and a 3' homology arm at the 3' end of a mouse genomic sequence containing an endogenous H-2Kα3 exon. In one embodiment, the nucleotide construct comprises, from 5' to 3', a 5' homology arm at the 5' end of a mouse genomic sequence containing an endogenous mouse H-2K locus, a human genomic sequence including an HLA-A leader, an HLA-A leader / α1 intron sequence, an HLA-A α1 exon, an HLA-A α1-α2 intron, an HLA-A α2 exon, a first 5' portion of an α2-α3 intron, a selection gene expression cassette with recombination sites on both sides, a second 3' portion of an α2-α3 intron, an HLA-A α3 exon, and a 3' homology arm at the 3' end of a non-mouse genomic sequence containing an endogenous mouse H-2K α3 exon. In one embodiment, the 5' homology arm sequence is as shown in SEQ ID NO: 1 and the 3' homology arm sequence is as shown in SEQ ID NO: 2.

[0100] After gene targeting is completed, ES cells or genetically modified non-human animals are screened to confirm successful insertion of the desired exogenous nucleotide sequence or expression of the exogenous polypeptide. A variety of techniques are well known to those skilled in the art, including (but not limited to) Southern blotting, long PCR, quantitative PCR (e.g., using Real-time PCR), fluorescent in situ hybridization, Northern blot, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (e.g., mice) carrying the target gene modification can be identified by screening for the loss of mouse alleles and / or the gain of human alleles, using the revised allele detection described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6): 652-659. Other tests for identifying specific nucleotide or amino acid sequences in genetically modified animals are well known to those skilled in the art.

[0101] The present disclosure also provides a method for modifying the MHC I locus of a non-human animal to express a human / non-human chimeric MHC I polypeptide described in the present application. In one embodiment, the present application provides a method for modifying the MHC I locus of a mouse to express a human / mouse chimeric MHC I polypeptide, wherein the method includes replacing a nucleotide sequence encoding a mouse MHC polypeptide peptide binding domain at an endogenous MHC I locus with a nucleotide sequence encoding a human MHC I polypeptide peptide binding domain. In some aspects, the nucleotide sequence of the mouse MHC I extracellular domain is replaced by a nucleotide sequence of a human MHC I extracellular domain. The mouse may be unable to express a peptide binding or extracellular domain of the mouse MHC I from an endogenous MHC I locus. In some embodiments, the nucleotide sequence of the mouse H-2K extracellular domain is replaced by a nucleotide sequence of a human HLA-A2 extracellular domain so that the modified mouse MHC I locus expresses a chimeric HLA-A2 / H-2K polypeptide.

[0102] In one aspect, the present application provides a method for preparing a human HLA class I / non-human MHC class I chimeric molecule, the method comprising expressing a chimeric HLA-A / H-2K protein from a nucleotide construct in a single cell, wherein the nucleotide construct comprises a cDNA sequence encoding the α1, α2 and α3 domains of the HLA-A protein and the transmembrane and cytoplasmic domains of a non-human H-2K protein, such as a mouse H-2K protein. 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 CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., PERC.6 TM cell).

[0103] In one aspect, the present application provides a cell expressing a human HLA class I / non-human MHC I chimeric protein (e.g., HLA-A / H-2K protein). In one embodiment, the cell comprises an expression vector containing a chimeric MHC class I gene, wherein the chimeric MHC class I gene comprises a human HLA-A gene sequence operably linked to a non-human H-2K gene sequence, such as a mouse H-2K gene. In one embodiment, the human HLA-A gene sequence comprises exons encoding the α1, α2, and α3 domains of the HLA-A protein. In one embodiment, the non-human H-2K gene sequence comprises exons encoding the transmembrane and cytoplasmic domains of the H-2K protein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and retinal cells expressing viral nucleic acid sequences (e.g., PERC.6 TM cell).

[0104] The present application also provides a chimeric MHC class I molecule prepared using a non-human animal described herein, wherein the chimeric MHC class I molecule comprises α1, α2, and α3 domains from human HLA-A protein and transmembrane and cytoplasmic domains from non-human, such as mouse H-2K protein. The chimeric MHC I polypeptide described in the present application can be detected by anti-HLA-A antibodies. Therefore, cells displaying human / non-human chimeric MHC I polypeptides can be detected and / or selected using anti-HLA-A antibody molecules. In some examples, anti-HLA-A2 antibodies can be used to detect the chimeric MHC I polypeptides described in the present application.

[0105] Although the following examples describe a genetically modified animal whose genome contains a nucleotide sequence encoding a mouse H-2K polypeptide extracellular domain at the endogenous mouse H-2K locus replaced by a sequence encoding a human HLA-A extracellular domain, a person skilled in the art will understand that a similar strategy can be used to replace other mouse MHC I loci (H-2D, H-2L, etc.) with corresponding human HLA loci (HLA-B, HLA-C, etc.). Therefore, the present application also provides a non-human animal, the animal comprising in its genome a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide, wherein the human portion of the polypeptide is derived from another HLA class I protein. It also relates to the replacement of multiple MHC I loci.

[0106] Animals with genetically modified β2-microglobulin

[0107] The present invention generally relates to a genetically modified non-human animal comprising in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide; thus, the animal expresses a human or humanized β2 microglobulin polypeptide.

[0108] β2 microglobulin or the light chain of the MHC class I complex (also referred to as "β2M") is a small (12 kDa) non-glycosylated protein whose main function is to stabilize the MHC α chain. The human β2 microglobulin gene encodes a protein of 119 amino acids with 20 N-terminal amino acids encoding a leader sequence. Its mature protein contains 99 amino acids. Its gene contains 4 exons, the first exon contains the 5' untranslated region, the entire leader sequence and the first two amino acids of the mature polypeptide; the second exon encodes most of the mature protein; the third exon encodes the last four amino acids of the mature protein and the stop codon; and the fourth exon contains the 3' untranslated region. Gussow et al., (1987) The β2-Microglobulin Gene. Primary Structure and Definition of the Transcriptional Unit, J. Immunol. 139: 3131-38. β2 microglobulin binds non-covalently to MHC I. Unbound β2 microglobulin is present in body fluids, such as plasma, and is excreted by the kidneys. Renal dysfunction leads to accumulation of β2 microglobulin, which can be pathogenic (e.g., dialysis-associated amyloidosis); the accumulated protein forms filamentous, fibrillar amyloid plaques in joints and connective tissues.

[0109] In addition to dialysis-related amyloidosis, β2 microglobulin is also involved in a variety of other diseases. Elevated β2 microglobulin levels have been detected in lymphatic malignancies such as non-Hodgkin's lymphoma and multiple myeloma. See, for example, Shi et al., (2009) β2Microglobulin:Emerging as a Promising Cancer Therapeutic Target, Drug Discovery Today 14:25-30. Some other malignancies with elevated β2 microglobulin levels include breast cancer, prostate cancer, lung cancer, kidney cancer, gastrointestinal tract and nasopharyngeal carcinoma. Overexpression of β2 microglobulin has been shown to have a tumor growth promoting effect, the same as above. Recently, it has also been found that β2 microglobulin drives the transition from epithelium to mesenchyme, which promotes cancer metastasis to bone and soft tissue in breast, prostate, lung and kidney cancers. Josson et al., (2011) β2 microglobulin Induces Epitelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells. Cancer Res. 71(7): 1-11. β2 microglobulin interacts with non-classical MHC I member hemochromatosis protein (HFE) and with transferrin receptor and regulates iron homeostasis, ibid. β2 microglobulin is widely documented in the prior art to be involved in other features of malignant tumors (self-renewal, enhanced angiogenesis, treatment resistance).

[0110] β2 microglobulin-deficient mice have been reported. See Koller et al. (1990) Normal development of mice deficient in β2m, MHC class I proteins, and CD8+ T cells, Science 248: 1227-1230. As reported by Koller et al., these mice appear healthy, but no MHC class I expression is detected. Moreover, in some tissues, most T cell populations are normal, while in other tissues a significant reduction in CD8+ T cells is observed. This so-called lack of MHC I expression is consistent with the previous study by Allen et al. ((1986) β2 microglobulin Is Not Required for Cell Surface Expression of the Murine Class I Histocompatibility Antigen H-2D b or of a Truncated H-2D b, Proc. Natl. Acad. Sci. USA 83:7447-7451). Allen et al. reported that β2-microglobulin is not necessary for all MHC I complexes to be expressed on the cell surface, because cells lacking β2-microglobulin can express H-2D b However, it is speculated that H-2D b The function is impaired, and H-2D b The conformation of the protein is different from that of the native protein, which explains why Koller and his colleagues used a b However, the absence of β 2 β2-microglobulin-deficient cells may present endogenous antigens to CD8+ T cells (including endogenous CD8+ T cells from normal mice), and mice reportedly do not require β2-microglobulin to produce high levels of H-2 in response to antigenic challenge. d MHC class I-restricted CD8+ CTLs, although they are required to maintain an effective immune response. Quinn et al., (1997) Virus-Specific, CD8+ Major Histocompatibility Complex Class I-Restricted Cytotoxic T Lymphocytes in Lymphocytic Choriomeningitis Virus-Infected β2-Microglobulin-Deficient Mice, J. Virol. 71: 8392-8396. It is noteworthy that the ability to generate high levels of such T cells in the absence of β2 microglobulin is reported to be limited to H-2 d MHC class I-restricted response. Mice deficient in β2 microglobulin have been reported to have a variety of notable characteristics, such as increased susceptibility to some parasitic diseases, increased susceptibility to hepatitis infection, defects in iron metabolism, and impaired reproductive phenotype. Cooper et al. (2007) An impaired breeding phenotype in mice with a genetic deletion of Beta-2 microglobulin and diminished MHC class I expression: Role in reproductive fitness, Biol. Reprod. 77: 274-279.

[0111] Mice expressing both human β2 microglobulin and human HLA class I molecules (i.e., HLA-B7) on randomly inserted transgenes have been reported. Chamberlain et al., (1988) Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (β2-microglobulin) chain genes in transgenic mice, Proc. Natl. Acad. Sci. USA 85:7690-7694. The expression of human HLA class I was consistent with a significant reduction in endogenous class I in the liver, supra. The expression of human β2 microglobulin was also consistent with endogenous β2 microglobulin, while the expression of human HLA class I molecules was increased 10- to 17-fold in double transgenic mice, supra. However, the authors did not attempt to replace the endogenous β2 microglobulin locus with the human β2 microglobulin locus.

[0112] Therefore, disclosed in the present application is a genetically modified non-human animal (e.g., a rodent, such as a mouse or a rat), comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide in its genome. In one aspect, the animal does not express endogenous non-human β2 microglobulin from an endogenous non-human β2 microglobulin locus. In some embodiments, the nucleotide sequence encoding the β2 microglobulin polypeptide is partially human and partially non-human, for example, it contains some amino acids corresponding to humans and some amino acids corresponding to non-human β2 microglobulin. In one aspect, the non-human animal does not express endogenous non-human β2 microglobulin polypeptides from endogenous non-human loci, and only expresses human or humanized β2 microglobulin polypeptides. In one example, the non-human animal does not express a complete endogenous non-human β2 microglobulin polypeptide from an endogenous β2 microglobulin locus, and only expresses a portion of a non-human endogenous β2 microglobulin polypeptide. Therefore, in various embodiments, the animal does not express a functional non-human β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In a specific aspect, the nucleotide sequence encoding a human or humanized β2 microglobulin is located at an endogenous non-human β2 microglobulin locus. In one aspect, the animal comprises two copies of a β2 microglobulin locus, comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In another aspect, the animal comprises a copy of a β2 microglobulin locus, comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. Therefore, the animal may be homozygous or heterozygous for a β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. The nucleotide sequence of the human or humanized β2 microglobulin may be derived from a collection of β2 microglobulin sequences naturally present in the population. In different embodiments, the genetically modified non-human animal of the present invention comprises a nucleotide sequence encoding a human or humanized β2 microglobulin in its germ cells. In one embodiment, the nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide comprising a human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to an MHC I protein.

[0113] The nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may include nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may include nucleic acid residues encoding the amino acid sequence shown in amino acids 21-119 of human β2 microglobulin (i.e., amino acid residues corresponding to mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may include nucleic acid residues encoding the amino acid sequence shown in amino acids 23-115 of human β2 microglobulin, such as the amino acid sequence shown in amino acids 23-119 of human β2 microglobulin. The nucleic acid and amino acid sequence of human β2 microglobulin are as described in Gussow et al., supra, which are incorporated into this application by reference.

[0114] Therefore, the human or humanized β2 microglobulin may comprise the amino acid sequence shown in amino acids 23-115 of a human β2 microglobulin polypeptide, such as the amino acid sequence shown in amino acids 23-119 of a human β2 microglobulin polypeptide, such as the amino acid sequence shown in amino acids 21-119 of a human β2 microglobulin polypeptide. Alternatively, the human β2 microglobulin may comprise amino acids 1-119 of a human β2 microglobulin polypeptide.

[0115] In some embodiments, the nucleotide sequence encoding human or humanized β2 microglobulin comprises the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises the nucleotide sequence shown in exons 2, 3, and 4 of the human β2 microglobulin gene. In this embodiment, the nucleotide sequences shown in exons 2, 3, and 4 are operably linked so that the gene is transcribed and translated normally. Therefore, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to exon 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to approximately 267 bp after exon 2 to exon 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence comprises approximately 2.8 kb of the human β2 microglobulin gene.

[0116] Therefore, the human or humanized β2 microglobulin polypeptide can be encoded by a nucleotide sequence comprising the nucleotide sequence shown in exon 2 to exon 4 of human β2 microglobulin, such as a nucleotide sequence corresponding to exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the polypeptide can be encoded by a nucleotide sequence comprising the nucleotide sequence shown in exons 2, 3 and 4 of the human β2 microglobulin gene. In a specific embodiment, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to about 267bp after exon 2 to exon 4 of the human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising about 2.8kb of the human β2 microglobulin gene. Because exon 4 of the β2 microglobulin gene contains a 5' untranslated region, the human or humanized polypeptide can be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.

[0117] One of ordinary skill in the art will appreciate that, although specific nucleic acid and amino acid sequences for generating genetically modified animals are described in this example, the present application also provides sequences with one or more conservative or non-conservative amino acid substitutions or sequences that differ from those described in the present application due to the degeneracy of the genetic code.

[0118] Therefore, the present application provides a non-human animal expressing a human β2 microglobulin sequence, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the human β2 microglobulin sequence. In a specific embodiment, the β2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the human β2 microglobulin sequence described in the Examples. In one embodiment, the human β2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence comprises one or more non-conservative substitutions.

[0119] In addition, the present application provides a non-human animal, wherein the nucleotide sequence encoding human or humanized β2 microglobulin also comprises the nucleotide sequence shown in exon 1 of a non-human β2 microglobulin gene. Therefore, in a specific embodiment, the non-human animal comprises a nucleotide sequence encoding human or humanized β2 microglobulin in its genome, wherein the nucleotide sequence comprises exon 1 of a non-human β2 microglobulin and exons 2, 3, and 4 of a human β2 microglobulin gene. Therefore, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of a non-human β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene (e.g., exons 2 and 3 of a human β2 microglobulin gene).

[0120] Similar to the non-human animals comprising nucleotide sequences encoding human / non-human chimeric MHC I polypeptides, the non-human animals comprising nucleotide sequences encoding human or humanized β2 microglobulin can be selected from the following groups: mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques). In some embodiments of the present invention, the non-human animal is a mammal. In a specific embodiment, the non-human animal is a rodent (e.g., mouse or rat) of murine origin. In one embodiment, the animal is a mouse.

[0121] Therefore, in some aspects, the present application provides a genetically modified mouse, wherein the mouse comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide as described in the present application. The present application provides a genetically modified mouse, wherein the mouse comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide (e.g., a human or substantially human β2 microglobulin polypeptide) in its endogenous β2 microglobulin locus. In some embodiments, the mouse does not express an endogenous β2 microglobulin polypeptide (e.g., a functional endogenous β2 microglobulin polypeptide) from an endogenous β2 microglobulin locus. In some embodiments, the genetically modified mouse comprises a nucleotide sequence containing exon 1 of a mouse β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene. In some embodiments, the mouse expresses a human or humanized β2 microglobulin polypeptide.

[0122] In one aspect, the present application provides a modified non-human β2 microglobulin gene locus, which comprises a heterologous β2 microglobulin sequence. In one embodiment, the heterologous β2 microglobulin sequence is a human or humanized sequence.

[0123] In one embodiment, the modified locus is a rodent locus. In a specific embodiment, the rodent locus is selected from a mouse or rat locus. In one embodiment, the non-human locus is modified with at least one human β2 microglobulin coding sequence.

[0124] In one embodiment, the heterologous β2 microglobulin sequence is operably linked to endogenous regulatory elements, such as endogenous promoters and / or expression control sequences. In a specific embodiment, the heterologous β2 microglobulin sequence is a human sequence and the human sequence is operably linked to an endogenous promoter and / or expression control sequence.

[0125] In one aspect, the present application provides a modified non-human β2 microglobulin locus comprising a human sequence operably linked to an endogenous promoter and / or expression control sequence.

[0126] In various aspects, the human or humanized β2 microglobulin expressed by the genetically modified non-human animal, or the cell, embryo or tissue from the non-human animal retains all functional aspects of the endogenous and / or human β2 microglobulin. For example, it is preferred that the human or humanized β2 microglobulin binds to the α chain of an MHC I polypeptide (e.g., an endogenous non-human or human MHC I polypeptide). The human or humanized β2 microglobulin polypeptide can bind, recruit or otherwise interact with any other molecule, such as a receptor, anchor or signaling molecule (e.g., HFE, etc.) associated with endogenous non-human and / or human β2 microglobulin.

[0127] In addition to genetically modified animals (e.g., rodents, such as mice or rats), the present application also provides tissues or cells, wherein the tissues or cells are derived from non-human animals as described in the present application, and contain exogenous β2 microglobulin genes or β2 microglobulin sequences, i.e., nucleotide and / or amino acid sequences. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or a human or humanized β2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express an MHC I complex, such as an antigen presenting cell. The human or humanized β2 microglobulin polypeptide expressed by the cell can interact with endogenous non-human MHC I (e.g., rodent MHC I) to form a functional MHC I complex. The resulting MHC I complex may be able to interact with T cells, such as cytotoxic T cells. Therefore, the present application also provides an in vitro complex of cells and T cells of a non-human animal as described in the present application.

[0128] The present application also provides a non-human cell comprising a human or humanized β2 microglobulin gene or sequence, and an additional human or humanized sequence such as a chimeric MHC I polypeptide disclosed in the present application. In this case, the human or humanized β2 microglobulin polypeptide can interact with, for example, a human / non-human chimeric MHC I polypeptide and form a functional MHC I complex. In some aspects, this complex can interact with a T cell such as a TCR on a human or non-human T cell. Therefore, the present application also provides an in vitro complex formed by a cell of a non-human animal as described in the present application and a non-human T cell.

[0129] Another aspect of the present disclosure is a rodent embryo (e.g., a mouse or rat embryo) comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence as described herein. In one embodiment, the embryo comprises an ES donor cell comprising the heterologous β2 microglobulin gene or β2 microglobulin sequence, and a host embryonic cell. The heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or β2 microglobulin sequence.

[0130] The present invention also includes a non-human animal cell, which comprises a chromosome or a fragment thereof of a non-human animal as described herein (e.g., wherein the chromosome or a fragment thereof comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide). The non-human cell may comprise a cell nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or a fragment thereof as a result of nuclear transfer.

[0131] In one aspect, the present application provides a non-human induced pluripotent stem cell comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence. In one embodiment, the induced pluripotent stem cell is derived from a non-human animal as described in the present application. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized gene or sequence.

[0132] The present application also provides a hybridoma or quadrivalent tumor, which is derived from a non-human animal cell as described in the present application. In one embodiment, the non-human animal is a mouse or a rat.

[0133] The present disclosure also provides a method for preparing a genetically modified non-human animal (e.g., a genetically modified rodent, such as a mouse or a rat) described in the present application. The method ultimately produces an animal whose genome contains a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the method ultimately produces a genetically modified mouse, which contains a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide in the endogenous β2 microglobulin locus in its genome. In some cases, the mouse does not express functional mouse β2 microglobulin from the endogenous mouse β2 microglobulin locus. In some aspects, the method utilizes the use of The targeting construct is prepared using the technology, the construct is introduced into ES cells, and the The technology introduces the targeted ES cell clone into a mouse embryo, as described in the examples. In one embodiment, the ES cell is a mixture of 129 and C57BL / 6 mouse strains; in another embodiment, the ES cell is a mixture of BALB / c and 129 mouse strains.

[0134] The present application also provides a nucleotide construct for generating genetically modified non-human animals. The nucleotide construct may include: 5' and 3' non-human homology arms, a human DNA fragment containing a human β2 microglobulin sequence, and a screening gene expression cassette with recombination sites on both sides. In one embodiment, the human DNA fragment is a genomic fragment containing introns and exons of a human β2 microglobulin gene. In one embodiment, the non-human homology arms have homology with a non-human β2 microglobulin locus. The genomic fragment may include exons 2, 3, and 4 of a human β2 microglobulin gene. In one example, the genomic fragment includes, from 5' to 3': exon 2, intron, exon 3, intron and exon 4, and the entire human β2 microglobulin sequence. The screening gene expression cassette may be located at any position outside the β2 microglobulin coding region in the construct, for example, it may be located at the 3' end of exon 4 of human β2 microglobulin. The 5' and 3' non-human homology arms may respectively comprise the 5' and 3' genomic sequences of the endogenous non-human β2 microglobulin gene. In another embodiment, the 5' and 3' non-human homology arms may respectively comprise the 5' and 3' genomic sequences of exon 2 and exon 4 of the endogenous non-human gene.

[0135] Another aspect of the present invention relates to a method for modifying the β2 microglobulin locus of a non-human animal (e.g., a rodent, such as a mouse or a rat) to express a human or humanized β2 microglobulin polypeptide as described herein. A method for modifying the β2 microglobulin locus of a mouse to express a human or humanized β2 microglobulin polypeptide includes replacing the nucleotide sequence encoding the mouse β2 microglobulin with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide at the endogenous β2 microglobulin locus. In one embodiment of this method, the mouse does not express a functional β2 microglobulin polypeptide from the endogenous mouse β2 microglobulin locus. In some specific embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises the nucleotide sequence shown in exons 2 to 4 of the human β2 microglobulin gene. In other embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises the nucleotide sequence shown in exons 2, 3, and 4 of the human β2 microglobulin gene.

[0136] Animals with genetically modified MHC I / β2 microglobulin

[0137] In various embodiments, the present invention generally provides a genetically modified non-human animal comprising in its genome a nucleotide sequence encoding a human or humanized MHC I and β2 microglobulin polypeptide; thus, the animal expresses a human or humanized MHC I and β2 microglobulin polypeptide.

[0138] Functional differences arise when mixed human / non-human system components are used. HLA class I binds more tightly to β2 microglobulin than mouse class I. Bernabeu (1984) β2-microgobulin from serum associates with MHC class I antigens on the surface of cultured cells, Nature 308:642-645. Attempts to eliminate functional differences have been made to construct specific humanized MHC mice. H-2 class I and class 2 knockout mice (on a mouse β2 microglobulin KO background) have been developed that express human α1 and α2 as well as mouse H-2D b A random integration of human HLA-A2.1 / HLA-DR1 chimeric transgene of α3, which is connected to the C-terminus of human β2-microglobulin by a linker at the N-terminus. See, for example, Pajot et al., (2004) A mouse model of human adaptive immune functions: HLA-A2.1- / HLA-DR1-transgenic H-2class I- / class II-knockout mice, Eur. J. Immunol. 34: 3060-3069. It is reported that these mice generate antigen-specific antibodies and CTL responses to hepatitis B virus, while transgenic mice with only HLA-A2.1 or mice with only H-2 class I / class II genes knocked out do not generate antigen-specific antibodies and CTL responses to hepatitis B virus. It is speculated that the reason for the defects of transgenic mice with only HLA-A2.1 is that such mice have the ability to introduce endogenous class I and / or class II genes to avoid the use of any transgenics, while MHC knockout mice do not have this option. However, the mice can at least express H-2D b , which is presumably due to breeding into mice in a mouse β2-microglobulin knockout background (see Pajot et al., supra; which apparently contains intact endogenous class I and class II loci).

[0139] Chimeric MHC fused to human β2 microglobulin was reported to be expressed at a lower cell surface level than endogenous MHC, but survival / NK killing and NK suicide rates were not reported. Pajot et al., supra. Some improvement in CD8+ T cell counts was observed in MHC class I-deficient β2 microglobulin knockout mice (2-3% of total splenocytes, compared to 0.6-1% in β2KO mice). However, T cell variable region usage was altered for BV 5.1, BV 5.2, and BV 11 gene segments. CD8+ and CD4+ T cell responses were reported to be limited to the appropriate hepatitis B antigens used to immunize the mice. However, when mice were immunized with only one of the two antigens, at least two mice killed cells carrying either of the two antigens. This may be due to a lack of NK cell inhibition or a lack of NK cell selectivity.

[0140] As described above, mice transgenic for human MHC I and human β2 microglobulin contain nucleotide sequences encoding chimeric MHC I / β2 microglobulin, wherein the MHC I and β2 microglobulin portions are contained in a single polypeptide chain, such that the MHC I α chain and the β2 microglobulin are covalently linked to each other to tether them to the cell surface. The present application provides a mouse comprising two independent nucleotide sequences in its genome, one of which encodes a human or humanized MHC I polypeptide and the other encodes a human or humanized β2 microglobulin polypeptide. The present application provides a mouse that will express an MHC I complex that is more like a naturally occurring MHC I complex, wherein the MHC I α chain and the β2 microglobulin are provided on two separate polypeptide chains that are non-covalently bound to the β2 microglobulin and the MHC I α chain.

[0141] Therefore, the present disclosure provides a non-human animal, the animal comprising in its genome: a first nucleotide sequence encoding a human or humanized MHC I polypeptide, and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the present application provides a non-human animal, the animal comprising in its genome: (a) a first nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises a peptide binding domain or an extracellular domain of human MHC I (e.g., HLA-A, HLA-B or HLA-C; e.g., HLA-A2), and (b) a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide.

[0142] The first nucleotide sequence may be located at an endogenous non-human MHC I locus, so that the animal comprises all or part of an endogenous MHC I gene (e.g., a portion encoding a peptide binding domain or an extracellular domain) at the MHC I locus of its genome replaced by a corresponding human MHC I sequence. Thus, the animal may comprise a nucleotide sequence encoding a human MHC I (e.g., HLA-A, HLA-B, or HLA-C; e.g., HLA-A2) extracellular domain and an endogenous non-human MHC I (e.g., H-2K, H-2D, etc., e.g., H-2Kb) transmembrane and cytoplasmic domain at the endogenous MHC I locus. In one aspect, the animal is a mouse, and the first nucleotide sequence comprises a nucleotide sequence encoding a human HLA-A2 (e.g., HLA-A2.1) extracellular domain and a mouse H-2K (e.g., H-2Kb) transmembrane and cytoplasmic domain.

[0143] The second nucleotide sequence may be located at an endogenous non-human β2 microglobulin locus so that the animal contains an endogenous β2 microglobulin gene in the β2 microglobulin locus of its genome, and all or part of the endogenous β2 microglobulin gene is replaced by a corresponding human β2 microglobulin sequence. The second nucleotide sequence may include the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the second nucleotide sequence may include the nucleotide sequence shown in exons 2, 3, and 4 of the human β2 microglobulin gene. In this embodiment, the nucleotide sequences are operably linked to each other. The second nucleotide sequence may further include a sequence of non-human β2 microglobulin exon 1.

[0144] In one aspect, the animal does not express functional MHC I from an endogenous non-human MHC I locus (e.g., does not express peptide binding domains and extracellular domains of endogenous MHC I), and the animal does not express functional β2 microglobulin polypeptides from an endogenous non-human β2 microglobulin locus. In some aspects, the animal is homozygous for an MHC I locus comprising a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide and a β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin. In other aspects, the animal is heterozygous for an MHC I locus comprising a nucleotide sequence encoding a human / non-human chimeric MHC I polypeptide and a β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin.

[0145] Preferably, the first and second nucleotide sequences are operably linked to endogenous expression regulatory elements (eg, promoters, enhancers, silencers, etc.).

[0146] Various other embodiments of the first and second nucleotide sequences (and polypeptides encoded therein) contained in the present application are easily understood based on the embodiments described in the specification, such as those described in the sections concerning genetically modified MHC I animals and genetically modified β2 microglobulin animals.

[0147] In one aspect, the present disclosure provides a mouse, the mouse comprising in its genome (a) a first nucleotide sequence encoding a human / mouse chimeric MHC I polypeptide (particularly an HLA-A2 / H-2K polypeptide), wherein the human portion of the chimeric polypeptide comprises the extracellular domain of human HLA-A2 and the mouse portion comprises the transmembrane and cytoplasmic domains of mouse H-2K, and (b) a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide (e.g., wherein the nucleotide sequence comprises the nucleotide sequence shown in exon 2 to exon 4 of the human β2 microglobulin gene or the nucleotide sequence shown in exons 2, 3 and 4 of the human β2 microglobulin gene), wherein the first nucleotide sequence is located at the endogenous H-2K locus, and the second sequence is located at the endogenous β2 microglobulin locus. In one embodiment, the mouse does not express functional H-2K and mouse β2 microglobulin polypeptides from their respective endogenous loci. In one embodiment, the mouse expresses a human / mouse chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide.

[0148] As described in the Examples below, genetically modified animals that co-express human or humanized MHC I and β2 microglobulin show increased expression of chimeric MHC class I on the cell surface compared to animals that are humanized only for MHC I. In some embodiments, the co-expression of human or humanized MHC I and β2 microglobulin increases the expression of human or humanized MHC I on the cell surface by more than about 10%, such as more than about 20%, such as more than about 50% or more, such as about 70%, compared to the expression of human or humanized MHC I in the absence of human or humanized β2 microglobulin.

[0149] The present disclosure also provides a method for preparing a genetically modified non-human animal (e.g., a rodent, such as a rat or a mouse), whose genome comprises the first and second nucleotide sequences described in the present application. The method generally includes generating a first genetically modified non-human animal, whose genome comprises the first nucleotide sequence described in the present application (i.e., a human or humanized MHC I sequence), generating a second genetically modified non-human animal, whose genome comprises the second nucleotide sequence described in the present application (i.e., a human or humanized β2 microglobulin sequence), and mating the first and second animals to obtain offspring whose genomes contain these two nucleotide sequences. In one embodiment, the first and second animals are heterozygous for the first and second nucleotide sequences, respectively. In one embodiment, the first and second animals are homozygous for the first and second nucleotide sequences, respectively. In one embodiment, the first and second animals are produced by replacing endogenous non-human loci with the first and second nucleotide sequences, respectively. In one aspect, the first and second animals are produced by utilizing The constructs were generated by The ES cell clone of interest carrying such a construct is introduced into an embryo (eg, a rodent embryo, such as a mouse or rat embryo).

[0150] Uses of genetically modified animals

[0151] In various embodiments, the genetically modified non-human animals described herein generate APCs with human or humanized MHC I and / or β2 microglobulin on the cell surface, and as a result present peptides derived from cytoplasmic proteins as epitopes for CTL in a human-like manner, because substantially all components of the complex are human or humanized. The genetically modified non-human animals of the present invention can be used to study the function of the human immune system in humanized animals; to identify antigens and antigenic epitopes that stimulate immune responses (e.g., T cell epitopes, such as unique human cancer epitopes), such as for the development of vaccines; to identify high-affinity T cells (i.e., T cells that bind to antigens in the context of a human MHC I complex with high affinity) against human pathogens or cancer antigens, such as for adaptive T cell therapy; to evaluate vaccine candidates and other vaccine strategies; to study human autoimmunity; to study human infectious diseases; and to design other better therapeutic strategies based on human MHC expression.

[0152] The MHC I complex binds to the peptide and presents it to the cell surface. Once presented to the surface in the context of this complex, the peptide is recognized by T cells. For example, when the peptide is derived from a pathogen or other antigen of interest (e.g., a tumor antigen), T cell recognition can result in T cell activation, macrophage killing of cells carrying the presented peptide sequence, and B cell activation of antibodies that bind to the presented sequence.

[0153] T cells interact with cells expressing MHC I complexes through peptide binding to the extracellular domain of MHC class I and the CD8 extracellular domain of T cells. CD8+T cells will form cytotoxic T cells when they encounter APCs with antigens suitable for binding to MHC class I molecules. Therefore, antigens that bind to T cell receptors with high affinity in the context of MHC class I are potentially important in the research and development of human disease treatment. However, antigen presentation in the context of mouse MHC I is only slightly relevant to human diseases because human and mouse MHC complexes recognize different antigens, such as mouse MHC I and human MHC I may not recognize the same antigen or may present different epitopes. Therefore, most of the relevant data for human pathology is obtained through studies of antigen epitopes presented by human MHC I.

[0154] Thus, in various embodiments, the genetically modified animals of the invention are useful, among other things, in evaluating the ability of antigens to elicit an immune response in humans, and for generating diverse antigens and identifying specific antigens that can be used in the development of vaccines for human use.

[0155] In one aspect, the present application provides a method for determining antigenicity in humans having a certain peptide sequence, the method comprising contacting a genetically modified non-human animal as described herein with a molecule comprising the peptide sequence, causing the non-human animal to produce an immune response, and detecting cells in the non-human animal that bind to the peptide sequence presented by the human / non-human chimeric MHC I or humanized MHC I complex described herein (comprising human / non-human chimeric MHC I and human or humanized β2 microglobulin).

[0156] In one aspect, the present application provides a method for determining whether a certain peptide will stimulate cellular immunity in the human body, the method comprising contacting a genetically modified non-human animal as described in the present application with the peptide, causing the non-human animal to produce an immune response, and detecting cells in the non-human animal that are bound to the sequence of the peptide by the human / non-human chimeric MHC class I molecule described in the present application. In one embodiment, the non-human animal after contact contains MHC class I-restricted CD8+ cytotoxic T lymphocytes (CTLs) bound to the peptide. In one embodiment, CTLs kill cells carrying the peptide.

[0157] In one aspect, the present application provides a method for identifying human CTL epitopes, the method comprising contacting a non-human animal as described herein with an antigen comprising a predicted CTL epitope, allowing the non-human animal to produce an immune response, isolating MHC class I-restricted CD8+CTLs that bind to the epitope from the non-human animal, and identifying the epitope that binds to the MHC class I-restricted CD8+CTLs.

[0158] In one aspect, the present application provides a method for identifying HLA class I-restricted peptides, which peptides are presented by human cells and bound by human lymphocytes (e.g., human T cells) and result in cytotoxicity to cells carrying the peptides. The method comprises contacting a non-human animal as described herein (or a cell thereof expressing MHC class I) with a molecule comprising the peptide of interest, isolating non-human animal cells expressing human / non-human chimeric class I molecules that bind to the peptide of interest, contacting the cells with human lymphocytes capable of producing HLA class I-restricted cytotoxicity, and detecting peptide-induced cytotoxicity.

[0159] In one aspect, the present application provides a method for identifying an antigen that generates a cytotoxic T cell response in humans, the method comprising contacting a predicted antigen with a mouse as described herein, allowing the mouse to produce an immune response, and identifying an antigen that binds to an HLA-A-restricted molecule.

[0160] In one embodiment, the antigen comprises a bacterial or viral surface or envelope protein. In one embodiment, the antigen comprises an antigen on the surface of a human tumor cell. In one embodiment, the antigen comprises a vaccine that is hypothesized to be used in the human body. In one embodiment, the antigen comprises a human epitope that produces antibodies in the human body. In another embodiment, the antigen comprises a human epitope that produces a high affinity CTL targeting an epitope / MHC I complex.

[0161] In one aspect, the present application provides a method for determining whether a predicted antigen contains an epitope that produces an HLA-A-restricted immune response (e.g., an HLA-A2-restricted response) when exposed to the human immune system, the method comprising contacting a mouse as described herein with the predicted antigen and determining the antigen-specific HLA-A-restricted (e.g., HLA-A2-restricted) immune response in the mouse.

[0162] In one embodiment, the predicted antigen is selected from a biopharmaceutical or a fragment thereof, a non-self protein, a surface antigen of a non-self cell, a surface antigen of a tumor cell, a surface antigen of a bacterial or yeast or fungal cell, a surface antigen of a virus or an envelope protein.

[0163] In addition, the genetically modified non-human animals described herein can be used to identify T cell receptors, such as high affinity T cell receptors, which recognize an antigen of interest, such as a tumor or other disease antigen. The method may include: contacting the non-human animal described herein with an antigen, causing the non-human animal to produce an immune response to the antigen, isolating from the non-human animal a T cell comprising a T cell receptor that binds to an antigen presented by a human or humanized MHC I, and determining the sequence of the T cell receptor.

[0164] In one aspect, the present application provides a method for identifying a T cell receptor variable domain with high affinity for a human tumor antigen, the method comprising contacting a mouse comprising humanized MHC Iα1, α2 and α3 domains (e.g., HLA-A2α1, α2 and α3 domains) with a human tumor antigen; allowing the mouse to produce an immune response; and isolating a nucleic acid sequence encoding the T cell receptor variable domain from the mouse, wherein the Kp1 binding affinity of the T cell receptor variable domain to the human tumor antigen is 0. D No higher than about 1 nanomolar.

[0165] In one embodiment, the mouse further comprises a replacement of multiple unrearranged human T cell receptor variable gene segments at the endogenous mouse T cell receptor variable region locus, wherein the unrearranged human T cell receptor variable region gene segments are recombined with genes encoding human-mouse chimeric T cell receptors comprising human variable regions and mouse constant regions. In one embodiment, the mouse comprises a human CD8 transgene, and the mouse expresses a functional human CD8 protein.

[0166] T cell receptors with high affinity for tumor antigens are used for cell-based therapy. T cell populations with high affinity for human tumor antigens have been prepared by contacting human T cells with HLA-A2, which has been mutated to minimize the binding of CD8 to the α3 subunit to select only those T cells with extremely high affinity for tumor antigens (i.e., T cell clones that recognize the antigen despite the inability of CD8 to bind to α3). See Pittet et al., (2003) α3 Domain Mutants of Peptide / MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells, J. Immunol. 171: 1844-1849. The non-human animals and non-human animal cells are used to identify peptides that form complexes with human HLA class I that bind to T cell receptors with high affinity or activate T cell receptor-bearing lymphocytes.

[0167] Any suitable method known in the art can be used to detect antigen / HLA class I binding to or activation of T cells. Peptide-specific APC-T cell binding and activation is detectable. For example, it has been reported that binding of T cells to antigen presenting cells expressing HLA-A2 leads to accumulation of PIP2 in the immune synapse, whereas cross-linked MHC class I molecules do not. See Fooksman et al., (2009) Cutting Edge: Phosphatidylinositol 4,5-Bisphosphate Concentration at the APC Side of the Immunological Synapse Is Required for Effector T Cell Function, J. Immunol. 182: 5179-5182.

[0168] Functional consequences of the interaction of TCR-bearing lymphocytes with class I-expressing APCs are also detectable and include cell killing by the lymphocytes. For example, it has been reported that the contact point of the HLA-A2α2 subunit generates a Fas-independent killing signal by CD8+CTLs. When contacted (by antibodies) on an epitope of the HLA-A2 molecule known (from crystallographic studies) to contact CD8, Jurkat cells expressing HLA-A2- undergo apoptosis, which does not have any significant dependence on the cytoplasmic domain. See Pettersen et al., (1998) The TCR-Binding Region of the HLA Class Iα2DomainSignals Rapid Fas-Independent Cell Death: A Direct Pathway for TCell-Mediated Killing of Target Cells? J.Immunol.160:4343-4352. It is speculated that the rapid killing induced by contact of HLA-A2α2 with CD8+ CTLs may be mainly due to this Fas-independent HLA-A2-mediated pathway (supra), because it is different from the killing mediated by the α3 domain of TCR, which is independent of TCR, which can itself induce apoptosis (see Woodle et al., (1997) Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway, J. Immunol. 158: 2156-2164).

[0169] The consequences of the interaction between T cells and peptides displayed by APCs in the context of MHC I can also be determined by T cell proliferation assays. Alternatively, it can be determined by detecting the release of cytokines that are generally associated with the activation of immune responses. In one embodiment, the activation of CD8+ T cells can be monitored and quantified using IFNγ ELISPOT.

[0170] As described in the present application, in the genetically modified non-human animals described in the present application, the activation of CD8+T cells can be prevented due to the species-specific binding of CD8 to MHC I. For species-specific CD8 interactions, it is an ideal embodiment that the cells of genetically modified animals (e.g., rodents, such as mice or rats) as described in the present application are contacted with human cells (e.g., in vitro), such as cells of CD8 carried by humans, such as human T cells. In one embodiment, cells expressing MHC I classes of mice as described in the present application are contacted with T cells comprising human CD8 and T cell receptors in vitro. In a specific embodiment, the T cells are human T cells. In one embodiment, the cells expressing MHC I classes of mice include peptides bound to human / mouse chimeric MHC I or humanized MHC I complexes (including human β2 microglobulin), and the T cells are human T cells, and the ability of T cells to bind to mouse cells displaying the peptides is determined. In one embodiment, the activation of human T cells by mouse cells displaying the peptides is determined. In one embodiment, the present application provides an in vitro method for detecting human T cell activation by cells displaying the peptide, the method comprising contacting a mouse or mouse cell as described in the present application with an antigen of interest, contacting a cell from the mouse or the mouse cell (assuming that it carries a peptide derived from an antigen that forms a complex with a human or humanized MHC I) with a human T cell, and determining the activation of the human T cell. In one embodiment, the method is used to identify T cell epitopes of human pathogens or human tumors. In one embodiment, the method is used to identify epitopes of vaccines.

[0171] In one embodiment, the present application provides a method for determining T cell activation caused by a putative human treatment, the method comprising contacting a genetically modified animal as described herein with a putative human treatment (or, for example, contacting cells expressing human or humanized MHC I derived from such an animal with a peptide sequence of the putative treatment), contacting cells derived from the genetically modified animal displaying human or humanized MHC I / peptide complexes with T cells comprising a human T cell receptor and CD8 capable of binding to cells of the genetically modified animal, and detecting the activation of human T cells induced by cells of the genetically modified animal displaying the peptide.

[0172] In various embodiments, T cells containing human CD8 sequences, such as human T cells or non-human animal T cells containing a transgene encoding human CD8, are used to prepare complexes with cells expressing human or humanized MHC class I from an animal as described herein. Transgenic mice for human CD8 are well known in the art. Tishon et al., (2000) Trangenic Mice Expressing Human HLA and CD8 Molecules Generate HLA-Restricted Measles Virus Cytotoxic T Lymphocytes of the Same Specificity as Humans with Natural Measles Virus Infection, Virology 275 (2): 286-293; LaFace et al., (1995) Human CD8 Transgene Regulation of HLA Recognition by Murine T Cells, J. Exp. Med. 182: 1315-1325.

[0173] In addition to being able to identify antigens and antigenic epitopes of human pathogens or tumors, the genetically modified animals of the present invention are also used to identify autoantigens associated with human autoimmune diseases, such as type I diabetes, multiple sclerosis, etc. For example, Takaki et al. ((2006) HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes, J. Immunol. 176: 3257-65) describe the use of NOD mice carrying HLA / β2 microglobulin single chains to identify autoantigens of type 1 diabetes. The genetically modified animals of the present invention are also used to study different aspects of human autoimmune diseases. Because some polymorphic human MHC I alleles are known to be associated with the progression of certain diseases, such as autoimmune diseases (e.g., Graves' disease, myasthenia gravis, psoriasis, etc.; see Bakker et al., (2006) A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC, Nature Genetics 38: 1166-72 and Supplementary Information and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases, Proc. Natl. Acad. Sci. USA 106: 18680-85, both of which are incorporated herein by reference), the genetically modified animals of the present invention comprising a humanized MHC I locus including such alleles can be used as models of autoimmune diseases. In one embodiment, the disease allele is HLA-B27, and the disease is ankylosing spondylitis or reactive arthritis; thus, in one embodiment, the animals used to study these diseases comprise human or humanized HLA-B27.

[0174] Other aspects of cellular immunity involving the MHC I complex are well known in the art; therefore, the genetically modified non-human animals described herein can be used to study these aspects of immunobiology. For example, in vivo binding of the TCR to MHC class I is regulated by other factors. Leukocyte immunoglobulin-like receptor subfamily B member (LILRB1 or LIR-1) is expressed on MHC class I-restricted CTLs and downregulates T cell stimulation by binding to specific determinants on the α3 subunit of the MHC class I molecule on APC. Structural studies have shown that the binding sites of LIR-1 and CD8 overlap, suggesting that the inhibitory LIR-1 competes with the stimulatory CD8 for binding to the MHC class I molecule. Willcox et al., (2003) Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor, Nature Immunology 4(9):913-919; Shirioshi et al., (2003) Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G, Proc. Natl. Acad. Sci. USA 100(15):8856-8861. LIR-1 transduces inhibitory signals through its (intracellular) immunoreceptor tyrosine-based inhibitory motif (ITIM). In NK cells, studies have shown that KIR (inhibitory killer cell Ig-like receptor) lacking ITIM (normally unable to inhibit) can inhibit binding to the α3 domain of MHC class I molecules in the presence of LIR-1 (presumably through the LIR-1 ITIM) (see Kirwin et al., (2005) Killer Cell Ig-Like Receptor-Dependent Signaling by Ig-Like Transcript 2 (ILT2 / CD85j / LILRB1 / LIR-1) J. Immunol. 175: 5006-5015), suggesting that LIR-1 binding to MHC class I acts synergistically with KIR, thereby playing a role in regulating the binding of the HLA α3 domain for NK cell inhibition.

[0175] As mentioned above, MHC molecules interact with cells that do not express TCRs. These cells are NK cells. NK cells are cytotoxic lymphocytes (different from CTLs or cytotoxic T lymphocytes) that play a central role in cellular immune responses, especially innate immunity. NK cells are the first line of defense against invasion by microorganisms, viruses, and other non-self (e.g., tumors) entities. NK cells are activated or inhibited via surface receptors, and they express CD8 but not TCRs. NK cells are able to interact with cells expressing MHC class I, but the interaction is through the CD8-binding α3 domain rather than the TCR-binding, peptide-carrying α 1 and α 2 The main function of NK cells is to destroy cells that lack sufficient MHC I surface proteins.

[0176] Cross-linking of MHC class I molecules on the surface of natural killer (NK) cells results in downregulation of intracellular tyrosine phosphorylation, migration of MHC class I molecules from the immune synapse, and tumor cell killing. Rubio et al., (2004) Cross-linking of MHC class I molecules on human NK cells inhibits NK cell function, segregates MHC I from the NK cell synapse, and induces intracellular phosphotyrosines, J. Leukocyte Biol. 76: 116-124.

[0177] Another function of MHC Class I in NK cells is apparently to prevent suicide. NK cells carry the activating receptor 2B4 and the ligand CD48 for 2B4; MHC Class I may bind to 2B4 and prevent CD48 from activating it. Betser-Cohen (2010) The Association of MHC Class I Proteins with the 2B4 Receptor Inhibits Self-Killing of Human NK Cells, J. Immunol. 184: 2761-2768.

[0178] Therefore, the genetically modified non-human animals described herein can be used to study these non-TCR or non-CTL mediated processes and to design methods for their modulation. Example

[0179] The application will further illustrate the invention by the following non-limiting examples. These embodiments are listed to assist in explaining the present invention, but are not intended to and should not be construed as limiting its scope in any way. The embodiments do not include the description of conventional methods (molecular cloning techniques, etc.) known to those of ordinary skill in the art. Unless otherwise expressly stated, parts are parts by weight, molecular weight is average molecular weight, and temperature is expressed in degrees Celsius and pressure is at or near atmospheric pressure.

[0180] Example 1: Construction and characterization of genetically modified HLA-A2 mice

[0181] Example 1:1: Expression of HLA-A2 / H-2K in MG87 cells.

[0182] Standard molecular cloning techniques known to those skilled in the art were used to prepare a chimeric gene containing the HLA-A2 / H-2K gene sequence ( Figure 4 A) to analyze the expression of human / mouse chimeric MHC I in transfected cells.

[0183] Briefly, human HLA-A / mouse H-2K chimeric virus constructs were prepared using exon sequences encoding the α1, α2, and α3 domains of the α chain and cloned in frame with the transmembrane and cytoplasmic domain coding sequences of the mouse H-2K gene ( Figure 4 A, pMIG-HLA-A2 / H2K). Figure 4 As shown, the construct contains an IRES-GFP receptor sequence, which can be used to determine whether the construct can be expressed in transfected cells.

[0184] Prepare and amplify the virus containing the chimeric construct described above in human embryonic kidney 293 (293T) cells. 293T cells are inoculated in 10cm culture dishes and grown to 95% fusion. Use 25μg pMIG-HLA-A2 / H2K, pMIG-human HLA-A2 or pMIG-humanized β2 microglobulin and 5μg pMDG (envelope plasmid), 15μg pCL-Eco (packaging construct without packaging signal Ψ), 1mL Opti-MEM (Invitrogen) to prepare DNA transfection mixture. 1mL of this DNA mixture that has been mixed together in advance and incubated at room temperature for 5 minutes is added to 1mL Opti-MEM containing 80μL Lipofectamine-2000 (Invitrogen). The Lipofectamine / DNA mixture is continued to be incubated at room temperature for 20 minutes, then added to a 10cm culture dish, and the culture dish is incubated at 37°C. After 24 hours, the cell culture medium was collected and 10 mL of fresh R10 (RPMI 1640 + 10% FBS) medium was added to the cells. The medium was replaced twice. After a total of 4 days, the collected culture medium was mixed, centrifuged and filtered using a sterile filter to remove cell debris.

[0185] MG87 (mouse fibroblast) cells were transduced using the amplified virus prepared above. MG87 cells from a single T-75 culture flask were washed once with PBS. 3 mL of 0.25% trypsin + EDTA was added to the cells and incubated at room temperature for 3 minutes. 7 mL of D10 (high glucose DMEM; 10% fetal bovine serum) was added to the cell / trypsin mixture and transferred to a 15 mL test tube and centrifuged at 1300 rpm for 5 minutes. After centrifugation of the cells, the culture medium was aspirated and the cells were resuspended in 5 mL of D10. The cells were counted and the concentration was ~3.0 x 10 5 Cells were seeded per well in a 6-well plate. pMIG-human HLA-A2 or pMIG-HLA-A2 / H-2K were added to the wells alone or together with pMIG-humanized β2 microglobulin virus, and non-transduced cells were used as controls. The cells were incubated at 37°C 5% CO2 for 2 days. Cells were prepared for FACS analysis of HLA-A2 expression in the presence or absence of β2 microglobulin (using anti-HLA-A2 antibody, clone BB7.2).

[0186] picture( Figure 4 B) and a table summarizing the data in the graph ( Figure 4 The results in C) show that co-transfection with humanized β2 microglobulin increases the expression of human HLA-A2 or human / non-human chimeric HLA-A2 / H-2K, as shown by the rightward shift of the curve.

[0187] Example 1.2: Engineering a chimeric HLA-A2 / H-2K locus.

[0188] Use The mouse H-2K gene was humanized in one step using a unique targeting vector derived from human and mouse bacterial artificial chromosome (BAC) DNA using the technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659). DNA from the mouse BAC clone RP23-173k21 (Invitrogen) was modified by homologous recombination with human genomic DNA encoding the α1, α2, and α3 subunits of the human HLA-A gene to replace the genomic DNA encoding the mouse H-2K gene α1, α2, and α3 domains. Figure 5 ).

[0189] Briefly, human genomic DNA encoding the α1, α2, and α3 domains of the human HLA-A*0201 gene was used to replace the genomic sequences encoding the α1, α2, and α3 subunits of the mouse H-2K gene in a single targeting event using a targeting vector comprising a hygromycin expression cassette flanked by loxP sites, the hygromycin expression cassette having a 5' mouse homology arm comprising the 5' sequence of the mouse H-2K locus including the 5' untranslated region (UTR; the 5' homology arm is set forth in SEQ ID NO: 1), and a 3' mouse homology arm comprising the 3' genomic sequence of the mouse H-2K α3 coding sequence (the 3' homology arm is set forth in SEQ ID NO: 2).

[0190] The final construct for targeting the endogenous H-2K locus included, from 5' to 3': (1) a 5' homology arm containing ∼200 bp of mouse 5' genomic sequence of the endogenous H-2K gene including the 5' UTR, (2) ∼1339 bp of human genomic sequence including the HLA-A*0201 leader sequence, the HLA-A*0201 leader / α1 intron, the HLA-A*0201α1 exon, the HLA-A*0201α1-α2 intron, the HLA-A *0201α2 exon, the 5' end of the ~316 bp α2-α3 intron, (3) a 5' loxP site, (4) a hygromycin expression cassette, (5) a 3' loxP site, (6) a ~580 bp human genomic sequence including the 3' end of the ~304 bp α2-α3 intron, the HLA-A*0201α3 exon, and (7) a 3' homology arm comprising ~200 bp of mouse genomic sequence including the intron between the mouse H-2Kα3 and transmembrane coding sequences (see Figure 5 Schematic diagram of the targeting vector for H-2K). The sequence of 140 nucleotides at the mouse / human sequence junction at the 5' end of the targeting vector is shown in SEQ ID NO: 3, and the sequence of 159 nucleotides at the human / mouse sequence junction at the 3' end of the targeting vector is shown in SEQ ID NO: 4. The targeting vector was used for homologous recombination to obtain a modified mouse H-2K locus, which contains human genomic DNA encoding the α1, α2 and α3 domains of the HLA-A*0201 gene operably linked to the coding sequence of the endogenous mouse H-2K transmembrane and cytoplasmic domains, which will form a human / mouse chimeric MHC class I protein after translation.

[0191] Mouse F1H4 ES cells were electroporated with the BAC DNA of interest to form modified ES cells for generating mice with chimeric MHC class I proteins expressed on the surface of nucleated cells (e.g., T and B lymphocytes, macrophages, neutrophils). TM Detection and identification of ES cells containing human HLA sequence insertion. Specific primer pairs and probes were designed to detect the insertion of human HLA sequences and related screening gene expression cassettes (gain allele GOA) and the deletion of endogenous mouse sequences (deletion allele LOA). Table 1 shows the names of the probes used in the quantitative PCR test and the detection sites.

[0192] Table 1: Probes used to detect genotypes

[0193]

[0194] The selection gene expression cassette can be removed by methods known in the art. For example, ES cells carrying a human / mouse chimeric MHC class I locus can be transfected with a construct expressing Cre to remove the "loxed" hygromycin expression cassette introduced by the targeting construct containing the human HLA-A*0201 gene sequence (see Figure 5 ). The hygromycin expression cassette can optionally be removed by mating with a mouse expressing Cre recombinase. Optionally, the hygromycin expression cassette is retained in the mouse.

[0195] The targeted ES cells described above were used as donor ES cells and The chimeric MHC class I gene was introduced into 8-cell stage mouse embryos using a modified allele detection method (Valenzuela et al., supra) (see, e.g., U.S. Pat. No. 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). (F0 mice were derived entirely from donor ES cells) and were genotyped for the presence of the unique human HLA-A*0201 gene sequence.

[0196] Example 1.3: In vivo expression of chimeric HLA-A / H-2K in genetically modified mice

[0197] Heterozygous mice carrying the genetically modified H-2K locus described in Example 1.2 were analyzed to detect expression of the chimeric HLA-A / H-2K protein in the cells of said animals.

[0198] Blood samples were collected from wild-type and HLA-A / H-2K chimeric heterozygous (A2 / H2K) mice. Human HLA-A2 in cells was stained using phycoerythrin (PE)-conjugated anti-HLA-A antibody, and cells were exposed to allophycocyanin-conjugated anti-H-2Kb antibody for 1 hour at 4°C. Cell expression was analyzed by flow cytometry using antibodies specific for HLA-A and H-2Kb. Fig. 6A Shown are the expression of H-2Kb and HLA-A2 in wild type and chimeric heterozygotes expressing both proteins. Figure 6B The expression of H-2Kb and chimeric HLA-A2 / H2K in heterozygous mice is shown.

[0199] Example 2: Construction and characterization of genetically modified β2 microglobulin mice

[0200] Example 2.1: Engineering a humanized β2 microglobulin locus.

[0201] Use The mouse β2 microglobulin (β2m) gene was humanized in one step using a unique targeting vector derived from human and mouse bacterial artificial chromosome (BAC) DNA using the PCR technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., supra).

[0202] Briefly, the targeting vector was generated by bacterial homologous recombination containing the mouse β2m upstream and downstream homology arms from the RPCI-23 library (Invitrogen) BAC clone 89C24. The mouse homology arms were engineered to flank the 2.8 kb human β2m DNA fragment ( Figure 7 ). A drug selection gene expression cassette (neomycin) flanking the recombinase recognition site (e.g., loxP site) is engineered into the targeting vector for subsequent selection. The final targeting vector is linearized and electroporated into the F1H4 mouse ES cell line (Valenzuela et al., supra).

[0203] pass Targeted ES cell clones that have been depleted of the drug expression cassette (by introduction of Cre recombinase) are introduced into 8-cell stage mouse embryos (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al., supra). Flocks carrying the humanized β2m gene are identified by screening for loss of the mouse allele and gain of the human allele using a modified allele assay (Valenzuela et al., supra). (F0 mice are derived entirely from donor ES cells).

[0204] Example 2.2: Characterization of humanized β2 microglobulin mice

[0205] Expression of the humanized β2-microglobulin (β2m) gene in mice heterozygous for the gene was assessed using flow cytometry. Figure 8 and Fig. 9 ).

[0206] Briefly, blood samples were isolated from groups of wild-type, humanized β2m, humanized MHC (i.e., human HLA) class I, and β2m and MHC class I double humanized mice (n=4 per group) using techniques known in the art. Blood samples from each mouse in each group were treated with ACK lysis buffer (Lonza Walkersville) to remove red blood cells. The remaining cells were stained with fluorescent dye-conjugated anti-CD3 (17A2), anti-CD19 (1D3), anti-CD11b (M1 / 70), anti-human HLA class I, and anti-human β2 microglobulin (2M2) antibodies. BD-FACSCANTO TM (BD Biosciences) for flow cytometry.

[0207] Human HLA class I expression was measured in cells from singly and doubly humanized animals, whereas β2-microglobulin expression was measured only in cells from doubly humanized mice ( Figure 8 Compared to the expression of human HLA class I in the absence of human β2m, co-expression of human β2m and human HLA class I increased the amount of human HLA class I detectable on the cell surface ( Fig. 9 , mean fluorescence intensity 2370 vs. 1387).

[0208] Example 3: Immune responses to Flu and Epstein-Barr virus (EBV) peptides presented by APCs from genetically modified mice expressing HLA-A2 / H-2K and humanized β2 microglobulin.

[0209] PBMCs from several human donors were screened for expression of HLA-A2 and their ability to mount responses to flu and EBV peptides. A single donor was selected for subsequent experiments.

[0210] Human T cells were isolated from PBMCs of selected donors using counter selection. Splenic non-T cells were isolated from mice that were heterozygous for chimeric HLA-A2 / H-2K and heterozygous for humanized β2-microglobulin genes, as well as wild-type mice. Approximately 50,000 splenic non-T cells from mice were added to Elispot plates coated with anti-human IFNγ antibodies. Flu peptide (10 μM) or a mixture of EBV peptides (5 μM each) was added. Poly IC, 25 μg / well, was added and the wells were incubated at 37°C with 5% CO. 2 Human T cells (50,000) and anti-human CD28 were added to the spleen non-T cells and the peptides, and the wells were incubated at 37°C and 5% CO2 for 40 hours, followed by IFNγ Elispot detection.

[0211] like Fig.10As shown, human T cells were able to mount responses against flu and EBV peptides when presented by mouse APCs expressing chimeric HLA-A2 / H-2K and humanized β2 microglobulin on their surface.

[0212] Equivalent

[0213] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0214] The entire contents of non-patent documents, patent applications, and patents cited throughout this application are hereby incorporated by reference into this application in their entirety. Sequence Listing <110> Razerne Pharmaceuticals <120> Method for preparing major histocompatibility complex gene-modified mice <130> 0825A-WO <150> 61 / 552,582 <151> 2011-10-28 <150> 61 / 552,587 <151> 2011-10-28 <150> 61 / 700,908 <151> 2012-09-14 <160> 8 <170> PatentIn Version 3.5 <210> 1 <211> 200 <212> DNA <213> Artificial <220> <223> 5' homology arm of the MHC I targeting construct <400> 1 ggattcccca tctccacagt ttcacttctg cacctaacct gggtcaggtc cttctgtccg 60 gacactgttg acgcgcagtc agctcttacc cccattgggt ggcgcgatca cccaagaacc 120 aatcagtgtc gccgcggacg ctggatataa agtccacgca gcccgcagaa ctcagaagtc 180 gcgaatcgcc gacaggtgcg 200 <210> 2 <211> 200 <212> DNA <213> Artificial <220> <223> 3' homology arm of the MHC I targeting construct <400> 2 gtaaggagag tgtgggtgca gagctggggt cagggaaagc tggagctttc tgcagaccct 60 gagctgctca gggctgagag ctggggtcat gaccctcacc ttcatttctt gtacctgtcc 120 ttcccagc ctcctccatc cactgtctcc aacatggcga ccgttgctgt tctggttgtc 180 cttggagctg caatagtcac 200 <210> 3 <211> 149 <212> DNA <213> Artificial <220> <223> Sequence of the human / mouse chimeric MHC I locus at the 5' junction of the mouse / human sequence <400> 3 agtgtcgccg cggacgctgg atataaagtc cacgcagccc gcagaactca gaagtcgcga 60 atcgccgaca ggtgcgatgg ccgtcatggc gccccgaacc ctcgtcctgc tactctcggg 120 ggctctggcc ctgacccaga cctgggcgg 149 <210> 4 <211> 159 <212> DNA <213> Artificial <220> <223> Sequence of the human / mouse chimeric MHC I locus at the 3' junction of the mouse / human sequence <400> 4 ggtggtgcct tctggacagg agcagagata cacctgccat gtgcagcatg agggtttgcc 60 caagcccctc accctgagat ggggtaagga gagtgtgggt gcagagctgg ggtcagggaa 120 agctggagct ttctgcagac cctgagctgc tcagggctg 159 <210> 5 <211> twenty one <212> DNA <213> Artificial <220> <223> Probe for detection of hygromycin expression cassette <400> 5 acgagcgggt tcggcccatt c 21 <210> 6 <211> 27 <212> DNA <213> Artificial <220> <223> Probe for detecting the human HLA-A2 α2-α3 intron <400> 6 agtccttcag cctccactca ggtcagg 27 <210> 7 <211> 25 <212> DNA <213> Artificial <220> <223> Probe for detecting human HLA-A2 α2-exon <400> 7 taccaccagt acgcctacga cggca 25 <210> 8 <211> 18 <212> DNA <213> Artificial <220> <223> Probe for detecting the human HLA-A2 α2-α3 intron <400> 8 atcctgtacc agagagtg 18

Claims

1. A rodent genome, said genome comprising a nucleic acid sequence at an endogenous β2 microglobulin locus, said nucleic acid sequence comprising exon 1 of a rodent β2 microglobulin gene and exons 2, 3 and 4 of a human β2 microglobulin gene, wherein said nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of human β2 microglobulin, wherein said rodent is a rat or a mouse. 2 . The rodent genome according to claim 1 , wherein the nucleic acid sequence comprises a human genomic sequence from exon 2 to exon 4 of the human β2 microglobulin gene.

3. The rodent genome of claim 1, wherein the nucleic acid sequence encodes a polypeptide comprising amino acids 23-119 of human β2 microglobulin.

4. The rodent genome of claim 1, wherein the nucleic acid sequence is operably linked to endogenous rodent β2 microglobulin regulatory elements.

5. The rodent genome of claim 1, wherein the rodent genome does not express a functional endogenous β2 microglobulin polypeptide from an endogenous β2 microglobulin locus.

6. A mouse genome comprising a nucleic acid sequence at an endogenous β2 microglobulin gene locus, the nucleic acid sequence (1) comprising exon 1 of a mouse β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene, and (2) operably linked to a mouse β2 microglobulin regulatory element, The nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of human β2 microglobulin.

7. The mouse genome according to claim 6, wherein the nucleic acid sequence comprises the human genomic sequence of exon 2 to exon 4 of the human β2 microglobulin gene.

8. The mouse genome of claim 6, wherein the nucleic acid sequence encodes a polypeptide of amino acids 23-119 of human β2 microglobulin.

9. The mouse genome of any one of claims 6-8, wherein the mouse genome does not encode a functional endogenous mouse β2 microglobulin polypeptide at the endogenous β2 microglobulin locus.

10. A method for genetically modifying mice, comprising replacing a nucleic acid sequence comprising exons 2, 3, and 4 of a mouse β2 microglobulin gene with a nucleic acid sequence comprising exons 2, 3, and 4 of a human β2 microglobulin gene at an endogenous β2 microglobulin locus, wherein exon 1 of the mouse β2 microglobulin gene is operably linked to the nucleic acid sequence comprising exons 2, 3, and 4 of the human β2 microglobulin gene.

11. The method according to claim 10, wherein the method comprises replacing the genomic sequence of exon 2 to exon 4 of the mouse β2 microglobulin gene with a nucleic acid sequence comprising the genomic sequence of exon 2 to exon 4 of the human β2 microglobulin gene at the endogenous β2 microglobulin locus.

12. The method of claim 10, wherein the mouse does not express a functional mouse β2 microglobulin polypeptide from the endogenous β2 microglobulin locus.

13. The method according to any one of claims 10-12, wherein the replacement is performed in a single embryonic stem (ES) cell, and the single ES cell is introduced into a mouse embryo to produce a mouse.

14. A nucleic acid encoding a functional β2 microglobulin polypeptide, wherein the nucleic acid comprises exon 1 of a rodent β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene, and wherein the rodent is a rat or a mouse. 15 . The nucleic acid according to claim 14 , wherein the nucleic acid comprises the genomic sequence of exon 2 to exon 4 of the human β2 microglobulin gene.

16. The nucleic acid of claim 14, wherein the functional β2 microglobulin polypeptide comprises amino acids 23-119 of human β2 microglobulin.

17. The nucleic acid of claim 14, wherein the nucleic acid is operably linked to a rodent β2 microglobulin regulatory element.

18. The nucleic acid of claim 14, wherein the nucleic acid is operably linked to a rat β2 microglobulin regulatory element.

19. The nucleic acid of claim 14, wherein the nucleic acid is operably linked to a mouse β2 microglobulin regulatory element.

20. A functional β2 microglobulin polypeptide encoded by the nucleic acid of any one of claims 14-19, comprising amino acids 23-119 of human β2 microglobulin protein.

21. The functional β2 microglobulin polypeptide according to claim 20, wherein the functional β2 microglobulin polypeptide is bound to an MHC I protein.

22. An isolated cell comprising the nucleic acid of any one of claims 14-19.

23. The isolated cell of claim 22, wherein the cell is an embryonic stem cell.

24. The isolated cell of claim 22, wherein the cell expresses the functional β2 microglobulin polypeptide on its surface.

25. An in vitro composition comprising the isolated cells of any one of claims 22-24.

26. The in vitro composition of claim 25, wherein the isolated cells express the functional β2 microglobulin polypeptide bound to an MHC I protein, and wherein the cells are complexed with human or rodent T cells.

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