RU2783984C2 - Genetically modified mhistocompatibility complex mice - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a rodent containing in the endogenous microglobulin β2 locus a nucleic acid sequence containing exon 1 of the endogenous rodent β2 microglobulin gene, functionally linked to a nucleic acid containing sequences presented in exon 2, exon 3 and exon 4 genes β2 human microglobulin.

EFFECT: invention is effective as a biological system that can create an immune system that exhibits components that mimic the function of the human immune system.

6 cl, 10 dwg, 1 tbl, 3 ex

Description

Links to related applications

This application claims priority under U.S. Provisional Applications Nos. 61/552,582 and 61/552,587, filed October 28, 2011, and U.S. Provisional Application No. 61/700,908, filed September 14, 2012, the entire contents of which are incorporated herein by reference.

The field of technology to which the present invention relates

The present invention relates to a genetically modified non-human animal, such as a rodent (eg, mouse or rat), that expresses a human or humanized major histocompatibility complex (MHC) class I molecule. The present invention also relates to a genetically modified non-human animal, such as a mouse or rat, that expresses a human or humanized MHC I protein (eg MHC I α chain) and/or a human or humanized β2 microglobulin; as well as embryos, tissues and cells expressing them. The present invention further relates to methods for producing a genetically modified non-human animal that expresses a human or humanized MHC class I protein (eg MHC I α chain) and/or β2 microglobulin. Also provided are methods for identifying and evaluating peptides in the context of a humanized cellular immune system in vitro or in a genetically modified non-human animal, and methods for modifying the MHC I locus and/or the microglobulin β2 locus of a non-human animal, e.g., mouse or rat, to expression of human or humanized MHC I and/or β2 microglobulin.

Background of the Invention

In the acquired immune response, foreign antigens are recognized by receptor molecules on B-lymphocytes (eg, immunoglobulins) and T-lymphocytes (eg, the T-cell receptor, or TCR). These foreign antigens are presented on the cell surface as peptide fragments by specialized proteins collectively referred to as the major histocompatibility complex (MHC) molecule. MHC molecules are encoded by multiple loci that occur as a connected gene cluster that spans approximately 4 Mb. In mice, the MHC genes are located on chromosome 17 and historically they are called histocompatibility 2 (H-2) genes. In humans, the genes are located on chromosome 6 and are called human leukocyte antigen (HLA) genes. Loci in mice and humans are polygenic; they include three highly polymorphic classes of MHC genes (class I, II and III) that exhibit similar organization in the human and mouse genomes (see Fig. 2 and Fig. 3, respectively).

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

Both humans and mice contain MHC class I genes (see Figures 2 and 3). In humans, the classic MHC I genes are called HLA-A, HLA-B, and HLA-C, while in mice they are H-2K, H-2D, and H-2L. Class I molecules are composed of two polypeptide chains: a polymorphic α chain (called a heavy chain in some cases) and a smaller chain called β2 microglobulin (also known as a light chain), which is mostly non-polymorphic (Fig. 1). These two chains form a non-covalent heterodimer 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 groove comprising α1 and α2 domains (which resemble Ig-like domains) followed by an α3 domain, which is analogous to β2 microglobulin.

β2 microglobulin is a non-glycosylated 12 kDa protein; one of its functions is to stabilize the α chain of MHC class I. Unlike the α chain, β2 microglobulin does not cross the membrane. The human microglobulin β2 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 biological fluids; thus, non-covalently bound to MHC I β2 microglobulin can be exchanged for circulating β2 microglobulin under physiological conditions.

Class I MHC molecules are expressed on all nucleated cells, including tumor cells. They are expressed specifically on T and B lymphocytes, macrophages, dendritic cells and neutrophils, among other cells, and function to present peptide fragments (typically 8-10 amino acids in length) on the surface of CD8+ cytotoxic T lymphocytes (CTLs). CTLs are specialized in the cytolysis of any cell that carries an MHC I-related peptide recognized by its own membrane-bound TCR. When a cell presents peptides derived from cellular proteins not normally present (eg, viral, tumor, or other non-self-originating), such peptides are recognized by CTLs, which activate and lyse the peptide-presenting cell.

As a rule, the presentation of normal (ie self) proteins in the context of MHC I molecules does not cause CTL activation due to tolerance mechanisms. However, in some diseases (eg, cancer, autoimmune diseases), self-derived peptides are targeted by the cellular component of the immune system, resulting in destruction of the cells presenting such peptides. Despite advances in recognition of self antigens that elicit a cellular immune response (e.g., antigens associated with various cancers), there remains a need for both in vivo and in vitro that mimic aspects of the human cellular immune system. Systems that mimic the human cellular immune system can be used in the identification of disease-associated antigens to develop human therapeutics, such as vaccines and other biological agents. Antigen recognition scoring systems in the context of the human immune system can assist in the identification of therapeutically useful CTL populations (eg, those useful for human disease research and control). Such systems can also assist in enhancing the activity of human CTL populations to more effectively fight infections and foreign antigen-bearing compounds. Thus, there is a need for biological systems (eg, genetically engineered animals) that can create an immune system that exhibits components that mimic the function of the human immune system.

Brief summary of the present invention

A biological system is provided for generating or identifying peptides that associate with human MHC class I proteins and their chimeras and bind to CD8+ T cells. Non-human animals are provided containing non-human cells that express human or humanized molecules that function in a cellular immune response. Also provided are humanized rodent loci that encode human or humanized MHC I and microglobulin β2 proteins. Also provided are humanized rodent cells that express human or humanized MHC and β2 microglobulin molecules. In vivo and in vitro systems are provided that contain humanized rodent cells, wherein the rodent cells express one or more human or humanized immune system molecules.

Provided herein is a non-human animal, e.g., a rodent (e.g., mouse or rat), having in its genome a nucleotide sequence encoding a chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide, wherein the human portion of the chimeric polypeptide contains the extracellular domain of the human MHC I polypeptide. In particular, provided herein is a non-human animal comprising, at an endogenous MHC I locus, a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of a human MHC I polypeptide, and wherein the animal expresses a chimeric human/non-human MHC I polypeptide. In one aspect, the animal does not express the extracellular domain of an endogenous non-human MHC I polypeptide from an endogenous non-human MHC I locus. In one aspect of the present invention, a non-human animal (e.g. , rodent, e.g., mouse or rat) contains two copies of the MHC I locus containing a nucleotide sequence encoding a chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide In another aspect of the present invention, the animal contains one copy of the MHC I locus containing a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide. chimeric human/non-human MHC I polypeptide. In various embodiments, the nucleotide sequence encoding the chimeric human/non-human MHC I polypeptide is contained in the germline of a non-human animal (e.g., a rodent, e.g., a rat or mouse) .

In one aspect, the nucleotide sequence encoding the chimeric human/non-human MHC I is operably linked to endogenous non-human regulatory elements, e.g., a promoter, enhancer, silencer, etc. In one embodiment, the human portion of the chimeric polypeptide contains a human leader sequence. In a further 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.

In one aspect, the genetically engineered non-human animal is a rodent. In one embodiment, the rodent is a mouse. Thus, in one embodiment, the endogenous non-human locus is a mouse locus, eg, a mouse H-2K, H-2D, or H-2L locus. In one embodiment, the non-human portion of the chimeric human/non-human MHC I polypeptide comprises the transmembrane and cytoplasmic domains of the endogenous non-human MHC I polypeptide. Thus, in the embodiment wherein the non-human animal is a mouse , the endogenous non-human MHC I locus may be an H-2K locus (eg, the H-2Kb locus), and the endogenous non-human MHC I polypeptide may be an H-2K polypeptide; therefore, a chimeric human/non-human MHC I polypeptide may contain 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; therefore, a chimeric human/non-human MHC I polypeptide may contain the transmembrane and cytoplasmic domains of the H-2D polypeptide. Similarly, in another embodiment, the endogenous non-human MHC I locus may be an H-2L locus and the endogenous non-human MHC I polypeptide may be an H-2L polypeptide; therefore, a chimeric human/non-human MHC I polypeptide may contain the transmembrane and cytoplasmic domains of the H-2L polypeptide.

Also provided herein is a mouse comprising, at an endogenous H-2K locus, a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the human portion of the chimeric polypeptide contains the HLA-A extracellular domain of a human polypeptide (e.g., HLA-A2), and related to the mouse portion contains the transmembrane and cytoplasmic domains of a mouse H-2K polypeptide, and wherein the mouse expresses a chimeric human/mouse 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 the chimeric human/mouse MHC I polypeptide is operably linked to endogenous mouse regulatory elements. The human portion of the chimeric polypeptide may contain a human leader sequence. It may also contain the α1, α2 and α3 domains of a human MHC I polypeptide. The human MHC I polypeptide may be an HLA-A polypeptide, for example an HLA-A2.1 polypeptide. In one aspect, the mouse H-2K locus is the H-2Kb locus.

Another aspect of the present invention relates to a non-human animal, such as a rodent (eg mouse or rat), containing in its genome a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide. Thus, provided herein is a non-human animal comprising, in an endogenous non-human microglobulin β2 locus, a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide, wherein the animal expresses a human or humanized microglobulin β2 polypeptide. In one aspect, the animal does not express a functional endogenous non-human microglobulin β2 polypeptide from an endogenous non-human β2 microglobulin locus. In one aspect, the animal contains two copies of the β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide; in another embodiment, the animal contains one copy of the β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. Thus, an 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 germline of a non-human animal (eg, a rodent, eg, a rat or mouse). In one embodiment, the nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to the MHC I protein.

In some embodiments, a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide is operably linked to endogenous non-human microglobulin β2 regulatory elements. In one aspect, the nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide comprises a nucleotide sequence present in exon 2 to exon 4 of the human β2 microglobulin gene. In another aspect, a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide comprises nucleotide sequences present in exons 2, 3, and 4 of the human β2 microglobulin gene. In a further aspect, the nucleotide sequence also comprises a nucleotide sequence present in exon 1 of the non-human microglobulin β2 gene. In some embodiments, the non-human animal is a rodent (eg, a mouse or rat); thus, a non-human microglobulin β2 locus is a rodent (eg, mouse or rat) β2 microglobulin locus.

Also provided is a mouse comprising, at an endogenous microglobulin β2 locus, a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide, wherein the mouse expresses the human or humanized microglobulin β2 polypeptide. In some embodiments, the mouse does not express functional endogenous mouse β2 microglobulin from the endogenous β2 microglobulin locus. The nucleotide sequence can be linked to endogenous mouse regulatory elements. In one aspect, the nucleotide sequence comprises a nucleotide sequence present in exon 2 - exon 4 of the human microglobulin β2 gene. Alternatively, the nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide may comprise nucleotide sequences present in exons 2, 3, and 4 of the human β2 microglobulin gene. The nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide may further comprise the nucleotide sequence of exon 1 of the murine β2 microglobulin gene. In one embodiment, a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to the MHC I protein.

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

Therefore, the present invention provides a mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of human HLA-A (e.g., HLA-A2), and the murine portion contains a transmembrane and mouse H-2K cytoplasmic domains; and a second nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide, wherein the first nucleotide sequence is located at the endogenous H-2K locus and the second nucleotide sequence is located at the mouse endogenous β2 microglobulin locus, and wherein the mouse expresses the chimeric human/mouse MHC I polypeptide and human or a humanized β2 microglobulin polypeptide. In one embodiment, a non-human animal (e.g., a mouse) containing both a chimeric MHC I polypeptide and a human or humanized microglobulin β2 polypeptide does not express the extracellular domain of an endogenous non-human MHC I polypeptide (e.g., an H-2K polypeptide mice) and/or functional endogenous non-human (eg, murine) microglobulin β2 polypeptides from their respective endogenous loci. In one aspect, the animal (eg, mouse) contains two copies of each of the first and second nucleotide sequences. According to another aspect, the animal (eg, mouse) contains one copy of the first and one copy of the second nucleotide sequences. Thus, the animal may be homozygous or heterozygous for both the first and second nucleotide sequences.

In one aspect, the first nucleotide sequence is operably linked to endogenous non-human (eg, murine) MHC I regulatory elements and the second nucleotide sequence is operably linked to endogenous non-human (eg, murine) microglobulin β2 elements. The human portion of the chimeric polypeptide may contain the α1, α2 and α3 domains of a human MHC I polypeptide. The second nucleotide sequence may comprise a nucleotide sequence present in exon 2 to exon 4 of the human microglobulin β2 gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene. In one aspect, a mouse comprising both a chimeric MHC I polypeptide and a human or humanized β2 microglobulin polypeptide can be such that expression of the human or humanized β2 microglobulin increases expression of the chimeric human/mouse MHC I polypeptide relative to expression of the chimeric human/mouse polypeptide MHC I in the absence of human or humanized β2 microglobulin polypeptide expression.

Also provided are methods for producing genetically engineered non-human animals (eg, rodents, eg, mice or rats) as described herein. Thus, in one embodiment, a method is provided for modifying a rodent (e.g., mouse or rat) MHC I locus to express a chimeric human/rodent (e.g., human/mouse or human/rat) MHC I polypeptide, the method comprising a substitution in an endogenous the MHC I locus of a nucleotide sequence encoding the extracellular domain of a rodent MHC I polypeptide, a nucleotide sequence encoding the extracellular domain of a human MHC I polypeptide. In another embodiment, a method is provided for modifying a rodent (e.g., mouse or rat) β2 microglobulin locus to express a human or humanized β2 microglobulin polypeptide, the method comprising replacing an endogenous rodent (e.g., mouse or rat) β2 microglobulin locus with a nucleotide sequence encoding a β2 polypeptide a rodent (eg, mouse or rat) microglobulin), a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide. According to such methods, replacement can be carried out in one ES (embryonic stem) cell, and one ES cell can be introduced into a rodent (eg, mouse or rat) to produce an embryo. The resulting rodent (eg, mouse or rat) can be crossed to produce a doubly humanized animal.

Thus, the present invention also relates to methods for producing doubly humanized animals, such as rodents (eg, mice or rats). In one embodiment, a method of producing a genetically modified mouse is provided, comprising (a) modifying a first mouse MHC I locus to express a chimeric human/mouse MHC I polypeptide, comprising replacing, in an endogenous mouse MHC I locus, a nucleotide sequence encoding an extracellular domain of a mouse MHC I polypeptide, a nucleotide sequence encoding the extracellular domain of a human MHC I polypeptide, (b) modifying a second mouse β2 microglobulin locus to express a human or humanized β2 microglobulin polypeptide, comprising substituting in the endogenous murine β2 microglobulin locus a nucleotide sequence encoding a mouse β2 microglobulin polypeptide with a nucleotide sequence encoding human or humanized β2 microglobulin polypeptide; and (c) crossing the first and second mice to produce a genetically modified mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide and a second nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide, wherein the genetically modified mouse expresses a chimeric human/mouse 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 an 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 polypeptide /H-2K). In one aspect, a chimeric HLA-A2/H-2K polypeptide comprises an extracellular domain of an HLA-A2 polypeptide and cytoplasmic and transmembrane domains of an H-2K polypeptide. In one aspect, the second nucleotide sequence comprises nucleotide sequences present in exons 2, 3, and 4 (eg, exon 2 to exon 4) of the human β2 microglobulin gene and a nucleotide sequence present in exon 1 of the mouse β2 microglobulin gene.

Also provided herein are cells, eg, isolated antigen-presenting cells, derived from non-human animals described herein (eg, rodents, eg, mice or rats). Also contemplated are tissues and embryos derived from non-human animals described herein.

According to another embodiment, the present invention relates to methods for identifying antigens or antigenic epitopes that elicit an immune response, methods for evaluating a vaccine candidate, methods for identifying high affinity T cells for human pathogens or cancer antigens.

Any of the embodiments and aspects described herein may be used in conjunction with each other unless otherwise indicated or evident from the context. Other embodiments will become apparent to those skilled in the art from an overview of the following detailed disclosure. The following detailed disclosure includes illustrative representations of various embodiments of the present invention, which do not limit the claimed present invention. The accompanying figures form part of the present description of the invention and together with the description serve solely to illustrate the embodiments and not to limit the present invention.

Brief description of graphic materials

In FIG. 1 is a schematic representation of the four domains of the MHC class I molecule: an α chain containing α1, α2 and α3 domains and a non-covalently bound fourth domain, β2 microglobulin (β2m). The gray circle represents the peptide bound in the peptide-binding groove.

In FIG. 2 is a schematic representation (not to scale) of the relative genomic structure of human HLA showing class I, II and III genes.

In FIG. 3 is a schematic representation (not to scale) of the relative genomic structure of mouse MHC showing class I, II and III genes.

In FIG. 4 illustrates a viral vector construct containing a cDNA encoding a chimeric HLA-A/H-2K polypeptide with an IRES-GFP reporter (A); and histograms comparing human HLA-A2 expression in MG87 cells transduced with HLA-A2 (dashed line), HLA-A2/H-2K (dotted line) or without transduction (solid line) or alone (left), or cotransduced with humanized β2 microglobulin (right) (B). Data from the horizontal intervals plotted in (B) are shown as the percentage of cells expressing the construct in the table in (C).

In FIG. 5 is a schematic diagram (not to scale) of the targeting strategy used to generate a chimeric H-2K locus that expresses the extracellular region of the human HLA-A2 protein. Mouse sequences are represented in black and human sequences are represented in white. L=leader sequence, UTR=untranslated region, TM=transmembrane domain, CYT=cytoplasmic domain, HYG=hygromycin.

In FIG. 6A shows expression (% of total cells) of HLA-A2 (left) and H-2K (right) in cells isolated from either wild-type (WT) or heterozygous (HET) mice carrying the HLA-A2 chimeric locus. /H-2K (HLA-A/H-2K NO).

In FIG. 6B is a dot plot of in vivo expression of the HLA-A2/H-2K chimeric protein in a heterozygous mouse carrying the HLA-A2/H-2K chimeric protein.

In FIG. 7 shows a targeted (not to scale) strategy for humanizing the microglobulin β2 gene at the mouse β2 microglobulin locus. Mouse sequences are represented in black and human sequences are represented in white. NEO=neomycin.

In FIG. 8 shows a representative dot plot of HLA class I and human β2 microglobulin expression in cells isolated from the blood of wild-type (WT) mice, mice heterozygous for chimeric HLA-A2/H-2K, and mice heterozygous for chimeric HLA-A2. /H-2K and heterozygous for humanized β2 microglobulin (double heterozygotes; HET for class I /β2m).

In FIG. 9 shows a representative histogram of human HLA class I expression (X-axis) in cells isolated from the blood of wild-type (WT) mice, mice heterozygous for chimeric HLA-A2/H-2K (HET class I), and double heterozygous for chimeric HLA-A2/H2K/humanized β2 microglobulin (HET class I/β2m).

In FIG. 10 shows the results of IFNγ ELISA staining of human T cells exposed to antigen presenting cells (APCs) from wild type (APC WT) mice or mice heterozygous for both chimeric HLA-A2/H-2K and humanized β2 microglobulin ( double HET APCs) in the presence of influenza peptides (left) or EBV (Epstein-Barr virus) (right). Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test of multiple comparisons.

Detailed disclosure of the present invention

Definitions

The present invention relates to genetically modified non-human animals (eg, mice, rats, rabbits, etc.) that express human or humanized MHC I and/or β2 microglobulin polypeptides; embryos, cells and tissues containing them; ways to get them; as well as how they are used. Unless otherwise indicated, all terms and phrases used herein include the meanings that are meant by the terms and phrases in the present technical field, unless the opposite is clearly indicated or clearly follows from the context in which the term or phrase is used.

The term "conservative", used to describe a conservative amino acid substitution, includes the replacement of an amino acid residue with another amino acid residue with 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 so to introduce a nucleotide change that will encode a conservative substitution.In general, a conservative amino acid substitution will not significantly alter the functional properties of the protein of interest, such as the ability of MHC I to present the peptide of interest.Examples of amino acid groups that contain side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; such aromatic other side chains like phenylalanine, tyrosine and tryptophan; major side chains such as lysine, arginine and histidine; acid 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, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution may be a replacement of any native residue in a protein with an alanine, as used, for example, in alanine-scanning mutagenesis. In some embodiments, a conservative substitution is made, which is characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), incorporated herein by reference. In some embodiments, is a moderately conservative substitution, wherein the substitution is characterized by a non-negative value in the PAM250 log-likelihood matrix.

Thus, the present invention also provides a genetically modified non-human animal whose genome contains a nucleotide sequence encoding a human or humanized MHC I polypeptide and/or a microglobulin β2 polypeptide, wherein the polypeptide(s) contain(s) conservative amino acid substitutions of the described(s). ) in this document amino acid(s) sequence(s).

The person skilled in the art will appreciate that, in addition to nucleic acid residues encoding the human or humanized MHC I and/or β2 microglobulin polypeptide described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the polypeptide(s) of the present invention. Therefore, in addition to a genetically modified non-human animal that contains in its genome a nucleotide sequence encoding an MHC I and/or β2 microglobulin polypeptide(s) with conservative amino acid substitutions, a non-human animal whose genome contains a nucleotide sequence is also provided. (s) sequence(s) that(s) differ(s) from those described(s) herein due to the degeneracy of the genetic code.

The term “identity” when used in connection with a sequence includes identity as determined by a variety of algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are determined using ClustalW v. 1.83 (slow) alignment using a deletion opening penalty of 10.0, a deletion extension penalty of 0.1, and using a comparison matrix according to Gonnet (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared for sequence identity will depend on the particular sequences. In various embodiments, identity is determined by comparing the sequence of a mature protein in the direction from its N-terminus to its C-terminus. In various embodiments, when comparing a chimeric human/non-human sequence with a human sequence, the human portion of the chimeric human/non-human sequence (but not the non-human portion) is used in performing the comparison to determine the level of identity between the human sequence. and the human portion of the chimeric human/non-human sequence (eg, comparing the human ectodomain of a chimeric human/mouse protein with the human ectodomain of a human protein).

The terms "homology" or "homologous" in relation to sequences, for example, nucleotide or amino acid sequences, means two sequences that, when optimally aligned and compared, are identical in 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 nucleotides or amino acids, for example, more than 97% of nucleotides or amino acids. One of skill in the art will appreciate that for optimal gene targeting, the targeting construct must comprise arms that are homologous to endogenous DNA sequences (i.e., “homology arms”); thus, homologous recombination between the targeting construct and the targeted endogenous sequence can occur.

The term "operably linked" refers to a contiguous position, and the components thus described are in interaction, allowing them to function as intended for them. In this regard, a protein-coding nucleic acid sequence can be operably linked to regulatory sequences (eg, a promoter, enhancer, silencer, etc.) sequence so as to maintain proper transcriptional regulation. In addition, various parts of the chimeric or humanized protein of the present invention may be operably linked to maintain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless otherwise indicated, the various domains of a chimeric or humanized protein of the present invention are operably linked to one another.

As used herein, the term "MHC I complex" or the like includes a complex between an MHC I chain α polypeptide and a microglobulin β2 polypeptide. As used herein, the term "MHC I polypeptide" or the like includes the MHC I α chain polypeptide alone. Generally, the terms "human MHC" and "HLA" can be used interchangeably.

The term “substitution” in relation to gene replacement refers to the placement of exogenous genetic material at an endogenous genetic locus, thereby replacing all or part of the endogenous gene with an orthologous or homologous nucleic acid sequence. As shown in the examples below, the nucleic acid sequences of endogenous loci encoding portions of the MHC I and β2 mouse microglobulin polypeptides were replaced with nucleotide sequences encoding portions of the MHC I and β2 human microglobulin polypeptides, respectively.

As used herein, the term "functional", for example, in relation to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the present invention, a substitution at an endogenous locus (eg, a substitution at an endogenous non-human MHC I locus and/or β2 microglobulin) results in a locus that is incapable of expressing a functional endogenous polypeptide.

Some of the aspects described herein below for non-human animals genetically modified for MHC I, such as the type of animal; animal lines; cell types; screening, detection and other methods; methods of application, etc., will be applicable to β2 microglobulin and MHC I/β2 microglobulin genetically engineered animals.

Animals genetically modified for MHC I

According to various embodiments, the present invention generally relates to genetically modified non-human animals that contain in their genome a nucleotide sequence encoding a human or humanized MHC I polypeptide; thus, animals express a human or humanized MHC I polypeptide.

The MHC genes belong to three classes: class I, class II, and class III, all of which are encoded on either human chromosome 6 or mouse chromosome 17. A schematic illustration of the relative organization of the human and mouse MHC classes is presented in Figures 2 and 3, respectively. The MHC genes are among the most polymorphic genes in the mouse and human genomes. MHC polymorphisms are thought to be important in providing evolutionary advantage; changes in sequence can lead to differences in peptide binding, allowing better presentation of pathogens to cytotoxic T cells.

The class I MHC protein contains an extracellular domain (which contains three domains: α ₁ , α ₂ and α ₃ ), a transmembrane domain and a cytoplasmic tail. α ₁ and α ₂ domains form a peptide-binding groove, while α ₃ interacts with β2 microglobulin.

In addition to its interaction with β2 microglobulin, the α ₃ domain interacts with the CD8 TCR co-receptor to facilitate antigen-specific activation. Although MHC class I binding to CD8 is about 100 times weaker than TCR binding to MHC class I, CD8 binding enhances TCR binding affinity. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366. Interestingly, increased MHC class I binding to CD8 abolishes antigen specificity in CTL activation (ibid.).

Binding of CD8 to class I MHC molecules is species-specific; showed that the mouse homologue of CD8, Lyt-2, binds H-2Dd molecules on the α3 domain but does not bind 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. The different binding is thought to be due to CDR-like determinants (CDR1- and CDR2-like) on CD8, which are not conserved in both 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 CD8αα and HLA-A2, Nature 387:630-634. CD8 has been reported to bind HLA-A2 in a conserved region of the α3 domain (at position 223-229). One substitution (V245A) in HLA-A reduced CD8 binding to HLA-A with a concomitant strong reduction in T-cell mediated lysis. Salter et al. (1989), Polymorphism in the α ₃ domain of HLA-A molecules affects binding to CD8, Nature 338:345-348. In general, polymorphisms in the α3 domain of HLA-A molecules also affected CD8 binding (ibid.). In mice, the amino acid substitution at residue 227 in H-2D ^d affected the binding of mouse Lyt-2 to H-2D ^d and cells transfected with the mutant H-2D ^d were 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.

Therefore, due to the species-specific interaction between the α3 domain of MHC class I and CD8, the MHC I complex containing the replacement of the α3 domain of H-2K on the α3 domain of human HLA-A2 was non-functional in the mouse (i.e., in vivo) in the absence of human CD8. In animals transgenic for HLA-A2, replacement of the human α3 domain with the mouse α3 domain resulted in the restoration of the T-cell response. 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), formerly.

The transmembrane domain and cytoplasmic tail of mouse class I MHC proteins also have important functions. One function of the MHC I transmembrane domain is to facilitate HLA-A2 modulation of homotypic cell adhesion (to enhance or inhibit adhesion), presumably as a result of cross-linking (or ligation) of MHC surface 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 monoclonal antibodies that bind to different epitopes of the HLA-A2 molecule, suggesting that there are multiple sites on HLA-A2 involved in modulating homotypic cell adhesion; depending on the associated epitope, exposure may enhance or inhibit HLA-A2 dependent adhesion (ibid.).

It has been reported that the cytoplasmic tail, encoded by exons 6 and 7 of the MHC I gene, is required for proper expression on the cell surface and for LIR1-mediated inhibition of NK cell cytotoxicity. 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 required for the multimerization of at least some MHC I molecules through the formation of disulfide bonds on its cysteine residues and thus may play a role in clustering and in NK cell recognition. Lynch et al. (2009) Novel MHC Class I Structures on Exosomes, J. Immunol. 183:1884-1891.

The cytoplasmic domain of HLA-A2 contains a constitutively phosphorylated serine residue and a phosphorylated tyrosine, although - in Jurkat T cells - mutant HLA-A2 molecules lacking the cytoplasmic domain were found to be normal in terms of expression, cytoskeletal association, aggregation, and endocytotic internalization. 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. Apparently, processed HLA-A2 molecules that do not contain a cytoplasmic domain were in most cases expressed and associated with β2 microglobulin (ibid.).

However, some studies have shown that the cytoplasmic tail is critical for intracellular transport, dendritic cell (DC) mediated antigen presentation and CTL priming. The tyrosine residue encoded by exon 6 has been shown to be required for transport of MHC I across endosomal compartments, presentation of exogenous antigens, and CTL priming; while deletion of exon 7 caused an increase in CTL antiviral responses. Lizee et al. (2003) Control of Dendritic Cross-Presentation by the Major Histocompatibility 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 Tail Enhances Dendritic Cell-Induced CD8+ T-Cell Responses and Boosts Anti-Tumor Immunity, PLoS ONE 6:e22939.

According to various embodiments, the present invention relates to a genetically modified non-human animal (eg, mouse, rat, rabbit, etc.) that contains in its genome a nucleotide sequence encoding a human or humanized MHC class I polypeptide. A non-human animal may contain in its genome a nucleotide sequence that encodes an MHC I polypeptide that is part human and part non-human, e.g., a non-human animal that expresses a chimeric human/non-human MHC I polypeptide In one aspect, the non-human animal only expresses a human or humanized MHC I polypeptide, e.g., a chimeric human/non-human MHC I polypeptide, and does not express an endogenous non-human MHC I protein from an endogenous MHC I locus.

In one embodiment, the chimeric human/non-human MHC I polypeptide comprises, in its human portion, the peptide-binding domain of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises the extracellular domain of human MHC I. According to the present 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 contains the α1 and α2 domains of human MHC I. In another embodiment, the human portion of the chimeric polypeptide contains the α1, α2 and α3 domains of human MHC I.

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

In one specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (eg, an HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, such as an HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is widespread in the North American population. While the present examples describe this particular HLA sequence, any suitable HLA-A sequence is contemplated herein, e.g., HLA-A2 polymorphic variants occurring in the human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences acids that differ from the sequence described herein due to the degeneracy of the genetic code, etc.

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

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

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, eg HLA-B27.

In one aspect, the non-human portion of the chimeric human/non-human MHC I polypeptide comprises the transmembrane and/or cytoplasmic domains of the non-human MHC I polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human the 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, eg H-2Kb. Although specific H-2K sequences are described in the examples, any suitable H-2K sequences are contemplated herein, eg, polymorphic variants, conservative/non-conservative amino acid substitutions, etc.

The non-human animal described herein may contain in its genome a nucleotide sequence encoding a human or humanized MHC I polypeptide, e.g. human MHC I locus (eg H-2K locus). In one aspect, this results in the replacement of an endogenous MHC I gene, or a portion thereof, with a nucleotide sequence encoding a human or humanized MHC I polypeptide, e.g., a chimeric gene encoding a chimeric human/non-human MHC I polypeptide described herein. In one embodiment, the substitution involves replacing an endogenous nucleotide sequence encoding a non-human MHC I peptide-binding domain or a non-human MHC I extracellular domain with a human nucleotide sequence (e.g., an HLA-A2 nucleotide sequence) encoding said domain. In the present embodiment, the substitution does not include replacement of the MHC I sequence encoding the transmembrane and/or cytoplasmic domains of a non-human MHC I polypeptide (eg, an H-2K polypeptide). Thus, a non-human animal contains a chimeric human/non-human nucleotide sequence at an endogenous non-human MHC I locus, and expresses a chimeric human/non-human MHC polypeptide from an endogenous non-human MHC I locus.

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

The chimeric human/non-human MHC I polypeptide may comprise in its human portion the complete or substantially complete extracellular domain of the human MHC I polypeptide. Thus, the human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g. 95% or more of the amino acids encoding the extracellular domain of a human MHC I polypeptide (e.g., an HLA-A2 polypeptide ). In one example, the substantially complete extracellular domain of a human MHC I polypeptide does not contain a human MHC I leader sequence. In another example, a chimeric human/non-human MHC I polypeptide contains a human MHC I leader sequence.

Moreover, 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 an arrangement will facilitate proper expression of the chimeric MHC I polypeptide in a non-human animal, for example, during an immune response in a non-human animal.

The genetically modified non-human animal may be selected from the group consisting of mouse, rat, rabbit, pig, cattle (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g. marmoset, rhesus monkey). For non-human animals in which suitable genetically modified ES cells are not commonly available, other methods are used to obtain a non-human animal containing the genetic modification. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblast or induced pluripotent cell) and using nuclear transport to transfer the modified genome to a suitable cell, such as an oocyte, and gestating the modified cell (e.g., a modified oocyte) in a non-human animal under suitable conditions for the formation of an embryo.

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, for example, from the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, hairy hamsters). ), Nesomyidae (ripidomys, rock rats, white-tailed rats, Magadascar rats and mice), Platacanthomyidae (eg spiny dormouse), and Spalacidae (eg mole rats, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), pygmy gerbil, spiny mouse, and hairy hamster. In one embodiment, the genetically modified mouse is derived from a member of the Muridae family. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent which is a C57BL mouse selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola. In another embodiment, the mouse is a mouse of strain 129 selected from the group consisting of strain 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, see also 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 combination of the aforementioned 129 line and the aforementioned C57BL/6 line. According to another specific embodiment, the mouse is a combination of the aforementioned 129 lines or a combination of the aforementioned BL/6 lines. In a particular embodiment, the combination line 129 is the 129S6 (129/SvEvTac) line. In another embodiment, the mouse is a BALB mouse, eg BALB/c. According to another embodiment, the mouse is a combination of the BALB lineage and the other lineage mentioned above.

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

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

Thus, according to one embodiment, a genetically modified mouse is provided, wherein the mouse comprises, at an endogenous H-2K (e.g., H-2Kb) locus, a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of an HLA polypeptide -A2 human (eg, HLA-A2.1), and the murine part contains the transmembrane and cytoplasmic domains of the H-2K polypeptide (eg, H-2Kb) mouse. In one aspect, the mouse does not express the extracellular domain of a mouse H-2K polypeptide (e.g., H-2Kb) from an endogenous MHC I locus. In one embodiment, the mouse expresses a chimeric HLA-A2/H-2K polypeptide (e.g., chimeric HLA-A2.1 /H-2Kb) from the endogenous H-2K locus (eg H-2Kb). In various embodiments, expression of the chimeric gene is under the control of endogenous murine class I MHC regulatory elements. In some aspects, the mouse contains two copies of the chimeric MHC I locus containing the nucleotide sequence encoding the chimeric HLA-A2/H-2K polypeptide; while in other aspects, the mouse contains one copy of the chimeric MHC I locus containing the nucleotide sequence encoding the chimeric HLA-A2/H-2K polypeptide. Thus, the mouse may be homozygous or heterozygous for the nucleotide sequence encoding the chimeric HLA-A2/H-2K polypeptide.

In some embodiments described herein, a mouse is provided that contains a chimeric MHC I locus located in the mouse endogenous H-2K locus. The chimeric locus contains a nucleotide sequence that encodes for the extracellular domain of the human HLA-A2 protein, such as the α1, α2 and α3 domains of the human HLA-A2 gene. The chimeric locus does not contain a nucleotide sequence that encodes for the extracellular domain of the mouse H-2K protein (eg, mouse H-2K α1, α2, and α3 domains). In one aspect, the chimeric locus does not contain a nucleotide sequence that encodes the leader peptide, α1, α2 and α3 domains of mouse H-2K; and contains the leader peptide, α1, α2 and α3 domains of human HLA-A2, and transmembrane and cytoplasmic domains of mouse H-2K. The various domains of the chimeric locus are operably linked to each other such that the chimeric locus expresses a functional chimeric human/mouse MHC I protein.

In various embodiments, a non-human animal, such as a rodent (eg, mouse or rat), that expresses a functional MHC I chimeric protein from the MHC I chimeric locus described herein presents the chimeric protein on the cell surface. In one embodiment, the non-human animal has expressed the chimeric MHC I protein on the cell surface in a cellular localization that is similar to that observed in humans. In one aspect, the cell is a peptide fragment (antigen fragment) associated with an extracellular portion (e.g., human HLA-A2 extracellular portion) of an MHC I chimeric protein. In an embodiment, the extracellular portion of such chimeric protein interacts with other proteins on the surface of said cell, e.g., β2 microglobulin.

In various embodiments, a cell representing a chimeric MHC I protein, such as an HLA-A2/H-2K protein, is a nucleated cell. In various aspects, the cell is an antigen presenting cell (APC). Although most cells in the body can present an antigen in the context of MHC I, 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 known in the art and provided herein. In some embodiments, the cell presenting the MHC I chimeric protein is a tumor cell and the peptide fragment presented by the chimeric protein is from a tumor. In other embodiments, the peptide fragment presented by the MHC I chimeric protein is from a pathogen, such as a bacterium or virus.

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

On the same cell, HLA class I molecules can functionally interact with both non-human (eg, rodent, eg, mouse or rat) β2 microglobulin and human β2 microglobulin. Thus, in one embodiment, a chimeric MHC I protein, eg HLA-A2/H-2K protein, interacts with mouse β2 microglobulin. Although the interaction between some human HLA class I molecules and mouse β2 microglobulin is possible, it can nevertheless be greatly reduced compared to the interaction between human HLA class I and human β2 microglobulin. Thus, in the absence of human β2 microglobulin, expression of human MHC I on the cell surface can 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 reports that HLA-B27 function in transgenic mice require both human β2 microglobulin and human CD8. Therefore, according to another embodiment, the chimeric MHC I protein interacts with human or humanized β2 microglobulin. In such embodiments as described herein below, a non-human animal, e.g., a rodent (e.g., mouse or rat), contains a human or humanized microglobulin β2 gene in its genome, and the animal expresses a functional human or humanized microglobulin β2 polypeptide. ; therefore, the chimeric MHC I protein interacts with a human or humanized microglobulin β2 polypeptide.

In various aspects, the chimeric protein (eg, HLA-A2/H-2K protein) also interacts with proteins on the surface of the second cell (via its extracellular portion). The second cell may be a cell derived from a non-human animal, such as a mouse, or from a human. The second cell may be derived from the same non-human animal or the same species of non-human animal as the cell expressing the chimeric MHC I polypeptide. Non-limiting examples of proteins with which the extracellular portion of the chimeric protein (eg, HLA-A2/H-2K) can interact include the T cell receptor (TCR) and its CD8 co-receptor. Thus, the second cell may be a T cell. In addition, the extracellular portion of the MHC I chimeric protein can bind to a protein on the surface of natural killer (NK) cells, such as the killer cell immunoglobulin-like receptor (KIR) on the surface of NK cells.

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

In one example, the chimeric MHC I polypeptide described herein, for example, the chimeric HLA-A2/H-2K polypeptide, interacts with the CD8 protein on the surface of a second cell. In one embodiment, the chimeric HLA-A2/H-2K polypeptide interacts with endogenous rodent (eg, mouse or rat) CD8 protein on the surface of a second cell. In one embodiment, the second cell is a T cell. In another embodiment, the second cell is engineered to express CD8. In certain aspects, the chimeric HLA-A2/H-2K polypeptide interacts with human CD8 on the surface of a second cell (eg, human or rodent cells). In some such embodiments, a non-human animal, such as a mouse or rat, contains a human CD8 transgene, and the mouse or rat expresses a functional human CD8 protein.

The chimeric MHC I polypeptide described herein can also interact with a non-human (eg, mouse or rat) TCR, a human TCR, or a humanized TCR on a second cell. The chimeric MHC I polypeptide can interact with an endogenous TCR (eg, mouse or rat TCR) on the surface of a second cell. The chimeric MHC I polypeptide can also interact with a human or humanized TCR expressed on the surface of a second cell, the cell being from the same animal or species (e.g., mouse or rat) as the cell expressing the chimeric MHC I polypeptide. The chimeric MHC I polypeptide can interact with the human TCR expressed on the surface of a human cell.

In addition to genetically engineered non-human animals, a non-human embryo (e.g., a rodent embryo, e.g., a mouse or rat fetus) is also provided, wherein the embryo contains an ES donor cell that is derived from a non-human animal (e.g., rodent, such as mice or rats) described herein. According to one aspect, the embryo contains a donor ES cell containing a chimeric MHC I gene and host germ cells.

A tissue is also provided, wherein the tissue is derived from a non-human animal (eg, mouse or rat) described herein and expresses a chimeric MHC I polypeptide (eg, an HLA-A2/H-2K polypeptide).

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

Also provided is a non-human cell containing a chromosome or a fragment of a non-human animal described herein. In one embodiment, the non-human cell comprises the nucleus of a non-human animal as described herein. In one embodiment, the non-human cell contains a chromosome or fragment thereof as a result of nuclear transport.

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

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

Also provided is a method for producing a genetically engineered non-human animal (eg, a genetically engineered rodent, such as a mouse or rat) as described herein. A method for producing a genetically engineered non-human animal results in an animal whose genome contains a nucleotide sequence encoding a chimeric MHC I polypeptide. In one embodiment, the method results in a genetically engineered mouse whose genome contains an endogenous MHC I locus, H-2K, a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the human portion of the chimeric MHC I polypeptide contains the extracellular domain of human HLA-A2, and the murine portion contains the mouse H-2K transmembrane and cytoplasmic domains. In some embodiments, the method uses a targeting construct made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using the VELOCIMOUSE® technology described in the examples. In one embodiment, the ES cells are a combination of 129 and C57BL/6 mouse lines; in another embodiment, the ES cells are a combination of the BALB/c and 129 mouse lines.

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

In one embodiment, the genomic fragment contains a human HLA-A leader sequence encoding an α1 domain, an α2 domain, and an α3 domain sequence. In one embodiment, the human DNA fragment contains, in the 5'-3' direction: HLA-A leader sequence, intron, HLA-A/α1 leader sequence, HLA-A α1 exon, HLA-Aα1-α2 intron, HLA-A exon α2, HLA-A intron α2-α3 and HLA-A α3 exon.

A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate selection of cells (eg, ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. Typically, the selection cassette provides a positive selection in the presence of a particular antibiotic (eg, Neo, Hyg, Pur, CM, Spec, etc.). In addition, the selection cassette can be flanked by recombination sites that allow for the deletion of the selection cassette when treated with recombinase enzymes. The most widely used recombination sites are loxP and Frt, recognized by the enzymes Cre and Flp, respectively, but others are known in the art.

In one embodiment, the selection cassette is located at the 5' end of a human DNA fragment. In another embodiment, the selection cassette is located at the 3' end of the human DNA fragment. In another embodiment, the selection cassette is located within a human DNA fragment. In another embodiment, the selection cassette is located within the intron of a human DNA fragment. In another embodiment, the selection cassette is located within the α2-α3 intron.

In one embodiment, the 5' and 3' non-human homology arms contain the genomic sequence at the 5' and 3' positions of the endogenous non-human (e.g., murine) class I MHC gene locus, respectively (e.g., 5' with respect to the first leader sequence and 3' to the α3 exon of the non-human MHC I gene). In one embodiment, the endogenous MHC class I locus is selected from H-2K, H-2D, and H-2L mice. In a specific embodiment, the endogenous class I MHC locus is a mouse H-2K.

In one aspect, a nucleotide construct is provided comprising, in the 5'-3' direction: the 5' homology arm comprising the mouse genomic sequence 5' to the mouse endogenous H-2K locus, the first human DNA fragment containing the first genomic sequence of the HLA- A, 5' recombination sequence site (eg, loxP), selection cassette, 3' recombination sequence site (eg, loxP), second human DNA fragment containing the second HLA-A gene genomic sequence, and 3' homology arm containing the genomic sequence mouse 3' in relation to the endogenous exon H-2K α3. In one embodiment, the nucleotide construct comprises, in the 5'-3' direction: the 5' homology arm comprising a mouse genomic sequence 5' to the mouse endogenous H-2K locus, a human genomic sequence comprising an HLA-A leader sequence, an intron, HLA-A/α1 leader sequence, HLA-A α1 exon, HLA-A α1-α2 intron, HLA-A α2 exon, first 5' part of α2-α3 intron, selection cassette flanked by recombination sites, second 3' part of α2 intron -α3, an HLA-A exon α3 and a 3' homology arm containing a non-mouse genomic sequence 3' to the mouse endogenous H-2K exon α3. In one embodiment, the homology arm 5' sequence is shown in SEQ ID NO:1 and the homology arm 3' sequence is shown in SEQ ID NO:2.

After targeting the ES gene, cells or genetically modified non-human animals are screened to confirm successful insertion of the exogenous nucleotide sequence of interest or expression of the exogenous polypeptide. Various techniques are known to those skilled in the art and include, but are not limited to, Southern blot, long fragment PCR, quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blot, flow cytometry, Western blotting, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (eg, mice) bearing a genetic modification of interest can be identified by screening for loss of the mouse allele and/or acquisition of the human allele using the modified allelic assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659. Those skilled in the art are aware of other assays that identify a particular nucleotide or amino acid sequence in genetically modified animals.

The present disclosure also provides a method for modifying the MHC I locus of a non-human animal to express the chimeric human/non-human MHC I polypeptide described herein. In one embodiment, the present invention provides a method for modifying the MHC I locus of a mouse to express a chimeric human /mouse MHC I polypeptide, and the method involves the replacement in the endogenous MHC I locus of the nucleotide sequence encoding the peptide-binding domain of the mouse MHC polypeptide, the nucleotide sequence encoding the peptide-binding domain of the human MHC I polypeptide. In some aspects, a mouse MHC I extracellular domain nucleotide sequence is replaced with a human MHC I extracellular domain nucleotide sequence. The mouse may not be able to express a mouse MHC I peptide-binding or extracellular domain from an endogenous MHC I locus. HLA-A2/H-2K polypeptide.

In one aspect, a method is provided for producing a chimeric human HLA class I/non-human MHC class I molecule, comprising expressing in one cell an HLA-A/H-2K chimeric protein from a nucleotide construct, the nucleotide construct comprising a cDNA sequence that encodes for α1, the α2 and α3 domain of the HLA-A protein; and the transmembrane and cytoplasmic domain of a non-human H-2K protein, eg mouse H-2K protein. In one embodiment, the nucleotide construct is a viral vector; in a particular 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 (eg, PERC.6™ cells).

In one aspect, a cell is provided that expresses a chimeric human HLA class I/non-human MHC I protein (eg, HLA-A/H-2K protein). In one embodiment, the cell comprises an expression vector comprising a class I chimeric MHC gene, wherein the class I chimeric MHC gene comprises a human HLA-A gene sequence fused in operable relationship to a non-human H-2K gene sequence, e.g., the H- 2K mice. In one embodiment, the human HLA-A gene sequence contains exons that encode for the α1, α2, and α3 domains of the HLA-A protein. In one embodiment, the non-human H-2K gene sequence contains exons that encode for the transmembrane and cytoplasmic domains of the H-2K protein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (eg, PERC.6™ cells).

Also provided is a class I chimeric MHC molecule produced by non-human animals as described herein, wherein the class I chimeric MHC molecule contains α1, α2, and α3 domains from human HLA-A protein and transmembrane and cytoplasmic domains from non-human, for example, mouse protein H-2K. The chimeric MHC I polypeptide described herein can be detected using anti-HLA-A antibodies. Thus, a cell representing a chimeric human/non-human MHC I polypeptide can be detected and/or selected using an anti-HLA-A antibody. In some instances, the chimeric MHC I polypeptide described herein can be detected using an anti-HLA-A2 antibody.

Although the following examples describe a genetically engineered animal whose genome contains a substitution of a nucleotide sequence encoding the extracellular domain of a mouse H-2K polypeptide with a sequence encoding an extracellular domain of human HLA-A at the endogenous murine H-2K locus, one skilled in the art It is understood by the art that a similar strategy can be used to replace other mouse MHC I loci (H-2D, H-2L, etc.) with their corresponding human HLA loci (HLA-B, HLA-C, etc.). Thus, a non-human animal is also contemplated, comprising in its genome a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein the human portion of the polypeptide is derived from another HLA class I protein. Substitution of multiple MHC I loci is also contemplated.

Animals genetically modified for β2 microglobulin

The present invention generally relates to genetically modified non-human animals that contain in their genome a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide; thus, the animals express the human or humanized β2 microglobulin polypeptide.

The β2 microglobulin or light chain of the MHC class I complex (also abbreviated as “β2M”) is a small (12 kDa) non-glycosylated protein that functions predominantly to stabilize the α chain of MHC I. The human β2 microglobulin gene encodes a 119 amino acid protein, with 20 N -terminal amino acids encoding the leader sequence. A mature protein contains 99 amino acids. The gene contains 4 exons, with the first exon containing the 5' untranslated region, the complete leader sequence, and the first two amino acids of the mature polypeptide; the second exon encodes the bulk of the mature protein; the third exon encodes the last four amino acids of the mature protein and a 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 is non-covalently associated with MHC I. Unbound β2 microglobulin is found in biological fluids such as plasma and is transported to the kidneys for excretion from the body. Impaired renal function causes accumulation of β2 microglobulin, which may be pathogenic (eg, cause dialysis amyloidosis); accumulated protein forms filamentous fibrils resembling amyloid plaques in joints and connective tissues.

In addition to dialysis amyloidosis, β2 microglobulin has been implicated in a number of other disorders. Elevated levels of β2 microglobulin have been found in lymphocytic malignancies such as non-Hodgkin's lymphoma and multiple myeloma. See, for example, Shi et al. (2009) β2 Microglobulin: Emerging as a Promising Cancer Therapeutic Target, Drug Discovery Today 14:25-30. Some other β2 microglobulin-enhanced cancers include breast cancer, prostate cancer, lung cancer, kidney cancer, gastrointestinal cancer, and nasopharyngeal cancer. It has been suggested that overexpression of β2 microglobulin is characterized by stimulatory effects on tumor growth (ibid.). It has also recently been shown that β2 microglobulin controls the transformation of epithelial cells into mesenchymal cells, stimulating the formation of metastases in bones and soft tissues in breast cancer, prostate cancer, lung cancer and kidney cancer. 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 the non-classical representative of MHC I, the hemochromatosis protein (HFE), and with the transferrin receptor, and modulates iron homeostasis (ibid.). The involvement of β2 microglobulin in other hallmarks of malignancy (self-renewal, increased angiogenesis, resistance to treatment) has been extensively documented in the art.

Mice deficient in β2 microglobulin 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 appeared to be healthy, however, MHC class I expression was not detected. In addition, the majority of T cell populations in some tissues were found to be normal, while in other tissues there was a significant decrease in the number of CD8+ T cells. This putative lack of MHC I expression contradicts previous results reported 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. showed that β2 microglobulin was not absolutely necessary for the expression of all MHC I complexes on the cell surface, since cells lacking β2 microglobulin were able to express H-2D ^b . However, the function of H-2D ^b in these cells was presumably defective and the conformation of H-2D ^b differed from the native protein, which explains why Koller and colleagues were unable to detect this protein using antibodies against native H-2D. ^b . However, it has been reported that cells lacking β2 microglobulin can present endogenous antigen to CD8+ T cells (including exogenous CD8+ T cells from normal mice), and it has been reported that β2 microglobulin is not necessary to produce high levels of restricted H-2 ^d MHC class I CD8+ CTL in response to antigen challenge in mice, although it is required to provide 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 should be noted that the ability to produce a high abundance of such T cells in the absence of β2 microglobulin is reportedly limited by the H-2 restricted ^d MHC class I response. β2 microglobulin deficient mice have been reported to have prominent characteristics such as, for example, an increased susceptibility to certain parasitic diseases, an increased susceptibility to hepatitis infections, iron metabolism deficiency, and an impaired breeding 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. reproduction. 77:274-279.

Mice have been reported to express human β2 microglobulin as well as human HLA class I molecules (ie HLA-B7) in a randomly inserted transgene. 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. Human class I HLA expression was comparable to endogenous class I expression with a significant decrease in the liver (ibid.). The expression of human β2 microglobulin was also comparable to the expression of endogenous β2 microglobulin, while the expression of the human HLA class I molecule increased 10-17-fold in double transgenic mice (ibid.). However, the authors made no attempt to replace the endogenous mouse β2 microglobulin locus with the human β2 microglobulin locus.

Therefore, disclosed herein is a genetically engineered non-human animal (eg, a rodent, eg, mouse or rat) whose genome contains a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. 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 encodes a β2 microglobulin polypeptide that is part human and part non-human, e.g., it contains some amino acids that correspond to human β2 microglobulin and some amino acids that correspond to non-human β2 microglobulin. In one aspect, the non-human animal does not express an endogenous non-human β2 microglobulin polypeptide from an endogenous non-human locus, and only expresses a human or humanized β2 microglobulin polypeptide. In one example, the non-human animal does not express the entire endogenous non-human β2 microglobulin polypeptide, but expresses only a portion of the non-human endogenous β2 microglobulin polypeptide from the endogenous β2 microglobulin locus. Thus, 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 contains two copies of the β2 microglobulin locus containing a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In another aspect, the animal contains one copy of the microglobulin β2 locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. Thus, an animal may be homozygous or heterozygous for a β2 microglobulin locus containing a nucleotide sequence that encodes a human or humanized β2 microglobulin polypeptide. The nucleotide sequence of a human or humanized β2 microglobulin can be derived from a set of β2 microglobulin sequences that occur naturally in human populations. In various embodiments, the genetically engineered non-human animal of the present invention contains in its germline a nucleotide sequence encoding a human or humanized β2 microglobulin. In one embodiment, the nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide comprises a nucleotide sequence encoding a polypeptide comprising the human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to the MHC I protein.

The nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide may contain nucleic acid residues corresponding to the complete human β2 microglobulin gene. Alternatively, the nucleotide sequence may comprise nucleic acid residues encoding the amino acid sequence represented at amino acids 21-119 of the human β2 microglobulin protein (ie amino acid residues corresponding to mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may comprise nucleic acid residues encoding the amino acid sequence represented at amino acids 23-115 of the human β2 microglobulin protein, e.g., the amino acid sequence represented at amino acids 23-119 of the human β2 microglobulin protein. The nucleic acid sequences and amino acid sequences of human β2 microglobulin are described in Gussow et al., previously incorporated herein by reference.

Thus, a human or humanized microglobulin β2 polypeptide may comprise the amino acid sequence represented at amino acids 23-115 of a human microglobulin β2 polypeptide, e.g. -119 human microglobulin β2 polypeptide. Alternatively, the human β2 microglobulin may comprise amino acids 1-119 of the human β2 microglobulin polypeptide.

In some embodiments, the nucleotide sequence encoding a human or humanized β2 microglobulin comprises a nucleotide sequence present in exon 2 to exon 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene. In the present embodiment, the nucleotide sequences present in exons 2, 3, and 4 are operably linked to ensure normal transcription and translation of the gene. Thus, 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 contains a nucleotide sequence corresponding to exon 2 up to about 267 bp. after exon 4 of the human β2 microglobulin gene. In a specific embodiment, the human sequence contains approximately 2.8 kb. human microglobulin β2 gene.

Thus, a human or humanized β2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising the nucleotide sequence present in exon 2-exon 4 of human β2 microglobulin, e.g., the nucleotide sequence corresponding to exon 2-exon 4 of the human β2 microglobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising the nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene. In a specific embodiment, the human or humanized microglobulin β2 polypeptide is encoded by a nucleotide sequence corresponding to exon 2 up to about 267 bp. after exon 4 of the human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence containing approximately 2.8 kb. human microglobulin β2 gene. Since exon 4 of the microglobulin β2 gene contains a 5' untranslated region, a human or humanized polypeptide can be encoded by a nucleotide sequence containing exons 2 and 3 of the microglobulin β2 gene.

Those skilled in the art will appreciate that while the present examples describe specific nucleic acid and amino acid sequences for the creation of genetically engineered animals, sequences of one or more conservative or non-conservative amino acid substitutions, or sequences that deviate from those described herein due to degeneracy, are also provided. genetic code.

Therefore, a non-human animal is provided that expresses a human β2 microglobulin sequence, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human β2 microglobulin sequence. . In a specific embodiment, the microglobulin β2 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 contains one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence contains one or more non-conservative substitutions.

In addition, non-human animals are contemplated, wherein the nucleotide sequence encoding a human or humanized microglobulin β2 protein also comprises a nucleotide sequence present in exon 1 of the non-human microglobulin β2 gene. Thus, in a particular embodiment, the non-human animal comprises in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin, wherein the nucleotide sequence comprises exon 1 of the non-human β2 microglobulin gene and exons 2, 3, and 4 of the human β2 microglobulin gene . Thus, a human or humanized β2 microglobulin polypeptide is encoded by exon 1 of the non-human β2 microglobulin gene and by exons 2, 3 and 4 of the human β2 microglobulin gene (eg, exons 2 and 3 of the human β2 microglobulin gene).

Similarly to a non-human animal containing a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, a non-human animal containing a nucleotide sequence encoding a human or humanized β2 microglobulin may be selected from the group consisting of a mouse, rat, rabbit, pig, cattle (eg cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (eg marmoset, rhesus monkey). According to some embodiments of the present invention, the non-human animal is a mammal. In a specific embodiment, the non-human animal is a member of the murine family, such as a rodent (eg, mouse or rat). In one embodiment, the animal is a mouse.

Thus, in some aspects, a genetically engineered mouse is provided, wherein the mouse comprises a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide described herein. A genetically engineered mouse is provided, wherein the mouse comprises, at its endogenous microglobulin β2 locus, a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide (eg, a human or substantially human microglobulin β2 polypeptide). In some embodiments, the mouse does not express an endogenous β2 microglobulin polypeptide (eg, a functional endogenous β2 microglobulin polypeptide) from an endogenous β2 microglobulin locus. In some embodiments, the genetically engineered mouse comprises a nucleotide sequence comprising exon 1 of the mouse β2 microglobulin gene and exons 2, 3, and 4 of the human β2 microglobulin gene. In some embodiments, the mouse expresses a human or humanized β2 microglobulin polypeptide.

In one aspect, a modified non-human microglobulin β2 locus is provided that contains a heterologous microglobulin β2 sequence. In one embodiment, the heterologous microglobulin β2 sequence is a human or humanized sequence.

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

In one embodiment, the heterologous microglobulin β2 sequence is operably linked to endogenous regulatory elements, eg, an endogenous promoter and/or an expression control sequence. In a specific embodiment, the heterologous microglobulin β2 sequence is a human sequence and the human sequence is operably linked to an endogenous promoter and/or expression control sequence.

In one aspect, a modified non-human microglobulin β2 locus is provided that contains a human sequence operably linked to an endogenous promoter and/or an expression control sequence.

In various aspects, a human or humanized β2 microglobulin expressed by genetically modified non-human animals, or cells, embryos or tissues derived from a non-human animal, retains all of the functional aspects of the endogenous and/or human β2 microglobulin. For example, it is preferred that a human or humanized β2 microglobulin binds to the α chain of an MHC I polypeptide (eg, an endogenous non-human or human MHC I polypeptide). A human or humanized β2 microglobulin polypeptide may bind, recruit, or otherwise associate with any other molecule, e.g., a receptor, anchor, or signal transduction molecule, that associates with endogenous non-human and/or human β2 microglobulin (e.g., HFE, etc.).

In addition to genetically modified animals (e.g., rodents, e.g., mice or rats), a tissue or cell is also provided, wherein the tissue or cell is derived from a non-human animal described herein and contains a heterologous microglobulin β2 gene or β2 sequence. microglobulin, i.e. nucleotide and/or amino acid sequence. In one embodiment, the heterologous microglobulin β2 gene or microglobulin β2 sequence is a human or humanized microglobulin β2 gene or a human or humanized microglobulin β2 sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express the MHC I complex, such as an antigen presenting cell. A human or humanized microglobulin β2 polypeptide expressed by said cell may interact with endogenous non-human MHC I (e.g., rodent MHC I) to form a functional MHC I complex. The resulting MHC I complex may be capable of interacting with a T cell, e.g. , cytotoxic T-cell. Thus, an in vitro complex of a cell from a non-human animal described herein and a T cell is also provided.

Also contemplated are non-human cells that contain a human or humanized microglobulin β2 gene or sequence and an additional human or humanized sequence, such as the chimeric MHC I polypeptide disclosed herein. In such a case, the human or humanized microglobulin β2 polypeptide may interact, for example , with a chimeric human/non-human MHC I polypeptide, and a functional MHC I complex can be formed. In some aspects, such a complex is capable of interacting with a TCR on a T cell, e.g., a human or non-human T cell. Thus, an in vitro cell complex from a non-human animal described herein and a human or non-human T cell is also provided.

Another aspect of the present disclosure relates to a rodent (eg, mouse or rat) fetus containing a heterologous microglobulin β2 gene or microglobulin β2 sequence as described herein. In one embodiment, the embryo contains an ES donor cell that contains a heterologous microglobulin β2 gene or microglobulin β2 sequence and a host cell. The heterologous microglobulin β2 gene or microglobulin β2 sequence is a human or humanized microglobulin β2 gene or microglobulin β2 sequence.

The present invention also provides a non-human cell comprising a chromosome or fragment thereof of a non-human animal described herein (eg, wherein the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide). The non-human cell may contain the nucleus of a non-human animal as described herein. In one embodiment, the non-human cell contains a chromosome or fragment thereof as a result of nuclear transport.

In one aspect, a non-human induced pluripotent cell is provided comprising a heterologous microglobulin β2 gene or microglobulin β2 sequence. In one embodiment, the induced pluripotent cell is from a non-human animal as described herein. In one embodiment, the heterologous microglobulin β2 gene or microglobulin β2 sequence is a human or humanized gene or sequence.

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

The present disclosure also relates to methods for producing a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or rat) described herein. The methods result in an animal whose genome contains a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide . In one aspect, the methods result in a genetically engineered mouse whose genome contains, at the endogenous β2 microglobulin locus, a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In some cases, the mouse does not express functional mouse β2 microglobulin from the endogenous mouse β2 microglobulin locus. In some embodiments, the method uses a targeting construct made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse fetus using the VELOCIMOUSE® technology described in the examples. In one embodiment, the ES cells are a combination of the 129 and C57BL/6 mouse lines; in another embodiment, the ES cells are a combination of BALB/c and 129 mouse lines.

Also provided is a nucleotide construct used to create genetically engineered non-human animals. The nucleotide construct may comprise: 5' and 3' non-human homology arms, a human DNA fragment containing human microglobulin β2 sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that contains both introns and exons of the human microglobulin β2 gene. In one embodiment, the non-human homology arms are homologous to the non-human microglobulin β2 locus. The genomic fragment may contain exons 2, 3, and 4 of the human β2 microglobulin gene. In one instance, the genomic fragment contains, in the 5'-3' direction: exon 2, intron, exon 3, intron and exon 4, all of the human β2 microglobulin sequences. The selection cassette can be located anywhere in the construct outside of the β2 microglobulin coding region, for example, it can be located 3' to exon 4 of the human β2 microglobulin gene. The 5' and 3' non-human arms of the homology may contain the genomic sequence 5' and 3' from the endogenous non-human β2 microglobulin gene, respectively. In another embodiment, the 5' and 3' non-human homology arms comprise the genomic sequence 5' to exon 2 and 3' to exon 4 of the endogenous non-human gene, respectively.

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

Animals genetically modified for MHC I/β2 microglobulin

According to various embodiments, the present invention generally relates to genetically modified non-human animals that contain in their genome nucleotide sequences encoding human or humanized polypeptides of both MHC I and β2 microglobulin; thus, animals express human or humanized polypeptides of both MHC I and β2 microglobulin.

When using mixed human/non-human systemic components, functional differences arise. Class I HLA binds to β2 microglobulin more strongly 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 are reflected in the design of specific MHC humanized mice. Class I and class II H-2 knockout mice (in mouse β2 microglobulin knockout genetic background) have been developed that express a randomly integrated human HLA-A2.1/HLA-DR1 chimeric transgene containing HLA-α1 and α2. A2.1 human, and α3 H-2D ^b mouse, attached at its N-terminus through a linker to the C-terminus of β2 human microglobulin. See, for example, Pajot et al. (2004) A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice, Eur. J. Immunol. 34:3060-3069. These mice were reported to produce antigen-specific antibody and CTL responses against hepatitis B virus, which was not observed in HLA-A2.1-only transgenic or H-2 class I/class II knockout mice. The lack of mice that are transgenic exclusively for these genes is presumably due to the ability of such mice to use endogenous class I and/or class II genes to avoid the occurrence of any transgene, a capability not available to MHC knockout mice. However, mice can express at least H-2D ^b , presumably due to crosses to a mouse β2 microglobulin knockout mouse genetic background (see Pajot et al., previously; which likely contained an intact class I and class II endogenous locus) .

Cell surface expression of a chimeric fusion with human β2 microglobulin has been reported to be lower than expression of endogenous MHC, but the survival/rate of NK cytotoxicity as well as the rate of NK autocytotoxicity has not been reported previously by Pajot et al. Some increase in CD8+ T cells was observed in MHC class I deficient β2 microglobulin knockout mice (2-3% of total splenocytes versus 0.6-1% in β2 knockout mice). However, the frequency of T cell variable region utilization showed altered profiles for BV 5.1, BV 5.2, and BV 11 gene segments. CD8+ and CD4+ T cell responses were reported to be restricted to the corresponding hepatitis B antigen used to immunize mice, although at least two mice that showed cell lysis carrying either antigen were mice immunized with only one antigen, which may be due to lack of NK cell inhibition or lack of NK cell selectivity.

As mentioned above, mice transgenic for both human MHC I and human β2 microglobulin contain the nucleotide sequence encoding the MHC I/β2 microglobulin chimeric protein, with the MHC I and β2 microglobulin portions contained within the same polypeptide chain, resulting in α chain MHC I and β2 microglobulin covalently linked to each other and thus attached to the cell surface. A mouse is provided that contains in its genome two independent nucleotide sequences, one encoding a human or humanized MHC I polypeptide and the other encoding a human or humanized microglobulin β2 polypeptide. The mouse provided herein will express an MHC I complex that more closely resembles the naturally occurring MHC I complex, wherein the MHC I α chain and β2 microglobulin are present on two separate polypeptide chains, the β2 microglobulin being non-covalently linked to the MHC I α chain.

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

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

The second nucleotide sequence may be located at an endogenous non-human microglobulin β2 locus such that the animal contains in its genome a substitution at the β2 microglobulin locus for all or part of the endogenous microglobulin β2 gene with the corresponding human β2 microglobulin sequence. The second nucleotide sequence may comprise a nucleotide sequence present in exon 2 to exon 4 of the human microglobulin β2 gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene. According to the present embodiment, the nucleotide sequences are operably linked to each other. The second nucleotide sequence may further comprise the exon 1 sequence of the non-human microglobulin β2 gene.

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

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

Various other embodiments of the first and second nucleotide sequences (and the polypeptides they encode) provided herein can be easily understood from the embodiments described herein, for example, the embodiments described in the sections relating to genetically engineered against MHC I animals and genetically engineered against β2 microglobulin animals.

In one aspect, the disclosure relates to a mouse comprising in its genome (a) a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide (in particular, an HLA-A2/H-2K polypeptide), wherein the human portion of the chimeric polypeptide contains an HLA extracellular domain -A2 human, and the mouse part contains the transmembrane and cytoplasmic domains of mouse H-2K, and (b) a second nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide (for example, and the nucleotide sequence contains a nucleotide sequence represented in exon 2 - exon 4 of the gene β2 human microglobulin, or nucleotide sequences present in exon 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 mouse microglobulin H-2K and β2 polypeptides from their respective endogenous loci. In one embodiment, the mouse expresses both a chimeric human/mouse MHC I polypeptide and a human or humanized microglobulin β2 polypeptide.

As shown in the following examples, animals genetically engineered to co-express both human or humanized MHC I and β2 microglobulin exhibit increased expression of class I chimeric MHC on the cell surface compared to animals humanized for MHC I alone. In some embodiments, co-expression of human or humanized MHC I and β2 microglobulin increases cell surface expression of human or humanized MHC I by more than about 10%, for example, more than about 20%, for example, about 50% or more, for example, about 70% , compared with the expression of human or humanized MHC I in the absence of human or humanized β2 microglobulin.

The present disclosure also relates to a method for obtaining genetically engineered non-human animals (eg, rodents, such as rats or mice), the genome of which contains the first and second nucleotide sequences described herein. Basically, the method involves obtaining a first genetically engineered non-human animal, the genome of which contains the first nucleotide sequence described herein (i.e., a human or humanized MHC I sequence), obtaining a second genetically engineered non-human animal, the genome of which contains a second nucleotide sequence described herein (ie, a human or humanized β2 microglobulin sequence), and crossing the first and second animal to produce offspring whose genome contains both nucleotide sequences. In one embodiment, the first and second animal are heterozygous for the first and second nucleotide sequence, respectively. In one embodiment, the first and second animal are homozygous for the first and second nucleotide sequence, respectively. In one embodiment, the first and second animals are obtained by replacing endogenous non-human loci with the first and second nucleotide sequences, respectively. In one aspect, a first and second animal is obtained by using constructs generated by VELOCIGENE® technology and by introducing targeted ES cell clones bearing such constructs into an embryo (eg, a rodent, such as a mouse or rat fetus) by the VELOCIMOUSE® method.

Application of genetically modified animals

In various embodiments, the genetically modified non-human animals described herein produce APCs with human or humanized MHC I and/or β2 microglobulin on the cell surface and, as a result, present peptides derived from cytosolic proteins as epitopes for CTL characteristic of in a human way, since essentially 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 a humanized animal; to identify antigens and antigenic epitopes that elicit an immune response (eg, T-cell epitopes, eg, unique human cancer epitopes), eg, for use in vaccine development; to identify high affinity T cells for human pathogens or cancer antigens (i.e., T cells that bind to an antigen in the context of high avidity human MHC I complex), e.g. for use in adaptive T-based therapy cells; to evaluate candidate vaccines and other vaccine development strategies; to study human autoimmunity; for the study of human infectious diseases; and in other cases to develop better therapeutic strategies based on human MHC expression.

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

T cells interact with cells expressing the MHC I complex via the MHC class I peptide-bound ectodomain and the CD8 T cell ectodomain. CD8+ T cells that encounter APCs containing appropriate antigens bound to an MHC class I molecule will become cytotoxic T cells. Thus, antigens that, in the context of MHC class I, bind with high avidity to the T cell receptor are potentially important in the development of treatments for human pathologies. However, antigen presentation in the context of mouse MHC I is only marginally relevant to human disease because human and mouse MHC complexes recognize antigens in different ways, e.g., mouse MHC I may not recognize the same antigens or may present different epitopes compared to MHC I human. Thus, the most relevant data for human pathologies is obtained by examining the presentation of human MHC I antigenic epitopes.

Thus, according to various embodiments, the genetically engineered animals of the present invention are useful, among other things, for evaluating the ability of an antigen to initiate an immune response in a human, and for obtaining a variety of antigens and identifying a specific antigen that can be used to develop a human vaccine.

In one aspect, a method is provided for determining the antigenicity of a peptide sequence in a human, comprising exposing a genetically modified non-human animal described herein to a molecule comprising the peptide sequence, allowing the non-human animal to mount an immune response, and detecting in the non-human animal a cell that binds a peptide sequence presented by the herein described chimeric human/non-human MHC I, or a humanized MHC I complex (comprising chimeric human/non-human MHC I and human or humanized β2 microglobulin).

In one aspect, a method is provided for determining whether a peptide will elicit an immune response in a human, comprising exposing the peptide to a genetically modified non-human animal described herein, allowing the non-human animal to develop an immune response, and detecting in the non-human animal cell that binds the peptide sequence of the chimeric human/non-human MHC class I molecule described herein. In one embodiment, the non-human animal, after exposure, contains an MHC class I restricted CD8+ cytotoxic T lymphocyte (CTL) that binds the peptide. In one embodiment, the CTLs are lysed with the peptide-bearing cell.

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

In one aspect, a method is provided for identifying an HLA class I restricted peptide whose presentation by a human cell and binding by a human lymphocyte (e.g., a human T cell) will result in cytotoxicity of a peptide-bearing cell, comprising exposure to a non-human animal described herein ( or its class I MHC-expressing cell) of a molecule containing the peptide of interest, isolating a non-human animal cell that expresses a chimeric human/non-human class I molecule that binds the peptide of interest, exposing a human lymphocyte cell that is capable of to carry out HLA class I restricted cytotoxicity, and measurement of peptide-induced cytotoxicity.

In one aspect, a method is provided for identifying an antigen that elicits a cytotoxic T cell response in a human, comprising exposing a putative antigen to a mouse as described herein, allowing the mouse to mount an immune response, and identifying an antigen associated with an HLA-A restricted molecule.

In one embodiment, the antigen comprises a bacterial or viral surface or envelope protein. In one embodiment, the antigen comprises an antigen on the surface of a human tumor cell. In one embodiment, the antigen provides for a putative vaccine for use in humans. In one embodiment, the antigen contains a human epitope that induces antibodies in the human. In another embodiment, the antigen contains a human epitope that induces the formation of high affinity CTLs targeted to the epitope/MHC I complex.

In one aspect, a method is provided for determining whether a putative antigen contains an epitope that, when exposed to the human immune system, would elicit an HLA-A restricted immune response (e.g., an HLA-A2 restricted response) comprising exposing the mouse described herein to the putative antigen and measuring the antigen-specific HLA-A-restricted (eg, HLA-A2-restricted) immune response in the mouse.

In one embodiment, the putative antigen is selected from a biopharmaceutical or fragment thereof, a foreign protein, a foreign cell surface antigen, a tumor cell surface antigen, a bacterial or yeast or fungal cell surface antigen, a surface antigen, or a viral envelope protein.

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

In one aspect, a method is provided for identifying a T cell receptor variable domain with high affinity for a human tumor antigen, comprising exposing a mouse to a human tumor antigen containing humanized α1, α2, and α3 domains (e.g., HLA-A2 α1, α2, and α3 domains ) MHC I; allowing the mouse to develop an immune response; and isolating from a mouse a nucleic acid sequence encoding a T cell receptor variable domain, wherein the T cell receptor variable domain binds a human tumor antigen with a K _D of not greater than about 1 nM.

In one embodiment, the mouse further comprises replacing, at an endogenous mouse T cell receptor variable region gene locus, with a plurality of unrearranged human T cell receptor variable region gene segments, wherein the unrearranged human T cell receptor variable region gene segments are recombined to encode the chimeric human/mouse gene A T-cell receptor containing a human variable region and a mouse constant region. In one embodiment, the mouse contains transgenic human CD8 and the mouse expresses functional human CD8 protein.

T-cell receptors with high avidity for tumor antigens are useful in cell-based therapeutics. T cell populations with high avidity for human tumor antigens have been generated by exposing human T cells to HLA-A2, which has been mutated to minimize CD8 binding to the α3 subunit to select only those T cells that have the highest avidity for the tumor antigen ( ie, 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. Non-human animals and non-human animal cells are useful for identifying peptides that will complex with human HLA class I that will bind with high avidity to the T cell receptor or activate a lymphocyte bearing the T cell receptor.

Class I antigen/HLA complex binding to a T cell, or T cell activation, can be measured by any suitable method known in the art. Peptide-specific APC and T cell binding and activation can be measured. For example, it has been reported that the involvement of T cells with antigen-presenting cells that express HLA-A2 causes the accumulation of PIP2 at the immune synapse, while cross-linking of MHC class I molecules does not. See Fooksman et al. (2009) Cutting Edge: Phosphatidylinositol 4,5-Bisphosphate Concentration at the APC Side of the Immunological Synapse Is Required for Effector T Cell Function, J. Immunol. 182:5179-5182.

It is also possible to measure the functional consequences of the interaction of a TCR-bearing lymphocyte and expressing an APC class I molecule, and these include lymphocyte-assisted cytolysis. For example, contact points on the α2 subunit of HLA-A2 have been reported to generate a signal for Fas-independent cytolysis by CD8+ CTL. HLA-A2 expressing Jurkat T cells undergo apoptosis upon contact (by antibodies) of epitopes on the HLA-A2 molecule known (from crystallographic studies) to contact CD8, without any apparent dependence on the cytoplasmic domain. See Pettersen et al. (1998) The TCR-Binding Region of the HLA Class I α2 Domain Signals Rapid Fas-Independent Cell Death: A Direct Pathway for T Cell-Mediated Killing of Target Cells? J. Immunol. 160:4343-4352. It has been suggested that the rapid cytolysis induced by contact of HLA-A2 α2 with CD8 CD8+ CTL may predominantly be mediated by this Fas-independent HLA-A2 mediated pathway (ibid.), which differs from the TCR-independent α3-mediated domain of cytolysis, which itself can induce apoptosis (see Woodle et al. (1997) Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway, J. Immunol. 158:2156-2164).

The consequence of the interaction between a T cell and an APC presenting a peptide in the context of MHC I can also be measured using a T cell proliferation assay. Alternatively, it can be determined by measuring the release of cytokines, often associated with the activation of the immune response. In one embodiment, an IFNγ ELISPOT (enzymatic immunostain method) can be used to monitor and quantify CD8+ T cell activation.

As described herein, activation of CD8+ T cells may be hindered in the genetically modified non-human animals described herein due to the species-specific binding of CD8 to MHC I. the modified animal (eg, a rodent, eg, mouse or rat) is exposed (eg, in vitro) to a human cell, eg, a human CD8-bearing cell, eg, a human T cell. In one embodiment, an MHC class I expressing cell of a mouse described herein is exposed in vitro to a T cell that contains human CD8 and a T cell receptor. In a specific embodiment, the T cell is a human T cell. In one embodiment, an MHC class I expressing mouse cell contains a peptide linked to a chimeric human/mouse MHC I complex or a humanized MHC I complex (which includes human β2 microglobulin), the T cell is a human T cell, and the ability to T cells bind the mouse peptide presenting cell. In one embodiment, activation of a human T cell by a mouse peptide presenting cell is determined. In one embodiment, there is provided an in vitro method for measuring the activation of a human T cell by a peptide presenting cell, comprising exposing a mouse or mouse cell of interest to an antigen of interest, exposing a human T cell to a cell from said mouse, or said mouse cell (presumably carrying a peptide derived from an antigen complexed with human or humanized MHC I) and measuring human T-cell activation. In one embodiment, the method is used to identify a T cell epitope of a human pathogen or human tumor. In one embodiment, the method is used to identify an epitope for a vaccine.

In one embodiment, a method is provided for determining T-cell activation by a putative human therapeutic agent, comprising exposing a genetically modified animal described herein to a putative human therapeutic agent (or, for example, exposing a human or humanized MHC I-expressing cell of such animal to using the peptide sequence of the putative therapeutic agent), exposing a cell of a genetically modified animal that represents a human or humanized MHC I/peptide complex with a T cell containing a human T cell receptor and CD8 capable of binding to a cell of a genetically modified animal, and measuring activation of a human T cell that is induced by a peptide presenting cell of a genetically modified animal.

In various embodiments, a complex formed between a human or humanized MHC class I-expressing cell from an animal described herein is formed with a T cell that contains a human CD8 sequence, e.g., a human T cell, or a T cell from a non-human animal , which contains a transgene that encodes human CD8. Mice transgenic for human CD8 are 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; and also, LaFace et al. (1995) Human CD8 Transgene Regulation of HLA Recognition by Murine T Cells, J. Exp. Med. 182:1315-1325.

In addition to being able to identify antigens and antigenic epitopes from human pathogens or tumors, the genetically modified animals of the present invention can be used to identify self antigens related to 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) describes the use of NOD mice bearing HLA/β2 microglobulin monochain in identification of type 1 diabetes autoantigens. In addition, genetically modified animals of the present invention can be used to study various aspects of human autoimmune disease. Because certain human MHC I polymorphic alleles are known to be associated with the development of certain diseases, e.g., autoimmune diseases (e.g., diffuse toxic goiter, 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 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, incorporated herein by reference), a genetically modified animal of the present invention containing a humanized MHC I locus, including such an allele may be useful as a model for an autoimmune disease. In one embodiment, the disease allele is HLA-B27 and the disease is ankylosing spondylitis or reactive arthritis; thus, in one embodiment, the animal used to study said diseases contains human or humanized HLA-B27.

Other aspects of cellular immunity are known in the present technical field, which include complexes MHC I; therefore, the genetically engineered non-human animals described herein can be used to study these aspects of the biology of immunity. For example, TCR binding to MHC class I is modulated in vivo by additional factors. A member of the B subfamily of leukocyte immunoglobulin-like receptors (LILRB1, or LIR-1) is expressed on MHC class I restricted CTLs and negatively regulates T cell stimulation by binding a specific determinant on the α3 subunit of the MHC class I molecule to APC. Structural studies show that the binding site for LIR-1 and CD8 overlap, indicating that inhibitory LIR-1 competes with stimulatory CD8 for binding to class I MHC molecules. 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; and also, 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 transmits inhibitory signals through its (intracellular) immunoreceptor tyrosine inhibitory motif (ITIM). Studies have shown that in NK cells, KIRs (inhibiting Ig-like killer cell receptors) that do not contain ITIM (normally unable to inhibit) can be inhibited in the presence of LIR-1 (presumably via LIR-1 ITIM) associated with the α3 domain by a molecule MHC class I (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), pointing to an interaction between LIR-1 associated with MHC class I and KIR and thus pointing to the role of HLA α3 domain binding in modulating NK cell inhibition.

As described above, MHC molecules interact with cells that do not express TCR. Among these cells are NK cells. NK cells are cytotoxic lymphocytes (which are distinct from CTL, or cytotoxic T lymphocytes) that play a central role in the cellular immune response and in particular innate immunity. NK cells represent the first line of defense against invasive microorganisms, viruses, and other "foreign" (eg, tumor) compounds. NK cells are activated or inhibited through surface receptors and they express CD8 but do not express TCR. NK cells can interact with cells that express MHC class I, but the interaction occurs through the CD8-binding α3 domain, and not through the TCR-binding, peptide-bearing α ₁ and α ₂ domains. The main function of NK cells is to destroy cells that do not contain sufficient surface protein MHC class I.

Cross-linking of MHC class I molecules on the surface of human natural killer (NK) cells leads to intracellular tyrosine phosphorylation, migration of the MHC class I molecule from the immune synapse, and downregulation of tumor cell cytolysis. 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.

Another function of MHC class I in NK cells is probably to prevent autocytotoxicity. NK cells carry both the activating 2B4 receptor and the 2B4 CD48 ligand; it appears that class I MHC binds 2B4 and prevents its activation by CD48. 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.

Thus, the genetically engineered non-human animals described herein can be used to investigate these non-TCR or CTL mediated processes and to develop approaches to modulate them.

Examples

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

Example 1 Construction and Characterization of HLA-A2 Genetically Modified Mice

Example 1.1 Expression of HLA-A2/H-2K in MG87 cells.

A viral construct containing the chimeric HLA-A2/H-2K gene sequence (FIG. 4A) was generated using standard molecular cloning techniques known to those skilled in the art for analyzing chimeric human/mouse MHC I expression in transfected cells.

Briefly, a murine HLA-A/H-2K chimeric viral construct was generated using the exon sequences encoding the α1, α2, and α3 domains of the α chain and cloning them in-frame with the murine coding sequences for the transmembrane and cytoplasmic domains from the H-2K gene. (Fig. 4A, pMIG-HLA-A2/H2K). As shown in FIG. 4, the construct contained the IRES-GFP reporter sequence, which allowed it to be determined whether the construct retained the ability to be expressed in cells after transfection.

Received containing the above-described chimeric construct viruses and propagated them in cells of human embryonic kidney 293 (293T). 293T cells were placed on 10 cm dishes and grown to 95% confluence. DNA transfection mixture was prepared with 25 µg pMIG-HLA-A2/H2K, pMIG-human HLA-A2, or pMIG-humanized β2 microglobulin, and 5 µg pMDG (enveloped plasmid), 15 µg pCL-Eco (packaging construct without signal packaging Ψ), 1 ml Opti-MEM (Invitrogen). To this 1 ml DNA mixture was added 80 μl of Lipofectamine-2000 (Invitrogen) in 1 ml of Opti-MEM, which were pre-mixed and incubated at room temperature for 5 minutes. The Lipofectamine/DNA mixture was incubated for an additional 20 minutes at room temperature and then added to 10 cm dishes and the plates were incubated at 37°C. The medium from the cells was collected after 24 hours and 10 ml of R10 (RPMI 1640 + 10% FBS) fresh medium was added to the cells. This medium change was repeated twice. After a full four days, the collected media were pooled, centrifuged and passed through a sterile filter to remove cell debris.

The propagated viruses obtained above were used to transduce MG87 cells (mouse fibroblast). MG87 cells from one T-75 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 D10 (DMEM high glucose; 10% fetal bovine serum) was added to the mixture of cells and trypsin and transferred to a 15 ml centrifuge tube at 1300 rpm for five minutes. After centrifugation of the cells, the medium was aspirated and the cells were resuspended in 5 ml D10. Cells were counted and ~3.0×10 ⁵ cells per well were placed in a 6-well plate. pMIG-HLA-A2 human or pMIG-HLA-A2/H-2K either alone or with virus pMIG-humanized β2 microglobulin was added to the wells, with non-transduced cells as a control. Cells were incubated at 37°C with 5% CO ₂ for 2 days. Cells were prepared for FACS analysis (using anti-HLA-A2 antibody, clone BB7.2) for HLA-A2 expression with or without β2 microglobulin.

The graphs (FIG. 4B), as well as a table summarizing the data obtained from the graphs (FIG. 4C), show that co-transduction with humanized β2 microglobulin increases the expression of human HLA-A2 or chimeric human/non-human HLA-A2/ H-2K, as shown by shifting the curves to the right.

Example 1.2. Construction of the chimeric HLA-A2/H-2K locus.

The mouse H-2K gene was humanized in one step by constructing a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, for example, US 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 mouse BAC clone RP23-173k21 (Invitrogen) was modified by homologous recombination to replace the genomic DNA encoding the α1, α2, and α3 domains of the mouse H-2K gene with human genomic DNA encoding the α1, α2, and α3 subunits of the human HLA-A gene. (Fig. 5).

Briefly, the genomic sequence encoding the mouse α1, α2, and α3 subunits of the H-2K gene was replaced with human genomic DNA encoding the α1, α2, and α3 domains of the human HLA-A*0201 gene in a single targeting event using a targeting vector containing a hygromycin cassette. , flanked by loxP sites with a 5' mouse homology arm containing a sequence 5' from the mouse H-2K locus, including a 5' untranslated region (UTR; 5' homology arm is shown in SEQ ID NO: 1) and a 3' mouse homology arm containing the 3' genomic sequence from the mouse H-2K α3 coding sequence (the 3' homology arm is shown in SEQ ID NO: 2).

The final construct for targeting the endogenous H-2K gene locus in the 5'-3' direction included (1) a 5' homology arm containing ~200 bp. mouse genomic sequence 5' from the endogenous H-2K gene, including the 5'UTR, (2) ~1339 bp. human genomic sequence, including HLA-A*0201 leader sequence, 1 HLA-A*0201/α intron leader sequence, HLA-A*0201 α1 exon, HLA-A*0201 α1-α2 intron, HLA-A* exon 0201 α2, ~316 b.p. 5' end of α2-α3 intron, (3) 5' loxP site, (4) hygromycin cassette, (5) 3' loxP site, (6) ~580 bp human genomic sequence, which includes ~304 bp. 3' end of intron α2- α3, exon HLA-A*0201 α3 and (7) 3' homology arm containing ~200 b.p. a mouse genomic sequence comprising an intron between mouse H-2K α3 and transmembrane coding sequences (see FIG. 5 for a schematic representation of the H-2K targeting vector). The 149 nucleotide sequence at the mouse/human junction 5' from the targeting vector is shown in SEQ ID NO: 3, and the 159 nucleotide sequence at the mouse/human junction 3' from the targeting vector is shown in SEQ ID NO: 4 . Homologous recombination with the indicated targeting vector created a modified mouse H-2K locus containing human genomic DNA encoding the α1, α2 and α3 domains of the HLA-A*0201 gene, operably linked to endogenous sequences encoding the mouse H-2K transmembrane and cytoplasmic domains, which upon translation leads to the formation of a chimeric human/mouse class I MHC protein.

Targeted BAC DNA was used to electroporate mouse F1H4 ES cells to generate modified ES cells to generate mice that express the MHC class I chimeric protein on the surface of nucleated cells (eg, T and B lymphocytes, macrophages, neutrophils). ES cells containing the insertion of human HLA sequences were identified by TAQMAN™ assay. Specific primer sets and probes were designed to detect insertion of human HLA sequences and associated selection cassettes (allele gain, GOA) and loss of endogenous mouse sequences (allele loss, LOA). Table 1 lists the names and positions found for each of the probes used in the quantitative PCR assays.

Table 1: Probes used for genotyping

Probe Analysis Area detected by the probe Subsequence SEQID NO HYG GOA Hygromycin cassette ACGAGCGGGT TCGGCCCATT C 5 1665H1 GOA Human HLA-A2 α2-α3 intron AGTCCTTCAG CCTCCACTCA GGTCAGG 6 1665H2 GOA Exon HLA-A2 human α2 TACCACCAGT ACGCCTACGA CGGCA 7 5112H2 GOA Human HLA-A2 α2-α3 intron ATCCTGTACCAGAGAGTG eight

The selection cassette can be removed by methods known to those skilled in the art. For example, ES cells harboring a chimeric human/mouse MHC class I locus can be transfected with a construct that expresses Cre to remove the lox-site flanked hygromycin cassette introduced by inserting a targeting construct containing human HLA-A*0201 gene sequences (see Figure 5) . The hygromycin cassette can optionally be removed by breeding mice that express the Cre recombinase. Optionally, a hygromycin cassette is retained in the mouse.

The targeted ESs described above were used as donor ES cells and introduced into the mouse embryo at the 8 cell stage by the VELOCIMOUSE® method (see, for example, US 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). VELOCIMICE® (F0 mice derived entirely from a donor ES cell) independently carrying a class I chimeric MHC gene were identified by genotyping using a modified allelic analysis (Valenzuela et al., formerly) that detects the presence of unique HLA-A*0201 gene sequences person.

Example 1.3. In vivo expression of chimeric HLA-A/H-2K in genetically modified mice.

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

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

Example 2 Construction and Characterization of β2 Microglobulin Genetically Modified Mice

Example 2.1: Construction of a humanized β2 microglobulin locus

The mouse β2 microglobulin (β2m) gene was humanized in one step by constructing a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, for example, US Pat. No. 6,586,251 and Valenzuela et al., formerly).

Briefly, a targeting vector containing the upstream and downstream mouse β2m homology arms from BAC clone 89C24 from the RPCI-23 library (Invitrogen) was generated by bacterial homologous recombination. Mouse homology arms were designed to flank 2.8 kb. human β2m DNA fragment extending from exon 2 to approximately 267 nucleotides downstream of non-coding exon 4 (FIG. 7). A drug selection cassette (neomycin) flanked by recombinase recognition sites (eg, loxP sites) was introduced into a targeting vector to allow subsequent selection. The final targeting vector was linearized and electroporated into the F1H4 mouse ES cell line (Valenzuela et al., formerly).

Targeted ES cell clones with the drug cassette removed (by introducing Cre recombinase) were introduced into the mouse embryo at the 8 cell stage by the VELOCIMOUSE® method (see, for example, US Pat. No. 7,294,754 and Poueymirou et al., formerly). VELOCIMICE® (F0 mice derived entirely from donor ES cells) carrying the humanized β2m gene were identified by screening for mouse allele loss and human allele gain using modified allelic analysis (Valenzuela et al., formerly).

Example 2.2 Characterization of Humanized β2 Microglobulin Mice

Mice heterozygous for the humanized β2 microglobulin gene (β2m) were evaluated for expression using flow cytometry (Figures 8 and 9).

Briefly, blood was obtained from a group (n=4 per group) of wild-type mice humanized for β2m, humanized for MHC (i.e. human HLA) class I, and dual humanized for β2m and MHC class I mice using methods known in the present technical field. Blood from each group of mice was treated with ACK lysis buffer (Lonza Walkersville) to remove erythrocytes. The remaining cells were stained with fluorochrome-conjugated anti-CD3 (17A2), anti-CD19 (1D3), anti-CD11b (M1/70), anti-human HLA class I and anti-human β2 microglobulin (2M2) antibodies. Flow cytometry was performed using BD-FACSCANTO™ (BD Biosciences).

Human class I HLA expression was detected on cells from singly humanized and double humanized animals, while β2 microglobulin expression was only detected on cells from doubly humanized mice (FIG. 8). Co-expression of human β2m and human class I HLA resulted in an increase in the detectable amount of human class I HLA on the cell surface compared to the expression of human class I HLA in the absence of human β2m (Figure 9; mean fluorescence intensity 2370 versus 1387).

Example 3 Immune response to influenza virus (Flu) and Epstein-Barr virus (EBV) peptides presented by APC from genetically modified mice expressing HLA-A2/H-2K and humanized β2 microglobulin.

PBMCs from multiple human donors were screened for both HLA-A2 expression and their ability to develop responses to influenza and EBV peptides. One donor was chosen for subsequent experiments.

Human T cells were isolated from PBMCs of a selected donor using negative selection. Non-T cell spleen cells were isolated from a mouse heterozygous for the HLA-A2/H-2K chimeric and heterozygous for the humanized microglobulin β2 gene and wild-type mice. Approximately 50,000 non-T cell spleen cells from mice were added to an anti-human IFNγ antibody-coated ELISA plate. A Flu peptide (10 μM) or a pool of EBV peptides (5 μM each) was added. Poly IC was added at a concentration of 25 μg/well and the wells were incubated for three hours at 37°C in 5% CO ₂ . Human T cells (50,000) and human anti-CD28 were added to non-T spleen cells and peptides, and the wells were incubated for 40 hours at 37° C. in 5% CO ₂ , after which IFNγ ELISA stains were analyzed.

As shown in FIG. 10, human T cells were able to develop a response to flu and EBV peptides presented by mouse APCs that expressed chimeric HLA-A2/H-2K and humanized β2 microglobulin on their surface.

Equivalents

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

The entire contents of all non-patent documents, patent applications and patents cited herein are incorporated by reference herein in their entirety.

In particular aspects, the present invention may relate to the following options:

Option 1. A non-human animal containing, at an endogenous major histocompatibility complex I (MHC I) locus, a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide,

wherein the human portion of the chimeric polypeptide contains the extracellular domain of the human MHC I polypeptide, and

wherein the non-human animal expresses a chimeric human/non-human MHC I polypeptide.

Option 2. A non-human animal according to Option 1, wherein the animal is a rodent, the rodent having at its endogenous major histocompatibility complex I (MHC I) locus a nucleotide sequence encoding a chimeric human/rodent MHC I polypeptide,

wherein the human portion of the chimeric polypeptide comprises the extracellular domain of the human MHC I polypeptide, and

wherein the rodent expresses a chimeric human/rodent MHC I polypeptide.

Option 3 A rodent according to option 2, wherein the rodent does not express the extracellular domain of the endogenous rodent MHC I polypeptide from the endogenous rodent MHC I locus.

Option 4. A rodent according to option 2, in which the nucleotide sequence is operably linked to endogenous rodent regulatory elements.

Embodiment 5 The rodent of Embodiment 2, wherein the rodent is a mouse.

Option 6 A mouse according to option 5 in which the endogenous locus is the mouse H-2K locus.

Option 7 A rodent according to Option 2 wherein the human portion of the chimeric polypeptide contains a human leader sequence.

Option 8. A rodent according to option 2, in which the human part of the chimeric polypeptide contains the α1, α2 and α3 domains of the human MHC I polypeptide.

Option 9 A rodent according to option 2, wherein the rodent portion of the chimeric polypeptide contains the transmembrane and cytoplasmic domains of the endogenous rodent MHC I polypeptide.

Embodiment 10 A rodent according to Embodiment 9, wherein the rodent is a mouse, the endogenous rodent MHC I locus is an H-2K locus, and the endogenous rodent MHC I polypeptide is H-2K.

Option 11 A rodent according to option 2 wherein the human MHC I polypeptide is selected from the group consisting of HLA-A, HLA-B and HLA-C.

Embodiment 12 A rodent according to Embodiment 11 wherein the human MHC I polypeptide is an HLA-A polypeptide.

Option 13. A mouse containing in the endogenous H-2K locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide,

moreover, the human part of the chimeric polypeptide contains the extracellular domain of the human HLA-A2 polypeptide, and the mouse part contains the transmembrane and cytoplasmic domains of the mouse H-2K polypeptide, and

wherein the mouse expresses a chimeric HLA-A2/H-2K polypeptide.

Option 14 A mouse according to option 13, wherein the mouse does not express the extracellular domain of the mouse H-2K polypeptide from the endogenous H-2K locus.

Option 15 A mouse according to Option 13 wherein the human portion of the chimeric polypeptide contains a human leader sequence.

Option 16 A mouse according to Option 13 in which the nucleotide sequence is operably linked to mouse endogenous regulatory elements.

Option 17 A mouse according to option 13 wherein the human portion of the chimeric polypeptide contains the α1, α2 and α3 domains of the human HLA-A2 polypeptide.

Embodiment 18 A mouse according to Embodiment 13 wherein the human HLA-A2 polypeptide is an HLA-A2.1 polypeptide.

Embodiment 19 A mouse according to embodiment 13 in which the mouse H-2K locus is the H-2Kb locus.

Option 20. A method for modifying the mouse MHC I locus to express a chimeric human/mouse MHC I polypeptide, comprising replacing, in the endogenous MHC I locus, a nucleotide sequence encoding the extracellular domain of a mouse MHC I polypeptide with a nucleotide sequence encoding the extracellular domain of a human MHC I polypeptide.

Option 21 The method of option 20 wherein the mouse does not express the extracellular domain of the mouse MHC I polypeptide from the endogenous mouse MHC I locus.

Option 22 The method of option 20 wherein the mouse MHC I locus is an H-2K locus and the mouse MHC I polypeptide is an H-2K polypeptide.

Option 23 The method of option 20 wherein the human MHC I polypeptide is an HLA-A polypeptide.

Option 24 The method of option 20 wherein the mouse expresses the α1, α2 and α3 domains of a human MHC I polypeptide.

Option 25 The method of option 20 wherein the mouse expresses the transmembrane and cytoplasmic domains of a mouse MHC I polypeptide.

Embodiment 26 The method of Embodiment 20 wherein the substitution is carried out in one ES cell and one ES cell is introduced into a mouse fetus to produce a mouse.

Option 27 A non-human animal comprising, at an endogenous non-human microglobulin β2 locus, a nucleotide sequence encoding a polypeptide comprising the human β2 microglobulin amino acid sequence, wherein the non-human animal expresses a human or humanized microglobulin β2 polypeptide.

Embodiment 28 A non-human animal according to Embodiment 27, wherein the animal is a rodent, wherein the rodent comprises, at the endogenous rodent microglobulin β2 locus, a nucleotide sequence encoding a polypeptide comprising the human microglobulin β2 amino acid sequence, and wherein the rodent expresses a human or humanized microglobulin β2 polypeptide .

Embodiment 29 A rodent according to embodiment 28, wherein the rodent does not express a functional endogenous rodent microglobulin β2 polypeptide from the endogenous rodent β2 microglobulin locus.

Option 30. A rodent according to option 28, in which the nucleotide sequence is operably linked to endogenous rodent microglobulin β2 regulatory elements.

Option 31. A rodent according to option 28, in which the nucleotide sequence contains the nucleotide sequence presented in exon 2 - exon 4 of the human β2 microglobulin gene.

Option 32. A rodent according to option 28, in which the nucleotide sequence contains the nucleotide sequences present in exons 2, 3 and 4 of the human microglobulin β2 gene.

Option 33 A rodent according to option 31, wherein the nucleotide sequence further comprises a nucleotide sequence present in exon 1 of the rodent microglobulin β2 gene.

Option 34 A rodent according to option 32, wherein the nucleotide sequence further comprises a nucleotide sequence present in exon 1 of the rodent microglobulin β2 gene.

Embodiment 35 A rodent according to Embodiment 28, wherein the rodent is a mouse.

Option 36 A mouse comprising, at an endogenous microglobulin β2 locus, a nucleotide sequence encoding a polypeptide comprising the human β2 microglobulin amino acid sequence, wherein the mouse expresses a human or humanized microglobulin β2 polypeptide.

Embodiment 37 A mouse according to Embodiment 36, wherein the mouse does not express functional endogenous mouse β2 microglobulin from the endogenous mouse β2 microglobulin locus.

Option 38 A mouse according to option 36 in which the nucleotide sequence is operably linked to mouse endogenous regulatory elements.

Option 39 A mouse according to option 36, wherein the nucleotide sequence contains the nucleotide sequence present in exon 2-exon 4 of the human microglobulin β2 gene.

Option 40 A mouse according to option 36, in which the nucleotide sequence contains the nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene.

Option 41 A mouse according to option 39, wherein the nucleotide sequence further comprises a nucleotide sequence present in exon 1 of the mouse microglobulin β2 gene.

Option 42 A mouse according to option 40, wherein the nucleotide sequence further comprises a nucleotide sequence present in exon 1 of the mouse microglobulin β2 gene.

Option 43. A method of modifying a mouse β2 microglobulin locus to express a human or humanized β2 microglobulin polypeptide, comprising replacing a nucleotide sequence encoding a mouse β2 microglobulin polypeptide in an endogenous murine β2 microglobulin locus with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide.

Option 44 The method of option 43 wherein the mouse does not express a functional murine microglobulin β2 polypeptide from the endogenous β2 microglobulin locus.

Option 45. The method according to option 43, wherein the nucleotide sequence encoding the human or humanized microglobulin β2 polypeptide comprises a nucleotide sequence present in exon 2 to exon 4 of the human β2 microglobulin gene.

Option 46 The method of option 43 wherein the nucleotide sequence encoding the human or humanized microglobulin β2 polypeptide comprises nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene.

Option 47 The method of option 43 wherein the modified locus retains the nucleotide sequence of exon 1 of the mouse microglobulin β2 gene.

Embodiment 48 The method of Embodiment 43 wherein the substitution is carried out in one ES cell and one ES cell is introduced into a mouse fetus to produce a mouse.

Option 49: A non-human animal that contains in its genome:

a first nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of the human MHC I polypeptide; and

a second nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide,

wherein the first nucleotide sequence is located at the endogenous non-human MHC I locus and the second nucleotide sequence is located at the endogenous non-human microglobulin β2 locus, and

wherein the non-human animal expresses a chimeric human/non-human MHC I polypeptide and a human or humanized microglobulin β2 polypeptide.

Embodiment 50. A non-human animal according to Embodiment 49, wherein the animal is a rodent, and wherein the rodent contains in its genome:

a first nucleotide sequence encoding a chimeric human/rodent MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of the human MHC I polypeptide; and

a second nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide,

wherein the first nucleotide sequence is located at the endogenous rodent MHC I locus and the second nucleotide sequence is located at the endogenous rodent microglobulin β2 locus, and

wherein the rodent expresses a chimeric human/rodent MHC I polypeptide and a human or humanized microglobulin β2 polypeptide.

Embodiment 51 A rodent according to embodiment 50, wherein the rodent does not express the extracellular domain of an endogenous rodent MHC I polypeptide and a functional endogenous rodent microglobulin β2 polypeptide from their endogenous rodent loci.

Option 52 A rodent according to option 50 wherein the first nucleotide sequence is operably linked to endogenous rodent MHC I regulatory elements and the second nucleotide sequence is operably linked to endogenous rodent microglobulin β2 regulatory elements.

Embodiment 53 A rodent according to Embodiment 50, wherein the rodent is a mouse.

Embodiment 54 A mouse according to embodiment 53 in which the endogenous MHC I locus is the mouse H-2K locus.

Embodiment 55 A rodent according to embodiment 50 wherein the human portion of the chimeric polypeptide contains the α1, α2 and α3 domains of a human MHC I polypeptide.

Embodiment 56 A rodent of embodiment 50 wherein the rodent portion of the chimeric human/rodent MHC I polypeptide contains the cytoplasmic and transmembrane domains of the rodent MHC I polypeptide.

Embodiment 57 A rodent according to embodiment 50 wherein the human MHC I polypeptide is selected from HLA-A, HLA-B and HLA-C.

Embodiment 58 A rodent according to embodiment 57 wherein the human MHC I polypeptide is an HLA-A polypeptide.

Option 59 A rodent according to option 50, wherein the second nucleotide sequence comprises the nucleotide sequence present in exon 2-exon 4 of the human β2 microglobulin gene.

Option 60. A rodent according to option 50, wherein the second nucleotide sequence contains the nucleotide sequences present in exons 2, 3, and 4 of the human β2 microglobulin gene.

Option 61. A mouse containing in its genome:

a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises the human HLA-A2 extracellular domain and the murine portion comprises the mouse H-2K transmembrane and cytoplasmic domains; and

a second nucleotide sequence encoding a human or humanized microglobulin β2 polypeptide,

wherein the first nucleotide sequence is located at the endogenous H-2K locus and the second nucleotide sequence is located at the endogenous mouse microglobulin β2 locus, and

wherein the mouse expresses a chimeric human/mouse MHC I polypeptide and a human or humanized β2 microglobulin polypeptide.

Embodiment 62 A mouse according to embodiment 61 wherein the mouse does not express endogenous mouse microglobulin H-2K and β2 polypeptides from their endogenous loci.

Option 63 A mouse of option 61 wherein the first nucleotide sequence is operably linked to endogenous mouse H-2K regulatory elements and the second nucleotide sequence is operably linked to endogenous mouse microglobulin β2 regulatory elements.

Option 64 A mouse according to option 61 wherein the human portion of the chimeric polypeptide contains the α1, α2 and α3 domains of the human MHC I polypeptide.

Option 65 A mouse according to option 61, wherein the second nucleotide sequence contains the nucleotide sequence present in exon 2-exon 4 of the human microglobulin β2 gene.

Option 66 A mouse according to option 61, in which the second nucleotide sequence contains the nucleotide sequences present in exons 2, 3, and 4 of the human microglobulin β2 gene.

Option 67 A mouse according to option 61 in which expression of a human or humanized β2 microglobulin polypeptide increases the expression of a chimeric human/mouse MHC I polypeptide compared to expression of a chimeric human/mouse MHC I polypeptide in the absence of expression of a human or humanized β2 microglobulin polypeptide.

Option 68. A method for obtaining a genetically modified mouse, comprising:

modifying the first mouse MHC I locus to express a chimeric human/mouse MHC I polypeptide comprising replacing, in the endogenous mouse MHC I locus, a nucleotide sequence encoding the extracellular domain of the mouse MHC I polypeptide with a nucleotide sequence encoding the extracellular domain of the human MHC I polypeptide;

modifying a second mouse β2 microglobulin locus to express a human or humanized β2 microglobulin polypeptide, comprising replacing, at the endogenous murine β2 microglobulin locus, a nucleotide sequence encoding a mouse β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide; and

crossing a first and a second mouse to produce a genetically modified mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the genetically modified mouse expresses the chimeric human/mouse an MHC I polypeptide; and a human or humanized β2 microglobulin polypeptide.

Option 69 The method of option 68 wherein the MHC I locus is an H-2K locus, the human MHC I polypeptide is HLA-A2, and the mouse expresses a chimeric HLA-A2/H-2K polypeptide.

Option 70. The method according to option 69, wherein the chimeric HLA-A2/H-2K polypeptide comprises the extracellular domain of the HLA-A2 polypeptide and the cytoplasmic and transmembrane domains of the H-2K polypeptide.

Option 71 The method of option 68 wherein the second nucleotide sequence comprises nucleotide sequences present in exons 2, 3 and 4 of the human β2 microglobulin gene and a nucleotide sequence present in exon 1 of the mouse β2 microglobulin gene.

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Sequence listing

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<---

Claims (9)

1. A rodent containing in the endogenous microglobulin β2 locus a nucleic acid sequence containing exon 1 of the endogenous rodent β2 microglobulin gene, functionally linked to a nucleic acid containing sequences presented in exon 2, exon 3 and exon 4 of the human β2 microglobulin gene, and the nucleic acid sequence acid encodes a β2 microglobulin polypeptide containing the amino acid sequence of human β2 microglobulin, wherein the nucleic acid sequence is operably linked to endogenous rodent microglobulin β2 regulatory elements, wherein the rodent expresses a β2 microglobulin polypeptide comprising the amino acid sequence of human β2 microglobulin, and wherein the rodent is a rat or a mouse. 2. A rodent according to claim 1, wherein the nucleic acid sequence contains a sequence presented in exon 2 - exon 4 of the human microglobulin β2 gene. 3. A rodent according to claim 1 or 2, wherein the .beta.2 microglobulin polypeptide comprises amino acids 23-119 of the human .beta.2 microglobulin protein. 4. A rodent according to any one of the preceding claims, wherein the rodent does not express a functional endogenous β2 microglobulin polypeptide from the endogenous β2 microglobulin locus. 5. A rodent according to any one of the preceding claims, wherein the rodent is a rat. 6. The rodent according to any one of paragraphs. 1-4, wherein the rodent is a mouse.

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