mice with genetically modified major histocompatibility complex

专利摘要:
  METHODS OF PRODUCTION OF GENETICALLY MODIFIED MICE EXPRESSING MAJOR HISTOCOMPATIBILITY COMPLEX I (MHC I), AS WELL AS NUCLEIC ACID AND POLYPEPTIDE.The present invention relates to methods for producing a genetically modified non-human animal comprising the step of modifying a non-human animal genome to comprise, at an endogenous non-human Major Histocompatibility Complex I (MHC I) gene locus, a sequence nucleotide encoding a human/non-human MHC I chimeric polypeptide, wherein a human portion of the chimeric polypeptide comprises (ALFA)1, (ALFA)2, and (ALFA)3 domains of a human MHC I polypeptide, wherein the animal expresses polypeptide of chimeric human/non-human MHC I. The present invention further relates to nucleic acid and polypeptide.
公开号:BR112014009259A2
申请号:R112014009259-1
申请日:2012-10-26
公开日:2020-10-27
发明作者:Lynn MacDonald；Andrew J. Murphy；Cagan Gurer；John McWhirter；Vera VORONINA；Faith HARRIS；Sean Stevens
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

[001] [001] This patent application claims priority benefit to Provisional Patent Application US 61/552,582 and 61/552,587, both filed October 28, 2011, and Provisional Patent Application US 61/700,908, filed September 14, 2012, all of which are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION
[002] [002] The present invention relates to a genetically modified non-human animal, eg a rodent (eg a mouse or rat), which expresses a molecule of the class | of the human or humanized Major Histocompatibility Complex (MHC). The invention also relates to a genetically modified non-human animal, for example a mouse or rat, which expresses an MHC | human or humanized (e.g., MHC I α-chain) and/or a human or humanized B2 microglobulin; as well as embryos, tissues, and cells expressing the same. The invention additionally provides methods for making a non-human genetically modified animal that expresses the protein of the class | of human or humanized MHC (e.g. α-chain of MHC1) and/or —B2 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 of modifying an MHC | and/or a B2 microglobulin from a non-human animal, for example a mouse or rat, to express an MHC | human or humanized and/or B2 microglobulin. BACKGROUND OF THE INVENTION
[003] [003] In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (eg, immunoglobulins) and T lymphocytes (eg, T cell receptor or TCR) These foreign antigens are presented on the surface of cells as peptide fragments via specialized proteins, generically referred to as major histocompatibility complex (MHC) molecules. MHC molecules are encoded by multiple loci that are found as a linked cluster of genes spanning about 4 Mb. In mice, the MHC genes are found on chromosome 17, and for historical reasons are referred to as the MHC genes. of histocompatibility 2 (H-2). In humans, the genes are found on chromosome 6 and are called human leukocyte antigen (HLA) genes. The loci in mice and humans are polygenic; they include three highly polymorphic classes of MHC genes (class I, II, and III) that exhibit similar organization in the human and murine genomes (see FIG. 2 and FIG. 3, respectively).
[004] [004] MHC loci exhibit the highest polymorphism in the genome; some genes are represented by >300 alleles (eg, human HLA-DRB and human HLA-B). All class MHC genes | and l may have peptide fragments, but each gene expresses a protein with different binding characteristics, reflecting polymorphisms and allelic variants. Any given individual has a unique range of peptide fragments that can be presented on the cell surface to B and T cells in the course of an immune response.
[005] [005] Humans and mice have class MHC genes | (see FIG. 2 and FIG. 3). In humans, the genes of the class | classics are called HLA-A, HLA-B and HLA-C, while in mice they are H-2K, H-2D and H-2L. Class molecules | they consist of two chains: a polymorphic a-chain (sometimes referred to as the heavy chain) and a smaller chain called microglobulin B2 (also known as the light chain) which is usually non-polymorphic (FIG. 1). These two chains form a non-covalent heterodimer on the cell surface. Chain a contains three domains (a1, a2, and a3). Exon 1 of the a-chain gene encodes the leader sequence, exons 2 and 3 encode the a1 and a2 domains, exon 4 encodes the a3 domain, exon 5 encodes the transmembrane domain, and exons 6 and 7 encode the cytoplasmic tail. The a-chain forms a peptide bond cleft that surrounds the a1 and a2 domains (which are similar to Ig-like domains) followed by the a3 domain, which is similar to 82 microglobulin.
[006] [006] Microglobulin B2 is a 12 kDa non-glycosylated protein; one of its functions is to stabilize the MHC a-chain of the class
[007] [007] Class MHC Molecules | are expressed in all nucleated cells, including tumor cells. They are specifically expressed on T and B lymphocytes, macrophages, dendritic cells and neutrophils, among other cells, and function to display peptide fragments (typically 8-10 amino acids in length) on the surface to CD8+ cytotoxic T lymphocytes (CTLs) . CTLs specialize in killing any cell that carries an MHC I-linked peptide recognized by its own membrane-bound TOR. When a cell displays peptides derived from cellular proteins not normally present (eg, of viral, tumor, or other non-autonomous origin), such peptides are recognized by CTLs that are activated and kill the cell displaying the peptide.
[008] [008] Typically, presentation of normal (ie, autonomous) proteins in the context of MHC molecules | does not elicit any CTL activation due to tolerance mechanisms. However, in some diseases (eg cancer, autoimmune diseases), peptides derived from autoproteins become a target of the cellular component of the immune system which results in the destruction of cells that present such peptides. Although progress has been made in recognizing some self-derived antigens that elicit a cellular immune response (e.g., antigens associated with various cancers) to improve the identification of peptides recognized by human CTLs through MHC molecules of the | class, there remains a need for systems 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 to identify disease-associated antigens to develop human therapeutics, eg vaccines and other biological agents. Systems to assess antigen recognition in the context of the human immune system can help identify therapeutically useful populations of CTLs (eg, useful for studying and combating human disease). Such systems can also help to enhance the activity of human CTL populations to more effectively fight infections and entities carrying foreign antigens. Thus, there is a need for biological systems (eg, genetically engineered animals) that can generate an immune system exhibiting components that mimic the function of the human immune system. SUMMARY OF THE INVENTION
[009] [009] A biological system for generating or identifying peptides that associate with MHC class proteins | and chimeras thereof, and bind to CD8+ T cells, is provided. Non-human animals comprising non-human cells that express human or humanized molecules that function in the cellular immune response are provided. Humanized rodent loci encoding B2 microglobulin and MHC proteins | human or humane or humanized are also provided. Humanized rodent cells that express human or humanized 82 microglobulin and MHC molecules are also provided. In vivo and in vitro systems are provided comprising humanized rodent cells, wherein the rodent cells express one or more human or humanized molecules of the immune system.
[0010] [0010] Provided herein is a non-human animal, eg a rodent (eg a mouse or rat), comprising in its genome a nucleotide sequence encoding an MHC polypeptide | chimeric human/non-human (e.g. human/rodent, e.g. human/mouse or human/rat), wherein a human portion of the chimeric polypeptide comprises an extracellular domain of an MHC polypeptide | human. Specifically, provided here is a non-human animal comprising at an MHC | a nucleotide sequence encoding an MHC polypeptide | chimeric human/non-human, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of an MHC polypeptide | human, and in which the animal expresses the MHC polypeptide | chimeric human/non-human. In one aspect, the animal does not express an extracellular domain of an MHC | non-human and endogenous MHC locus | endogenous non-human. In one aspect of the invention, the non-human animal (e.g. a rodent, e.g. a mouse)
[0011] [0011] In one aspect, the nucleotide sequence encoding the MHC | chimeric human/non-human is operably linked to endogenous non-human regulatory elements, eg promoter, enhancer, silencer, etc. In one embodiment, a human portion of the chimeric polypeptide comprises a human leader sequence. In a further embodiment, the human portion of the chimeric polypeptide comprises domains a1, a2, and a3 of the MHC | human. The MHC polypeptide | human can be selected from the group consisting of HLA-A, HLA-B, and HLA-C. In one embodiment, the MHC | human is an HLA-A2 polypeptide, for example, an HLA-A2.1 polypeptide.
[0012] [0012] 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
[0013] [0013] Also provided herein is a mouse comprising at an endogenous H-2K locus a nucleotide sequence encoding an MHC polypeptide | human/mouse chimeric, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A polypeptide (e.g., HLA-A2) and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K polypeptide, and in which the mouse expresses MHC polypeptide | chimeric human/mouse. In some embodiments, the mouse does not express an extracellular domain of the
[0014] [0014] Another aspect of the invention pertains to a non-human animal, e.g. a rodent (e.g. a mouse or rat), comprising in its genome a nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide. . Thus provided herein is a non-human animal comprising at an endogenous non-human B2 microglobulin B2 locus a nucleotide sequence encoding a human or humanized B2 microglobulin B2 polypeptide, wherein the animal expresses the human or humanized B2 microglobulin polypeptide. In one aspect, the animal does not express a functional endogenous non-human B2 microglobulin B2 polypeptide from an endogenous non-human B2 microglobulin B2 locus. In one aspect, the animal comprises two copies of the B2 microglobulin locus that encode human or humanized B2 microglobulin polypeptide; in another embodiment, the animal comprises a copy of the B2 microglobulin locus that encodes human or humanized B2 microglobulin polypeptide. Thus, the animal may be homozygous or heterozygous for the B2 microglobulin locus that encodes the human or humanized B2 microglobulin polypeptide. In various embodiments, the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide is comprised in the germ line of the non-human animal.
[0015] [0015] In some embodiments, the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide is operably linked to endogenous non-human B2 microglobulin B2 regulatory elements. In one aspect, the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide comprises a nucleotide sequence exposed in exon 2 to exon 4 of a human B2 microglobulin gene. In another aspect, the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide comprises nucleotide sequences exposed in exons 2, 3, and 4 of a human B2 microglobulin gene. In another aspect, the nucleotide sequence also comprises a nucleotide sequence exposed in exon 1 of a non-human B2 microglobulin gene. In some embodiments, the non-human animal is a rodent (eg, mouse or rat); thus, the non-human B2 microglobulin locus is a rodent (eg, a mouse or rat) microglobulin B2 locus.
[0016] [0016] Also provided is a mouse comprising at an endogenous microglobulin B2 locus a nucleotide sequence encoding a human or humanized microglobulin B2 polypeptide, wherein the mouse expresses the human or humanized microglobulin B2 polypeptide. In some embodiments, the mouse does not express an endogenous mouse B2 microglobulin.
[0017] [0017] The invention further provides a non-human animal (eg a rodent, eg a mouse or rat) comprising in its genome a nucleotide sequence encoding an MHC polypeptide | chimeric human/non-human and a nucleotide sequence encoding a human or humanized microglobulin B2 polypeptide. In one embodiment, the invention provides a non-human animal comprising in its genome a first nucleotide sequence encoding an MHC | chimeric human/non-human, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of an MHC polypeptide | human; and a second nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide, wherein the first nucleotide sequence is located
[0018] [0018] Therefore, the invention provides a mouse comprising in its genome a first nucleotide sequence encoding an MHC polypeptide | chimeric human/mouse, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A (e.g., HLA-A2) and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K; and a second nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous H-2K locus and the second nucleotide sequence is located at an encoded B2 microglobulin locus. gene, and in which the mouse expresses the MHC polypeptide | human/chimeric mouse and human or humanized B2 microglobulin polypeptide. In one embodiment, the non-human animal (e.g., the mouse) comprising both
[0019] [0019] In one aspect, the first nucleotide sequence is operably linked to MHC regulatory elements | endogenous non-human (eg, mouse), and the second nucleotide sequence is operably linked to endogenous (eg, mouse) non-human B2 microglobulin elements. The human portion of the chimeric polypeptide may comprise domains a1, a2 and a3 of the MHC | human. The second nucleotide sequence may comprise a nucleotide sequence exposed in exon 2 to exon 4 of a human B2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences exposed in exons 2, 3, and 4 of a human microglobulin B2 gene. In one aspect, the mouse comprising both the MHC | chimeric as human or humanized B2 microglobulin polypeptide may be such that human or humanized B2 microglobulin expression increases MHC polypeptide expression | human/mouse chimeric when compared to MHC polypeptide expression | human-—in the chimeric/mouse in the absence of human or humanized B2 microglobulin polypeptide expression.
[0020] [0020] Also provided are methods of making the genetically engineered non-human animals (eg rodents, eg mice or rats) described herein. Thus, in one embodiment, provided is a method of modifying an MHC | of a rodent (eg, a mouse or rat) to express an MHC polypeptide | chimeric human/rodent (eg human/mouse or human/rat), wherein the method comprises substituting at the MHC locus | a nucleotide sequence encoding an extracellular domain of an MHC Polypeptide | of rodent with a nucleotide sequence encoding an extracellular domain of an MHC polypeptide | human. In another embodiment, provided is a method of modifying a rodent microglobulin B2 locus (e.g., a mouse or rat) to express a human or humanized microglobulin B2 polypeptide, wherein the method comprises substituting at the microglobulin B2 locus. endogenous rodent (e.g., mouse or rat) croglobulin B2 a nucleotide sequence encoding a rodent (e.g., a mouse or rat) microglobulin B2 polypeptide with a nucleotide sequence encoding a human B2 microglobulin polypeptide, or humanized. In such methods, the replacement can be done in a simple ES cell, and the simple ES cell can be introduced into a rodent (eg, a mouse or rat) to make an embryo. The resulting rodent (eg a mouse or rat) can be bred to generate a doubly humanized animal.
[0021] [0021] Accordingly, the invention also provides a method of making doubly humanized animals, e.g. rodents (e.g. mice or rats). In one embodiment, provided is a method of making a genetically modified mouse with
[0022] [0022] Also provided here are cells, eg isolated antigen presenting cells, derived from the non-human animals (eg rodents, eg mice or rats) described herein. Tissues and embryos derived from the non-human animals described herein are also provided.
[0023] [0023] In yet another embodiment, the invention provides methods for identifying antigens or antigen epitopes that elicit an immune response, methods for evaluating a vaccine candidate, methods for identifying T cells of high affinity for human pathogens or cancer antigens.
[0024] [0024] Any of the modalities and aspects described here may be used together with each other unless otherwise indicated or evident from the context. Other embodiments will become apparent to those skilled in the art from a review of the following detailed description. The following detailed description includes exemplary representations of the various embodiments of the invention which are not restrictive of the invention so claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate the embodiments and not to limit the invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] [0025] FIG. 1 is a schematic drawing of the four domains of a class β MHC molecule: a chain containing the a1, a2 and a3 domains and the fourth non-covalently associated domain, microglobulin B2 (B2m). The gray circle represents a peptide bound in the peptide bond cleft.
[0026] [0026] FIG. 2 is a schematic (unscaled) representation of the relative genomic structure of human HLA, showing the genes of classes I, II, and III.
[0027] [0027] FIG. 3 is a schematic (unscaled) representation of the relative genomic structure of the mouse MHC, showing the genes of classes I, II, and III.
[0028] [0028] FIG. 4 illustrates a viral vector construct containing a cCDNA 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 (dotted line), HLA-A2/H-2K (dotted line), or no transduction (solid line) or alone (left) or cotransduced with humanized microglobulin B2 (right) (B). Data from the horizontal gates graphically presented in (B) are shown as a percentage of cells that express the construction in the table in (C).
[0029] [0029] FIG. 5 is a schematic (unscaled) diagram of the targeting strategy used to make a chimeric H-2K locus expressing an extracellular region of a human HLA-A2 protein. Mouse sequences are shown in black and human sequences are shown in white. L = leader, UTR = non-translated region, TM = transmembrane domain, CYT = cytoplasmic domain, HYG = hygromycin.
[0030] [0030] FIG. 6A demonstrates the expression (% total cells) of HLA-A2 (left) and H-2K (right) in cells or isolated from a wild-type (WT) mouse or a heterozygous mouse carrying the HLA-A2 locus /H-2K chimeric (HLA-A/H-2K HET).
[0031] [0031] FIG. 6B is a dot diagram of in vivo expression of chimeric HLA-A2/H-2K protein in a heterozygous mouse harboring a chimeric HLA-A2/H-2K locus.
[0032] [0032] FIG. 7 shows a targeting strategy (no scale)
[0033] [0033] FIG. 8 shows a representative dot diagram of the HLA expression of the class | and human B2 microglobulin 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 B2 microglobulin (double heterozygote; class I/B2m HET).
[0034] [0034] FIG. 9 shows a representative histogram of human HLA expression of the | (geometric X axis) in cells isolated from the blood of wild-type (WT) mice, heterozygous for
[0035] [0035] FIG. 10 shows the results of IFNy Elispot assays for human T cells exposed to antigen presenting cells (APCs) from wild-type mice (WT APCs) or mice heterozygous for chimeric HLA-A2/H-2K and microglobulin B2 (double HET APCs) in the presence of influenza peptides (left) or EBV (right). Statistical analysis was performed using one-way ANOVA with Tukey's Multiple Comparison Post-Test. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[0036] [0036] The present invention provides genetically modified non-human animals (eg, mice, rats, rabbits, etc.) that express MHC polypeptides | human or humanized and/or B2 microglobulin; embryos, cells, and tissues comprising the same; methods of doing the same; as well as methods of using them. Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have obtained in the art, unless otherwise clearly indicated or clearly evident from the context in which the term or phrase it is used.
[0037] [0037] The term "conservative", when used to describe a conservative amino acid substitution, includes substitution of an amino acid residue for another amino acid residue having a side-chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying a nucleotide sequence to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially alter the functional properties of interest of a protein, for example, the ability of MHC | to present a peptide of interest. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acid substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, — alanine/valine, — glutamate/aspartate, and asparagine/glutamine. In some embodiments, an amino acid substitution
[0038] [0038] Accordingly, also encompassed by the invention is a genetically modified non-human animal whose genome comprises a nucleotide sequence encoding an MHC | human or humanized and/or B2 microglobulin polypeptide, wherein the polypeptide(s) comprise(s) conservative amino acid substitutions of the amino acid sequence(s) described herein.
[0039] [0039] One skilled in the art would understand that in addition to nucleic acid residues encoding an MHC | human or humanized and/or the B2 microglobulin described herein, due to the degeneration of the genetic code, other nucleic acids may encode the polypeptide(s) of the invention. Therefore, in addition to a genetically modified non-human animal comprising in its genome a nucleotide sequence that encodes the MHC | and/or B2 microglobulin with conservative amino acid substitutions, a non-human animal whose genome comprises a nucleotide sequence(s) that differs from that described herein due to degeneracy of the genetic code is also provided.
[0040] [0040] The term "identity" when used with respect to sequence identity includes as determined by several different algorithms known in the art that can be used to measure the identity of the sequence.
[0041] [0041] The terms "homology" or "homologous" in reference to sequences, e.g. nucleotide or amino acid sequences, mean two sequences that, in optimal alignment and comparison, are identical in at least about 75% of the way. nucleotides or amino acids, at least about 80% nucleotides or amino acids, at least about 90-95% nucleotides or amino acids, eg greater than 97% nucleotides or amino acids. One skilled in the art would understand that for optimal gene targeting, the targeting construct should contain arms homologous to endogenous DNA sequences (ie, "homology arms"); thus, homologous recombination can occur between the target construct and the target endogenous sequence.
[0042] [0042] The term "operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in their intended manner. As such, a nucleic acid sequence encoding a protein can be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer, etc. sequence) to retain proper transcriptional regulation. In addition, various portions of the chimeric or humanized protein of the invention can be operably linked to retain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless otherwise stated, various domains of the chimeric or humanized proteins of the invention are operably linked together.
[0043] [0043] The term "MHC complex |" or others, as used herein, includes the complex between the MHC polypeptide | chain and the B2 microglobulin polypeptide. The term "MHC polypeptide |" or others, as used herein, includes the MHC polypeptide | from jail to alone. Typically, the terms "human MHC" and "HLA" may be used interchangeably.
[0044] [0044] The term "replacement" in reference to gene substitution refers to placing exogenous genetic material at an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. . As demonstrated in the Examples below, nucleic acid sequences from endogenous loci encoding mouse MHC portions and B2 microglobulin polypeptides were replaced with nucleotide sequences encoding portions of MHC | human and B2 microglobulin polypeptides, respectively.
[0045] [0045] "Functional" as used herein, for example, in reference to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a substitution at an endogenous locus (e.g., substitution at an endogenous non-human MHC| and/or microglobulin 82 locus) results in a locus that does not express a fungal endogenous polypeptide. national.
[0046] [0046] Various aspects described here below for MHC gene non-human animals | genetically modified, for example, type of animals; animal strains; cell types; screening, detection and other methods; usage methods; etc., will apply to genetically engineered microglobulin B2 and MHC I/microglobulin B2 animals. MHC animals | Genetically Modified
[0047] [0047] In various embodiments, the invention generally provides genetically modified non-human animals comprising in their genome a nucleotide sequence that encodes an MHC polypeptide | human or humanized; thus, the animals express an MHC polypeptide | human or humanized.
[0048] [0048] MHC genes are categorized into three classes: class |, class |1, and class III, all of which are encoded on human chromosome 6 or mouse chromosome 17. A schematic of the relative organization of the human and mouse MHC classes is presented in Figures 2 and 3, respectively. MHC genes are among the most polymorphic genes in the mouse and human genomes. MHC polymorphisms are presumed to be important in providing evolutionary advantage; Sequence alterations can result in differences in peptide binding that allow better presentation of pathogens to cytotoxic T cells.
[0049] [0049] MHC Protein Class | comprises an extracellular domain (comprising three domains: a1, a2, and a3), a transmembrane domain, and a cytoplasmic tail. The a1 and a2 domains form the cleaved peptide bond, while a3 interacts with microglobulin 82.
[0050] [0050] In addition to its interaction with microglobulin B2, the a3 domain interacts with the TCR CD8 co-receptor, facilitating antigen-specific activation. Although class binding | of MHC to CD8 is about 100 times weaker than TCR binding to the class | of MHC, CD8 binding enhances the affinity of TOR binding. Wooldridge et al. (2010) MHC Class | Molecules with Superenbhanced CD8 Binding Properties Bypass the Requirement for Cognate TOR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366. Interestingly, increasing class bonding | CD8 MHC abrogates antigen specificity in CTL activation. /d.
[0051] [0051] Binding of CD8 to Class MHC Molecules | is species-specific; the mouse CD8 homolog, Lyt-2, has been shown to bind H-2° molecules to the a3 domain, but not bind HLA-AAS molecules. Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class | a3 Domain in Allogeneic Cytotoxic T Cell Response, J. Exp. Med. 168:325-341. Differential binding was presumably due to CDR-like determinants (CDR1 and CDR2-like) on CD8 which was not conserved between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class | 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 | Major Histocompatibility Complex, J. Exp. Med. 173:1007-1015; and, Gao et al. (1997) Crystal structure of the complex between human CD8aa and HLA-A2, Nature 387:630-634. CD8 has been reported to bind HLA-A2 in a conserved region of the a3 domain (at position 223-229). A single substitution (V245A) in the binding of reduced HLA-AA from CD8 to HLA-A,
[0052] [0052] Therefore, due to the species specificity of the interaction between the a3 domain of the class | of MHC and CD8, an MHC complex | comprising a replacement of an a3 domain of H-2K with an a3 domain of human HLA-A2 was non-functional in a mouse (i.e., in vivo) in the absence of a human CD8. In HLA-A2 transgenic animals, replacement of the human a3 domain with the mouse a3 domain resulted in restoration of the T cell response. Irwin et al. (1989) Species-restricted interactions between CD8 and the a3 domain of class | influence the magnitude of the xenogeneic response, J. Exp. Med. 170:1091-1101; Vitiello et al. (1991), supra.
[0053] [0053] The transmembrane domain and cytoplasmic tail of class proteins | mouse MHC also have important functions. A function of the MHC transmembrane domain | is to facilitate HLA-A2 modulation of homotypic cell adhesion (to enhance or inhibit adhesion), presumably as a result of crosslinking (or binding) surface MHC molecules. Wagner et al. (1994) Connection of MHC Class | and Class Il Molecules Can Lead to Heterologous Desensitization of Signal Transduction Pathways That Regulate Homotypic Adhesion in Human Lymphocytes, J. Immunol. 152:5275-5287. Cell adhesion can be affected by mAbs that bind to diverse epitopes of the HLA-A2Z molecule, suggesting that there are multiple sites on HLA-A2 implicated in modulating homotypic cell adhesion; depending on the epitope bound, the effect may be to enhance or inhibit HLA-A2-dependent adhesion. /d.
[0054] [0054] The cytoplasmic tail, encoded by exons 6 and 7 of the β-MHC gene, is purportedly necessary for proper cell surface expression and for LIR1-mediated inhibition of NK cell cytotoxicity. Gruda et al. (2007) Intracellular Cysteine Residues in the Tail of MHC Class | Proteins Are Crucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1, J. Immunol. 179:3655-3661. A cytoplasmic tail is required for multimerization of at least some MHC molecules | through the formation of disulfide bonds in their cysteine residues, and thus may play a role in clustering and recognition by NKA cells.
[0055] [0055] The cytoplasmic domain of HLA-A2 contains a constitutively phosphorylated serine residue and a phosphorylatable tyrosine, although - in Jurkat cells - mutant HLA-A2 molecules that are devoid of a cytoplasmic domain appear normal with respect to expression, cytoskeletal association, aggregation, and endocytic internalization. Gur et al. (1997) Structural Analysis of Class | MHC Molecules: The Cytoplasmic Domain Is Not Required for Cytoskeletal Association, Aggregation, and Internalization, Mol. Immunol. 34(2):125-132.
[0056] [0056] However, several studies have demonstrated that the cytoplasmic tail is critical in intracellular trafficking, dendritic cell (DC) mediated antigen presentation, and CTL preparation. a residue
[0057] [0057] In various embodiments, the invention provides a genetically modified non-human animal (eg, mouse, rat, rabbit, etc.) that comprises in its genome a nucleotide sequence that encodes a polypeptide of the class | by MHC | human or humanized. The non-human animal may comprise in its genome a nucleotide sequence encoding an MHC polypeptide | which is partly human and partly non-human, e.g., a non-human animal expressing an MHC polypeptide | chimeric human/non-human. In one aspect, the non-human animal only expresses the MHC | or humanized, eg MHC polypeptide | human/non-human chimeric, and does not express an MHC protein | non-human endogenous MHC locus | endogenous.
[0058] [0058] In one embodiment, the MHC polypeptide | chimeric human/non-human comprises in its human portion a peptide binding domain of an MHC polypeptide | human. In one aspect, the human portion of the chimeric polypeptide comprises an extracellular domain of an MHC | human. In this mode, the
[0059] [0059] The MHC polypeptide | human or humanized may be derived from a functional human HLA molecule encoded by any HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci.
[0060] [0060] In a specific aspect, the MHC | human or humanized is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (eg, HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., HLA-A*02:01:01:01 allele. The HLA-
[0061] [0061] In one aspect, there is provided a non-human animal that expresses a human HLA-A2 sequence, wherein the human HLA-A2 sequence comprises one or more conservative or non-conservative modifications.
[0062] [0062] In one aspect, there is provided a non-human animal 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% identical to a human HLA-A2 sequence. In a specific embodiment, the human HLA-A2 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human HLA-A2 sequence described in the Examples. In one embodiment, the human HLA-A2 sequence comprises one or more conservative substitutions. In one embodiment, the human HLA-A2 sequence comprises one or more non-conservative substitutions.
[0063] [0063] In another specific aspect, the MHC | or humanized is derived from MHC | human selected from HLA-BeHLA-C. In one aspect, the MHC | human or humanized is derived from HLA-B, for example HLA-B27.
[0064] [0064] In one aspect, the non-human portion of the MHC | human/non-human chimeric comprises transmembrane and/or cytoplasmic domains of MHC polypeptide | non-human. In one embodiment, the non-human animal is a mouse,
[0065] [0065] The non-human animal described here may comprise in its genome a nucleotide sequence that encodes an MHC polypeptide | human or humanized, e.g. chimeric human/non-human MHC I polypeptide, wherein the nucleotide sequence encoding such a polypeptide is located at an MHC locus | endogenous non-human (eg, H-2K locus). In one aspect, this results in a replacement of an MHC gene | or a portion thereof with a nucleotide sequence that encodes an MHC polypeptide | or humanized, e.g. a chimeric gene encoding an MHC polypeptide | chimeric human/non-human described here. In one embodiment, the substitution comprises a substitution of an endogenous nucleotide sequence that encodes a non-human MHCI peptide binding domain or an extracellular MHC | non-human with a human nucleotide sequence (eg, HLA-A2 nucleotide sequence) encoding the same. In this modality, the substitution does not comprise a substitution of an MHC sequence | encoding the transmembrane and/or cytoplasmic domains of an MHC polypeptide | non-human (eg, H-2K polypeptide). Thus, the non-human animal contains chimeric human/non-human nucleotide sequence at an MHC | endogenous non-human, and expresses chimeric human/non-human MHC polypeptide from the MHC locus | non-humanendogenous.
[0066] [0066] A chimeric human/non-human polypeptide may be such that it comprises a human or a non-human leader (signal) sequence. In one embodiment, the chimeric polypeptide comprises a non-human leader sequence of an MHC protein | endogenous. In another embodiment, the chimeric polypeptide comprises a leader sequence from an MHC | human, e.g., HLA-A2 protein (e.g., HLA-A2.1 leader sequence). Thus, the nucleotide sequence encoding the MHC | chimeric can be operably linked to a nucleotide sequence encoding an MHC leader sequence | human.
[0067] [0067] An MHC polypeptide | chimeric human/non-human may comprise in its human portion a complete or substantially complete extracellular domain of an MHC polypeptide | human. Thus, the human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, for example 95% or more, of the amino acids encoding an extracellular domain of an MHC | human (e.g., HLA-A2 polypeptide). In one example, extracellular domain of the MHC | substantially complete human is devoid of an MHC leader sequence | human. In another example, the MHC | chimeric human/non-human comprises an MHC leader sequence | human.
[0068] [0068] In addition, the MHC polypeptide | chimeric can be expressed under the control of endogenous non-human regulatory elements, e.g. rodent MHC γ regulatory animals. Such an arrangement will facilitate proper expression of the MHC | chimeric in the non-human animal, for example during the immune response in the non-human animal.
[0069] [0069] The genetically modified non-human animal may be selected from a group consisting of a mouse, rat, co-
[0070] [0070] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, for example of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a selected family of Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, wild rats), Muridae (mice and rats). common, gerbils, spiny mice, crested mice), Nesomyidae (tree mice, stone mice, tailed mice, rats and Malagasy mice), Platacanthomyidae (e.g. spiny voles), and Spalacidae (e.g. e.g. mole rats, bamboo rats, and Chinese zokors). In one specific embodiment, the genetically modified rodent is selected from a common mouse or rat (the family Muridae), a gerbil/spiny mouse, and a crested rat. In a modality
[0071] [0071] In a specific embodiment, the non-human animal is a rodent that is a mouse of a C57BL strain selected from C57BL/A, C57BL/Ann C57BL/GrFay C57BL/KalwN, C57BL/6, C57BL/6J, C57BL/ 6ByJ, C57BL/GNJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 12981 (eg, 129S1/SV, 129S1/Svim), 12982, 129S4, 12985, 129S9/ SvEvH, 7129S6 (129/SvEvTac), 12987, 12988, 129T1, 129T2 (see, for example, Festingetal. (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 mixture of an above-mentioned 129 strain and an above-mentioned C57BL/6 strain. In another specific embodiment, the mouse is a mixture of the aforementioned 129 strains, or a mixture of the aforementioned BL/6 strains. In a specific embodiment, the 129 strain in the mixture is a 129S6 strain (129/SvEvTac). In another embodiment, the mouse is a BALB strain, eg BALB/c strain. In yet another embodiment, the mouse is a mixture of a BALB strain and another strain mentioned above.
[0072] [0072] In one embodiment, the non-human animal is a mouse. In one embodiment, the mouse is selected from a Wistar mouse, a LEA strain, a Sprague-Dawley strain, a Fischer strain, F344, F6, and A-
[0073] [0073] Thus, in one embodiment, the invention relates to a genetically modified mouse comprising in its genome a nucleotide sequence encoding an MHC | chimeric mouse/human, wherein a human portion of the chimeric polypeptide comprises a peptide-binding domain or an extracellular domain of an MHC | human (e.g., human HLA-A, e.g., human HLA-A2, e.g., human HLA-A2.1). In some embodiments, the mouse does not express a peptide-binding domain or an extracellular one of an endogenous mouse polypeptide from its endogenous mouse locus. The MHC peptide binding domain | human can understand domains a1 and a2. Alternatively, the peptide-binding domain of MHC | human can understand domains there, a2, and a3. In one aspect, the extracellular domain of the MHC | human comprises an extracellular domain of an MHC a-chain | human. In one embodiment, the endogenous mouse locus is an H-2K locus (e.g., H-2K°), and the mouse portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of an H-2K polypeptide from mouse (eg H-2K).
[0074] [0074] Thus, in one embodiment, a genetically modified mouse is provided, wherein the mouse comprises at an endogenous H-2K locus (e.g., H-2K") a nucleotide sequence encoding an MHC polypeptide. | chimeric human/mouse, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 polypeptide (e.g., HLA-A2.1) and a mouse portion comprises transmembrane domains and cytoplasmic from a mouse H-2K polypeptide (eg, H-2K”). In one aspect, the mouse does not express an extracellular domain of the mouse H-2K polypeptide (eg, H-2K°) from an endogenous MHC I locus. In one embodiment, the mouse expresses a chimeric HLA-A2/H-2K polypeptide (eg, a chimeric HLA-A2.1/H-2K°) from an endogenous H-2K locus (eg, H-2K ”). In various embodiments, chimeric gene expression is under the control of regulatory elements of the | of endogenous mouse MHC. In some respects, the mouse comprises two copies of the MHC | chimeric containing a nucleotide sequence encoding a chimeric HLA-A2/H-2K polypeptide; while in other respects the mouse comprises a copy of the MHC locus | chimeric containing a nucleotide sequence encoding a chimeric HLA-A2/H-2K polypeptide. Thus, the mouse can be homozygous or heterozygous for the nucleotide sequence that encodes the chimeric HLA-A2/H-2K polypeptide.
[0075] [0075] In some embodiments described here, a mouse is provided that comprises an MHC | chimeric located at an endogenous mouse H-2K locus. The chimeric locus comprises a nucleotide sequence that encodes an extracellular domain of a human HLA-A2 protein, for example, a1, a2, and a3 domains of a human HLA-A2 gene. The chimeric locus is devoid of a nucleotide sequence that encodes an extracellular domain of a mouse H-2K protein (e.g., α, a2, and a3 domains of mouse H-2K). In one aspect, the chimeric locus is devoid of a nucleotide sequence encoding a leader peptide, domains a1, a2, and a3 of a mouse H-2K; and comprises a leader peptide, domains a1, a2, and a3 from a human HLA-A2, and transmembrane and cytoplasmic domains from a mouse H-2K. The various domains of the chimeric locus are operably linked to each other so that the chimeric locus expresses an MHC protein | human/mouse functional chimeric.
[0076] [0076] In various embodiments, a non-human animal, eg a rodent (eg a mouse or rat), which expresses an MHC protein | functional chimeric of an MHC locus | chimeric as described herein displays the chimeric protein on a cell surface. In one embodiment, the non-human animal expressed the MHC protein | chimeric on a cell surface in a cell distribution equal to that seen in a human. In one aspect, the cell exhibits a peptide fragment (an antigen fragment) bound to an extracellular portion (eg, extracellular portion of human HLA-A2) of the MHC protein | chimerical. In one embodiment, the extracellular portion of such a chimeric protein interacts with other proteins on the surface of said cell, e.g. 82 microglobulin.
[0077] [0077] In various embodiments, a cell that exhibits the chimeric MHCI protein, eg HLA-A2/H-2K protein, is a nucleated cell. In many respects, the cell is an antigen presenting cell (APC). Although most cells in the body can present an antigen in the context of MHC1, some non-limiting examples of antigen-presenting cells include macrophages, dendritic cells, and B cells. Other antigen-presenting cells, including professional APCs and non-professionals, are known in the art, and are encompassed herein. In some embodiments, the cell that exhibits the MHC protein | chimeric is a tumor cell, and a peptide fragment presented by the chimeric protein is derived from a tumor. In other embodiments, the peptide fragment presented by the MHC | chimeric is derived from a pathogen, for example a bacterium or a virus.
[0078] [0078] The MHC protein | chimeric described here can interact with other proteins on the surface of the same cell or a second cell. In some embodiments, the MHC | chimeric interacts with endogenous non-human proteins on the surface of said cell. The MHC protein | chimeric can also interact with human or humanized proteins on the surface of the same cell or a second cell.
[0079] [0079] In the same cell, molecules of the class | antibodies can functionally interact with both non-human (eg rodent, mouse or rat) and human B2 microglobulin. Thus, in one embodiment, the MHC | chimeric, eg HLA-A2/H-2K protein, interacts with a mouse B2 microglobulin. Although the interaction between some of the molecules in the class | of human HLA and mouse B2 microglobulin is possible, however it may be greatly reduced compared to the class interaction | of human HLA and human B2 microglobulin. Thus, in the absence of human B2 microglobulin, MHC | human on the cell surface can be reduced. Perarnau et al. (1988) Human g2-microglobulin Specifically Enhances Cell-Surface Expression of HLA Class | Molecules in Transfected Murine Cells, J. Immunol. 141:1383-89. Other HLA molecules, eg HLA-B27, do not interact with mouse microglobulin B2; see, for example, Tishon et al. (2000) Transgenic Mice Ex-pressing 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 the function of HLA-B27 in Transgenic mice require CD8 for human and humanized B2 microglobulin. Therefore,
[0080] [0080] In several aspects, the chimeric protein (eg, HLA-A2/H-2K protein) also interacts with proteins on the surface of a second cell (through its extracellular portion). The second cell can be a cell of a non-human origin, for example, a mouse, or a human. The second cell may be derived from the same non-human animal or the same non-human animal species as the cell expressing the MHC polypeptide | chimerical. Non-limiting examples of proteins with which the extracellular portion of a chimeric protein (e.g., HLA-A2/H-2K) can interact include T cell receptor (TOR) and its CD8 co-receptor. Thus, a second cell can be a T cell. Furthermore, the extracellular portion of the MHC | chimeric can bind a protein on the surface of Natural Killer (NK) cells, for example, killer immunoglobulin receptors (KIRs) on the surface of NK cells.
[0081] [0081] A T cell or NK cell can bind a complex formed between the MHC polypeptide | chimeric and its displayed peptide fragment. Such binding may result in T cell activation or inhibition of NK-mediated cell death, respectively. One hypothesis is that NK cells have evolved to kill either infected or tumor cells that evade T cell-mediated cytotoxicity by down-regulating their MHC complex |. However, when the MHC | is expressed on the cell surface, NK cell receptors recognize it, and NK-mediated cell death is inhibited. Thus, in some respects, when an NK cell binds an MHC | chimeric (e.g., HLA-A2/H-2K polypeptide) and a peptide fragment displayed on the surface of the infected or tumor cell, NK-mediated cell death is inhibited.
[0082] [0082] In one example, the MHC polypeptide | chimeric described herein, for example, a 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 a human CD8 on the surface of the second cell (eg, a human cell or a rodent cell). In some such embodiments, the non-human animal, for example, a mouse or rat, comprises a human CD8 transgene, and the mouse or rat expresses a functional human CD8 protein.
[0083] [0083] The MHC polypeptide | chimeric described here may also interact with a non-human TCR (eg, a mouse or rat), a human TOR, or a humanized TCR in a second cell. The MHC polypeptide | chimeric can interact with an endogenous TCR (eg, mouse or rat TOR) on the surface of a second cell. The MHC polypeptide | chimeric may also interact with a human or humanized TCR expressed on the surface of a second cell, where the cell is derived from the same animal or animal species (eg, mouse or rat) as the cell expressing the MHC polypeptide | chimerical. The MHC polypeptide | chimeric can interact with a human TCR expressed on the surface of a human cell.
[0084] [0084] In addition to genetically engineered non-human animals, a non-human embryo (e.g. a rodent embryo, e.g. a mouse or rat embryo) is also provided, wherein the embryo comprises an ES cell donor that is derived from a non-human animal (eg a rodent, eg a mouse or rat) as described here. In one aspect, the embryo comprises a donor ES cell comprising the MHC gene | chimeric, and the cells of the host embryo.
[0085] [0085] Also provided is a tissue, wherein the tissue is derived from a non-human animal (eg, a mouse or rat) as described herein, and expresses the MHC polypeptide | chimeric (e.g., HLA-A2/H-2K polypeptide).
[0086] [0086] In addition, a non-human cell isolated from a non-human animal as described herein is provided. In one embodiment, the cell is an ES cell. In one embodiment, the cell is an antigen presenting cell, eg, dendritic cell, macrophage, B cell. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is a lymphocyte.
[0087] [0087] Also provided is a non-human cell comprising a chromosome or fragment thereof from a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus from a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of nuclear transfer.
[0088] [0088] In one aspect, a non-human induced pluripotent cell comprising gene encoding an MHC polypeptide | chimeric (e.g., HLA-A2/H-2K polypeptide) as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.
[0089] [0089] In one aspect, a hybridoma or quadroma is provided, derived from a cell of a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or rat.
[0090] [0090] Also provided is a method for making a genetically engineered non-human animal (eg, a genetically engineered rodent, eg, a mouse or rat) described herein. The method for making a non-human animal genetically engineered results in the animal whose genome comprises a nucleotide sequence encoding an MHC polypeptide | chimerical. In one embodiment, the method results in a genetically engineered mouse whose genome comprises at an MHC | endogenous, eg H-2K locus, a nucleotide sequence —encoding an MHC polypeptide | human/mouse chimeric, in which a human portion of the MHC polypeptide | chimeric comprises an extracellular domain from a human HLA-A2 and a mouse portion comprises transmembrane and cytoplasmic domains from a mouse H-2K. In some embodiments, the method utilizes a targeting construct made using VELOCIGENES technology, introducing the construct into ES cells, and introducing the target ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples. In one embodiment, the ES cells are a mixture of mouse strains 129 and C57BL/6; in another embodiment, the ES cells are a mixture of mouse BALB/c and 129 strains.
[0091] [0091] Accordingly, a nucleotide construct used to generate genetically engineered non-human animals described herein is also provided. In one aspect, the nucleotide construct comprises: 5' and 3' non-human homology arms, a human DNA fragment comprising human HLA-A gene sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment comprising introns and exons of a human HLA-A gene. In one embodiment, the non-human homology arms are homologous to an MHC locus of class | non-human (eg, a mouse H-2K locus).
[0092] [0092] In one embodiment, the genomic fragment comprises a human HLA-A leader sequence, an a1 domain coding sequence, a2 domain and a3 domain. In one embodiment, the human DNA fragment comprises, from 5' to 3: an HLA-AA leader sequence, an HLA-A a1 leader/intron, an a1 HLA-A exon, an a1-a2 intron of HLA-A, an a2 exon of HLA-A, an intron of a2-a3 of HLA-A, and an exon of a3 of HLA-A.
[0093] [0093] A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate the selection of cells (eg, ES cells) that have integrated the construct of interest. Various suitable selection cassettes are known in the art. Commonly, a selection cassette allows positive selection in the presence of a particular antibiotic (eg Neo, Hyg, Pur, CM, Spec, etc.). Furthermore, a selection cassette can be flanked by recombination sites that allow deletion of the selection cassette under treatment with recombinase enzymes. Commonly used recombination sites are loxP and Frt, recognized by the Cre and Flp enzymes, respectively, but others are known in the art.
[0094] [0094] In one embodiment, the selection cassette is located at the 5' end of the human DNA fragment. In another modality
[0095] [0095] In one embodiment, the 5' and 3rd non-human homology arms comprise genomic sequence at the 5' and 3' locations of a class gene locus | of endogenous (eg, murine) non-human MHC, respectively (eg, 5' of the first leader sequence and 3' of the a3 exon of the non-human MHC gene). In one embodiment, the locus of the class | of endogenous MHC is selected from mouse H-2K, H-2º and H-2L. In a specific modality, the locus of the class | of endogenous MHC is mouse H-2K.
[0096] [0096] In one aspect, there is provided a nucleotide construct comprising, from 5' to 3': a 5' homology arm containing 5' mouse genomic sequence from the endogenous mouse H-2K locus, a first human DNA fragment comprising a first genomic sequence of an HLA-A gene, a 5' recombination sequence site (e.g. /oxP), a selection cassette, a 3' recombination sequence site ' (eg, 1/oxP), a second fragment of human DNA comprising a second genomic sequence of an HLA-A gene and a 3' homology arm containing mouse genomic sequence 3' of an exon of a3 of endogenous H-2K. In one embodiment, the nucleotide construct comprises, from 5' to 3': a 5' homology arm containing 5' mouse genomic sequence from the endogenous mouse H-2K locus, a human genomic sequence including an HLA-A leader sequence, an HLA-AA a1 leader/intron, an HLA-A a1 exon, an HLA-A a1-a2 intron, an HLA-A a2 exon , a first 5' portion of an a2-03 intron, a selection cassette flanked by recombination sites, a second 3' portion of an a2-a03 intron, an a3 exon of HLA-A, and a 3' homology arm containing 3' non-mouse genomic sequence of endogenous mouse H-2K a3 exon. In one embodiment, a 5' homology arm sequence is set forth in SEQ ID NO: 1, and a 3' homology arm sequence is set forth in SEQ ID NO: 2.
[0097] [0097] Upon completion of gene targeting, ES cells or the genetically modified non-human animals are screened to confirm successful incorporation of the exogenous nucleotide sequence of interest or exogenous polypeptide expression. Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCT (e.g., real-time PCR using TAQMAN®, fluorescence in situ hybridization, Northern blotting, cytometry flow analysis, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (eg, mice) carrying the genetic modification of interest can be identified by screening for mouse allele loss and/or gain. of human allele using an allele assay modification 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.Others assays which identify a specific nucleotide or amino acid sequence in the genetically modified animals are known to those skilled in the art.
[0098] [0098] The description also provides a method of modifying an MHC locus | of a non-human animal to express an MHC polypeptide | chimeric human/non-human described here. In one embodiment, the invention provides a method of modifying a locus of
[0099] [0099] In one aspect, a method for making a molecule of the class | Class/Chimeric Human HLA Test | of non-human MHC is provided, comprising expressing in a single cell a chimeric HLA-A/H-2K protein of a nucleotide construct, wherein the nucleotide construct comprises a cDNA sequence encoding an a1 domain , a2, and a3 of an HLA-AA protein and a transmembrane and cytoplasmic domain of a non-human H-2K protein, e.g. mouse H-2K protein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell that expresses a viral nucleic acid sequence (e.g., a PERC.6"Y cell).
[00100] [00100] In one aspect, a cell expressing MHC for a Class | Chimeric/MHC Human HLA Test | non-human (eg, HLA-A/H-2K protein) is provided. In one embodiment, the cell comprises an expression vector comprising an MHC class gene | chimeric, in which the MHC class gene | chimeric comprises a sequence from a human HLA-A gene fused in operable linkage to a sequence from a non-human H-2K gene, e.g. mouse H-2K gene. In one embodiment, the human HLA-A gene sequence comprises exons encoding the a1, a2, and a3 domains of an HLA-AA protein. In one embodiment, the non-human H-2K gene sequence comprises exons encoding the transmembrane and cytoplasmic domains of an H-2K protein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell that expresses a viral nucleic acid sequence (eg, a PERC.6"" cell).
[00101] [00101] A molecule of the class | of chimeric MHC made by a non-human animal as described herein is also provided, wherein the class molecule | chimeric MHC comprises a1, a2, and aqa3 domains of a human HLA-A protein and transmembrane and cytoplasmic domains of a non-human, e.g., mouse, H-2K protein. The MHC polypeptide | chimeric described herein can be detected by anti-HLA-A antibodies. Thus, a cell that exhibits MHC | chimeric human/non-human can be detected and/or selected using anti-HLA-A antibody. In some circumstances, the MHC | chimeric described herein can be detected by an anti-HLA-A2 antibody.
[00102] [00102] “Although the Examples below describe a genetically engineered animal whose genome comprises a replacement of a nucleotide sequence encoding a mouse H-2K polypeptide extracellular domain with the sequence encoding a mouse extracellular domain. human HLA-A at the endogenous mouse H-2K locus, one skilled in the art would understand that a similar strategy could be used to replace other mouse MHCI loci (H-2D, H-2L, etc.) of corresponding human HLA (HLA-B, HLA-C, etc.). Thus, a non-human animal comprising in its genome a nucleotide sequence encoding an MHC | chimeric human/non-human in which a human portion of the polypeptide is derived from another protein of the class | of HLA is also provided. The MHC loci replacement | multiples is also contemplated. Genetically Modified Microglobulin B2 Animals
[00103] [00103] The invention in general provides genetically modified non-human animals that comprise in their genome a nucleotide sequence that encodes a human or humanized B2 microglobulin polypeptide; thus, the animals express a human or humanized B2 microglobulin polypeptide.
[00104] [00104] “Microglobulin B2 or the light chain of the class | of MHC (also abbreviated "B2M") is a small (12 kDa) non-glycosylated protein that functions primarily to stabilize the α-chain of MHC . The human B2 microglobulin gene encodes a protein of 119 amino acids, with 20 N-terminal amino acids encoding a leader sequence. The mature protein comprises 99 amino acids. The gene contains 4 exons, with the first exon containing the 5th untranslated region, the entire leader sequence, and the first two amino acids of the mature polypeptide; the second exon encoding most of the mature protein; the third exon encoding the last four amino acids of the mature protein and a termination codon; and the fourth exon containing the 3' untranslated region. Gussow et al. (1987) The gB2-Microglobulin Gene. Primary Structure and Definition of the Transcriptional Unit, J. Immunol. 139:3131-38. Microglobulin B2 is non-covalently associated with MHC |. Unbound microglobulin B2 is found in body fluids, such as plasma, and is carried to the kidney for excretion. Renal dysfunction causes accumulation of microglobulin B2 which can be pathogenic (eg, Dialysis-Related Amyloidosis); the accumulated protein forms filamentous fibrils that resemble amyloid plaques in joints and connective tissues.
[00105] [00105] In addition to Dialysis-Related Amyloidosis, B2 microglobulin has been implicated in several other disorders. Elevated levels of microglobulin B2 have been detected in lymphocytic malignancies, eg non-Hodgkin's lymphoma and multiple myeloma. See, for example, Shi et al. (2009) 82 Microglobulin: Emerging as a Promising Cancer Therapeutic Target, Drug Discovery Today 14:25-30. Some other malignancies with elevated B2 microglobulin levels include breast cancer, prostate cancer, lung cancer, kidney cancer, gastrointestinal and nasopharyngeal cancers. Overexpression of microglobulin B2 has been suggested to have tumor growth promoting effects. Id. It was also recently shown that microglobulin B2 triggers epithelial to mesenchymal transition, promoting bone and soft tissue metastasis in breast, prostate, lung and kidney cancers. Josson et al. (2011) 82 microglobulin Induces Epithelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells. Cancer Res. 71(7): 1-11. B2 microglobulin interacts with an MHC member | non-classical, the hemochromatosis protein (HFE), and with the transferrin receptor, and modulates iron homeostasis. /d. Involvement of microglobulin B2 in other malignancy benchmarks (self-renewal, enhancement of angiogenesis, resistance to treatment) is widely documented in the art.
[00106] [00106] Mice deficient in microglobulin B2 have been reported. See, Koller et al. (1990) Normal development of deficient mice in B2m, MHC class | proteins, and CD8+ T cells, Science 248: 1227-
[00107] [00107] “Mice expressing human B2 microglobulin as well as molecules of the class | of human HLA (i.e., HLA-B7) in a randomly inserted transgene have been reported. Chamberlain et al. (1988) Tissue-specific and cell surface expression of human major histocompatibility complex class | heavy (HLA-B7) and light (B2-microglobulin) chain genes in transgenic mice, Proc. natl. academy Sci. USA 85:7690-7694. The class expression | of human HLA was consistent with that of the class | endogenous with a noticeable decrease in the liver. /d. Human B2 microglobulin expression was also consistent with endogenous B2 microglobulin, while expression of the class | of human HLA was increased 10-17 fold in doubly transgenic mice. /d. However, the authors did not attempt a replacement of an endogenous mouse microglobulin B2 locus with a human microglobulin B2 locus.
[00108] [00108] Therefore, described here is a genetically engineered non-human animal (e.g. a rodent, e.g. a mouse or a rat) whose genome comprises a nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide . In one aspect, the animal does not express an endogenous non-human B2 microglobulin from an endogenous non-human B2 microglobulin locus.
[00109] [00109] The nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide may comprise nucleic acid residues that correspond to the entire human B2 microglobulin gene. Alternatively, the nucleotide sequence may comprise nucleic acid residues encoding the exposed amino acid sequence at amino acids 21-119 of a human B2 microglobulin protein (i.e., amino acid residues that correspond to mature human B2 microglobulin). . In an alternative embodiment, the nucleotide sequence may comprise nucleic acid residues encoding the amino acid sequence displayed at amino acids 23-115 of a human B2 microglobulin protein, for example, amino acid sequence displayed at amino acids 23-119 of a human B2 microglobulin protein. The nucleic acid and amino acid sequences of human microglobulin B2 are described in Gussow et al., supra, incorporated herein by reference.
[00110] [00110] Thus, the human or humanized microglobulin B2 polypeptide may comprise amino acid sequence displayed at amino acids 23-115 of a human microglobulin B2 polypeptide, for example, amino acid sequence displayed at amino acids 23-119 of a human B2 microglobulin polypeptide, for example, amino acid sequence displayed at amino acids 21-119 of a human B2 microglobulin polypeptide. Alternatively, human B2 microglobulin may comprise amino acids 1-119 of a human B2 microglobulin polypeptide.
[00111] [00111] In some embodiments, the nucleotide sequence encoding a human or humanized B2 microglobulin comprises a nucleotide sequence exposed in exon 2 to exon 4 of a human B2 microglobulin gene. Alternatively, the nucleotide sequence comprises nucleotide sequences set forth in exons 2, 3, and 4 of a human B2 microglobulin gene. In this embodiment, the exposed nucleotide sequences in exons 2, 3, and 4 are operably linked to allow normal transcription and gene translation. Thus, in one embodiment, the human sequence comprises a nucleotide sequence that corresponds to exon 2 to exon 4 of a human B2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence that corresponds to exon 2 at about 267 bp after exon 4 of a human B2 microglobulin gene. In a specific embodiment, the human sequence comprises about 2.8 kb of a human B2 microglobulin gene.
[00112] [00112] Thereby, the human or humanized B2 microglobulin polypeptide can be encoded by a nucleotide sequence comprising nucleotide sequence exposed in exon 2 to exon 4 of a human B2 microglobulin, for example, nucleotide sequence corresponding to the exon 2 to exon 4 of a human B2 microglobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequences set forth in exons 2, 3, and 4 of a human B2 microglobulin gene. In a specific embodiment, the human or humanized B2 microglobulin polypeptide is encoded by a nucleotide sequence that corresponds to exon 2 at about 267 bp after exon 4 of a human B2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising about 2.8 kb of a human B2 microglobulin gene. As exon 4 of the microglobulin B2 gene contains the 5' untranslated region, the human or humanized polypeptide can be encoded by a nucleotide sequence comprising exons 2 and 3 of the microglobulin 82 gene.
[00113] [00113] It would be understood by those of ordinary skill in the art that while specific nucleic acid and amino acid sequences for generating genetically engineered animals are described in the present examples, the sequences of one or more conservative or non-conservative amino acid substitutions, or sequences that differ from those described here due to degeneration of the genetic code, are also provided.
[00114] [00114] Therefore, a non-human animal that expresses a human B2 microglobulin sequence is provided, wherein the B2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical to a human B2 microglobulin sequence. In a specific embodiment, the B2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human B2 microglobulin sequence described in the Examples. In one embodiment, the human B2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human B2 microglobulin sequence comprises one or more non-conservative substitutions.
[00115] [00115] Further provided are non-human animals in which the nucleotide sequence encoding a human or humanized B2 microglobulin protein also comprises a nucleotide sequence exposed in exon 1 of a non-human B2 microglobulin gene. Thus, in a specific embodiment, the non-human animal comprises in its genome a nucleotide sequence that encodes a human or humanized microglobulin B2 in which the nucleotide sequence comprises exon 1 of a non-human microglobulin fb2 and exons 2,3, e4 of a human B2 microglobulin gene. Thus, human or humanized microglobulin B2 polypeptide is encoded by exon 1 of a non-human microglobulin B2 gene and by exons 2, 3, and 4 of a human microglobulin B2 gene (e.g., exons 2 and 3 of a human B2 microglobulin gene).
[00116] [00116] Similarly to a non-human animal comprising a nucleotide sequence encoding an MHC | chimeric, the non-human animal comprising a nucleotide sequence encoding a human or humanized B2 microglobulin may be selected from the group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (eg marmoset, rhesus monkey). In some embodiments of the invention, the non-human animal is a mammal. In a specific embodiment, the non-human animal is a murine, eg, a rodent (eg, a mouse or rat). In one embodiment, the animal is a mouse.
[00117] [00117] Thus, in some aspects, a genetically engineered mouse is provided, wherein the mouse comprises a nucleotide sequence that encodes a humanized or humanized B2 microglobulin polypeptide as described herein. A genetically engineered mouse is provided, wherein the mouse comprises in its endogenous B2 microglobulin locus a nucleotide sequence encoding a human or humanized microglobulin B2 polypeptide (e.g., a human or substantially human B2 microglobulin polypeptide). . In some embodiments, the mouse does not express an endogenous microglobulin B2 polypeptide (eg, a functional endogenous microglobulin B2 polypeptide) from an endogenous microglobulin B2 locus In some embodiments, the genetically engineered mouse comprises a nucleotide sequence comprising exon 1 of a mouse microglobulin gene and exons 2, 3, and 4 of a human microglobulin B2 gene. In some modalities, the mouse expresses human or humanized B2 microglobulin polypeptide.
[00118] [00118] In one aspect, a non-human B2 microglobulin locus is modified so long as it comprises a heterologous B2 microglobulin sequence. In one embodiment, the heterologous B2 microglobulin sequence is a human or humanized sequence.
[00119] [00119] 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 B2 microglobulin coding sequence.
[00120] [00120] In one embodiment, the heterologous B2 microglobulin sequence is operably linked to endogenous regulatory elements, eg, endogenous promoter and/or expression control sequence. In a specific embodiment, the heterologous B2 microglobulin sequence is a human sequence and the human sequence is operably linked to an endogenous promoter and/or expression control sequence.
[00121] [00121] In one aspect, a non-human B2 microglobulin locus is modified so long as it comprises a human sequence operably linked to an endogenous promoter and/or expression control sequence.
[00122] [00122] In many respects, human or humanized microglobulin B2 expressed by a genetically modified non-human animal, or cells, embryos, or tissues derived from a non-human animal, preserves all functional aspects of microglobulin B2 endogenous and/or human. For example, human or humanized B2 microglobulin is preferred to bind the α-chain of MHC | (eg, non-human or human endogenous MHC polypeptide). Human or humanized B2 microglobulin B2 polypeptide can bind, recruit or otherwise associate with any other molecule, e.g. receptor, anchor or signaling molecules that associate with endogenous non-human B2 microglobulin B2 and/or human (eg HFE, etc.).
[00123] [00123] In addition to genetically modified animals (eg rodents, eg mice or rats), also provided is a tissue or cell, wherein the tissue or cell is derived from a non-human animal as described here , and comprises a heterologous B2 microglobulin gene or B2 microglobulin sequence, i.e., nucleotide and/or amino acid sequence. In one embodiment, the heterologous B2 microglobulin gene or BRB2 microglobulin sequence is a human or humanized B2 microglobulin gene or human or humanized B2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell can be any cell known to express the MHC complex, for example an antigen presenting cell. The human or humanized B2 microglobulin polypeptide expressed by said cell can interact with MHC | endogenous non-human (eg, rodent | MHC), to form an MHC | functional. The MHC complex | the resulting one may be able to interact with a T cell, for example, a cytotoxic T cell. Thus, also provided is an in vitro complex of a cell from a non-human animal as described herein and a T cell.
[00124] [00124] Also provided are non-human cells comprising the human or humanized B2 microglobulin gene or sequence, and an additional human or humanized sequence, eg MHC | chimerical presently revealed. In such a circumstance, the human or humanized B2 microglobulin polypeptide may interact, for example, with an MHC | human/non-human chimeric, and an MHC complex | functional can be formed. In some aspects, such a complex is capable of interacting with a TCR on a T cell, for example, a human or non-human T cell. Thus, also provided in an in vitro complex of a cell from a non-human animal as described herein and a human or non-human T cell.
[00125] [00125] Another aspect of the description is a rodent embryo (e.g., a mouse or rat embryo) comprising a heterologous B2 microglobulin gene or B2 microglobulin sequence as described herein. In one embodiment, the embryo comprises a donor ES cell comprising the heterologous B2 microglobulin gene or B2 microglobulin sequence, and host embryo cells. The heterologous B2 microglobulin gene or B2 microglobulin sequence is a human or humanized B2 microglobulin gene or B2 microglobulin sequence.
[00126] [00126] This invention also encompasses a non-human cell comprising a chromosome or fragment thereof from a non-human animal as described herein (e.g., wherein the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide). The non-human cell may comprise a nucleus from a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of nuclear transfer.
[00127] [00127] In one aspect, a non-human induced pluripotent cell comprising a heterologous B2 microglobulin gene or B2 microglobulin sequence is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein. In one embodiment, the heterologous B2 microglobulin gene or B2 microglobulin sequence is a human or humanized gene or sequence.
[00128] [00128] Also provided is a hybridoma or quadroma, derived from a cell from a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or rat.
[00129] [00129] The description also provides methods for making a genetically engineered non-human animal (eg a genetically engineered rodent, eg a mouse or rat) described here. The methods result in an animal whose genome comprises a nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide. In one aspect, the methods result in a genetically engineered mouse whose genome comprises at an endogenous B2 microglobulin locus a nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide. In some circumstances, the mouse does not express a functional mouse B2 microglobulin from an endogenous mouse B2 microglobulin locus. In some respects, the methods utilize a targeting construct made using VELOCIGENES technology, introducing the construct into ES cells, and introducing the target ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples . In one embodiment, the ES cells are a mixture of mouse 129 and C57BL/6 strains; in another embodiment, the ES cells are a mixture of mouse BALB/c and 129 strains.
[00130] [00130] Also provided is a nucleotide construct used to generate genetically engineered non-human animals. The nucleotide construct may comprise: non-human 5' and 3 homology arms, a human DNA fragment comprising human B2 microglobulin sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment comprising introns and exons of a human B2 microglobulin gene. In one embodiment, the non-human homology arms are homologous to a non-human B2 microglobulin locus. The genomic fragment may comprise exons 2, 3, and 4 of the human B2 microglobulin gene. In one instance, the genomic fragment comprises, from 5' to 3': exon 2, intron, exon 3, intron, and exon 4, all of the human B2 microglobulin sequence. The selection cassette may be located anywhere in the construct outside the B2 microglobulin coding region, for example it may be located 3' of human B2 microglobulin exon 4. The 5' and 3' non-human homology arms may comprise 5' and 3' genomic sequence of the endogenous non-human B2 microglobulin gene, respectively. In another embodiment, the 5' and 3' non-human homology arms comprise the 5' genomic sequence of exon 2 and 3' of exon 4 of the endogenous non-human gene, respectively.
[00131] [00131] Another aspect of the invention pertains to a method of modifying a B2 microglobulin locus of a non-human animal (e.g., a rodent, e.g., a mouse or rat) to express a human or human B2 microglobulin polypeptide. - manizada described here. One method of modifying a mouse microglobulin B2 locus to express a human or humanized microglobulin B2 polypeptide comprises substituting a nucleotide sequence that encodes a mouse microglobulin B2 microglobulin with a sequence into an endogenous microglobulin B2 locus. nucleotide sequence encoding human or humanized B2 microglobulin polypeptide. In one embodiment of such a method, the mouse does not express a functional B2 microglobulin polypeptide from an endogenous mouse B2 microglobulin locus. In some specific embodiments, the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide comprises the nucleotide sequence displayed in exons 2 to 4 of the human B2 microglobulin gene. In other embodiments, the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide comprises the nucleotide sequences set forth in exons 2, 3, and 4 of the human B2 microglobulin gene. Genetically Modified MHC W/Microglobulin B2 Animals
[00132] [00132] In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their nucleotide sequences a genome encoding both MHC | human or humanized such as B2 microglobulin; thus, the animals express both MHC | human or humanized as of microglobulin B2.
[00133] [00133] Functional differences arise in the use of mixed human/non-human system component. Class | of HLA binds microglobulin B2 more tightly than the class | of mouse. Bernabeu (1984) g2-microgobulin from serum associates with MHC class | antigens on the surface of cultured cells, Nature 308:642-645. Attempts to abrogate functional differences are reflected in the construction of particular humanized MHC mice. Class 1 and class 2 H-2 knockout mice (on a mouse B2 microglobulin KO basis) that express chimeric transgene having a human HLA-A2.1 a1 and a2 to an HLA- Fortuitously integrated human A2.1/HLA-DR1, and mouse H-2Db a3, linked at its N-terminus via a linker to the C-terminus of human microglobulin B2 were developed. See, for example, Pajot et al. (2004) A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class |- fclass Il-knockout mice, Eur. J. Immunol. 34:3060-3069. These mice supposedly generate antigen-specific antibody and CTL responses against the hepatitis B virus, whereas the mice merely transgenic for HLA-A2.1 or H-2 class I/class II knockout mice they don't. The deficiency of mice that are merely transgenic for genes presumably stems from the ability of such mice to employ the endogenous genes of the class | and/or class II to avoid any transgene, an option not available for knockout mice.
[00134] [00134] Cell surface expression of chimeric fusion with human B2 microglobulin is supposedly lower than endogenous MHC expression, but survival/death rate of NK is not reported, nor is the rate of NK self-kill. Pajot et al., supra. Some improvement in CD8+ T cell numbers was observed in the class | of MHC 1 (2-3% of total splenocytes, vs. 0.6-1% in 82 KO mice). However, the use of variable region T cells exhibited altered profiles for BV 5.1, BV 5.2, and BV 11 gene segments. CD8+ and CD4+ T cell responses were supposedly restricted to the appropriate hepatitis B antigen used to immunize the mice,
[00135] [00135] As mentioned above, both MHC | human and human microglobulin B2 comprise a nucleotide sequence encoding a chimeric MHC I/microglobulin B2 protein, wherein the MHC | and microglobulin B2 are contained within a single polypeptide chain, resulting in an MHC a-chain | and B2 microglobulin being covalently linked together and thus attached to the cell surface. A mouse comprising in its genome two independent nucleotide sequences, one encoding an MHC polypeptide | human or humanized and the other encoding a human or humanized B2 microglobulin polypeptide is provided. The mouse provided here would express an MHC complex | that would most closely resemble an MHC complex | present in nature, where the a chain of MHC | and microglobulin B2 are provided on two separate polypeptide chains with microglobulin B2 noncovalently associating with the a-chain of MHC 1.
[00136] [00136] Thus, the present description provides a non-human animal comprising in its genome: a first nucleotide sequence encoding an MHC polypeptide | human or humanized, and a second nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide. In one aspect, provided is a non-human animal comprising in its genome: (a) a first nucleotide sequence encoding an MHC polypeptide | chimeric human/non-human, wherein the human portion of the chimeric polypeptide comprises a peptide binding domain or an extracellular domain of an MHC | (for example, HLA-A, HLA-B, or HLA-C; for example, HLA-A2), and (b) a second nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide.
[00137] [00137] The first nucleotide sequence may be located at an MHC locus | endogenous non-human so that the animal comprises in its genome a substitution at the MHC locus | of all or a portion of MHC gene | endogenous (e.g., a portion encoding a peptide binding domain or an extracellular domain) with the MHC sequence | corresponding human. In this way, the animal can understand at an MHC locus | a nucleotide sequence encoding an extracellular domain of an MHC | (e.g., HLA-A, HLA-B, or HLA-C; e.g., HLA-A2) and transmembrane and cytoplasmic domains of endogenous non-human MHC 1 (e.g., H-2K, H-2D, etc. ., e.g. H-2K”). In one aspect, the animal is a mouse, and the first nucleotide sequence comprises a nucleotide sequence encoding an extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) and transmembrane and cytoplasmic domains. - mouse cosdeumH-2K (eg H-2K”).
[00138] [00138] The second nucleotide sequence can be located at an endogenous non-human microglobulin B2 locus such that the animal comprises in its genome a substitution at the microglobulin B2 locus of all or a portion of the endogenous microglobulin B2 gene with the corresponding human B2 microglobulin sequence. The second nucleotide sequence may comprise a nucleotide sequence exposed in exon 2 to exon 4 of a human B2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences exposed in exons 2,3, and 4 of a human B2 microglobulin gene.
[00139] [00139] In one aspect, the animal does not express an MHC | function of an MHC locus | endogenous non-human (e.g., either does not express an endogenous peptide-binding domain or extracellular MHC | domain), and the animal does not express a functional microglobulin B2 polypeptide from an endogenous non-human microglobulin B2 locus . In some respects, the animal is homozygous for both an MHC | comprising a nucleotide sequence encoding an MHC polypeptide | chimeric human/non-human as for a B2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized B2 microglobulin. In other respects, the animal is heterozygous for both an MHC | comprising a nucleotide sequence encoding an MHC polypeptide | chimeric human/non-human as for a B2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized B2 microglobulin.
[00140] [00140] Preferably, the first and second nucleotide sequences are operably linked to endogenous expression control elements (e.g., promoters, enhancers, silencers, etc.).
[00141] [00141] Various other modalities of the first and second nucleotide sequences (and the polypeptides they encode) encompassed herein can be easily understood from the modalities described throughout the specification, for example those described in the sections relating to MHC animals | genetically engineered and genetically engineered B2 microglobulin animals.
[00142] [00142] In one aspect, the description provides a mouse comprising in its genome (a) a first nucleotide sequence encoding an MHC polypeptide | chimeric human/mouse (specifically, HLA-A2/H-2K polypeptide), wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 and the mouse portion comprises transmembrane domains and cytoplasmic from a mouse H-2K, and (b) a second nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide (e.g., wherein the nucleotide sequence comprises a nucleotide sequence exposed in exon 2 to the exon 4 of the human microglobulin B2 gene or exposed nucleotide sequences in exon 2, 3, and 4 of the human microglobulin B2 gene), where the first nucleotide sequence is located at an endogenous H-2K locus, and the second sequence is located at an endogenous B2 microglobulin locus. In one embodiment, the mouse does not express functional H-2K and B2 microglobulin polypeptide from mice from their respective endogenous loci. In one embodiment, the mouse expresses both the MHC | chimeric human/mouse as for human or humanized microglobulin B2 polypeptide.
[00143] [00143] As shown in the following Examples, animals engineered to co-express both MHC | and humanized or genetically humanized B2 microglobulin exhibited increased cell surface expression of chimeric MHC classaldehyde compared to MHC humanized animals | alone. In some embodiments, MHC | or humanized and B2 microglobulin increases cell surface expression of MHC | human or humanized by more than about 10%, for example, more than about 20%, for example, about 50% or more, for example, about 70%,
[00144] [00144] The description also provides a method of making genetically engineered non-human animals (eg rodents, eg rats or mice) whose genome comprises a first and a second nucleotide sequence as described here. The method generally comprises generating a first genetically engineered non-human animal whose genome comprises a first nucleotide sequence described herein (i.e., a human or humanized MHC sequence), generating a second genetically engineered non-human animal whose genome comprises a second nucleotide sequence described here (ie, a human or humanized B2 microglobulin sequence), and breed the first and second animals to obtain progeny whose genomes contain both nucleotide sequences. In one embodiment, the first and second animals are heterozygous for the first and second nucleotide sequence, respectively. In one embodiment, the first and second animals are homozygous for the first and second nucleotide sequence, respectively. In one embodiment, the first and second animals are generated by replacing endogenous non-human loci with the first and second nucleotide sequence, respectively. In one aspect, the first and second animals are generated using constructs generated through VELOCIGENES technology, and introducing the target ES cell clones by carrying such constructs into an embryo (e.g. rodent, e.g. a mouse or rat embryo) using the VELOCIMOUSE method. Use of Genetically Modified Animals
[00145] [00145] In various embodiments, the genetically modified non-human animals described here produce APCs with MHC | and/or human or humanized B2 microglobulin on the cell surface and, as a result, present peptides derived from cytosolic proteins as epitopes for CTLs in a human-like manner, because substantially all components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit an immune response (eg T cell epitopes, eg unique human cancer epitopes), eg for use in vaccine development; for identification of T cells of high affinity for human pathogens or cancer antigens (i.e., T cells that bind antigen in the context of the human MHC | complex with high avidity), e.g. for use in adaptive cancer therapy. T cells; for evaluation of vaccine candidates and other vaccine strategies; to study human autoimmunity; to study human infectious diseases; and otherwise to devise better therapeutic strategies based on human MHC expression.
[00146] [00146] The MHC complex | binds the peptides and presents them on the cell surface. Once presented on the surface in the context of such a complex, the peptides are recognizable by T cells. For example, when the peptide is derived from a pathogen or other antigen of interest (eg, a tumor antigen), it will be recognized. - T cell activation can result in T cell activation, death of T cell macrophages carrying the presented peptide sequence, and B cell activation of antibodies that bind to the presented sequence.
[00147] [00147] T cells interact with cells that express the MHC complex | through the ectodomain class of the class | of MHC bound to the peptide and the CD8 ectodomain of T cells.
[00148] [00148] “Thus, in various embodiments, the genetically engineered animals of the present invention are useful, among other things, for assessing the ability of an antigen to initiate an immune response in a human, and for generating a diversity of antigens. - genos and identify a specific antigen that can be used in the development of a human vaccine.
[00149] [00149] In one aspect, a method for determining antigenicity in a human of a peptide sequence is provided, comprising exposing a non-human genetically modified animal as described herein to a molecule comprising the peptide sequence, allowing the non-human animal to mount an immune response, and to detect in the non-human animal a cell that binds a peptide sequence presented by an MHC | human/non-human chimeric, or an MHC complex | humanized (comprising a chimeric human/non-human MHC and a human or humanized B2 microglobulin) as described herein.
[00150] [00150] In one aspect, a method for determining whether a peptide
[00151] [00151] In one aspect, there is provided a method for identifying a human CTL epitope, comprising exposing a non-human animal as described herein to an antigen comprising a putative CTL epitope, allowing the non-human animal to mount an immune response, to isolate a class-restricted CD8+ CTL from the non-human animal | binding epitope, and identifying the epitope bound by the class-restricted CD8+ CTL | of MHC.
[00152] [00152] In one aspect, a method is provided to identify a class-restricted peptide | of HLA whose presentation by a human cell and binding by a human lymphocyte (e.g., human T cell) will result in cytotoxicity of the cell carrying the peptide, comprising exposing a non-human animal (or expression cell of the | class of MHC thereof) as described herein to a molecule comprising a peptide of interest, isolating a cell from the non-human animal expressing a molecule of the class | chimeric human/non-human class that binds the peptide of interest, exposing the cell to a human lymphocyte that is capable of conducting class-restricted cytotoxicity | of HLA, and measure peptide-induced cytotoxicity.
[00153] [00153] In one aspect, a method is provided for identifying an antigen that generates a cytotoxic T cell response in a human, comprising exposing a putative antigen to a mouse as described herein, allowing the mouse to generate an immune response, and identifying the antigen bound by the HLA-A restricted molecule.
[00154] [00154] In one embodiment, the antigen comprises a bacterial or viral surface or envelope protein. In one embodiment, the antigen comprises an antigen on the surface of a human tumor cell. In one embodiment, the antigen comprises a putative vaccine for use in a human. In one embodiment, the antigen comprises a human epitope that generates antibodies in a human. In another embodiment, the antigen comprises a human epitope that generates high-affinity CTLs that target the β-epitope/MHC complex.
[00155] [00155] In one aspect, a method is provided for determining whether a putative antigen contains an epitope that upon exposure to a human immune system will generate an HLA-A-restricted immune response (e.g., HLA-A2-restricted response) , comprising exposing a mouse as described herein to the putative antigen and measuring an antigen-specific HLA-A-restricted (eg, HLA-A2-restricted) immune response in the mouse.
[00156] [00156] In one embodiment, the putative antigen is selected from a biopharmaceutical or fragment thereof, a non-autoprotein, a surface antigen of a non-autocell, a surface antigen of a tumor cell, a surface antigen of a bacterial or yeast or fungal cell, a surface antigen or envelope protein of a virus.
[00157] [00157] In addition, the genetically engineered non-human animals described here may be useful for identifying T cell receptors, e.g., high avidity T cell receptors that recognize an antigen of interest, e.g., a T cell receptor that recognizes an antigen of interest. tumor or other disease antigen. The method may comprise: exposing the non-human animal described herein to an antigen, allowing the non-human animal to mount an immune response to the antigen, isolating from the non-human animal a T cell comprising a T cell receptor that binds to the antigen presented by a MHC | human or humanized, and determining the sequence of said T cell receptor.
[00158] [00158] In one aspect, a method for identifying a T cell receptor variable domain having high affinity for a human tumor antigen is provided, comprising exposing a mouse comprising the MHC | humanized a1, a2, and a3 (e.g., HLA-A2 domains a1, a2, and a3) to a human tumor antigen; allowing the mouse to generate an immune response; and, isolating from the mouse a nucleic acid sequence encoding a T cell receptor variable domain, wherein the T cell receptor variable domain binds human tumor antigen with a Kp of no higher than about 1 nanomolar.
[00159] [00159] In one embodiment, the mouse further comprises a substitution at the endogenous mouse T cell receptor variable region gene locus with a plurality of unarranged human T cell receptor variable region gene segments, in that unarranged human T cell receptor variable region gene segments recombine to encode a chimeric human T cell receptor gene comprising a human variable region and a mouse constant region. In one embodiment, the mouse comprises a human CD8 transgene, and the mouse expresses a functional human CD8 protein.
[00160] [00160] T cell receptors that have high avidity for tumor antigens are useful in cell-based therapies. T cell populations with high avidity for human tumor antigens were prepared by exposing human T cells to HLA-A2 that had been mutated to minimize CD8 binding to the a3 subunit, in order to select only those T cells with extremely high avidity. high for tumor antigen (ie, T cell clones that recognize the antigen despite the inability of CD8 to bind a3). See, Pittet et al. (2003) n3 Domain Mutants of Peptide/MHC Class | Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells, J. Immunol. 171:1844-1849. Non-human animals, and cells from non-human animals, are useful for identifying peptides that will form a classed complex | of human HLA that will bind with high avidity to a T cell receptor, or activate a lymphocyte that carries a T cell receptor.
[00161] [00161] Class Antigen/HLA Binding | to a T cell, or activation of a T cell, can be measured by any suitable method known in the art. Peptide-specific APC-T cell binding and activation are measurable. For example, T cell binding of antigen-presenting cells that express HLA-A2 supposedly causes PIP2 to accumulate in the immunosynapse, while cross-linking of MHC class | do not. See, Fooksman et al. (2009) Cutting Edge: Phosphatidylinositol 4,5-Bisphosphate Concentration at the APC Side of the Immunological Synapse Is Required for Effector T Cell Function, J. Immunol. 182:5179-5182.
[00162] [00162] Functional consequences of the interaction of a lymphocyte that carries a TCR, and a class I expression APC, are also measurable and include cell death by the lymphocyte. For example, contact points on the a2 subunit of HLA-A2 by CD8+ CTLs supposedly generate a signal for Fas-independent killing. Jurkat cells expressing HLA-A2 perform apoptosis when contacted (via antibodies) with epitopes on the HLA-A2 molecule known (from crystallographic studies) to contact CD8,
[00163] [00163] The consequence of the interaction between a T cell and an APC that displays a peptide in the MHC context | it can also be measured by a T cell proliferation assay. Alternatively, it can be determined by measuring cytokine release commonly associated with the activation of an immune response. In one embodiment, IFNy ELISPOT can be used to monitor and quantify CD8+ T cell activation.
[00164] [00164] As described herein, CD8+ T cell activation can be prevented in the genetically modified non-human animals described herein due to species-specific binding of CD8 to the β-MHC. For modalities where a species-specific CD8 interaction is desired, a cell from a genetically modified animal as described herein (e.g., a rodent, e.g., a mouse or rat) is exposed (e.g., in vitro) to a human cell, eg a cell carrying human CD8, eg a human T cell. In one embodiment, an expression cell of class | of a mouse MHC as described herein is exposed in vitro to a T cell comprising a human CD8 and a T cell receptor. In a specific embodiment, the T cell is a human T cell. In one embodiment, the expression cell of class | of mouse MHC comprises a peptide linked to an MHC | human/mouse chimeric or an MHC complex | (which includes human B2 microglobulin), the T cell is a human T cell, and the ability of the T cell to bind to the peptide-presenting mouse cell is determined. In one embodiment, activation of the human T cell by the peptide-presenting mouse cell is determined. In one embodiment, an in vitro method for measuring activation of a human T cell by the peptide-presenting cell is provided, comprising exposing a mouse or a mouse cell as described herein to an antigen of interest, exposing a cell from said mouse or said mouse cell (presumably carrying a peptide derived from the antigen in the complex with human or humanized MHC1) to a human T cell, and measure human T cell activation. In one embodiment, the method is used to identify a T cell epitope of a human pathogen or a human neoplasm. In one embodiment, the method is used to identify an epitope for a vaccine.
[00165] [00165] In one embodiment, a method is provided for determining T cell activation by a putative human therapeutic, comprising exposing a genetically modified animal as described herein to a putative human therapeutic (or for example, exposing an expression cell of human or humanized MHC | from such an animal to a peptide sequence of the putative therapy), exposing a cell of the genetically modified animal that exhibits a MHC | human or humanized/peptide to a T cell comprising a human T cell receptor and a CD8 capable of binding to the cell of the genetically modified animal, and measuring human T cell activation.
[00166] [00166] In various embodiments, a complex formed between an expression cell of class | of human or humanized MHC from an animal as described herein is made with a T cell comprising a human CD8 sequence, for example, a human T cell, or a T cell from a non-human animal comprising a transgene that encodes human CD8. Human CD8 transgenic mice are known in the art. 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(2):286-293; also, LaFace et al. (1995) Human CD8 Transgene Regulation of HLA Recognition by Murine T Cells, J. Exp. Med. 182:1315-1325.
[00167] [00167] In addition to the ability to identify antigens and antigen epitopes from human pathogens or neoplasms, the genetically modified animals of the invention can be used to identify autoantigens of relevance to human autoimmune diseases, e.g. type I diabetes, multiple sclerosis , etc. For example, Takaki et al. ((2006) HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes, J. Immunol. 176:3257-65 ) describe the utility of NOD mice carrying HLA monochain /microglobulin B2 identifying type 1 diabetes autoantigens. Also, the genetically modified animals of the invention can be used to study various aspects of human autoimmune disease. How some polymorphic MHC alleles | human are known to be associated with the development of certain diseases, e.g. autoimmune diseases (e.g. Graves disease, mias-
[00168] [00168] Other aspects of cellular immunity involving MHC complexes | are known in the art; therefore, genetically engineered non-human animals described here can be used to study these aspects of immune biology. For example, TCR binding to class | of MHC is modulated in vivo through additional factors. A member of the leukocyte immunoglobulin-like receptor subfamily B (LILRB1, or LIR-1) is expressed in class-restricted CTLs | of MHC and down-regulates T cell stimulation by binding a specific determinant on the a3 subunit of molecules of the class | of MHC in APCs. Structural studies show that the binding site for LIR-1 and CD8 overlap, suggesting that the LIRA inhibitor competes with stimulant CD8 to bind with the β-class 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; also, Shirioshi et al. (2003) Human inhibitory lg-like transcript 2 (ILT2) and ILT4 receptors compete with CD8 for MHC class | binding and bind preferentially to
[00169] [00169] — As described above, MHC molecules interact with cells that do not express a TCR. Among these cells are NK cells. NK cells are cytotoxic lymphocytes (as distinguished from C-TLs, or cytotoxic T lymphocytes) that play a central role in the cellular immune response, and in particular innate immunity. NK cells are the first line of defense against invading microorganisms, viruses, and other non-autonomous entities (eg, tumor). NK cells are activated or inhibited by surface receptors, and they express CD8 but do not express TCRs. NK cells can interact with cells that express the class | of MHC, but interaction is through the a3 domain of CD8 binding rather than TCR binding, domains carrying a1 and a2 peptide. A primary function of NK cells is to destroy cells that lack the surface protein of the class | enough MHC.
[00170] [00170] Crosslinking of class molecules | of MHC on the surface of human natural killer (NK) cells results in intracellular tyrosine phosphorylation, migration of the class molecule | of MHC of the immunosynapse, and downregulation of tumor cell killing.
[0017] [0017]] Another function of the |MHC class in NK cells is apparently to prevent automation. NK cells carry the 2B4 activating receptor and the 2B4 CD48 ligand; the class | of MHC appears to bind 2B4 and prevent its activation by CD48. Betser-Cohen (2010) The Association of MHC Class | Proteins with the 2B4 Receptor Inhibits Self-Killing of Human NK Cells, J. Immunol. 184:2761-2768.
[00172] [00172] In this way, the genetically engineered non-human animals described here can be used to study these non-TCR or non-CTL mediated processes and design approaches for their modulation. EXAMPLES
[00173] [00173] The invention will be further illustrated by the following non-limiting examples. These Examples are set out to aid understanding of the invention, but are not intended, and should not be interpreted, to limit its scope. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Centigrade, and pressure is atmospheric or nearly so.
[00174] [00174] Example 1. Construction and Characterization of Genetically Modified HLA-A2 Mice Example 1.1: Expression of HLA-A2/H-2K in MG87 Cells.
[00175] [00175] A viral construct containing a chimeric HLA-A2/H-2K gene sequence (FIG. 4A) was made using cloning techniques.
[00176] [00176] Briefly, a chimeric human HLA-A/mouse H-2K viral construct was made using the exon sequences encoding the a1, a2 and a3 domains of the a-chain and cloning them in structure with the sequences coding for the transmembrane and cytoplasmic domains of the H-2K gene (FIG. 4A, pMIG-HLA-A2/H2K). As illustrated in FIG. 4, the construct contained an IRES-GFP reporter sequence that allowed to determine whether the construct was able to express itself in the cells under transfection.
[00177] [00177] Virus containing 797979797979797979797979797979797979797979797979797979797979797979 797979797979797979797979797979797979797979797979797979797979797979 797979797979797979797979797979797979797979797979797979a chimeric construct described above were made and propagated in 293 human embryonic kidney cells (293T). 293 T cells were plated in 10 cm dishes and allowed to grow to 95% confluence. A DNA transfection mix was prepared with 25 µg of pMIG-HLA-A2/H2K, human pMIG-HLA-A2, or pMIG humanized microglobulin B2, and 5 µg of pMDG (envelope plasmid), 15 µg of pMIG. pCL-Eco (packaging construct without packaging the tv signal, 1 ml of Opti-MEM (Invitrogen). Added to this 1 ml of DNA mix was 80 ul of Lipofectamine-2000 (Invitrogen) in 1 ml of Opti-MEM which was previously mixed and allowed to incubate at room temperature for 5 minutes. The Lipofectamine/DNA mixture was allowed to incubate for an additional 20 minutes at room temperature, then added to 10 cm dishes, and the plates were incubated at 37°C .Medium was harvested from the cells after 24 hours and a fresh 10 ml R10 medium (RPMI 1640 + 10% FBS) was added.
[00178] [00178] The propagated viruses made above were used to transduce MG87 (mouse fibroblast) cells. MG87 cells from a single T-75 flask were washed once with PBS. 3 ml of 0.25% Trypsin + EDTA was added to the cells and allowed to incubate at room temperature for three minutes. 7 ml of D10 (high glucose DMEM; 10% Fetal Bovine Serum) was added to the cell/trypsin mixture and transferred to a 15 ml tube to centrifuge at 1300 rpm for five minutes. After centrifuging the cells, the media were aspirated and the cells resuspended in 5 ml of D10. Cells were counted and -3.0x10° cells were placed per well in a 6-well plate. Human pMIG-HLA-A2 or pMIG-HLA-A2/H-2K either alone or with humanized B2 microglobulin virus with pMIG was added to the wells, with untransduced cells as a control. Cells were incubated at 37°C with 5% CO for 2 days. Cells were prepared for FACS analysis (using anti-HLA-A2 antibody, clone BB7.2) for HLA-A2 expression with or without 82 microglobulin.
[00179] [00179] The graphs (FIG. 4B), as well as the table that summarizes the data obtained from the graphs (FIG. 4C) demonstrate that cotransduction with humanized B2 microglobulin increases the expression of human HLA-A2 or HLA-A2/H -2K human/non-human chimeric, as demonstrated by shifting the curves to the right.
[00180] [00180] The mouse H-2K gene was humanized in a simple step by building a single targeting vector from mouse and human bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (See, for example, US Pat. 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 RP23-173Kk21 (Invitrogen) was modified through homologous recombination to replace the genomic DNA encoding the α, a2 and a3 domains of the mouse H-2K gene with genomic DNA encoding the a1, a2 and a3 subunits of the human HLA-A gene (FIG. 5).
[00181] [00181] Briefly, the genomic sequence that encodes the a1, a2 and a3 subunits of the mouse H-2K gene is replaced by the human genomic DNA that encodes the a1, a2 and a3 domains of the human HLA-A*0201 gene in a single targeting event using a targeting vector comprising a hygromycin cassette flanked by /oxP sites with a mouse 5' homology arm containing the 5' mouse H-2K locus sequence including the non 5' homology arm (UTR; 5th homology arm is set forth in SEQ ID NO: 1) and a 3' mouse homology arm containing the 3' genomic sequence of the a3 coding sequence of H-2K from mouse (3' homology arm is set forth in SEQ ID NO: 2).
[00182] [00182] The final construct to target the endogenous H-2K locus of the 5' to 3' gene included (1) a 5' homology arm containing -200 bp of the 5' mouse genomic sequence of the H- gene. Endogenous 2K including the SUTR, (2) -1339 bp of human genomic sequence including the HLA-A*0201 leader sequence, the a1 leader/intron of HLA-A*0201, the a1 exon of HLA-A*0201, the a1-a2 intron of HLA-A*0201, the a2 exon of HLA-A*0201, -316 bp from the 5' end of the intron of a2-03,(3)a 5' site of loxP, ( 4) a hygromycin cassette, (5) a 3' site of /oxP, (6) -580 bp of human genomic sequence including -304 bp from the 3' end of the a2-03 intron, the a3 exon of HLA-A*0201, and (7) a 3' homology arm containing —-200 bp of mouse genomic sequence including the intron between the H-2K a3 coding sequences and the mouse transmembrane coding sequence. mundongo (see FIG. 5 for schematic representation of the H-2K aim vector). The 149-nucleotide sequence at the junction of the mouse/human sequences at the 5' of the targeting vector is shown in SEQ ID NO: 3, and the 159-nucleotide sequence at the junction of the human/mouse sequences at the 3' of the target vector. The targeting vector is set forth in SEQ ID NO: 4. Homologous recombination with this targeting vector created a modified mouse H-2K locus containing human genomic DNA encoding the al, a2, and a3 domains of the HLA-A gene. *0201 operably linked to endogenous mouse H-2K transmembrane and cytoplasmic domain coding sequences which, upon translation, lead to the formation of a protein of the class | of human/mouse chimeric MHC.
[00183] [00183] The targeted BAC DNA was used to electroporate mouse F1H4 ES cells to create ES cells modified to generate mice expressing a protein of the class | of chimeric MHC on the surface of nucleated cells (eg, TeB lymphocytes, macrophages, neutrophils). ES cells containing an insert of human HLA sequences were identified by a quantitative TAQMAN'Y 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 identifies the names and locations detected for each of the probes used in quantitative PCR assays.
[00184] [00184] The selection cassette can be removed by methods known to the skilled artisan. For example, ES cells that carry the locus of class | MHC human/chimeric mouse can be transfected with a construct expressing Cre to remove the "lox-treated" hygromycin cassette introduced by inserting the targeting construct containing the human HLA-A*0201 gene sequences (See Fig. 5). The hygromycin cassette can optionally be removed by breeding to mice expressing Cre recombinase. Optionally, the hygromycin cassette is retained in the mice.
[00185] [00185] Target ES cells described above were used as donor ES cells and introduced into an 8 cell stage mouse embryo by the VELOCIMOUSE™ method (see, for example, US Pat. 7,294,754 and Poueymirou et al. (2007) ) FO generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyzes Nature Biotech. 25(1):91-99). SPEED (FU mice derived completely from the donor ES cell) independently carrying a chimeric MHC class I gene was identified by genotyping.
[00186] [00186] Example 1.3. In vivo Expression of Chimeric HLA-A/H-2K in Genetically Modified Mice.
[00187] [00187] A heterozygous mouse carrying a genetically modified H-2K locus as described in Example 1.2 was analyzed for expression of the chimeric HLA-A/H-2K protein in the cells of the animal.
[00188] [00188] Blood was obtained separately from a wild type mouse and a chimeric HLA-A/H-2K heterozygote (A2/H2K) mouse. Cells were stained for human HLA-A2 with a phycoerythrin (PE)-conjugated anti-HLA-A antibody, and exposed to an allophycocyanin-conjugated anti-H-2K" antibody for one hour at 4°C. analyzed for expression by flow cytometry using antibodies specific for HLA-A and H-2K.” FIG. 6A shows expression of H-2Kº” and HLA-A2 in wild-type and chimeric heterozygote, with chimeric heterozygote expressing both Fig. 6B shows expression of both H-2K” and chimeric HLA-A2/H2K in the heterozygous mouse.
[00189] [00189] Example 2: Construction and Characterization of Genetically Modified Microglobulin B2 Mice Example 2.1: Engineering a Humanized Microglobulin B2 Locus
[00190] [00190] The mouse B2 microglobulin (B2m) gene was humanized in a simple step by constructing a unique targeting vector of mouse and human bacterial artificial chromosome (BAC) DNA using VELOCIGENE (See, for example, U.S. Pat. No. 6,586,251 and Valenzuela et al., supra).
[00191] [00191] Briefly, a targeting vector was generated by recom-
[00192] [00192] Targeted ES cell clones with drug cassette removed (by introduction of Cre recombinase) were introduced into an 8 cell-stage mouse embryo by the VELOCIMOUSE (See, for example, U.S. Pat. 7,294,754 and Poueymirou et al., supra). SPEED (FO mice completely derived from the donor ES cell) carrying the humanized B2m gene were identified by screening for mouse allele loss and human allele gain using an allele assay modification (Valenzuela et al., supra). Example 2.2: Characterization of Humanized Microglobulin B2 Mice
[00193] [00193] “Mice heterozygous for a humanized B2 microglobulin gene (B2m) were evaluated for expression using flow cytometry (Figs 8.and 9).
[00194] [00194] Briefly, blood was isolated from groups (n = 4 per group) of wild type mice, humanized B2m, class | of humanized MHC (ie, human HLA), and doubly humanized B2m and class | of MHC using techniques known in the art. Blood from each of the mice was treated in each group with ACK lysis buffer (Lonza Walkersville) to eliminate red blood cells. The remaining cells were stained using fluorochrome conjugated to anti-CD3 (17A2), anti-CD19 (1D3), anti-CD11b (M1/70), class | anti-human HLA, and anti-human B2 microglobulin antibodies (2M2). Flow cytometry was performed using BD-FACSCANTO” (BD Biosciences).
[00195] [00195] Class expression | Human HLA was detected in cells from single humanized animals and doubly humanized animals, while expression of microglobulin B2 was only detected in cells from doubly humanized mice (FIG. 8). Co-expression of human and class B2m | of human HLA resulted in an increase in the detectable amount of the class | of human HLA on the cell surface compared to human HLA expression of the | in the absence of human B2m (FIG. 9; mean fluorescent intensity 2370 versus 1387).
[00196] [00196] Example 3. Immune Response to Influenza and Epstein-Barr Virus (EBV) Peptides Displayed by Genetically Modified Mouse APCs Expressing HLA-A2/H-2K and Humanized Microglobulin B2.
[00197] [00197] PBMCs were screened from various human donors for HLA-A2 expression and their ability to mount a response to influenza and EBV peptides. One donor was selected only for subsequent experiments.
[00198] [00198] Human T cells are isolated from selected donor PBMCs using negative selection. Splenic non-T cells were isolated from a mouse heterozygous for a chimeric HLA-A2/H-2K and heterozygous for a humanized B2 microglobulin gene, and a wild-type mouse. About 50,000 splenic non-T cells from the mice were added to an Elispot plate coated with anti-human IFNy antibody. influence peptide
[00199] [00199] “As shown in FIG. 10, human T cells were able to mount a response to influenza and EBV peptides when presented by mouse APCs that expressed chimeric HLA-A2/H-2K and humanized B2 microglobulin on their surface. EQUIVALENTS
[00200] [00200] — Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein.
[00201] [00201] The entire contents of all non-patent documents, patent applications and patents cited throughout this patent application are hereby incorporated by reference in their entirety.

权利要求:
Claims (29)
[1]
1. Method for producing a genetically modified non-human animal, characterized by the fact that it comprises the step of modifying a non-human animal genome to comprise a Major Histocompatibility Complex gene locus | (MHC 1) endogenous non-human, a nucleotide sequence encoding a chimeric human/non-human MHC β polypeptide, wherein a human portion of the chimeric polypeptide comprises domains α1, α2 and α3 of an MHC β polypeptide. human, and where the animal expresses MHC polypeptide | chimerical human/non-human.
[2]
2. Method according to claim 1, characterized in that the modification step comprises replacing, at the MHC | endogenous non-human, a nucleotide sequence encoding a1, a2, and a3 domains of an MHC polypeptide | non-human by a nucleotide sequence encoding a1, a2 and a3 domains of an MHC polypeptide | not human.
[3]
3. Method according to claim 1 or 2, characterized by the fact that the non-human animal does not express 0a1l,0a2eas domains of an MHC polypeptide | non-human from the MHC locus | endogenous non-human.
[4]
4. Method according to any one of claims 1 to 3, characterized in that the nucleotide sequence encoding the human/non-human chimeric MHC | it is operably linked to non-human endogenous regulatory elements.
[5]
5. Method according to any one of claims 1 to 4, characterized in that the human portion of the human/non-human chimeric MHC | comprises a human leader sequence.
[6]
6. Method according to any one of claims 1a, characterized in that the MHC | human is selected from the group consisting of HLA-A, HLA-B, and HLA-C.
[7]
7. Method according to claim 6, characterized in that the MHC | human is an HLA-A polypeptide.
[8]
Method according to any one of claims 1 to 7, characterized in that a non-human portion of the chimeric human/non-human MHC | comprises transmembrane and cytoplasmic domains of an MHC polypeptide | endogenous non-human.
[9]
9. Method according to any one of claims 1 to 8, characterized in that the non-human animal is a rodent.
[10]
10. Method according to claim 9, characterized in that the rodent is a mouse.
[11]
11. Method according to claim 10, characterized in that the endogenous locus is a mouse H-2K locus.
[12]
12. Method according to claim 10 or 11, characterized in that the MHC | endogenous non-human is H-2K.
[13]
A method according to any one of claims 10 to 12, characterized in that the modification step is carried out in a single ES cell and the single ES cell is introduced into a rodent embryo to produce a mouse.
[14]
Method according to any one of claims 1 to 13, characterized in that it further comprises modifying a non-human animal genome to comprise, at an endogenous non-human B2 microglobulin B2 locus, a sequence of — nucleotide encoding a human or humanized B2 microglobulin polypeptide, wherein the non-human animal expresses the human or humanized B2 microglobulin polypeptide.
[15]
15. Method according to any one of the claims | to 13, characterized in that the modification step comprises replacing, at the endogenous non-human B2 microglobulin B2 locus, a nucleotide sequence encoding a non-human B2 microglobulin B2 polypeptide with the nucleotide sequence encoding the B2 microglobulin polypeptide human or humanized.
[16]
16. Method according to claim 14 or 15, characterized by the fact that the non-human animal does not express a functional endogenous non-human B2 microglobulin B2 polypeptide from the endogenous non-human B2 microglobulin B2 locus.
[17]
A method according to any one of claims 14 to 16, characterized in that the nucleotide sequence encoding the human or humanized microglobulin B2 polypeptide is operably linked to non-reactive microglobulin B2 regulatory elements. endogenous human.
[18]
Method according to any one of claims 14 to 17, characterized in that the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide comprises a nucleotide sequence exposed in exon 2 to exon 4 of a human B2 microglobulin gene.
[19]
Method according to any one of claims 14 to 18, characterized in that the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide comprises nucleotide sequences exposed in exons 2, 3 and 4. of a human B2 microglobulin gene.
[20]
20. Method according to claim 18 or 19, characterized in that the nucleotide sequence encoding the human or humanized B2 microglobulin polypeptide further comprises a nucleotide sequence exposed in exon 1 of a B2 microglobulin gene not human.
[21]
21. Method for producing a genetically modified mouse, characterized in that it comprises the steps of: modifying an MHC locus | of a first mouse to express an MHC polypeptide | chimeric human/mouse comprising substituting, at the MHC locus | endogenous mouse, a nucleotide sequence encoding domains a1, a2 and a3 of an MHC polypeptide | of mouse by a nucleotide sequence encoding a1, a2 and a3 domains of an MHC polypeptide | human; modifying a second mouse microglobulin B2 locus to express a human or humanized microglobulin B2 polypeptide comprising replacing, at the endogenous mouse microglobulin B2 locus, a nucleotide sequence encoding a mouse microglobulin B2 polypeptide with a nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide; and crossing the first and second mice to generate a genetically modified mouse comprising in its genome a first nucleotide sequence encoding an MHC polypeptide | human/chimeric mouse and a second nucleotide sequence encoding a human or humanized B2 microglobulin polypeptide, in which the genetically modified mouse expresses MHC polypeptide | human/chimeric mouse and human or humanized B2 microglobulin polypeptide.
[22]
22. Method according to claim 21, characterized by the fact that the MHC | is an H-2K locus, the human MHCI polypeptide is HLA-A2, and the mouse expresses a poly-
chimeric HLA-A2/H-2K peptide.
[23]
23. Method according to claim 21 or 22, characterized in that the chimeric HLA-A2/H-2K polypeptide comprises an extracellular domain of the HLA-A2 polypeptide and cytoplasmic and transmembrane domains of the HLA-A2 polypeptide. -2K.
[24]
A method according to any one of claims 21 to 23, characterized in that the second nucleotide sequence comprises nucleotide sequences exposed in exons 2, 3, and 4 of a human B2 microglobulin gene, and one if - nucleotide sequence exposed in exon 1 of a mouse microglobulin B2 gene.
[25]
25. Nucleic acid, characterized in that it comprises a sequence encoding an MHC polypeptide | chimeric/mouse, wherein a human portion of the chimeric polypeptide comprises the a1, a2, and a3 domains of an MHC polypeptide | human.
[26]
26. Nucleic acid according to claim 25, characterized in that it further comprises non-human regulatory elements operably linked to the sequence encoding the MHC polypeptide | chimeric human/mouse.
[27]
27. MHC polypeptide | chimeric human/non-human animal, characterized in that a human portion of the chimeric polypeptide comprises the a1, a2 and a3 domains of an MHC polypeptide | and a non-human portion of the chimeric polypeptide comprises cytoplasmic and transmembrane domains of an MHC polypeptide | non-human animal.
[28]
28. Polypeptide according to claim 27, characterized by the fact that the MHC | is non-covalently linked to a human or humanized B2 microglobulin.
[29]
29. Invention, characterized in any form of its embodiment or in any applicable category of claim, for example, of product or process or use encompassed by the matter initially described, disclosed or illustrated in the patent application.

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CN104039132A|2014-09-10|

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CY1119657T1|2018-04-04|

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法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-12-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-15| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-04-06| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161552587P| true| 2011-10-28|2011-10-28|

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US201161552582P| true| 2011-10-28|2011-10-28|

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US61/552,582|2011-10-28|

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US61/552,587|2011-10-28|

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US201261700908P| true| 2012-09-14|2012-09-14|

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US61/700,908|2012-09-14|

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PCT/US2012/062042|WO2013063346A1|2011-10-28|2012-10-26|Genetically modified major histocompatibility complex mice|

---