Genetically modified non-human animals for generating therapeutic antibodies to peptide-MHC complexes and methods of making and using the same

Abstract

To provide genetically modified non-human animals for generating therapeutic antibodies against peptide-MHC complexes, and methods of making and using the same.SOLUTION: The non-human animal is genetically modified with a sequence encoding a human or humanized MHC molecule or a sequence encoding a related molecule, e.g., β 2 microglobulin, and the non-human animal expresses the sequence to induce tolerance to the corresponding human HLA from which the human or human MHC molecule is derived. The tolerance exhibited by these non-human animals allows these animals to generate a specific antibody response against the corresponding human HLA when such HLA presents a peptide that is antigenic to the non-human animal.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Disclosed herein are genetically modified non-human animals (e.g., rodents (e.g., rats, mice, etc.)) that are tolerant to human (humanized) MHC molecules (such as, but not limited to, empty human (humanized) MHC molecules) or peptide-binding portions thereof (e.g., the peptide-binding groove of an MHC molecule), thereby enabling the non-human animal to generate a stable B cell response against the human (humanized) MHC molecule when the human (humanized) MHC molecule is complexed, e.g., presented with a peptide foreign to the non-human animal, e.g., as part of a peptide / MHC (pMHC) complex, where the peptide is xenogeneic to the non-human animal. Such animals may be useful for generating therapeutic antigen-binding proteins against pathogenic pMHC complexes, e.g., autoimmunogenic human self-peptides presented in the context of human HLA.

[0002] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 647,720, filed March 24, 2018, and U.S. Provisional Patent Application No. 62 / 647,724, filed March 24, 2018, each of which is incorporated by reference herein in its entirety. Sequence Listing

[0003] This specification makes reference to a Sequence Listing that has been submitted electronically as an ascii.txt file entitled "10116WO01_ST25_AsFiled", which was created on March 22, 2019, has a file size of 38.9 kilobytes, and is incorporated by reference in its entirety. [Background technology]

[0004] T cells play an important role in adaptive anti-infective and anti-tumor responses, but also function in inappropriate immune responses, such as autoimmune and graft rejection. Generally, T cell-mediated immune responses involve close contact between T cells and antigen-presenting cells (APCs). The formation of an immune synapse that triggers T cell activation involves several molecular pairs, including but not limited to: (a) the T cell receptor (TCR) on the T cell, which specifically binds to peptides presented in the peptide-binding groove of major histocompatibility complex (MHC) molecules on the APC, and (b) CD28 (on the T cell), which pairs with B7 molecules on the APC. The TCR forms a TCR complex with CD3 molecules, and pairing of the TCR with a peptide-MHC (pMHC) complex sends a signal through CD3. Signaling through both the TCR complex and CD28 on the T cell results in T cell activation.

[0005] Immunotherapies for treating diseases act to regulate T cell activity in vivo, for example, to downregulate autoimmune responses and transplant rejection. However, because many immunotherapies target TCR complex signaling by binding to CD3 and / or costimulatory molecule pairs, these approaches often lack specificity. These approaches often result in undesirable side effects, such as excessive immune responses or generalized immunosuppression. Therefore, therapeutic approaches that exploit the unique interactions between TCR and pMHC complexes may offer the ability to regulate the activity of specific T cells in vivo and provide new therapies based on T cell modulation. Summary of the Invention [Means for solving the problem]

[0006] Disclosed are non-human animals (e.g., mammals, e.g., rodents, e.g., rats or mice) that have been genetically modified to be tolerant to human HLA molecules and / or β2-microglobulin and capable of producing human or humanized antibodies. Both (1) the tolerance of these non-human animals to human HLA molecules and (2) the ability of these non-human animals to provide humanized antigen-binding proteins allow them to be used as unique platforms for the isolation of cells that may be useful in in vivo experiments, e.g., alloresponses, and / or for the production of human or humanized antigen-binding proteins that specifically bind to peptide-MHC complexes of interest, poised to become useful therapeutic agents in their own right. Accordingly, also provided herein are methods of making and using the non-human animals disclosed herein, as well as cells, tissues, nucleic acids, and antigen-binding proteins isolated from the animals.

[0007] In some embodiments, the genetically modified non-human animal comprises (a) a nucleotide sequence encoding a human or humanized MHC molecule, or at least a peptide-binding portion thereof, and (b) a (non-)rearranged human or humanized immunoglobulin heavy chain locus, and / or a (non-)rearranged human or humanized immunoglobulin light chain locus, optionally wherein at least one of the (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or the (non-)rearranged human or humanized immunoglobulin light chain locus is unrearranged, and the genetically modified non-human animal comprises a nucleotide sequence encoding a human or humanized MHC molecule, or at least a peptide-binding portion thereof. In some embodiments, the genetically modified non-human animal expresses a peptide-binding portion thereof, the genetically modified non-human animal expresses immunoglobulins containing a human or humanized heavy chain variable domain and / or a human or humanized light chain variable domain, and the non-human animal is tolerant to the human or humanized MHC molecule or at least a peptide-binding portion thereof, thereby generating a specific B cell response when immunized with an antigenic peptide-MHC (pMHC) complex containing (i) a peptide that is heterologous to the non-human animal, complexed with (ii) a human HLA molecule, or portion thereof, from which the human or humanized MHC molecule is derived. In some embodiments, the genetically modified non-human animal further contains an antigenic peptide-MHC (pMHC) complex containing a peptide that is heterologous to the non-human animal, associated with the human HLA molecule from which the human or humanized MHC molecule is derived. In some embodiments, the genetically modified non-human animal further contains (c)(ii) an antigenic peptide-MHC (pMHC) complex comprising an HLA molecule, or portion thereof, from which the human or humanized MHC molecule is derived, a peptide that is heterologous to the non-human animal and associated with (i)(ii), and (d) a human or humanized antigen binding protein that specifically binds to the antigenic pMHC and does not bind to the human HLA molecule from which the human or humanized MHC is derived.

[0008] In some embodiments, the human or humanized MHC molecule is selected from the group consisting of a human or humanized MHC class I molecule, a human or humanized MHC class II α molecule, a human or humanized MHC class II β molecule, or any combination thereof. In some embodiments, the human or humanized MHC molecule is a human or humanized MHC class I molecule. In some embodiments, the human or humanized MHC molecule is derived from an HLA class I molecule selected from the group consisting of an HLA-A molecule, an HLA-B molecule, an HLA-C molecule, and any combination thereof. In some embodiments, the genetically modified non-human animal further contains a nucleotide sequence encoding human or humanized β2 microglobulin in its genome, optionally at an endogenous β2 microglobulin locus, wherein the non-human animal expresses the human or humanized β2 microglobulin, thereby rendering the non-human animal tolerant to the β2 microglobulin itself or to β2 microglobulin associated with a human or humanized class I molecule.

[0009] In some embodiments, the human or humanized MHC molecule is a human or humanized MHC class II molecule, optionally derived from the α and / or β chain, or at least the peptide binding groove, of an HLA class II molecule selected from the group consisting of an HLA-DP molecule, an HLA-DQ molecule, an HLA-DR molecule, and any combination thereof.

[0010] In some embodiments, the human or humanized MHC molecule is a human HLA molecule. In some embodiments, the non-human animal contains a nucleotide sequence encoding a human HLA molecule at an endogenous MHC locus, optionally replacing an endogenous nucleic acid sequence encoding the endogenous MHC molecule. In some embodiments, the non-human animal contains a nucleotide sequence encoding a human HLA molecule at an ectopic locus. In some embodiments, the non-human animal contains a nucleotide sequence encoding a human HLA molecule at the ROSA26 locus. In some embodiments, the non-human animal is homozygous for the nucleotide sequence at the endogenous MHC locus, the ectopic locus, or the ROSA26 locus. In some embodiments, the non-human animal is heterozygous for the nucleotide sequence at the endogenous MHC locus, the ectopic locus, or the ROSA26 locus.

[0011] In some embodiments, the human or humanized MHC molecule is a humanized MHC molecule, e.g., a chimeric MHC molecule. In some embodiments, the non-human animal contains a nucleotide sequence encoding the chimeric MHC molecule at an endogenous MHC locus, where the nucleotide sequence optionally replaces an endogenous nucleic acid sequence encoding the endogenous MHC molecule. In some embodiments, the non-human animal contains a nucleotide sequence encoding the chimeric MHC molecule at an ectopic locus. In some embodiments, the non-human animal contains a nucleotide sequence encoding the chimeric MHC molecule at the ROSA26 locus. In some embodiments, the non-human animal is homozygous for the nucleotide sequence encoding the chimeric MHC molecule at the endogenous MHC locus, an ectopic locus, or the ROSA26 locus. In some embodiments, the non-human animal is heterozygous for the nucleotide sequence encoding the chimeric MHC molecule at the endogenous MHC locus, an ectopic locus, or the ROSA26 locus.

[0012] In some embodiments, the nucleotide sequence encodes a chimeric human / non-human MHC molecule comprising the extracellular domain of a human HLA molecule operably linked to the transmembrane and cytoplasmic domains of an endogenous MHC molecule. In some embodiments, the nucleotide sequence encodes (i) a chimeric human / non-human MHC class I molecule comprising the α1, α2, and α3 domains of a human MHC class I molecule selected from the group consisting of HLA-A, HLA-B, and HLA-C operably linked to the transmembrane and cytoplasmic domains of an endogenous non-human MHC class molecule, such as, for example, an endogenous mouse H-2K polypeptide, an endogenous mouse H-2D polypeptide, or an endogenous mouse H-DL polypeptide, and / or (ii) a chimeric human / non-human MHC class I molecule comprising the α1, α2, and α3 domains of a human MHC class I molecule selected from the group consisting of, for example, an endogenous mouse H-2Aα polypeptide or an endogenous mouse H-2Aα polypeptide. and / or the β1 and β2 domains of a human HLA class I β polypeptide operably linked to the transmembrane and cytoplasmic domains of an endogenous non-human MHC class II β molecule, such as an endogenous mouse H-2Aα polypeptide or an endogenous mouse H-2Eα polypeptide.

[0013] In some embodiments, the nucleotide sequence encoding a human or humanized MHC molecule does not disrupt an endogenous non-human MHC locus. In some embodiments, the nucleotide sequence encoding a human or humanized MHC molecule is integrated into a locus outside of the endogenous MHC locus. In some embodiments, the integration does not disrupt the function of any other endogenous genes. In one embodiment, the nucleotide sequence is placed within the endogenous ROSA26 locus.

[0014] In some embodiments, the non-human animal is heterozygous for the nucleotide sequence encoding a human or humanized MHC molecule, or at least a peptide-binding portion thereof.

[0015] In some embodiments, the genetically modified non-human animal contains an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region at the endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal contains a restricted unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region at the endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal contains a consensus heavy chain coding sequence at the endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal contains a histidine-modified unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region at the endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal contains a heavy chain-only immunoglobulin coding sequence at the endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal contains an unrearranged human (humanized) hybrid heavy chain sequence encoding a hybrid immunoglobulin chain at the endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal contains an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region at the endogenous light chain locus. In some embodiments, the genetically modified non-human animal contains a consensus light chain coding sequence at the endogenous light chain locus. In some embodiments, the genetically modified non-human animal contains a constrained unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region at the endogenous light chain locus. In some embodiments, the genetically modified non-human animal contains a histidine-modified unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region at the endogenous light chain locus. In some embodiments, the genetically modified non-human animal contains a histidine-engineered, reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region at the endogenous light chain locus.

[0016] In some embodiments, the genetically modified non-human animal contains a functional ADAM6 gene, optionally wherein the functional ADAM6 gene is an endogenous ADAM6 gene.

[0017] In some embodiments, the genetically modified non-human animal expresses an exogenous terminal deoxynucleotidyl transferase (TdT) gene.

[0018] In some embodiments, the method of making the genetically modified non-human animal of any of the preceding claims comprises modifying the genome of a non-human animal to contain (a) a nucleotide sequence encoding a human or humanized MHC molecule, or at least a peptide-binding portion thereof, and (b) a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus, optionally wherein at least one of the (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or the (non-)rearranged human or humanized immunoglobulin light chain locus is unrearranged, and wherein the genetically modified non-human animal is (ii) is tolerant to a human or humanized MHC molecule, or at least a peptide-binding portion thereof, thereby generating a specific B cell response when immunized with a peptide-MHC complex containing a human HLA molecule, or portion thereof, from which the human or humanized MHC molecule is derived, (i) a peptide that is xenogeneic to the non-human animal, complexed with (ii), and is capable of providing a human or humanized antigen-binding protein containing a human or humanized heavy chain variable domain and / or a human or humanized light chain variable domain. (a) (i) inserting a nucleotide sequence encoding a human or humanized MHC molecule, or at least a peptide-binding portion thereof, into a first ectopic locus; or (ii) substituting a nucleotide sequence encoding a non-human animal MHC I polypeptide with a nucleotide sequence encoding a human (humanized) MHC I polypeptide at the endogenous non-human animal MHC I locus, and / or substituting a nucleotide sequence encoding a non-human animal MHC II molecule with a nucleotide sequence encoding a human (humanized) MHC II molecule at the endogenous non-human animal MHC II locus, Optionally, the human (humanized) MHC I molecule contains the α1, α2, and α3 domains of human MHC I and at least the transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide; Optionally, the human (humanized) MHC II molecule contains or replaces the α1, α2, β1, and β2 domains of human MHC II and at least the transmembrane and cytoplasmic domains of an endogenous rodent MHC II polypeptide; and (b) (i) inserting a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus into a second ectopic locus; or (ii) (A) Endogenous non-human immunoglobulin variable (V) sequences at the endogenous non-human heavy chain locus. H ) gene segments and unrearranged human immunoglobulin variable (V H ) gene segments, and optionally, endogenous non-human immunoglobulin diversity (D H ) gene segments and / or endogenous non-human joining (J H ) gene segments and unrearranged human immunoglobulin diversity (D H ) gene segments and / or unrearranged human immunoglobulin binding (J H ) gene segments, respectively, H gene segment, and optionally D H Gene and J H The gene segment is operably linked to, replaces, and / or is an endogenous heavy chain constant region gene sequence. (B) Endogenous non-human light chain locus, endogenous non-human light chain variable (V L ) gene segments and endogenous non-human light chain joining (J L ) gene segment and the human light chain variable (V L ) gene segments and human light chain joining (J L) gene segments, which are optionally rearranged to form V L / J L Gene sequence is formed, and the human V L Gene segments and joints L the gene segment is operably linked to, or replaces, an endogenous light chain constant region gene sequence; (a) a nucleotide sequence encoding a non-human MHC I molecule and / or a non-human MHC II molecule, respectively; and (b) a V H , D H , J H , V L , and J. L The gene segment is (I) inserted or replaced by sequential homologous recombination in a single non-human embryonic stem (ES) cell; or (II) a first non-human animal and a second non-human animal are generated using the first ES cell and the second ES cell, respectively, and the method further includes mating the first and second non-human animals.

[0019] In some embodiments, a method for producing a genetically modified non-human animal comprises administering to the non-human animal an antigenic pMHC complex containing a peptide heterologous to the non-human animal, associated with a human HLA molecule from which a human or humanized MHC molecule is derived. In some embodiments, the antigenic pMHC complex is linked to a helper T cell epitope. In some embodiments, the helper T cell epitope comprises PADRE, e.g., as set forth in SEQ ID NO: 28.

[0020] In some embodiments, a method of producing an antigen binding protein, or a nucleic acid sequence encoding the protein, that specifically binds to an antigenic pMHC complex of interest comprises maintaining a genetically modified non-human animal as described herein under conditions sufficient for the non-human animal to mount an immune response to an antigenic pMHC complex of interest, wherein the antigenic pMHC complex of interest comprises a peptide that is xenogeneic to the non-human animal and is presented in the context of a human HLA, or portion thereof, from which a human or humanized MHC molecule is derived. In some embodiments, the method comprises, as a first step, immunizing the non-human animal with the antigenic pMHC complex of interest and optionally boosting the immune response of the immunized non-human animal, optionally wherein immunizing and / or boosting comprises administering to the non-human animal the pMHC complex of interest linked to a helper T cell epitope, such as, for example, PADRE (SEQ ID NO: 28).

[0021] In some embodiments, a method for obtaining nucleic acids encoding human immunoglobulin heavy chain variable domains and / or human immunoglobulin light chain variable domains comprises isolating from a non-human animal described herein a nucleic acid comprising a rearranged human immunoglobulin variable region gene sequence encoding a human immunoglobulin variable domain expressed by a lymphocyte of the non-human animal or a hybridoma generated from the lymphocyte, wherein the human immunoglobulin variable domain expressed by the lymphocyte or hybridoma generated from the lymphocyte associates with its cognate variable domain to form an antigen-binding domain specific for an antigenic pMHC complex. In some embodiments, the method further comprises immunizing the non-human animal with an antigenic pMHC complex of interest and mounting an immune response to the antigen in the non-human animal, followed by obtaining the nucleic acid. In some embodiments, the obtained rearranged human immunoglobulin variable region gene sequence comprises at least one somatic hypermutation.

[0022] In some embodiments, the nucleic acids described herein comprise a human constant region gene sequence operably linked to a rearranged human immunoglobulin variable region gene sequence. In some embodiments, the human heavy chain constant region gene sequence comprises a modification that increases the affinity of the CH2-CH3 region of the IgG heavy chain constant region amino acid sequence for the fetal Fc receptor (FcRn) at a pH ranging from 5.5 to 6.0, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and combinations thereof. Further described herein are mammalian host cells for expressing nucleic acids encoding human immunoglobulin heavy and / or light chains specific for, for example, antigenic pMHC complexes.

[0023] In some embodiments, a method for obtaining cells expressing a human immunoglobulin heavy chain variable domain and / or a human immunoglobulin light chain variable domain comprises isolating lymphocytes from a non-human animal described herein, wherein the lymphocytes express a human immunoglobulin variable domain that forms an antigen-binding domain specific for an antigenic pMHC complex, hi some embodiments, the method comprises producing a hybridoma from the isolated lymphocytes.

[0024] In some embodiments, an isolated cell described herein, e.g., a germ cell, embryonic stem cell, somatic cell (e.g., B cell), etc., comprises (a) a nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof, and (b) a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus. In some embodiments, at least one of the (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or the (non-)rearranged human or humanized immunoglobulin light chain locus is unrearranged. In some embodiments, the isolated cell is obtained according to a method described herein.

[0025] In some embodiments, a method of producing a human immunoglobulin variable domain in vitro comprises expressing in a cell a first nucleic acid comprising a rearranged human immunoglobulin variable region gene sequence encoding a human immunoglobulin variable domain expressed by a lymphocyte of a non-human animal described herein or a hybridoma produced from said lymphocyte, wherein the human immunoglobulin variable domain expressed by said lymphocyte or hybridoma produced from said lymphocyte associates with its cognate variable domain to form an antigen-binding domain specific for an antigenic pMHC complex. In some embodiments, the first nucleic acid further comprises a human immunoglobulin constant region gene sequence operably linked to the rearranged human immunoglobulin variable region gene sequence. In some embodiments, the human immunoglobulin constant region gene sequence is a heavy chain constant region gene sequence and comprises a modification that increases the affinity of the CH2-CH3 region of the IgG heavy chain constant region amino acid sequence for the fetal Fc receptor (FcRn) at a pH in the range of 5.5 to 6.0, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and combinations thereof. [Brief explanation of the drawings]

[0026] [Figure 1]1A-1C are exemplary embodiments of the present invention and provide schematic diagrams (not to scale) of chimeric MHC I and MHC II loci, e.g., chimeric HLA-A2 / H-2K at the endogenous H-2K locus (FIG. 1A), chimeric HLA-DR2 / H-2E at the endogenous H-2E locus (FIG. 1B), and humanized β2M at the endogenous β2M locus (FIG. 1C). Unless otherwise indicated, human sequences are shown in open text and mouse sequences are shown in solid text. Stripes indicate exon 1 and portions of the downstream intron of the H-2E gene sequence from a mouse strain different from the endogenous locus. The floxed neomycin phosphotransferase cassette is indicated by appropriately labeled arrows before Cre-mediated cassette removal (FIG. 1C) and after Cre-mediated cassette removal (FIGS. 1A and 1B).

[0027] [Figure 2] Figure 2 presents a schematic diagram (not to scale) of an exemplary transgene of the invention (SEQ ID NO: 22) encoding a single-chain MHC molecule (SEQ ID NO: 23) comprising the full-length mature HLA-A2 (A2) polypeptide (e.g., amino acids 25-365 of HLA-A2) associated with human β2-microglobulin (B2m) at the ROSA26 (Gt(ROSA)26 Sor) locus. Unless otherwise indicated, human sequences are shown in open text, mouse sequences in solid text, and non-human and non-mouse sequences are shown in various patterns. Solid arrows indicate exons of the endogenous mouse ROSA26 locus. 5'HB and 3'HB: homology boxes of the ROSA26 gene used for insertion of the B2m-G4Sx4-HLA-A2 transgene (SEQ ID NO: 22) encoding the single-chain HLA-A2 / β2M complex (SEQ ID NO: 23) by homologous recombination. SA: consensus splice acceptor. ROR: mouse ROR signal sequence. G4Sx4: GGGS linker (SEQ ID NO: 21), SV40PA: polyadenylation signal derived from SV40 virus. LoxP-New-LoxP: floxed neomycin phosphotransferase cassette before Cre-mediated removal of the cassette.

[0028] [Figure 3] FIG. 3 shows the results of an exemplary embodiment of the invention, in which serum from test mice (comprising nucleotide sequences encoding a humanized MHC I molecule (HLA-A2 / H-2K), humanized β2 microglobulin, an unrearranged humanized immunoglobulin heavy chain locus, and a humanized consensus light chain locus Vκ1-39 / Jκ; ●) or control mice (comprising a functional (e.g., murine) ADAM6 gene and humanized heavy and light chain loci; ■) immunized with a nucleotide sequence encoding an immunogen comprising peptide B presented in the peptide-binding groove of HLA-A associated with human β2 microglobulin was tested for titers (y-axis) of antibodies binding to an unrelated or related peptide (peptide A (unrelated), peptide B (related), or peptide C (unrelated)) presented as a single-chain pMHC complex in the peptide-binding groove of HLA-A associated with human β2 microglobulin. Titers of antibodies in serum binding to individual pMHC complexes are calculated as the interpolated serum dilution factor at which the binding signal is twice background.

[0029] [Figure 4]Figure 4 shows the results of an exemplary embodiment of the invention, in which serum from test mice (comprising nucleotide sequences encoding a humanized MHC I molecule (HLA-A2 / H-2K), humanized β2 microglobulin, an unrearranged humanized immunoglobulin heavy chain locus, and the humanized common light chain locus Vκ1-39 / Jκ; ●) or control mice (comprising a functional (e.g., murine) ADAM6 gene and humanized heavy chain and light chain loci; ■) immunized with a single-chain pMHC complex immunogen comprising peptide B presented in the peptide-binding groove of HLA-A associated with human β2 microglobulin was tested for antibody titers (y-axis) binding to an unrelated peptide or related peptides (peptide A (unrelated), peptide B (related), or peptide C (unrelated)) presented as a single-chain pMHC complex in the peptide-binding groove of HLA-A associated with human β2 microglobulin. The titers of antibodies in the serum that bind to individual pMHC complexes (y-axis) are calculated as the interpolated serum dilution factor at which the binding signal is twice background.

[0030] [Figure 5]FIG. 5 shows the results of an exemplary embodiment of the invention, in which serum from test mice (comprising nucleotide sequences encoding a humanized MHC I molecule (HLA-A2 / H-2K), humanized β2 microglobulin, an unrearranged humanized immunoglobulin heavy chain locus, and a humanized common light chain locus Vκ1-39 / Jκ) immunized with an immunogen comprising peptide B presented in the peptide-binding groove of HLA-A associated with human β2 microglobulin and boosted with another recombinant polypeptide comprising peptide B presented in the peptide-binding groove of HLA-A associated with a helper T cell epitope (PADRE) was tested for binding to an unrelated peptide or related peptides (peptide A (unrelated), peptide B (related), or peptide C (unrelated)) presented as a single-chain pMHC complex in the peptide-binding groove of HLA-A associated with human β2 microglobulin. Antibody titers (y-axis) of antibodies in the serum that bind to individual pMHC complexes are calculated as the interpolated serum dilution at which the binding signal is twice background. DETAILED DESCRIPTION OF THE INVENTION

[0031] As shown herein, control non-human animals that are not tolerant to human HLA and human β2-microglobulin molecules but are immunized with a peptide of interest, i.e., a pMHC complex of interest, presented in the context of HLA molecules, elicit antibody titers against the pMHC complex of interest. Figures 3-4 (square symbols). However, sera from non-tolerant, immunized control animals possessed comparable antibody titers against an unrelated pMHC complex (e.g., an unrelated peptide presented in the same context) to which the non-human animal was not immunized. This suggests that the response generated against the pMHC complex of interest is not a specific response. See Figures 3-4 (square symbols).

[0032] In contrast, non-human animals that are tolerant to human HLA and human β2 microglobulin molecules, or at least their peptide-binding grooves (e.g., their extracellular portions), and immunized with a target pMHC complex derived from a human HLA molecule elicited higher antibody titers against the target pMHC complex than against an unrelated pMHC complex. See Figures 3-5. Thus, it is shown herein that non-human animals that are tolerant to human HLA and human β2 microglobulin molecules (or at least their peptide-binding grooves (e.g., their extracellular portions)) provide an in vivo platform for the generation of a specific immune response against a target pMHC, from which lead compounds can be selected. This platform represents an improvement over platforms involving non-tolerant animals that generate immune responses non-specific to the target pMHC. Thus, it is shown herein that non-human animals genetically modified to be tolerant to human HLA molecules can generate specific B cell responses against a target pMHC complex when immunized with a target pMHC complex containing a target peptide presented in the context of the human HLA molecule.

[0033] In exemplary embodiments, non-human animals are genetically modified to be tolerant to human HLA molecules, portions thereof, and / or single-chain derivatives thereof, but not tolerant to antigenic peptide-MHC (pMHC) complexes comprising antigenic peptides (e.g., peptides that are xenogeneic to the genetically modified non-human animal) associated with (e.g., presented by) the human HLA molecules to which the non-human animal has been tolerized. Because the non-human animal is not tolerant to the antigenic pMHC complexes, genetically modified animals as disclosed herein may be useful not only for the isolation of immune cells that are unreactive to human HLA molecules, portions thereof, and / or single-chain derivatives thereof, e.g., for in vitro experiments, but also for the generation of antigen binding proteins, particularly human or humanized antigen binding proteins, that specifically bind to antigenic pMHC complexes of interest. Such specific (human or humanized) antigen-binding proteins may be useful in therapies for the treatment of human diseases, such as preventing the formation of an immune synapse between an autoreactive TCR and a pMHC complex containing an autoreactive antigen (e.g., preventing and / or treating autoimmune disorders, graft-versus-host disease, graft rejection, etc.), targeting cells infected with pathogens (e.g., viruses), and presenting viral peptides via pMHC complexes expressed on the cell surface. In addition to describing non-human animals that are tolerant to human or humanized HLA molecules, portions thereof, and / or single-chain derivatives thereof, and methods for producing such non-human animals, methods for administering antigenic pMHC complexes to such non-human animals to generate anti-pMHC antibody responses, as well as methods for producing such antigenic pMHC complexes, are also disclosed.

[0034] Definition of Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0035] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0036] The term "about" or "approximately" includes within a meaningful range of values. The allowable variation encompassed by the term "about" or "approximately" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0037] The terms "major histocompatibility complex" and "MHC" encompass such terms as "human leukocyte antigen" or "HLA" (the latter two generally referring to human MHC molecules), native MHC molecules, individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2 microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., the α1, α2, and / or α3 subunits of the MHC class I α chain, the α1-α2 subunits of the MHC class II α chain, the β1-β2 subunits of the MHC class II β chain), as well as portions thereof (e.g., peptide-binding portions, e.g., peptide-binding grooves), variants and various derivatives thereof (including fusion proteins), where the portions, variants and derivatives retain the ability to present antigenic peptides for recognition by a T cell receptor (TCR), e.g., an antigen-specific TCR. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the α heavy chain that can accommodate peptides of approximately 8-10 amino acids. Despite the fact that both classes of MHC bind to a core of approximately 9 amino acids (e.g., 5-17 amino acids) in peptides, the open-ended nature of the MHC class II peptide-binding groove (the α1 domain of an MHC class II α polypeptide associated with the β1 domain of an MHC class II β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II typically vary between 13-17 amino acids in length, although shorter or longer amino acid lengths are not uncommon. Consequently, peptides may shift within the peptide-binding groove of MHC class II, altering the 9-mer directly residing in the groove at any given time. Conventional methods for identifying specific MHC variants are used herein. In some embodiments, the genetically modified non-human animal comprises nucleotides encoding a human (humanized) MHC molecule comprising at least a human peptide-binding groove (e.g., a peptide-binding portion), and in further embodiments, comprises nucleotides encoding at least a human HLA class I / human β2 microglobulin molecule, and / or an extracellular domain of a human HLA class II molecule.

[0038] The terms "tolerized," "tolerance," "tolerization," and the like refer to the inability or reduced ability of an animal, such as a genetically modified non-human animal disclosed herein, to mount an immune response to a substance. Generally, an animal is tolerized, tolerates, or undergoes tolerization to its own proteins or molecules expressed during embryonic development and / or at birth. For example, the animal does not mount or is unlikely to mount an immune response to self-proteins or molecules expressed from its own genome, such as its own germline genome. By genetically modifying an animal to contain human (humanized) MHC molecules in its genome, such as its germline genome, when an "empty" human (humanized) MHC molecule is expressed, the animal becomes tolerant, tolerates, or undergoes tolerization to the empty human (humanized) MHC molecule as if it were a self-protein. In the context of HLA molecules, MHC molecules, human (humanized) MHC molecules, etc., "empty" includes HLA molecules, MHC molecules, human (humanized) MHC molecules, etc. that are expressed without a peptide in the peptide-binding groove, or that are expressed with a peptide that is endogenous to the animal expressing the HLA molecule, MHC molecule, human (humanized) MHC molecule, etc. For example, an empty MHC molecule can include a human (humanized) MHC molecule that is expressed in an animal from the animal's genome, e.g., germline genome, and that presents an endogenous animal self-protein or portion thereof.

[0039] The genome of a non-human animal may be considered a "somatic genome." For example, it may be a genome present in the somatic cells of the non-human animal. Alternatively, the genome of a non-human animal may be considered a "germline genome." For example, it may be a genome present in the germ cells of the non-human animal and passed on to the offspring of the non-human animal. Those skilled in the art will readily recognize that unrearranged immunoglobulin heavy and / or light chain variable region loci in a germline genome have the ability to rearrange in somatic cells (e.g., B cells) of the non-human animal to form rearranged immunoglobulin variable region loci encoding immunoglobulin variable domains. Thus, in exemplary embodiments, unrearranged heavy and / or light chain loci may be present in the germline genome of a non-human animal, and rearranged sequences derived therefrom may be present in, for example, B cells of the non-human animal.

[0040] Terms such as "non-human animal" refer to any vertebrate organism that is not a human. In some embodiments, the non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, or a bird. In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, the non-human animal is a rodent such as a rat or a mouse.

[0041] The terms "humanized," "chimeric," "human / non-human," and the like refer to molecules (e.g., nucleic acids, proteins, and the like) that are non-human in origin and in which portions thereof have been replaced with corresponding portions of a corresponding human molecule such that the modified (e.g., humanized, chimeric, human / non-human, etc.) molecule retains its biological function and / or maintains a structure that performs the retained biological function. A humanized molecule may be considered to be derived from a human molecule, in which case the humanized molecule is encoded by nucleotides that include a nucleic acid sequence that encodes a human molecule (or a portion thereof). In contrast, "human" and the like encompasses molecules that are exclusively human in origin, e.g., have human nucleotides or proteins that contain only human nucleotide and amino acid sequences, respectively. The term "human (humanized)" is used to reflect that the human (humanized) molecule can be (a) a human molecule or (b) a humanized molecule.

[0042] In some embodiments, the human (humanized) MHC molecule contains, or the non-human animal expresses, and the non-human animal is tolerized to, a human (humanized) MHC molecule containing at least the human peptide-binding groove of a human HLA molecule, wherein the human (humanized) MHC molecule retains the ability to present antigen in the human peptide-binding groove and / or the human (humanized) MHC molecule maintains the structure of the human peptide-binding groove of the human HLA molecule. In some embodiments, the human (humanized) MHC molecule contains, or the non-human animal expresses, and the non-human animal is tolerized to, a human (humanized) MHC molecule containing at least the human extracellular domain of a human HLA molecule, wherein the human (humanized) MHC molecule retains the ability to present antigen and / or the human (humanized) MHC molecule maintains the structure of the human extracellular domain of the human HLA molecule, allowing the peptide-binding groove to form. In some embodiments, the human (humanized) MHC molecule contains, or the non-human animal expresses, and the non-human animal is tolerized to, a human (humanized) MHC molecule containing a human peptide-binding groove of a human HLA class I polypeptide (e.g., at least the α1 and α2 domains of a human HLA class I polypeptide, e.g., the extracellular portion of a human HLA class I polypeptide, such as at least a full-length mature human HLA class I polypeptide), in which the human (humanized) MHC class I polypeptide retains the ability to present antigen and / or retains the structure necessary for presenting antigen in the human peptide-binding groove. In some embodiments, the human (humanized) MHC molecule contains, or the non-human animal expresses, and the non-human animal is tolerized to, a human (humanized) MHC molecule containing a human peptide-binding groove of a human HLA class I polypeptide (e.g., at least the α1 and α2 domains of a human HLA class I polypeptide, e.g., the extracellular portion of a human HLA class I polypeptide, such as at least a full-length mature human HLA class I polypeptide).In this case, the human (humanized) MHC class I molecule retains the ability to present antigen and / or retains the structure necessary to present antigen in the human peptide-binding groove, and in this case, the human (humanized) MHC class I molecule further contains human or humanized β2 microglobulin, which stabilizes the MHC class I molecule.

[0043] The term "antigen" refers to any substance (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portion thereof, or combination thereof) that, when introduced into an immunocompetent host, is recognized by the host's immune system and elicits an immune response by the host. T cell receptors recognize peptides presented in the context of major histocompatibility complexes (MHC) as part of the immune synapse. Peptide-MHC (pMHC) complexes are recognized by TCRs, and the peptide (antigenic determinant) and the TCR idiotype provide specificity for the interaction. Thus, the term "antigen" encompasses peptides presented in the context of MHC, e.g., peptide-MHC complexes, e.g., pMHC complexes. Peptides presented on MHC are sometimes referred to as "epitopes" or "antigenic determinants." Terms such as "peptide," "antigenic determinant," and "epitope" not only encompass those naturally presented by antigen-presenting cells (APCs), but also any desired peptide, as long as it is recognized by immune cells of a genetically modified non-human animal when appropriately presented to cells of the immune system. For example, a peptide having an artificially prepared amino acid sequence can also be used as an epitope.

[0044] "Peptide-MHC complex", "pMHC complex", "peptide-in-groove", etc. (i) an MHC molecule, e.g., a human and / or humanized (e.g., human (humanized)) MHC molecule, or a portion thereof (e.g., its peptide-binding groove, and e.g., its extracellular portion), and (ii) an antigenic peptide, In this case, the MHC molecule and the antigenic peptide are complexed, and the pMHC complex can specifically bind to a T cell receptor. pMHC complexes include pMHC complexes expressed on the cell surface and soluble pMHC complexes. In an exemplary embodiment, a non-human animal that contains in its genome, e.g., a germline genome, a nucleotide sequence encoding a human (humanized) MHC molecule or at least its human peptide-binding groove is tolerant to an empty human (humanized) MHC molecule or tolerant to the empty human peptide-binding groove. Upon administration of an antigenic pMHC complex to the non-human animal, e.g., a complex comprising a human (humanized) MHC molecule complexed with a peptide foreign to the host non-human animal to which the pMHC complex is administered, the non-human animal has the ability to generate an antibody response to the antigenic pMHC complex. Such specific antigen-binding proteins may then be isolated and used as therapeutic agents that specifically modulate the interaction of antigenic pMHC with a specific T cell receptor. In an exemplary embodiment, soluble pMHC complexes comprising a peptide (exogenous to the host non-human animal to which the pMHC complex is administered) complexed with an MHC molecule to which the non-human animal has been tolerized may not elicit a T cell immune response due to the soluble nature of the administered pMHC complex, but such soluble pMHC complexes may still be considered antigenic in that they can elicit a B cell-mediated immune response that produces an antigen-binding protein that specifically binds to the pMHC complex.

[0045] The term "gene segment" or "segment" refers to a variable (V) gene segment (e.g., an immunoglobulin light chain variable (V L ) gene segment or immunoglobulin heavy chain variable (V H ) gene segment), immunoglobulin heavy chain diversity (D H ) gene segment, or joining (J) gene segment, such as an immunoglobulin light chain joining (J L ) gene segment or immunoglobulin heavy chain joining (J L) gene segments that participate in a rearrangement (e.g., mediated by an endogenous recombinase) to form a rearranged light chain V L / J L A heavy chain V rearrangement or V H / D H / J H Immunoglobulin loci that can form sequences, including unrearranged sequences. Unless otherwise indicated, unrearranged V, D, and J segments are V sequences that follow the 12 / 23 rule. L / J L Recombinant, or V H / D H / J H It contains a recombination signal sequence (RSS) that enables recombination.

[0046] The terms "antigen-binding protein," "immunoglobulin," "antibody," "binding protein," and the like refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), as well as epitope-binding fragments of any of the above. The terms "antibody" and "antibodies" also refer to covalently linked diabodies, such as those disclosed in U.S. Patent No. 20070004909, and Ig-DARTS, such as those disclosed in U.S. Patent No. 20090060910, both of which are incorporated by reference in their entireties. pMHC-binding protein refers to antigen-binding proteins, immunoglobulins, antibodies, and the like that specifically bind to pMHC complexes.

[0047] The terms "specifically bind," "bind in a specific manner," "antigen-specific," and the like indicate that the molecules involved in specific binding (1) are capable of stably binding, e.g., stably associating, e.g., forming stable intermolecular non-covalent bonds, under physiological conditions, and (2) are incapable of forming stable bonds with other molecules outside of the specific binding pair under physiological conditions. Thus, an antigen-binding protein (e.g., an immunoglobulin, antibody, etc.) that binds to a pMHC complex in a specific manner suggests that the pMHC-binding protein forms a stable intermolecular non-covalent bond with the pMHC complex. Thus, a pMHC-binding protein that specifically binds, e.g., binds in a specific manner, to a particular pMHC complex comprising a first peptide complexed with a first MHC molecule is (i) does not stably bind under physiological conditions to a pMHC complex containing a second peptide presented in the context of a first MHC molecule, wherein the first and second peptides are not identical and exhibit different idiotypes (conformations), e.g., when associated with the peptide-binding groove of the first MHC molecule; (ii) capable of interfering with (e.g., disrupting) the interaction between (a) a first TCR that specifically recognizes a first peptide presented in the context of a first MHC molecule, and (b) a pMHC complex comprising the first peptide and the first MHC molecule; (iii) does not interfere with the interaction between (a) a second TCR that specifically recognizes a second peptide presented in the context of a first MHC molecule, or (b) a pMHC complex comprising a second peptide presented in the context of a first MHC molecule, where the first and second peptides are different and, for example, exhibit different idiotypes (conformations) when associated with the peptide-binding groove of the first MHC molecule. A pMHC binding protein that specifically binds to a pMHC complex comprising a first peptide and a first MHC molecule may additionally or independently bind to a first cell presenting the first peptide in the context of a first MHC molecule, but not to a second cell presenting the second peptide in the context of the first MHC molecule, where the first and second peptides are different and exhibit different idiotypes (conformations) when associated with the peptide-binding groove of the MHC molecule. Specific binding also occurs with an equilibrium dissociation constant (K) in the low micromolar to picomolar range. D ) can be characterized by specificity. High specificity can be in the low nanomolar range, while very high specificity can be in the picomolar range. Methods for determining whether two molecules specifically bind are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0048] "Individual" or "subject" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In one embodiment, the subject is a human.

[0049] The term "protein" as used herein encompasses all types of natural and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins including, but not limited to, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation).

[0050] The terms "nucleic acid" and "nucleotide" encompass both DNA and RNA, unless otherwise specified.

[0051] The terms "titer," "antibody titer," "antibody titer," and the like refer to the ability of an antibody sample (e.g., in serum obtained from a subject) to share a characteristic ability, e.g., isotype, to bind to an antigen in a specific or non-specific manner. Methods for determining the titer of an antibody in serum taken from a subject are known in the art. In some embodiments, the titer of an antibody in serum taken from a subject is calculated as the interpolated serum dilution at which the binding signal is twice background. In some embodiments, a non-human animal that expresses and is tolerant to a human (humanized) MHC molecule can or does generate a specific response (e.g., a specific immune response, such as a specific B-cell response, such as a specific antibody response) when immunized with a pMHC complex of interest (e.g., a peptide of interest presented in the context of HLA molecules from which the human (humanized) MHC molecule was derived). In some embodiments, the response is considered a "specific response" or the like. In this case, the non-human animal, when immunized with the target pMHC complex, generates a higher antibody titer to the target pMHC complex than the antibody titer to an unrelated pMHC complex. In some embodiments, the response is considered a specific response or the like. In this case, the non-human animal, when immunized with the target pMHC complex, generates an antibody titer that is at least two-fold higher to the target pMHC complex than the antibody titer to an unrelated pMHC complex. In some embodiments, the response is considered a specific response or the like. In this case, the non-human animal, when immunized with the target pMHC complex, generates an antibody titer that is at least five-fold higher to the target pMHC complex than the antibody titer to an unrelated pMHC complex. In some embodiments, the response is considered a specific response or the like. In this case, the non-human animal, when immunized with the target pMHC complex, generates an antibody titer that is at least ten-fold higher to the target pMHC complex than the antibody titer to an unrelated pMHC complex. In some embodiments, the response is considered a specific response or the like.In this case, the non-human animal, when immunized with the pMHC complex of interest, develops a significantly higher antibody titer against the pMHC complex of interest than against an unrelated pMHC complex.

[0052] The terms "operably linked" and like terms refer to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. For example, an unrearranged variable region gene segment is "operably linked" to a contiguous constant region gene if it can be rearranged to form a rearranged variable region gene, and the rearranged variable region is joined to the constant region gene and expressed as a polypeptide chain of an antigen-binding protein. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences that act in trans or remotely to regulate the gene (or sequence of interest) of interest. The term "expression control sequence" includes polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are linked. "Expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak sequences), sequences that enhance polypeptide stability, and, if desired, sequences that enhance polypeptide secretion. The nature of such control sequences will vary depending on the host organism. For example, in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, and transcription termination sequence, while in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include elements whose presence is essential for expression and processing, and can also include additional elements whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0053] Terms such as "administration" refer to and include administration of a composition to a subject or system (e.g., a cell, organ, tissue, organism, or related component or set of components thereof). One of skill in the art will recognize that the route of administration can vary depending, for example, on the subject or system to which the composition is administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., a human or rodent) may be bronchial (including bronchial infusion), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal infusion), transdermal, vaginal, and / or intravitreal administration. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (eg, perfusion) for at least a selected period of time.

[0054] The term "derived from," when used in reference to a rearranged variable region gene or rearranged variable domain "derived from" an unrearranged variable region and / or unrearranged variable region gene segment, refers to the ability to trace the sequence of the rearranged variable region gene or rearranged variable domain back to a set of rearranged, unrearranged variable region gene segments (accounting for splice differences, and somatic mutation, where applicable), to form a rearranged variable region gene that expresses a variable domain. For example, a rearranged variable region gene that has undergone somatic mutation remains the fact that it was derived from an unrearranged variable region gene segment.

[0055] The term "endogenous locus" or "endogenous gene" refers to a genetic locus present in a parent or reference organism prior to the introduction of a disruption, deletion, substitution, alteration, or modification described herein. In some embodiments, an endogenous locus has a sequence that occurs in nature. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is an engineered locus.

[0056] The term "heterologous" refers to a substance or entity from a different source. For example, when used in reference to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the related polypeptide, gene, or gene product: 1) has been genetically manipulated by the hand of man; 2) has been introduced into the cell or organism (or a precursor thereof) by the hand of man (e.g., by genetic manipulation); and / or 3) is not naturally produced by or occurs in the related cell or organism (e.g., related cell type or organism type). "Heterologous" also includes polypeptides, genes, or gene products that are normally present in a particular native cell or organism but that are altered or modified by mutation or substitution, e.g., not naturally associated with, and in some embodiments, under the control of non-endogenous regulatory elements (e.g., promoters).

[0057]

[0058] In accordance with the disclosure herein there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being explained fully in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein referred to as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed. 1985); Oligonucleotide Synthesis (MJGait ed.1984);Nucleic Acid Hybridization [BDHames & SJHiggins eds.(1985)];Transcription And Translation [BDHames & SJHiggins,eds.(1984)];Animal Cell Culture [RIFreshney,ed.(1986)];Immobilized Cells And Enzymes [IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning(1984);Ausubel,FMet al.(eds.).Current See Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. Each of these publications is incorporated herein by reference in its entirety.These techniques include site-directed mutagenesis, e.g., Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Patent No. 5,071,743; Fukuoka et al., Biochem. Biophys. Res. Commun. 263:357-360 (1999); Kim and Maas, BioTech. 28:196-198 (2000); Parikh and Guengerich, BioTech. 24:4 28-431 (1998); Ray and Nickoloff, BioTech. 13:342-346 (1992); Wang et al. al., BioTech. 19:556-559 (1995); Wang and Malcolm, BioTech. 26:680-682 (1999); Xu and Gong, BioTech. 26:639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3:74-75 (2001), U.S. Patent Nos. 5,702,931, 5,780,270 and 6,242,222, Angag and Schutz, Biotech. 30:486-488 (2001), Wang and Wilkinson, Biotech. 29:976-978 (2000), Kang et al., Biotech. 20:44-46 (1996), Ogel and McPherson, Protein Engineer. 5:467-468 (1992), Kirsch and See Joly, Nucl. Acids. Res. 26:1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178:3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nucl. Acids. Res. 22:541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57:229-237, and Pons et al., Meth. Molec. Biol. 67:209-218, each of which publications is incorporated herein by reference in its entirety.

[0059] Tolerization of human or humanized MHC molecules In exemplary embodiments, a genetically modified non-human animal, e.g., a mammal, e.g., a rodent, e.g., a rat or mouse, expresses and is tolerant to at least one empty human or humanized MHC molecule, or at least an empty human peptide-binding groove thereof. However, when complexed with an antigenic, e.g., heterologous, peptide, it is capable of producing an antigen-binding protein, e.g., an antigen-binding protein comprising a human or humanized variable domain, against a human (humanized) MHC molecule. In some embodiments, tolerance of a non-human animal to an empty human (humanized) MHC molecule is achieved by genetically modifying the non-human animal to contain in its genome a nucleotide sequence encoding the human (humanized) MHC molecule or at least the human peptide-binding groove thereof, such that the non-human animal expresses the human (humanized) MHC molecule or at least the human peptide-binding groove thereof as an empty human (humanized) MHC molecule or as the empty human peptide-binding groove thereof. Such animals genetically modified to contain nucleotides encoding human (humanized) MHC molecules may be further modified to contain humanized immunoglobulin heavy and / or light chain loci and to express human or humanized antigen binding proteins, e.g., antigen binding proteins having human or humanized variable domains.

[0060] MHC molecules are generally classified into two categories: class I and class II MHC molecules. MHC class I molecules are integral membrane proteins containing glycoprotein heavy chains, also referred to herein as α chains, which have three extracellular domains (i.e., α1, α2, and α3) and two intracellular domains (i.e., transmembrane (TM) and cytoplasmic (CYT) domains). The heavy chains are non-covalently associated with a soluble subunit called β2-microglobulin (β2m or β2M). MHC class II molecules or proteins are heterodimeric integral membrane proteins containing one non-covalently associated α chain and one β chain. The α chain has two extracellular domains (α1 and α2) and two intracellular domains (TM and CYT). The β chain contains two extracellular domains (β1 and β2) and two intracellular domains (TM and CYT).

[0061] The domain structures of class I and class II MHC molecules form the antigen-determining binding site of the MHC molecule, e.g., the peptide-binding portion or peptide-binding groove. The peptide-binding groove refers to the portion of the MHC protein that forms a cavity to which a peptide, e.g., an antigenic determinant, can bind. The structure of the peptide-binding groove has the ability to change upon peptide binding, allowing proper alignment of amino acid residues important for TCR binding to the peptide-MHC (pMHC) complex.

[0062] In some embodiments, the MHC molecule contains a fragment of an MHC chain sufficient to form a peptide-binding groove. For example, the peptide-binding groove of a class I protein contains portions of the α1 and α2 domains of the heavy chain and can form two β-pleated sheets and two α-helices. The inclusion of a portion of the β2-microglobulin chain stabilizes the MHC class I molecule. While for most versions of MHC class II molecules, α-chain and β-chain interactions can occur even in the absence of peptide, the two-chain MHC class I molecule is unstable until the binding groove is filled with peptide. The peptide-binding groove of a class II protein contains portions of the α1 and β1 domains and can form two β-pleated sheets and two α-helices. The first portion of the α1 domain forms a first β-pleated sheet, and the second portion of the α1 domain forms a first α-helix. The first portion of the β1 domain forms a second β-pleated sheet, and the second portion of the β1 domain forms a second α-helix. X-ray crystallographic analysis of class II proteins with peptides associated in the protein's binding groove has shown that one or both ends of the associated peptide can protrude beyond the MHC protein (Brown (E. al., pp. 33-39, 1993, Nature, Vol. 364, incorporated herein by reference in its entirety). Thus, the ends of the α1 and β1 α-helices of class II form an open cavity, and the end of the peptide bound in the binding groove is not buried within the cavity. Furthermore, X-ray crystallography of class II proteins has revealed that MHC It has been shown that the N-terminus of the β-chain clearly protrudes from the side of the MHC protein in an unsystematic manner, and the first four amino acid residues of the β-chain could not be assigned by X-ray crystallography.

[0063] Many human and other mammalian MHCs are known in the art.

[0064] In some embodiments, the non-human animal contains at least one of a first, second, and / or third nucleotide sequence, each of which encodes a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, and a human or humanized MHC Iα polypeptide. The non-human animal may further contain, for example, a human or humanized β2 microglobulin in embodiments containing a nucleotide sequence encoding a human or humanized MHC Iα polypeptide. The use of the designations first, second, and third herein should not be construed as limiting the non-human animals disclosed herein as requiring all three nucleotide sequences in any particular order or as requiring the presence of any of the human or humanized MHC polypeptides in any particular order.

[0065] In various embodiments herein, a genetically modified non-human animal, e.g., a mammal, e.g., a rodent (e.g., a mouse or rat), is provided that contains in its genome a nucleotide sequence encoding a human or humanized MHC I polypeptide, and / or a nucleotide sequence encoding a human or humanized MHC II protein, or at least the human peptide-binding groove thereof. The MHC I nucleotide sequence may encode a fully human MHC I polypeptide (e.g., a human HLA class I molecule), or a partially human, partially non-human humanized MHC I polypeptide (e.g., a chimeric human / non-human MHC I polypeptide), and the MHC II nucleotide sequence may encode a fully human MHC II protein (e.g., a human HLA class II molecule), or a partially human, partially non-human humanized MHC class II protein (e.g., a chimeric human / non-human MHC II protein, including, e.g., chimeric human / non-human MHC II α and β polypeptides).

[0066] In the Examples herein, we demonstrate that genetically modified animals that express, from their endogenous loci, chimeric human / non-human MHC I molecules comprising a human extracellular portion of a human HLA class I molecule (containing a human peptide-binding domain) operably linked to the transmembrane and cytoplasmic domains of a non-human MHC I molecule are tolerant to the human extracellular domain, e.g., the human peptide-binding domain, of the human HLA class I molecule when empty, and can mount a specific immune response to the human peptide-binding domain when complexed with an antigen, e.g., a peptide, that is heterologous to the non-human animal. See, e.g., Examples. Thus, in some embodiments, a non-human animal, e.g., a rodent such as a rat or mouse, (1) expressing, e.g., from an endogenous MHC locus, (b) a chimeric human / non-human MHC molecule containing (a) at least a human peptide-binding groove, e.g., a human extracellular portion, of a human HLA molecule operably linked to a non-human transmembrane and cytoplasmic domain of a non-human MHC I molecule, and (2) Tolerance to at least the extracellular portion of human HLA molecules.

[0067] Genetically modified non-human animals comprising nucleotide sequences in their genomes, e.g., at endogenous loci, encoding chimeric human / non-human MHC II polypeptides, are disclosed in U.S. Patent Nos. 9,591,835 and 9,615,550, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals comprising nucleotide sequences in their genomes, e.g., at endogenous loci, encoding humanized, e.g., chimeric human / non-human MHC II polypeptides, are disclosed in U.S. Patent Nos. 8,847,005 and 9,043,996, each of which is incorporated herein by reference in its entirety. Genetically modified non-human animals comprising in their genome, e.g., at an endogenous locus, a nucleotide sequence encoding a humanized, e.g., chimeric human / non-human MHC I polypeptide, and comprising in their genome, e.g., at an endogenous locus, a nucleotide sequence encoding a humanized, e.g., chimeric human / non-human MHC II polypeptide, are disclosed in U.S. Pat. No. 10,154,658, which is incorporated herein by reference in its entirety.

[0068] In various embodiments herein, in its genome, e.g., in its germline genome, e.g., at one or more endogenous MHC loci: (i) a first nucleotide sequence encoding a chimeric human / non-human MHC I polypeptide, comprising a human portion comprising an extracellular portion of a human MHC I polypeptide (or a portion thereof, e.g., one or more extracellular domains, e.g., a peptide-binding groove), operably linked to a non-human portion comprising a transmembrane domain and a cytoplasmic domain of a non-human MHC I polypeptide, e.g., an endogenous MHC I polypeptide; and / or (ii) a genetically modified non-human animal is provided that comprises a second nucleotide sequence encoding a chimeric human / non-human MHC IIα polypeptide comprising a human portion operably linked to a non-human portion, wherein the human portion of the chimeric MHC IIα polypeptide comprises an extracellular portion (or portion thereof, e.g., one or more extracellular domains, e.g., the α1 domain) of an α polypeptide of a human MHC class II molecule, and a third nucleotide sequence encoding a chimeric human / non-human MHC IIβ polypeptide comprising a human portion operably linked to a non-human portion, wherein the human portion of the chimeric MHC IIβ polypeptide comprises an extracellular portion (or portion thereof, e.g., one or more extracellular domains, e.g., at least the β1 domain) of a β polypeptide of a human MHC class II molecule; The non-human animal may contain chimeric human / non-human MHC I proteins and / or MHC The non-human animal expresses an MHC I protein and / or MHC II protein and is tolerant to the chimeric human / non-human MHC I protein and / or MHC II protein. In one embodiment, the first, second, and / or third nucleotide sequence are located at the endogenous non-human MHC I, MHC IIα, and MHC IIβ loci, respectively. In one embodiment, the non-human animal is a mouse, and the first, second, and / or third nucleotide sequence are located at the endogenous mouse MHC locus on mouse chromosome 17. In one embodiment, the first nucleotide sequence is located at the endogenous non-human MHC I locus. In one embodiment, the second nucleotide sequence is located at the endogenous non-human MHC IIα locus. In one embodiment, the third nucleotide sequence is located at the endogenous non-human MHC IIβ locus.

[0069] In one embodiment, a chimeric human / non-human MHC I polypeptide comprises a human portion operably linked to a non-human portion, wherein the human portion comprises at least the peptide-binding groove of a human MHC I polypeptide. In one embodiment, the human portion of the chimeric polypeptide comprises the extracellular portion of a human MHC I molecule. In this embodiment, the human portion of the chimeric polypeptide comprises the extracellular domain of the α chain of a human MHC I molecule. In one embodiment, the human portion of the chimeric polypeptide comprises the α1 and α2 domains of a human MHC I molecule. In another embodiment, the human portion of the chimeric polypeptide comprises the α1, α2, and α3 domains of a human MHC I molecule.

[0070] In one embodiment, the human portion of the chimeric MHC IIα polypeptide and / or the human portion of the chimeric MHC IIβ polypeptide comprise the peptide-binding domain of a human MHC IIα polypeptide and / or a human MHC IIβ polypeptide, respectively, such as the MHC IIα and β polypeptides of a human MHC II protein. In one embodiment, the human portion of the chimeric MHC IIα and / or β polypeptide comprises the extracellular portion of a human MHC IIα and / or β polypeptide, respectively, such as the MHC IIα and β polypeptides of a human MHC II protein. In one embodiment, the human portion of the chimeric MHC IIα polypeptide comprises the α1 domain of a human MHC IIα polypeptide, and in another embodiment, the human portion of the chimeric MHC IIα polypeptide comprises the α1 domain and the α2 domain of a human MHC IIα polypeptide. In a further embodiment, the human portion of the chimeric MHC IIβ polypeptide comprises the β1 domain of a human MHC IIβ polypeptide, and in another embodiment, the human portion of the chimeric MHC IIβ polypeptide comprises the β1 domain and the β2 domain of a human MHC IIβ polypeptide.

[0071] In exemplary embodiments, the non-human animal expresses and is tolerant to chimeric human / non-human MHC molecules. In one embodiment, the human portions comprise the extracellular domains of human MHC I, MHC IIα, and / or MHC IIβ molecules, respectively, and the human portions are operably linked to non-human portions, and the non-human portions of the chimeric human / non-human MHC I, MHC IIα, and / or MHC IIβ polypeptides comprise the transmembrane and / or cytoplasmic domains of endogenous non-human (e.g., rodent, e.g., mouse, rat) MHC I, MHC IIα, and / or MHC IIβ polypeptides, respectively. Thus, the non-human portions of the chimeric human / non-human MHC I polypeptides may comprise the transmembrane and / or cytoplasmic domains of endogenous non-human MHC I polypeptides. The non-human portions of the chimeric MHC IIα polypeptides may comprise the transmembrane and / or cytoplasmic domains of endogenous non-human MHC IIα polypeptides. The non-human portion of the chimeric human / non-human MHC IIβ polypeptide can comprise the transmembrane and / or cytoplasmic domains of an endogenous non-human MHC IIβ polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human portion of the chimeric MHC I polypeptide is derived from a mouse H-2K protein. In one embodiment, the non-human animal is a mouse, and the non-human portion of the chimeric MHC IIα and β polypeptides is derived from a mouse H-2E protein. Thus, the non-human portion of the chimeric MHC I polypeptide can comprise the transmembrane and cytoplasmic domains derived from a mouse H-2K, and the non-human portion of the chimeric MHC IIα and β polypeptides can comprise the transmembrane and cytoplasmic domains derived from a mouse H-2E protein. While specific H-2K and H-2E sequences are contemplated in this example, any suitable sequences, such as polymorphic variants, conservative / non-conservative amino acid substitutions, etc., are encompassed herein.

[0072] In one embodiment, the human portion of the chimeric human / mouse MHC I polypeptide comprises the peptide-binding domain or extracellular domain of human MHC I (e.g., human HLA-A, e.g., human HLA-A2, e.g., human HLA-A2.1). The peptide-binding groove of human MHC I can comprise the α1 and α2 domains. Alternatively, the peptide-binding groove of human MHC I can comprise the α, α2, and α3 domains. In one embodiment, the extracellular domain of human MHC I comprises the extracellular domain of the human MHC Iα chain. In one embodiment, the endogenous mouse MHC I locus is the H-2K (e.g., H-2Kb) locus, and the mouse portion of the chimeric MHC I polypeptide comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. Thus, in one embodiment, a mouse of the invention comprises, at its endogenous mouse MHC I locus, a nucleotide sequence encoding a chimeric human / mouse MHC I, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide and the mouse portion comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide (see, e.g., SEQ ID NO: 24), and the mouse expresses the chimeric human / mouse HLA-A2 / H-2K protein. In other embodiments, the mouse portion of the chimeric MHC I polypeptide may be derived from other mouse MHC I, e.g., H-2D, H-2L, etc., and the human portion of the chimeric MHC I polypeptide may be derived from other human MHC I, e.g., HLA-B, HLA-C.

[0073] In one embodiment, the human portion of the chimeric human / mouse MHC IIα polypeptide comprises the peptide-binding domain or extracellular domain of human MHC IIα, and the human portion of the chimeric human / mouse MHC IIβ polypeptide comprises the peptide-binding domain or extracellular domain of human MHC IIβ. The peptide-binding domain of the human MHC IIα polypeptide may comprise the α1 domain, and the peptide-binding domain of the human MHC IIβ polypeptide may comprise the β1 domain. Thus, the peptide-binding domain of the chimeric MHC II molecule may comprise the human α1 domain and β1 domain. Human MHC The extracellular domain of the IIα polypeptide may comprise the α1 domain and the α2 domain, and the extracellular domain of the human MHC IIβ polypeptide may comprise the β1 domain and the β2 domain. Thus, the extracellular domain of the chimeric MHC II molecule may comprise human α1 domain, α2 domain, β1 domain, and β2 domain. In one embodiment, the mouse portion of the chimeric MHC II molecule comprises the transmembrane and cytoplasmic domains of mouse MHC II, e.g., mouse H-2E (e.g., the transmembrane and cytoplasmic domains of mouse H-2E α and β chains). Thus, in one embodiment, the mouse of the present invention comprises a chimeric human / mouse MHC II at its endogenous mouse MHC II locus. The chimeric MHC IIα polypeptide comprises a nucleotide sequence encoding IIα, wherein the human portion of the chimeric MHC IIα polypeptide comprises an extracellular domain derived from the α chain of human MHC II (e.g., the α chain of HLA-DR2), and the mouse portion comprises a transmembrane and cytoplasmic domain derived from the α chain of mouse MHC II (e.g., H-2E). The mouse then comprises, at its endogenous mouse MHC II locus, a nucleotide sequence encoding a chimeric human / mouse MHC IIβ, wherein the human portion of the chimeric MHC IIβ polypeptide comprises an extracellular domain derived from the β chain of human MHC II (e.g., the β chain of HLA-DR2), and the mouse portion comprises a transmembrane and cytoplasmic domain derived from the β chain of mouse MHC II (e.g., H-2E). For example, in this case, the mouse expresses a chimeric human / mouse HLA-DR2 / H-2E protein. In other embodiments, the mouse portion of the chimeric MHC II protein may be derived from other mouse MHC IIs, such as H-2A, and the human portion of the chimeric MHC II protein may be derived from other human MHC IIs, such as HLA-DQ.

[0074] In some embodiments, chimeric human / non-human polypeptides may comprise a human or non-human leader (signal) sequence. In one embodiment, a chimeric MHC I polypeptide comprises the non-human leader sequence of an endogenous MHC I polypeptide. In one embodiment, a chimeric MHC IIα polypeptide comprises the non-human leader sequence of an endogenous MHC IIα polypeptide. In one embodiment, a chimeric MHC IIβ polypeptide comprises the non-human leader sequence of an endogenous MHC IIβ polypeptide. In another embodiment, a chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC The chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide may comprise a non-human leader sequence of an MHC I polypeptide, an MHC IIα polypeptide, and / or an MHC IIβ polypeptide, respectively, derived from another non-human animal, e.g., another rodent or another mouse strain. Thus, the nucleotide sequence encoding the chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide may comprise a non-human MHC I leader sequence, an MHC IIα polypeptide, and / or an MHC IIβ polypeptide, respectively. The chimeric MHC I polypeptide may be operably linked to a nucleotide sequence encoding an IIα leader sequence, and / or an MHC IIβ leader sequence. In yet another embodiment, the chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide comprises a human leader sequence for a human MHC I polypeptide, a human MHC IIα polypeptide, and / or a human MHC IIβ polypeptide, respectively (e.g., the leader sequence for human HLA-A2, human HLA-DRα, and / or human HLA-DRβ1*1501, respectively).

[0075] In some embodiments, a chimeric human / non-human MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide may comprise, in its human portion, the complete or substantially complete extracellular domain of a human MHC I polypeptide, human MHC IIα polypeptide, and / or human MHC IIβ polypeptide, respectively. Thus, the human portion may comprise at least 80%, at least 85%, at least 90%, e.g., 95% or more of the amino acids encoding the extracellular domain of a human MHC I polypeptide, human MHC IIα polypeptide, and / or human MHC IIβ polypeptide (e.g., human HLA-A2, human HLA-DRα, and / or human HLA-DRβ1*1501). In one example, the substantially complete extracellular domain of a human MHC I polypeptide, human MHC IIα polypeptide, and / or human MHC IIβ polypeptide lacks a human leader sequence. In another example, the chimeric human / non-human MHC I polypeptide, the chimeric human / non-human MHC IIα polypeptide, and / or the chimeric human / non-human MHC IIβ polypeptide comprises a human leader sequence.

[0076] Furthermore, in some embodiments, the chimeric MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide may be operably linked to (expressed under the control of) an endogenous non-human promoter and regulatory elements, such as, for example, mouse MHC I, MHC IIα, and / or MHC IIβ regulatory elements, respectively. Such an arrangement facilitates proper expression of the chimeric MHC I polypeptide and / or chimeric MHC II polypeptide in the non-human animal, for example, during an immune response in the non-human animal.

[0077] While the examples provided herein demonstrate tolerance to human peptide-binding domains of chimeric human / non-human MHC molecules expressed from endogenous MHC loci, such tolerance also occurs in non-human animals that express human MHC molecules (or functional peptide-binding domains thereof) from ectopic loci (data not shown). Furthermore, non-human animals that express and are tolerized to empty human MHC molecules (or empty peptide-binding domains thereof) from ectopic loci are able to mount a specific immune response against the human HLA molecule (or peptide-binding domain, or derivative thereof) from which the expressed human MHC molecule is derived when the non-human animal is immunized with a human HLA molecule (or peptide-binding domain, and / or derivative thereof) complexed with an antigenic peptide, e.g., a peptide xenogeneic to the non-human animal.

[0078] Without wishing to be bound by theory, tolerance of the non-human animal is believed to occur upon expression of human or humanized MHC molecules. Thus, the human or humanized MHC molecules do not necessarily need to be expressed from endogenous loci. Accordingly, various embodiments herein provide genetically modified non-human animals comprising in their genome a first nucleotide sequence encoding a human (humanized) MHC I polypeptide (or a portion and / or derivative thereof), a second nucleotide sequence encoding a human (humanized) MHC IIα polypeptide (or a portion and / or derivative thereof), and / or a third nucleotide sequence encoding a human (humanized) MHC IIβ polypeptide (or a portion and / or derivative thereof), wherein the non-human animal expresses the human (humanized) MHC I polypeptide, MHC IIα polypeptide, and / or MHC IIβ polypeptide (or a portion and / or derivative thereof) and is tolerant to the human (humanized) polypeptide or a portion and / or derivative thereof. In one embodiment, the first, second, and / or third nucleotide sequences are each encoded by an endogenous non-human MHC The MHC I locus, MHC IIα locus, and MHC IIβ locus are not disrupted. For example, they are located at an ectopic locus, such as the ROSA26 locus. In some embodiments, the genetically modified mouse comprises a nucleotide sequence encoding a chimeric human / mouse MHC I at an ectopic locus, such as the ROSA26 locus, wherein the human portion of the chimeric polypeptide comprises the extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide, and the mouse portion comprises the transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide (see, e.g., SEQ ID NO: 24), and the mouse expresses and is tolerant to the chimeric human / mouse HLA-A2 / H-2K protein.

[0079] Additionally, if from an ectopic locus, a fully human MHC molecule, or a fully human portion and / or derivative thereof, may be expressed. Thus, in various embodiments herein, a genetically modified non-human animal is provided that comprises in its genome a first nucleotide sequence encoding a fully human MHC I polypeptide (or a fully human portion and / or derivative thereof), a second nucleotide sequence encoding a fully human MHC IIα polypeptide (or a fully human portion and / or derivative thereof), and / or a third nucleotide sequence encoding a fully human MHC IIβ polypeptide (or a fully human portion and / or derivative thereof), wherein the non-human animal expresses and is tolerant to the fully human MHC I polypeptide, MHC IIα polypeptide, and / or MHC II β polypeptide (or a fully human portion and / or derivative thereof). In one embodiment, the first, second, and / or third nucleotide sequences each do not disrupt an endogenous non-human MHC I locus, MHC IIα locus, and MHC IIβ locus, and optionally do not disrupt any endogenous locus, and are located at an ectopic locus, such as the ROSA26 locus.

[0080] In some embodiments, a human or humanized MHC I polypeptide may be derived from, for example, a nucleic acid encoding or comprising a portion of a nucleotide sequence encoding a functional human HLA molecule selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof. A human or humanized MHC II α or β polypeptide may be derived from an α or β polypeptide of a functional human HLA molecule encoded by any of the HLA-DP locus, HLA-DQ locus, and HLA-DR locus. A list of commonly used HLA antigens and alleles is provided in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), which is incorporated herein by reference in its entirety. Shankarkumar et al. also provide a brief explanation of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) *The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1 / 3 / 4 / 5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens*, Tissue Antigens 73:95-170, and recently updated by Marsh et al. (2010) *Nomenclature for factors of the HLA system*, 2010, Tissue Antigens 75:291-455. These publications are incorporated herein by reference in their entirety. In some embodiments, the MHC I or MHC II polypeptide may be derived from any functional human HLA-A, B, C, DR, or DQ molecule.Thus, human or humanized MHC I and / or II polypeptides may, in exemplary embodiments, be derived from any functional human HLA molecule, hi some embodiments, all MHC I and II polypeptides expressed on the cell surface comprise a portion derived from a human HLA molecule.

[0081] Of particular interest are polymorphic human HLA alleles, which are known to be associated with many human diseases, such as human autoimmune diseases. Indeed, specific polymorphisms in the HLA locus have been identified that correlate with the development of rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune disorders. For example, Wong and Wen (2004) What can the HLA transgenic mouse tell us? about autoimmune diabetes?, Diabetologia 47:1476-87;Taneja and David(1998)HLA Transgenic Mice as Humanized Mouse Models of Disease and Immunity,J.Clin.Invest.101:921-26;Bakker et al.(2006),A high-resolution HLA and SNP haplotype map for disease association studies in the extended See, e.g., "The Human MHC," Nature Genetics 38:1166-72, and supplemental information; and "International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases," Proc. Natl. Acad. Sci. USA 106:18680-85, each of which publications is incorporated herein by reference in its entirety. Thus, in some embodiments, human or humanized MHC I and / or MHC II polypeptides may be derived from human HLA molecules known to be associated with particular diseases, such as, for example, autoimmune diseases.

[0082] In one particular embodiment, the human or humanized MHC I polypeptide is derived from human HLA-A. In a particular embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., an HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., an HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is widely used among North American populations. While this example describes this particular HLA sequence, any suitable HLA-A sequence is encompassed herein. For example, polymorphic variants of HLA-A2 exhibited in human populations, sequences with one or more conservative or non-conservative amino acid substitutions, nucleotide sequences that differ from the sequences of the exemplary embodiments herein due to degeneracy of the genetic code, etc.

[0083] In another particular embodiment, the human or humanized MHC I polypeptide is derived from a human MHC I selected from HLA-B and HLA-C. In one embodiment, it is derived from HLA-B, e.g., HLA-B27. In another embodiment, it is derived from HLA-A3, HLA-B7, or HLA-Cw6.

[0084] In one particular embodiment, the human or humanized MHC II α and β polypeptides are derived from human HLA-DR, e.g., HLA-DR2. Typically, the HLA-DR α chain is monomorphic. For example, the α chain of the HLA-DR protein is encoded by the HLA-DRA gene (e.g., the HLA-DRα*01 gene). On the other hand, the HLA-DR β chain is polymorphic. Thus, HLA-DR2 contains a β chain encoded by the HLA-DRA gene and a β chain encoded by the HLA-DR1β*1501 gene. Any suitable HLA-DR sequence is encompassed herein, including, for example, polymorphic variants exhibited in the human population, sequences with one or more conservative or non-conservative amino acid substitutions.

[0085] In some embodiments, the human or humanized MHC II α and / or β polypeptides may be encoded by the nucleotide sequence, or portions thereof, of HLA alleles known to be associated with common human diseases. Such HLA alleles include, but are not limited to, HLA-DRB1*0401, -DRB1*0301, -DQA1*0501, -DQB1*0201, DRB1*1501, -DRB1*1502, -DQB1*0602, -DQA1*0102, -DQA1*0201, -DQB1*0202, -DQA1*0501, and combinations thereof. For a review of HLA allele / disease associations, see Bakker et al. (2006), supra, which is incorporated herein by reference in its entirety.

[0086] In further embodiments, the non-human animal of the invention, e.g., a rodent, e.g., a rat or mouse, comprises a nucleotide sequence encoding human or humanized β2 microglobulin (e.g., at the endogenous β2 microglobulin locus). The MHC class I protein β2 microglobulin or light chain (also abbreviated as "β2M") is a small (12 kDa), non-glycosylated protein that functions primarily to stabilize the MHC Iα chain. The generation of human or humanized β2 microglobulin animals is detailed in U.S. Patent No. 9,615,550, which is incorporated herein by reference in its entirety.

[0087] In some embodiments, a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide may contain nucleotide residues corresponding to only a portion of the human β2 microglobulin gene, e.g., only a portion that assists in stabilizing a human (humanized) MHC I molecule. In some embodiments, a nucleotide sequence encoding a human β2 microglobulin polypeptide comprises the entire human β2 microglobulin gene. Alternatively, in some embodiments, the nucleotide sequence may include nucleotide residues encoding the amino acid sequence set forth in amino acids 21-119 of the human β2 microglobulin protein (i.e., the amino acid residues corresponding to mature human β2 microglobulin). In another embodiment, the nucleotide sequence may include nucleotide residues encoding the amino acid sequence set forth in amino acids 23-115 of the human β2 microglobulin protein, e.g., the amino acid sequence set forth in amino acids 23-119 of the human β2 microglobulin protein. The nucleic acid and amino acid sequences of human β2-microglobulin are described in Gussow et al. (1987) The β2-Microglobulin Gene. Primary Structure and Definition of the Transcriptional Unit, J. Immunol. 139:3131-38, which is incorporated herein by reference in its entirety.

[0088] Thus, in some embodiments, a human or humanized β2 microglobulin polypeptide may comprise the amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin polypeptide, such as the amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin polypeptide, such as the amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin polypeptide. Alternatively, the human β2 microglobulin may comprise amino acids 1-119 of a human β2 microglobulin polypeptide.

[0089] In some embodiments, the nucleotide sequence encoding human or humanized β2 microglobulin comprises the nucleotide sequence set forth in exons 2 through 4 of the human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises the nucleotide sequence set forth in exons 2, 3, and 4 of the human β2 microglobulin gene. In this embodiment, the nucleotide sequences set forth in exons 2, 3, and 4 are operably linked to permit normal transcription and translation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exons 2 through 4 of the human β2 microglobulin gene. In a particular embodiment, the human sequence comprises a nucleotide sequence corresponding to approximately 267 bp after exons 2 through 4 of the human β2 microglobulin gene. In a particular embodiment, the human sequence comprises approximately 2.8 kb of the human β2 microglobulin gene.

[0090] Thus, in some embodiments, a human or humanized β2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising the nucleotide sequence set forth in exons 2 through 4 of human β2 microglobulin, e.g., the nucleotide sequence corresponding to exons 2 through 4 of the human β2 microglobulin gene. Alternatively, in some embodiments, the polypeptide may be encoded by a nucleotide sequence comprising the nucleotide sequence set forth in exons 2, 3, and 4 of the human β2 microglobulin gene. In particular embodiments, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to approximately 267 bp after exons 2 through 4 of the human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising approximately 2.8 kb of the human β2 microglobulin gene. In cases where exon 4 of the β2 microglobulin gene contains a 5' untranslated region, the human or humanized polypeptide may be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.

[0091] Furthermore, in some embodiments, the non-human animal comprising a nucleotide sequence encoding human or humanized β2 microglobulin also comprises the nucleotide sequence set forth in exon 1 of the non-human β2 microglobulin gene. Thus, in particular embodiments, the non-human animal comprises in its genome a nucleotide sequence encoding human or humanized β2 microglobulin, wherein the nucleotide sequence comprises exon 1 of the non-human β2 microglobulin and exons 2, 3, and 4 of the human β2 microglobulin gene. Thus, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of the non-human β2 microglobulin gene and exons 2, 3, and 4 of the human β2 microglobulin gene (e.g., exons 2 and 3 of the human β2 microglobulin gene).

[0092] In some embodiments, a nucleotide sequence encoding human or humanized β2 microglobulin is located at the endogenous non-human animal β2 microglobulin locus. In some embodiments, the nucleotide sequence of human β2 microglobulin replaces the corresponding nucleotide sequence encoding endogenous non-human β2 microglobulin at the endogenous non-human animal β2 microglobulin locus. For example, in some embodiments, a nucleotide sequence corresponding to exons 2 to 4 of the human β2 microglobulin gene replaces the endogenous mouse sequence corresponding to exons 2 to 4 of the mouse β2 microglobulin gene at the endogenous mouse β2 microglobulin locus (see Figure 1C). In some embodiments, a nucleotide sequence comprising the nucleotide sequence set forth in exons 2, 3, and 4 of the human β2 microglobulin gene replaces the nucleotide sequence set forth in exons 2, 3, and 4 of the mouse β2 microglobulin gene.

[0093] In some embodiments, the nucleotide sequence encoding human or humanized β2 microglobulin does not disrupt the endogenous non-human animal β2 microglobulin locus, but is located at an ectopic locus, such as, for example, the ROSA26 locus.

[0094] In some embodiments, the genetically modified non-human animal comprises in its genome, at an ectopic locus, e.g., the ROSA26 locus, a first nucleotide sequence and / or a second nucleotide sequence, wherein the first nucleotide sequence encodes a single-chain polypeptide comprising a functional peptide-binding portion of an HLA class I molecule (e.g., at least the α1 and α2 domains of a human MHC class I molecule), optionally operably linked, e.g., fused, to human or humanized β2 microglobulin (or a portion thereof), and wherein the second nucleotide sequence encodes a single-chain polypeptide comprising a functional peptide-binding portion of an HLA class II molecule (e.g., at least the α1 and β1 domains of a human MHC class II molecule). In some embodiments, the genetically modified non-human animal comprises, e.g., at an ectopic locus, a first nucleotide sequence encoding a single-chain polypeptide comprising at least a functional peptide-binding portion of a human HLA-A2 polypeptide (e.g., a full-length mature HLA-A2 polypeptide) fused to human β2 microglobulin (see, e.g., Figure 2). In some embodiments, a single-chain polypeptide comprising at least a functional peptide-binding portion of a human HLA-A2 polypeptide (e.g., a full-length mature HLA-A2 polypeptide) fused to human β2 microglobulin comprises the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 23. In some embodiments, a genetically modified non-human animal described herein comprises, at an ectopic locus, e.g., the ROSA26 locus, a nucleotide sequence encoding a human (humanized) MHC class I molecule comprising at least a functional peptide-binding portion of a human HLA-A2 molecule fused to human (humanized) β2 microglobulin, e.g., a nucleotide sequence set forth as SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, or a degenerate variant thereof. In some embodiments, a genetically modified non-human animal described herein comprises, at an ectopic locus, e.g., the ROSA26 locus, a nucleotide sequence encoding a chimeric MHC polypeptide, e.g., an HLA-A2 / H2-K polypeptide set forth as SEQ ID NO: 24.

[0095] Those skilled in the art will understand that while certain embodiments include specific nucleotide and amino acid sequences for generating genetically engineered animals, sequences with one or more conservative or non-conservative amino acid substitutions, or sequences that differ from the embodiments described herein due to the degeneracy of the genetic code, are also considered within the scope of the invention.

[0096] Thus, in some embodiments, non-human animals are provided that express a human (humanized) MHC class Iα polypeptide nucleic acid sequence, where the human (humanized) MHC class Iα polypeptide nucleic acid sequence, or a portion thereof, is not identical to the human MHC class Iα polypeptide nucleic acid sequence due to the degeneracy of the genetic code, but is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In particular embodiments, the human (humanized) MHC class Iα polypeptide nucleic acid sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the human MHC class Iα polypeptide nucleic acid sequence of the embodiments exemplified herein. In one embodiment, the expressed human (humanized) MHC class Iα polypeptide sequence comprises one or more conservative substitutions. In one embodiment, the human (humanized) MHC class Iα polypeptide sequence comprises one or more non-conservative substitutions.

[0097] Further, in some embodiments, non-human animals are provided that express a human (humanized) β2 microglobulin sequence, where the human (humanized) β2 microglobulin sequence or a portion thereof is not identical to the human β2 microglobulin sequence due to the degeneracy of the genetic code, but is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In particular embodiments, the human (humanized) β2 microglobulin sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence of the embodiments exemplified herein. In one embodiment, the human (humanized) β2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human (humanized) β2 microglobulin sequence comprises one or more non-conservative substitutions.

[0098] In some embodiments, a non-human animal is provided that expresses a human (humanized) MHC class IIα polypeptide nucleic acid sequence, where the human (humanized) MHC class IIα polypeptide nucleic acid sequence, or a portion thereof, is not identical to, but is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, the nucleic acid sequence of a human MHC class Iα polypeptide due to degeneracy of the genetic code. In one embodiment, the expressed human (humanized) MHC class IIα polypeptide sequence contains one or more conservative substitutions. In one embodiment, the human (humanized) MHC class IIα polypeptide sequence contains one or more non-conservative substitutions.

[0099] In some embodiments, a non-human animal is provided that expresses a nucleic acid sequence of a human (humanized) MHC class II β polypeptide, where the nucleic acid sequence of the human (humanized) MHC class II β polypeptide, or a portion thereof, is not identical to the nucleic acid sequence of a human MHC class Iα polypeptide due to degeneracy of the genetic code, but is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In one embodiment, the sequence of the expressed human (humanized) MHC class II α polypeptide contains one or more conservative substitutions. In one embodiment, the sequence of the human (humanized) MHC class II α polypeptide contains one or more non-conservative substitutions.

[0100] In some embodiments, the non-human animal is heterozygous for the first, second, and / or third nucleotide sequence, wherein each sequence encodes a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, and a human or humanized MHC Iα polypeptide, or a portion thereof. In some embodiments, the non-human animal is homozygous for the first, second, and / or third nucleotide sequence, wherein each sequence encodes a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, and a human or humanized MHC Iα polypeptide, or a portion thereof.

[0101] Human or humanized B cell immune response In some embodiments, the genetically modified non-human animal, in addition to comprising first, second and / or third nucleotide sequences, each sequence encoding a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC IIα polypeptide, a human or humanized MHC IIβ polypeptide, and a human or humanized MHC Iα polypeptide, or a portion thereof, also comprises, e.g., a human or humanized immunoglobulin heavy chain and / or light chain locus, such that the non-human animal is capable of providing a human or humanized antigen binding protein comprising a human or humanized antigen binding domain, e.g., a human or humanized variable domain.

[0102] Immunoglobulin loci comprising human variable region gene segments are known in the art and are described, e.g., in U.S. Pat. Nos. 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,232,449, Nos. 8,502,018, 8,697,940, 8,703,485, 8,754,287, 8,791,323, 8,809,051, 8,907,157, 9,035,128, 9,145,588, 9,206,263, 9,447,177, 9,551,124, 9,580,491, and 9,475,5 No. 59, each of which is incorporated herein by reference in its entirety, as well as U.S. Patent Application Publications 20100146647, 20110195454, 20130167256, 20130219535, 20130326647, 20130096287, and 2015 / 0113668, each of which is incorporated herein by reference in its entirety. and WO2014093908, which are incorporated by reference in their entireties.

[0103] In some embodiments, the non-human animals disclosed herein comprise, in addition to nucleotide sequences encoding human or humanized MHC molecules, exogenously introduced fully human immunoglobulin transgenes that can rearrange in mouse B cell precursors (Alt et al., 1985, Immunoglobulin genes in transgenic mice, Trends Genet 1:231-236, incorporated herein by reference in its entirety). In these embodiments, fully human immunoglobulin transgenes may be inserted (randomly) and endogenous immunoglobulin genes may be knocked out (Green et al., 1994, Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs, Nat Genet 7:13-21; Lonberg et al., 1994, Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368:856-859; Jakobovits et al., 2007, From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice, Nat Biotechnol 25:1134-1143; each of these publications is incorporated herein by reference in its entirety). For example, in this case, the endogenous immunoglobulin heavy chain and kappa light chain loci are inactivated, e.g., by targeted deletion of a small but essential portion of each endogenous locus, followed by introduction of human immunoglobulin gene loci as a randomly integrated large transgene, or minichromosome (Tomizuka et al., 2000, Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies, PNAS USA 97:722-727, incorporated herein by reference in its entirety).

[0104] In some embodiments, the human or humanized immunoglobulin heavy and light chain loci are located at the endogenous immunoglobulin heavy and light chain loci, respectively. Large-scale in situ gene replacement of mouse germline immunoglobulin variable gene loci with human germline immunoglobulin variable gene loci while maintaining the reproductive ability of the mouse has been previously described. See, e.g., U.S. Patent Nos. 6,596,541 and 8,697,940, which are incorporated herein by reference in their entirety. Specifically, precise replacement of 6 megabases of both the mouse heavy and kappa light chain immunoglobulin variable gene loci with their human counterparts while leaving the mouse constant regions intact has been described. As a result, mice have been generated in which the entire germline immunoglobulin variable repertoire has been precisely replaced with the equivalent human germline immunoglobulin variable sequences while maintaining the mouse constant regions. The human variable regions are linked to mouse constant regions, forming a chimeric human-mouse immunoglobulin locus that rearranges and expresses at physiologically appropriate levels. The expressed antibody is a "reverse chimera," i.e., the antibody contains human variable region sequences and mouse constant region sequences. These mice with humanized immunoglobulin variable regions that express antibodies with human or humanized variable regions and mouse constant regions are called VELOCIMMUNE® mice.

[0105] VELOCIMMUNE® humanized mice exhibit fully functional humoral immune systems essentially indistinguishable from wild-type mice. These mice exhibit normal cell populations at all stages of B cell development. These mice exhibit normal lymphoid organ morphology. Antibody sequences in VELOCIMMUNE® mice exhibit normal V(D)J rearrangements and normal somatic hypermutation frequencies. The antibody population in these mice reflects the isotype distribution resulting from normal class switching (e.g., normal isotype cis-switching). Immunization of VELOCIMMUNE® mice generates a stable humoral immune response, generating a large and diverse antibody repertoire with human immunoglobulin variable domains suitable for use as therapeutic candidates. This platform provides a rich source of natural affinity-matured human immunoglobulin variable region sequences for the generation of pharmaceutically acceptable antibodies and other antigen-binding proteins. Furthermore, one endogenous V H Gene segments and human V H It has also been shown that even gene segment replacement can result in an immune response involving humanized immunoglobulin variable domains. See, e.g., Tien et al. (2016) Cell 166:1471-84, which is incorporated herein by reference in its entirety. By precisely substituting mouse immunoglobulin variable sequences for human immunoglobulin variable sequences, the human immunoglobulin variable sequences are operably linked to endogenous non-human constant region gene sequences in a reverse chimeric manner, creating VELOCIMMUNE® mice.

[0106] Mice modified in a reverse chimeric manner include mice modified to contain a human (humanized) variable region (e.g., comprising a (D), J, and one or more human V gene segments) operably linked to an endogenous constant region at an endogenous immunoglobulin locus, e.g., (a) at the endogenous heavy chain locus; (i) an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the unrearranged human (humanized) immunoglobulin heavy chain variable region comprises a plurality of unrearranged human heavy chain variable region Vs; H Gene segments (e.g., all functional human unrearranged non-V H gene segment), one or more unrearranged immunoglobulin heavy chains D H gene segment, and one or more unrearranged immunoglobulin heavy chain J H comprising a gene segment, Optionally, the one or more unrearranged immunoglobulin heavy chains D H gene segment, and one or more unrearranged immunoglobulin heavy chain J H The gene segment may comprise one or more unrearranged human immunoglobulin heavy chain D H gene segments (e.g., all functional human D H gene segments) and / or one or more unrearranged human immunoglobulin heavy chain J H gene segments (e.g., all functional human J H gene segment), (ii) a constrained unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, the constrained unrearranged human (humanized) heavy chain variable region comprising one or more unrearranged immunoglobulin heavy chain Ds. H a gene segment and one or more unrearranged immunoglobulin heavy chain J H One unrearranged human heavy chain variable region V operably linked to a gene segment H and optionally, the one or more unrearranged immunoglobulin heavy chain D gene segments. H gene segment, and one or more unrearranged immunoglobulin heavy chain J H Each gene segment encodes one or more unrearranged human immunoglobulin heavy chain D H gene segments, and / or one or more unrearranged human immunoglobulin heavy chain J H a gene segment, (iii) a consensus heavy chain coding sequence comprising a reshaped human (humanized) heavy chain variable region sequence operably linked to an endogenous heavy chain constant region, the reshaped human (humanized) heavy chain variable region sequence being an immunoglobulin heavy chain D H rearranged with gene segments and immunoglobulin heavy chain J H Human heavy chain variable region V rearranged with gene segment H comprising a gene segment, Optionally, the immunoglobulin heavy chain J H Gene segment, or immunoglobulin heavy chain D H The gene segments are human immunoglobulin heavy chain D H gene segments, and / or human immunoglobulin heavy chain J H a gene segment, (iv) a histidine-engineered, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, the histidine-engineered, unrearranged human (humanized) heavy chain variable region comprising an unrearranged immunoglobulin heavy chain variable gene sequence comprising a substitution of at least one non-histidine codon for a histidine codon, or an insertion of at least one histidine codon, in a complementarity-determining region 3 (CDR3)-encoding sequence; (v) a heavy chain-only immunoglobulin coding sequence comprising an unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, the endogenous heavy chain constant region comprising (1) an intact endogenous IgM gene encoding an IgM isotype associated with a light chain, and (2) a non-IgM gene, e.g., an IgG gene, lacking sequences encoding a functional CH1 domain, wherein the non-IgM gene encodes a non-IgM isotype lacking a CH1 domain capable of covalently linking to a light chain constant domain; or (vi) an unrearranged human (humanized) hybrid heavy chain sequence encoding a hybrid immunoglobulin chain, the unrearranged human (humanized) hybrid heavy chain sequence comprising an unrearranged human light chain variable (V) operably linked to an endogenous heavy chain constant region; L ) gene segments and unrearranged junctions (J L ) gene segments, Optionally, the endogenous heavy chain constant region comprises (1) an intact endogenous IgM gene encoding an IgM isotype associated with a light chain, and (2) a non-IgM gene, e.g., an IgG gene, lacking sequences encoding a functional CH1 domain, wherein the non-IgM gene encodes a non-IgM isotype lacking a CH1 domain capable of covalently linking to a light chain constant domain; and / or (b) at the endogenous light chain locus; (i) an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region, the unrearranged human (humanized) immunoglobulin light chain variable region comprising a plurality of unrearranged human light chain variable region Vs; L Gene segments (e.g., all functional human unrearranged non-V L gene segment), and one or more unrearranged immunoglobulin light chain J L comprising a gene segment, Optionally, the one or more unrearranged immunoglobulin light chains J L The gene segment may comprise one or more unrearranged human immunoglobulin light chain J L gene segments (e.g., all functional human J H L gene segment), Optionally, the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain kappa (κ) locus, and the unrearranged human (humanized) immunoglobulin light chain variable region is a human variable κ (V κ ) gene segments and combined kappa (J κ ) gene segments, and wherein the endogenous light chain constant region is an endogenous κ chain constant region sequence, and / or the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain lambda (λ), and the unrearranged human (humanized) immunoglobulin light chain variable region is a human variable λ (V λ ) gene segments and combined λ(J λ) gene segments, and the endogenous light chain constant region is an endogenous λ chain constant region sequence, optionally wherein the endogenous immunoglobulin light chain λ locus comprises (a) one or more human V λ (b) one or more human J gene segments; λ gene segment, and (c) one or more human C λ gene segments, wherein (a) and (b) are linked to (c) and a rodent immunoglobulin light chain constant (C λ gene segments), and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ, and one or more human immunoglobulin λ light chain enhancers (Eλ), optionally comprising three human Eλ; (ii) a consensus light chain coding sequence comprising a reshaped human (humanized) light chain variable region sequence operably linked to an endogenous light chain constant region, the reshaped human (humanized) light chain variable region sequence comprising an immunoglobulin light chain J L Human light chain variable region V rearranged with gene segments L comprising a gene segment, (iii) a constrained unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region, wherein the constrained unrearranged human (humanized) light chain variable region comprises one or more unrearranged human immunoglobulin light chain binding (J) chains. L operably linked to a non-rearranged human immunoglobulin light chain variable (V) gene segment; L ) gene segments, (iv) a histidine-engineered, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region, the histidine-engineered, unrearranged human (humanized) light chain variable region comprising an unrearranged human (humanized) immunoglobulin light chain variable gene sequence comprising a substitution of at least one non-histidine codon for a histidine codon, or an insertion of at least one histidine codon, in the complementarity-determining region 3 (CDR3) coding sequence; or (v) A mouse has been previously reported that has been modified to contain a histidine-engineered reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region, the histidine-engineered reshaped human (humanized) light chain variable region comprising a reshaped human (humanized) immunoglobulin light chain variable gene sequence that includes a substitution of at least one non-histidine codon for a histidine codon or an insertion of at least one histidine codon in the complementarity-determining region 3 (CDR3) coding sequence; Optionally, the mouse further comprises: (i) comprises a human (humanized) immunoglobulin heavy chain locus containing a functional ADAM6 gene, such that the mouse exhibits wild-type fertility of a non-human animal; and / or (ii) Inclusion of an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity, optionally whereby at least 10% of the reconstructed variable region genes contain non-templated additions. See, e.g., U.S. Patent Nos. 8,697,940, 8,754,287, 9,204,624, 9,334,334, 9,801,362, 9,332,742, and 9,516,868; U.S. Patent Application Publications 20110195454, 20120021409, 20120192300, 20130045492, 20150289489, 20180125043, 20180244804; PCT Patent Application Publications WO2017210586 and WO2011163314; Lee et al. (2014) Nature Biotechnology 32:356. Each of these publications is incorporated herein by reference in its entirety.

[0107] In some embodiments, the present invention provides a method for detecting a cell whose genome, e.g., a germline genome, is: Human V H Gene segment, human D H gene segments, and human J H an endogenous immunoglobulin locus comprising an immunoglobulin heavy chain variable region comprising a gene segment, the immunoglobulin heavy chain variable region being operably linked to a constant region; and / or The present invention also includes a genetically modified non-human animal comprising an endogenous chain locus comprising an immunoglobulin light chain variable region comprising a human V gene segment, and a human J gene segment, wherein the immunoglobulin light chain variable region is operably linked to a constant region.

[0108] In some embodiments, the present invention provides a method for detecting a cell whose genome, e.g., a germline genome, is: Human V H Gene segment, human D H gene segments, and human J H an endogenous immunoglobulin heavy chain locus comprising an immunoglobulin heavy chain variable region comprising a gene segment, the immunoglobulin heavy chain variable region being operably linked to a constant region; and / or and a genetically modified non-human animal comprising an endogenous immunoglobulin light chain locus comprising an immunoglobulin light chain variable region comprising a human V gene segment, and a human J gene segment, wherein the immunoglobulin light chain variable region is operably linked to a constant region.

[0109] In some embodiments, a non-human animal, such as a rodent, e.g., a rat or mouse, contains in its genome one or more endogenous VH chains at endogenous immunoglobulin heavy chain loci in addition to a nucleotide sequence encoding a human or humanized MHC. H , D H , and J. H segment and one or more human V H , D H , and J. H segment substitution, wherein the one or more human V H , D H , and J. H The segments are operably linked to an endogenous immunoglobulin heavy chain gene and, optionally, to an unrearranged or rearranged human V L Segment and human J L The segments may be non-human, e.g., rodent, e.g., mouse or rat, or human immunoglobulin light chain constant (C L) region gene, e.g., at the endogenous non-human light chain locus, such that the non-human animal is tolerant to human or humanized MHC molecules and produces reverse chimeric antibodies in response to an immunogen, e.g., an antigenic pMHC complex.

[0110] In certain embodiments, a genetically modified non-human animal that expresses and tolerates human or humanized MHC molecules further comprises, in its genome, e.g., a germline genome, an immunoglobulin locus (exogenous or endogenous) that contains an immunoglobulin variable region comprising one or more unrearranged human immunoglobulin variable region gene segments, and an immunoglobulin constant region comprising an immunoglobulin constant region gene, and at the locus, the one or more unrearranged human immunoglobulin variable region gene segments are operably linked to the immunoglobulin constant region gene. In some embodiments, a non-human animal that expresses and tolerates human or humanized MHC molecules comprises a plurality of such immunoglobulin loci in its genome, e.g., a germline genome. For example, in some embodiments, the genetically modified non-human animal comprises in its genome, e.g., in its germline genome, nucleotide sequences encoding human or humanized MHC molecules and one or more immunoglobulin loci (including genetically modified rearranged or unrearranged immunoglobulin loci), such that the mouse produces human antibodies, humanized antibodies, partially human antibodies, and / or reverse chimeric antibodies (human variable regions and non-human constant regions).

[0111] Generally, a genetically modified immunoglobulin locus comprises an immunoglobulin variable region (including an immunoglobulin variable region gene segment) operably linked to an immunoglobulin constant region. In some embodiments, a genetically modified immunoglobulin locus comprises one or more human unrearranged immunoglobulin heavy chain variable region gene segments operably linked to a heavy chain constant region gene. In some embodiments, a genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin light chain, such as a κ gene segment, operably linked to a heavy chain constant region gene. See, e.g., U.S. Pat. No. 9,516,868, which is incorporated herein by reference in its entirety. In some embodiments, a genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin heavy chain variable region gene segment operably linked to a κ chain constant region gene. In some embodiments, a genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region κ gene segment operably linked to a κ chain constant region gene. In some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region λ gene segment operably linked to a κ chain constant region gene, hi some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region λ gene segment operably linked to a λ chain constant region gene.

[0112] In certain embodiments, the non-human animal comprises an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region at an endogenous heavy chain locus, wherein the immunoglobulin variable region comprises one or more unrearranged human Ig heavy chain variable region gene segments. In some embodiments, the one or more unrearranged human Ig variable region gene segments comprise at least one human immunoglobulin heavy chain variable (V H ) segments, one or more immunoglobulin heavy chain diversity (D H ) segment (optionally one or more unrearranged human D H segment), and one or more immunoglobulin heavy chain joining (J H ) segments (optionally one or more unrearranged human JH In some embodiments, the unrearranged human Ig variable region gene segments comprise a plurality of unrearranged human V H Segment, one or more unrearranged (human) D H segment, and one or more unrearranged (human) J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least three V segments. H Gene segment, at least 18 V H Gene segment, at least 20 V H Gene segment, at least 30 V H Gene segment, at least 40 V H Gene segment, at least 50 V H Gene segment, at least 60 V H Gene segment, at least 70 V H gene segments, or at least 80 V H In some embodiments, the unrearranged human Ig gene segment comprises a functional human D H In some embodiments, the unrearranged human Ig gene segments comprise all of the functional human J gene segments. H Exemplary variable region, including Ig heavy chain gene segments, are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplementary information, which are incorporated herein by reference in their entirety.

[0113] In some embodiments, the non-human animals provided herein comprise, at an endogenous heavy chain locus, a constrained, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region comprising at least a non-human IgM gene, wherein the constrained, unrearranged human (humanized) heavy chain variable region is one human V H Gene segments, multiple D H Gene segments (e.g., human D H gene segments), and multiple JH Gene segments (e.g., human J H a constrained immunoglobulin heavy chain locus characterized by a plurality of distinct V gene segments, wherein the constrained immunoglobulin heavy chain locus can undergo rearrangement and form multiple distinct rearrangements, wherein each rearrangement is associated with one human V gene segment. H Gene segment, D H One of the segments, and J H In some embodiments, one human V is derived from one of the V segments, and in this case each rearrangement encodes a different heavy chain variable domain (e.g., as described in U.S. Patent Application Publication No. 20130096287, which is incorporated herein by reference in its entirety). H The gene segment is V H 1-2 or V H It is 1-69.

[0114] In certain embodiments, the non-human animal contains an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region at an endogenous light chain locus. In some embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region contains unrearranged human Ig κ variable region gene segments. In some embodiments, the unrearranged human (humanized) immunoglobulin variable region contains a plurality of unrearranged human Vκ segments and one or more unrearranged human J κ segments. κIn some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all of the human Jκ segments. In some embodiments, the immunoglobulin variable region gene segments comprise four functional Vκ segments and all of the human Jκ segments. In some embodiments, the immunoglobulin variable region gene segments comprise 16 functional Vκ segments and all of the human Jκ segments (e.g., all of the functional human Vκ segments and Jκ segments). In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all of the human Jκ segments and all of the human Jκ segments. Exemplary variable regions comprising Igκ gene segments are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 1 11:5147-52 and supplementary information, which are incorporated herein by reference in their entireties.

[0115] In some embodiments, a constrained unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region is one in which the unrearranged human (humanized) light chain variable region comprises no more than two human V L Gene segments, and multiple J L In some embodiments, the V L The gene segment is a Vκ gene segment. LThe gene segments are Vλ gene segments. In some embodiments, the Vκ gene segments are IGKV3-20 and IGKV1-39. In some embodiments, the non-human animal comprises exactly two unrearranged human Vκ gene segments and five unrearranged human Jκ gene segments operably linked to a mouse light chain constant region at an endogenous κ light chain locus of the mouse, optionally wherein the exact two unrearranged human Vκ gene segments are a human Vκ1-39 gene segment and a human Vκ3-20 gene segment, and the five unrearranged human Jκ gene segments are a human Jκ1 gene segment, a human Jκ2 gene segment, a human Jκ3 gene segment, a human Jκ4 gene segment, and a human Jκ5 gene segment, and the unrearranged human kappa light chain gene segments are capable of rearranging and encoding a human variable domain of an antibody, and optionally further, the non-human animal does not comprise endogenous Vκ gene segments capable of rearranging to form an immunoglobulin light chain variable region.

[0116] In certain embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to the endogenous light chain constant region contains an unrearranged human Igλ variable region gene segment. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises multiple human Vλ segments and one or more human Jλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises one or more human Vλ segments, one or more human Jλ segments, and one or more human Cλ constant region sequences. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of the human Vλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of the human Jλ segments. Exemplary variable regions comprising Igλ gene segments are provided, for example, in U.S. Patent Nos. 9,035,128 and 6,998,514, each of which is incorporated herein by reference in its entirety. In some embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region comprises (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) and an endogenous (e.g., rodent) Cλ gene segment, and wherein the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ), and one or more human immunoglobulin λ light chain enhancers (Eλ), optionally including three human Eλ.

[0117] In certain embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region comprises an unrearranged human Igλ variable region gene segment operably linked to an endogenous (e.g., rodent, e.g., rat or mouse) Cκ gene, such that the non-human animal expresses an immunoglobulin light chain comprising a human λ variable domain sequence derived from Vλ and Jλ gene segments fused to an endogenous κ constant domain. See, e.g., U.S. Pat. No. 9,226,484, which is incorporated herein by reference in its entirety.

[0118] In some embodiments, the immunoglobulin variable region comprising an unrearranged human immunoglobulin variable region gene segment also comprises a human immunoglobulin variable region intergenic sequence. In some embodiments, the immunoglobulin variable region comprises a non-human (e.g., rodent, rat, mouse) Ig variable region intergenic sequence. In some embodiments, the intergenic sequence is an intergenic sequence of endogenous species origin.

[0119] In some embodiments, the immunoglobulin variable region is a rearranged heavy chain variable region (universal heavy chain variable region, or common heavy chain coding sequence). In some embodiments, the rearranged Ig heavy chain variable region gene is a human rearranged Ig heavy chain variable region gene. Examples of rearranged Ig heavy chain variable regions are provided in U.S. Patent Publication 20140245468 and U.S. Patent Nos. 9,204,624 and 9,930,871, each of which is incorporated by reference in its entirety. In some embodiments, a bispecific antibody is produced using a non-human organism comprising a universal heavy chain variable region. In some embodiments, the non-human animal comprises a common heavy chain coding sequence at an endogenous immunoglobulin heavy chain locus, such as a rearranged human immunoglobulin heavy chain variable region nucleotide sequence operably linked to an endogenous immunoglobulin constant region gene sequence, e.g., a rearranged human immunoglobulin heavy chain variable region nucleotide sequence operably linked to an endogenous heavy chain constant region gene sequence, wherein the rearranged heavy chain variable region nucleotide sequence is H 3-23 / X1X2 / JH wherein X1 is any amino acid and X2 is any amino acid, wherein the non-human animal encodes a rearranged human immunoglobulin heavy chain variable region nucleotide sequence, e.g., V, linked to an endogenous heavy chain constant region gene sequence. H 3-23 / X1X2 / J H The antibody expresses an immunoglobulin heavy chain variable domain derived from a gene and optionally homologous to a human immunoglobulin light chain variable domain.

[0120] In some embodiments, the immunoglobulin variable region is a rearranged light chain variable region (universal light chain variable region). In some embodiments, the rearranged Ig light chain variable region gene is a human rearranged Ig light chain variable region gene. Examples of rearranged Ig light chain variable regions are provided, for example, in U.S. Pat. Nos. 9,969,814, 10,130,181, and 10,143,186, and U.S. Patent Application Publications 20120021409, 20120192300, 20130045492, 20130185821, 20130302836, and 20150313193, each of which is incorporated by reference in its entirety. In some embodiments, bispecific antibodies are produced using a non-human organism comprising a universal light chain variable region (a "universal light chain" organism). In some embodiments, the common light chain coding sequence comprises one rearranged human immunoglobulin light chain Vκ / Jκ sequence operably linked to an endogenous light chain constant region, wherein the one rearranged human immunoglobulin light chain Vκ / Jκ sequence is either (i) a human Vκ1-39 / Jκ5 sequence comprising a human Vκ1-39 gene segment fused to a human Jκ5 gene segment, or (ii) a human Vκ3-20 / Jκ1 sequence comprising a human Vκ3-20 gene segment fused to a human Jκ1 gene segment.

[0121] In some embodiments, the immunoglobulin variable region is a light and / or heavy chain immunoglobulin variable region that includes a histidine codon insertion and / or substitution designed to introduce pH-dependent binding properties into antibodies produced in such non-human organisms. In some such embodiments, the histidine codon is inserted and / or substituted into the nucleic acid sequence encoding CDR3. Various such light and / or heavy chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, and 9,801,362, and U.S. Patent Application Publication No. 20140013456. Each of these patent documents is incorporated herein by reference in its entirety. In some embodiments, the histidine-engineered reshaped human (humanized) light chain variable region operably linked to the endogenous light chain constant region comprises a reshaped human immunoglobulin light chain variable region gene sequence comprising a human Vκ segment sequence and a human Jκ segment sequence, optionally wherein the Vκ segment sequence is derived from a human Vκ1-39 or a human Vκ3-20 gene segment, and wherein the reshaped human immunoglobulin light chain variable region gene sequence comprises a substitution of at least one non-histidine codon of the Vκ segment sequence expressed at a position selected from the group consisting of 105, 106, 107, 108, 109, 111, and combinations thereof (according to IMGT numbering) with a histidine codon. In some embodiments, the histidine-engineered, unrearranged human (humanized) heavy chain variable region operably linked to the endogenous heavy chain constant region comprises an unrearranged human (humanized) immunoglobulin heavy chain variable gene sequence comprising a substitution of at least one non-histidine codon with a histidine codon, or an insertion of at least one histidine codon, in the complementarity-determining region 3 (CDR3)-encoding sequence. In some embodiments, the unrearranged human (humanized) immunoglobulin heavy chain variable gene sequence comprises an unrearranged human V H , unrecombined human D H , or composite D H , and unrecombined human J H and optionally, in this case, the unrearranged human D H , or composite D HThe gene segment comprises a substitution of at least one non-histidine codon with a histidine codon, or an insertion of at least one histidine codon. In some embodiments, the histidine-modified unrearranged human (humanized) light chain variable region operably linked to an endogenous heavy chain constant region comprises an unrearranged V L , and non-reconstructed J L In some embodiments, the histidine-engineered unrearranged human (humanized) light chain variable region comprises no more than two unrearranged human V gene segments. L (e.g., two or fewer Vκ gene segments), and one or more unrearranged human J L (e.g., Jκ) gene segments, wherein the two or less human V L Each of the gene segments comprises a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon in the CDR3-encoding sequence. In some embodiments, the no more than two unrearranged human Vκ gene segments are human Vκ1-39 and human Vκ3-20 gene segments, each comprising one or more substitutions of one or more non-histidine codons with histidine codons, wherein the human Vκ and Jκ gene segments have the ability to rearrange, and the human Vκ and human Jκ gene segments encode a human light chain variable domain comprising one or more histidines at positions selected from the group consisting of 105, 106, 107, 108, 109, 111 (according to IGMT numbering), and combinations thereof, wherein the one or more histidines are derived from the one or more substitutions.

[0122] In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region gene. In some embodiments, the heavy chain constant region is a human heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is a heavy chain constant region gene of endogenous species origin. In some embodiments, the heavy chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the constant region gene is a mixture of human and non-human sequences. For example, in some embodiments, the constant region gene encodes a human CH1 region and a non-human (e.g., endogenous species origin, mouse, rat) CH2 and / or CH3 region. In some embodiments, the heavy chain constant region gene is a Cμ, Cδ, Cγ (Cγ1, Cγ2, Cγ3, Cγ4), Cα, or Cε constant region gene. In some embodiments, the constant region gene is an endogenous constant region gene. In some embodiments, the constant region gene encodes a mutated CH1 region, thereby causing the non-human animal to express a heavy chain-only antibody (see, e.g., U.S. Pat. No. 8,754,287; U.S. Patent Application Publication No. 2015 / 0289489, each of which is incorporated by reference in its entirety). For example, in some embodiments where the goal is to generate heavy chains for making bispecific antibodies (in universal or dual light chain organisms), the Fc domain of the heavy chain contains modifications that promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Pat. Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Application Publication No. 2013 / 0195849, each of which is incorporated by reference in its entirety.

[0123] In some embodiments, the immunoglobulin constant region comprises a light chain constant region gene. In some embodiments, the light chain constant region gene is a kappa constant region gene. In some embodiments, the light chain constant region gene is a lambda constant region gene. In some embodiments, the light chain constant region gene is a light chain constant region gene of endogenous species origin. In some embodiments, the light chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the light chain constant region gene is a mixture of human and non-human sequences.

[0124] In some embodiments, the immunoglobulin variable region comprising a human variable region gene segment and the immunoglobulin constant region gene to which the variable region gene segment is operably linked are located at an endogenous immunoglobulin locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous heavy chain locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous κ locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous λ locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably linked is an endogenous constant region gene.

[0125] In some embodiments, one or more of the endogenous immunoglobulin loci in the genome of a non-human animal provided herein, or portions of the one or more endogenous loci (e.g., variable and / or constant regions), are inactivated. Endogenous immunoglobulin variable region gene loci and portions thereof can be inactivated using any method known in the art, including, but not limited to, deleting the locus or portions thereof from the genome of the organism, replacing the locus or portions thereof with a different nucleic acid sequence, inverting the portion of the locus, and / or moving the portion of the locus to another location in the genome of the non-human organism. In some embodiments, the inactivation of the locus is only partial. In some embodiments, the variable regions of the locus are inactivated, but the constant regions remain functional (e.g., because they are operably linked to non-endogenous variable region gene segments).

[0126] In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin heavy chain locus. In some embodiments, the endogenous immunoglobulin heavy chain locus, or a portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of the endogenous variable region of the endogenous heavy chain locus. In some embodiments, at least a portion of the variable region of the endogenous heavy chain locus that is deleted, replaced, moved, and / or inverted comprises a variable region J segment. In some embodiments, the endogenous immunoglobulin heavy chain locus, or a portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of the endogenous constant region of the endogenous heavy chain locus. In some embodiments, at least a portion of the constant region of the endogenous heavy chain locus that is deleted, replaced, moved, and / or inverted comprises the Oμ gene of the endogenous constant region.

[0127] In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous immunoglobulin κ chain locus, or portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of an endogenous variable region of the endogenous κ chain locus. In some embodiments, the deleted, replaced, moved, and / or inverted at least a portion of the variable region of the endogenous κ chain locus comprises a variable region J segment. In some embodiments, the endogenous immunoglobulin κ chain locus, or portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of the endogenous constant region of the endogenous κ chain locus. In some embodiments, the deleted, replaced, moved, and / or inverted at least a portion of the constant region of the endogenous κ chain locus comprises an endogenous constant region CK gene.

[0128] In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin λ chain locus. In some embodiments, the endogenous immunoglobulin λ chain locus, or a portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of an endogenous variable region of the endogenous λ chain locus. In some embodiments, at least a portion of at least one VJC gene cluster in the endogenous λ chain locus is deleted, replaced, moved, and / or inverted. In some embodiments, the endogenous immunoglobulin λ chain locus, or a portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of an endogenous constant region of the endogenous λ chain locus. In some embodiments, at least a portion of the constant region of the endogenous λ chain locus that is deleted, replaced, moved, and / or inverted comprises an endogenous constant region C gene.

[0129] In various embodiments, modification of the immunoglobulin locus does not affect the fertility of the non-human animal. In some embodiments, the heavy chain locus comprises a functional, e.g., endogenous ADAM6a gene, ADAM6b gene, or both, and the genetic modification does not affect the expression and / or function of the endogenous ADAM6a gene, ADAM6b gene, or both. In some embodiments, the genome of the genetically modified non-human animal further comprises an ectopically positioned functional, e.g., endogenous ADAM6a gene, ADAM6b gene, or both. Examples of non-human animals that express exogenous ADAM6a and / or ADAM6b are described in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety.

[0130] In some embodiments, the genetically modified non-human animal further contains and expresses an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity. An example of a non-human animal expressing exogenous TdT is described in PCT Patent Application Publication WO2017210586, which is incorporated herein by reference in its entirety.

[0131] In some embodiments, the genetically modified non-human animal contains and expresses a nucleotide sequence encoding a human or humanized MHC molecule and expresses antibodies having a human variable domain (e.g., a human variable domain derived from (e.g., encoded by) a rearranged human variable region gene segment), but lacks antibodies that specifically bind to empty human or humanized MHC. In some embodiments, the human or humanized variable domain is a human or humanized heavy chain variable domain. In some embodiments, the antibody is a heavy chain-only antibody. In some embodiments, the human or humanized variable domain is a human or humanized light chain variable domain. In some embodiments, the antibody produced by the non-human animal has both a human or humanized heavy chain variable domain and a human or humanized light chain variable domain. In some embodiments, the antibody has a human or humanized heavy chain constant domain. In some embodiments, the antibody has a human or humanized light chain constant domain. In some embodiments, the heavy and / or light chain constant domains are constant domains of non-human origin. For example, in some embodiments, the heavy chain constant domain is a heavy chain constant domain of endogenous species origin. In some embodiments, the heavy chain constant domain is of mouse or rat origin. In some embodiments, the light chain constant domain is of endogenous species origin. In some embodiments, the light chain constant domain is of mouse or rat origin.

[0132] Non-human animals, tissues and cells In some embodiments, the genetically modified non-human animals of the present invention may be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., dairy cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to generate non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell) and using nuclear transfer to introduce the modified genome into a suitable cell, such as an oocyte, and gestation of the modified cell (e.g., the modified oocyte) into a non-human animal under conditions suitable for the formation of an embryo.

[0133] In one embodiment, the non-human animal is a mammal. In one embodiment, the non-human animal is a small mammal, for example, of the superfamily Jerboidea or Murine superfamily. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the superfamily Murine. In one embodiment, the genetically modified animal is from a family selected from the family Odontoidea (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, and voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, and maned mice), Tetragnathidae (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), Dormiceidae (e.g., spiny dormice), and Moleratidae (e.g., mole rats, bamboo rats, and plateau mole rats). In a particular embodiment, the genetically modified rodent is selected from a pure breed mouse or rat (Muridae), a gerbil, a spiny mouse, and a maned mouse. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a rat. In one embodiment, the non-human animal is a mouse.

[0134] In a particular embodiment, the non-human animal is a rodent that is a mouse of the C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of the following strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836; see also Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In one embodiment, the genetically modified mouse is a mix of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another particular embodiment, the mouse is a mix of the aforementioned 129 strains, or a mix of the aforementioned BL / 6 strains. In a particular embodiment, the 129 strain in the mix is ​​the 129S6 (129 / SvEvTac) strain. In another embodiment, the mouse is a BALB strain, e.g., a BALB / c strain. In yet another embodiment, the mouse is a mix of the BALB strain and another of the aforementioned strains. The non-human animals provided herein may be mice derived from any combination of the aforementioned strains.

[0135] Germline inheritance of targeted modified alleles in rat ES cells has been established over the past decade. See, for example, U.S. Patent Application Publications 20140235933 and 20140310828; Tong et al. (2010) Nature 467:211-215, Tong et al. (2011) Nat Protoc. 6(6):doi:10.1038 / nprot.2011.338, each of which is incorporated herein by reference in its entirety. Thus, in one embodiment, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0136] Thus, in one embodiment of the present invention, a genetically modified mouse is provided, wherein the mouse has in its genome, e.g., its germline genome: (a) a nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof, and (b) (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci, optionally wherein at least one of the (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci is an unrearranged locus; the genetically modified non-human animal expresses a human or humanized MHC molecule or at least a peptide-binding portion thereof; The genetically modified non-human animal expresses immunoglobulins comprising a human or humanized heavy chain variable domain and / or a human or humanized light chain variable domain, and The non-human animal is tolerant to the human or humanized MHC molecule, or at least a peptide-binding portion thereof, thereby generating a specific B cell response when immunized with an antigenic peptide-MHC (pMHC) complex containing a peptide that is heterologous to the non-human animal complexed with (i)(ii), the human HLA molecule or portion thereof from which the human or humanized MHC molecule is derived.

[0137] In some embodiments, the mouse comprises a first nucleotide sequence encoding a first fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIα), a second nucleotide sequence encoding a second fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIβ), and / or a third nucleotide sequence encoding a third fully human or chimeric human / mouse MHC polypeptide (e.g., MHC I), and optionally a β2 microglobulin locus encoding human or humanized β2 microglobulin; and (a) at the endogenous heavy chain locus; (i) an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region; (ii) a constrained, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (iii) a consensus heavy chain coding sequence; (iv) a histidine-engineered, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (v) a heavy chain-only immunoglobulin coding sequence, or (vi) a non-rearranged human (humanized) hybrid heavy chain sequence encoding a hybrid immunoglobulin chain, and / or (b) at the endogenous light chain locus; (i) an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region; (ii) a consensus light chain coding sequence; (iii) a constrained, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; (iv) a histidine-engineered, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; or (v) a histidine-engineered reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region; Optionally, the mouse further comprises: (i) a human (humanized) immunoglobulin heavy chain locus containing a functional ADAM6 gene, such that the mouse exhibits wild-type fertility; and / or (ii) Inclusion of an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity, optionally whereby at least 10% of the reconstructed variable region genes contain non-templated additions.

[0138] In addition to genetically modified animals (e.g., rodents such as mice or rats), tissues or cells are also provided, where the tissues or cells are derived from the non-human animals of some embodiments, e.g., the tissues or cells are derived from (a) a first nucleotide sequence encoding a first fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIα), a second nucleotide sequence encoding a second fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIβ), and / or a third fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIβ). I), and optionally a β2 microglobulin locus encoding human or humanized β2 microglobulin, and (b) when the cell is not a B cell, a (non-) rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-) rearranged human or humanized immunoglobulin light chain locus, optionally in which case at least one of the (non-) rearranged human or humanized immunoglobulin heavy chain locus and / or the (non-) rearranged human or humanized immunoglobulin light chain locus is unrearranged.

[0139] In some embodiments, the tissue or cells express a human or humanized MHC molecule and a human or humanized antigen binding protein and / or a nucleic acid encoding one or more variable domains of the human or humanized antigen binding protein, wherein the antigen binding protein specifically binds to a pMHC complex that is antigenic to the non-human animal from which the tissue or cell is derived, e.g., the pMHC complex comprises an antigenic peptide complexed with human MHC (or a portion thereof) to which the non-human animal is generally tolerant. In some embodiments, the cell is a B cell. In some embodiments, the cell is a hybridoma or quadroma derived from the fusion of a B cell isolated from the non-human animal of some embodiments and a myeloma cell. In some embodiments, the tissue is an antigen binding protein, or a nucleic acid sequence encoding the antigen binding protein, wherein the antigen binding protein specifically binds to a pMHC complex that is antigenic to the non-human animal from which the antigen binding protein is derived, e.g., the pMHC complex comprises an antigenic peptide complexed with human MHC (or a portion thereof) to which the non-human animal is generally tolerant.

[0140] In addition to genetically engineered non-human animals, non-human embryos (e.g., rodent, e.g., mouse or rat embryos) are also provided, wherein the non-human embryos comprise donor ES cells that can be used to generate the non-human animals (e.g., rodents, e.g., mice or rats) of exemplary embodiments. In one embodiment, the non-human animal embryo comprises an ES donor cell that comprises a human or humanized MHC I (e.g., MHC Iα) nucleotide sequence, a human or humanized MHC II (e.g., MHC IIα and / or MHC IIβ) nucleotide sequence, a (non-)rearranged human or humanized immunoglobulin locus (e.g., a heavy and / or light chain variable locus), and / or a human or humanized β2 microglobulin gene sequence, and a host embryonic cell.

[0141] In some embodiments, cells or genomes of non-human animals (e.g., rodents, e.g., rats or mice) can be used to generate non-human animals, such as pluripotent cells, embryonic stem (ES) cells, germ cells, etc., in which case the cells or genomes are (a) a nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof, and (b) (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci, optionally wherein at least one of the (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci is an unrearranged locus; the resulting genetically modified non-human animal expresses a human or humanized MHC molecule or at least a peptide-binding portion thereof; The resulting genetically modified non-human animal expresses immunoglobulins comprising a human or humanized heavy chain variable domain and / or a human or humanized light chain variable domain, and the resulting human animal is tolerant to the human or humanized MHC molecule, or at least a peptide-binding portion thereof, thereby generating a specific B cell response when immunized with an antigenic peptide-MHC (pMHC) complex containing (i) a peptide heterologous to the non-human animal complexed with (ii), the human HLA molecule or portion thereof from which the human or humanized MHC molecule was derived.

[0142] In some exemplary embodiments, the cell or genome comprises a first nucleotide sequence encoding a first fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIα), a second nucleotide sequence encoding a second fully human or chimeric human / mouse MHC polypeptide (e.g., MHC IIβ), and / or a third nucleotide sequence encoding a third fully human or chimeric human / mouse MHC polypeptide (e.g., MHC I), and optionally a β2 microglobulin locus encoding human or humanized β2 microglobulin; and (a) at the endogenous heavy chain locus; (i) an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region; (ii) a constrained, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (iii) a consensus heavy chain coding sequence; (iv) a histidine-engineered, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (v) a heavy chain-only immunoglobulin coding sequence, or (vi) a non-rearranged human (humanized) hybrid heavy chain sequence encoding a hybrid immunoglobulin chain, and / or (b) at the endogenous light chain locus; (i) an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region; (ii) a consensus light chain coding sequence; (iii) a constrained, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; (iv) a histidine-engineered, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; or (v) a histidine-engineered reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region; Optionally, the cell or genome further comprises: (i) a human (humanized) immunoglobulin heavy chain locus containing a functional ADAM6 gene, such that the mouse exhibits wild-type fertility; and / or (ii) Inclusion of an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity, whereby, optionally, at least 10% of the rearranged variable region genes contain non-templated additions.

[0143] Although the following examples describe genetically engineered animals whose genomes contain a nucleic acid sequence encoding a mouse H-2K protein substituted with a nucleic acid sequence encoding a chimeric human / mouse HLA-A2 / H-2K, those skilled in the art will understand that similar strategies can be used to introduce other human (humanized) MHC I and MHC II genes (other HLA-A, -B, and -C genes, as well as other HLA-DR, -DP, and -DQ genes, e.g., at endogenous loci or at ectopic loci, e.g., the ROSA26 locus). Also provided are animals that contain multiple chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) MHC I and MHC II genes at endogenous MHC loci. Examples of such chimeric MHC I and MHC II proteins are described in U.S. Patent Application Publication Nos. 20130111617, 20130185819, 20130185820, and 20140245467, and U.S. Patent No. 8,847,005, each of which is incorporated by reference in its entirety.

[0144] Also provided is a non-human cell comprising a chromosome, or a fragment thereof, of a non-human animal embodiment of the invention. In one embodiment, the non-human cell comprises a nucleus of a non-human animal embodiment of the invention. In one embodiment, the non-human cell comprises the chromosome, or a fragment thereof, as a result of nuclear transfer.

[0145] Creation of genetically modified non-human animals Also provided are methods for producing genetically engineered non-human animals (e.g., genetically engineered rodents, e.g., mice or rats). Generally, the methods involve modifying the genome, e.g., germline genome, of a non-human cell to include (a) a first nucleic acid sequence encoding a first chimeric human / non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human / non-human MHC polypeptide, a third nucleic acid sequence encoding a third chimeric human / non-human MHC polypeptide, and / or a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide, and (b) immunoglobulin heavy chain and immunoglobulin light chain loci encoding a human or humanized antibody. In some embodiments, the methods involve targeting and replacing sequences encoding an endogenous MHC polypeptide extracellular domain, all or a portion of β2 microglobulin, and an immunoglobulin variable region with a human MHC extracellular domain, all or a portion of human β2 microglobulin, and a human immunoglobulin variable region, respectively.

[0146] In some embodiments, the modification may involve mating with animals of the same species, e.g., breeding. In other embodiments, the modification involves sequential homologous recombination in one or more ES cells. In some embodiments, the ES cells are derived from a non-human animal that has been genetically modified to contain one or more but not all of the desired genetic modifications, and homologous recombination in the ES cells completes the genetic modification. In other embodiments, the modification may involve a combination of mating, e.g., mating an animal with another animal(s) of the same species, and homologous recombination in ES cells, in which case some or all of the non-human animals may be generated from ES cells that have been genetically modified via a single homologous recombination event or genetically modified via sequential homologous recombination events, and in which case some ES cells may be isolated from a non-human animal containing one or more of the genetic modifications disclosed herein. In some embodiments, the modification involves sequential homologous recombination in a single ES cell.

[0147] In some embodiments, the method utilizes a targeting construct generated using VELOCIGENE® technology, introduces the construct into ES cells, and introduces targeted ES cell clones into mouse embryos using VELOCIMOUSE® technology (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) Nature Biotech 25:91-99, each of which is incorporated by reference in its entirety). The targeting construct may include 5' and / or 3' homology arms that target the endogenous sequence to be replaced, an insertion sequence (that replaces the endogenous sequence), and one or more selection cassettes. A selection cassette is a nucleotide sequence inserted into the targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. Many suitable selection cassettes are known in the art. Typically, the selection cassette allows for positive selection in the presence of a specific antibiotic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). Additionally, the selection cassette may be flanked by recombination sites, which allow for deletion of the selection cassette upon treatment with a recombinase enzyme. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, respectively, although others are known in the art. The selection cassette may be located anywhere in the construct outside of the coding region. In one embodiment, the selection cassette is located at the 5' end of the human DNA fragment. In another embodiment, the selection cassette is located at the 3' end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment. In another embodiment, the selection cassette is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located at the junction of the human and mouse DNA fragments. F0 generation mice derived essentially entirely from donor gene-targeted ES cells are readily available for phenotypic analysis.Independently, VELOCIMICE® (F0 mice derived entirely from donor ES cells) carrying human or humanized MHC class I genes, human or humanized β2 microglobulin genes, and / or human or humanized MHC class II genes, and humanized immunoglobulin loci are identified by genotyping using a modified version of an allelic assay that detects the presence of these unique gene sequences (Valenzuela et al. (2003) High-throughput engineering of the mouse genome). coupled with high-resolution expression (analysis, Nature Biotech. 21(6):652-659, which is incorporated herein by reference in its entirety.) Heterozygous mice produced by this method can be bred to homozygosity.

[0148] In some embodiments, human or humanized MHC I, β2 microglobulin, MHC A non-human animal containing MHC I, β2-microglobulin, MHC II molecules may be bred with a second non-human animal of the same species, where the second non-human animal contains unrearranged human or humanized immunoglobulin heavy chain loci, and / or unrearranged human or humanized immunoglobulin light chain loci. For example, a mouse containing human or humanized MHC I, β2-microglobulin, MHC II molecules may be bred with a second mouse containing: (a) at the endogenous heavy chain locus; (i) an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region; (ii) a constrained, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (iii) a consensus heavy chain coding sequence; (iv) a histidine-engineered, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (v) a heavy chain-only immunoglobulin coding sequence, or (vi) a non-rearranged human (humanized) hybrid heavy chain sequence encoding a hybrid immunoglobulin chain; and / or (b) at the endogenous light chain locus; (i) an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region; (ii) a consensus light chain coding sequence; (iii) a constrained, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; (iv) a histidine-engineered, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; or (v) a histidine-engineered reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region. Optionally, the second mouse further comprises: (i) a human (humanized) immunoglobulin heavy chain locus containing a functional ADAM6 gene, such that the mouse exhibits wild-type fertility; and / or (ii) Inclusion of an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity, whereby, optionally, at least 10% of the rearranged variable region genes contain non-templated additions.

[0149] In another embodiment, the construct inserting human or humanized MHC I, MHC II, and / or β2 microglobulin is involved in homologous recombination (at the endogenous MHC I, MHC II, and / or β2 microglobulin locus or at an ectopic locus) in non-human animal ES cells genetically modified to contain unrearranged human or humanized immunoglobulin heavy chain loci and / or unrearranged human or humanized immunoglobulin light chain loci. Alternatively, the insertion construct is involved in homologous recombination in non-human animal ES cells genetically modified to contain unrearranged human or humanized immunoglobulin heavy chain loci and / or unrearranged human or humanized immunoglobulin light chain loci and a nucleotide sequence encoding human or humanized MHC I, MHC II, and / or β2 microglobulin. In one embodiment, constructs for targeting and replacing endogenous MHC I, MHC II and / or β2 microglobulin sequences with nucleic acid sequences encoding chimeric human / mouse MHC I, MHC II and / or β2 microglobulin, or constructs for insertion of single chain MHC I / β2 microglobulin and / or single chain MHC II proteins, involve homologous recombination in ES cells, which may further comprise: (a) at the endogenous heavy chain locus; (i) an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region; (ii) a constrained, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (iii) a consensus heavy chain coding sequence; (iv) a histidine-engineered, unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region; (v) a heavy chain-only immunoglobulin coding sequence, or (vi) a non-rearranged human (humanized) hybrid heavy chain sequence encoding a hybrid immunoglobulin chain; and / or (b) at the endogenous light chain locus; (i) an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region; (ii) a consensus light chain coding sequence; (iii) a constrained, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; (iv) a histidine-engineered, unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region; or (v) a histidine-engineered reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region. Optionally, the ES cells are further (i) a human (humanized) immunoglobulin heavy chain locus containing a functional ADAM6 gene, and / or (ii) containing an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity;

[0150] In various embodiments of the present invention, sequences encoding chimeric human / non-human MHC I and MHC II polypeptides are located at endogenous non-human MHC loci (e.g., mouse H-2K and / or H-2E loci). In one embodiment, this results in a configuration such as the replacement of an endogenous MHC gene, or a portion thereof, with a nucleic acid sequence encoding a human or humanized MHC I polypeptide. To maximize humanization of both MHC I and MHC II in a single animal, the nucleic acid sequences encoding MHC I, MHC IIα, and MHC IIβ polypeptides are located adjacent to each other on the chromosome, so that the MHC I and MHC II loci are targeted consecutively, if desired. Accordingly, methods are also provided herein for producing genetically modified non-human animals comprising nucleic acid sequences encoding chimeric human / non-human MHC I, MHC IIα, and MHC IIβ polypeptides.

[0151] Thus, in some embodiments, a nucleotide construct for generating a genetically modified animal comprising a chimeric human / non-human MHC is provided. In one embodiment, the nucleic acid construct comprises 5' and 3' non-human homologous arms, a human DNA fragment comprising a human MHC gene sequence (e.g., a human HLA-A2 gene sequence or a human HLA-DR gene sequence), and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment comprising both introns and exons of a human MHC gene (e.g., a human HLA-A2 gene or an HLA-DR2 gene). In one embodiment, the non-human homologous arms are homologous to a non-human MHC locus (e.g., an MHC I locus or an MHC II locus).

[0152] In one embodiment, the 5' and 3' non-human homology arms comprise genomic sequences at the 5' and 3' positions, respectively, of an endogenous non-human (e.g., mouse) MHC class I or class II gene locus (e.g., 5' of the first leader sequence and 3' of the α3 exon of the mouse MHC I gene, or upstream of the mouse H-2Ab1 gene and downstream of the mouse H-2Ea gene). In one embodiment, the endogenous MHC class I locus is selected from mouse H-2K, H-2D, and H-2L. In a particular embodiment, the endogenous MHC class I locus is mouse H-2K. In one embodiment, the endogenous MHC II locus is selected from mouse H-2E and H-2A. In one embodiment, the engineered MHC II construct replaces both the mouse H-2E gene and the mouse H-2A gene. In one embodiment, the mouse does not express a functional endogenous MHC polypeptide from its H-2D locus. In some embodiments, the mouse is engineered to lack all or part of the endogenous H-2D locus. In other embodiments, the mouse does not express functional endogenous MHC I and MHC II polypeptides on the cell surface. In one embodiment, the only MHC I and MHC II expressed on the cell surface by the mouse are chimeric human / mouse MHC I and MHC II.

[0153] The present disclosure further provides a method for producing a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or rat) whose genome comprises a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In one embodiment, the method results in a genetically engineered rodent, e.g., a mouse, whose genome comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide at the endogenous β2 microglobulin locus. In some examples, the mouse does not express functional mouse β2 microglobulin from the endogenous mouse β2 microglobulin locus.

[0154] Nucleotide constructs for use in generating genetically engineered non-human animals are also provided. In some embodiments, the nucleotide construct may include 5' and 3' non-human homology arms, a human DNA fragment containing a human β2 microglobulin sequence, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment containing both introns and exons of the human β2 microglobulin gene. In one embodiment, the non-human homology arms are homologous to the non-human β2 microglobulin locus. The genomic fragment may include exons 2, 3, and 4 of the human β2 microglobulin gene. In one example, the genomic fragment includes, from 5' to 3', all of the human β2 microglobulin sequence: exon 2, intron, exon 3, intron, and exon 4. The selection cassette may be located anywhere outside the β2 microglobulin coding region in the construct, for example, 3' to exon 4 of human β2 microglobulin. The 5' and 3' non-human homologous arms may comprise genomic sequences 5' and 3', respectively, of the endogenous non-human β2 microglobulin gene. In another embodiment, the 5' and 3' non-human homologous arms comprise genomic sequences 5' to exon 2 and 3' to exon 4, respectively, of the endogenous non-human gene.

[0155] Another embodiment of the present invention relates to methods for modifying the β2 microglobulin locus of a non-human animal (e.g., a rodent, such as a mouse or rat) to express a human or humanized β2 microglobulin polypeptide. One method for modifying the β2 microglobulin locus of a non-human animal, e.g., a mouse, to express a human or humanized β2 microglobulin polypeptide comprises replacing a nucleotide sequence encoding mouse β2 microglobulin with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide at the endogenous β2 microglobulin locus. In one embodiment of such a method, the non-human animal, e.g., a mouse, does not express functional β2 microglobulin polypeptide from the endogenous non-human β2 microglobulin locus, e.g., a mouse. In some particular embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises the nucleotide sequence set forth in exons 2 through 4 of the human β2 microglobulin gene. In other embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises the nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.

[0156] The present disclosure further provides a method for producing a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or rat) whose genome comprises a sequence encoding a single-chain β2 microglobulin / MHC complex comprising human (humanized) β2 microglobulin and a human (humanized) MHC class Iα polypeptide, a human (humanized) MHC class Iα polypeptide, and / or a human (humanized) β2 microglobulin at an ectopic locus, e.g., the ROSA26 locus. In one embodiment, the method results in a genetically engineered rodent, e.g., a rat or mouse, whose genome comprises a nucleotide sequence encoding a single-chain β2 microglobulin / MHC complex comprising human (humanized) β2 microglobulin and a human (humanized) MHC complex, a human (humanized) MHC class Iα polypeptide, and / or a human (humanized) β2 microglobulin at an endogenous locus that is not an endogenous MHC I locus or β2 microglobulin locus, e.g., the endogenous ROSA26 locus. Methods for targeting the ROSA locus are known in the art. See, for example, Stefano Casola, "Mouse Models for miRNA expression: the ROSA26 Locus," in Methods in Molecular Biology vol. 667: 145-163 (S. Monticelli ed. 2010), which is incorporated herein by reference in its entirety.

[0157] Nucleotide constructs for use in generating genetically engineered non-human animals are also provided. In some embodiments, the nucleotide construct may comprise 5' and 3' non-human homology arms, a nucleotide sequence encoding a single-chain β2 microglobulin / MHC complex comprising a human (humanized) β2 microglobulin and a human (humanized) MHC class Iα polypeptide (e.g., a single-chain β2 microglobulin / HLA-A2 complex set forth in SEQ ID NO: 23), and a selection cassette flanked by recombination sites. In some embodiments, the nucleotide construct may comprise 5' and 3' non-human homology arms, a nucleotide sequence encoding a human (humanized) MHC class Iα polypeptide, and a selection cassette flanked by recombination sites. In some embodiments, the nucleotide construct may comprise 5' and 3' non-human homology arms, a nucleotide sequence encoding a human (humanized) β2 microglobulin, and a selection cassette flanked by recombination sites. The 5' and 3' non-human homology arms may comprise genomic sequences adjacent to an endogenous ROSA26 intron.

[0158] In some embodiments, the genetically modified non-human animal (e.g., mouse) comprises a human or humanized MHC I, a human or humanized β2 microglobulin; a human or humanized MHC MHC II (e.g., MHC IIα and / or MHC IIβ); and one or two copies of human or humanized immunoglobulin heavy and light chains. Thus, in some embodiments, the non-human animal may be heterozygous or homozygous for any or all of these genes. Because a non-limiting purpose of modifying a non-human animal to contain human or human MHC I, human or humanized β2-microglobulin; and human or humanized MHC II (e.g., MHC IIα and / or MHC IIβ) is to tolerize the non-human animal to the human or humanized MHC molecule, homozygosity for these genes is not required. Thus, in some embodiments, the non-human animal may be heterozygous for the nucleotide sequence encoding the human or humanized MHC molecule. In contrast, because a non-limiting purpose of modifying the immunoglobulin locus of a non-human animal is to generate human or humanized antibodies against an antigenic pMHC complex of interest, in some embodiments, the non-human animal may be homozygous for the modified immunoglobulin locus.

[0159] Once gene targeting is complete, the ES cells or genetically modified non-human animals are screened to confirm successful integration of the exogenous nucleotide sequence or expression of the exogenous polypeptide of interest. Numerous techniques are known to those skilled in the art, including (but not limited to) Southern blotting, long-range PCR, quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescent in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, and the like. In one example, non-human animals (e.g., mice) carrying the genetic modification of interest can be identified by screening for loss of mouse alleles and / or gain of human alleles using a modified version of the allele assay described in Valenzuela et al. (2003), supra. Other assays for identifying specific nucleotide or amino acid sequences in genetically modified animals are known to those skilled in the art.

[0160] Antigenic peptide-MHC complex of interest Mice tolerized to the MHC proteins described herein are immunized with antigenic peptide-MHC complexes to produce pMHC-specific antigen-binding proteins. In some embodiments, MHC useful as part of an antigenic peptide-MHC (pMHC) complex includes native, full-length MHC, as well as individual chains of MHC (e.g., MHC class I α (heavy) chain, β2-microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC (e.g., α1-α3 subunits of the MHC class I α chain, α1-α2 subunits of the MHC class II α chain, and β1-β2 subunits of the MHC class II β chain), and fragments, variants, and various derivatives (including fusion proteins) thereof, where the fragments, variants, and derivatives retain the ability to present antigenic determinants for recognition by an antigen-specific TCR.

[0161] Native MHC is encoded by a gene cluster on human chromosome 6. MHC includes, but is not limited to, HLA specificities such as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9), and DP (e.g., DP1-DP6). HLA specificities include A1, A2, A3, A11, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR11.

[0162] Native MHC class I molecules bind peptides derived from proteolytically degraded proteins, particularly endogenously synthesized proteins, by cells, and the resulting small peptides are transported into the endoplasmic reticulum, where they associate with nascent MHC class I molecules, then pass through the Golgi apparatus and are presented on the cell surface for recognition by cytotoxic T lymphocytes.

[0163] Native MHC class I molecules consist of an α (heavy) chain associated with β2 microglobulin. The heavy chain consists of subunits α1-α3. The β2 microglobulin protein and the α3 subunit of the heavy chain are associated. In certain embodiments, the β2 microglobulin and α3 subunits are covalently associated. In certain embodiments, the β2 microglobulin and α3 subunits are non-covalently associated. The α1 and α2 subunits of the heavy chain fold to form a groove for peptides, e.g., antigenic determinants, to be presented and recognized by the TCR.

[0164] Class I molecules generally associate with, e.g., bind to, peptides that are, e.g., about 8-9 amino acids (e.g., 7-11 amino acids) in length. Every human has 3-6 different Class I molecules, each capable of binding many different types of peptides.

[0165] In some embodiments, the pMHC complex comprises (i) a Class I MHC polypeptide, or a fragment, variant, or derivative thereof, and optionally (ii) a β2 microglobulin polypeptide, or a fragment, variant, or derivative thereof. In one particular embodiment, the Class I MHC polypeptide is associated, e.g., linked, to the β2 microglobulin polypeptide by a peptide linker.

[0166] In one particular embodiment, the MHC class I polypeptide is a human MHC class I polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another particular embodiment, the MHC class I polypeptide is a mouse MHC class I polypeptide selected from the group consisting of H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0167] In some embodiments, the MHC class I α heavy chain of the antigenic pMHC complex is fully human. In some embodiments, the MHC class I α heavy chain of the antigenic pMHC complex is humanized. Humanized MHC class I α heavy chains are described, for example, in U.S. Patent Application Publications 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467. In some embodiments, the MHC class I α heavy chain comprises a human extracellular domain (human α1 domain, α2 domain, and / or α3 domain) and a cytoplasmic domain of another species. In some embodiments, the class I α heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence may be the HLA-A*0201 sequence. In various embodiments, the peptide-MHC may comprise all domains of an MHC class I heavy chain.

[0168] In some embodiments, the antigenic pMHC complex comprises β2 microglobulin. In some embodiments, the β2 microglobulin is fully human. In some embodiments, the β2 microglobulin is humanized. Humanized β2 microglobulin polypeptides are described, for example, in U.S. Patent Application Publications 2013 / 0111617 and 2013 / 0185819, each of which is incorporated by reference in its entirety.

[0169] In some embodiments, the MHC class I molecule of the antigenic pMHC complex comprises a mutation in a human (humanized) β2 microglobulin (β2m or B2M) polypeptide and in a human (humanized) MHC class I α heavy chain, such that a disulfide bond can form between the human (humanized) B2M and the human (humanized) MHC class I α heavy chain. In some embodiments, the disulfide bond links one of the following pairs of residues: human (humanized) B2M residue 12, human (humanized) MHC class I α heavy chain residue 236; human (humanized) B2M residue 12, human (humanized) MHC class I α heavy chain residue 237; human (humanized) B2M residue 8, human (humanized) MHC class I α heavy chain residue 234; human (humanized) B2M residue 10, human (humanized) MHC class I α heavy chain residue 235; human (humanized) B2M residue 24, human (humanized) MHC class I α heavy chain residue 236; human (humanized) B2M residue 28, human (humanized) MHC class I α heavy chain residue 232; human (humanized) B2M residue 98, human (humanized) MHC class I α-heavy chain residue 192; human (humanized) B2M residue 99; human (humanized) MHC class I α-heavy chain residue 234; human (humanized) B2M residue 3; human (humanized) MHC class I α-heavy chain residue 120; human (humanized) B2M residue 31; human (humanized) MHC class I α-heavy chain residue 96; human (humanized) B2M residue 53; human (humanized) MHC class I α-heavy chain residue 35; human (humanized) B2M residue 60; human (humanized) MHC class I α-heavy chain residue 96; human (humanized) B2M residue 60; human (humanized) MHC class I α-heavy chain residue 122; human (humanized) B2M residue 63; human (humanized) MHC class I α-heavy chain residue 27; human (humanized) B2M residue Arg3; human (humanized) MHC class I α-heavy chain residue Gly 120; human (humanized) B2M residue His31, human (humanized) MHC class I α heavy chain residue Gln96; human (humanized) B2M residue Asp53, human (humanized) MHC class I α heavy chain residue Arg35; human (humanized) B2M residue Trp60, human (humanized) MHC class I α heavy chain residue Gln96; human (humanized) B2M residue Trp60, human (humanized) MHC class I α heavy chain residue Asp 122; human (humanized) B2M residue Tyr63, human (humanized) MHC class I α heavy chain residue Tyr27;Human (humanized) B2M residue Lys6, human (humanized) MHC class I α heavy chain residue Glu232; human (humanized) B2M residue Gln8, human (humanized) MHC class I α heavy chain residue Arg234; human (humanized) B2M residue Tyr10, human (humanized) MHC class I α heavy chain residue Pro235; human (humanized) B2M residue Ser11, human (humanized) MHC class I α heavy chain residue Gln242; human (humanized) B2M residue Asn24, human (humanized) MHC class I α heavy chain residue Ala236; human (humanized) B2M residue Ser28, human (humanized) MHC class I α heavy chain residue Glu232; human (humanized) B2M residue Asp98, human (humanized) MHC class I α heavy chain residue His 192; human (humanized) B2M residue Met99, human (humanized) MHC class I α heavy chain residue Arg234, and / or human (humanized) B2M residue Arg 12, human (humanized) MHC class I α heavy chain residue Gly237. See, e.g., International Patent Application Publication No. WO / 2015195531, which is incorporated herein by reference in its entirety.

[0170] In some embodiments, the amino acid sequence of the antigenic determinant may be the amino acid sequence of a peptide that can be associated with, e.g., presented by, an MHC class I molecule. In certain embodiments, the sequence may comprise 6 to 20 contiguous amino acids. In certain embodiments, the peptide sequence may be the peptide sequence of a protein fragment, where the protein is derived from a portion of a cellular protein, e.g., a protein associated with an autoimmune disorder, where the peptide may be bound to an MHC class I heavy chain.

[0171] In some embodiments, at least one chain of the MHC and the peptide are associated as a fusion protein. In one embodiment, the MHC and peptide are associated by a linker sequence. For example, a single-chain molecule may comprise, from the amino to the carboxyl terminus, an antigenic determinant, a β2-microglobulin sequence, and a class I α (heavy) chain sequence. Alternatively, a single-chain molecule may comprise, from the amino to the carboxyl terminus, an antigenic determinant, a class I α (heavy) chain sequence, and a β2-microglobulin sequence. The generation and use of pMHC complexes as single-chain trimers has been previously reported. See, e.g., U.S. Patent No. 8,895,020, U.S. Patent No. 8,992,937; Hansen et al. (2010) Trends Immunol. 31:363-69; Truscott et al. (2007) J. Immunol. 178:6280-89; Mitaksov et al. (2007) Chem Biol 14:909-22, each of which is incorporated herein by reference in its entirety. The single-chain pMHC complex may further comprise a signal peptide sequence at the amino terminus. In certain embodiments, there may be a linker sequence between the peptide sequence and the β2 microglobulin sequence. In certain embodiments, there may be a linker sequence between the β2 microglobulin sequence and the class I α (heavy) chain sequence. The single-chain molecule may further comprise a signal peptide sequence at the amino terminus, and a first linker sequence extending between the peptide sequence and the β2 microglobulin sequence, and / or a second linker sequence extending between the β2 microglobulin sequence and the class I heavy chain sequence.

[0172] In some embodiments, the single-chain pMHC complex may include a first flexible linker between the peptide ligand segment and the β2 microglobulin sequence. For example, the linker may extend from the carboxyl terminus of the peptide ligand segment and connect it to the amino terminus of the β2 microglobulin segment. In some embodiments, the linker is constructed so that the linked peptide ligand folds into the binding groove to generate a functional MHC-antigen peptide. In some embodiments, the linker may include at least 3 amino acids and up to about 15 amino acids (e.g., 20 amino acids). In some embodiments, the single-chain molecule may include a second flexible linker inserted between the β2 microglobulin segment and the MHC I heavy chain segment. For example, the linker may extend from the carboxyl terminus of the β2 microglobulin segment and connect it to the amino terminus of the MHC I heavy chain segment. In certain embodiments, the β2 microglobulin and the MHC I heavy chain fold into the binding groove to generate a molecule that can function in promoting T cell expansion.

[0173] Suitable linkers for use in pMHC complexes may be any of a number of suitable lengths, including, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids. Exemplary linkers include glycine polymers (Gn), glycine-serine polymers (e.g., (GS)n, (GSGGS) (SEQ ID NO: 1), and (GGGS) (SEQ ID NO: 2), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may also be used. Both Gly and Ser are relatively unstructured and therefore may serve as neutral linkers between components. Glycine polymers may also be used. Glycine has access to much more φ-ψ space than the equivalent alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 1 1173-142 (1992), which is incorporated herein by reference in its entirety). Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), GSSSG (SEQ ID NO: 8), GCGASGGGGSGGGGS (SEQ ID NO: 9), GCGASGGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGS (SEQ ID NO: 11), GGGASGGGGSGGGGS (SEQ ID NO: 12), GGGGSGGGGSGGGGGS (SEQ ID NO: 13), or GGGASGGGGS (SEQ ID NO: 14), GGGGSGGGGSGGGGS (SEQ ID NO: 15), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16), GCGGS (SEQ ID NO: 21). In some embodiments, the linker polypeptide comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in a second polypeptide.

[0174] In certain embodiments, a single-chain pMHC complex may comprise a peptide covalently linked to an MHC class I α(heavy) chain via a disulfide bond (i.e., a disulfide bond between two cysteines). See, e.g., U.S. Pat. Nos. 8,992,937 and 8,895,020, each of which is incorporated herein by reference in its entirety. In certain embodiments, the disulfide bond comprises a first cysteine ​​located within a linker extending from the carboxy terminus of the antigenic peptide and a second cysteine ​​located within the MHC class I heavy chain (e.g., the MHC class I α(heavy) chain carrying the covalent binding site for the antigenic peptide). In certain embodiments, the second cysteine ​​may be a mutation (addition or substitution) in the MHC class I α(heavy) chain. In certain embodiments, a single-chain molecule may comprise one continuous polypeptide chain as well as a disulfide bond. In certain embodiments, a single-chain molecule may comprise two continuous polypeptide chains linked via a disulfide bond as the only covalent bond. In some embodiments, the linking sequence may contain at least one amino acid, including one or more glycines, one or more alanines, and / or one or more serines, in addition to a cysteine. In some embodiments, the single-chain molecule comprises, from N- to C-terminus, an MHC class I peptide (e.g., an antigenic peptide), a first linker containing a first cysteine, a human (humanized) β2-microglobulin sequence, a second linker, and a human (humanized) MHC class I heavy chain sequence containing a second cysteine, wherein the first cysteine ​​and the second cysteine ​​comprise a disulfide bond. In some embodiments, the second cysteine ​​is a human (humanized) MHC class I heavy chain amino acid substitution selected from the group consisting of T80C, Y84C, and N86C (Y84C refers to a mutation at position 108 in the mature protein, which lacks the signal sequence; alternatively, if the protein still contains the 24-mer signal sequence, this position is referred to instead as Y108C).

[0175] In certain embodiments, a disulfide bond may link an antigenic peptide in the class I groove of a pMHC complex when the pMHC complex contains a first cysteine ​​in a Gly-Ser linker extending between the C-terminus of the peptide and β2 microglobulin and a second cysteine ​​in an adjacent heavy chain.

[0176] In some embodiments, the β2 microglobulin sequence may comprise a full-length (human or non-human) β2 microglobulin sequence. In certain embodiments, the β2 microglobulin sequence lacks a leader peptide sequence. Thus, the β2 microglobulin sequence may comprise approximately 99 amino acids and may be the human β2 microglobulin sequence (Genebank AF072097.1).

[0177] In some embodiments, the pMHC complex comprises a human HLA class I molecule fused to human β2 microglobulin. In some embodiments, the human HLA class I molecule fused to human β2 microglobulin comprises one or more linkers, e.g., a linker connecting the HLA molecule to the β2 microglobulin and / or a linker connecting the peptide to the human HLA class I molecule fused to human β2 microglobulin. In some embodiments, the nucleotide sequence encoding the pMHC complex may comprise sequences encoding human HLA and β2 microglobulin. In some embodiments, the nucleotide sequence encoding the pMHC complex may comprise sequences encoding human HLA and β2 microglobulin and one or more linkers. In some embodiments, the nucleotide sequence encoding the pMHC complex may comprise sequences encoding human HLA and β2 microglobulin and sequences encoding a label (e.g., green fluorescent protein) or tag (e.g., c-myc, histidine tag, etc.). In some embodiments, the nucleotide sequence encoding the pMHC complex may include sequences encoding human HLA and β2 microglobulin, sequences encoding a linker, and sequences encoding a label or tag. Non-limiting examples of nucleotide sequences encoding exemplary human HLA and β2 microglobulin, and one or more linkers, are set forth as SEQ ID NO: 17 and SEQ ID NO: 19. The amino acid sequences encoded therefrom are set forth as SEQ ID NO: 18 and SEQ ID NO: 20, respectively.

[0178] The peptide of interest may be linked to the N-terminal GCGGS linker sequence (SEQ ID NO: 21) of SEQ ID NO: 18 and SEQ ID NO: 20, wherein the cysteine ​​of the linker forms a disulfide bond with the Y108C amino acid of the human HLA-A2 polypeptide. Thus, in some embodiments, non-human animals are immunized and / or boosted with a pMHC complex comprising an amino acid sequence comprising the sequence set forth as SEQ ID NO: 18 or the sequence set forth as SEQ ID NO: 20. In some embodiments, non-human animals are immunized with DNA encoding a pMHC complex having an amino acid sequence comprising the sequence set forth as SEQ ID NO: 18 or SEQ ID NO: 20.

[0179] In some embodiments, a helper T cell epitope, such as PADRE, may be linked to the C-terminus of a single-chain pMHC complex. See, e.g., U.S. Patent No. 6,413,935 and Alexander J. et al. (1994) Immunity 1:751-61, each of which is incorporated by reference in its entirety. In some embodiments, PADRE is linked directly to the C-terminus of a single-chain pMHC complex. In several embodiments, PADRE is linked to the C-terminus of a single-chain pMHC complex via a linker. In some embodiments, the immunization protocols described herein comprise administering to a non-human animal a single-chain pMHC complex linked to PADRE at its C-terminus. In some embodiments, the single-chain pMHC complex linked to PADRE at its C-terminus comprises the amino acid sequence set forth in SEQ ID NO:25.

[0180] In some embodiments, the pMHC complexes are prepared using methods described in U.S. Patent Nos. 4,478,82, 6,011,146, 8,895,020, 8,992,937, WO 96 / 04314, Mottez et al. J. Exp. Med. 181:493-502, 1995, Madden et al. Cell 70:1035-1048, 1992, Matsumura et al., Science 257:927-934, 1992, Mage et al., Proc. Natl. Acad. Sci. USA 89:10658-10662, 1992, Toshitani et al., Proc. Nat'l Acad. Sci. 93:236-240, 1996, Chung et al. al., J. Immunol. 163:3699-3708, 1999; Uger and Barber, J. Immunol. 160:1598-1605, 1998; Uger et al., J. Immunol. 162, pp. 6024-6028, 1999; White et al., J. Immunol. 162:2671-2676, 1999.

[0181] In some embodiments, the pMHC complex comprises a Class II MHC polypeptide, or a fragment, variant, or derivative thereof. In one particular embodiment, the MHC comprises an α polypeptide and a β polypeptide of a Class II MHC molecule, or a fragment, variant, or derivative thereof. In one particular embodiment, the α polypeptide and the β polypeptide are linked by a peptide linker. In one particular embodiment, the MHC comprises an α polypeptide and a β polypeptide of a human Class II MHC molecule selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO.

[0182] MHC class II molecules generally consist of two polypeptide chains, α and β. The chains can be derived from the DP, DQ, or DR gene clusters. There are approximately 40 known different human MHC class II molecules. All have the same basic structure, but their molecular architectures vary slightly. MHC class II molecules bind peptides 13 to 18 amino acids in length.

[0183] In some embodiments, the antigenic pMHC complex comprises one or more MHC class II α chains. In some embodiments, the MHC class II α chain is fully human. In some embodiments, the MHC class II α chain is humanized. Humanized MHC class II α chains are described, for example, in U.S. Patent Nos. 8,847,005 and 9,043,996 and U.S. Patent Application Publication No. 2014 / 0245467. In some embodiments, the humanized MHC class II α chain polypeptide comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, the class II α chain polypeptide is humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA.

[0184] In some embodiments, the viral particle comprises one or more MHC class II β chains. In some embodiments, the MHC class II β chain is fully human. In some embodiments, the MHC class II β chain polypeptide is humanized. Humanized MHC class II β chain polypeptides are described, for example, in U.S. Patent Nos. 8,847,005 and 9,043,996 and U.S. Patent Application Publication No. 2014 / 0245467. In some embodiments, the humanized MHC class II β chain comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some embodiments, the class II β chain is humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB.

[0185] In some embodiments, the antigenic determinant, e.g., a peptide, included in the antigenic pMHC complex may include any peptide capable of binding to an MHC protein, such that the pMHC complex can bind, e.g., in a specific manner, to a TCR.

[0186] Examples include peptides produced by hydrolysis, most typically synthetically produced peptides, randomly generated peptides, specifically designed peptides, and peptides in which at least some of the amino acid positions are conserved among some peptides, with the remaining positions being random.

[0187] In nature, peptides generated by hydrolysis undergo hydrolysis and then bind to antigens via MHC proteins. Class I MHC typically presents peptides derived from proteins actively synthesized in the cytoplasm of cells. In contrast, class II MHC typically presents peptides derived from either exogenous proteins that enter the endocytic pathway of cells or proteins synthesized in the ER. Upon intracellular transport, peptides associate with MHC proteins.

[0188] Binding of peptides to the MHC peptide-binding groove can control the spatial arrangement of amino acid residues of MHC and / or peptides recognized by the TCR, or can control the pMHC-binding proteins produced by the genetically modified animals disclosed herein. Such spatial control relies, in part, on hydrogen bonds formed between the peptide and the MHC protein. Based on knowledge of how peptides bind to various MHCs, key MHC anchor amino acids and surface-exposed amino acids that vary between different peptides can be determined. In some embodiments, the length of the MHC-binding peptide is 5 to 40 amino acid residues, e.g., 6 to 30 amino acid residues, e.g., 8 to 20 amino acid residues, e.g., 9 to 11 amino acid residues, including peptides of any size between 5 and 40 amino acids in all integer increments (i.e., 5, 6, 7, 8, 9, ... 40). Natural MHC class II-binding peptides vary from approximately 9 to 40 amino acids. However, in almost all cases, peptides can be shortened to the 9 to 11 amino acid core without loss of MHC-binding ability or T cell recognition.

[0189] Peptides include, for example, peptides comprising at least a portion of an antigenic determinant of a protein selected from the group consisting of a human self-protein associated with an autoimmune disorder, a protein of an infectious agent (e.g., a bacterium, virus, or parasite), an allergen, and a tumor-associated protein. In one embodiment, the pMHC complex comprises an antigenic determinant of a human self-protein associated with an autoimmune disorder. In another embodiment, the pMHC complex comprises an antigenic determinant of an allergen. In another embodiment, the pMHC complex comprises an antigenic determinant of a bacterium. In another embodiment, the pMHC complex comprises an antigenic determinant of a virus. In another embodiment, the pMHC complex comprises an antigenic determinant of a parasite.

[0190] Attaching the peptide to an MHC class I or II molecule via a flexible linker has the advantage of ensuring that the peptide occupies and remains associated with the MHC during biosynthesis, transport, and presentation. As an alternative approach, in some embodiments, the MHC and peptide are expressed separately.

[0191] Antigen-binding proteins that specifically bind to antigenic pMHC complexes of interest, nucleic acid constructs, cells and methods for producing the same. In one embodiment, a nucleic acid encoding a variable domain of an antigen binding domain that specifically binds to an antigenic pMHC complex, and a cell expressing the nucleic acid, is provided.

[0192] In one embodiment, there is provided the use of a nucleic acid sequence derived from a non-human animal to generate a cell line for the production of a human therapeutic agent, in one embodiment, the human therapeutic agent is a binding protein comprising a human antigen-binding domain and a human Fc region.

[0193] In one embodiment, an expression system is provided, the system comprising human C H a nucleic acid encoding a polypeptide comprising a somatically mutated human heavy chain variable domain fused to a human C L a mammalian host cell comprising a nucleic acid encoding a polypeptide comprising a somatically mutated human light chain variable domain fused to a human light chain variable domain, wherein the V H Domains and V L The domains are cognate.

[0194] In one embodiment, suitable host cells are selected from B cells, hybridomas, quadromas, CHO cells, COS cells, 293 cells, HeLa cells, and human retinal cells expressing viral nucleic acid sequences (e.g., PERC.6™ cells (Creative Biolabs)).

[0195] In one embodiment, a method of making a binding protein is provided, the method comprising isolating cells or nucleic acid from a non-human animal as disclosed herein, wherein the cells or nucleic acid comprise or encode an antigen binding protein that specifically binds to a pMHC complex of interest. In some embodiments, the method further comprises isolating a human heavy or light chain variable region sequence (which may encode a histidine-modified human heavy chain variable domain and / or a histidine-modified human light chain variable domain, and may simultaneously or independently be a universal light chain variable domain) from a human C H or C L cloning a nucleotide sequence encoding the region in frame with a gene encoding the region to form a human binding protein sequence, and expressing the human binding protein sequence in a suitable cell.

[0196] In one embodiment, a non-human animal is immunized with a pMHC complex of interest, and the human antigen-binding domain specifically binds to an epitope of the pMHC complex of interest (K in the micromolar, nanomolar, or picomolar range). D In one embodiment, V H Domain and / or V L The nucleotide sequence encoding the domain is somatically mutated in the non-human animal.

[0197] In one embodiment, there is provided a method of making an antigen binding protein that binds to a pMHC complex of interest, the method comprising: (1) Immunizing a non-human animal with a pMHC complex of interest, wherein the non-human animal has in its genome: (i) a nucleotide sequence encoding a human (humanized) MHC molecule or at least a peptide-binding portion thereof, and (ii) a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus, whereby the non-human animal is capable of providing a human or humanized antigen-binding protein comprising a human or humanized antigen-binding domain, e.g., a human or humanized variable domain; optionally, in which case at least one of the (un)rearranged human or humanized immunoglobulin heavy chain loci and / or the (un)rearranged human or humanized immunoglobulin light chain loci is unrearranged; (2) initiating an immune response in the non-human animal against the subject pMHC complex or the subject pMHC complex linked to a carrier; (3) isolating cells (e.g., lymphocytes) from the immunized non-human animal, wherein the cells comprise first and second immunoglobulin variable region nucleic acid sequences encoding a human heavy chain variable domain and a human light chain variable domain (each of which may be independently histidine modified, and the light chain variable domain may be a common light chain variable domain) that form an antigen-binding domain that specifically binds to a pMHC complex of interest; (4) identifying immunoglobulin heavy and light chain variable region nucleic acid sequences that, when paired, encode immunoglobulin heavy and light chain variable domains that specifically bind to a pMHC complex of interest or that are linked to a carrier; and (5) expressing the nucleic acid sequence of (d) in an expression system suitable for expressing an antigen-binding protein to form an antigen-binding protein comprising a dimer of a heavy chain variable domain and a light chain variable domain that binds to a pMHC complex of interest.

[0198] In some embodiments, there is provided a method of making an antigen binding protein that binds to a pMHC complex of interest, the method comprising: (1) Immunizing a non-human animal with a pMHC complex of interest, wherein the non-human animal has in its genome: (i) a nucleotide sequence encoding a human (humanized) MHC molecule or at least a peptide-binding portion thereof, and (ii) a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus, whereby the non-human animal is capable of providing a human or humanized antigen-binding protein comprising a human or humanized antigen-binding domain, e.g., a human or humanized variable domain; optionally, in which case at least one of the (un)rearranged human or humanized immunoglobulin heavy chain loci and / or the (un)rearranged human or humanized immunoglobulin light chain loci is unrearranged; (2) obtaining a human (humanized) immunoglobulin heavy chain variable region sequence and / or a human (humanized) immunoglobulin light chain variable region sequence encoding a human (humanized) immunoglobulin heavy chain variable domain and / or a human (humanized) immunoglobulin light chain variable domain, respectively, of an antibody that specifically binds to a pMHC complex of interest; (c) employing the human (humanized) immunoglobulin heavy chain variable region sequence and / or the human (humanized) immunoglobulin light chain variable region sequence to generate an antibody that binds to pMHC.

[0199] In some embodiments, cells (e.g., B cells) are harvested from the non-human animal (e.g., from the spleen or lymph nodes). The cells may be fused with a myeloma cell line to prepare immortalized hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies containing a hybrid heavy chain specific for the antigen used for immunization.

[0200] In one embodiment, immunization involves priming (e.g., administering) a non-human animal with a target pMHC complex, allowing the non-human animal to rest for a period of time, and re-immunizing the non-human animal with the target pMHC complex (e.g., to boost the non-human animal's immune response). In some embodiments, the method involves immunizing and / or boosting the non-human animal with a helper T cell epitope, such as a pan-DR T helper epitope (PADRE). See, e.g., U.S. Patent No. 6,413,935 and Alexander J. et al. (1994) Immunity 1:751-61, each of which is incorporated herein by reference in its entirety. In some embodiments, the method involves priming a non-human animal with a target pMHC complex and boosting the immunized animal with a target pMHC complex linked to a helper T cell epitope, such as PADRE. In some embodiments, the method includes both priming and boosting a non-human animal with a pMHC complex of interest linked to a helper T cell epitope. In embodiments involving priming and / or boosting with PADRE, the non-human animal is a mouse on a C57 / B16 genetic background. For example, the non-human animal is a C57BL strain mouse selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10C, ​​and C57BL / Ola, or a hybrid of the aforementioned C57BL / 6 strain with another strain, such as 129 or BALB. In some embodiments, the period between priming the non-human animal and boosting the non-human animal is several days, at least one week, at least two weeks, at least three weeks, at least four weeks, or at least one month.

[0201] In one embodiment, immunoglobulin variable regions (VRs) (e.g., rearranged human Vs) generated in a non-human animal are used. H / D H / JH Gene sequence, or rearranged human V L / J L The gene sequences each independently represent a histidine-modified reshaped human V H / D H / J H Gene sequence or rearranged human V L / J L and the latter may further comprise, or independently comprise, a consensus rearranged human V L / J L In one embodiment, a rearranged V H / D H / J H The gene sequence may comprise one or more human heavy chain constant region sequences (e.g., human or mouse C H 1. Hinge, C H 2. C H 3, and combinations thereof) and fused or reconstituted V L / J L The gene sequence is fused to a human light chain constant region sequence. Also provided herein are amino acid sequences of immunoglobulin variable domains of binding proteins encoded by nucleic acid sequences produced in and / or isolated from non-human animals of embodiments of the invention.

[0202] In one embodiment, a binding protein or antigen-binding fragment thereof (e.g., Fab, F(ab)2, scFv) is provided that is derived from a sequence produced in a non-human animal embodiment of the invention or produced in a mouse embodiment of the invention.

[0203] Bispecific Binding Proteins Immunoglobulin-like binding proteins comprising human variable domains that specifically bind to a pMHC complex of interest are provided. Cells expressing such binding proteins, mice producing the cells, and related methods and compositions are also provided.

[0204] In some embodiments, the binding proteins and nucleotide sequences encoding them can be used to generate multispecific binding proteins, such as bispecific binding proteins. In this embodiment, a first polypeptide comprising a first heavy chain variable domain can be associated with a second polypeptide comprising a second heavy chain variable domain. When the first heavy chain variable domain and the second heavy chain variable domain specifically bind to different epitopes, a bispecific binding molecule can be generated using two heavy chain variable domains. H The regions may be the same or different. In one embodiment, for example, C H One of the regions may be modified to remove Protein A binding determinants, while the other heavy chain constant region is not so modified (see, e.g., U.S. Pat. No. 8,586,713 B2, which is incorporated herein by reference in its entirety). This unique configuration simplifies isolation of the bispecific binding protein from, for example, a mixture of homodimers (e.g., homodimers of the first or second polypeptide). In some embodiments, the bispecific pMHC binding protein may be heterodimeric with respect to Protein A binding, and a first polypeptide comprising, from N-terminus to C-terminus, a first epitope-binding region that selectively binds to a first epitope, an immunoglobulin constant region comprising a first CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, wherein the first CH3 region binds to Protein A; and b. A second polypeptide comprising, from N-terminus to C-terminus, a second epitope-binding region that selectively binds a second epitope, an immunoglobulin constant region comprising a second CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, wherein the second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 region to Protein A. In some embodiments, the modification is selected from the group consisting of (a) 95R, and (b) 95R and 96F of the IMGT exon numbering system, or (a') 435R, and (b') 435R and 436F of the EU numbering system. In some embodiments, the second CH3 region further comprises one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I according to the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I according to the EU numbering system.

[0205] In one embodiment, the methods and compositions are used to generate bispecific binding proteins. H The first V to be merged into the realm H , and C H A second V merged into the realm H are each independently cloned in-frame with a human IgG sequence of the same isotype (e.g., human IgG1, IgG2, or IgG4). H specifically binds to the first pMHC complex and the second V H specifically binds to a second pMHC complex. The first and second epitopes may be on different pMHC complexes or may be on the same pMHC complex.

[0206] In one embodiment, the first V H C to be fused H IgG isotype of the region, and the second V H C to be fused HThe IgG isotypes of the regions are of the same isotype, but differ in that one IgG isotype contains at least one amino acid substitution that, in one embodiment, renders the heavy chain bearing that substitution unable or substantially unable to bind Protein A compared to a heavy chain lacking that substitution.

[0207] In one embodiment, the first C H The region is a first C region of a human IgG selected from IgG1, IgG2, and IgG4. H 3 domains, and the second C H The region is a second C region of a human IgG selected from IgG1, IgG2, and IgG4. H 3 domain, in which case the second C H The third domain is the second C domain to protein A. H These include modifications that reduce or eliminate binding of the three domains (see US Pat. No. 8,586,713 B2, which is incorporated herein by reference in its entirety).

[0208] In one embodiment, the second C H In another embodiment, the second C3 domain contains a 435R modification numbered according to the EU numbering system. H The 3 domain further comprises the 436F modification, numbered according to the EU numbering system.

[0209] In one embodiment, the second C H The C3 domain of human IgG1 contains modifications selected from the group consisting of D356E, L358M, N384S, K392N, V397M, and V422I, numbered according to the EU numbering system. H There are three domains.

[0210] In one embodiment, the second C HThe C3 domain of human IgG2 contains an alteration selected from the group consisting of N384S, K392N, and V422I, numbered according to the EU numbering system. H There are three domains.

[0211] In one embodiment, the second C H The C3 domain of human IgG4 contains modifications selected from the group consisting of Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I, numbered according to the EU numbering system. H There are three domains.

[0212] In one embodiment, the binding protein has one or more of the modifications listed herein. H In particular embodiments, the constant region of the binding protein comprises a C H In another particular embodiment, the binding protein comprises an amino acid sequence that is free of human immunogenic epitopes. H The area includes, and the C H The region does not contain sequences that generate T cell epitopes.

[0213] In one embodiment, the Fc domain may be modified to alter Fc receptor binding, thereby affecting effector function. An engineered heavy chain constant region (C) containing an Fc domain may be used. H ) may be chimeric. Thus, chimeric C H The region contains C -derived from multiple immunoglobulin isotypes. H domain combinations, e.g., chimera C H The region is a C region derived from a human IgG1, human IgG2, or human IgG4 molecule. H C derived from human IgG1, human IgG2, or human IgG4 molecules combined with some or all of the three domains H Contains part or all of the 2 domains. Chimeric C HThe region may also contain a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence derived from a human IgG1, IgG2, or IgG4 hinge region (amino acid residues 216-227 according to EU numbering) combined with a "lower hinge" sequence derived from a human IgG1, IgG2, or IgG4 hinge region (amino acid residues 228-236 according to EU numbering). In one embodiment, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.

[0214] For a particular therapy, the Fc domain can be engineered to activate all, some, or none of the normal Fc effector functions without affecting the desired pharmacokinetic properties of the Fc-containing protein (e.g., an antibody). For examples of proteins containing chimeric CH regions and with altered effector function, see U.S. Patent No. 9,359,437, which is incorporated herein by reference in its entirety. In some embodiments, a pMHC-binding protein comprises a recombinant polypeptide comprising a heavy chain constant (CH) region comprising, from N-terminus to C-terminus, a CH1 domain, a chimeric hinge, a CH2 domain, and a CH3 domain, wherein (a) the CH1 domain comprises the amino acid sequence of DKKV or DKRV at positions 212-215 (EU numbering); (b) the chimeric hinge comprises the amino acid sequence of a human IgG1 or human IgG4 upper hinge at positions 216-227 (EU numbering) and the amino acid sequence of a human IgG2 lower hinge of PCPAPPVA (SEQ ID NO: 29) at positions 228-236 (EU numbering); and (c) the CH2 domain comprises the amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of a human IgG4 (d) the CH3 domain comprises the amino acid sequence of a CH2 domain of human IgG1 or human IgG4 at positions 341 to 447 (EU numbering).

[0215] In some embodiments, the bispecific antibody comprises a first V H and the second V H each having its own isogenic V L They share a common light chain domain.

[0216] Breaking tolerance to endogenous peptides Obtaining specific pMHC-binding proteins by immunizing a non-human animal (e.g., a rodent, e.g., a mouse or rat) with antigenic pMHC relies on sequence differences between the endogenous protein in the non-human animal and the presented foreign protein, allowing the non-human animal's immune system to recognize the target pMHC complex as non-self (i.e., foreign). Generating antibodies against pMHC highly homologous to self pMHC can be challenging due to immune tolerance to self pMHC. Methods for breaking tolerance to self peptides homologous to a target peptide are known to those skilled in the art. See, e.g., U.S. Patent Application Publication No. 20170332610, which is incorporated herein by reference in its entirety. In some embodiments, a method for breaking tolerance to an endogenous peptide involves modifying a non-human animal described herein to include a deletion, e.g., a knockout mutation, of a self peptide highly homologous to a target peptide.

[0217] Pharmaceutical Composition In certain embodiments, provided herein are compositions, e.g., pharmaceutical compositions, comprising at least one pMHC-binding protein formulated together with a pharmaceutically acceptable carrier.

[0218] The pharmaceutical compositions provided herein may be specifically formulated for administration in solid or liquid form, including forms adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., tablets, pills, powders, granules, pastes for application to the tongue, for buccal, sublingual, and systemic absorption, or (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution, or sterile suspension, or sterile sustained-release formulation.

[0219] In some embodiments, pharmaceutical compositions provided herein suitable for parenteral administration comprise one or more therapeutic agents of particular embodiments of the invention combined with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0220] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (for example, but not limited to, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0221] In certain embodiments, the composition comprises a pMHC-binding protein at a concentration that results in a w / v suitable for the desired administration. The pMHC-binding protein is at least 1 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, at least 35 mg / mL, at least 40 mg / mL, at least 45 mg / mL, at least 50 mg / mL, at least 55 mg / mL, at least 60 mg / mL, at least 65 mg / mL, at least 70 mg / mL, at least 75 mg / mL, at least 80 mg / mL, at least 85 mg / mL, at least 90 mg / mL, at least 95 mg / mL, at least 100 mg / mL, at least 10 ... The pMHC-binding protein may be present in the composition at a concentration of 10 mg / mL, at least 115 mg / mL, at least 120 mg / mL, at least 125 mg / mL, at least 130 mg / mL, at least 135 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 200 mg / mL, at least 250 mg / mL, or at least 300 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 1 mg / mL to 300 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 5 mg / mL to 250 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 10 mg / mL to 200 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 15 mg / mL to 150 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 20 mg / mL to 140 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 25 mg / mL to 135 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 30 mg / mL to 130 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 35 mg / mL to 125 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 40 mg / mL to 120 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 45 mg / mL to 115 mg / mL. In some embodiments, the pMHC-binding protein may be present in the composition at a concentration of 50 mg / mL to 110 mg / mL.

[0222] In some embodiments, the composition includes one or more active compounds necessary for the particular condition being treated, typically one or more compounds with complementary activities that do not adversely affect each other, and such additional active compounds are suitably present in combination in amounts that are effective for the purpose intended. In some embodiments, compositions are prepared by mixing a pMHC-binding protein with any physiologically acceptable carrier, excipient, or stabilizer, including but not limited to, buffers, sugars, salts, surfactants, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelators, preservatives, etc., at a desired final concentration in the form of a lyophilized composition or in the form of an aqueous solution (see Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th edition, L. Brunton, et al. and Remington's Pharmaceutical Sciences, 16th edition). edition, Osol, A. Ed. (1999)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, for example, buffers such as histidine, phosphate, citrate, glycine, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 10-15 residues) polypeptides; e.g., serum albumin, gelatin hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including trehalose, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, polysorbate 80, PLURONICS®, or polyethylene glycol (PEG).

[0223] In some embodiments, the buffering agent is histidine, citrate, phosphate, glycine, or acetate. The sugar excipient may be trehalose, sucrose, mannitol, maltose, or raffinose. The surfactant may be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic® F68. The salt may be NaCl, KCl, MgCl2, or CaCl2.

[0224] In some embodiments, the compositions include a buffer or pH adjuster to provide improved pH control. Such compositions may have a pH of about 3.0 to about 9.0, about 4.0 to about 8.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 8.0, about 5.5 to about 7.0, or about 5.5 to about 6.5. In further embodiments, such compositions have a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In a particular embodiment, the composition has a pH of about 6.0.

[0225] In some embodiments, the compositions include a buffering agent or pH adjuster to provide improved pH control. Such compositions may have a pH of 3.0-9.0, 4.0-8.0, 5.0-8.0, 5.0-7.0, 5.0-6.5, 5.5-8.0, 5.5-7.0, or 5.5-6.5. In further embodiments, such compositions have a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.5, 8.0, 8.5, or 9.0. In particular embodiments, the compositions have a pH of 6.0.

[0226] Those skilled in the art will understand that the pH of a composition generally will not be equal to the isoelectric point of the particular pMHC-binding protein used in the composition. Typically, buffers are salts prepared from organic or inorganic acids or bases. Representative buffers include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris; tromethamine hydrochloride; or phosphate buffers. Additionally, amino acid components may also function in a buffering capacity. Representative amino acid components that may be utilized as buffers in the composition include, but are not limited to, glycine and histidine. In certain embodiments, the buffer is selected from histidine, citrate, phosphate, glycine, and acetate. In a particular embodiment, the buffer is histidine. In another particular embodiment, the buffer is citrate. In yet another particular embodiment, the buffer is glycine. The purity of the buffer should be at least 98%, or at least 99%, or at least 99.5%. As used herein, the term "purity" in the context of histidine and glycine refers to the chemical purity of histidine or glycine as understood in the art and as described, for example, in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001), which is incorporated herein by reference in its entirety.

[0227] In certain embodiments, the composition comprises histidine as a buffering agent, hi certain embodiments, the histidine is present in the composition at a histidine concentration of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM. In another embodiment, the composition comprises about 1 mM to about 200 mM, about 1 mM to about 150 mM, about 1 mM to about 100 mM, about 1 mM to about 75 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 75 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 75 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM histidine. In further embodiments, the composition comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In particular embodiments, the composition may comprise about 10 mM, about 25 mM, or no histidine.

[0228] In certain embodiments, the composition comprises histidine as a buffering agent, hi certain embodiments, the histidine is present in the composition at a histidine concentration of at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 75 mM, at least 100 mM, at least 150 mM, or at least 200 mM. In another embodiment, the composition comprises between 1 mM and 200 mM, between 1 mM and 150 mM, between 1 mM and 100 mM, between 1 mM and 75 mM, between 10 mM and 200 mM, between 10 mM and 150 mM, between 10 mM and 100 mM, between 10 mM and 75 mM, between 10 mM and 50 mM, between 10 mM and 40 mM, between 10 mM and 30 mM, between 20 mM and 75 mM, between 20 mM and 50 mM, between 20 mM and 40 mM, or between 20 mM and 30 mM histidine. In further embodiments, the composition comprises 1 mM, 5 mM, 10 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, or 200 mM histidine. In particular embodiments, the composition may comprise 10 mM, 25 mM, or no histidine.

[0229] In some embodiments, the composition includes a carbohydrate excipient. The carbohydrate excipient may act, for example, as a viscosity enhancer, stabilizer, bulking agent, solubilizer, and / or the like. The carbohydrate excipient is generally present in an amount of about 1% to about 99% by volume or weight, e.g., about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 5%, about 1% to about 20%, about 5% to about 15%, about 8% to about 10%, about 10% to about 15%, about 15% to about 20%, 0.1% to 20%, 5% to 15%, 8% to 10%, 10% to 15%, 15% to 20%, about 0.1% to about 5%, about 5% to about 10%, or about 15% to about 20%. In still other particular embodiments, the carbohydrate excipient is present at 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%.

[0230] In some embodiments, the composition includes a carbohydrate excipient. The carbohydrate excipient may act, for example, as a viscosity enhancer, stabilizer, bulking agent, solubilizer, and / or the like. The carbohydrate excipient is generally present at 1% to 99% by volume or weight, e.g., 0.1% to 20%, 0.1% to 15%, 0.1% to 5%, 1% to 20%, 5% to 15%, 8% to 10%, 10% to 15%, 15% to 20%, 0.1% to 20%, 5% to 15%, 8% to 10%, 10% to 15%, 15% to 20%, 0.1% to 5%, 5% to 10%, or 15% to 20%. In still other particular embodiments, the carbohydrate excipient is present at 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%.

[0231] In some embodiments, the composition comprises a carbohydrate excipient.Carbohydrate excipients suitable for use in the composition include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol).In certain embodiments, the carbohydrate excipient for use in the compositions provided herein is selected from sucrose, trehalose, lactose, mannitol, and raffinose.In a specific embodiment, the carbohydrate excipient is trehalose.In another specific embodiment, the carbohydrate excipient is mannitol.In yet another specific embodiment, the carbohydrate excipient is sucrose.In yet another specific embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient should be at least 98%, or at least 99%, or at least 99.5%.

[0232] In some embodiments, the composition comprises trehalose. In specific embodiments, the composition comprises at least about 1%, at least about 2%, at least about 4%, at least about 8%, at least about 20%, at least about 30%, or at least about 40% trehalose. In other embodiments, the composition comprises about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 2% to about 40%, about 2% to about 30%, about 2% to about 20%, about 4% to about 40%, about 4% to about 30%, or about 4% to about 20% trehalose. In further embodiments, the composition comprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% trehalose. In individual embodiments, the composition comprises about 4%, about 6%, or about 15% trehalose.

[0233] In some embodiments, the composition comprises trehalose. In specific embodiments, the composition comprises at least 1%, at least 2%, at least 4%, at least 8%, at least 20%, at least 30%, or at least 40% trehalose. In other embodiments, the composition comprises 1% to 40%, 1% to 30%, 1% to 20%, 2% to 40%, 2% to 30%, 2% to 20%, 4% to 40%, 4% to 30%, or 4% to 20% trehalose. In further embodiments, the composition comprises 1%, 2%, 4%, 6%, 8%, 15%, 20%, 30%, or 40% trehalose. In particular embodiments, the composition comprises 4%, 6%, or 15% trehalose.

[0234] In certain embodiments, the composition includes an excipient. In a separate embodiment, the composition includes at least one excipient selected from sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. In certain embodiments, the composition includes a salt, such as a salt selected from NaCl, KCl, CaCl, and MgCl. In a separate embodiment, the composition includes NaCl.

[0235] In some embodiments, the composition comprises an amino acid, such as, for example, lysine, arginine, glycine, histidine, or an amino acid salt. The composition may comprise at least about 1 mM, at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, or at least about 400 mM of an amino acid. In another embodiment, the composition may contain about 1 mM to about 100 mM, about 10 mM to about 150 mM, about 25 mM to about 250 mM, about 25 mM to about 300 mM, about 25 mM to about 350 mM, about 25 mM to about 400 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 100 mM to about 250 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 150 mM to about 250 mM, about 150 mM to about 300 mM, or about 150 mM to about 400 mM of amino acids. In further embodiments, the composition comprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of amino acid.

[0236] In some embodiments, the composition comprises an amino acid, such as, for example, lysine, arginine, glycine, histidine, or an amino acid salt. The composition may comprise at least 1 mM, at least 10 mM, at least 25 mM, at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, or at least 400 mM of an amino acid. In another embodiment, the composition may comprise 1 mM to 100 mM, 10 mM to 150 mM, 25 mM to 250 mM, 25 mM to 300 mM, 25 mM to 350 mM, 25 mM to 400 mM, 50 mM to 250 mM, 50 mM to 300 mM, 50 mM to 350 mM, 50 mM to 400 mM, 100 mM to 250 mM, 100 mM to 300 mM, 100 mM to 400 mM, 150 mM to 250 mM, 150 mM to 300 mM, or 150 mM to 400 mM of amino acids. In further embodiments, the composition comprises 1 mM, 1.6 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, or 400 mM of amino acid.

[0237] In some embodiments, the composition includes a surfactant. The term "surfactant" as used herein refers to an organic substance with an amphiphilic structure. That is, surfactants are composed of groups with opposing solubility tendencies, typically consisting of an oil-soluble hydrocarbon chain and a water-soluble ionic group. Depending on the charge of the surface-active moiety, surfactants can be classified into anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants for various pharmaceutical compositions and preparations of biomaterials. Polysorbates (e.g., Polysorbate 20 or 80); poloxamers (e.g., Poloxamer 188); Triton; sodium octyl glycoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearomidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearomidopropyl dimethylamine; sodium methyl cocoyl taurate, or disodium methyl oleyl taurate; and the MONAQUA® series (Mona Pharmaceutically acceptable surfactants, such as polysorbate 20, polysorbate 40, polysorbate 60, and copolymers of ethylene and propylene glycol (e.g., Polysorbate Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS® PF68), can optionally be added to the composition to reduce aggregation. In certain embodiments, the composition comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Surfactants are particularly useful when the composition is administered using a pump or plastic container. The presence of a pharmaceutically acceptable surfactant reduces the tendency of the protein to aggregate.The composition may contain polysorbate at a concentration ranging from about 0.001% to about 1%, or from about 0.001% to about 0.1%, or from about 0.01% to about 0.1%. In other particular embodiments, the composition contains polysorbate at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%. The composition may contain polysorbate at a concentration ranging from 0.001% to 1%, or from 0.001% to 0.1%, or from 0.01% to 0.1%. In other particular embodiments, the composition comprises a concentration of polysorbate of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.

[0238] In some embodiments, the composition comprises other excipients and / or additives, including but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable excipients and / or additives may be used in the compositions provided herein. Commonly used excipients / additives, such as pharmaceutically acceptable chelators (e.g., but not limited to, EDTA, DTPA, or EGTA), can optionally be added to the composition to reduce aggregation. These additives are particularly useful when the composition is administered using a pump or plastic container.

[0239] In some embodiments, the composition contains a preservative. Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (including, but not limited to, hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, can be optionally added to the composition at any suitable concentration, such as 0.001% to 5%, or any range or value therebetween. The concentration of the preservative used in the composition is sufficient to produce a microbial effect. The concentration will depend on the preservative selected and can be easily determined by one of ordinary skill in the art.

[0240] In some embodiments, the composition is isotonic with human blood, meaning that the composition has essentially the same osmotic pressure as human blood. Such isotonic compositions generally have an osmotic pressure of 250 mOSm to 350 mOSm. Isotonicity can be measured, for example, using a vapor pressure or ice-freezing osmometer. The tonicity of the composition is adjusted by using a tonicity adjusting agent. A "tonicity adjusting agent" is a pharmaceutically acceptable inert substance that can be added to a composition to provide isotonicity to the composition. Tonicity adjusting agents suitable for the compositions provided herein include, but are not limited to, sugars, salts, and amino acids.

[0241] In certain embodiments, the composition is pyrogen-free, i.e., substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins encapsulated within microorganisms that are released only upon destruction or death of the microorganism. Pyrogens also include fever-inducing, heat-stable substances derived from the outer membranes of bacteria and other microorganisms. Both of these substances can cause fever, hypotension, and shock when administered to humans. Due to potential adverse effects, even small amounts of endotoxin must be removed from intravenously administered drug solutions. The Food and Drug Administration (FDA) has established a limit of 5 endotoxin units (EU) per kilogram of body weight per undivided hour for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223(2000)). When therapeutic proteins are administered in amounts of hundreds to thousands of milligrams per kilogram of body weight, as may be the case for proteins of interest (e.g., antibodies), even trace amounts of harmful and dangerous endotoxins must be removed. In some embodiments, the endotoxin and pyrogen levels in the composition are less than 10 EUmg, or less than 5 EUmg, or less than 1 EUmg, or less than 0.1 EUmg, or less than 0.01 EUmg, or less than 0.001 EUmg.

[0242] In some embodiments, pharmaceutical compositions used for in vivo administration must be sterile. The compositions may be sterilized by various sterilization methods, including sterile filtration and irradiation. In certain embodiments, the compositions are sterile filtered through a pre-sterilized 0.22 micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005), which is incorporated herein by reference in its entirety.

[0243] Compositions comprising pMHC-binding proteins, such as those disclosed herein, are typically stored in lyophilized form or in solution. Sterile compositions comprising pMHC-binding proteins are placed into a container having a sterile access port, such as an intravenous solution bag or vial, having an adapter that allows for withdrawal of the composition, such as a stopper pierceable by a hypodermic injection needle. In certain embodiments, the composition is provided as a pre-filled syringe.

[0244] In certain embodiments, the composition is a lyophilized formulation. The term "lyophilized" or "freeze-dried" includes the state of a material that has been subjected to a drying procedure, such as lyophilization, in which at least 50% of the water has been removed.

[0245] Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions provided herein, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0246] While the present invention has been particularly shown and described with reference to numerous embodiments, those skilled in the art will understand that changes may be made in form and detail to the various embodiments disclosed herein without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to serve as limitations on the scope of the claims. [Example]

[0247] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Example 1

[0248] Using either or both of breeding techniques and sequential homologous recombination in ES cells, mice are generated that contain (1) a human or humanized MHC I locus, a human or humanized β2 microglobulin locus, and / or a human or humanized MHC II locus (see, e.g., Figures 1-2 for non-limiting examples of such humanized loci), and (2) a humanized immunoglobulin heavy chain locus and / or a humanized light chain locus (see, e.g., Macdonald (See, e.g., Wang et al., (2014) Proc. Natl. Acad. Sci. USA 111:5147-52, which is incorporated herein by reference in its entirety.) These mice express and are tolerant to human or humanized MHC I / β2 microglobulin molecules and / or human or humanized MHC II molecules. Mice are immunized with a peptide-MHC (pMHC) complex of interest comprising a peptide antigenic to the mouse and a human or humanized MHC to which the mouse is tolerant. The mice are additionally and optionally boosted with a pMHC complex of interest, which is optionally further linked to a helper T cell epitope. Human or humanized antibodies expressed from the humanized immunoglobulin heavy and / or light chain loci are isolated from the serum of the immunized mice and tested for binding specificity to the pMHC complex.

[0249] Humanized MHC I molecules (HLA-A2 / H-2K) associated with humanized β2 microglobulin (e.g., Figure 1A) were identified using a C57BL background. and 1C. See also U.S. Patent Nos. 9,591,835 and 9,615,550, which are incorporated by reference in their entireties), a humanized immunoglobulin heavy chain locus (see, e.g., Macdonald (2014) supra), and a humanized common light chain locus (see, e.g., U.S. Patent Nos. 10,143,186, 10,130,081, and 9,969,814; U.S. Patent Application Publication Nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836, and 20150313193, each of which is incorporated by reference in its entirety). Test mice were generated containing nucleotide sequences encoding a humanized immunoglobulin heavy chain locus (see, e.g., U.S. Patent Nos. 10,143,186, 10,130,081, and 9,969,814; U.S. Patent Application Publication Nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836, and 20150313193, each of which is incorporated by reference in its entirety). These test mice, as well as control mice containing a functional (e.g., murine) ADAM6 gene (see, e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940, which are incorporated herein by reference in their entireties) and humanized immunoglobulin heavy and light chain loci, were immunized with a single-chain pMHC complex containing a heterologous peptide (peptide B) presented in the context of HLA-A2 / β2m molecules, either as a protein immunogen set forth in SEQ ID NO:26 (FIG. 4) or as DNA encoding a single-chain pMHC complex containing the amino acid sequence set forth in SEQ ID NO:27 (FIG. 3). Mice were boosted at various intervals and via various routes using pMHC complexes with standard adjuvants (FIGS. 3-4) or pMHC complex immunogens linked to T-helper peptides (PADRE, FIG. 5). Preimmune serum was collected from the mice, after which immunizations were initiated. Mice were bled periodically and antiserum titers against each antigen were analyzed.

[0250] Serum antibody titers against unrelated antigens (peptide A or peptide C) and related antigens presented in the context of HLA-A2 were determined using ELISA. 96-well microtiter plates (Thermo Scientific) were coated overnight with 5 μg / ml of anti-myc antibody in phosphate-buffered saline (PBS, Irvine Scientific). Plates were washed with 0.05% Tween® 20 in phosphate-buffered saline (PBS-T, Sigma-Aldrich) and blocked with 250 μl of 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 hour at room temperature. Plates were washed with PBS-T and coated with 2 μg / ml of c-myc-tagged single-chain pMHC complexes containing related peptide B, or unrelated antigen peptide A or peptide C, presented in the context of HLA-A2.

[0251] Preimmune serum and immune antisera were serially diluted 3-fold in 0.5% BSA-PBS and added to the plate for 1 hour at room temperature. The plate was washed, and goat anti-mouse IgG-Fc-horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immunoresearch) was added and incubated for 1 hour at room temperature. The plate was washed, and color was developed using 3,3',5,5'-tetramethylbenzidine (TMB) / H2O2 as substrate according to the manufacturer's recommended procedure. Absorbance at 450 nm was recorded using a spectrophotometer (Victor, Perkin Elmer). Antibody titers were calculated using Graphpad PRISM software. Antibody titers were calculated as the interpolated serum dilution at which the binding signal was twice background.

[0252] Control mice immunized with either a DNA immunogen encoding peptide B in an HLA-A context or a protein immunogen containing the peptide elicited antibody titers binding to peptide B-containing single-chain pMHC (Figures 3-4). Control mice not tolerant to chimeric HLA-A2 / H-2K polypeptides and / or human or humanized β2-microglobulin were included in the cohort. However, the sera of these control mice also contained antibody titers against unrelated peptides (peptide A or peptide C) presented in an HLA-A context, which were comparable to the antibody titers against peptide B-containing single-chain pMHC (Figures 3-4). Thus, although non-tolerant animals can generate an immune response to the target pMHC complex, such an immune response can be considered a nonspecific immune response, since the antibody titers against peptide B-containing single-chain pMHC are comparable to those against unrelated peptide-containing single-chain pMHC.

[0253] In contrast, serum from test mice tolerant to human (humanized) HLA-A and β2-microglobulin molecules immunized with single-chain peptide B / HLA-A / β2M complexes elicited antibody titers against peptide B-containing single-chain pMHC (Figure 4). The antibody titers against peptide B-containing pMHC complexes were higher than those against HLA-A / β2M complexes containing unrelated peptides A or C (Figure 4). Similarly, when DNA encoding the peptide B / HLA-A / β2M complex was used as the immunogen, higher antibody titers against the relevant peptide B single-chain protein (compared to unrelated pMHC complexes) were observed (Figure 3). High titers against the immunogen were also elicited in a third cohort. In this cohort, test mice received a prime injection of a single-chain pMHC complex containing peptide B, followed by a boost with a single-chain pMHC complex containing peptide B linked to the PADRE helper T cell epitope, and antibody titers were compared to those against an unrelated pMHC complex (Figure 5). Furthermore, mice expressing a single-chain HLA-A2 / β2M polypeptide (SEQ ID NO: 23) from the ROSA26 locus (see, e.g., Figure 2) were similarly tolerant to empty HLA-A2 / β2M molecules, and when immunized and boosted with pMHC / peptide B complex protein (in the presence or absence of PADRE), generated higher antibody titers against pMHC complexes presenting peptide B compared to those against pMHC complexes presenting an unrelated peptide (data not shown). Example 2

[0254] Mice that express MHC class I, MHC class II, and / or β2M molecules from loci other than the corresponding endogenous loci, such as the ROSA26 locus, are tolerant to empty MHC class I, MHC class II, and / or β2M molecules and generate higher antibody titers to immunogens, such as DNA encoding single-chain pMHC complexes, compared with mice that are not tolerant to empty MHC class I, MHC class II, and / or β2M molecules.

[0255] The data herein demonstrate that tolerizing a non-human animal to human HLA class I molecules and human β2 microglobulin molecules or portions thereof enhances the ability of the modified non-human animal to generate specific antibody responses against the target pMHC compared to a control non-human animal that is not tolerant to human HLA class I molecules and human β2 microglobulin molecules. The present invention provides, for example, the following items. (Item 1) A genetically modified non-human animal, the genome of which is (a) a nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof, and (b) (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci, optionally wherein at least one of the (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci is an unrearranged locus; the genetically modified non-human animal expresses the human or humanized MHC molecule or at least a peptide-binding portion thereof; the genetically modified non-human animal expresses an immunoglobulin comprising a human or humanized heavy chain variable domain and / or a human or humanized light chain variable domain, and (i) a human HLA molecule or portion thereof from which the human or humanized MHC molecule is derived, or a portion thereof, and (ii) a peptide that is heterologous to the non-human animal, wherein the non-human animal is tolerant to the human or humanized MHC molecule, or at least a peptide-binding portion thereof, thereby generating a specific B cell response when immunized with an antigenic peptide-MHC (pMHC) complex containing a peptide that is heterologous to the non-human animal complexed with (i)(ii). (Item 2) the human or humanized MHC molecule is selected from the group consisting of a human or humanized MHC class I molecule, a human or humanized MHC class II α molecule, a human or humanized MHC class II β molecule, or any combination thereof; and / or The genetically modified non-human animal is (a) at the endogenous heavy chain locus; (i) an unrearranged human or humanized immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region; (ii) a constrained unrearranged human or humanized heavy chain variable region operably linked to an endogenous heavy chain constant region; (iii) a consensus heavy chain coding sequence; (iv) a histidine-engineered, unrearranged human or humanized heavy chain variable region operably linked to an endogenous heavy chain constant region; (v) a heavy chain-only immunoglobulin coding sequence, or (vi) an unrearranged human or humanized hybrid heavy chain sequence encoding a hybrid immunoglobulin chain; and / or (b) at the endogenous light chain locus; (i) an unrearranged human or humanized immunoglobulin light chain variable region operably linked to an endogenous light chain constant region; (ii) a consensus light chain coding sequence; (iii) a constrained unrearranged human or humanized light chain variable region operably linked to an endogenous light chain constant region; (iv) a histidine-engineered, unrearranged human or humanized light chain variable region operably linked to an endogenous light chain constant region; or (v) a histidine-engineered, reshaped human or humanized light chain variable region operably linked to an endogenous light chain constant region. (Item 3) 3. The genetically modified non-human animal of item 1 or 2, wherein the non-human animal further comprises a functional ADAM6 gene, and optionally the functional ADAM6 gene is an endogenous ADAM6 gene. (Item 4) 4. The genetically modified non-human animal according to any one of items 1 to 3, wherein the non-human animal further expresses an exogenous terminal deoxynucleotidyl transferase (TdT) gene. (Item 5) 5. The genetically modified non-human animal according to any one of items 1 to 4, wherein the human or humanized MHC molecule is a human or humanized MHC class I molecule, and optionally the human or humanized MHC molecule is derived from an HLA class I molecule selected from the group consisting of an HLA-A molecule, an HLA-B molecule, an HLA-C molecule, and any combination thereof. (Item 6) further comprising a nucleotide sequence encoding human or humanized β2 microglobulin in its genome, optionally at an endogenous β2 microglobulin locus; 6. The genetically modified non-human animal of item 5, wherein the non-human animal expresses the human or humanized β2 microglobulin, thereby making the non-human animal tolerant to the β2 microglobulin itself or tolerant to the β2 microglobulin associated with the human or humanized MHC class I molecule. (Item 7) 5. The genetically modified non-human animal according to any one of items 1 to 4, wherein the human or humanized MHC molecule is a human or humanized MHC class II molecule, and optionally the human or humanized MHC molecule is derived from the α chain and / or β chain, or at least the peptide-binding groove thereof, of an HLA class II molecule selected from the group consisting of an HLA-DP molecule, an HLA-DQ molecule, an HLA-DR molecule, and any combination thereof. (Item 8) the nucleotide sequence encodes a complete human HLA molecule; 8. The genetically modified non-human animal of any of items 1 to 7, wherein optionally the nucleotide sequence does not disrupt an endogenous non-human MHC locus, and optionally the nucleotide sequence is located at an endogenous ROSA26 locus. (Item 9) the nucleotide sequence encodes a chimeric human / non-human MHC molecule comprising the extracellular domain of a human HLA molecule operably linked to the transmembrane and cytoplasmic domains of an endogenous MHC molecule, and optionally the nucleotide sequence encodes (i) a chimeric human / non-human MHC class I molecule comprising the α1, α2, and α3 domains of a human MHC class I molecule selected from the group consisting of HLA-A, HLA-B, and HLA-C, operably linked to the transmembrane and cytoplasmic domains of an endogenous non-human MHC class I molecule, e.g., an endogenous mouse H-2K polypeptide, an endogenous mouse H-2D polypeptide, or an endogenous mouse H-DL polypeptide; and / or (ii) The genetically modified non-human animal according to any one of items 1 to 7, encoding a chimeric human / non-human MHC class II molecule comprising the α1 and α2 domains of a human HLA class II α polypeptide operably linked to the transmembrane and cytoplasmic domains of an endogenous non-human MHC class II α molecule, e.g., an endogenous mouse H-2Aα polypeptide or an endogenous mouse H-2Eα polypeptide, and / or the β1 and β2 domains of a human HLA class I β polypeptide operably linked to the transmembrane and cytoplasmic domains of an endogenous non-human MHC class II β molecule, e.g., an endogenous mouse H-2Aα polypeptide or an endogenous mouse H-2Eα polypeptide. (Item 10) 10. The genetically modified non-human animal according to any of items 1 to 9, further comprising an antigenic peptide-MHC (pMHC) complex containing a peptide that is heterologous to the non-human animal, associated with a human HLA molecule from which the human or humanized MHC molecule is derived. (Item 11) (c)(ii) an antigenic peptide-MHC (pMHC) complex containing a human HLA molecule or portion thereof from which the human or humanized MHC molecule is derived, a peptide that is heterologous to the non-human animal and associated with (i)(ii), and (d) a human or humanized antigen-binding protein that specifically binds to the antigenic peptide-MHC and does not bind to the human HLA molecule from which the human or humanized MHC molecule is derived. (Item 12) 12. The genetically modified non-human animal according to any of items 1 to 11, wherein the non-human animal is heterozygous for the nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof. (Item 13) 13. The genetically modified non-human animal according to any one of items 1 to 12, wherein the non-human animal is a rodent such as a rat or a mouse. (Item 14) 14. The genetically modified non-human animal according to any one of items 1 to 13, wherein the non-human animal is a mouse. (Item 15) A method for producing a genetically modified non-human animal according to any one of Items 1 to 14, (a) a nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof, and (b) modifying its genome to include (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci, optionally wherein at least one of said (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci is unrearranged; The genetically modified non-human animal comprises: A. is tolerant to said human or humanized MHC molecule or at least a peptide-binding portion thereof, thereby generating a specific B cell response when immunized with a peptide-MHC (pMHC) complex comprising (i) a peptide xenogeneic to said non-human animal complexed with (ii) a human HLA molecule or portion thereof from which said human or humanized MHC molecule is derived, and B. A method capable of providing a human or humanized antigen-binding protein comprising a human or humanized heavy chain variable domain and / or a human or humanized light chain variable domain (Item 16). The method comprises: (a) (i) inserting a nucleotide sequence encoding a human or humanized MHC molecule, or at least a peptide-binding portion thereof, into a first ectopic locus; or (ii) substituting a nucleotide sequence encoding a non-human animal MHC I polypeptide and a nucleotide sequence encoding a chimeric human / non-human MHC I polypeptide at the endogenous non-human animal MHC I locus, and / or substituting a nucleotide sequence encoding a non-human animal MHC II molecule and a nucleotide sequence encoding a chimeric human / non-human MHC II molecule at the endogenous non-human animal MHC II locus, the chimeric human / non-human MHC I molecule contains the α1, α2, and α3 domains of human MHC I and at least the transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide; the chimeric human / non-human MHC II molecule contains the α1, α2, β1, and β2 domains of human MHC II and at least the transmembrane and cytoplasmic domains of an endogenous rodent MHC II polypeptide; and (b) (i) inserting a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus into a second ectopic locus; or (ii) (A) Endogenous non-human immunoglobulin variable (V) sequences at the endogenous non-human heavy chain locus. H ) gene segments and unrearranged human immunoglobulin variable (V H ) gene segments, and optionally replacing endogenous non-human immunoglobulin diversity (D H ) gene segments and / or endogenous non-human junctions (J H ) gene segments and unrearranged human immunoglobulin diversity (D H ) gene segments and / or unrearranged human immunoglobulin binding (J H ) gene segments, respectively, HGene segments, as well as any D H Gene segments and J H The gene segment is operably linked to, replaces, and / or is an endogenous heavy chain constant region gene sequence. (B) Endogenous non-human light chain locus, endogenous non-human light chain variable (V L ) gene segments and endogenous non-human light chain joining (J L ) gene segment and the human light chain variable (V L ) gene segments and human light chain joining (J L ) gene segments, which are optionally rearranged to form V L / J L forming a gene sequence of the human V L Gene segments and joints L the gene segment is operably linked to, or replaces, an endogenous light chain constant region gene sequence; (a) the nucleotide sequence encoding a non-human MHC I molecule and / or a non-human MHC II molecule, respectively; and (b) the V H , D H , J H , V L , and J. L The gene segment is (I) inserted or replaced by sequential homologous recombination in a single non-human embryonic stem (ES) cell; or (II) The method according to Item 15, wherein a first ES cell and a second ES cell are used to generate a first non-human animal and a second non-human animal, respectively, and the method further comprises mating the first and second non-human animals. (Item 17) 17. The method of claim 15 or 16, further comprising administering to the non-human animal an antigenic pMHC complex comprising a peptide xenogeneic to the non-human animal associated with a human HLA molecule from which the human or humanized MHC molecule is derived, optionally wherein the antigenic pMHC complex is linked to a helper T cell epitope, and optionally wherein the helper T cell epitope is PADRE. (Item 18) 18. A method of producing an antigen-binding protein that specifically binds to an antigenic pMHC complex of a subject, or a nucleic acid sequence encoding said protein, comprising maintaining a non-human animal according to any one of items 1 to 14, or a non-human animal produced according to the method of any one of items 15 to 17, under conditions sufficient for the non-human animal to mount an immune response to said antigenic pMHC complex of the subject, wherein said antigenic pMHC complex of the subject is xenogeneic to the non-human animal and comprises a peptide presented in the context of human HLA, or a part thereof, from which said human or humanized MHC molecule is derived. (Item 19) 19. The method of claim 18, comprising, as a first step, immunizing the non-human animal with an antigenic pMHC complex of the subject and optionally boosting the immune response of the immunized non-human animal, wherein optionally immunizing and / or boosting comprises administering to the non-human animal the pMHC complex of the subject linked to a helper T cell epitope, and optionally the helper T cell epitope comprises PADRE as set forth in SEQ ID NO: 28. (Item 20) 1. A method for obtaining nucleic acids encoding a human immunoglobulin heavy chain variable domain and / or a human immunoglobulin light chain variable domain, comprising: isolating, from the non-human animal according to any one of Items 10 to 14, a nucleic acid comprising a rearranged human immunoglobulin variable region gene sequence encoding a human immunoglobulin variable domain expressed by a lymphocyte of the non-human animal or a hybridoma prepared from the lymphocyte; wherein the human immunoglobulin variable domain expressed by the lymphocyte, or a hybridoma produced from the lymphocyte, associates with its cognate variable domain to form an antigen-binding domain specific for the antigenic pMHC complex. (Item 21) 21. The method of claim 20, further comprising immunizing the non-human animal with an antigenic pMHC complex of interest and allowing the non-human animal to mount an immune response to the antigen, and then obtaining the nucleic acid. (Item 22) 22. The method of claim 20 or 21, wherein the obtained rearranged human immunoglobulin variable region gene sequence comprises at least one somatic hypermutation. (Item 23) A nucleic acid comprising a rearranged human immunoglobulin heavy chain variable region gene sequence produced by the method according to any one of items 20 to 22. (Item 24) 24. The nucleic acid of Item 23, wherein the nucleic acid further comprises a human constant region gene sequence operably linked to the rearranged human immunoglobulin variable region gene sequence. (Item 25) The human heavy chain constant region gene sequence has a C IgG heavy chain constant region amino acid sequence for fetal Fc receptor (FcRn) at a pH in the range of 5.5 to 6.0. H 2-C H 25. The nucleic acid of item 22 or item 24, comprising a modification that increases affinity in three regions, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and a combination thereof. (Item 26) 26. A host cell comprising the nucleic acid according to any one of items 23 to 25. (Item 27) A method for obtaining cells expressing a human immunoglobulin heavy chain variable domain and / or a human immunoglobulin light chain variable domain, comprising: 15. A method comprising isolating lymphocytes from the non-human animal according to any one of Items 10 to 14, wherein the lymphocytes express a human immunoglobulin variable domain that forms an antigen-binding domain specific for the antigenic pMHC complex. (Item 28) 28. The method of claim 27, further comprising producing hybridomas from the isolated lymphocytes. (Item 29) An isolated cell, such as a germ cell, an embryonic stem cell, or a somatic cell (e.g., a B cell), (a) a nucleotide sequence encoding a human or humanized MHC molecule or at least a peptide-binding portion thereof, and (b) a (non-)rearranged human or humanized immunoglobulin heavy chain locus and / or a (non-)rearranged human or humanized immunoglobulin light chain locus, wherein the non-human animal is capable of providing a human or humanized antigen-binding protein comprising a human or humanized antigen-binding domain, optionally wherein the human or humanized antigen-binding domain comprises a human or humanized variable domain; optionally, at least one of said (un)rearranged human or humanized immunoglobulin heavy chain loci and / or (un)rearranged human or humanized immunoglobulin light chain loci is unrearranged; Optionally, the isolated cell is obtained according to the method of item 27 or item 28. (Item 30) 1. A method for producing human immunoglobulin variable domains in vitro, comprising: expressing in a cell a first nucleic acid comprising a rearranged human immunoglobulin variable region gene sequence encoding a human immunoglobulin variable domain expressed by the lymphocyte of the non-human animal according to any one of Items 10 to 14 or a hybridoma prepared from the lymphocyte; wherein the human immunoglobulin variable domain expressed by the lymphocyte, or a hybridoma produced from the lymphocyte, associates with its cognate variable domain to form an antigen-binding domain specific for the antigenic pMHC complex. (Item 31) 32. The method of claim 30, wherein the first nucleic acid further comprises a human immunoglobulin constant region gene sequence operably linked to the rearranged human immunoglobulin variable region gene sequence. The human immunoglobulin constant region gene sequence is a heavy chain constant region gene sequence, and has a C IgG heavy chain constant region amino acid sequence for fetal Fc receptor (FcRn) at a pH in the range of 5.5 to 6.0. H 2-C H32. The method of claim 31, comprising a modification that increases affinity in three regions, wherein the modification is a mutation in the IgG heavy chain constant region amino acid sequence selected from the group consisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E, T250Q, H433K, N434Y, and a combination thereof. (Item 33) A human immunoglobulin heavy chain variable domain produced according to the method of any one of items 30 to 32.

Claims

[Claim 1] A composition as described in the specification.

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