Genetically modified non-human animals with humanized immunoglobulin loci

Abstract

Genetically modified animals and cells are provided that have humanized heavy chain immunoglobulin loci and / or humanized light chain immunoglobulin loci.

Description

[Technical field]

[0001] Claiming priority This application claims priority to PCT / CN Patent Application Publication No. 2019 / 075406, filed on February 18, 2019, and PCT / CN Patent Application Publication No. 2019 / 106320, filed on September 18, 2019. The contents of the above patent applications are incorporated herein by reference in their entireties.

[0002] The present disclosure relates to genetically modified animals and cells that have humanized heavy chain immunoglobulin loci and / or humanized light chain immunoglobulin loci. [Background technology]

[0003] Therapeutic antibodies are one of the fastest growing classes of therapeutic compounds and are rapidly overtaking the growth of small molecule drugs. These therapeutic antibodies are usually human or humanized antibodies. Human or humanized antibodies can be generated by humanizing rodent antibodies (e.g., murine antibodies) or by using phage libraries. Antibodies generated by these methods often have suboptimal binding affinity and biophysical attributes, leading to manufacturing difficulties and poor pharmacokinetics. In particular, the humanization process can adversely affect binding affinity and can introduce immunogenic epitopes into the antibodies, and antibodies discovered using phage libraries exhibit limited diversity and non-natural immunoglobulin heavy and light chain pairings. Often, repeated experiments are required to spend time to improve the properties. In addition, in some cases, such antibodies can be immunogenic in patients, leading to their efficacy waning over time. Summary of the Invention [Problem to be solved by the invention]

[0004] One possible approach to creating fully human antibodies is to use transgenic animals engineered to express a human antibody repertoire. The creation of transgenic animals, such as mice with altered immunoglobulin loci, has enabled the use of such transgenic animals in a variety of research and development applications, including drug discovery and basic research on various biological systems. Many of the early generations of transgenic animals had incomplete human antibody repertoires, subnormal rates of antibody production due to inefficient V(D)J recombination, endogenous antibody repertoires that may carry immunogenic epitopes, and a variety of other problems. There is a need for efficient and cost-effective methods of generating human antibodies, and there is a need for non-human animals that contain humanized immunoglobulin loci capable of responding to antigens to give rise to humanized antibodies. [Means for solving the problem]

[0005] The present disclosure relates to genetically modified animals and cells having humanized heavy and light chain immunoglobulin loci.

[0006] In some aspects, the present disclosure relates to a genetically modified non-human animal that comprises one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at an endogenous heavy chain immunoglobulin locus. In some embodiments, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and capable of undergoing VDJ rearrangement.

[0007] In some embodiments, the animal comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes selected from Table 1, about or at least 20, 21, 22, 23, 24, 25, 26 or 27 human IGHD genes selected from Table 2, and about or at least 5, 6, 7, 8 or 9 human IGHJ genes selected from Table 3. In some embodiments, the animal comprises all of the human IGHV genes in Table 1 except IGHV2-10, IGHV3-9 and IGHV1-8, all of the human IGHD genes in Table 2, and all of the human IGHJ genes in Table 3. In some embodiments, the animal comprises all of the human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, all of the human IGHD genes in Table 2, and all of the human IGHJ genes in Table 3. In some embodiments, the animal comprises all of the human IGHV genes, all of the human IGHD genes, and all of the human IGHJ genes at the endogenous heavy chain immunoglobulin locus of human chromosome 14 of a human subject. In some embodiments, the animal comprises all of the human IGHV genes, all of the human IGHD genes, and all of the human IGHJ genes at the endogenous heavy chain immunoglobulin locus of human chromosome 14 of a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell lacking any V(D)J rearrangement).

[0008] In some embodiments, the animal comprises a disruption of the animal's endogenous heavy chain immunoglobulin locus.

[0009] In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and / or one or more mouse IGHJ genes in Table 6.

[0010] In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of a contiguous sequence beginning with the mouse IGHV1-85 gene through the mouse IGHJ4 gene.

[0011] In some embodiments, the animal comprises one or more endogenous IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.

[0012] In some embodiments, the animal comprises unmodified human sequences derived from a human heavy chain immunoglobulin locus.

[0013] In some embodiments, the unmodified human sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

[0014] In some embodiments, the animal comprises unmodified human sequences derived from a human heavy chain immunoglobulin locus from human IGHV(III)-82 through human IGHV1-2. In some embodiments, the animal comprises unmodified human sequences derived from a human heavy chain immunoglobulin locus from human IGHV(III)-82 through human IGHV6-1. In some embodiments, the animal comprises unmodified human sequences derived from a human heavy chain immunoglobulin locus from human IGHD1-1 through human IGHJ6.

[0015] In some embodiments, the animal comprises unmodified human sequences derived from the human heavy chain immunoglobulin locus beginning with human IGHV(III)-82 through human IGHJ6.

[0016] In some embodiments, the animal is homozygous for the heavy chain immunoglobulin locus. In some embodiments, the animal is heterozygous for the heavy chain immunoglobulin locus.

[0017] In some embodiments, the animal further comprises one or more human IGKV genes and one or more human IGKJ genes at the endogenous light chain immunoglobulin locus.

[0018] In some embodiments, the animal comprises a disruption of the animal's endogenous λ light chain immunoglobulin locus.

[0019] In some embodiments, the animal is a rodent (eg, a mouse).

[0020] In some aspects, the disclosure relates to a genetically modified animal comprising at an endogenous heavy chain immunoglobulin locus: a first sequence comprising one or more human IGHV genes; a second sequence comprising an endogenous sequence; and a third sequence comprising one or more human IGHD genes and one or more human IGHJ genes, wherein the first sequence, the second sequence, and the third sequence are operably linked.

[0021] In some embodiments, the first sequence comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 human IGHV genes selected from Table 1. In some embodiments, the first sequence comprises about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2.

[0022] In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus. In some embodiments, the first sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

[0023] In some embodiments, the second sequence comprises an endogenous sequence that is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kb.

[0024] In some embodiments, the third sequence comprises about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2. In some embodiments, the third sequence comprises about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3. In some embodiments, the third sequence comprises all of the human IGHD genes in Table 2 and all of the human IGHJ genes in Table 3.

[0025] In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus, hi some embodiments, the third sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kb.

[0026] In some embodiments, the animal comprises a disruption of the animal's endogenous heavy chain immunoglobulin locus.

[0027] In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and one or more mouse IGHJ genes in Table 6.

[0028] In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of sequences beginning with mouse IGHV1-85 through mouse IGHJ4.

[0029] In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of an IGHM, an IGHδ, an IGHG3, an IGHG1, an IGHG2b, an IGHG2a, an IGHE, and an IGHA gene.

[0030] In some embodiments, the animal is homozygous for the heavy chain immunoglobulin locus. In some embodiments, the animal is heterozygous for the heavy chain immunoglobulin locus.

[0031] In some embodiments, the animal further comprises one or more human IGKV genes and one or more human IGKJ genes at the endogenous light chain immunoglobulin locus.

[0032] In some embodiments, the animal comprises a disruption of the animal's endogenous λ light chain immunoglobulin locus.

[0033] In some embodiments, the animal is a rodent (eg, a mouse).

[0034] In some aspects, the disclosure relates to genetically modified non-human animals that comprise one or more human IGKV genes and one or more human IGKJ genes at their endogenous light chain immunoglobulin loci.

[0035] In some embodiments, the animal comprises about or at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 human IGKV genes in Table 7 and / or about or at least 1, 2, 3, 4 or 5 human IGKJ genes in Table 8.

[0036] In some embodiments, the animal comprises an unmodified sequence derived from a human light chain immunoglobulin locus beginning with human IGKV3D-7 through human IGKJ5.

[0037] In some embodiments, the animal comprises a disruption of the animal's endogenous light chain immunoglobulin locus.

[0038] In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous light chain immunoglobulin locus comprises a deletion of one or more mouse IGKV genes in Table 9 and one or more mouse IGKJ genes in Table 10. In some embodiments, the animal comprises all of the human IGKV genes and all of the human IGKJ genes at an endogenous kappa chain immunoglobulin locus on human chromosome 2 in a human subject. In some embodiments, the animal comprises all of the human IGKV genes and all of the human IGKJ genes at an endogenous kappa immunoglobulin locus on human chromosome 2 in a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell lacking any V(D)J rearrangement).

[0039] In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous light chain immunoglobulin locus comprises a deletion of sequences beginning with mouse IGKV2-137 through mouse IGKJ5.

[0040] In some embodiments, the animal comprises an endogenous IGKC.

[0041] In some embodiments, the animal is homozygous for the light chain immunoglobulin locus. In some embodiments, the animal is heterozygous for the light chain immunoglobulin locus.

[0042] In some embodiments, the animal further comprises one or more human IGHV genes, one or more human IGHD genes and one or more human IGHJ genes at the endogenous heavy chain immunoglobulin locus.

[0043] In some embodiments, the animal comprises a disruption of the animal's endogenous λ light chain immunoglobulin locus.

[0044] In some embodiments, the animal is a rodent (eg, a mouse).

[0045] In some aspects, the disclosure relates to a genetically modified non-human animal, the genome of which comprises an endogenous heavy chain immunoglobulin locus comprising a replacement of one or more endogenous IGHV, IGHD and IGHJ genes with one or more human IGHV, IGHD and IGHJ genes. In some embodiments, the human IGHV, IGHD and IGHJ genes are operably linked to one or more endogenous genes selected from the group consisting of IGHM, IGHδ, IGHG, IGHE and IGHA genes.

[0046] In some embodiments, one or more of the endogenous IGHV, endogenous IGHD and endogenous IGHJ genes are replaced by about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes in Table 1, about or at least 20, 21, 22, 23, 24, 25, 26 or 27 human IGHD genes in Table 2, and about or at least 5, 6, 7, 8 or 9 human IGHJ genes in Table 3.

[0047] In some embodiments, the animal is a mouse, and about or at least 180 mouse IGHV genes in Table 4, all of the mouse IGHD genes in Table 5, and all of the mouse IGHJ genes in Table 6 have been replaced.

[0048] In some aspects, the disclosure relates to a genetically modified non-human animal, the genome of which comprises an endogenous light chain immunoglobulin locus comprising a replacement of one or more endogenous IGKV and IGKJ genes with one or more human IGKV and IGKJ genes. In some embodiments, the human IGKV and IGKJ genes are operably linked to an endogenous IGKC gene.

[0049] In some embodiments, one or more endogenous IGKV and endogenous IGKJ genes are replaced by about or at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 human IGKV genes in Table 7 and about or at least 1, 2, 3, 4 or 5 human IGKJ genes in Table 8.

[0050] In some embodiments, the animal is a mouse, and all of the mouse IGKV genes in Table 9 and all of the mouse IGKJ genes in Table 10 are replaced.

[0051] In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain (e.g., a murine heavy chain variable domain).

[0052] In some embodiments, the animal lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain (e.g., a murine light chain variable domain).

[0053] In some embodiments, the animals are capable of producing humanized antibodies.

[0054] In some aspects, the disclosure relates to a cell obtained from an animal as described herein.

[0055] In some embodiments, the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, in some embodiments, the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region.

[0056] In some embodiments, the cell is a B cell that expresses a chimeric immunoglobulin light chain comprising an immunoglobulin light chain variable domain derived from a rearrangement of one or more human IGKV genes and one or more human IGKJ genes, wherein the immunoglobulin light chain variable domain is operably linked to a non-human light chain constant region.

[0057] In some embodiments, the cells are embryonic stem (ES) cells.

[0058] In some aspects, the disclosure relates to a method of making a chimeric antibody that specifically binds to an antigen, the method comprising exposing an animal to the antigen as described herein, producing a hybridoma from cells recovered from the animal, and recovering the chimeric antibody produced by the hybridoma. In some embodiments, the cells of interest are isolated and sequencing is performed to determine the sequence of the rearranged heavy and light chain variable regions.

[0059] In some embodiments, the method further comprises sequencing the genome of the hybridoma.

[0060] In some aspects, the disclosure relates to a method of modifying the genome of a cell, the method comprising modifying a human chromosome; introducing the modified human chromosome into a cell of an animal; and inducing recombination between the modified human chromosome and an endogenous chromosome, thereby replacing one or more endogenous genes with one or more human genes.

[0061] In some embodiments, the modified human chromosome comprises two or more exogenous recombination sites.

[0062] In some embodiments, the endogenous chromosome comprises two or more exogenous recombination sites.

[0063] In some embodiments, about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes selected from Table 1, about or at least 20, 21, 22, 23, 24, 25, 26 or 27 human IGHD genes selected from Table 2, and about or at least 5, 6, 7, 8 or 9 human IGHJ genes selected from Table 3 are recombinably integrated into an endogenous chromosome.

[0064] In some embodiments, about or at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 human IGKV genes in Table 7 and about or at least 1, 2, 3, 4 or 5 human IGKJ genes in Table 8 are recombinably integrated into an endogenous chromosome.

[0065] In some embodiments, the human sequence is recombination integrated into an endogenous chromosome, and the human sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

[0066] In one aspect, the disclosure provides a method of making an antibody that specifically binds to an antigen, the method comprising obtaining nucleic acid sequences encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen, the cell being obtained by exposing an animal as described herein to the antigen; operably linking a nucleic acid encoding a human heavy chain immunoglobulin variable region to a nucleic acid encoding a human heavy chain immunoglobulin constant region and a nucleic acid encoding a human light chain immunoglobulin variable region to a nucleic acid encoding a human light chain immunoglobulin constant region; and expressing the nucleic acid in the cell, thereby obtaining the antibody.

[0067] In one aspect, the disclosure provides a method for obtaining nucleic acids encoding an antibody binding domain that specifically binds to an antigen, the method comprising exposing an animal as described herein to the antigen; and sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in cells expressing a hybrid antibody that specifically binds to the antigen.

[0068] In one aspect, the disclosure provides a method of obtaining a sample, comprising exposing an animal to an antigen as described herein; and recovering a sample from the animal. In some embodiments, the sample is spleen tissue, spleen cells, or B cells.

[0069] In one aspect, the disclosure provides a method of making an antibody that specifically binds to an antigen, the method comprising exposing an animal as described herein to the antigen; obtaining (e.g., by sequencing) sequences of nucleic acids encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen; and operably linking, in the cell, the nucleic acid encoding the human heavy chain immunoglobulin variable region to a nucleic acid encoding a human heavy chain immunoglobulin constant region, and the nucleic acid encoding the human light chain immunoglobulin variable region to a nucleic acid encoding a human light chain immunoglobulin constant region.

[0070] The present disclosure also relates to the progeny of the non-human mammals.

[0071] In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0072] The present disclosure also provides a cell comprising a targeting vector as described herein.

[0073] The present disclosure also relates to cells (e.g., stem cells, embryonic stem cells, immune cells, B cells, T cells, or hybridomas) or cell lines or primary cell cultures thereof derived from the non-human mammal or its progeny. The present disclosure further relates to tissues, organs, or cultures thereof derived from the non-human mammal or its progeny.

[0074] The disclosure further relates to the use of non-human mammals or their progeny, animal models generated by the methods as described herein in the development of products related to immunization processes, the production of human antibodies or model systems for research in pharmacology, immunology, microbiology and medicine.

[0075] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall control.

[0076] Other features and advantages of the invention will become apparent from the following detailed description and figures, and from the claims. [Brief description of the drawings]

[0077] [Figure 1A] 1 is a flow chart of a method for introducing human immunoglobulin genes into the mouse genome. [Figure 1B] 1 is a schematic of the replacement of mouse immunoglobulin heavy chain variable regions with human immunoglobulin heavy chain variable regions. [Figure 1C] 1 is a schematic of the replacement of mouse immunoglobulin light chain variable regions with human immunoglobulin light chain variable regions. [Diagram 2] FIG. 1 is a schematic diagram showing the mouse heavy chain immunoglobulin locus. [Figure 3A] FIG. 1 is a schematic diagram showing the mouse heavy chain immunoglobulin locus following introduction of two recombination sites into the genome. [Figure 3B] FIG. 1 is a schematic diagram showing the mouse heavy chain immunoglobulin locus after recombination with a targeting vector. [Figure 4] 1 shows a targeting strategy for modification of the mouse heavy chain immunoglobulin locus. [Figure 5A] 1 shows the results of a PCR assay using the mIgHV-5'loxP-L-GT-F / mIGHV-005-L-GT-R2 primer pair. [Figure 5B] 1 shows the results of a PCR assay using the primer pair mIGHV-005-5'loxP-R-GT-F2 / mIgHV-5'loxP-R-GT-R. [Figure 6A] 1 shows the results of a PCR assay using the mIGHV-3'lox-L-GT-F2 / mIGHV-3'lox-L-GT-R2 primer pair. [Figure 6B] 1 shows the results of a PCR assay using the mIGHV3'lox-R-GT-F2 / mIGHV-3'lox-R-GT-R1 primer pair. [Figure 7] Schematic diagram of human chromosome 14, highlighting the heavy chain immunoglobulin locus (not to scale). The heavy chain immunoglobulin locus has variable regions (VH, DH, JH) and constant regions (CH). VH represents a segment of the IGHV gene cluster, DH represents a segment of the IGHD gene cluster, JH represents a segment of the IGHJ gene cluster, and CH represents the gene cluster expressing the constant domain. [Figure 8] FIG. 1 is a schematic diagram showing human chromosome 14 after modification. [Figure 9] Modifications on human chromosome 14 by two vectors are shown. [Figure 10] 1 shows modified human chromosome 14. [Figure 11]8 shows the results of a PCR assay for loxP site 301 on chromosome hChr14-mut3 using primer pairs hIGHV-5'loxP-L-GT-F1 and hIGHV-5'loxP-R-GT-R. 8-D7 is a positive control clone. [Figure 12] 8 shows the results of a PCR assay for loxP site 302 on chromosome hChr14-mut3. 8-D7 is a negative control clone. [Figure 13] Fluorescence in situ hybridization (FISH) images of cells before modification of human chromosome 14. [Figure 14] This is a FISH image of cells after modifying human chromosome 14. [Figure 15] FIG. 1 is a schematic diagram showing modified mouse chromosome 12. [Figure 16] FIG. 1 is a schematic diagram showing Cre-mediated recombination to replace the mouse heavy chain variable region locus with the corresponding human genomic DNA sequence. [Figure 17] 1 shows the results of a PCR assay using the M5-L and M5-R primer pairs. [Figure 18] 4 shows the results of a PCR assay using the M3 primer pair. [Figure 19] 1 shows the results of a PCR assay using the H5-L and H5-R primer pairs. [Figure 20] 1 shows the results of a PCR assay using the H3-L and H3-R primer pairs. [Figure 21] FISH images. White arrows (1) and (2) indicate mouse chromosome 12. White arrow (3) indicates a human chromosome fragment labeled with a human-specific IGH break-apart probe. [Figure 22] FIG. 1 is a schematic diagram showing Flp-mediated recombination. [Diagram 23] FIG. 1 is a schematic diagram showing the mouse light chain immunoglobulin locus. [Figure 24A] FIG. 1 is a schematic diagram showing the mouse light chain immunoglobulin locus following introduction of two recombination sites into the genome. [Figure 24B] FIG. 1 is a schematic diagram showing the mouse light chain immunoglobulin locus after recombination with a targeting vector. [Diagram 25] FIG. 1 is a schematic diagram showing the gene targeting strategy for mouse chromosome 6. [Figure 26] Shown are PCR assay results using the IGKV-005-C-5G-L-GT-F / IGKV-005-C-5G-L-GT-R2 primer pair. [Figure 27] Shown are PCR assay results using the IGKV-005-C-5G-R-GT-F1 / IGKV-005-C-5G-R-GT-R primer pair. [Figure 28] Schematic diagram of human chromosome 2 highlighting the light chain immunoglobulin locus (not to scale). VHK represents a segment of the IGKV gene cluster, JHK represents a segment of the IGKJ gene cluster, and CHK represents the IGKC gene. [Figure 29] FIG. 1 is a schematic diagram showing modified human chromosome 2. [Diagram 30] FIG. 1 is a schematic diagram showing the gene targeting strategy for human chromosome 2. [Diagram 31] The results of PCR assay after the first recombination (after introduction of vector 2702) are shown. WT is wild-type H9 cells. [Diagram 32] FISH image results. The white arrow indicates the correctly recombined modified human chromosome 2. [Diagram 33] The results of a PCR assay after the second recombination (after introduction of vector 2701) are shown. [Diagram 34] FIG. 1 is a schematic showing Cre-mediated recombination, in which a human light chain variable region genomic DNA sequence was added to the corresponding mouse locus. [Diagram 35] Fluorescence in situ hybridization (FISH) images. Arrows (1) and (3) indicate mouse chromosome 6. Arrow (2) indicates a human chromosome fragment labeled with a human-specific IGK break-apart probe. [Diagram 36] FIG. 1 is a schematic diagram showing Flp-mediated recombination. [Figure 37] FIG. 1 is a schematic diagram showing the human immunoglobulin heavy chain (IGH) locus on chromosome 14 (14q32.33). [Figure 38A] FIG. 1 is a schematic diagram showing the mouse (Mus musculus) IGH locus on chromosome 12 (12F2) (strain C57BL / 6). [Figure 38B] FIG. 1 is a schematic diagram showing the mouse (Mus musculus) IGH locus on chromosome 12 (12F2) (strain C57BL / 6). [Figure 39] FIG. 1 is a schematic diagram showing the human immunoglobulin kappa chain (IGK) locus on chromosome 2 (2p11.2). [Figure 40A] FIG. 1 is a schematic diagram showing the mouse (Mus musculus) IGK locus on chromosome 6 (6C1). [Figure 40B] FIG. 1 is a schematic diagram showing the mouse (Mus musculus) IGK locus on chromosome 6 (6C1). [Figure 41A] 1 shows a list of the IMGT repertoire of the human heavy chain immunoglobulin locus (IGH). [Figure 41B] 1 shows a list of the IMGT repertoire of the human heavy chain immunoglobulin locus (IGH). [Figure 41C] 1 shows a list of the IMGT repertoire of the human heavy chain immunoglobulin locus (IGH). [Figure 41D] 1 shows a list of the IMGT repertoire of the human heavy chain immunoglobulin locus (IGH). [Figure 41E] 1 shows a list of the IMGT repertoire of the human heavy chain immunoglobulin locus (IGH). [Fig.41F] 1 shows a list of the IMGT repertoire of the human heavy chain immunoglobulin locus (IGH). [Figure 42A] The IMGT repertoire of mouse IGH is shown. [Figure 42B]The IMGT repertoire of mouse IGH is shown. [Figure 42C] The IMGT repertoire of mouse IGH is shown. [Fig.42D] The IMGT repertoire of mouse IGH is shown. [Figure 42E] The IMGT repertoire of mouse IGH is shown. [Fig.42F] The IMGT repertoire of mouse IGH is shown. [Figure 42G] The IMGT repertoire of mouse IGH is shown. [Fig. 42H] The IMGT repertoire of mouse IGH is shown. [Fig.42I] The IMGT repertoire of mouse IGH is shown. [Figure 43A] 1 shows a list of the IMGT repertoire of the human κ chain immunoglobulin locus (IGK). [Figure 43B] 1 shows a list of the IMGT repertoire of the human κ chain immunoglobulin locus (IGK). [Figure 43C] 1 shows a list of the IMGT repertoire of the human κ chain immunoglobulin locus (IGK). [Fig. 43D] 1 shows a list of the IMGT repertoire of the human κ chain immunoglobulin locus (IGK). [Figure 44A] The IMGT repertoire of mouse IGK is shown. [Figure 44B] The IMGT repertoire of mouse IGK is shown. [Figure 44C] The IMGT repertoire of mouse IGK is shown. [Fig.44D] The IMGT repertoire of mouse IGK is shown. [Figure 44E] The IMGT repertoire of mouse IGK is shown. [Fig.44F] The IMGT repertoire of mouse IGK is shown. [Figure 44G] The IMGT repertoire of mouse IGK is shown. [Diagram 45] The percentage of leukocytes detected in peripheral blood is shown. [Diagram 46] The percentage of leukocytes detected in spleen cells is shown. [Figure 47] The percentage of leukocytes detected in the lymph nodes is shown. [Figure 48] The percentage of splenic B cells according to developmental stage is shown. [Figure 49] The percentage of lymph node B cells by developmental stage is shown. [Figure 50] The percentage of splenic B cells in the splenic marginal zone (MZ-B) and follicular zone (FO-B) is shown. [Figure 51A] Figure 1 shows flow cytometry analysis of B cells at different developmental stages in bone marrow obtained from wild type mice: region 1 represents pro-B cells, region 2 represents pre-B cells, and region 3 represents immature B cells. [Figure 51B] 1 shows flow cytometry analysis of B cells at different developmental stages in bone marrow obtained from humanized heavy chain heterozygous mice: region 1 represents pro-B cells, region 2 represents pre-B cells, and region 3 represents immature B cells. [Figure 51C] 1 shows flow cytometry analysis of B cells at different developmental stages in bone marrow obtained from humanized heavy chain homozygous mice: region 1 represents pro-B cells, region 2 represents pre-B cells, and region 3 represents immature B cells. [Figure 52] IgA isotype levels in serial dilutions of mouse serum are shown. [Figure 53] IgG1 isotype levels in serial dilutions of mouse serum are shown. [Figure 54] IgG2b isotype levels in serial dilutions of mouse serum are shown. [Figure 55] IgG2c isotype levels in serial dilutions of mouse serum are shown. [Figure 56] IgG3 isotype levels in serial dilutions of mouse serum are shown. [Figure 57] IgM isotype levels in serial dilutions of mouse serum are shown. [Figure 58]1 shows the distribution of detected IGKV gene expression after VJ recombination among individual mice. [Figure 59] Flow cytometry results of wild type mice after immunization with human BTLA are shown. [Figure 60] 1 shows flow cytometry results of humanized heavy chain homozygous mice after immunization with human BTLA. [Figure 61] Flow cytometry results of wild-type mice after immunization with canine PD-1 (dPD-1) are shown. [Figure 62] 1 shows flow cytometry results of humanized heavy chain homozygous mice after immunization with canine PD-1 (dPD-1). [Figure 63] A summary of ELISA results of wild-type mice (black bars; mice are designated 1-5) and humanized heavy chain homozygous mice (grey bars; mice are designated 6-10) after immunization with ovalbumin (OVA) is shown. [Figure 64] A list of human distal Vκ cluster IGKV genes and a list of human proximal Vκ cluster IGKV genes are shown. [Figure 65A] The body weights of naive wild-type mice and hVH / hVL mice are shown. [Figure 65B] Spleen weights of naive wild-type mice and hVH / hVL are shown. [Figure 66] The percentage of immune cells in the spleens of naive wild-type and hVH / hVL mice is shown. [Figure 67A] Percentages of type 1 transitional (T1, B220+IgM+IgD-), type 2 transitional (T2, B220+IgM+IgD+) and mature (M, B220+IgMlowIgD+) B cell populations in splenic B cells from naive wild type or hVH / hVL mice. [Figure 67B] The percentages of marginal zone (MZ) and follicular (FO) B cell populations among splenic B cells are shown. Splenic B cells were from naive wild type or hVH / hVL mice. [Figure 68A]The percentages of pro-B cell (B220lowCD43highIgMlow), pre-B cell (B220lowCD43intIgMlow) and immature B cell (B220highCD43lowIgMhigh) populations in bone marrow B cells are from naive wild type or hVH / hVL mice. [Figure 68B] The percentages of plasma cell (B220lowIgM-IgD-CD138-) and memory B cell (B220+IgM+IgD-CD38+) populations in bone marrow B cells are shown from naive wild type or hVH / hVL mice. [Figure 68C] The percentages of plasma cell (B220lowIgM-IgD-CD138-) and memory B cell (B220+IgM+IgD-CD38+) populations in splenic B cells are shown from naive wild type or hVH / hVL mice. [Figure 69] Serum immunoglobulin (Ig) subtype concentrations in naive wild-type or hVH / hVL mice are shown. Ig subtype concentrations were quantitatively measured by ELISA. [Figure 70A] IGHV usage (frequency >1%) in naive hVH / hVL mice is shown. [Figure 70B] IGHV usage in naive hVH / hVL mice (frequency <1%) is shown. [Figure 70C] IGHD use of naive hVH / hVL mice is shown. [Fig. 70D] 1 shows IGHJ usage in naive hVH / hVL mice. [Figure 71A] IGKV usage (frequency >1%) in naive hVH / hVL mice is shown. [Figure 71B] IGKV usage in naive hVH / hVL mice (frequency <1%) is shown. [Figure 71C] IGKJ use in naive hVH / hVL mice is shown. [Figure 72] 1 is a histogram showing the amino acid length distribution of the heavy chain CDR3 of naive hVH / hVL mice. [Figure 73] 1 shows the amino acid frequency in the heavy chain CDR3 of naive hVH / hVL mice. [Figure 74] 1 shows the frequency of HCDR3 containing cysteine ​​residues in hVH / hVL mice. [Figure 75] A is a histological image of the spleen of an naive wild-type mouse, B is a histological image of the inguinal lymph node of an naive wild-type mouse, C is a histological image of the Peyer's patch of an naive wild-type mouse, D is a histological image of the spleen of an naive hVH / hVL mouse, E is a histological image of the inguinal lymph node of an naive hVH / hVL mouse, and F is a histological image of the Peyer's patch of an naive hVH / hVL mouse. [Figure 76A] Figure 1 shows BCMA-specific antibody titers after the second and third immunizations in wild type and hVH / hVL mice using human BCMA (B cell maturation antigen) as antigen. [Figure 76B] IL4R-specific antibody titers after the second and third immunizations in wild-type and hVH / hVL mice using human IL4R (interleukin-4 receptor) as the antigen are shown. [Figure 76C] PD-1 specific antibody titers after the second and third immunizations in wild type and hVH / hVL mice using human PD-1 (programmed cell death protein 1) as antigen. [Fig. 76D] Figure 1 shows Siglec-15 specific antibody titers after the second and third immunizations in wild type and hVH / hVL mice using human Siglec-15 (sialic acid-binding immunoglobulin-like lectin 15) as antigen. [Figure 76E] SIRPα (signal regulatory protein α)-specific antibody titers after the second and third immunizations in wild-type and hVH / hVL mice using human SIRPα as antigen. [Figure 77A] The body weights of wild-type and hVH / hVL mice after immunization are shown. [Fig. 77B] Spleen weights of wild-type and hVH / hVL mice after immunization are shown. [Figure 78] The percentage of immune cells in the spleens of wild-type and hVH / hVL mice after immunization is shown. [Figure 79A]The percentages of type 1 transitional (T1, B220+IgM+IgD-), type 2 transitional (T2, B220+IgM+IgD+) and mature (M, B220+IgMlowIgD+) B cell populations in splenic B cells are shown from wild-type or hVH / hVL mice after immunization. [Fig. 79B] The percentages of marginal zone (MZ) and follicular (FO) B cell populations among splenic B cells are shown from wild type or hVH / hVL mice after immunization. [Figure 80A] The percentages of pro-B cell (B220lowCD43highIgMlow), pre-B cell (B220lowCD43intIgMlow) and immature B cell (B220highCD43lowIgMhigh) populations in bone marrow B cells are from wild type or hVH / hVL mice after immunization. [Figure 80B] The percentages of plasma cell (B220lowIgM-IgD-CD138-) and memory B cell (B220+IgM+IgD-CD38+) populations in bone marrow B cells are shown from wild type or hVH / hVL mice after immunization. [Fig. 80C] The percentages of plasma cell (B220lowIgM-IgD-CD138-) and memory B cell (B220+IgM+IgD-CD38+) populations in splenic B cells are shown from wild-type or hVH / hVL mice after immunization. [Figure 81] Serum immunoglobulin (Ig) subtype concentrations in naive wild-type or hVH / hVL mice or after the third immunization are shown. Ig subtype concentrations were determined by ELISA. [Figure 82] Serum total IgG concentrations in naive wild type or hVH / hVL mice or after immunization are shown. IgG concentrations were determined by ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] The present disclosure relates to genetically modified animals and cells that have a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus (eg, a κ chain locus).

[0079] Genetically modified animals can be produced by introducing human immunoglobulin genes into the genome of a non-human animal to produce an animal capable of expressing humanized or chimeric antibodies. FIG. 1A shows a method for producing a humanized mouse. In some embodiments, the method first involves modifying a human immunoglobulin region on a human chromosome. The modified human chromosome is then introduced into a mouse recipient cell. The human immunoglobulin variable region is then introduced by direct replacement (e.g., in a one-step replacement) into the corresponding region of the mouse genome. The recipient cells are then screened, preferably for cells that do not contain human chromosomes. The cells are then injected into blastocysts to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals that contain intact humanized immunoglobulin loci.

[0080] The genetically modified animals described herein may have various advantages. For example, in some cases, the genetically modified animals described herein have a fully human antibody repertoire. Thus, the variable domains produced by such animals may have a diversity very similar to that of human variable domains. Furthermore, because the entire sequence of the human immunoglobulin locus (without or limited modifications) is introduced into the animal genome, the genes can undergo V(D)J recombination very similar to that which occurs in humans. In addition, antibody production may be very efficient, and because V(D)J recombination is efficient, the production rate is comparable to the normal production rate. In addition, because V(D)J recombination may occur between endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes and human genes, when endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes are incorporated into rearranged heavy chain VDJ segments or rearranged light chain VJ segments, the antibodies produced by the antibody repertoire are likely to have epitopes that are immunogenic in humans. This immunogenicity can lead to the production of anti-drug antibodies and can include efficacy. Here, endogenous IGHV, IGHD, IGHJ, IGKV and IGKJ genes are effectively deleted. The antibodies generated by this antibody repertoire are less likely to be immunogenic in humans. Thus, the antibodies are more suitable for use as therapeutic agents in humans. Thus, genetically modified animals provide an advantageous platform for the production of humanized antibodies.

[0081] As used herein, the term "antibody" refers to an immunoglobulin molecule that comprises four polypeptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable (VH) domain and a heavy chain constant region (CH). Each light chain comprises a light chain variable (VL) domain and a light chain constant region (CL). The VH and VL domains can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody that has a K D About 10 -9 M or less (for example, about 1×10 -9 M, 1×10 -10 M, 1×10 -11 M or 1×10 -12 In some embodiments, the antibody is an antibody having a molecular weight of K D can be measured by surface plasmon resonance, for example BIACORE™ or ELISA.

[0082] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, where this portion of the antibody has the ability to specifically bind to an antigen. In some embodiments, the antigen-binding fragment comprises at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2 and Fv fragments.

[0083] As used herein, the term "human antibody" refers to an antibody encoded by nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in a human cell culture (e.g., a human hybridoma cell). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a mouse) that includes unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0084] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody that contains a variable domain sequence of a human antibody (e.g., all or a portion of a light and / or heavy chain variable domain sequence) and a constant domain of a non-human antibody. Further examples of chimeric antibodies are described herein and known in the art.

[0085] As used herein, the term "humanized antibody" refers to non-human antibodies that contain sequences derived from non-human (e.g., murine) immunoglobulin and that contain sequences derived from human immunoglobulin.

[0086] As used herein, the term "single-chain antibody" refers to a single polypeptide comprising at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) capable of specific binding to an antigen.

[0087] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human or non-human. Veterinary and non-veterinary applications are contemplated by the present disclosure. A human patient can be an adult human or a juvenile human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, minipigs), equines, canines, felines, bovines and other domestic, farm and zoo animals.

[0088] As used herein, the phrases "specifically binding" and "specifically bind," when referring to an antibody, mean that the antibody interacts preferentially with its target molecule over other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule. In other words, the reagent does not recognize and bind to any molecule in general, but rather recognizes and binds to a molecule that contains a specific structure. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody.

[0089] As used herein, the terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0090] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to nucleotide polymers of any length, at least two nucleotides, and include, without limitation, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0091] As used herein, the term "unmodified human sequence" refers to a sequence derived from a human subject, a human cell, a cultured human cell, or a human cell line, where the sequence is identical to a gene sequence in the human subject, human cell, cultured human cell, or human cell line.

[0092] Genetically modified heavy chain immunoglobulin locus The heavy chain immunoglobulin locus (also known as IGH or immunoglobulin heavy chain locus) is a region on a chromosome (eg, human chromosome 14) that contains the genes for the heavy chain of human antibodies (or immunoglobulins).

[0093] This region represents the germline configuration of the heavy chain locus. This locus contains V (variable), D (diversity), J (joining) and C (constant) segments. The genes of the V region form the V gene cluster (also known as the IGHV gene cluster). The genes of the D region form the D gene cluster (also known as the IGHD gene cluster). The genes of the J region form the J gene cluster (also known as the IGHJ gene cluster).

[0094] During B cell development, a recombination event at the DNA level joins a single D segment (also known as the IGHD gene) to a J segment (also known as the IGHJ gene). The fused DJ exons of this partially rearranged DJ region are then joined to a V segment (also known as the IGHV gene). The rearranged VDJ region, including the fused VDJ exons, is then transcribed and fused to the IGHM constant region at the RNA level. This transcript encodes a μ heavy chain. Later in development, B cells produce a VDJ-Cμ-Cδ pre-messenger RNA that, upon alternative splicing, encodes either a μ or a δ heavy chain. Mature B cells in lymph nodes undergo switch recombination, whereby the fused VDJ gene segment is brought into close proximity with one of the IGHG, IGHA or IGHE gene segments, and each cell expresses either a gamma, alpha or epsilon heavy chain. Recombination can occur between many different IGHV genes and several IGHJ genes, conferring a broad range of antigen recognition. Further diversity is achieved through junction diversity generated by random nucleotide addition by terminal deoxynucleotide convertase and through somatic hypermutation that occurs during B cell maturation in the spleen and lymph nodes. Several V, D, J and C segments are known to have no protein-coding capacity and are considered pseudogene segments (often simply referred to as pseudogenes).

[0095] The human heavy chain immunoglobulin locus is located on human chromosome 14. Table 1 lists the IGHV genes at this locus and their relative order.

[0096] [Table 1]

[0097] RPS8P1, ADAM6 and KIAA0125 are also located at this locus. The relative order of RPS8P1 is 160, the relative order of ADAM6 is 161 and the relative order of KIAA0125 is 164. Table 2 shows a list of all IGHD genes on human chromosome 14 and their relative order. Table 3 shows a list of all IGHJ genes on human chromosome 14 and their relative order. After the IGHV, IGHD and IGHJ genes are located the genes for the immunoglobulin constant domain. These genes include (as shown in the following order): immunoglobulin heavy chain constant μ (IGHM), immunoglobulin heavy chain constant δ (IGHδ), immunoglobulin heavy chain constant gamma 3 (IGHG3), immunoglobulin heavy chain constant gamma 1 (IGHG1), immunoglobulin heavy chain constant εP1 (pseudogene) (IGHEP1), immunoglobulin heavy chain constant α1 (IGHA1), immunoglobulin heavy chain constant gamma P (non-functional) (IGHGP), immunoglobulin heavy chain constant gamma 2 (IGHG2), immunoglobulin heavy chain constant gamma 4 (IGHG4), immunoglobulin heavy chain constant ε (IGHE), and immunoglobulin heavy chain constant α2 (IGHA2). These genes and the order of these genes are also shown in Figure 37, Figure 41A, Figure 41B, Figure 41C, Figure 41D, Figure 41E, and Figure 41F.

[0098] [Table 2]

[0099] [Table 3]

[0100] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12. Table 4 lists the IGHV genes at this locus and their relative order.

[0101] [Table 4]

[0102] Table 5 lists all IGHD genes on mouse chromosome 12 and their relative order. Table 6 lists all IGHJ genes on mouse chromosome 12 and their relative order. Following the IGHV, IGHD and IGHJ genes are the genes for the immunoglobulin constant domain. These genes include (as shown in the following order): immunoglobulin heavy chain constant μ (IGHM), immunoglobulin heavy chain constant delta (IGHδ), immunoglobulin heavy chain constant gamma 3 (IGHG3), immunoglobulin heavy chain constant gamma 1 (IGHG1), immunoglobulin heavy chain constant gamma 2b (IGHG2b), immunoglobulin heavy chain constant gamma 2a (IGHG2a), immunoglobulin heavy chain constant epsilon (IGHE) and immunoglobulin heavy chain constant alpha (IGHA) genes. These genes and the order of these genes are also shown in Figures 38A, 38B, 42A, 42B, 42C, 42D, 42E, 42F, 42G, 42H, and 42I.

[0103] [Table 5]

[0104] [Table 6]

[0105] The present disclosure provides a genetically modified non-human animal comprising one or more human IGHV genes, one or more human IGHD genes and / or one or more human IGHJ genes. In some embodiments, the human IGHV genes, the human IGHD genes and the human IGHJ genes are operably linked to each other and can undergo VDJ rearrangement. In some embodiments, the human IGHV genes, the human IGHD genes and the human IGHJ genes are in an endogenous heavy chain immunoglobulin locus.

[0106] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes (e.g., genes as shown in Table 1).

[0107] In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76 and IGHV3-75.

[0108] In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1 and IGHV6-1.

[0109] In some embodiments, the animal comprises an unmodified human sequence beginning with a gene selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75, and ending with a gene selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus beginning with human IGHV(III)-82 through human IGHV1-2. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 through human IGHV(II)-1-1. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 through human IGHV-6-1.

[0110] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes (e.g., genes as shown in Table 2). In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27.

[0111] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes (e.g., genes as shown in Table 3). In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6.

[0112] In some embodiments, the animal comprises an unmodified human sequence that includes a sequence beginning with a gene selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27 and ending with a gene selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus beginning with human IGHD1-1 through human IGHJ6.

[0113] In some embodiments, the sequence is unmodified human derived from the human heavy chain immunoglobulin locus beginning at human IGHD1-1 through human IGHD7-27.

[0114] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHJ1P through human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHJ1 through human IGHJ6.

[0115] In some embodiments, the sequence is unmodified human derived from the human heavy chain immunoglobulin locus starting from human IGHV(III)-82 through human IGHJ6.

[0116] In some embodiments, the human heavy chain immunoglobulin locus is an unmodified human sequence from human IGHV1-2 through human IGHJ6. In some embodiments, the human heavy chain immunoglobulin locus is an unmodified human sequence from human IGHV(II)-1-1 through human IGHJ6. In some embodiments, the human heavy chain immunoglobulin locus is an unmodified human sequence from human IGHV6-1 through human IGHJ6.

[0117] In some embodiments, the animal may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified human sequences. In some embodiments, the unmodified human sequences are about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb in length.

[0118] In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of immunoglobulin heavy chain constant μ (IGHM), immunoglobulin heavy chain constant δ (IGHδ), immunoglobulin heavy chain constant gamma 3 (IGHG3), immunoglobulin heavy chain constant gamma 1 (IGHG1), immunoglobulin heavy chain constant gamma 2b (IGHG2b), immunoglobulin heavy chain constant gamma 2a (IGHG2a), immunoglobulin heavy chain constant ε (IGHE) and immunoglobulin heavy chain constant alpha (IGHA) genes. In some embodiments, these endogenous genes are operably linked to each other. In some embodiments, these endogenous genes are in the same order as in a wild-type animal. In some embodiments, isotype switching (immunoglobulin class switching) can occur in the animal.

[0119] In some embodiments, the IGHV, IGHD and / or IGHJ genes are operably linked to each other. VDJ recombination can occur between these genes to produce functional antibodies. In some embodiments, these genes are arranged in an order similar to that of the human heavy chain immunoglobulin locus. This arrangement provides various advantages, for example, the arrangement of these genes allows the production of heavy chain variable domains with a diversity that is very similar to that of heavy chain variable domains in humans. In some embodiments, a fully human antibody repertoire with no or only minimal modifications may reduce the likelihood of non-human sequences being inserted during VDJ recombination, since some random sequences may be inserted into the sequence during VDJ recombination.

[0120] In some embodiments, the IGHV gene, IGHD gene and / or IGHJ gene are operably linked to one or more genes (e.g., all of the genes) selected from the IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE and IGHA genes.

[0121] In some embodiments, the animal comprises a disruption of the animal's endogenous heavy chain immunoglobulin locus, in some embodiments, the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of one or more endogenous IGHV genes, one or more endogenous IGHD genes, and one or more endogenous IGHJ genes.

[0122] In some embodiments, the animal is a mouse. The disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182 mouse IGHV genes (e.g., genes as shown in Table 4). In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78 and IGHV1-77 (e.g., IGHV1-86).

[0123] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1. In some embodiments, the mouse still comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1.

[0124] In some embodiments, the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mouse IGHD genes (e.g., genes as shown in Table 5). In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2 and IGHD4-1.

[0125] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4. In some embodiments, the mouse still comprises about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.

[0126] In some embodiments, the disruption of an animal's endogenous heavy chain immunoglobulin locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, or 3000 kb of endogenous sequence.

[0127] In some embodiments, the deleted sequence is from IGHV1-86 to IGHJ4, from IGHV1-85 to IGHJ4, from IGHV1-84 to IGHJ4, from IGHV1-83 to IGHJ4 or from IGHV1-82 to IGHJ4 (e.g., from IGHV1-85 to IGHJ4).

[0128] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence in a human heavy chain immunoglobulin locus. In some embodiments, the sequences are about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb in length. In some embodiments, the sequence is human IGHV(III)-82 to IGHV1-2. In some embodiments, the sequence is human IGHV7-81 to IGHV1-2. In some embodiments, the sequence is human IGHV(II)-1-1 to IGHVJ6. In some embodiments, the sequence is human IGHV6-1 to IGHVJ6.

[0129] The human IGHV gene, the human IGHD gene and the human IGHJ gene are operably linked to each other and can undergo VDJ rearrangement. In some embodiments, the modified mouse has a complete human IGHV, IGHD and IGHJ gene repertoire (e.g., including all non-pseudohuman IGHV, IGHD and IGHJ genes). Thus, the modified mouse can produce a complete human antibody repertoire. In some embodiments, after VDJ recombination, one IGHV gene (e.g., IGHV3-21 or IGHV3-74) in Table 15 contributes to the sequence encoding the antibody heavy chain variable region. One IGHD gene in Table 15 contributes to the sequence encoding the antibody heavy chain variable region. Also, one IGHJ gene in Table 15 contributes to the sequence encoding the antibody heavy chain variable region. In some embodiments, the IGHV gene is IGHV3-21 or IGHV3-74.

[0130] In some embodiments, one IGHV gene in Figures 70A and 70B (e.g., IGHV3-30, IGHV3-33, IGHV4-39, or IGHV4-34) contributes sequence encoding an antibody heavy chain variable region. One IGHD gene in Figure 70C (e.g., IGHD6-19) contributes sequence encoding an antibody heavy chain variable region. And one IGHJ gene in Figure 70D (e.g., IGHJ4 or IGHJ6) contributes sequence encoding an antibody heavy chain variable region. In some embodiments, one IGKV gene in Figures 71A and 71B (e.g., IGKV4-1, IGKV1-33, IGKV2-30) contributes sequence encoding an antibody light chain variable region. One IGKJ gene in Figure 71C (e.g., IGKJ1, IGKJ2, or IGKJ4) contributes sequence encoding an antibody light chain variable region.

[0131] Furthermore, in some cases, the entire mouse IGHV, IGHD and IGHJ genes (e.g., including all non-pseudogenes) are knocked out, such that the heavy chain variable region does not have any sequences encoded by sequences derived from the mouse, thus minimizing immunogenicity in humans.

[0132] Genetically modified kappa light chain immunoglobulin locus The kappa chain immunoglobulin locus (also known as IGK or immunoglobulin kappa locus) is a region on a chromosome (e.g., human chromosome 2) that contains the genes for the human antibody (or immunoglobulin) light chain. Similarly, the immunoglobulin light chain genes can also undergo a series of rearrangements that lead to the production of a mature immunoglobulin light chain nucleic acid (e.g., a kappa chain).

[0133] When the V segment (also called IGKV gene) and the J segment (also called IGKJ gene) join, they create a continuous exon sequence that encodes the entire light chain variable domain. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far from the C region. The J gene segment (or IGKJ gene cluster) is located close to the C region. When the V segment joins to the J gene segment, the V gene also moves closer to the C region sequence. The J gene segment of the rearranged V region is separated from the C region sequence by only one intron. The V region exons are joined to the C region sequence by RNA splicing after transcription to create the complete immunoglobulin light chain messenger RNA.

[0134] The human light chain immunoglobulin locus is located on human chromosome 2. Table 7 lists the IGKV genes at this locus and their relative order. There are several different groups of human IGKV genes, including IGKV1 genes (includes all IGKV genes starting at IGKV1, also known as VκI), IGKV2 genes (includes all IGKV genes starting at IGKV2, also known as VκII), IGKV3 genes (includes all IGKV genes starting at IGKV3, also known as VκIII), IGKV4 genes (includes all IGKV genes starting at IGKV4, also known as VκIV), IGKV5 genes (includes all IGKV genes starting at IGKV5, also known as VκV), IGKV6 genes (includes all IGKV genes starting at IGKV6, also known as VκVI), and IGKV7 genes (includes all IGKV genes starting at IGKV7, also known as VκVII).

[0135] These IGKV genes on human chromosome 2 also form two clusters, the proximal Vκ cluster and the distal Vκ cluster (Figure 28). The sequences in these two clusters are similar but not identical. This large segmental duplication of sequences occurred after the divergence of the human lineage from the most recent shared ancestor that included other great apes. The related IGVK genes in each cluster are summarized in Figure 64.

[0136] [Table 7]

[0137] Table 8 lists all the IGKJ genes and their relative order on human chromosome 2. Following the IGKV and IGKJ genes is the immunoglobulin kappa constant (IGKC) gene, which encodes the light chain immunoglobulin constant domain. These genes and the order of these genes are also shown in Figure 39, Figure 43A, Figure 43B, Figure 43C, and Figure 43D.

[0138] [Table 8]

[0139] The mouse light chain immunoglobulin locus is located on mouse chromosome 6. Table 9 lists the IGKV genes at this locus and their relative order.

[0140] [Table 9]

[0141] Gm9728 and Amd-ps2 are also located at this locus. The relative order of Gm9728 is 4 and the relative order of Amd-ps2 is 134. Table 10 shows a list of all IGKJ genes on mouse chromosome 6 and their relative order. Following the IGKV and IGKJ genes is the IGKC gene, which encodes the light chain immunoglobulin constant domain. These genes and the order of these genes are also shown in Figure 40A, Figure 40B, Figure 44A, Figure 44B, Figure 44C, Figure 44D, Figure 44E, Figure 44F, and Figure 44G.

[0142] [Table 10]

[0143] The present disclosure provides genetically modified non-human animals comprising one or more human IGKV genes and / or one or more human IGKJ genes. In some embodiments, the human IGKV genes and the human IGKJ genes are operably linked to each other and capable of undergoing VJ rearrangement. In some embodiments, the human IGKV genes and the human IGKJ genes are in an endogenous light chain immunoglobulin locus.

[0144] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 human IGKV genes (e.g., genes as shown in Table 7).

[0145] In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes selected from IGKV3D-7, IGKV1D-8, IGKV1D-43, IGKV1D-42, IGKV2D-10, IGKV3D-11, IGKV1D-12, IGKV1D-13, IGKV2D-14 and IGKV3D-15.

[0146] In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes selected from IGKV2-10, IGKV1-9, IGKV1-8, IGKV3-7, IGKV1-6, IGKV1-5, IGKV2-4, IGKV7-3, IGKV5-2 and IGKV4-1.

[0147] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, or 5 human IGKJ genes (e.g., genes as shown in Table 3). In some embodiments, the animal comprises 1, 2, 3, 4, or 5 human IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.

[0148] In some embodiments, the animal comprises an endogenous IGKC. In some embodiments, the IGKV and / or IGKJ genes are operably linked to each other. VJ recombination can occur between these genes to produce functional antibodies. In some embodiments, the genes are arranged in an order similar to the order of the human light chain immunoglobulin locus. This arrangement provides various advantages, for example, the arrangement of the genes allows for the production of light chain variable domains with a diversity that is very similar to that of light chain variable domains in humans.

[0149] In some embodiments, the IGKV gene and / or the IGKJ gene are operably linked to each other with an IGKC gene (eg, an endogenous IGKC gene).

[0150] In some embodiments, the animal comprises a disruption of the animal's endogenous light chain immunoglobulin locus, hi some embodiments, the disruption of the animal's endogenous light chain immunoglobulin locus comprises a deletion of one or more endogenous IGKV genes and one or more endogenous IGKJ genes.

[0151] In some embodiments, the animal is a mouse. The disruption of the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163 mouse IGKV genes (e.g., genes as shown in Table 9). In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130 and IGKV9-129. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130 and IGKV9-129.

[0152] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1.

[0153] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5).

[0154] In some embodiments, the disruption of an animal's endogenous kappa light chain immunoglobulin locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb of endogenous sequence.

[0155] In some embodiments, the deleted sequence is from IGKV2-137 to IGKJ4, from IGKV1-136 to IGKJ4, from IGKV1-135 to IGKJ4, from IGKV2-137 to IGKJ5, from IGKV1-136 to IGKJ5 or from IGKV1-135 to IGKJ5 (e.g. from IGKV2-137 to IGKJ5).

[0156] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence in a human light chain immunoglobulin locus. In some embodiments, the sequences are about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb in length.

[0157] In some embodiments, the animal may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified human sequences. In some embodiments, the unmodified human sequences are about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, or 3500 kb in length.

[0158] In some embodiments, the sequence is human IGKV3D-7 to IGKJ5. In some embodiments, the sequence is human IGKV3D-7 to IGKJ4. In some embodiments, the sequence is human IGKV1D-8 to IGKJ5. In some embodiments, the sequence is human IGKV1D-8 to IGKJ4.

[0159] The human IGKV gene and the human IGKJ gene are operably linked to each other and can undergo VJ rearrangement. In some embodiments, the modified mouse has a full human IGKV and IGKJ gene repertoire (e.g., including all non-pseudo-human IGKV and IGKJ genes). Thus, the modified mouse can produce a full human antibody repertoire. In some embodiments, after VJ recombination, one IGKV gene in Table 16 (e.g., IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12) contributes to the sequence encoding the antibody light chain variable region. One human IGKJ gene contributes to the sequence encoding the antibody light chain variable region. In some embodiments, the IGKV gene is IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12. Furthermore, in some cases, the entire mouse IGKV and IGKJ genes (all non-pseudogenes) are knocked out and the light chain variable region does not have any sequences encoded by sequences derived from the mouse, thus minimizing immunogenicity in humans.

[0160] In some embodiments, the modified chromosome comprises a human proximal VK cluster IGKV gene. In some embodiments, the modified chromosome comprises a human distal VK cluster IGKV gene. In some embodiments, the modified chromosome comprises both a human proximal VK cluster IGKV gene and a human distal VK cluster IGKV gene.

[0161] Genetically modified lambda light chain immunoglobulin locus The lambda chain immunoglobulin locus (also known as IGL or immunoglobulin lambda locus) is a region on a chromosome (e.g., human chromosome 22) that contains the genes for the light chain of human antibodies (or immunoglobulins). Similarly, immunoglobulin light chain genes can undergo a series of rearrangements that lead to the production of mature immunoglobulin light chain nucleic acids (e.g., lambda chains). In healthy human individuals, the overall kappa to lambda ratio is approximately 2:1 in serum (measured whole intact antibodies) or 1:1.5 when measuring free light chains. In mice, the overall kappa to lambda ratio is approximately 9:1.

[0162] In some embodiments, the animal comprises a human λ chain immunoglobulin locus.

[0163] In some embodiments, the animal comprises a disruption of the animal's endogenous λ light chain immunoglobulin locus. In some embodiments, the disruption of the animal's endogenous light chain immunoglobulin locus comprises a deletion of one or more endogenous IGLV genes, one or more endogenous IGLJ genes, and / or one or more immunoglobulin λ constant (IGLC) genes (e.g., IGLC1, IGLC2, IGLC3, and IGLC4).

[0164] The mouse lambda light chain immunoglobulin locus (IGL locus) is located on mouse chromosome 16. Table 11 lists the IGLV, IGLJ and IGLC genes at this locus and their relative order.

[0165] [Table 11]

[0166] The disruption of the animal's endogenous lambda light chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mouse IGLV, IGLJ, and IGLC genes (e.g., genes as shown in Table 11). In some embodiments, the deletion comprises about or at least 1, 2, 3, or 4 mouse IGKC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption comprises a deletion of about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3. In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ3, IGLJ3P, and IGLJ4.

[0167] In some embodiments, the disruption of an animal's endogenous lambda light chain immunoglobulin locus comprises a deletion of about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 210 kb, 220 kb, 230 kb, 240 kb, 250 kb, 260 kb, 270 kb, 280 kb, 290 kb, 300 kb, 350 kb, 400 kb, 450 kb, 500 kb, or 1000 kb of nucleotides. In some embodiments, there is no disruption in the animal's endogenous λ light chain immunoglobulin gene.

[0168] In some embodiments, the deleted sequence is from IGLV2 to IGLC1, from IGLV3 to IGLC1, or from IGLJ2 ​​to IGLC1.

[0169] Genetically modified animals In one aspect, the disclosure provides a genetically modified non-human animal comprising a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus. In some embodiments, the animal comprises one or more human IGHV genes, one or more human IGHD genes, one or more human IGHJ genes, one or more human IGKV genes and / or one or more human IGKJ genes. In some embodiments, these genes are located at endogenous immunoglobulin loci.

[0170] In some embodiments, the animal comprises a human λ chain immunoglobulin locus. In some embodiments, the animal comprises a disruption in the animal's endogenous λ light chain immunoglobulin locus. In some embodiments, the animal does not have a disruption in the animal's endogenous λ light chain immunoglobulin locus.

[0171] The genetically modified non-human animals can be various animals, such as mice, rats, rabbits, pigs, bovines (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are used to generate non-human animals that include genetic modifications. Such methods include, for example, modification of the non-ES cell genome (e.g., fibroblasts or induced pluripotent cells) and transfer of the modified genome into a suitable cell, such as an oocyte, using nuclear transfer and gestation of the modified cell (e.g., modified oocyte) in the non-human animal under suitable conditions to form an embryo. These methods are known in the art and described, for example, in A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)", Cold Spring Harbor Laboratory Press, 2003, which is incorporated herein by reference in its entirety. Thus, in various embodiments, human V, D and / or J segments can be operably linked to non-human animal (e.g., rodent, mouse, rat, hamster) constant region gene sequences, and during B cell development, these rearranged human V, D and / or J segments are linked to the non-human animal immunoglobulin constant region.

[0172] In one aspect, the animal is a mammal, such as a mammal of the superfamily Dipodoidea or the superfamily Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is of a family selected from Calomyscidae (e.g., kangaroo hamsters), Cricetidae (e.g., hamsters, New World rats and mice, and voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, and maned mice), Nesomyidae (tree mice, rock mice, white-tailed hamsters, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and mole rats). In some embodiments, the genetically modified rodent is selected from pure-breed mice or rats (Muridae), gerbils, spiny mice, and maned mice. In some embodiments, the non-human animal is a mouse.

[0173] In some embodiments, the animal is a mouse on a C57 background (e.g., a 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 some embodiments, the mouse is a 129 strain selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a cross between a 129 strain and a C57BL / 6 strain. In some embodiments, the mouse is a cross between a 129 strain or a BL / 6 strain. In some embodiments, the mouse is a BALB strain, e.g., a BALB / c strain. In some embodiments, the mouse is a cross between a BALB strain and another strain. In some embodiments, the mice are of a mixed strain (eg, 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129).

[0174] In some embodiments, the animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and dark agouti. In some embodiments, the rat strain is a cross of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and dark agouti.

[0175] The animal may have one or more other genetic and / or other modifications suitable for the particular purpose of generating a humanized animal.

[0176] A genetically modified non-human animal comprising a modification of an endogenous non-human immunoglobulin locus. In some embodiments, the modification may comprise a human nucleic acid sequence encoding at least a portion of a human protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a human heavy chain variable domain or light chain variable domain sequence). Genetically modified cells (e.g., ES cells, somatic cells) that may comprise the modifications described herein are also provided, although in many embodiments, the genetically modified non-human animal comprises a modification of an endogenous locus in the germline of the animal.

[0177] The genetically modified animals are capable of expressing humanized and / or chimeric antibodies from endogenous mouse loci in which one or more endogenous mouse immunoglobulin genes have been replaced with a human immunoglobulin gene and / or a nucleotide sequence at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a human immunoglobulin gene sequence (e.g., an IGHV, IGHD, IGHJ, IGKV and / or IGKJ gene). In various embodiments, the endogenous non-human immunoglobulin locus is modified in whole or in part to contain a human nucleic acid sequence.

[0178] Genetic, molecular and behavioral analysis can be performed on the non-human mammals described above. The present disclosure also relates to offspring produced by mating the non-human mammals provided by the present disclosure with the same or other genotypes. The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. The preferred non-human mammal is a mammal (e.g., rodent). In some embodiments, the non-human mammal is a mouse.

[0179] The present disclosure also provides cell lines or primary cell cultures derived from non-human mammals or their descendants. Models based on cell cultures can be prepared, for example, by the following methods. Cell cultures can be obtained through isolation from non-human mammals, or alternatively, cells can be obtained from established cell cultures using the same constructs and standard cell transfection techniques. Integration of gene constructs containing DNA sequences encoding human or humanized immunoglobulins can be detected by various methods.

[0180] There are many analytical methods that can be used to detect modifications on exogenous or genomic DNA, including nucleic acid level methods (including mRNA quantification techniques using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting and in situ hybridization) and protein level methods (including histochemistry, immunoblot analysis and in vitro binding assays). In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analytical methods can be used to achieve quantitative measurements. For example, transcript levels can be measured using RT-PCR and hybridization methods, including RNase protection, Southern blot analysis, and RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, and Western blot analysis can also be used to evaluate the presence of human or humanized proteins.

[0181] Antibodies and antigen-binding fragments The disclosure provides antibodies and antigen-binding fragments thereof (e.g., humanized or chimeric antibodies) produced by the methods described herein.

[0182] Generally, antibodies (also called immunoglobulins) are composed of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact four immunoglobulin chain antibody, including two heavy chains and two light chains. The heavy chain of the antibody can be any isotype, including IgM, IgG, IgE, IgA or IgD, or subclass, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can contain two identical copies of the light chain and two identical copies of the heavy chain. One variable domain (or variable region, V H ) and multiple constant domains (or constant regions) bound to each other by disulfide bonds within their constant domains to form the "stem" of an antibody. L Each light chain, which contains a constant domain (or constant region) and one light chain, is bound to one heavy chain by disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0183] These hypervariable regions, known as complementarity determining regions (CDRs), form the loops that comprise the major antigen-binding surface of an antibody. The four framework regions generally adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and contribute to the formation of the antigen-binding region with the CDRs of the other chain.

[0184] Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and several definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. For these methods and definitions, see, for example, Martin, "Protein sequence and structure analysis of antibody variable domains", Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275 (2): 269-94 (Jan. 1998); Chothia et al. al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated by reference in its entirety herein.

[0185] CDR is important for recognizing the epitope of an antigen. As used herein, "epitope" is the smallest part of a target molecule that has the ability to undergo specific binding by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6 or 7 amino acids, but these amino acids do not need to be a continuous linear sequence on the primary structure of the antigen, since the epitope can depend on the three-dimensional arrangement of the antigen based on the secondary and tertiary structure of the antigen.

[0186] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and described, for example, in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions". Frontiers in immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases". Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. "The human IgG subclasses: molecular analysis of structure, function and regulation". Elsevier, 2016 (each of which is incorporated by reference in its entirety herein).

[0187] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, rat, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies, including those in which an immunoglobulin binding domain is fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that has the ability to specifically bind to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single chain antibody molecule, multispecific antibody, and any polypeptide that includes a binding domain that is or is homologous to an antibody binding domain formed from an antibody fragment. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, a full-length heavy or light chain of an intact antibody or individual CDRs from either the heavy or light chain of an intact antibody.

[0188] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR), which is a fusion of a single chain variable fragment (scFv) as described herein fused to a CD3-zeta transmembrane domain and endodomain.

[0189] In some embodiments, the scFV has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFV has two antigen-binding regions, each capable of binding to a target antigen.

[0190] The antibodies and antigen-binding fragments thereof (e.g., humanized or chimeric antibodies) produced by the methods described herein have various advantages: in some embodiments, they do not require further optimization to achieve the desired properties (e.g., binding affinity, thermal stability, and / or limited aggregation).

[0191] In some implementations, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or equal to 0.00001s -1 Specifically binds to a target with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Greater than or equal to 0.000001s -1 It's super.

[0192] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1×10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0193] Affinity can be estimated from the quotient of kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1×10 -10In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1×10 -12 In some embodiments, the antibody binds to a target with a KD of about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less.

[0194] In some embodiments, the thermal stability is determined. An antibody or antigen-binding fragment as described herein can have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

[0195] IgG can be described as a multi-domain protein and therefore the melting curve sometimes shows two or three transitions including a first denaturation temperature, Tm D1, and a second denaturation temperature, Tm D2, and optionally a third denaturation temperature, Tm D3.

[0196] In some embodiments, an antibody or antigen-binding fragment as described herein has a Tm D1 of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95° C. In some embodiments, an antibody or antigen-binding fragment as described herein has a Tm D2 of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C. In some embodiments, an antibody or antigen-binding fragment as described herein has a Tm D3 of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

[0197] In some embodiments, the Tm, Tm D1, Tm D2, Tm D3 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

[0198] In some embodiments, an antibody or antigen-binding fragment as described herein does not form aggregates when the temperature is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

[0199] Methods for producing genetically modified animals Genetically modified animals can be produced by introducing human immunoglobulin genes into the genome of a non-human animal to produce an animal capable of expressing humanized or chimeric antibodies. FIG. 1A shows a method for producing a humanized animal. In some embodiments, the method includes first modifying a human immunoglobulin locus on a human chromosome. The modified human chromosome is then introduced into a mouse recipient cell. Human immunoglobulin variable regions are then introduced by direct replacement into the corresponding regions of the mouse genome. The recipient cells are then screened. In some embodiments, the cells are free of human chromosomes. The cells are then injected into blastocysts to prepare chimeric mice. Subsequent breeding can be performed to obtain mice that contain intact humanized immunoglobulin loci.

[0200] Several other techniques may be used to create genetically modified animals, including, for example, non-homologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to create genetically modified animals. Many of these genome editing techniques are known in the art and described, for example, in Yin et al., "Delivery technologies for genome editing", Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated by reference in its entirety. Many other methods are provided and can be used for genome editing, such as, for example, microinjection of a genetically modified nucleus into an enucleated oocyte and fusion of the enucleated oocyte with another genetically modified cell.

[0201] The genetic modification process may include replacing endogenous sequences with human sequences by homologous recombination. In some embodiments, cleavage (e.g., by zinc finger nucleases, TALENs, or CRISPRs) upstream and downstream of the target site can generate DNA double-strand breaks, and endogenous sequences are replaced with human sequences using homologous recombination.

[0202] In some embodiments, methods of making a genetically modified humanized animal may include replacing a nucleic acid (e.g., a V, D, J region or a V, J region) with a corresponding region of a human sequence at an endogenous locus (or site). The sequence may include a region (e.g., a portion or the entire region) of an IGHV, IGHD, IGHJ, IGKV and / or IGKJ gene. In some embodiments, the replacement is mediated by homologous recombination. In some embodiments, the replacement is mediated by Cre recombinase.

[0203] Figure 9 shows a targeting strategy for adding functional genetic elements to human chromosomes. These vectors can be inserted upstream of the V region, between the J and C regions.

[0204] In some embodiments, the first vector has, from 5' to 3', one or more of the following: a DNA homology arm sequence upstream of the insertion site, a PGK promoter, a red fluorescent protein reporter gene (tdTomato), an FMDV (foot and mouth disease virus) self-cleaving peptide (2A), a zeomycin resistance gene (Zeo), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a LoxP recognition sequence, a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "3'HygR"), and an Flp recognition target ("FRT"), a downstream DNA homology arm sequence, and a DTA gene.

[0205] The second vector has, from 5' to 3', one or more of the following: a DNA homology arm sequence upstream of the insertion site, a LoxP recognition sequence, a PGK promoter, a partial sequence of a puromycin resistance gene (5'PuroR), a mammalian expression promoter derived from human elongation factor 1 alpha (EF-1a), a piggyBac transposase gene sequence (PBase), an internal ribosome entry site (IRES), a kanamycin resistance gene sequence (Neo), a transcription termination / polyadenylation signal sequence, a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0206] These vectors can integrate into the genome of cells and cells can be selected by drug resistance markers or combinations thereof (e.g., Zeocin, G418 and / or puromycin). In some embodiments, the PB transposase can be expressed to delete genetic elements between the transposase target sequences.

[0207] In some embodiments, these vectors are integrated into a modified human chromosome. The human chromosome may first be modified before integrating the first and second vectors into the genome. In some embodiments, one or more additional vectors may be added to various locations of the chromosome as needed. In some embodiments, the vectors are added between the C region and the centromere. The third vector may have one or more of the following from 5' to 3': a DNA homology arm sequence upstream of the insertion site, a PGK promoter, a puromycin resistance gene sequence (PuroR), a thymidine kinase gene sequence (TK), a LoxP recognition sequence, a PGK promoter, a puromycin resistance gene partial sequence (5'PuroR), a mammalian expression promoter (EF-1a), PBase, IRES, Neo, a transcription termination / polyadenylation signal sequence, a DNA homology arm sequence downstream of the insertion site, and a DTA. In some embodiments, these vectors may be inserted into a variable gene region or a constant region. In some embodiments, a portion of an endogenous variable gene region or an endogenous constant region is deleted. In some embodiments, a large fragment of a chromosome (e.g., between the constant region and the centromere) is deleted. The cells can also be treated with Cre enzyme, leading to recombination of loxP sites, thus removing the genomic DNA sequence between the J region and the centromere on human chromosome 14 or between the C region and the centromere on human chromosome 14. In some embodiments, natural chromosome breaks can be present. The modified human chromosome with the desired chromosome break can be selected for the experiment.

[0208] The human chromosomes can be obtained from human cell lines, cancer cells, primary cell cultures and / or human fibroblasts. In some embodiments, a first vector is introduced into a human cell, which is then fused with a recipient cell. The modified chromosome is then isolated and introduced into another suitable recipient cell. Cells containing only one human chromosome are obtained by selecting cells with the desired resistance. A second vector is then introduced into the cell, and cells are selected by resistance. A third and / or fourth vector can then be introduced, if desired. The recipient cell can be a mammalian cell, a human cell or a mouse cell. In some embodiments, the recipient cell is a CHO cell or preferably an A9 cell. In some embodiments, the modified chromosome is fluorescently labeled and isolated. The modified chromosome is also injected into the recipient cell by chromosome microinjection. In some embodiments, the donor cell is induced to multinucleate its chromosomes. These nuclei are then pushed through the cell membrane to create microcells that can be fused to the recipient cell. In some embodiments, microcell-mediated chromosome transfer may also be used.For chromosome manipulation techniques, see, for example, CN 1200014A; CN 109837307A; US 20120093785A1; and US 2009253902; Kuroiwa et al. "Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts". Nature Biotechnology 18.10(2000)):1086-1090; CN 1717483A; Paulis, Marianna. "Chromosome Transfer Via Cell Fusion". Methods in Molecular Biology 738(2011):57; Genes, Chromosomes & Cancer 14:126127(1995); Tomizuka et al. "Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice”. Nature Genetics 16.2(1997):133-143; Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284, each of which is incorporated by reference in its entirety herein.

[0209] In some embodiments, modifications may be made to mouse chromosomes. The targeting strategy is shown in FIG. 4. The first vector may have DNA homology arm sequences and LoxP sequences upstream and downstream of the insertion site. In some embodiments, the first vector has one or more of the following from 5' to 3': DNA homology arm sequence upstream of the insertion site, Flp recognition target (FRT), CAG promoter, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "5'HygR"), LoxP, FRT, 5'PB transposon sequence (PB5'), PGK promoter, blue fluorescent protein reporter gene (BFP), FMDV self-cleaving peptide (2A), hygromycin resistance gene (hygromycin phosphotransferase; HygR), 3'PB transposon sequence (PB3'), DNA homology arm sequence downstream of the insertion site, and DTA.

[0210] The second vector may have DNA homology arm sequences upstream and downstream of the insertion site and LoxP sequences. In some embodiments, the second vector has, from 5' to 3', one or more of the following: DNA homology arm sequence upstream of the insertion site, 5'PB transposon sequence (PB5'), PGK promoter, green fluorescent protein reporter gene sequence (EGFP), FMDV self-cleaving peptide (2A), puromycin resistance gene sequence (PuroR), 3'PB transposon sequence (PB3'), Flp recognition target (FRT), puromycin resistance gene partial sequence (3'PuroR), FMDV self-cleaving peptide (2A), DT receptor (DTR), LoxP recognition sequence, DNA homology arm sequence downstream of the insertion site and DTA.

[0211] Figure 30 shows a similar targeting strategy for the kappa light chain immunoglobulin locus. Two vectors can be integrated into a human chromosome initially. The first vector has DNA homology arm sequences upstream and downstream of the insertion site and a LoxP recognition sequence. In some embodiments, the first vector has, from 5' to 3', one or more of the following: a DNA homology arm sequence upstream of the insertion site, a PGK promoter, tdTomato, an FMDV self-cleaving peptide (2A), Bsr, a transcription termination / polyadenylation signal sequence, a LoxP recognition sequence, a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "3'HygR"), FRT, a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0212] The second vector has DNA homology arm sequences upstream and downstream of the insertion site and a LoxP recognition sequence. In some embodiments, the second vector has, from 5' to 3', one or more of the following: a DNA homology arm sequence upstream of the insertion site, a LoxP recognition sequence, a PGK promoter, a portion of the puromycin resistance gene sequence (5'PuroR), EF-1a, PBase, IRES, Neo, a transcription termination / polyadenylation signal sequence, a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0213] LoxP recognition sequences can also be added to human chromosomes (e.g., human chromosomes 2, 14, 22). Cells can also be treated with the Cre enzyme, leading to recombination of the loxP sites, thus removing genomic DNA sequences. In some embodiments, natural chromosomal breaks can be used to remove genomic DNA sequences as well.

[0214] Modifications on the mouse light chain immunoglobulin locus can be performed directly, in some embodiments, a vector is used directly to replace the entire mouse light chain immunoglobulin variable region. In some embodiments, the vector comprises, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, a Flp recognition target (FRT), a mammalian expression promoter derived from human elongation factor 1 alpha (EF-1a), a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "5'HygR"), a LoxP recognition sequence for Cre recombinase, a 5'PB transposon sequence (PB5'), a blue fluorescent protein reporter gene (BFP), a DT receptor (DTR), an FMDV self-cleaving peptide (2A), a kanamycin resistance gene sequence (Neo), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a 3'PB transposon sequence (PB3'), a puromycin resistance gene partial sequence (3'PuroR), an FMDV self-cleaving peptide (2A), a DT receptor (DTR), a LoxP recognition sequence for Cre recombinase, a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0215] A mouse immunoglobulin variable region can be replaced with a human immunoglobulin variable region by replacement (e.g., homologous recombination or Cre-mediated recombination). In some embodiments, the replacement can be mediated using Cre recombination. In some embodiments, the vector can add a LoxP recognition sequence to a human chromosome. A similar modification can be made to a mouse chromosome, where two LoxP recognition sequences can be added to the chromosome. For example, Cre recombinase can then mediate the replacement of a V, J region on a mouse chromosome with a V, J region on a human chromosome or a V, D, J region on a mouse chromosome with a V, D, J region on a human chromosome.

[0216] The cells can be further screened (e.g., by DT) for cells that do not have human chromosomes. In some cases, cells that are not screened by DT may contain recombinant human chromosome fragments, but these fragments are small and unstable in mouse cells (e.g., Shinohara et al. (2000) Chromosome Research, 8:713-725) and will be lost naturally during cell proliferation. In some embodiments, large fragments of modified human chromosomes are deleted, for example, by Cre-mediated deletion or by natural chromosome breakage.

[0217] The 5'-end homology arm and / or the 3'-end homology arm can be of any desired length to promote homologous recombination. In some embodiments, the homology arm is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb (e.g., about 3 kb). In some embodiments, the homology arm is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb.

[0218] In some embodiments, the vector may also optionally include a reporter protein, such as luciferase (eg, Gluc) or a fluorescent protein (eg, EGFP, BFP, etc.).

[0219] These modifications can be performed in a variety of cells, hi some embodiments, the cells are stem cells, embryonic stem cells or fertilized egg cells.

[0220] The present disclosure provides a method for establishing a humanized animal model, comprising the steps of: (a) providing a cell (e.g., a fertilized egg cell) according to the methods described herein; (b) culturing the cells in a liquid culture medium; (c) implanting the cultured cells into the fallopian tubes or uterus of a recipient female non-human mammal, thereby allowing the cells to develop within the uterus of the female non-human mammal; (d) identifying germline transmission in a pregnant female genetically modified humanized non-human mammal progeny of step (c). The present invention further provides a method comprising:

[0221] In some embodiments, the non-human mammal in the aforementioned methods is a mouse (eg, a C57 mouse, a BALB / c mouse, or a C57BL / 6 mouse).

[0222] In some embodiments, the non-human mammal of step (c) is a pseudo-pregnant (or pseudo-pregnant) female.

[0223] In some embodiments, the fertilized egg of the method described above is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method as described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0224] The fertilized egg can be from any non-human animal, for example, any non-human animal as described herein.In some embodiments, the fertilized egg cell is from rodent.The gene construct can be introduced into the fertilized egg by microinjection of DNA.For example, after microinjection, the fertilized egg is cultured, and the cultured fertilized egg can be implanted into a pseudopregnant non-human animal, which then gives birth to a non-human mammal, thus creating the non-human mammal referred to in the above-described manner.

[0225] Also provided are cells, tissues and animals (e.g., mice) that comprise a nucleotide sequence as described herein, as well as cells, tissues and animals (e.g., mice) that express humanized or chimeric antibodies from endogenous non-human loci.

[0226] The present disclosure also provides various targeting vectors (e.g., vectors useful for producing genetically modified animals). In some embodiments, the vectors can include a) a DNA fragment (5' homology arm) that is homologous to the 5' end of the region to be altered; b) a sequence that includes a desired genetic element (e.g., a LoxP recognition site, a drug resistance gene, and / or a reporter gene, etc.); and c) a second DNA fragment (3' homology arm) that is homologous to the 3' end of the region to be altered. The present disclosure also relates to cells that include the targeting vectors as described herein.

[0227] In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

[0228] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

[0229] The present disclosure further relates to a method for producing a genetically modified animal model comprising two or more human or chimeric genes. The animal can comprise one or more human or humanized immunoglobulin loci and sequences encoding additional human or chimeric proteins. In some embodiments, the additional human or chimeric proteins can be programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), lymphocyte activation 3 (LAG-3), B and T lymphocyte binding (BTLA), programmed cell death 1 ligand 1 (PD-L1), CD27, CD28, CD47, CD137, CD154, T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin and mucin domain containing 3 (TIM-3), glucocorticoid-inducible TNFR-related protein (GITR) or TNF receptor superfamily member 4 (TNFRSF4 or OX40).

[0230] A method for generating a genetically modified animal model that includes additional human or chimeric genes (e.g., humanized genes) comprises the following steps: (a) obtaining a genetically modified non-human animal using a method as described herein; (b) obtaining a genetically modified non-human animal that contains two or more human or chimeric genes by mating the genetically modified non-human animal with another genetically modified non-human animal and then screening the progeny. may include.

[0231] In some embodiments, in step (b) of the method, the genetically modified animal can be bred with a genetically modified non-human animal having human or chimeric PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40. Some of these genetically modified non-human animals are described in, for example, PCT / China Patent Application Publication No. 2017 / 090320, PCT / China Patent Application Publication No. 2017 / 099577, PCT / China Patent Application Publication No. 2017 / 099575, PCT / China Patent Application Publication No. 2017 / 099576, PCT / China Patent Application Publication No. 2017 / 099574, PCT / China Patent Application Publication No. 2017 / 106 No. 024, PCT / CHINA Patent Application Publication No. 2017 / 110494, PCT / CHINA Patent Application Publication No. 2017 / 110435, PCT / CHINA Patent Application Publication No. 2017 / 120388, PCT / CHINA Patent Application Publication No. 2018 / 081628, and PCT / CHINA Patent Application Publication No. 2018 / 081629 (each of which is incorporated by reference in its entirety herein).

[0232] In some embodiments, the genetically modified animal may have a human ADAM6 gene, an endogenous ADAM6 gene, or a modified ADAM6 gene. The ADAM6 protein is a member of the ADAM protein family, where ADAM is an acronym for a disintegrin and metalloprotease. The human ADAM6 gene is usually present between the human IGHV genes IGHV1-2 and IGHV6-1, and is a pseudogene (Figure 37). In mice, there are two ADAM6 genes, ADAM6a and ADAM6b. They are located in the intergenic region between the mouse IGHV and IGHD gene clusters. Mouse ADAM6a is located between mouse IGHV5-1 and mouse IGHD5-1. Mouse ADAM6b is located between mouse IGHD3-1 and mouse IGHD1-1. Thus, in some embodiments, the genetically modified animal may have a human ADAM6 gene. In some embodiments, the genetically modified animal does not have an endogenous ADAM6 gene.

[0233] In some embodiments, the genetically modified animal is a mouse. In some embodiments, the mouse is modified to include a nucleotide sequence encoding an ADAM6 protein (e.g., ADAM6a or ADAM6b). In some embodiments, this sequence is placed in any suitable location. It can be placed in an intergenic region or in any suitable location in the genome. In some embodiments, the nucleic acid encodes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse ADAM6a gene (e.g., 113539230-113547024 of NC_000078.6; SEQ ID NO: 53) or the mouse ADAM6b gene (e.g., 113486188-113492125 of NC_000078.6; SEQ ID NO: 54). In some embodiments, the nucleic acid further comprises regulatory elements (e.g., promoters) of the ADAM6a gene and the ADAM6b gene.

[0234] In some embodiments, a functional mouse ADAM6 locus may be placed in the center of the human IGHV gene cluster. In some embodiments, the mouse ADAM6 locus is between two human IGHV genes. In some embodiments, the mouse ADAM6 locus replaces the human ADAM6 pseudogene between human VH1-2 and human VH(II)-1-1. In some embodiments, the ADAM6a and ADAM6b genes are located between human IGHV1-2 and human VH(II)-1-1 in the genome of the animal. In some embodiments, the location of the mouse ADAM6 sequence within the human gene sequence may approximate the location of the human ADAM6 pseudogene (e.g., within the VD intergenic region) or may approximate the location of the mouse ADAM6 sequence. In some embodiments, the genetically modified mouse has a humanized heavy chain immunoglobulin locus. In some embodiments, the mouse ADAM6a and mouse ADAM6b are located between the human IGHV1-2 and IGHV6-1 genes. Placing mouse ADAM6a and mouse ADAM6b between the human IGHV1-2 and IGHV6-1 genes may have various advantages, for example, because these genes replace the human ADAM6 gene at the same locus, the effect of the replacement of the human ADAM6 gene on VDJ recombination is limited, and the mouse ADAM6a and mouse ADAM6b genes are likely to function normally (as they are located at the same site as the endogenous locus).

[0235] Thus, in one aspect, the disclosure provides a genetically modified animal comprising at an endogenous heavy chain immunoglobulin locus a first sequence comprising one or more human IGHV genes, a second sequence comprising an ADAM6 gene, and a third sequence comprising one or more human IGHD genes and one or more human IGHJ genes. In some embodiments, the first sequence, the second sequence, and the third sequence are operably linked.

[0236] In some embodiments, the first sequence comprises all of the human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, IGHV1-8, IGHV(II)-1-1 and IGHV6-1. In some embodiments, the first sequence comprises all of the human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, IGHV(II)-1-1 and IGHV6-1. In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus.

[0237] In some embodiments, the second sequence comprises either or both of the mouse ADAM6a gene and the mouse ADAM6b gene. In some embodiments, the animal is a fertile male mouse. In some embodiments, the second sequence does not have the mouse ADAM6a gene or the mouse ADAM6b gene.

[0238] In some embodiments, the third sequence comprises all of the human IGHD genes in Table 2 and all of the human IGHJ genes in Table 3. In some embodiments, the third sequence comprises human IGHV6-1. In some embodiments, the third sequence comprises human IGHV(II)-1-1. In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus.

[0239] In some embodiments, AMAM6a and / or ADAM6b are endogenous sequences. In some embodiments, AMAM6a and / or ADAM6b are not replaced and / or are in their endogenous or natural locations. In some embodiments, the mouse IGHV gene in front of the mouse IGHV1-2 at the heavy chain variable region locus is replaced with a human IGHV gene. In some embodiments, the mouse IGHV, IGHD and IGHJ gene in front of the mouse IGHV6-1 at the heavy chain variable region locus is replaced with one or more human IGHV, IGHD and / or IGHJ genes.

[0240] Thus, in some embodiments, mouse IGHV, IGHD and IGHJ genes can be replaced with human IGHV, IGHD and IGHJ by two or more replacements. In the first step, a selected number of mouse IGHV genes (e.g., all mouse IGHV genes in Table 4) located 5' of ADAM6a are replaced with human IGHV genes. In the second step, a selected number of mouse IGHD and IGHJ genes (e.g., all mouse IGHD genes in Table 5 except IGHD5-1 and IGHD3-1 and all IGHJ genes in Table 6) located 3' of ADAM6b are replaced with human IGHD and human IGHJ genes. This replacement can be performed by homologous recombination or Cre-mediated recombination.

[0241] In some embodiments, the mouse does not have a mouse ADAM6a or ADAM6b gene. In some embodiments, the mouse has a human ADAM6 gene.

[0242] Various methods can be used to increase mouse fertility. In some embodiments, female mice exhibiting superovulation can be used for mating. In some embodiments, in vitro fertilization can be used. Superovulation can be induced by injecting serum gonadotropins and chorionic gonadotropins (e.g., human or mouse CG) into mature female mice. Mature male mice can be sacrificed and their cauda epididymis can be removed. The tubules of the cauda epididymis can be incised to release the sperm. Mature female mice undergoing superovulation can then be sacrificed and their oviducts can be removed. Cumulus-oocyte complexes (COCs) can be released from the oviducts. A sperm suspension can then be added to the COCs and incubated to allow for insemination. Diseased oocytes containing only one pronucleus can be removed. After incubation, two-cell stage embryos can be transferred to recipient females. Methods for increasing mouse fertility are known in the art.

[0243] The present disclosure relates to nucleic acid sequences and methods that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences as described herein. and amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein.

[0244] In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0245] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

[0246] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

[0247] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95% or 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For purposes of the present invention, comparison of sequences and determination of percent identity between two sequences may be accomplished using a Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0248] The percentage of conserved residues with similar physicochemical properties (percent homology), e.g., leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The percentage of homology is often higher than the percentage of identity. Accordingly, the present disclosure also provides amino acid sequences having a percentage homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any amino acid sequence as described herein or a nucleic acid encoding those amino acid sequences.

[0249] Methods of using genetically modified animals Genetically modified animals can be used to generate humanized or chimeric antibodies capable of specifically binding to a target. In some embodiments, antibodies can be generated in such animals using the target (e.g., a protein or a fragment of that protein) as an immunogen using standard polyclonal and monoclonal antibody preparation techniques. In some embodiments, the genetically modified animals are exposed to an antigen of choice for a time and under conditions that allow the animal to produce antibodies specific to that antigen.

[0250] Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than once (e.g., 2, 3, or 4 times).

[0251] A full-length polypeptide or protein can be used as an immunogen, or alternatively, an antigenic peptide fragment thereof. An antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of its amino acid sequence and encompasses an epitope of the protein such that antibodies raised against the peptide will form a specific immune complex with the protein.

[0252] An immunogen is typically used to prepare antibodies by immunizing a suitable subject (e.g., a genetically modified animal as described herein). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide (e.g., a fragment of a protein). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0253] Polyclonal antibodies can be prepared by immunizing a suitable subject with a polypeptide or its antigenic peptide (e.g., a portion of a protein) as an immunogen, as described above. Antibody titers over time in immunized subjects can be monitored by standard techniques, such as by enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as obtaining the IgG fraction by protein A or protein G chromatography. At a suitable time after immunization, for example when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, eg, using a standard ELISA assay.

[0254] In one aspect, the disclosure provides a mouse comprising a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse produces B cells comprising a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human heavy chain V, D and / or J sequence. In some embodiments, the heavy chain constant region gene sequence comprises a human or mouse heavy chain sequence selected from the group consisting of a CH1, hinge, CH2, CH3 and combinations thereof.

[0255] In one aspect, the disclosure provides a mouse comprising a modification of an endogenous immunoglobulin light chain (e.g., κ or λ) locus, wherein the mouse produces B cells comprising a rearranged immunoglobulin sequence operably linked to a light chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operably linked to the light chain constant region gene sequence comprises a human light chain V and / or J sequence. In some embodiments, the light chain constant region gene sequence comprises a human or mouse light chain constant region.

[0256] The mouse B cells or spleen cells can, for example, contain rearranged non-mouse immunoglobulin variable gene sequences operably linked to mouse immunoglobulin constant region genes. The sequences encoding the human heavy chain variable region and the human light chain variable region are determined. These sequences can be determined, for example, by sequencing the hybridoma or B cells of interest. In some embodiments, single B cell screening is used. This can screen the natural antibody repertoire without the need for hybridoma fusion and combinatorial display. For example, B cells can be mixed with a panel of DNA barcoded antigens, allowing the single cell sequencing protocol to retrieve both one or more antigen barcodes and B cell receptor (BCR) sequences of individual B cells.

[0257] The antibodies can be further modified, for example, by operably linking a sequence encoding a human heavy chain variable region to a sequence encoding a human heavy chain constant region and / or a sequence encoding a human light chain variable region to a sequence encoding a human light chain constant region, to obtain a humanized or human antibody.

[0258] In some embodiments, when a mouse expresses a protein that is very similar to an antigen of interest, it may be difficult to elicit an immune response in the mouse. This is because during immune cell development, B cells and T cells that recognize MHC molecules bound to self-derived peptides are deleted from the immune cell repertoire. In such cases, the humanized mouse can be further modified. The corresponding gene in the mouse can be knocked out, and then the mouse is exposed to the antigen of interest. Since the mouse does not go through negative selection for the gene product, the mouse can easily generate antibodies that can specifically bind to the target.

[0259] The present disclosure also provides methods for making antibodies, nucleic acids, cells, tissues (e.g., spleen tissue). In some embodiments, the methods include exposing an animal as described herein to an antigen. From the animal, antibodies (e.g., hybrid antibodies), nucleic acids encoding antibodies, cells, and / or tissues (e.g., spleen tissue) can be obtained. In some embodiments, nucleic acids encoding human heavy and light chain immunoglobulin variable regions are determined, for example, by sequencing. In some embodiments, nucleic acids encoding human heavy chain immunoglobulin variable regions can be operably linked to nucleic acids encoding human heavy chain immunoglobulin constant regions. In some embodiments, nucleic acids encoding human light chain immunoglobulin variable regions can be operably linked to nucleic acids encoding human light chain immunoglobulin constant regions. In some embodiments, cells containing nucleic acids as described herein are cultured and antibodies are recovered.

[0260] In some embodiments, no mouse immunoglobulin V, D, J genes (e.g., no mouse IGHV, IGHD, IGHJ, IGKV, or IGKJ genes) contribute to the heavy and / or light chain variable region sequences. In some embodiments, the heavy and / or light chain variable region sequences produced by the animal are fully human, with fully contributed human immunoglobulin V, D, J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, and IGKJ genes).

[0261] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized or chimeric antibodies or antigen-binding fragments thereof described herein or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence that constitutes the antigen-binding site of the antibody or antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will exhibit increased affinity for the target protein. Any combination of deletions, insertions and / or combinations can be made to result in an antibody or antigen-binding fragment thereof with increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter and introduce new post-translational modifications to the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in the cell), or introducing new glycosylation sites.

[0262] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0263] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.

[0264] Further modifications to the antibody or antigen-binding fragment can be made. For example, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. Homodimeric antibodies thus generated may have some increased in vitro and / or in vivo half-lives. Homodimeric antibodies with increased in vitro and / or in vivo half-lives can also be prepared using heterobifunctional cross-linkers, e.g., as described in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies with dual Fc regions can be engineered (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0265] In some embodiments, covalent modifications can be made to the present antibodies or antigen-binding fragments thereof. Such covalent modifications can be made by chemical or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0266] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0267] Example 1: Overview An experiment was carried out to introduce human immunoglobulin genes into the mouse genome to generate mice expressing humanized antibodies. Figure 1A shows a method for producing humanized mice. The method involves first modifying a human immunoglobulin region on a human chromosome. The modified human chromosome was then introduced into a mouse recipient cell.

[0268] The mouse immunoglobulin variable region was replaced with a human immunoglobulin variable region by direct replacement (e.g., homologous recombination or Cre-mediated recombination). Optionally, the human immunoglobulin variable region can be introduced into the mouse genome by a stepwise approach. The recipient cells were then screened for correct replacement. The cells were then injected into blastocysts to prepare chimeric mice. Subsequent breeding was performed to obtain mice containing intact human immunoglobulin variable regions.

[0269] Because the mouse heavy chain gene and the two light chain genes are located on chromosomes 12, 6, and 16, respectively, mice containing human heavy chain variable regions or human light chain variable regions can be prepared separately (FIGS. 1B and 1C). These mice can then be crossed with each other to obtain mice capable of expressing both human heavy chain variable domains and human light chain variable domains.

[0270] Example 2: Modification of the mouse heavy chain immunoglobulin locus The heavy chain immunoglobulin locus is located on mouse chromosome 12. Figure 2 is a schematic diagram showing the mouse heavy chain immunoglobulin locus. Two recombination sites (1301, 1302) were introduced on either side of the variable region of the heavy chain immunoglobulin locus. The resulting modified chromosome is shown in Figures 3A-3B. One of these is a wild-type loxP site and the other is a heterospecific mutant lox site (lox2272). Recombination cannot occur between the wild-type loxP site and the heterospecific mutant lox site. The modification was performed in mouse embryonic stem cells. An overview of the targeting strategy is shown in Figure 4. The vector (V1401) has, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, an Flp recognition target (FRT), a CAG promoter, a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "5'HygR"), LoxP (1301), FRT, a 5'PB transposon sequence (PB5'), a PGK promoter, a blue fluorescent protein reporter gene (BFP), an FMDV self-cleaving peptide (2A), a hygromycin resistance gene (hygromycin phosphotransferase; HygR), a 3'PB transposon sequence (PB3'), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0271] The vector (V1402) has, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, a 5'PB transposon sequence (PB5'), a PGK promoter, a green fluorescent protein reporter gene sequence (EGFP), an FMDV self-cleaving peptide (2A), a puromycin resistance gene sequence (PuroR), a 3'PB transposon sequence (PB3'), an Flp recognition target (FRT), a partial puromycin resistance gene sequence (3'PuroR), an FMDV self-cleaving peptide (2A), a DT receptor (DTR), a LoxP recognition sequence (1302), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0272] The vectors (V1401 and V1402) were introduced into mouse embryonic stem cells. The cells were then screened with hygromycin B and puromycin. The integration of the exogenous gene into the mouse genome was confirmed by PCR. The results are shown in Figures 5A-5B and 6A-6B. Clones 030, 035, 036 and 037 were confirmed as positive.

[0273] PCR assays were performed using the following primers: mIgHV-5'loxP-L-GT-F:5'-gccaaggaatttaaaaggggattgaaagcaa-3' (SEQ ID NO: 1), mIGHV-005-L-GT-R2: 5'-gccctccatgtacagcttcatgtgc-3' (SEQ ID NO: 2); mIGHV-005-5'loxP-R-GT-F2: 5'-actgggcttgtcgagacagagaaag-3' (SEQ ID NO: 3), mIgHV-5'loxP-R-GT-R: 5'-ccacagcccgatctacttggctttt-3' (SEQ ID NO: 4); mIGHV-3'lox-L-GT-F2: 5'-gcaaggttttgactaagcggagcac-3' (SEQ ID NO:5); mIGHV-3'lox-L-GT-R2: 5'-tgacgcatgtgttttatcggtctgt-3' (SEQ ID NO: 6); mIGHV3'lox-R-GT-F2: 5'-gtgcctgacacgtgctacgagattt-3' (SEQ ID NO: 7); mIGHV-3'lox-R-GT-R1: 5'-ttcaacaataagcagggccagaggg-3' (sequence number 8).

[0274] Among these primers, mIgHV-5'loxP-L-GT-F and mIgHV-5'loxP-R-GT-R are located on the mouse chromosome, mIGHV-005-L-GT-R2 and mIGHV-005-5'loxP-R-GT-F2 are located on vector 1401, mIGHV-3'lox-L-GT-F2 and mIGHV-3'lox-R-GT-R1 are located on the mouse chromosome, and mIGHV-3'lox-L-GT-R2 and mIGHV3'lox-R-GT-F2 are located on vector 1402.

[0275] Example 3: Modification of human chromosome 14 The goal of this experiment was to create a modified human chromosome that contained at least two recombination sites: one on each side of the variable region of the heavy chain immunoglobulin locus.

[0276] The heavy chain immunoglobulin locus is located on human chromosome 14. Figure 7 is a schematic diagram of human chromosome 14, highlighting the heavy chain immunoglobulin locus.

[0277] The modified human variable regions are shown in Figure 8. The targeting strategy is outlined in Figure 9. As shown in Figure 9, sites 301 and 302 are recombination sites. Recombination site 1301 is identical to recombination site 301. Recombination site 1302 is identical to recombination site 302.

[0278] An experiment was carried out to insert a vector upstream of the V region and between the J and C regions of human chromosome 14. The first targeting vector (V401) has, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, a PGK promoter, a red fluorescent protein reporter gene (tdTomato), a self-cleaving peptide (2A) derived from FMDV (foot-and-mouth disease virus), a zeocin resistance gene sequence (Zeo), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a LoxP recognition sequence for Cre recombinase (301), a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "3'HygR") and an Flp recognition target ("FRT"), a downstream DNA homology arm sequence and a diphtheria toxin subunit A (DTA) gene.

[0279] The second vector (V402) has, from 5' to 3': a DNA homology arm sequence upstream of the insertion site, a LoxP recognition sequence for Cre recombinase (302), a PGK promoter, a partial sequence of the puromycin resistance gene (5'PuroR), a mammalian expression promoter derived from human elongation factor 1 alpha (EF-1a), a piggyBac transposase gene sequence (PBase), an internal ribosome entry site (IRES), a kanamycin resistance gene sequence (Neo), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0280] In some experiments, cells were transfected with vectors (V401, V402) and selected by appropriate drug resistance markers or combinations thereof (Zeocin, G418).

[0281] There are many ways to introduce a vector of interest into a human chromosome. Human chromosomes can be obtained from human cell lines, cancer cells, primary cell cultures and / or human fibroblasts. In one experiment, a first vector is introduced into the chromosome. This modified chromosome can be added to a recipient cell, and then a second vector can be inserted into the modified chromosome. In some experiments, V401 is first introduced into a human cell, then the chromosome is fluorescently labeled and then separated, and then the modified chromosome is injected into a recipient cell by microinjection. Then, V402 is introduced into the cell. In another experiment, human fibroblasts are selected and vector 402 is introduced. Then, the human fibroblasts are fused with recipient cells (A9 cells or CHO cells).

[0282] In some experiments, one or more vectors can be inserted into the desired site of human chromosome 14 by homologous recombination, for example at the same time. The vector can contain a drug resistance marker (Zeocin, G418), and the cells are then screened. The chromosomes are then labeled and separated, and the modified chromosomes are then injected into recipient cells by chromosome microinjection.

[0283] In some experiments, one or more additional vectors are inserted. These additional vectors can be inserted into different sites on human chromosome 14 as needed. In one experiment, the third vector can have, from 5' to 3', the following: DNA homology arm sequence upstream of the insertion site, PGK promoter, puromycin resistance gene sequence (PuroR), thymidine kinase gene sequence (TK), LoxP recognition sequence (302), PGK promoter, puromycin resistance gene partial sequence (5'PuroR), mammalian expression promoter from human elongation factor 1 alpha (EF-1a), piggyBac transposase gene sequence (PBase), internal ribosome entry site (IRES), kanamycin resistance gene sequence (Neo), transcription termination / polyadenylation signal sequence (polyA; "PA"), DNA homology arm sequence downstream of the insertion site, and DTA. The vector is inserted into the C region.

[0284] In one experiment, a third vector (V403) was inserted between the C region and the centromere, which contains, from 5' to 3', the following: a DNA homology arm sequence upstream of the insertion site, a PGK promoter, a puromycin resistance gene sequence (PuroR), a thymidine kinase gene sequence (TK), a LoxP recognition sequence (302), a PGK promoter, a puromycin resistance gene partial sequence (5'PuroR), a mammalian expression promoter derived from human elongation factor 1α (EF-1a), a piggyBac transposase gene sequence (PBase), an internal ribosome entry site (IRES), a kanamycin resistance gene sequence (Neo), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0285] In one experiment, human fibroblasts were selected and transfected with vector 402. Then, the human fibroblasts were fused with recipient cells (A9 cells or CHO cells). The modified chromosome was separated and transfected into another suitable recipient cell. Then, the cells were selected by G418 resistance to obtain cells containing only one human chromosome. Then, the cells were transfected with vector 401 and the cells were selected by Zeocin resistance. Then, the cells were transfected with vector 403 and the cells were selected by puromycin resistance. The positive clones selected after screening were treated with Cre enzyme. For chromosome techniques, see, for example, Kuroiwa et al. "Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts". Nature Biotechnology 18.10(2000)):1086-1090; China Patent Publication No. 1200014A; China Patent Publication No. 109837307A; U.S. Patent Publication No. 20120093785A1; U.S. Patent Publication No. 2009253902; China Patent Publication No. 1717483A; Paulis, Marianna. "Chromosome Transfer Via Cell Fusion". Methods in Molecular Biology 738(2011):57; Genes, Chromosomes & Cancer 14:126127(1995); Tomizuka et al. "Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice”. Nature Genetics 16.2(1997):133-143; and Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284, each of which is incorporated by reference in its entirety herein.

[0286] PCR was performed to confirm the presence on the chromosome of the 5'-end recombination site 301 and the 3'-end recombination site 302. The absence of random insertions in the cells was confirmed by Southern blot and analyzed by fluorescent in situ hybridization (FISH).

[0287] Figure 10 shows the modified human chromosome 14. Figure 11 shows the results of PCR identification of loxP site 301 on chromosome hChr14-mut3. Figure 12 shows the results of PCR identification of loxP site 302 on chromosome hChr14-mut3. As shown in these figures, 12 clones (1-B2, 1-B8, 1-D6, 1-D10, 1-F11, 1-G11, 2-A2, 3-E5, 3-G5, 3-H4, 5-C3 and 6-F11) were positive clones.

[0288] The following PCR primers were used for these experiments: hIGHV-5'loxP-L-GT-F1:5'-TCAAAGTCAATTTCCTCAGCGAGGCT-3' (SEQ ID NO: 9), hIGHV-5'loxP-R-GT-R:5'-AGGGAGGGAATGGAATGAGGGTGAT-3' (SEQ ID NO:10); hIGHV-3'loxP-L-GT-F1:5'-CCATGTGACCCATTCGAGTGTCCTG-3' (SEQ ID NO: 11), hIGHV-3'loxP-R-GT-R:5'-TTGTGAGGGCTCAAGTTCAGTGCAT-3' (Sequence number 12).

[0289] Positive clones were used to perform FISH analysis using a CCP14 FISH probe (CytoTest Inc., Rockville, MD, Catalog No. CT-CCP014). Representative FISH images of clone 1-D10 are shown in Figures 13 and 14. In Figure 13, the white arrow indicates the full-length human chromosome 14 (before modification). In Figure 14, the white arrow indicates the human chromosome 14 fragment after modification.

[0290] Example 4: Introduction of human chromosomes or fragments into mouse ES cells The modified chromosome obtained in Example 3 was introduced into the cells obtained in Example 2 by the method previously described. The cells were then screened by G418. Only cells containing only one human chromosome were selected. Figure 15 shows the modified mouse chromosome 12.

[0291] Then, the V, D, J regions on mouse chromosome mChr12-mut2 were replaced by the V, D, J regions on human chromosome hChr14-mut3 via Cre recombinase (Figure 16). Human chromosomal DNA sequences replaced the sequences between recombination sites 1301 and 1302. Hygromycin and puromycin were used to screen for positive cells. The cells were further screened by DT to obtain mouse cells without human chromosomes, and then injected into mouse blastocysts. In some cases, the cells were directly injected into blastocysts without DT screening.

[0292] The cells after Cre recombination were tested to confirm the integration of the human gene sequence in the mouse genome. The PCR results are shown in Figure 17, Figure 18, Figure 19 and Figure 20. All PCR results showed that cells 1-B4, 1-B10 and 2-A7 had the correct recombination and the human chromosome was lost in 1-B10 cells. 1-B10 cells were tested by FISH using a mouse whole chromosome painting probe (Cytocell Ltd, Cambridge, UK; Catalogue No. AMP12R) and a human specific IGH break-apart probe (Cytocell Ltd, Cambridge, UK; Catalogue No. LPH 014). The results are shown in Figure 21 and confirmed the presence of the human chromosome fragment in the mouse chromosome. These primers are listed in the table below.

[0293] [Table 12]

[0294] Example 5: Generating mice containing humanized heavy chain immunoglobulin loci Positive cloned cells were injected into blastocysts of BALB / c mice by microinjection. Embryo microinjection was performed according to the method described, for example, in A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)", Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient mice to produce genetically modified humanized mice (F0 generation). These mice were then mated with mice on a C57BL / 6 background. Black offspring were selected and mated with Flp tool mice (Figure 22). PCR analysis was performed on DNA obtained from the tails of the mice. The mice were further crossed with mice on a BALB / c background several times (e.g., at least five times) to obtain humanized heavy chain immunoglobulin locus heterozygous mice on a BALB / c background.

[0295] To confirm that the mice express human antibody heavy chains, blood was collected from chimeric mice (F0 generation) and black mice (F1 generation). RNA was extracted and reverse transcribed to obtain cDNA. The following PCR primers were used to amplify the sequence, which was then sequenced:

[0296] [Table 13]

[0297] Example 6: Modification of the Mouse Light Chain Immunoglobulin Locus The light chain immunoglobulin locus is located on mouse chromosome 6. Figure 23 was a schematic diagram showing the mouse light chain immunoglobulin locus. Two recombination sites were introduced on both sides of the variable region of the light chain immunoglobulin locus, and the resulting modified chromosome was shown in Figure 24A-B. The detailed targeting strategy was shown in Figure 25.

[0298] The modification was performed in mouse embryonic stem cells. The vector (V3901) contains, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, a Flp recognition target (FRT), a mammalian expression promoter derived from human elongation factor 1α (EF-1a), a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "5'HygR"), a LoxP recognition sequence for Cre recombinase (1101), a 5'PB transposon sequence (PB5'), a blue fluorescent protein reporter gene (BFP), and a DT It contained the FMDV self-cleaving peptide (2A), kanamycin resistance gene sequence (Neo), transcription termination / polyadenylation signal sequence (polyA; "PA"), 3'PB transposon sequence (PB3'), puromycin resistance gene partial sequence (3'PuroR), FMDV self-cleaving peptide (2A), DT receptor (DTR), LoxP recognition sequence for Cre recombinase (1102), DNA homology arm sequence downstream of the insertion site, and DTA.

[0299] A vector (V3901) was introduced into mouse embryonic stem cells. The cells were screened by the corresponding antibiotic resistance gene markers or their combination. It was confirmed by PCR that the vector V3901 was integrated into the correct locus of the mouse genome. The results are shown in Figures 26 to 27. The combination of these two PCR results confirmed that cells 208, 209, 215, 217 and 269 were positive clones.

[0300] PCR assays were performed using the following primers: IGKV-005-C-5G-L-GT-F:5'-TCACACACTACAGCTTCCACCACAA-3' (SEQ ID NO: 34); IGKV-005-C-5G-L-GT-R2: 5'-CGGGGAAAAGTCGACTCTAGAACGG-3' (SEQ ID NO: 35); IGKV-005-C-5G-R-GT-F1: 5'-ACTGCATTCTAGTTGTGGTTTGTCCA-3' (SEQ ID NO: 36); IGKV-005-C-5G-R-GT-R: 5'-GGCCTGGAAAACTCAGCTATCCTTT-3' (sequence number 37).

[0301] Of these primers, IGKV-005-C-5G-L-GT-F and IGKV-005-C-5G-R-GT-R were located on the mouse chromosome, and IGKV-005-C-5G-L-GT-R2 and IGKV-005-C-5G-R-GT-F1 were located on the vector V3901.

[0302] In this way, two recombination sites were introduced into mouse chromosome 6 in mouse embryonic stem cells.

[0303] Example 7: Modifying human chromosome 2 The human light chain immunoglobulin locus is located on human chromosome 2. Figure 28 is a schematic diagram of human chromosome 2, highlighting the light chain immunoglobulin locus.

[0304] Two recombination sites were introduced on either side of the variable region of the light chain immunoglobulin locus. HK The region between the chromosome 1 and the centromere was deleted to obtain a shorter artificial chromosome for subsequent experiments. A similar recombination site was introduced into the variable region of the mouse immunoglobulin locus on chromosome 6. A human chromosome was then introduced into mouse recipient cells to obtain a humanized light chain immunoglobulin locus.

[0305] The modified human chromosome 2 is shown in Figure 29. The targeting strategy is shown in Figure 30. The vector (V2701) has, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, a PGK promoter, a red fluorescent protein reporter gene sequence (tdTomato), an FMDV self-cleaving peptide (2A), a blasticidin S deaminase from Aspergillus terreus (Bsr), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a LoxP recognition sequence 2601, a hygromycin resistance gene (a partial sequence of hygromycin phosphotransferase; "3'HygR"), an Flp recognition target (FRT), a DNA homology arm sequence downstream of the insertion site, and a diphtheria toxin subunit A (DTA).

[0306] The vector (V2702) has, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, LoxP recognition sequence 2602, a PGK promoter, a portion of the puromycin resistance gene sequence (5'PuroR), EF-1a, PBase, IRES, a kanamycin resistance gene sequence (Neo), a transcription termination / polyadenylation signal sequence (polyA; "PA"), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0307] The sequence of the vector (V2702) was confirmed by sequencing. The vector was transfected into human H9 cells. The cells were then screened for G418 and ouabain resistance. The integration of the gene into the human genome was confirmed by PCR. The results are shown in Figure 31. Clones 01, 02, 03 and 04 were identified as positive clones.

[0308] PCR assays were performed using the following primers: 3'LL-GT-F: 5'-AAGGTGACTCTGCAATCAGCCTCTG-3' (SEQ ID NO: 38), 3'LL-GT-R1: 5'-TCATCTACAGCCACAACGTGAGCAG-3' (SEQ ID NO: 39); 3'LR-GT-F1: 5'-CCCATGTACAGGTTCCGCATGAACT-3' (SEQ ID NO: 40), 3'LR-GT-R: 5'-CTCCGTCCGCTTTTATTTCCCCTGT-3' (sequence number 41).

[0309] Cells with modified chromosomes suitable for further experiments were selected. The modified human chromosomes were introduced into recipient cells by chromosome techniques. Recipient cells A9 cells were screened for G418 resistance. Cells containing only one human chromosome were selected for further gene editing.

[0310] Upon screening, clone #03, shown in Figure 31, contained the correct recombination and was labeled with a human chromosome 2 counting probe (CCP2 FISH probe) (CytoTest Inc., Rockville, MD, Catalog No. CT-CCP002). The results confirmed that the cells contained a modified human chromosome 2 (Figure 32).

[0311] The cells were then further transfected with a vector (V2701). The cells were screened for G418 and blasticidin S resistance. Recombination was confirmed by PCR. The primers are shown in the table below. These results showed that 5-C3, 1-H2, 1-H9 and 1-G5 were positive clones (Figure 33).

[0312] [Table 14]

[0313] Example 8: Generating mice capable of producing humanized antibodies The mouse embryonic stem cells were fused with the cells obtained in Example 7 to introduce the modified human chromosome 2 into the mouse embryonic stem cells obtained in Example 6. Mouse ES cells containing only one modified human chromosome 2 were selected, and the cells were screened after Cre-mediated recombination (Cre-mediated recombination) as shown in FIG. 34. The cells after Cre recombination were tested to confirm the integration of the human gene sequence into the mouse genome. The PCR-confirmed positive clones were verified by fluorescence in situ hybridization (FISH) using a mouse whole chromosome painting probe (Cytocell Ltd, Cambridge, UK; Catalogue No. AMP06G) and a human-specific IGK break-apart probe (Cytocell Ltd, Cambridge, UK; Catalogue No. LPH 034). The results are shown in FIG. 35, confirming the presence of the human chromosome fragment in the mouse chromosome. These cells were injected into blastocysts. Mice containing a humanized light chain immunoglobulin locus can be obtained.

[0314] The chimeric mice were selected and mated with C57BL / 6 mice to produce gray and black offspring (F1 generation). PCR analysis was performed on DNA obtained from the tails of the black mice, and positive F1 generation mice were selected and mated with Flp-tool mice. Figure 36 shows a schematic diagram of Flp-mediated recombination. The mice prepared by this method included a C57BL / 6 background. Mice of different backgrounds can have different advantages, and the heterozygous or homozygous mice prepared by the methods herein can be used to create mice with some other background by backcrossing for several generations to obtain mice of the desired background (e.g., BALB / c mice have the advantage of humoral immunity).

[0315] Several mice were selected and crossed several times with BALB / c mice to obtain heterozygous mice on a BALB / c background, which were then crossed with each other to obtain homozygous mice.

[0316] Mice bearing humanized light chain immunoglobulin loci were crossed with mice bearing humanized heavy chain immunoglobulin loci to obtain mice bearing both humanized heavy and light chain immunoglobulin loci.

[0317] Example 9: B cell development in transgenic mice An experiment was conducted to compare the immune systems of humanized and wild-type mice. Three 9-10 week old wild-type (WT), three mice with heterozygous humanized heavy chain immunoglobulin loci, and three mice with homozygous humanized heavy chain immunoglobulin loci were selected. Among them, the heterozygous and homozygous mice had similar weight, appearance, and vitality when compared to the wild-type mice. Peripheral blood, spleen, lymph node, and bone marrow tissues of these mice were obtained, and no obvious anatomical changes were found (e.g., there was no observable difference in spleen size, morphology, and weight among these three groups of mice). Flow cytometry was performed to analyze the lymphocyte population and distribution in the peripheral blood, spleen, and lymph nodes of these mice (Figures 45-47) and the B cell population in the spleen, lymph node, and bone marrow (Figures 48-50). In these results, leukocytes included B cells (e.g., characterized by CD45+, CD19+, TCR-), T cells, and natural killer (NK) cells (e.g., characterized by CD45+, TCR-, NK1.1+). T cells were characterized by CD45+, CD19-, TCR+. CD4+ T cells (CD4) were characterized by CD45+, CD19-, TCR+, CD4+, CD8-, and CD8+ T cells (CD8) were characterized by CD45+, CD19-, TCR+, CD4-, CD8+. Only intact, single, viable leukocytes were included in the flow cytometry analysis.

[0318] The developmental stage of B cells in lymph nodes and spleen is T1 (transitional B cells type 1, B220 + IgM + IgD -), T2 (type 2 transitional B cells, B220 + IgM + IgD + ) and mature B cells (B220 + IgM low IgD + Figures 45-47 show the percentage of white blood cells in various tissue samples. Figures 48-49 show the percentage of B cells by developmental stage.

[0319] In addition, B cell development is conserved in the splenic marginal zone (MZ-B, B220 + CD21 + CD23 - follicular zone (characterized by follicular B cells, also called FO-B, B220 + CD21 low CD23 + Figure 50 shows the percentage of splenic B cells in the splenic marginal zone (MZ-B) and follicular zone (FO-B).

[0320] Based on different developmental stages, B cells in the bone marrow are classified as pro-B cells (B220 low CD43 high IgM low ), pre-B cells (B220 low CD43 int IgM low ) and immature B cells (B220 high CD43 low IgM high 51A to 51C show the proportions of B cells in the bone marrow according to the developmental stage.

[0321] Compared to wild-type mice, the proportions of immune cells and B cells in humanized mice were similar and not statistically different between the different groups. No significant defects in B cell differentiation were observed in either heavy chain humanized heterozygous or homozygous mice.

[0322] Example 10: Serum immunoglobulin isotype analysis Additionally, various immunoglobulin levels were analyzed in the serum of non-immunized mice from the above examples, including WT mice, mice with heterozygous humanized heavy chain immunoglobulin loci, and mice with homozygous humanized heavy chain immunoglobulin loci.

[0323] The experiment was performed using Clonotyping System-B6 / C57J-HRP (Southern Biotech, Catalog No. 5300-05B) kit. First, the capture antibody goat anti-mouse Ig, human ads-UNLB was diluted to 10ug / mL in PBS (Solarbio, Catalog No. P1020). Then, 0.1mL of antibody diluent was added to each well of the enzyme-linked immunosorbent assay (ELISA) plate and incubated at 37°C for 2 hours. The plate was then washed and blocked at 4°C for 12 hours. Serum samples were serially diluted in 1% BSA (Cell Signaling, Catalog No. 9998). 0.1mL of sample diluent was added to each well, followed by incubation at 37°C for 1 hour. 1% BSA was added to one well as a blank control.

[0324] The plate was then washed with PBS containing 0.05% Tween-20 (Amresco, Cat. No. M147). HRP-conjugated secondary antibodies (goat anti-mouse IgA, IgG (1, 2b, 2c, 3), IgM) (diluted 300-fold with 1% BSA, 0.1 mL per well) were added and incubated with the samples at 37°C for 1 hour. The plate was then washed and developed by adding 0.1 mL of TMB chromogen solution (Beyotime Biotechnology, Cat. No. P0209) to each well. After 8 minutes of incubation at room temperature in the dark, 0.1 mL of reaction solution (Beijing Dingguo Changsheng Biotechnology Co.LTD., Cat. No. EIA-0032) was added to each well. The absorbance at 450 nm and 570 nm was measured using a microplate reader (Thermo MULTISKAN GO, Thermo Fisher Scientific), and the standard OD value was calculated.

[0325] These results demonstrated that mice with humanized heavy chain immunoglobulin loci were capable of producing IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM antibody isotypes, with similar expression levels of each isotype compared to wild-type mice (Figures 52-57), indicating that humanization of the heavy chain variable region gene segments did not have significant adverse effects on antibody class switching, expression, or secretion of various antibody isotypes.

[0326] Example 11: V(D)J recombination of human variable region gene segments in mice The mRNA sequences of the heavy and light chain variable regions in mice were analyzed by next-generation sequencing.

[0327] One unimmunized (not exposed to a specific antigen) humanized heavy chain homozygous mouse was selected. Spleen cells were collected from this mouse for RNA extraction. Reverse transcription was performed using a 5'RACE kit (SMARTer RACE 5' / 3' kit, Takara Bio USA, Inc., Catalog No. 634858) to obtain cDNA. The obtained cDNA was PCR amplified using the IgM constant region specific primer and UPM primer of the 5'RACE kit to obtain a heavy chain variable region sequence fragment, which was then sequenced. The IgM constant region specific primer sequence was 5'-ccaagcttacgagggggaagacatttgggaa-3' (SEQ ID NO: 50).

[0328] In another experiment, 11 light chain humanized heterozygous mice were selected and RNA was extracted from retro-orbital blood. After reverse transcription as described above, primers VKF1 and IgKC tag were used to amplify the VκI family light chain gene, followed by sequencing. The following primer sequences were used: VKF1 sequence: 5'-cataagatctcgmcatccrgwtgacccagt-3' (SEQ ID NO:51); IgKC tag primer sequence: 5'-ctaacactcattcctgttgaagctcttgac-3' (SEQ ID NO:52).

[0329] By comparing the sequencing results with the NCBI Ig Blast tool for human immunoglobulin sequences, we found that the human V(D)J recombination H , D H , J H In the 135 clones analyzed, the expression of specific Vκ and Jκ genes was identified. H , D H Gene segments and all J HExpression of gene segments was detected (Table 15). Some of these gene segments were located near the modification site on the humanized fragment, and some were located far from the modification site. This suggests that the expression of human V sequences on the human chromosomal fragment integrated into the mouse was consistent with that of the human V sequences on the human chromosomal fragment integrated into the mouse after replacing the endogenous chromosomal fragment with the human immunoglobulin heavy chain sequence. H , D H and J H It was noted that human heavy chains could be expressed by genetic engineering.

[0330] Most of the VκI family light chain genes were detected in 441 clones derived from humanized light chain heterozygous mice (Table 16). Similar to the heavy chain detection results, some of these genes on the humanized fragments were located close to the modification site, and some were distant from the modification site. This indicated that after replacing the endogenous light chain immunoglobulin variable region locus with the human light chain immunoglobulin variable region locus, light chains with human light chain variable regions could be expressed by recombination of the human Vκ and Jκ genes integrated into the mouse genome. Further analysis of the results of 11 mice showed that there was no significant difference in the distribution of IGKV genes detected among the tested mice (Figure 58).

[0331] [Table 15]

[0332] [Table 16]

[0333] Example 12: Immunization and antibody production in humanized mice Five wild-type (WT) mice and five humanized heavy chain homozygous mice (9-10 weeks old) were randomly selected and immunized with exogenous antigens. The mice were repeatedly immunized once every two weeks for a total of three immunizations. Retroorbital blood was collected after the second and third immunizations. Serum was collected and then serum titers were measured by ELISA or FACS to determine and analyze antigen-specific antibody responses. Three antigens, hBTLA, dPD1, and OVA, were used in this study (Figures 59-63). These results showed that most of the wild-type (WT) and humanized heavy chain homozygous mice produced antigen-specific antibodies after the second immunization. After the third immunization, the antibody titers were 1×10 4 ~1×10 5 Immune titer test results were essentially the same in humanized mice compared to wild-type mice, indicating that the humanized immunoglobulin variable region loci in the mice are functional and capable of producing antigen-specific antibodies.

[0334] Example 13: B cell development in hVH / hVL mice Mice with homozygous humanized heavy chain immunoglobulin loci (humanized VH mice or hVH mice) and mice with humanized light chain immunoglobulin loci (humanized VL mice or hVL mice) were crossed with each other to obtain mice with both homozygous humanized heavy chain immunoglobulin loci and homozygous humanized light chain immunoglobulin loci (humanized VH / VL mice or hVH / hVL mice), which can be used for the production of humanized monoclonal antibodies in vivo.

[0335] An experiment was carried out to compare the immune systems of naive humanized VH / VL mice and naive wild-type mice. The body weight and spleen weight of wild-type and hVH / hVL mice were measured (Figures 65A-65B). No significant differences were detected between wild-type and hVH / hVL mice in the mean body weight and spleen weight.

[0336] Flow cytometry was performed to analyze lymphocyte populations and distribution in the mouse spleen (Figure 66) and B cell populations in the spleen and bone marrow (Figures 67A-67B, Figures 68A-68C). These results showed that the proportions of B cells, T cells, NK cells, CD4+ T cells, and CD8+ T cells in the spleen were similar in hVH / hVL mice to those in wild-type mice. In these results, leukocytes included B cells (e.g., characterized by CD45+, CD19+, TCR-), T cells, and natural killer (NK) cells (e.g., characterized by CD45+, TCR-, NK1.1+). T cells were characterized by CD45+, CD19-, TCR+. CD4+ T cells (CD4) were characterized by CD45+, CD19-, TCR+, CD4+, CD8-. CD8+ T cells (CD8) were also characterized by CD45+, CD19-, TCR+, CD4-, and CD8+. Only intact single viable leukocytes were included in the flow cytometry analysis.

[0337] Figure 67A shows the percentage of B cells by developmental stage. The developmental stage of B cells in the spleen is T1 (transitional B cells of type 1, B220 + IgM + IgD - ), T2 (type 2 transitional B cells, B220 + IgM + IgD + ) and mature B cells (B220 + IgM low IgD + In addition, B cell development was classified into the splenic marginal zone (marginal zone B cells, MZ-B, B220 + CD21 + CD23 - follicular zone (characterized by follicular B cells, also called FO-B, B220 + CD21 low CD23 +The percentage of splenic B cells in the splenic marginal zone (MZ-B) and follicular zone (FO-B) was also evaluated in wild-type mice and hVH / hVL mice.

[0338] Figure 68A shows the proportion of B cells in the bone marrow at each developmental stage. B cell precursors in the bone marrow were analyzed by flow cytometry. Based on the expression levels of B220 and CD43, B cell precursors in the bone marrow were divided into three cell populations: pro-B cells (B220 low CD43 high IgM low ), pre-B cells (B220 low CD43 int IgM low ) and immature B cells (B220 high CD43 low IgM high No significant differences were observed between wild-type and hVH / hVL mice.

[0339] In addition, B cell development was monitored by flow cytometry in the bone marrow or spleen to detect plasma cells (B220 low IgM - IgD - CD138 - ) and memory B cells (B220 + IgM + IgD - CD38 + ) was also evaluated by selective staining (FIGS. 68B-68C). No significant differences were observed between wild-type and hVH / hVL mice.

[0340] The different immunoglobulin (Ig) subtypes in the serum of hVH / hVL and wild-type mice were quantitatively measured by ELISA. A total of six mice were selected from each group. No significant differences were observed in the IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM levels (Figure 69).

[0341] These experiments demonstrated that the immune systems of hVH / hVL mice are functional and that the humanized immunoglobulin loci of hVH / hVL mice can interact properly with mouse immunoglobulin constant regions.

[0342] Example 14: Analysis of germline usage in hVH / hVL mice Heavy chain IGHV, IGHD, and IGHJ usage in hVH / hVL naive mice (unstimulated) was analyzed. The results are shown in Figures 70A-70D. In addition, kappa chain IGKV and IGKJ usage was also analyzed. The results are shown in Figures 71A-71C.

[0343] Germline usage of naive hVH / hVL mice was determined by next generation sequencing (NGS). For example, as shown in Figure 71C, IGKJ1, IGKJ2 and IGKJ4 were frequently used in naive hVH / hVL mice, while IGKJ3 and IGKJ5 were observed less frequently. Such IGKJ germline usage patterns are consistent with literature reports on human IGKJ germline usage.

[0344] The immune repertoire from splenocytes of naive hVH / hVL mice (n=2) was sequenced by NGS to determine the distribution of heavy chain CDR3 length. As shown in Figure 72, the median length of CDR3 was 14 amino acids. This result was consistent with the median length of human heavy chain CDR3 in the human immune system.

[0345] The amino acid types at each position of the heavy chain CDR3 (HCDR3) were analyzed (Figure 73). Several patterns were observed, including increased tyrosine usage and increased usage of the DH2 (IGHD2) germline family. These patterns are similar to the amino acid composition of human HCDR3.

[0346] Cysteine ​​residues can form disulfide bonds. Human HCDR3 can contain one or two cysteine ​​residues, while mouse HCDR3 typically does not contain cysteines. The results in Figure 74 show the frequency of HCDR3 in hVH / hVL mice that contain cysteine ​​residues and the increased frequency with increasing HCDR3 length. This result is consistent with the diversity of HCDR3 in human peripheral blood mononuclear cells (PBMCs).

[0347] Example 15: Histological analysis of lymphoid organs Spleens, inguinal lymph nodes, and Peyer's patches from naive wild-type or naive hVH / hVL mice were stained with H&E. Representative sections are shown in Figure 75. Wild-type (C57BL / 6) mice and hVH / hVL mice exhibited normal architecture, including distinct follicles, and no significant differences were observed in histological morphology.

[0348] Example 16: Antibody production in hVH / hVL mice After the second and third immunizations with BCMA, IL4R, PD-1, Siglec-15, and SIRPα antigens, blood was collected and the antigen-specific antibody titers of wild-type (C57BL / 6) and hVH / hVL mice were analyzed by ELISA (Figures 76A-76E). These results showed that hVH / hVL mice were able to produce antibodies that specifically bound to the antigens, and that the immune responses of wild-type and hVH / hVL mice were comparable.

[0349] Example 17: B cell development in hVH / hVL mice An experiment was carried out to compare the immune systems of humanized VH / VL mice and wild-type mice after immunization. The body weight and spleen weight of wild-type and hVH / hVL mice were measured (Figures 77A-77B). No significant differences were detected between wild-type and hVH / hVL mice in the average body weight and spleen weight.

[0350] Flow cytometry was performed to analyze lymphocyte populations and distribution in the mouse spleen (Figure 78) and B cell populations in the spleen and bone marrow (Figures 79A-B, 80A-C).

[0351] Figure 79A shows the percentage of B cells in the spleen at each developmental stage. In addition, B cell development was also evaluated in the splenic marginal zone and follicular zone. Figure 79B shows the percentage of splenic B cells in the splenic marginal zone (MZ-B) and follicular zone (FO-B). No significant difference was observed between wild-type and hVH / hVL mice.

[0352] Figure 80A shows the proportion of B cells in the bone marrow at each developmental stage. B cell precursor cells in the bone marrow were analyzed by flow cytometry. No significant difference was observed between wild-type mice and hVH / hVL mice.

[0353] In addition, B cell development was monitored by flow cytometry in the bone marrow or spleen to detect plasma cells (B220 low IgM - IgD - CD138 - ) and memory B cells (B220 + IgM + IgD - CD38 + ) was also evaluated by selective staining (FIGS. 80B-80C). No significant difference was observed between wild-type and hVH / hVL mice.

[0354] The different immunoglobulin (Ig) subtypes in the serum of hVH / hVL and wild-type mice were quantitatively measured by ELISA. A total of 6 mice were selected from each group. No significant differences were observed in the IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM levels (Figure 81). In addition, the total IgG amount in the serum of hVH / hVL and wild-type mice was quantitatively measured by ELISA. No significant differences were observed (Figure 82).

[0355] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, rather than limiting, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for producing a genetically modified non-human animal, comprising: (a) modifying a first human chromosome; wherein said modified first human chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a heavy chain immunoglobulin locus; (b) introducing the modified first human chromosome into a cell of the animal; and (c) inducing recombination between said modified first human chromosome and an endogenous chromosome, wherein said endogenous chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a heavy chain immunoglobulin locus, thereby replacing an endogenous sequence at the heavy chain immunoglobulin variable region locus in a one-step replacement with a contiguous human sequence comprising all of the non-pseudo human IGHV genes, all of the non-pseudo human IGHD genes and all of the non-pseudo human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14; Including, The method of claim 1, wherein the genetically modified non-human animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain.

2. (d) modifying a second human chromosome; wherein said modified second human chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a light chain immunoglobulin locus; (e) introducing the modified second human chromosome into cells of the animal; and (f) inducing recombination between the modified second human chromosome and an endogenous chromosome, wherein the endogenous chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a light chain immunoglobulin locus, thereby replacing the endogenous sequence at the light chain immunoglobulin variable region locus in a one-step replacement with a contiguous human sequence comprising all non-pseudo-human IGKV genes and all non-pseudo-human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2; Further comprising:

2. The method of claim 1, wherein the genetically modified non-human animal lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain.

3. The method of claim 1 , wherein the animal is a mouse.

4. 1. A method for producing a genetically modified non-human animal, comprising: (a) modifying a first human chromosome; wherein said modified first human chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a light chain immunoglobulin locus; (b) introducing the modified first human chromosome into a cell of the animal; and (c) inducing recombination between the modified first human chromosome and an endogenous chromosome, wherein the endogenous chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a light chain immunoglobulin locus, thereby replacing an endogenous sequence at the light chain immunoglobulin variable region locus with a contiguous human sequence comprising all non-pseudo-human IGKV genes and all non-pseudo-human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2 in a one-step replacement; Including, The method of claim 1, wherein the genetically modified non-human animal lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain.

5. (d) modifying a second human chromosome; wherein said modified second human chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a heavy chain immunoglobulin locus; (e) introducing the modified second human chromosome into cells of the animal; and (f) inducing recombination between said modified second human chromosome and an endogenous chromosome, wherein said endogenous chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a heavy chain immunoglobulin locus, thereby replacing an endogenous sequence at the heavy chain immunoglobulin variable region locus in a one-step replacement with a contiguous human sequence comprising all of the non-pseudo human IGHV genes, all of the non-pseudo human IGHD genes and all of the non-pseudo human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14; Further comprising:

5. The method of claim 4, wherein the genetically modified non-human animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain.

6. The method of claim 4 , wherein the animal is a mouse.

7. A genetically modified non-human animal, the genome of which comprises an endogenous heavy chain immunoglobulin locus comprising a replacement in a one-step replacement of an endogenous IGHV, endogenous IGHD and endogenous IGHJ gene with a contiguous human sequence comprising all non-pseudo-human IGHV, all non-pseudo-human IGHD and all non-pseudo-human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14, wherein the human IGHV, human IGHD and human IGHJ genes are operably linked to one or more of the endogenous IGHM, IGHδ, IGHG, IGHE and IGHA genes; The genetically modified non-human animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain.

8. The animal of claim 7, which is a mouse, and the continuous sequence starting from the mouse IGHV1-85 gene to the mouse IGHJ4 gene has been replaced by one-step replacement.

9. A genetically modified non-human animal, the genome of which comprises an endogenous light chain immunoglobulin locus comprising a replacement in a one-step replacement of an endogenous IGKV and endogenous IGKJ gene with a contiguous human sequence comprising all non-pseudo-human IGKV and non-pseudo-human IGKJ genes at an endogenous kappa chain immunoglobulin locus of human chromosome 2, the human IGKV and human IGKJ genes being operably linked to an endogenous IGKC gene; The genetically modified non-human animal lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain.

10. The animal of claim 9, which is a mouse, and the consecutive sequence starting from mouse IGKV2-137 to mouse IGKJ5 is replaced by one-step replacement.

11. A cell obtained from the animal according to any one of claims 7 to 10.

12. 1. A method for modifying the genome of a cell, comprising: (a) modifying a human chromosome; wherein said modified human chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a heavy chain immunoglobulin locus; (b) introducing the modified human chromosome into a cell of the animal; and (c) inducing recombination between the modified human chromosome and an endogenous chromosome, wherein the endogenous chromosome comprises two exogenous recombination sites introduced on either side of a variable region of a heavy chain immunoglobulin locus, thereby replacing an endogenous sequence at the heavy chain immunoglobulin variable region locus in a one-step replacement with a contiguous human sequence comprising all of the non-pseudo-human IGHV genes, all of the non-pseudo-human IGHD genes and all of the non-pseudo-human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14. Includes The method of claim 1, wherein the cell having the modified genome lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain.

13. A contiguous human sequence including all non-pseudo-human IGKV and non-pseudo-human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2 is also integrated into the endogenous chromosome by recombination in a one-step replacement, thereby replacing the endogenous sequence at the light chain immunoglobulin variable region locus.

13. The method of claim 12, wherein the cell having the modified genome lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain.

14. 1. A method for producing an antibody that specifically binds to an antigen, comprising the steps of: Obtaining nucleic acid sequences encoding human heavy and light chain immunoglobulin variable regions in cells expressing a hybrid antibody that specifically binds to said antigen, said cells being obtained by exposing an animal according to any one of claims 7 to 10 to said antigen; operably linking said nucleic acid encoding said human heavy chain immunoglobulin variable region with a nucleic acid encoding a human heavy chain immunoglobulin constant region, and said nucleic acid encoding said human light chain immunoglobulin variable region with a nucleic acid encoding a human light chain immunoglobulin constant region; and expressing said nucleic acid in a cell, thereby obtaining said antibody. The method includes:

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