Genetically modified non-human animals having humanized immunoglobulin loci

Abstract

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

Description

[0001] This application is a divisional application of application number 202080002761.4, filed February 18, 2020, entitled “Genetically Modified Non-Human Animals Having Humanized Immunoglobulin Loci.”

[0002] CLAIM OF PRIORITY

[0003] This application claims priority to PCT / CN2019 / 075406 filed February 18, 2019 and PCT / CN2019 / 106320 filed September 18, 2019. The entire contents of the foregoing are incorporated herein by reference. TECHNICAL FIELD

[0004] The present disclosure relates to genetically modified animals and cells having humanized heavy chain immunoglobulin loci and / or humanized light chain immunoglobulin loci. BACKGROUND

[0005] Therapeutic antibodies are one of the fastest growing classes of therapeutic compounds, growing faster than small molecule drugs. These therapeutic antibodies are often human or humanized antibodies. Human or humanized antibodies can be generated by humanization of rodent antibodies (e.g., mouse antibodies) or by using phage libraries. Antibodies generated by these methods often have suboptimal binding affinities and biophysical properties, leading to manufacturing difficulties and poor pharmacokinetics. In particular, the humanization process can adversely affect binding affinity and introduce immunogenic epitopes into the antibody, and antibodies discovered using phage libraries show limited diversity and non-native pairing of immunoglobulin heavy and light chains. Iterative and time-consuming experiments are often needed to improve the properties. In some cases, these antibodies can also be immunogenic in patients, leading to their efficacy waning over time.

[0006] One possible method to generate fully human antibodies is to use transgenic animals engineered to express human antibody lineages. The generation of transgenic animals, such as mice with different immunoglobulin loci, has allowed the use of such transgenic animals in various research and development applications, for example, in drug discovery and basic research on various biological systems. Many early transgenic animals had incomplete human antibody lineages, with lower than normal levels of antibody production due to lower V(D)J recombination efficiency, with endogenous antibody lineages that can introduce immunogenic epitopes, and various other problems. There is a need for efficient and cost-effective methods to produce human antibodies, and for non-human animals comprising humanized immunoglobulin loci that have the ability to respond to antigens to produce humanized antibodies. SUMMARY

[0007] The present disclosure relates to genetically modified animals and cells having humanized heavy chain and light chain immunoglobulin loci.

[0008] In some aspects, the present disclosure relates to a genetically modified non-human animal comprising 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, human IGHD genes, and human IGHJ genes are operably linked and can undergo VDJ recombination.

[0009] 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 human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, and IGHV1-8, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at an endogenous heavy chain immunoglobulin locus on human chromosome 14. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at an endogenous heavy chain immunoglobulin locus on human chromosome 14 in a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell that does not have any V(D)J recombination).

[0010] In some embodiments, the animal comprises a disruption in its endogenous heavy chain immunoglobulin locus.

[0011] In some embodiments, the animal is a mouse and the disruption in 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.

[0012] In some embodiments, the animal is a mouse and the disruption in the endogenous heavy chain immunoglobulin locus of the animal comprises a deletion of a continuous sequence beginning at the mouse IGHV1-85 gene to the mouse IGHJ4 gene.

[0013] In some embodiments, the animal comprises one or more endogenous IGHM, IGHd, IGHG3, IGHGl, IGHG2b, IGHG2a, IGHE, and IGH A genes.

[0014] In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus.

[0015] In some embodiments, the unmodified human sequence is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1000 kb.

[0016] In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus beginning at the human IGHV(III)-82 to the human IGV1-2. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus beginning at the human IGHV(III)-82 to the human IGHV6-1. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus beginning at the human IGHD1-1 to the human IGHJ6.

[0017] In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus beginning at the human IGHV(III)-82 to the human IGHJ6.

[0018] 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.

[0019] In some embodiments, the animal further comprises one or more human IGKV genes and one or more human IGKJ genes at an endogenous light chain immunoglobulin locus.

[0020] In some embodiments, the animal comprises a disruption in its endogenous lambda light chain immunoglobulin locus.

[0021] In some embodiments, the animal is a rodent (e.g., a mouse).

[0022] In some aspects, the present 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.

[0023] 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.

[0024] 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 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1000 kb.

[0025] In some embodiments, the second sequence comprises an endogenous sequence that is about or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, or 20 kb.

[0026] 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.

[0027] In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus. In some embodiments, the third sequence is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, or 100 kb.

[0028] In some embodiments, the animal comprises a disruption in its endogenous heavy chain immunoglobulin locus.

[0029] In some embodiments, the animal is a mouse and the disruption in 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 IGH D genes in Table 5, and one or more mouse IGHJ genes in Table 6.

[0030] In some embodiments, the animal is a mouse and the disruption in the animal’s endogenous heavy chain immunoglobulin locus comprises a deletion of sequences starting from mouse IGHV1-85 to mouse IGHJ4.

[0031] In some embodiments, the animal comprises one or more endogenous genes selected from IGHM, IGHd, IGHG3, IGHGl, IGHG2b, IGHG2a, IGHE, and IGH A genes.

[0032] 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.

[0033] In some embodiments, the animal further comprises one or more human IGKV genes and one or more human IGKJ genes on an endogenous light chain immunoglobulin locus.

[0034] In some embodiments, the animal comprises a disruption in its endogenous lambda light chain immunoglobulin locus.

[0035] In some embodiments, the animal is a rodent (e.g., a mouse).

[0036] In some aspects, the disclosure relates to a genetically modified non-human animal comprising one or more human IGKV genes and one or more human IGKJ genes on an endogenous light chain immunoglobulin locus.

[0037] 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 comprises about or at least 1, 2, 3, 4, or 5 human IGKJ genes in Table 8.

[0038] In some embodiments, the animal comprises unmodified human sequences derived from the human light chain immunoglobulin locus from human IGKV3D-7 to human IGKJ5.

[0039] In some embodiments, the animal comprises a disruption in its endogenous light chain immunoglobulin locus.

[0040] In some embodiments, the animal is a mouse and the disruption in 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 on the endogenous kappa chain immunoglobulin locus of human chromosome 2. In some embodiments, the animal comprises all of the human IGKV genes and all of the human IGKJ genes on the endogenous heavy chain immunoglobulin locus of human chromosome 2 in a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell without any V(D)J recombination).

[0041] In some embodiments, the animal is a mouse and the disruption in the animal’s endogenous light chain immunoglobulin locus comprises a deletion of sequences from mouse IGKV2-137 to mouse IGKJ5.

[0042] In some embodiments, the animal comprises an endogenous IGKC.

[0043] 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.

[0044] 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 on the endogenous heavy chain immunoglobulin locus.

[0045] In some embodiments, the animal comprises a disruption in its endogenous lambda light chain immunoglobulin locus.

[0046] In some embodiments, the animal is a rodent (e.g., a mouse).

[0047] In some aspects, the disclosure relates to a genetically modified non-human animal whose genome comprises an endogenous heavy chain immunoglobulin locus comprising: one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes replaced with one or more human IGHV, human IGHD, and human IGHJ genes. In some embodiments, the human IGHV, human IGHD, and human IGHJ genes are operably linked to one or more endogenous genes selected from the group consisting of IGHM, IGHd, IGHG, IGHE, and IGH A genes.

[0048] In some embodiments, one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes are replaced with 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 human IGHJ genes in Table 3.

[0049] In some embodiments, the animal is a mouse and about or at least 180 mouse IGHV genes in Table 4, all mouse IGHD genes in Table 5, and all mouse IGHJ genes in Table 6 are replaced.

[0050] In some aspects, the present disclosure relates to a genetically modified non-human animal whose genome comprises an endogenous light chain immunoglobulin locus comprising: one or more endogenous IGKV and endogenous IGKJ genes are replaced with one or more human IGKV and human IGKJ genes. In some embodiments, the human IGKV and human IGKJ genes are operably linked to an endogenous IGKC gene.

[0051] In some embodiments, one or more endogenous IGKV and endogenous IGKJ genes are replaced with 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.

[0052] In some embodiments, the animal is a mouse and all mouse IGKV genes in Table 9 and all mouse IGKJ genes in Table 10 are replaced.

[0053] In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable domain locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain (e.g., a mouse heavy chain variable domain).

[0054] In some embodiments, the animal lacks an endogenous immunoglobulin light chain variable domain locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain (e.g., a mouse light chain variable domain).

[0055] In some embodiments, the animal can produce humanized antibodies.

[0056] In some aspects, the present disclosure relates to a cell obtained from an animal as described herein.

[0057] In some embodiments, the cell is a B cell expressing 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.

[0058] In some embodiments, the cell is a B cell expressing 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, and wherein the immunoglobulin light chain variable domain is operably linked to a non-human light chain constant region.

[0059] In some embodiments, the cell is an embryonic stem (ES) cell.

[0060] 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 described herein to an antigen; producing a hybridoma from cells collected from the animal; and collecting the chimeric antibody produced by the hybridoma. In some embodiments, the cell of interest is isolated and sequenced to determine the sequence of the rearranged heavy chain variable region and light chain variable region.

[0061] In some embodiments, the method further comprises sequencing the genome of the hybridoma.

[0062] 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.

[0063] In some embodiments, the modified human chromosome comprises two or more exogenous recombination sites.

[0064] In some embodiments, the endogenous chromosome comprises two or more exogenous recombination sites.

[0065] 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 IGH D genes selected from Table 2, and about or at least 5, 6, 7, 8, or 9 human IGH J genes selected from Table 3 are integrated into the endogenous chromosome by recombination.

[0066] 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 IGK J genes in Table 8 are integrated into the endogenous chromosome by recombination.

[0067] In some embodiments, human sequences are integrated into the endogenous chromosome by recombination, and the human sequences are about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1000 kb.

[0068] In one aspect, the present disclosure provides a method of making an antibody that specifically binds to an antigen. The method comprises 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, wherein the cell is obtained by exposing an animal described herein to the antigen; operably linking nucleic acid encoding a human heavy chain immunoglobulin variable region to nucleic acid encoding a human heavy chain immunoglobulin constant region and operably linking nucleic acid encoding a human light chain immunoglobulin variable region to nucleic acid encoding a human light chain immunoglobulin constant region; and expressing the nucleic acids in a cell, thereby obtaining the antibody.

[0069] In one aspect, the present disclosure provides a method of obtaining nucleic acid encoding an antibody binding domain that specifically binds to an antigen. The method comprises exposing an animal described herein to the antigen; and sequencing nucleic acid encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen.

[0070] In one aspect, the present disclosure provides a method of obtaining a sample, the method comprising exposing an animal described herein to the antigen; and collecting a sample from the animal. In some embodiments, the sample is spleen tissue, spleen cells, or B cells.

[0071] In one aspect, the present disclosure provides a method of making an antibody that specifically binds to an antigen. The method comprises exposing an animal described herein to the antigen; obtaining (e.g., by sequencing) nucleic acid sequences encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a chimeric antibody that specifically binds to the antigen; and operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region to nucleic acid encoding a human heavy chain immunoglobulin constant region and the nucleic acid encoding the human light chain immunoglobulin variable region to nucleic acid encoding a human light chain immunoglobulin constant region in a cell.

[0072] The present disclosure also relates to offspring of the non-human mammal.

[0073] In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0074] The present disclosure also provides a cell comprising a targeting vector described herein.

[0075] The present disclosure also relates to a cell (e.g., a stem cell, an embryonic stem cell, an immune cell, a B cell, a T cell, or a hybridoma) or a cell line derived from the non-human mammal or offspring thereof, or a primary cell culture thereof. The present disclosure also relates to a tissue, an organ, or a culture thereof derived from the non-human mammal or offspring thereof.

[0076] The present disclosure also relates to the use of the non-human mammal or offspring thereof, an animal model produced by the methods described herein, in the development of products related to immune processes, in the manufacture of human antibodies, or in model systems for pharmacological, immunological, microbiological, and medical research.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials are described herein for use in the present application; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and 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, will control.

[0078] Other features and advantages of the present application will be apparent from the detailed description, the drawings, and the claims which follow, below. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1A is a flowchart of a method of introducing human immunoglobulin genes into a mouse genome.

[0080] Figure 1Bis a summary of replacing mouse immunoglobulin heavy chain variable regions with human immunoglobulin heavy chain variable regions.

[0081] Figure 1C is a summary of replacing mouse immunoglobulin light chain variable regions with human immunoglobulin light chain variable regions.

[0082] Figure 2 is a schematic showing the mouse heavy chain immunoglobulin locus.

[0083] Figure 3A is a schematic showing the mouse heavy chain immunoglobulin locus after introduction of two recombination sites into the genome.

[0084] Figure 3B is a schematic showing the mouse heavy chain immunoglobulin locus after recombination with a targeting vector.

[0085] Figure 4 shows a targeting strategy for modifying the mouse heavy chain immunoglobulin locus.

[0086] Figure 5A shows the results of PCR detection using the mIgHV-5'loxP-L-GT-F / mIGHV- 005-L-GT-R2 primer pair.

[0087] Figure 5B shows the results of PCR detection using the mIGHV-005-5'loxP-R-GT-F2 / mIgHV- 5'loxP-R-GT-R primer pair.

[0088] Figure 6A shows the results of PCR detection using the mIGHV-3'lox-L-GT-F2 / mIGHV-3'lox- L-GT-R2 primer pair.

[0089] Figure 6B shows the results of PCR detection using the mIGHV 3'lox-R-GT-F2 / mIGHV-3'lox- R-GT-Rl primer pair.

[0090] Figure 7 is a schematic of human chromosome 14 highlighting the heavy chain immunoglobulin locus (not drawn to scale). The heavy chain immunoglobulin locus has variable regions (V H ,D H ,J H ) and constant regions (C H ). V H represents segments of the IGHV gene cluster, D H represents segments of the IGHD gene cluster, J H represents segments of the IGHJ gene cluster, C H represents a gene cluster expressing constant domains.

[0091] Figure 8 is a schematic representation of the human chromosome 14 after modification.

[0092] Figure 9 shows the modification of human chromosome 14 with two vectors.

[0093] Figure 10 shows the modified human chromosome 14.

[0094] Figure 11 shows the results of PCR detection of loxP site 301 on chromosome hChr14-mut3 using the hIGHV-5'loxP-L-GT-F1 and hIGHV-5'loxP-R-GT-R primer pairs. 8-D7 is a positive control clone.

[0095] Figure 12 shows the results of PCR detection of loxP site 302 on chromosome hChr14-mut3. 8-D7 is a negative control clone.

[0096] Figure 13 is a fluorescence in situ hybridization (FISH) image of a cell before the human chromosome 14 has been modified.

[0097] Figure 14 is a FISH image of a cell after the human chromosome 14 has been modified.

[0098] Figure 15 is a schematic representation showing the modified mouse chromosome 12.

[0099] Figure 16 is a schematic representation showing Cre-mediated recombination that replaces the mouse heavy chain variable region locus with the corresponding human genomic DNA sequence.

[0100] Figure 17 shows the results of PCR detection using the M5-L primer pair and the M5-R primer pair.

[0101] Figure 18 shows the results of PCR detection using the M3 primer pair.

[0102] Figure 19 shows the results of PCR detection using the H5-L primer pair and the H5-R primer pair.

[0103] Figure 20 shows the results of PCR detection using the H3-L primer pair and the H3-R primer pair.

[0104] Figure 21is a FISH image. White arrows (1) and (2) indicate mouse chromosome 12. White arrow (3) indicates a human chromosome fragment labeled by the human specific IGH break apart probe.

[0105] Figure 22 is a schematic showing Flp-mediated recombination.

[0106] Figure 23 is a schematic showing the mouse light chain immunoglobulin locus.

[0107] Figure 24A is a schematic showing the mouse light chain immunoglobulin locus after introduction of two recombination sites in the genome.

[0108] Figure 24B is a schematic showing the mouse light chain immunoglobulin locus after recombination with the targeting vector.

[0109] Figure 25 is a schematic showing the gene targeting strategy for mouse chromosome 6.

[0110] Figure 26 Results of PCR detection using IGKV-005-C-5G-L-GT-F / IGKV-005-C-5G-L-GT-R2 primer pair are shown.

[0111] Figure 27 Results of PCR detection using IGKV-005-C-5G-R-GT-F1 / IGKV-005-C-5G-R-GT-R primer pair are shown.

[0112] Figure 28 is a schematic of human chromosome 2 highlighting the light chain immunoglobulin locus (not drawn to scale). V HK represents a segment of the IGKV gene cluster, J HK represents a segment of the IGKJ gene cluster, C HK represents an IGKC gene.

[0113] Figure 29 is a schematic showing the modified human chromosome 2.

[0114] Figure 30 is a schematic showing the gene targeting strategy for human chromosome 2.

[0115] Figure 31 Results of PCR assay after the first recombination (introduction of vector 2702) are shown. WT is wild type H9 cell.

[0116] Figure 32 is a FISH image result. White arrows indicate the modified human chromosome 2 with correct recombination.

[0117] Figure 33 Results of PCR detection after the second recombination (introduction of vector 2701) are shown.

[0118] Figure 34 is a schematic showing Cre-mediated recombination in which the human light chain variable region genomic DNA sequence is added to the corresponding mouse locus.

[0119] Figure 35 is a fluorescence in situ hybridization (FISH) image. Arrows (1) and (3) indicate mouse chromosome 6. Arrow (2) indicates the human chromosome fragment labeled by the human-specific IGK break-apart probe.

[0120] Figure 36 is a schematic showing Flp-mediated recombination.

[0121] Figure 37 is a schematic showing the human immunoglobulin heavy chain (IGH) locus on chromosome 14 (14q32.33).

[0122] Figure 38 is a schematic showing the mouse (Mus musculus) IGH locus on chromosome 12 (12F2) (strain C57BL / 6).

[0123] Figure 39 is a schematic showing the human immunoglobulin kappa chain (IGK) locus on chromosome 2 (2pl l.2).

[0124] Figure 40 is a schematic showing the mouse (Mus musculus) IGK locus on chromosome 6 (6C1).

[0125] Figure 41 Lists the IMGT lineage of the human heavy chain immunoglobulin locus (IGH).

[0126] Figure 42 Lists the IMGT lineage of mouse IGH.

[0127] Figure 43 Lists the IMGT lineage of the human kappa chain immunoglobulin locus (IGK).

[0128] Figure 44 Lists the IMGT lineage of mouse IGK.

[0129] Figure 45 Shows the percentage of white blood cells detected in the peripheral blood.

[0130] Figure 46Percentages of leukocytes detected in spleen cells are shown.

[0131] Figure 47 Percentages of leukocytes detected in lymph nodes are shown.

[0132] Figure 48 Percentages of spleen B cells at different developmental stages are shown.

[0133] Figure 49 Percentages of lymph node B cells at different developmental stages are shown.

[0134] Figure 50 Percentages of spleen B cells in marginal zone (MZ-B) and follicular zone (FO-B) are shown.

[0135] Figure 51A Flow cytometry analysis results of B cells at different developmental stages in bone marrow obtained from wild-type mice are shown. Zone 1 represents pro-B cells, zone 2 represents pre-B cells, and zone 3 represents immature B cells.

[0136] Figure 51B Flow cytometry analysis results of B cells at different developmental stages in bone marrow obtained from humanized heavy chain heterozygous mice are shown. Zone 1 represents pro-pre-B cells, zone 2 represents pre-B cells, and zone 3 represents immature B cells.

[0137] Figure 51C Flow cytometry analysis results of B cells at different developmental stages in bone marrow obtained from humanized heavy chain homozygous mice are shown. Zone 1 represents pro-B cells, zone 2 represents pre-B cells, and zone 3 represents immature B cells.

[0138] Figure 52 IgA isotype levels in gradient-diluted mouse serum are shown.

[0139] Figure 53 IgG1 isotype levels in gradient-diluted mouse serum are shown.

[0140] Figure 54 IgG2b isotype levels in gradient-diluted mouse serum are shown.

[0141] Figure 55 IgG2c isotype levels in gradient-diluted mouse serum are shown.

[0142] Figure 56 IgG3 isotype levels in gradient-diluted mouse serum are shown.

[0143] Figure 57 IgM isotype levels in gradient-diluted mouse serum are shown.

[0144] Figure 58 Distribution of IGKV gene expression detected after VJ recombination in individual mice is shown.

[0145] Figure 59 Flow cytometry results from wild type mice immunized with human BTLA are shown.

[0146] Figure 60 Flow cytometry results from humanized heavy chain homozygous mice immunized with human BTLA are shown.

[0147] Figure 61 Flow cytometry results from wild type mice immunized with canine PD-1 (dPD-1) are shown.

[0148] Figure 62 Flow cytometry results from humanized heavy chain homozygous mice immunized with canine PD-1 (dPD-1) are shown.

[0149] Figure 63 Summary of ELISA results from wild type mice (black bars; mice labeled 1-5) and humanized heavy chain homozygous mice (gray bars; mice labeled 6-10) immunized with ovalbumin (OVA) are shown.

[0150] Figure 64 A list of human distal VK cluster IGKV genes and a list of human proximal VK cluster IGKV genes are shown.

[0151] Figure 65A Body weights of unimmunized wild type mice and hVH / hVL mice are shown.

[0152] Figure 65B Spleen weights of unimmunized wild type mice and hVH / hVL are shown.

[0153] Figure 66 Percentages of immune cells in the spleen of unimmunized wild type mice and hVH / hVL mice are shown.

[0154] Figure 67A Percentages of transitional type 1 (T1, B220 + IgM + IgD - ), transitional type 2 (T2, B220 + IgM + IgD + ), and mature type (M, B220 + IgM 低 IgD + ) B cell populations in splenic B cells. Splenic B cells were from unimmunized wild type or hVH / hVL mice.

[0155] Figure 67BPercentages of marginal zone (MZ) and follicular (FO) B cell populations in splenic B cells are shown. Splenic B cells were from unimmunized wild-type or hVH / hVL mice.

[0156] Figure 68A Percentages of pro-B (B220 低 CD43 高 IgM 低 ), pre-B (B220 低 CD43 中 IgM 低 ), and immature B (B220 高 CD43 低 IgM 高 ) cell populations in bone marrow B cells are shown. Bone marrow B cells were from unimmunized wild-type or hVH / hVL mice.

[0157] Figure 68B Percentages of plasma (B220 低 IgM - IgD - CD138 - ) and memory B (B220 + IgM + IgD - CD38 + ) cell populations in bone marrow B cells are shown. Bone marrow B cells were from unimmunized wild-type or hVH / hVL mice.

[0158] Figure 68C Percentages of plasma (B220 低 IgM - IgD - CD138 - ) and memory B (B220 + IgM + IgD - CD38 + ) cell populations in splenic B cells are shown. Splenic B cells were from unimmunized wild-type or hVH / hVL mice.

[0159] Figure 69 Concentrations of serum immunoglobulin (Ig) subtypes in unimmunized wild-type or hVH / hVL mice are shown. Ig subtype concentrations were quantitatively determined by ELISA.

[0160] Figure 70A shows IGHV utilization (frequency > 1%) in unimmunized hVH / hVL mice.

[0161] Figure 70B shows IGHV utilization (frequency < 1%) in unimmunized hVH / hVL mice.

[0162] Figure 70C IGHD utilization in unimmunized hVH / hVL mice is shown.

[0163] Figure 70D IGHJ utilization in unimmunized hVH / hVL mice is shown.

[0164] FIG. 71A shows IGKV utilization (frequency > 1%) in unimmunized hVH / hVL mice.

[0165] FIG. 71B shows IGKV utilization (frequency < 1%) in unimmunized hVH / hVL mice.

[0166] Figure 71C IGKJ utilization in unimmunized hVH / hVL mice is shown.

[0167] Figure 72 is a histogram showing heavy chain CDR3 amino acid length distribution from unimmunized hVH / hVL mice.

[0168] Figure 73 Amino acid frequency on heavy chain CDR3 in unimmunized hVH / hVL mice is shown.

[0169] Figure 74 Frequency of cysteine residues in hVH / hVL mouse HCDR3 is shown.

[0170] FIG. 75A is a histology image of spleen from unimmunized wild-type mice.

[0171] FIG. 75B is a histology image of inguinal lymph node from unimmunized wild-type mice.

[0172] FIG. 75C is a histology image of Peyer's patch from unimmunized wild-type mice.

[0173] FIG. 75D is a histology image of spleen from unimmunized hVH / hVL mice.

[0174] FIG. 75E is a histology image of inguinal lymph node from unimmunized hVH / hVL mice.

[0175] FIG. 75F is a histology image of Peyer's patch from unimmunized hVH / hVL mice.

[0176] Figure 76A BCMA (B-cell maturation antigen)-specific antibody titers after secondary and tertiary immunizations using human BCMA as antigen in wild-type and hVH / hVL mice are shown.

[0177] Figure 76B Titers of IL4R-specific antibodies after the second and third immunizations using human IL4R (interleukin-4 receptor) as an antigen in wild-type and hVH / hVL mice are shown.

[0178] Figure 76C Titers of PD-1-specific antibodies after the second and third immunizations using human PD-1 (programmed cell death protein 1) as an antigen in wild-type and hVH / hVL mice are shown.

[0179] Figure 76D Titers of Siglec15-specific antibodies after the second and third immunizations using human Siglec15 (sialic acid-binding Ig-like lectin 15) as an antigen in wild-type and hVH / hVL mice are shown.

[0180] Figure 76E Titers of SIRPα-specific antibodies after the second and third immunizations using human SIRPα (signal-regulatory protein alpha) as an antigen in wild-type and hVH / hVL mice are shown.

[0181] Figure 77A Body weights of wild-type and hVH / hVL mice after immunization are shown.

[0182] Figure 77B Spleen weights of wild-type and hVH / hVL mice after immunization are shown.

[0183] Figure 78 Percentages of immune cells in the spleen of wild-type and hVH / hVL mice after immunization are shown.

[0184] Figure 79A Percentages of transitional type 1 (T1, B220 + IgM + IgD - ), transitional type 2 (T2, B220 + IgM + IgD + ), and mature type (M, B220 + IgM 低 IgD + ) B cell populations in splenic B cells from wild-type or hVH / hVL mice after immunization are shown.

[0185] Figure 79B Percentages of marginal zone (MZ) and follicular (FO) B cell populations in splenic B cells from wild-type or hVH / hVL mice after immunization are shown.

[0186] Figure 80AIt shows proto-B cells (B220) in bone marrow B cells. 低 CD43 高 IgM 低 ), pre-B cells (B220) 低 CD43 中 IgM 低 ) and immature B cells (B220) 高 CD43 低 IgM 高 The percentage of the group. Bone marrow B cells are derived from immunized wild-type or hVH / hVL mice.

[0187] Figure 80B Plasma cells (B220) were shown in bone marrow B cells. 低 IgM - IgD - CD138 - ) and memory B cells (B220) + IgM + IgD - CD38 + The percentage of the group. Bone marrow B cells are derived from immunized wild-type or hVH / hVL mice.

[0188] Figure 80C Plasma cells (B220) were shown in spleen B cells. 低 IgM - IgD - CD138 - ) and memory B cells (B220) + IgM + IgD - CD38 + The percentage of splenic B cells. Splenic B cells are derived from wild-type or hVH / hVL mice after immunization.

[0189] Figure 81 The concentrations of serum immunoglobulin (Ig) isoforms in unimmunized or post-third immunization wild-type or hVH / hVL mice are shown. Ig isoform concentrations were determined by ELISA.

[0190] Figure 82 Serum total IgG concentrations are shown in unimmunized or immunized wild-type or hVH / hVL mice. IgG concentrations were determined by ELISA. Detailed Implementation

[0191] This disclosure relates to genetically modified animals and cells having humanized heavy chain immunoglobulin loci and / or humanized light chain immunoglobulin loci (e.g., κ chain loci).

[0192] Genetically modified animals can be produced by introducing human immunoglobulin genes into the genome of a non-human animal, which can express humanized antibodies or chimeric antibodies. Figure 1A A method of producing a humanized mouse is shown. 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 regions are then introduced into the corresponding region of the mouse genome by direct replacement (e.g., in a one-step replacement). The recipient cells are then screened, preferably for cells that do not contain the human chromosome. The cells are then injected into a blastocyst to make a chimeric animal (e.g., a mouse). Subsequent breeding can be performed to obtain animals that contain a complete humanized immunoglobulin locus.

[0193] The transgenic animals described herein have various advantages. For example, in some cases, the genetically modified animals described herein have a complete human antibody repertoire. Thus, the variable domains produced by these animals can have a very similar diversity to the diversity of variable domains in humans. Furthermore, because the entire sequence on the human immunoglobulin locus (without modification or with limited modification) is introduced into the animal genome, these genes can undergo V(D)J recombination in a very similar manner to that which occurs in humans. Additionally, because of the efficient V(D)J recombination, antibodies can be produced very efficiently and at a rate similar to the normal rate. Additionally, because V(D)J recombination can occur between endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes and the human genes, if the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes are incorporated into a rearranged heavy chain VDJ segment or a rearranged light chain VJ segment, the antibodies produced by this antibody repertoire are likely to have immunogenic epitopes in humans. Immunogenicity can lead to the production of anti-drug antibodies and can be active. Here, the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes are effectively deleted. The antibodies produced by this antibody repertoire are less likely to have immunogenic epitopes in humans. Thus, the antibodies are more suitable for use as therapeutics in humans. Thus, the genetically modified animals provide an advantageous platform for the production of humanized antibodies.

[0194] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains (two heavy chains and two light chains) interconnected 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 subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed 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 can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody having a K D about 10 -9 M or lower (e.g., about or less than 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or 1 x 10 -12 M). In some embodiments, the K D may be measured by surface plasmon resonance (e.g., BIACORE TM ) or ELISA.

[0195] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen binding fragment comprises at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of a light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab', F(ab')2, and Fv fragments.

[0196] As used herein, the term "human antibody" refers to an antibody encoded by a nucleic acid present in the human body (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is collected 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) containing an unrearranged or rearranged human immunoglobulin locus (e.g., a heavy or light chain human immunoglobulin locus).

[0197] As used herein, the term "chimeric antibody" refers to an antibody that comprises sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as a human and a mouse). A non-limiting example of a chimeric antibody is an antibody that comprises variable domain sequences (e.g., all or a portion of light and / or heavy chain variable domain sequences) of a human antibody and constant domains of a non-human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0198] As used herein, the term "humanized antibody" refers to a non-human antibody that comprises sequences derived from a non-human (e.g., mouse) immunoglobulin and comprises sequences derived from a human immunoglobulin.

[0199] As used herein, the term "single-chain antibody" refers to a single polypeptide comprising at least two immunoglobulin variable domains (e.g., variable domains of a mammalian immunoglobulin heavy or light chain) that is capable of specific binding to an antigen.

[0200] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and are used to describe an animal, human or non-human. Veterinary and non-veterinary applications are contemplated by the present application. A human patient can be an adult or a juvenile (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, hamsters, gerbils, ferrets, rabbits), lagomorphs, swine (e.g., pigs, mini-pigs), equids, canids, felines, bovids, and other domestic, farm, and zoo animals.

[0201] As used herein, the phrases "specifically binds" and "specifically binds to" when referring to an antibody means that the antibody interacts with its target molecule better than it interacts with 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 agent recognizes and binds to molecules comprising the particular structure, rather than to all molecules generally. An antibody that specifically binds to a target molecule can be referred to as a target-specific antibody.

[0202] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to an amino acid polymer of any length.

[0203] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to a polymer of nucleotides of any length, including without limitation, DNA, RNA, DNA / RNA hybrids, and modifications of each. As used herein, the term "nucleotide" refers to a monomeric unit of a nucleic acid, including without limitation, DNA, RNA, DNA / RNA hybrids, and modifications of each. As used herein, the term "nucleic acid" refers to a polymer of nucleotides of any length, including without limitation, DNA, RNA, DNA / RNA hybrids, and modifications of each.

[0204] 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, wherein the sequence is identical to the genetic sequence of the human subject, the human cell, the cultured human cell, or the human cell line.

[0205] Genetically modified heavy chain immunoglobulin locus

[0206] A heavy chain immunoglobulin locus (also referred to as IGH or immunoglobulin heavy chain locus) is a region of a chromosome (e.g., human chromosome 14) that contains human antibody (or immunoglobulin) heavy chain genes.

[0207] This region represents the germline organization of the heavy chain locus. The locus includes V (variable), D (diversity), J (joining), and C (constant) segments. The genes in the V region form a V gene cluster (also referred to as an IGHV gene cluster). The genes in the D region form a D gene cluster (also referred to as an IGHD gene cluster). The genes in the J region form a J gene cluster (also referred to as an IGHJ gene cluster).

[0208] During B cell development, a recombination event at the DNA level joins a single D segment (also referred to as an IGHD gene) to a J segment (also referred to as an IGHJ gene); the fused D-J exons of this partially rearranged D-J region are then joined to a V segment (also referred to as an IGHV gene). The rearranged V-D-J region containing the fused V-D-J exons is then transcribed and fused at the RNA level to an IGHM constant region; this transcript encodes a mu heavy chain. At later stages of development, B cells produce a V-D-J-C mu -C delta pre-messenger RNA that is selectively spliced to encode either a mu or a delta heavy chain. Mature B cells in the lymph nodes undergo a switch recombination so that the fused V-D-J gene segment is proximal to one of the IGHG, IGH A, or IGHE gene segments, and each cell expresses a gamma, alpha, or epsilon heavy chain. Extensive antigen recognition is provided by the potential recombination of many different IGHV genes with several IGHJ genes. Additional diversity is obtained by junctional diversity, which results from the random addition of nucleotides by terminal deoxynucleotidyl transferase and somatic hypermutation, which occurs during B cell maturation in the spleen and lymph nodes. Several V, D, J, and C segments are known not to encode proteins and are considered to be pseudogene segments (often simply referred to as pseudogenes).

[0209] The human heavy chain immunoglobulin locus is located on human chromosome 14. Table 1 lists the IGHV genes and their relative order in the locus.

[0210] Table 1. List of IGHV genes on human chromosome 14

[0211]

[0212]

[0213] RPS8P1, ADAM6, and KIAA0125 are also located in 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 lists all of the IGHD genes on human chromosome 14 and their relative order. Table 3 lists all of the IGHJ genes on human chromosome 14 and their relative order. The genes for immunoglobulin constant domains are located after the IGHV, IGHD, and IGHJ genes. These genes include (in the following order): immunoglobulin heavy chain constant mu (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 epsilon P1 (pseudogene) (IGHEP1), immunoglobulin heavy chain constant alpha 1 (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 epsilon (IGHE), and immunoglobulin heavy chain constant alpha 2 (IGHA2). These genes and the order of these genes are also shown in Figure 37 and Figure 41 .

[0214] Table 2. List of IGHD genes on human chromosome 14

[0215] Gene Name Order Gene Name Order Gene Name Order Gene Name Order IGHD1-1 165 IGHD2-8 172 IGHD2-15 179 IGHD3-22 186 IGHD2-2 166 IGHD3-9 173 IGHD3-16 180 IGHD4-23 187 IGHD3-3 167 IGHD3-10 174 IGHD4-17 181 IGHD5-24 188 IGHD4-4 168 IGHD4-11 175 IGHD5-18 182 IGHD6-25 189 IGHD5-5 169 IGHD5-12 176 IGHD6-19 183 IGHD1-26 190 IGHD6-6 170 IGHD6-13 177 IGHD1-20 184 * IGHD1-7 171 IGHD1-14 178 IGHD2-21 185 IGHD7-27 192

[0216] Table 3. List of IGHJ genes on human chromosome 14

[0217] Gene Name Order Gene Name Order IGHJ1P 191 IGHJ4 197 IGHJ1 193 IGHJ5 198 IGHJ2 194 IGHJ3P 199 IGHJ2P 195 IGHJ6 200 IGHJ3 196

[0218] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12. Table 4 lists the IGHV genes and their relative order in the locus.

[0219] Table 4. List of IGHV genes on mouse chromosome 12

[0220]

[0221]

[0222] Table 5 lists all of the IGHD genes and their relative order on mouse chromosome 12. Table 6 lists all of the IGHJ genes and their relative order on mouse chromosome 12. The genes for immunoglobulin constant domains are located after the IGHV, IGHD, and IGHJ genes. These genes include (in the following order): immunoglobulin heavy chain constant mu (IGHM), immunoglobulin heavy chain constant delta (IGHD), 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 Figure 38 and Figure 42 .

[0223] Table 5. List of IGHD genes on mouse chromosome 12

[0224]

[0225]

[0226] Table 6. List of IGHJ genes on mouse chromosome 12

[0227] Gene Name Order Gene Name Order IGHJ1 203 IGHJ3 205 IGHJ2 204 IGHJ4 206

[0228] 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, human IGHD genes, and human IGHJ genes are operably linked together and can undergo VDJ recombination. In some embodiments, the human IGHV genes, human IGHD genes, and human IGHJ genes are located on an endogenous heavy chain immunoglobulin locus.

[0229] 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).

[0230] 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.

[0231] 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.

[0232] In some embodiments, the animal comprises an unmodified human sequence comprising a sequence starting at 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 at 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 the portion of the human heavy chain immunoglobulin locus starting at human IGHV(III)-82 to human IGHV1-2. In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus starting at human IGHV(III)-82 to human IGHV(II)-1-1. In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus starting at human IGHV(III)-82 to human IGHV-6-1.

[0233] 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.

[0234] 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.

[0235] In some embodiments, the animal comprises an unmodified human sequence comprising a sequence beginning at 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 at a gene selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6. In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus beginning at human IGHD1-1 to human IGHJ6.

[0236] In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus beginning at IGHD1-1 to human IGHD7-27.

[0237] In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus beginning at human IGHJ1P to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus beginning at human IGHJ1 to human IGHJ6.

[0238] In some embodiments, the unmodified human sequence is derived from a portion of the human heavy chain immunoglobulin locus starting at human IGHV (III)-82 to human IGHJ6.

[0239] In some embodiments, the unmodified human sequence is derived from a portion of the human heavy chain immunoglobulin locus starting at human IGHV1-2 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from a portion of the human heavy chain immunoglobulin locus starting at human IGHV (II)-1-1 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from a portion of the human heavy chain immunoglobulin locus starting at human IGHV6-1 to human IGHJ6.

[0240] In some embodiments, the animal can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified human sequences. In some embodiments, the unmodified human sequence has a length 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, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1000 kb.

[0241] In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of immunoglobulin heavy chain constant mu (IGHM), immunoglobulin heavy chain constant delta (IGHD), immunoglobulin heavy chain constant gamma 3 (IGHG3), immunoglobulin heavy chain constant gamma 1 (IGHGl), 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 (IGH A) genes. In some embodiments, these endogenous genes are operably linked together. In some embodiments, these endogenous genes have the same order as in a wild-type animal. In some embodiments, isotype switching (immunoglobulin class switching) can occur in the animal.

[0242] In some embodiments, the IGHV genes, IGH D genes, and / or IGH J genes are operably linked together. VDJ recombination can occur between these genes and result in a functional antibody. In some embodiments, these genes are arranged in a similar order as in the human heavy chain immunoglobulin locus. This arrangement provides various advantageous aspects, for example, the arrangement of these genes allows for the production of heavy chain variable domains with a diversity very similar to that of the heavy chain variable domains in humans. Since some random sequences can be inserted into the sequence during VDJ recombination, in some embodiments, an intact human antibody repertoire with no or only minimal modifications can reduce the likelihood of non-human sequences being inserted during VDJ recombination.

[0243] In some embodiments, the IGHV genes, IGH D genes, and / or IGH J genes are operably linked together with one or more genes (e.g., all genes) selected from the group consisting of IGHM, IGHd, IGHG3, IGHGl, IGHG2b, IGHG2a, IGHE, and IGH A genes.

[0244] In some embodiments, the animal comprises a disruption in its endogenous heavy chain immunoglobulin locus. In some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of one or more endogenous IGHV genes, one or more endogenous IGH D genes, and one or more endogenous IGH J genes.

[0245] In some embodiments, the animal is a mouse. The disruption in the endogenous heavy chain immunoglobulin locus of the animal 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).

[0246] 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.

[0247] In some embodiments, the disruption in the endogenous heavy chain immunoglobulin locus of the animal 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 IGH D genes (e.g., genes as set forth 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 of the mouse IGH D genes selected from IGH D5-1, IGH D3-1, IGH D1-1, IGH D6-1, IGH D2-3, IGH D2-7, IGH D2-8, IGH D5-6, IGH D3-2, and IGH D4-1. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGH D genes selected from IGH D5-1, IGH D3-1, IGH D1-1, IGH D6-1, IGH D2-3, IGH D2-7, IGH D2-8, IGH D5-6, IGH D3-2, and IGH D4-1.

[0248] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, or 4 mouse IGH J 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 IGH J genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.

[0249] In some embodiments, the disruption in the endogenous heavy chain immunoglobulin locus of the animal 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.

[0250] In some embodiments, the deleted sequence begins at IGHV1-86 to IGHJ4, at IGHV1-85 to IGHJ4, at IGHV1-84 to IGHJ4, at IGHV1-83 to IGHJ4, or at IGHV1-82 to IGHJ4 (e.g., at IGHV1-85 to IGHJ4).

[0251] 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 sequence is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb in length. In some embodiments, the sequence starts at human IGHV (III)-82 to IGHV1-2. In some embodiments, the sequence starts at human IGHV7-81 to IGHV1-2. In some embodiments, the sequence starts at human IGHV (II)-1-1 to IGHVJ6. In some embodiments, the sequence starts at human IGHV6-1 to IGHVJ6.

[0252] The human IGHV genes, human IGHD genes, and human IGHJ genes are operably linked together and are subject to VDJ recombination. In some embodiments, the modified mouse has a complete human IGHV, IGHD, and IGHJ gene repertoire (e.g., including all non-pseudo genes in the human IGHV, IGHD, and IGHJ genes). Thus, the modified mouse can produce a complete human antibody repertoire. In some embodiments, after VDJ recombination, one of the IGHV genes in Table 15 (e.g., IGHV3-21 or IGHV3-74) constitutes the sequence encoding the antibody heavy chain variable region. One of the IGHD genes in Table 15 constitutes the sequence encoding the antibody heavy chain variable region. One of the IGHJ genes in Table 15 constitutes the sequence encoding the antibody heavy chain variable region. In some embodiments, the IGHV gene is IGHV3-21 or IGHV3-74.

[0253] In some embodiments, one of the IGHV genes in Figures 70A and 70B (e.g., IGHV3-30, IGHV3-33, IGHV4-39, or IGHV4-34) constitutes the sequence encoding the antibody heavy chain variable region. Figure 70C In some embodiments, one of the IGHD genes in Table 15 (e.g., IGHD6-19) constitutes the sequence encoding the antibody heavy chain variable region. Figure 70Done IGHJ gene (e.g., IGHJ4 or IGHJ6) in FIG. 71A and FIG. 71B constitutes the sequence encoding the antibody heavy chain variable region. In some embodiments, one IGKV gene (e.g., IGKV4-1, IGKV1-33, IGKV2-30) in FIG. 71A and FIG. 71B constitutes the sequence encoding the antibody light chain variable region. Figure 71C one IGKJ gene (e.g., IGKJ1, IGKJ2, or IGKJ4) in FIG. 71A and FIG. 71B constitutes the sequence encoding the antibody light chain variable region.

[0254] In addition, in some cases, the entire mouse IGHV genes, IGHD genes, and IGHJ genes (e.g., including all non-pseudogenes) are knocked out, and the heavy chain variable region will not have any sequence encoded by sequence derived from a mouse, thereby minimizing immunogenicity in humans.

[0255] Genetically modified kappa light chain immunoglobulin loci

[0256] The kappa chain immunoglobulin locus (also referred to as IGK or immunoglobulin kappa locus) is a region on a chromosome (e.g., human chromosome 2) that contains human antibody (or immunoglobulin) light chain genes. Similarly, immunoglobulin light chain genes can also undergo a series of rearrangements leading to the production of mature immunoglobulin light chain nucleic acids (e.g., kappa chains).

[0257] The joining of the V segment (also referred to as an IGKV gene) and the J segment (also referred to as an IGKJ gene) produces a contiguous exon that encodes the entire light chain variable domain. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far away from the C region. The J gene segment (or IGKJ gene cluster) is located near the C region. The joining of the V segment to the J gene segment also brings the V gene in proximity 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. To produce the complete immunoglobulin light chain messenger RNA, the V region exon is joined to the C region sequence by RNA splicing after transcription.

[0258] The human light chain immunoglobulin locus is located on human chromosome 2. Table 7 lists the IGKV genes and their relative order in the locus. The human IGKV genes are organized into several different groups, including IGKV1 genes (including all IGKV genes beginning with IGKV1, also referred to as VKI), IGKV2 genes (including all IGKV genes beginning with IGKV2, also referred to as VKII), IGKV3 genes (including all IGKV genes beginning with IGKV3, also referred to as VKIII), IGKV4 genes (including all IGKV genes beginning with IGKV4, also referred to as VKIV), IGKV5 genes (including all IGKV genes beginning with IGKV5, also referred to as VKV), IGKV6 genes (including all IGKV genes beginning with IGKV6, also referred to as VKVI), and IGKV7 genes (including all IGKV genes beginning with IGKV7, also referred to as VKVII).

[0259] These IGKV genes in human chromosome 2 also form two clusters, a proximal VK cluster and a distal VK cluster Figure 28 . The sequences in the two clusters are similar, but not identical. Large segmental duplications of this sequence occurred at the time the human lineage diverged from the most recent common ancestor with other great apes. Figure 64 The relevant IGVK genes in each cluster are outlined.

[0260] Table 7. List of IGKV genes on human chromosome 2

[0261] Gene Name Order Gene Name Order Gene Name Order Gene Name Order IGKV3D-7 1 IGKV3D-25 21 IGKV2-36 41 IGKV1-16 61 IGKV1D-8 2 IGKV2D-26 22 IGKV1-35 42 IGKV3-15 62 IGKV1D-43 3 IGKV1D-27 23 IGKV3-34 43 IGKV2-14 63 IGKV1D-42 4 IGKV2D-28 24 IGKV1-33 44 IGKV1-13 64 IGKV2D-10 5 IGKV2D-29 25 IGKV1-32 45 IGKV1-12 65 IGKV3D-11 6 IGKV2D-30 26 IGKV3-31 46 IGKV3-11 66 IGKV1D-12 7 IGKV3D-31 27 IGKV2-30 47 IGKV2-10 67 IGKV1D-13 8 IGKV1D-32 28 IGKV2-29 48 IGKV1-9 68 IGKV2D-14 9 IGKV1D-33 29 IGKV2-28 49 IGKV1-8 69 IGKV3D-15 10 IGKV3D-34 30 IGKV1-27 50 IGKV3-7 70 IGKV1D-16 11 IGKV1D-35 31 IGKV2-26 51 IGKV1-6 71 IGKV1D-17 12 IGKV2D-36 32 IGKV3-25 52 IGKV1-5 72 IGKV6D-41 13 IGKV1D-37 33 IGKV2-24 53 IGKV2-4 73 IGKV2D-18 14 IGKV2D-38 34 IGKV2-23 54 IGKV7-3 74 IGKV2D-19 15 IGKV1D-39 35 IGKV1-22 55 IGKV5-2 75 IGKV3D-20 16 IGKV2D-40 36 IGKV6-21 56 IGKV4-1 76 IGKV6D-21 17 IGKV2-40 37 IGKV3-20 57 IGKV1D-22 18 IGKV1-39 38 IGKV2-19 58 IGKV2D-23 19 IGKV2-38 39 IGKV2-18 59 IGKV2D-24 20 IGKV1-37 40 IGKV1-17 60

[0262] Table 8 lists all of the IGKJ genes and their relative order on human chromosome 2. The immunoglobulin kappa constant region (IGKC) gene, which encodes the constant domain of light chain immunoglobulins, is located after the IGKV and IGKJ genes. These genes and the order of these genes are also shown in Figure 39 and Figure 43 .

[0263] Table 8. List of IGKJ genes on human chromosome 2

[0264] Gene Name Order Gene Name Order IGKJ1 77 IGKJ4 80 IGKJ2 78 IGKJ5 81 IGKJ3 79

[0265] The mouse light chain immunoglobulin locus is located on mouse chromosome 6. Table 9 lists the IGKV genes and their relative order in the locus.

[0266] Table 9. List of IGKV genes on mouse chromosome 6

[0267]

[0268]

[0269] Gm9728 and Amd-ps2 are also located in this locus. The relative order of Gm9728 is 4 and the relative order of Amd-ps2 is 134. Table 10 lists all IGKJ genes and their relative order on mouse chromosome 6. The IGKC genes, which encode the constant domain of the light chain immunoglobulin, are located after the IGKV and IGKJ genes. These genes and the order of these genes are also shown in Figure 40 and Figure 44 .

[0270] Table 10. List of IGKJ genes on mouse chromosome 6

[0271] Gene Name Order Gene Name Order IGKJ1 166 IGKJ4 169 IGKJ2 167 IGKJ5 170 IGKJ3 168

[0272] The present disclosure provides a genetically modified non-human animal comprising one or more human IGKV genes and / or one or more human IGKJ genes. In some embodiments, the human IGKV genes and human IGKJ genes are operably linked together and can undergo VJ rearrangement. In some embodiments, the human IGKV genes and human IGKJ genes are located in an endogenous light chain immunoglobulin locus.

[0273] 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).

[0274] 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.

[0275] 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.

[0276] 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.

[0277] In some embodiments, the animal comprises an endogenous IGKC. In some embodiments, the IGKV genes and / or IGKJ genes are operably linked together. VJ recombination can occur between these genes and produce functional antibodies. In some embodiments, these genes are arranged in a similar order as in the human light chain immunoglobulin locus. This arrangement provides various advantageous aspects, for example, the arrangement of these genes allows for the production of light chain variable domains with a diversity very similar to that of light chain variable domains in humans.

[0278] In some embodiments, the IGKV genes and / or IGKJ genes are operably linked together with an IGKC gene (e.g., an endogenous IGKC gene).

[0279] In some embodiments, the animal comprises a disruption in its endogenous light chain immunoglobulin locus. In some embodiments, the disruption in 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.

[0280] In some embodiments, the animal is a mouse. The disruption in the endogenous heavy chain immunoglobulin locus of the animal 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.

[0281] 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.

[0282] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from the group consisting of 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 the group consisting of IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5).

[0283] In some embodiments, the disruption in the endogenous kappa light chain immunoglobulin locus of the animal 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.

[0284] In some embodiments, the deleted sequence starts at IGKV2-137 to IGKJ4, at IGKV1-136 to IGKJ4, at IGKV1-135 to IGKJ4, at IGKV2-137 to IGKJ5, at IGKV1-136 to IGKJ5, or at IGKV1-135 to IGKJ5 (e.g., at IGKV2-137 to IGKJ5).

[0285] 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 sequence is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb in length.

[0286] In some embodiments, the animal can have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the unmodified human sequence is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb in length.

[0287] In some embodiments, the sequence starts from human IGKV3D-7 to IGKJ5. In some embodiments, the sequence starts from human IGKV3D-7 to IGKJ4. In some embodiments, the sequence starts from human IGKV1D-8 to IGKJ5. In some embodiments, the sequence starts from human IGKV1D-8 to IGKJ4.

[0288] The human IGKV genes and human IGKJ genes can be operably linked together and can undergo VJ rearrangement. In some embodiments, the modified mouse has a complete human IGKV and IGKJ gene repertoire (e.g., including all non-pseudo genes in the human IGKV and IGKJ genes). Thus, the modified mouse can produce a complete 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) constitutes the sequence encoding the antibody light chain variable region. One human IGKJ gene constitutes 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 genes and IGKJ genes (both non-pseudo genes) are knocked out, the light chain variable region will not have any sequence encoded by sequence derived from mouse, thereby minimizing immunogenicity in humans.

[0289] In some embodiments, the human proximal VK cluster IGKV genes are included in the modified chromosome. In some embodiments, the human distal VK cluster IGKV genes are included in the modified chromosome. In some embodiments, both the human proximal VK cluster IGKV genes and the human distal VK cluster IGKV genes are included in the modified chromosome.

[0290] Genetically modified lambda light chain immunoglobulin locus

[0291] The lambda chain immunoglobulin locus (also referred to as IGL or immunoglobulin lambda locus) is a region of a chromosome (e.g., human chromosome 22) that contains human antibody (or immunoglobulin) light chain genes. Similarly, immunoglobulin light chain genes can also undergo a series of rearrangements leading to the production of mature immunoglobulin light chain nucleic acids (e.g., lambda chains). In healthy human individuals, the total kappa to lambda ratio in serum is approximately 2: 1 (measuring intact whole antibodies), or 1 : 1.5 if free light chains are measured. In mice, the total kappa to lambda ratio is approximately 9: 1.

[0292] In some embodiments, the animal comprises a human lambda chain immunoglobulin locus.

[0293] In some embodiments, the animal comprises a disruption in its endogenous lambda light chain immunoglobulin locus. In some embodiments, the disruption in 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 lambda constant (IGLC) genes (e.g., IGLC1, IGLC2, IGLC3, and IGLC4).

[0294] The mouse lambda light chain immunoglobulin locus (IGL locus) is located on mouse chromosome 16. Table 11 lists IGLV, IGLJ, and IGLC genes and their relative order on the locus.

[0295] Table 11. List of genes on the mouse IGL locus

[0296]

[0297] The disruption in 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 IGLC 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.

[0298] In some embodiments, the disruption in the endogenous lambda light chain immunoglobulin locus of the animal 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, the animal has no disruption in the endogenous lambda light chain immunoglobulin gene.

[0299] In some embodiments, the deleted sequence begins at IGLV2 to IGLC1, at IGLV3 to IGLC1, or at IGLJ2 to IGLC1.

[0300] Genetically modified animals

[0301] In one aspect, the present 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 IGH D 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 in an endogenous immunoglobulin locus.

[0302] In some embodiments, the animal comprises a human lambda chain immunoglobulin locus. In some embodiments, the animal comprises a disruption in the endogenous lambda light chain immunoglobulin locus of the animal. In some embodiments, the animal has no disruption in the endogenous lambda light chain immunoglobulin locus of the animal.

[0303] The genetically modified non-human animal can be a variety of animals, e.g., mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, water 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 can be employed to make a genetically modified non-human animal. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell), and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art and are described in, e.g., Nagy et al. “Manipulating the Mouse Embryo: A Laboratory Manual (3rd ed.),” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein 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. During B cell development, these rearranged human V, D, and / or J segments are joined to non-human animal immunoglobulin constant regions.

[0304] In one aspect, the animal is a mammal, e.g., a mammal of the order Diprotodontia or Muroidea. In some embodiments, the transgenic animal is a rodent. The rodent can be selected from the group consisting of mice, rats, and hamsters. In some embodiments, the genetically modified animal is from a family selected from the group consisting of Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., moles, bamboo rats, and zokors). In some embodiments, the genetically modified rodent is selected from the group consisting of a mouse or a rat (Muridae), a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

[0305] In some embodiments, the animal is a mouse of 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 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 in, e.g., 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 by reference herein in their entireties. In some embodiments, the genetically modified mouse is a hybrid strain of a 129 strain and a C57BL / 6 strain. In some embodiments, the genetically modified mouse is a hybrid strain of a 129 strain or a hybrid strain of 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 hybrid strain of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129).

[0306] 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 hybrid strain of two or more strains selected from Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0307] The animals can have one or more other genetic modifications and / or other modifications that are suitable for the particular purpose of making a humanized animal.

[0308] Genetically modified non-human animals comprising a modification of an endogenous non-human immunoglobulin locus. In some embodiments, the modification can 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). Although genetically modified cells (e.g., ES cells, somatic cells) that can comprise the modifications described herein are also provided, in many embodiments, the genetically modified non-human animals comprise a modification of an endogenous locus in the germline of the animal.

[0309] Genetically modified animals can express humanized antibodies and / or chimeric antibodies from endogenous mouse loci, wherein one or more endogenous mouse immunoglobulin genes have been replaced with human immunoglobulin genes and / or nucleotide sequences that are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to human immunoglobulin gene sequences (e.g., IGHV, IGHD, IGHJ, IGKV, and / or IGKJ genes). In various embodiments, the endogenous non-human immunoglobulin loci are modified, in whole or in part, to comprise human nucleic acid sequences.

[0310] The non-human mammals described above can be subjected to genetic, molecular, and behavioral analyses. The disclosure also relates to offspring produced by mating a non-human mammal provided by the disclosure with the same genotype or other genotype. The non-human mammal can be any non-human animal known in the art and can be used in the methods described herein. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0311] The disclosure also provides cell lines or primary cell cultures derived from the non-human mammal or its offspring. Cell culture-based models can be made, for example, by the following methods. Cell cultures can be obtained by isolation from the non-human mammal, or cells can be obtained from cell cultures established using the same constructs and standard cell transfection techniques. Integration of the genetic construct comprising the DNA sequence encoding the human or humanized immunoglobulin can be detected by a variety of methods.

[0312] A number of analytical methods are available for detecting foreign DNA or modifications to genomic DNA, including methods at the nucleic acid level (including mRNA quantification methods using reverse transcription polymerase chain reaction (RT-PCR) or Southern blotting and in situ hybridization) and at the protein level (including histochemistry, immunoblot analysis, and in vitro binding studies). In addition, the expression level of a gene of interest can be quantified by ELISA techniques well known to those skilled in the art. A number of standard analytical methods are available for accomplishing quantitative measurements. For example, transcriptional levels can be measured using RT-PCR and hybridization methods, including RNase protection, Southern blot analysis, RNA dot analysis (RNA dot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized proteins.

[0313] Antibodies and antigen-binding fragments

[0314] The present disclosure provides antibodies and antigen-binding fragments thereof (e.g., humanized antibodies or chimeric antibodies) produced by the methods described herein.

[0315] Generally, antibodies (also known as immunoglobulins) are composed of two classes of polypeptide chains (light and heavy). Non-limiting antibodies of the present disclosure can be intact, four-chain immunoglobulin antibodies comprising two heavy chains and two light chains. The heavy chain of an antibody can be of any isotype (including IgM, IgG, IgE, IgA, or IgD) or subclass (including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2), etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. Each heavy chain comprises one variable domain (or variable region, V H ) and a plurality of constant domains (or constant regions), which are joined to each other by disulfide bonds within their constant regions, forming the "stalk" of the antibody. Each light chain comprises one variable domain (or variable region, V L ) and one constant domain (or constant region), each light chain being joined to a heavy chain by a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is joined. The variable regions of the light and heavy chains each comprise three hypervariable regions, which are referred to as complementarity determining regions (CDRs).

[0316] These hypervariable regions, which are referred to as complementarity determining regions (CDRs), form loops connecting, and in some cases forming part of, the beta-sheet structure of the framework regions. The CDRs of each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding region.

[0317] Methods for identifying CDR regions of antibodies by analyzing the amino acid sequence of the antibody are well known and generally use a variety of CDR definition schemes. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of structural loop regions. These methods and definitions are described in, e.g., 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, T.T. and Kabat, E.A. (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., Nature 342(6252): 877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

[0318] CDR pairs are important for recognizing the epitope of an antigen. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by an antigen binding domain of an antibody. The minimum size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids need not be in a contiguous linear sequence of the primary structure of the antigen, as the epitope can depend on the three-dimensional configuration of the antigen based on its secondary and tertiary structure.

[0319] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly their hinge and upper CH2 domains. Sequences and differences of IgG subclasses are known in the art and described, e.g., 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 herein in its entirety.

[0320] Antibodies can also be immunoglobulin molecules derived from any species (e.g., human, rodent, mouse, rat, camel). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term“antigen binding domain” or“antigen binding fragment” is a portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specifically binding 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, a scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising or homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, for example, a heavy and / or light chain CDR of an intact antibody, a heavy and / or light chain variable region of an intact antibody, a full-length heavy or light chain of an intact antibody, or a single CDR from a heavy or light chain of an intact antibody.

[0321] In some embodiments, the antigen binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single chain variable fragment (scFv) as described herein fused to a transmembrane and intracellular domain of CD3-zeta.

[0322] 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 that can bind to respective target antigens.

[0323] Antibodies and antigen binding fragments thereof produced by the methods described herein (e.g., humanized antibodies or chimeric antibodies) have various advantageous aspects. In some embodiments, no further optimization is required to obtain desirable properties (e.g., binding affinity, thermal stability, and / or limited aggregation).

[0324] In some embodiments, the antibody (or antigen binding fragment thereof) binds to a target antigen with a dissociation constant (Kd) of less than 0.1 s -1 less than 0.01 s -1 less than 0.001 s -1 less than 0.0001 s -1 or less than 0.00001 s -1The dissociation rate (koff) is related to target-specific binding. In some embodiments, the dissociation rate (koff) is greater than 0.01 s. -1 Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 or greater than 0.000001s -1 .

[0325] In some implementations, the kinetic association rate (kon) is greater than 1 x 10⁻⁶. 2 / Ms, greater than 1x 10 3 / Ms, greater than 1x10 4 / Ms, greater than 1x 10 5 / Ms or greater than 1x 10 6 / Ms. In some implementations, the kinetic association rate (kon) is less than 1 x 10⁻⁶. 5 / Ms, less than 1x 10 6 / Ms or less than 1x 10 7 / Ms.

[0326] The affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some implementations, KD is less than 1 x 10⁻⁶. -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M or less than 1 x 10 -10 M. In some implementations, 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 implementations, KD is greater than 1 x 10^6 nM. -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M, greater than 1 x 10 -10 M, greater than 1 x 10 -11 M or greater than 1 x 10 -12 M. In some embodiments, the antibody binds to the target with a KD of less than or equal to 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.

[0327] In some embodiments, thermal stability is determined. The Tm of an antibody or antigen binding fragment described herein can be higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0328] Since IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, or three transitions, with a first denaturation temperature Tm D1, a second denaturation temperature Tm D2, and optionally a third denaturation temperature Tm D3.

[0329] In some embodiments, the Tm D1 of an antibody or antigen binding fragment described herein is higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the Tm D2 of an antibody or antigen binding fragment described herein is higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the Tm D3 of an antibody or antigen binding fragment described herein is higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0330] In some embodiments, Tm, Tm D1, Tm D2, Tm D3 is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0331] In some embodiments, the antibody or antigen-binding fragment described herein does not form aggregates when the temperature is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0332] Methods of producing genetically modified animals

[0333] The 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 antibodies or chimeric antibodies. Figure 1A Methods of producing humanized animals are shown. In some embodiments, the methods first involve modifying a human immunoglobulin locus on a human chromosome. The modified human chromosome is then introduced into a mouse recipient cell. The human immunoglobulin variable regions are then introduced into the corresponding region of the mouse genome by direct replacement. The recipient cell is then screened. In some embodiments, the cell does not contain a human chromosome. The cell is then injected into a blastocyst to make a chimeric mouse. Subsequent breeding can be performed to obtain a mouse containing a complete humanized immunoglobulin locus.

[0334] Several other techniques can also be used to generate 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 the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate 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 herein in its entirety. Numerous other methods are also provided that can be used for genome editing, for example, microinjection of a genetically modified cell nucleus into an enucleated oocyte and fusion of the enucleated oocyte with another genetically modified cell.

[0335] The genetic modification process can involve replacing an endogenous sequence with a human sequence by homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by a zinc finger nuclease, TALEN, or CRISPR) can result in a DNA double-strand break and homologous recombination is used to replace the endogenous sequence with the human sequence.

[0336] In some embodiments, a method for generating a humanized animal can include the step of replacing a nucleic acid (e.g., a V, D, J region or a V, J region) at an endogenous locus (or site) with a corresponding region of a human sequence. The sequence can 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.

[0337] Figure 9 Targeting strategies for adding functional genetic elements into human chromosomes are shown. These vectors can be inserted upstream of the V region, between the J and C regions.

[0338] In some embodiments, the first vector has one or more of the following sequences from 5' to 3': 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 (Poly A; "PA"), a LoxP recognition sequence, a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "3'HygR"), and a Flp recognition target ("FRT"), a downstream DNA homology arm sequence, and a DTA gene.

[0339] The second vector has one or more of the following sequences from 5' to 3': 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 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.

[0340] These vectors can be integrated into the genome of a cell, and the cells can be selected by drug resistance markers or combinations thereof (e.g., Zeocin, G418, and / or puromycin). In some embodiments, a transposase is expressed, and the genetic elements between the transposase target sequences can be deleted.

[0341] In some embodiments, the vectors are integrated into a human chromosome that has been modified. The human chromosome can be first modified before the first vector and the second vector are integrated into the genome. In some embodiments, one or more additional vectors can be added at different locations of the chromosome as desired. In some embodiments, a vector is added between the C region and the centromere (kinetochore). The third vector can 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-la), a PBase, an IRES, a Neo, a transcription termination / polyadenylation signal sequence, a DNA homology arm sequence downstream of the insertion site, and a DTA. In some embodiments, the vectors can 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 can be deleted. In some embodiments, a large fragment of the chromosome can be deleted (e.g., between the constant region and the centromere). The cells can also be treated with Cre enzyme, resulting in recombination of the loxP sites, thereby 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, a spontaneous chromosome break can occur. The modified human chromosome with the desired chromosome break can be selected for experimentation.

[0342] 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, and then the cell is fused with a recipient cell. The modified chromosome is then isolated and introduced into another suitable recipient cell. Cells with the desired resistance are selected to obtain cells containing only one human chromosome. Then, a second vector is introduced into the cell, and the cell is selected by resistance. Then, if needed, a third vector and / or a fourth vector can be introduced. 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 labeled with a fluorescent marker and isolated. And the modified chromosome is injected into a recipient cell by chromosome microinjection. In some embodiments, donor cells are induced to multinucleate their chromosomes. Then, these nuclei are forced through the cell membrane to form minicells, which can be fused with recipient cells. In some embodiments, microcell-mediated chromosome transfer can also be used. Chromosome manipulation techniques are described in, for example, CN1200014A, CN109837307A, US20120093785A1, and US2009253902; Kuroiwa et al. “Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts.” Nature Biotechnology 18.10 (2000): 1086-1090; Chinese patent CN1717483A; 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, Volume 13, Number 3, 1987, pp.279-284; each of which is incorporated by reference in its entirety.

[0343] In some implementations, mouse chromosomes can be modified. Targeting strategies include... Figure 4 As shown. The first vector may have DNA homologous arm sequences upstream and downstream of the insertion site, as well as a LoxP sequence. In some embodiments, the first vector has one or more of the following sequences from 5' to 3': DNA homologous 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 homologous arm sequence downstream of the insertion site, and DTA.

[0344] The second vector may have DNA homologous arm sequences upstream and downstream of the insertion site, as well as a LoxP sequence. In some embodiments, the second vector has one or more of the following sequences from 5' to 3': a DNA homologous 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 sequence of the puromycin resistance gene (3'PuroR), an FMDV self-cleaving peptide (2A), a DT receptor (DTR), a LoxP recognition sequence, a DNA homologous arm sequence downstream of the insertion site, and a DTA.

[0345] Figure 30 A similar targeting strategy for the κ light chain immunoglobulin locus is shown. Two vectors are first integrated into the human chromosome. The first vector has DNA homologous arm sequences upstream and downstream of the insertion site, as well as a LoxP recognition sequence. In some embodiments, the first vector has one or more of the following sequences from 5' to 3': a DNA homologous 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 (partial sequence of hygromycin phosphotransferase; "3'HygR"), FRT, a DNA homologous arm sequence downstream of the insertion site, and DTA.

[0346] The second vector has DNA homology arm sequences upstream and downstream of the insertion site, and has LoxP recognition sequences. In some embodiments, the second vector has, from 5' to 3', one or more of the following sequences: a DNA homology arm sequence upstream of the insertion site, a LoxP recognition sequence, a PGK promoter, a portion of a 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 DTA.

[0347] LoxP recognition sequences can also be added to human chromosomes (e.g., human chromosome 2, 14, 22). Cells can also be treated with Cre enzyme, resulting in recombination of the loxP sites and removal of the genomic DNA sequence. In some embodiments, a spontaneous chromosomal break can also be used to remove the genomic DNA sequence.

[0348] Modifications to the mouse light chain immunoglobulin locus can be made directly. In some embodiments, a vector is used directly to replace the entire mouse light chain immunoglobulin variable region. In some embodiments, the vector has, from 5' to 3', a DNA homology arm sequence upstream of the insertion site, a Flp recognition target (FRT), a mammalian expression promoter from human elongation factor 1 alpha (EF-1a), a hygromycin resistance gene (a portion of sequence of hygromycin phosphotransferase; "5' HygR"), a LoxP recognition sequence for Cre recombinase, a 5' PB transposon sequence (PB 5'), 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 (PB 3'), a portion of a puromycin resistance gene 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 DTA.

[0349] The mouse immunoglobulin variable region can be replaced by a human immunoglobulin variable region through replacement (e.g., homologous recombination or Cre-mediated recombination). In some embodiments, Cre recombination can be used to mediate the replacement. In some embodiments, a vector can add LoxP recognition sequences to a human chromosome. Similar modifications can be made to a mouse chromosome, where two LoxP recognition sequences can be added to the chromosome. For example, a Cre recombinase can mediate replacement of a V, J region on a mouse chromosome with a V, J region on a human chromosome, or replacement of a V, D, J region on a mouse chromosome with a V, D, J region on a human chromosome.

[0350] Cells that do not have human chromosomes can be further screened (e.g., by DT). In some cases, cells that are not screened by DT can contain recombinant human chromosome fragments, but these fragments are small, unstable in mouse cells (e.g., Shinohara et al. (2000) Chromosome Research, 8:713-725), and will naturally disappear during cell proliferation. In some embodiments, large fragments of the modified human chromosomes are deleted, e.g., by Cre-mediated deletion or by spontaneous chromosome breakage.

[0351] The 5’ end homology arm and / or the 3’ end homology arm can have a desired length to facilitate homologous recombination. In some embodiments, the homology arm is about or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, or 50 kb (e.g., about 3 kb). In some embodiments, the homology arm is less than 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, or 50 kb.

[0352] In some embodiments, the vector can also optionally include a reporter protein, e.g., luciferase (e.g., Gluc) or a fluorescent protein (e.g., EGFP, BFP, etc.).

[0353] These modifications can be made in different cells. In some embodiments, the cell is a stem cell, an embryonic stem cell, or a zygote cell.

[0354] The present disclosure also provides methods of establishing a humanized animal model, comprising the steps of:

[0355] (a) providing a cell (e.g., a zygote cell) based on the methods described herein;

[0356] (b) culturing the cell in a liquid medium;

[0357] (c) transplanting the cultured cell into the oviduct or uterus of a recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

[0358] (d) identifying in the germline of a progeny of a pregnant female in step (c) a genetically modified humanized non-human mammal.

[0359] In some embodiments, the non-human mammal in the foregoing methods is a mouse (e.g., a C57 mouse, a BALB / c mouse, or a C57BL / 6 mouse).

[0360] In some embodiments, the non-human mammal in step (c) is a pseudo-pregnant (or pseudopregnant) female animal.

[0361] In some embodiments, the fertilized egg for use in the above-described methods is a C57BL / 6 fertilized egg. Other fertilized eggs that can be used in the methods 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.

[0362] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is from a rodent. The genetic construct can be introduced into the fertilized egg by microinjection of DNA. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which then produces a non-human mammal, thereby generating the non-human mammal referred to in the above-described methods.

[0363] Also provided are cells, tissues, and animals (e.g., mice) comprising the nucleotide sequences described herein, as well as cells, tissues, and animals (e.g., mice) expressing humanized or chimeric antibodies from endogenous non-human loci.

[0364] The present disclosure also provides various targeting vectors (e.g., vectors for use in generating genetically modified animals). In some embodiments, the vector can comprise: a) a DNA segment homologous to the 5' end of the region to be altered (5' homology arm); b) a sequence comprising the desired genetic element (e.g., a LoxP recognition site, a drug resistance gene, and / or a reporter gene, etc.); and c) a second DNA segment homologous to the 3' end of the region to be altered (3' homology arm). The present disclosure also relates to cells comprising the targeting vectors described herein.

[0365] In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

[0366] In some embodiments, the non-human mammal cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

[0367] The present disclosure also relates to methods of generating genetically modified animal models having two or more human or chimeric genes. The animals can comprise one or more human or humanized immunoglobulin loci and a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein can be Programmed Cell Death Protein 1 (PD-1), Cytotoxic T Lymphocyte-Associated Protein 4 (CTLA-4), Lymphocyte Activation Protein 3 (LAG-3), B and T Lymphocyte Associated Protein (BTLA), Programmed Cell Death Protein 1 Ligand 1 (PD-L1), CD27, CD28, CD47, CD137, CD154, T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glycocorticoid-Inducible TNFR-Related Protein (GITR), or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40).

[0368] Methods of generating genetically modified animal models having an additional human or chimeric gene (e.g., a humanized gene) can comprise the following steps:

[0369] (a) obtaining a genetically modified non-human animal using the methods described herein;

[0370] (b) mating the genetically modified non-human animal with another genetically modified non-human animal and then screening the offspring to obtain a genetically modified non-human animal having two or more human or chimeric genes.

[0371] In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal having a human or chimeric PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40. For example, some of these genetically modified non-human animals are described in PCT / CN2017 / 090320, PCT / CN2017 / 099577, PCT / CN2017 / 099575, PCT / CN2017 / 099576, PCT / CN2017 / 099574, PCT / CN2017 / 106024, PCT / CN2017 / 110494, PCT / CN2017 / 110435, PCT / CN2017 / 120388, PCT / CN2018 / 081628, PCT / CN2018 / 081629 (each of which is incorporated by reference herein in its entirety).

[0372] In some embodiments, the genetically modified animal can have a human ADAM6 gene, an endogenous ADAM6 gene, or a modified ADAM6 gene. ADAM6 proteins are members of the ADAM family of proteins, where ADAM is an abbreviation for a disintegrin and metalloproteinase. The human ADAM6 gene, typically found between human IGHV genes IGHV 1-2 and IGHV 6-1, is a pseudogene (ADAM6p) (see, e.g., FIG. 1). 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 IGHV 5-1 and mouse IGHD 5-1. Mouse ADAM6b is located between mouse IGHD 3-1 and mouse IGHD 1-1. Thus, in some embodiments, the genetically modified animal can have a human ADAM6 gene. In some embodiments, the genetically modified animal does not have an endogenous ADAM6 gene. 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 IGHV 5-1 and mouse IGHD 5-1. Mouse ADAM6b is located between mouse IGHD 3-1 and mouse IGHD 1-1. Thus, in some embodiments, the genetically modified animal can have a human ADAM6 gene. In some embodiments, the genetically modified animal does not have an endogenous ADAM6 gene.

[0373] 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, the sequence is placed in any suitable location. It can be placed in an intergenic region, or 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 a mouse ADAM6a gene (e.g., NC_000078.6 113539230-113547024; SEQ ID NO: 53) or a mouse ADAM6b gene (e.g., NC_000078.6 113486188-113492125; SEQ ID NO: 54). In some embodiments, the nucleic acid additionally includes regulatory elements (e.g., promoters) of the ADAM6a gene and the ADAM6b gene.

[0374] In some embodiments, a functional mouse ADAM6 locus can be placed in the middle of a human IGHV gene cluster. In some embodiments, the mouse ADAM6 locus is located between two human IGHV genes. In some embodiments, the human ADAM6 pseudogene between human VH1-2 and human VH(II)-1-1 is replaced with a mouse ADAM6 locus. In some embodiments, in the animal genome, the ADAM6a gene and the ADAM6b gene are located between human IGHV1-2 and human VH(II)-1-1. In some embodiments, the location of the mouse ADAM6 sequence in the human gene sequence can be close to the location of the human ADAM6 pseudogene, or can be close to the location of the mouse ADAM6 sequence (e.g., within the V-D intergenic region). In some embodiments, the genetically modified mouse has a humanized heavy chain immunoglobulin locus. In some embodiments, the mouse ADAM6a and the mouse ADAM6b are located between human IGHV1-2 and IGHV6-1 genes. Placing the mouse ADAM6a and the mouse ADAM6b between human IGHV1-2 and IGHV6-1 genes can have a number of advantageous aspects. For example, because these genes replace the human ADAM6 genes at the same locus, the replacement of the human ADAM6 genes has limited effect on VDJ recombination, and the mouse ADAM6a and the mouse ADAM6b genes can also function normally (as in a similar location to the endogenous locus).

[0375] Accordingly, in one aspect, the present 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.

[0376] In some embodiments, the first sequence comprises all 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 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.

[0377] In some embodiments, the second sequence comprises one 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.

[0378] In some embodiments, the third sequence comprises all of the human IGH D genes in Table 2 and all of the human IGH J 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 the human heavy chain immunoglobulin locus.

[0379] In some embodiments, AMAM6a and / or ADAM6b is an endogenous sequence. In some embodiments, AMAM6a and / or ADAM6b is not replaced, and / or is located in its endogenous or native position. In some embodiments, the mouse IGHV genes before mouse IGHV1-2 in the heavy chain variable region locus are replaced with human IGHV genes. In some embodiments, the mouse IGHV, IGH D, and IGH J genes after mouse IGHV6-1 in the heavy chain variable region locus are replaced with one or more human IGHV genes, IGH D, and / or IGH J genes.

[0380] Thus, in some embodiments, more than one mouse IGHV, IGH D, and IGH J gene can be replaced with human IGHV, IGH D, and IGH J genes. In a first step, a selected number of mouse IGHV genes (e.g., all of the mouse IGHV genes in Table 4) 5' to ADAM6a are replaced with human IGHV genes. In a second step, a selected number of mouse IGH D and IGH J genes 3' to ADAM6b (e.g., all of the mouse IGH D genes in Table 5 except IGH D5-1 and IGH D3-1 and all of the IGH J genes in Table 6) are replaced with human IGH D and human IGH J genes. The replacements can be performed by homologous recombination or Cre-mediated recombination.

[0381] In some embodiments, the mouse does not have a mouse ADAM6a or ADAM6b gene. In some embodiments, the mouse has a human ADAM6 gene.

[0382] Various methods can be used to enhance the fertility of mice. In some embodiments, female mice with superovulation can be used for mating. In some embodiments, in vitro fertilization can be used. Superovulation can be induced by injecting serum gonadotropin and chorionic gonadotropin (e.g., human or mouse CG) into mature female mice. Mature male mice can be sacrificed and their epididymal tails isolated. The epididymal tails can be cut open to release sperm. Next, the mature female mice with superovulation can be sacrificed and the oviducts isolated. Cumulus-oocyte complexes (COCs) can be released from the oviducts. Next, a sperm suspension can be added to the COCs and incubated for insemination. Pathogenic oocytes containing only one pronucleus can be removed. After incubation, 2-cell stage embryos can be transferred to recipient females. Methods of enhancing the fertility of mice are known in the art.

[0383] The present disclosure also provides nucleic 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 of the nucleotide sequences 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 of the amino acid sequences described herein.

[0384] In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein, or any of the amino acid sequences encoded by any of the nucleotide sequences 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.

[0385] In some embodiments, the amino acid sequence (i) comprises; or (ii) consists of, an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0386] In some embodiments, the nucleic acid sequence (i) comprises; or (ii) consists of, a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0387] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80%, in some embodiments at least 90%, 95%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions 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, then the molecules are identical at that position (as used herein, "identity" with respect to amino acid or nucleic acid sequences means the degree of sequence relatedness between amino acid or nucleotide sequences as determined by the number of identical positions shared between the sequences). The percent identity between two sequences is a function of the number of identical positions shared between the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present application, the comparison of sequences and determination of percent identity between two sequences is accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0388] The percentage of residues which are conservative substitutions (percent homology) of residues having similar physical chemical properties (e.g., leucine and isoleucine) can also be used in measuring the sequence similarity. Families of amino acid residues having similar physical chemical 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). In many instances, the percent homology is higher than the percent identity. Accordingly, the present disclosure also provides amino acid sequences having 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% percent homology to any of the amino acid sequences described herein, or nucleic acids encoding these amino acid sequences.

[0389] Methods of using genetically modified animals

[0390] The genetically modified animals can be used to produce humanized or chimeric antibodies that specifically bind to a target. In some embodiments, the target (e.g., a protein or fragment of a protein) can be used as an immunogen to produce antibodies in these animals using standard techniques for preparing polyclonal and monoclonal antibodies. In some embodiments, the genetically modified animals are exposed to an antigen of choice for a period of time under conditions that allow the animals to produce antibodies specific for the antigen.

[0391] Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an 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. The animal can be injected with the antigenic peptide or protein more than once (e.g., two, three, or four times).

[0392] Full-length polypeptides or proteins can be used, or alternatively, antigenic peptide fragments thereof can be used as immunogens. An antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence and contains an epitope of the protein, such that an antibody raised against the peptide forms a specific immune complex with the protein.

[0393] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a genetically modified animal described herein). Suitable immunogenic preparations can include, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of proteins). The preparations can also include adjuvants, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agents.

[0394] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide or an antigenic peptide thereof (e.g., a portion of a protein) as an immunogen. Antibody titers in the immunized subject can be monitored over time by standard techniques, such as 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 protein A chromatography, to obtain the IgG fraction. At an appropriate time after immunization, e.g., when specific antibody titers are 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 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 that produce monoclonal antibodies are detected by, for example, screening the hybridoma culture supernatants for antibodies that bind the target polypeptide or epitope using standard ELISA assay methods.

[0395] In one aspect, the present disclosure provides a mouse comprising a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse produces a B cell 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 CHI, hinge, CH2, CH3, and combinations thereof.

[0396] In one aspect, the present disclosure provides a mouse comprising a modification of an endogenous immunoglobulin light chain (e.g., kappa or lambda) locus, wherein the mouse produces a B cell 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.

[0397] The mouse B cells or splenocytes can comprise, for example, a rearranged non-mouse immunoglobulin variable gene sequence operably linked to a mouse immunoglobulin constant region gene. Sequences encoding human heavy chain variable regions and human light chain variable regions are determined. The sequences can be determined by, for example, sequencing a target hybridoma or B cell. In some embodiments, single B cell screening is used. This can screen for natural antibody repertoires without the need for hybridoma fusion and combinatorial display. For example, B cells can be mixed with a panel of DNA barcoded antigens, enabling recovery of one or more antigen barcodes and B cell receptor (BCR) sequences from a single B cell by single cell sequencing protocols.

[0398] The antibody can be further modified to obtain a humanized antibody or a human antibody, 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 operably linking a sequence encoding a human light chain variable region to a sequence encoding a human light chain constant region.

[0399] In some embodiments, it can be difficult to elicit an immune response in a mouse if the mouse expresses a protein that is very similar to a target antigen. This is because B cells and T cells that recognize MHC molecules bound to self-derived peptides are deleted from the lineage of immune cells during immune cell development. In these 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 target antigen. Because the mouse does not negatively select for the gene product, the mouse can produce antibodies that readily bind specifically to the target.

[0400] The present disclosure also provides methods of making antibodies, nucleic acids, cells, tissues (e.g., spleen tissue). In some embodiments, the methods comprise exposing an animal described herein to an antigen. Antibodies (e.g., chimeric antibodies), nucleic acids encoding antibodies, cells, and / or tissues (e.g., spleen tissue) can be obtained from the animal. In some embodiments, nucleic acids encoding human heavy chain and light chain immunoglobulin variable regions can be determined, e.g., 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 described herein are cultured and antibodies are collected.

[0401] In some embodiments, mouse immunoglobulin V, D, J genes (e.g., mouse IGHV, IGHD, IGHJ, IGKV, or IGKJ genes) do not contribute to the heavy chain and / or light chain variable region sequence. In some embodiments, the heavy chain and / or light chain variable region sequence produced by the animal is entirely human and is entirely contributed by human immunoglobulin V, D, J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, and IGKJ genes).

[0402] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human antibodies, humanized antibodies, or chimeric antibodies, or antigen-binding fragments thereof, described herein, or by peptide synthesis. Such variants include, for example, deletions from, insertions into, or substitutions of residues within the amino acid sequences that make up an antigen-binding site or antigen-binding domain of an antibody. Among the variants are some that exhibit increased affinity for the target protein. Any combination of deletion, insertion, and / or substitution can be made to obtain an antibody or antigen-binding fragment thereof with increased binding affinity for the target. Introducing amino acid changes into an antibody or antigen-binding fragment can also alter or introduce new post-translational modifications of the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation site (e.g., altering the amino acid sequence such that a different sugar is attached to the glycosylation site upon action of an enzyme in the cell), or introducing a new glycosylation site.

[0403] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), bovines, porcines, equines, ovines, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0404] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences or having amino acid sequences identical to variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation).

[0405] Additional modifications can be made to the antibody or antigen-binding fragment. For example, one or more cysteine residues can be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibodies thus produced can have any extended half-life in vitro and / or in vivo. Homodimeric antibodies with extended half-lives in vitro and / or in vivo can also be prepared using heterobifunctional cross-linkers, as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies can be engineered to have dual Fc regions (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0406] In some embodiments, the antibody or antigen-binding fragment thereof can be covalently modified. These covalent modifications can be produced by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications to the antibody or antibody fragment are introduced into the molecule by reacting target amino acid residues of the antibody or antibody fragment with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

[0407] Examples

[0408] The application is further described in the following examples, which do not limit the scope of the application described in the claims.

[0409] Example 1: Overview

[0410] Experiments were performed to introduce human immunoglobulin genes into the mouse genome to produce mice that express humanized antibodies. Figure 1A A method for producing humanized mice is shown. 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.

[0411] The mouse immunoglobulin variable regions are replaced with human immunoglobulin variable regions by direct substitution (e.g., homologous recombination or Cre-mediated recombination). In some cases, the human immunoglobulin variable regions can be introduced into the mouse genome by a stepwise approach. The correctly replaced recipient cells are then selected. The cells are then injected into blastocysts to make chimeric mice. Breeding is then performed to obtain mice containing the complete human immunoglobulin variable regions.

[0412] Because the mouse heavy chain gene and 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 made separately Figure 1B and Figure 1C ). These mice can then be mated with each other to obtain mice expressing both human heavy chain variable regions and human light chain variable regions.

[0413] Example 2: Modification of the mouse heavy chain immunoglobulin locus

[0414] 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 flanking the variable region of the heavy chain immunoglobulin locus. The resulting modified chromosome is shown in Figures 3A-3B One is a wild-type loxP site and the other is a heterospecific mutant lox site (lox2272). Recombination between the wild-type loxP site and the heterospecific mutant lox site does not occur. This 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, a Flp recognition target (FRT), a CAG promoter, a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "5' HygR"), a LoxP (1301), a FRT, a 5' PB transposon sequence (PB 5'), a PGK promoter, a blue fluorescent protein reporter gene (BFP), a FMDV self-cleaving peptide (2A), a hygromycin resistance gene (hygromycin phosphotransferase; HygR), a 3' PB transposon sequence (PB 3'), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0415] 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'), a Flp recognition target (FRT), a partial sequence of the puromycin resistance gene (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.

[0416] The vectors (V1401 and V1402) were introduced into mouse embryonic stem cells. The cells were then selected with hygromycin B and puromycin. Integration of the exogenous genes into the mouse genome was confirmed by PCR. The results are shown in Figures 5A-5B and Figures 6A-6B Clones numbered 030, 035, 036, and 037 were confirmed to be positive.

[0417] PCR assays were performed using the following primers:

[0418] 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);

[0419] mIGHV-3'lox-L-GT-R2: 5'-tgacgcatgtgttttatcggtctgt-3' (SEQ ID NO: 6);

[0420] mIGHV 3'lox-R-GT-F2: 5'-gtgcctgacacgtgctacgagattt-3' (SEQ ID NO: 7); mIGHV-3'lox-R-GT-Rl: 5'-ttcaacaataagcagggccagaggg-3' (SEQ ID NO: 8);

[0421] Among these primers, mlgHV-5'loxP-L-GT-F and mlgHV-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-Rl are located on the mouse chromosome, mIGHV-3'lox-L-GT-R2 and mIGHV 3'lox-R-GT-F2 are located on vector 1402.

[0422] Example 3: Modification of human chromosome 14

[0423] The purpose of this experiment was to produce a modified human chromosome with at least two recombination sites. These two recombination sites were introduced flanking the variable region of the heavy chain immunoglobulin locus.

[0424] The heavy chain immunoglobulin locus is located on human chromosome 14. Figure 7 is a schematic representation of human chromosome 14 highlighting the heavy chain immunoglobulin locus.

[0425] The modified human variable region is shown in Figure 8 . Figure 9 An overview of the targeting strategy is shown. As shown in Figure 9 , the 301 and 302 sites are recombination sites. Recombination site 1301 and recombination site 301 are identical. Recombination site 1302 and recombination site 302 are identical.

[0426] Experiments were performed to insert vectors 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 from FMDV (Foot and Mouth Disease Virus) (2A), a Zeocin resistance gene sequence (Zeo), a transcription termination / polyadenylation signal sequence (Poly A; "PA"), a LoxP recognition sequence for Cre recombinase (301), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "3'HygR"), and a Flp recognition target ("FRT"), a downstream DNA homology arm sequence, and a diphtheria toxin A subunit (DTA) gene.

[0427] 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 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 (Poly A; "PA"), a DNA homology arm sequence downstream of the insertion site, and DTA.

[0428] In some experiments, the vectors (V401, V402) were introduced into cells and the cells were selected by the appropriate drug resistance marker or combination thereof (Zeocin, G418).

[0429] 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, the first vector is introduced into a chromosome. The modified chromosome can be added to a recipient cell, and then the second vector can be inserted into the modified chromosome. In some experiments, V401 is first introduced into a human cell, then the chromosome is labeled with a fluorescent marker, followed by isolation of the chromosome, and then the modified chromosome is injected into a recipient cell by microinjection. V402 is then introduced into the cell. In another experiment, human fibroblasts are selected and vector 402 is introduced. The human fibroblasts are then fused with a recipient cell (A9 cell or CHO cell).

[0430] In some experiments, one or more vectors can be inserted into human chromosome 14 at the desired location by homologous recombination, e.g., simultaneously. The vectors can comprise a drug resistance marker (Zeocin, G418), and then the cells are selected. The chromosome is labeled, then isolated, and then the modified chromosome is injected into a recipient cell by chromosomal microinjection.

[0431] In some experiments, one or more additional vectors are inserted. These additional vectors can be inserted at different locations on human chromosome 14 as desired. In one experiment, a third vector has the following sequences 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 (302), a PGK promoter, a partial puromycin resistance gene sequence (5' PuroR), a mammalian expression promoter 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 (PolyA; "PA"), a DNA homology arm sequence downstream of the insertion site, and a DTA. The vector is inserted into the C region.

[0432] In one experiment, a third vector (V403) is inserted between the C region and the kinetochore. The vector has the following sequences 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 (302), a PGK promoter, a partial puromycin resistance gene sequence (5' PuroR), a mammalian expression promoter 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.

[0433] In one experiment, human fibroblasts were selected and introduced with vector 402. The human fibroblasts were then fused with recipient cells (A9 cells or CHO cells). The modified chromosome was isolated and introduced into another suitable recipient cell. Cells were then selected by G418 resistance to obtain cells containing only one human chromosome. Vector 401 was then introduced into the cells and cells were selected by resistance to Zeocin. After that, vector 403 was introduced into the cells and cells were selected by resistance to puromycin. The selected positive clones after screening were treated with Cre enzyme. Chromosome techniques are described in, for example, Kuroiwa et al. "Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts." Nature Biotechnology 18.10 (2000): 1086-1090; CN1200014A; CN109837307A; US20120093785A1; US2009253902; CN1717483A; 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, Volume 13, Number 3, 1987, pp. 279-284, each of which is incorporated by reference herein in its entirety.

[0434] PCR was performed to confirm the presence of 5'-end recombination site 301 and 3'-end recombination site 302 on the chromosome. Cells without random insertion were confirmed by Southern blotting and analyzed by fluorescence in situ hybridization (FISH).

[0435] Figure 10 A modified human chromosome 14 is shown. Figure 11 PCR identification of the loxP site 301 on the chromosome hChr14-mut3 is shown. Figure 12PCR identification results for the loxP site 302 on chromosome hChr14-mut3 are shown. As shown, 12 clones (numbered 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.

[0436] The following PCR primers were used in the experiments:

[0437] hIGHV-5' loxP-L-GT-F1 : 5'-TCAAAGTCAATTTCCTCAGCGAGGCT-3'

[0438] (SEQ ID NO: 9),

[0439] hIGHV-5' loxP-R-GT-R: 5'-AGGGAGGGAATGGAATGAGGGTGAT-3'

[0440] (SEQ ID NO: 10);

[0441] hIGHV-3' loxP-L-GT-F1 : 5'-CCATGTGACCCATTCGAGTGTCCTG-3'

[0442] (SEQ ID NO: 11),

[0443] hIGHV-3' loxP-R-GT-R: 5'-TTGTGAGGGCTCAAGTTCAGTGCAT-3' (SEQ ID NO: 12).

[0444] FISH analysis was performed using the positive clones and CCP14 FISH probe (CytoTest Inc., Rockville, MD, Cat. No. CT-CCP014). Representative FISH images of clone 1-D10 are shown in Figure 13 and Figure 14 In Figure 13 , the white arrow indicates the full length of human chromosome 14 (before modification). In Figure 14 , the white arrow indicates the modified human chromosome 14 fragment.

[0445] Example 4: Introduction of human chromosome or fragment into mouse ES cells

[0446] The modified chromosome obtained in Example 3 was introduced into the cells obtained in Example 2 by the aforementioned method. The cells were then selected by G418. Only cells containing only one human chromosome were selected. Figure 15 A modified mouse chromosome 12 is shown.

[0447] The V, D, J regions on human chromosome hChr14-mut3 were then replaced with the V, D, J regions on mouse chromosome mChr12-mut2 by Cre recombinase Figure 16 ). The human chromosomal DNA sequence was replaced with the sequence between recombination sites 1301 and 1302. Hygromycin and puromycin were used to screen for positive cells. Cells were further screened by DT to obtain mouse cells without human chromosomes before injection into mouse blastocysts. In some cases, cells were injected directly into blastocysts without DT screening.

[0448] Cells after Cre recombination were tested to confirm that the human gene sequence had been integrated into the mouse genome. The results of PCR assays are shown in Figure 17 , Figure 18 , Figure 19 and Figure 20 . The results of all PCR assays showed that cells numbered 1-B4, 1-B10 and 2-A7 had the correct recombination, and the human chromosome was absent in the 1-B10 cell. Murine Whole Chromosome Painting Probes (Cytocell Ltd, Cambridge, UK; Catalog No. AMP12R) and human specific IGH break apart probe (Cytocell Ltd, Cambridge, UK; Catalog No. LPH 014) were used to test the 1-B10 cell by FISH. The results, shown in Figure 21 , confirmed the presence of the human chromosome fragment in the mouse chromosome. These primers are shown in the following table.

[0449] Table 12

[0450]

[0451] Example 5: Generation of mice containing humanized heavy chain immunoglobulin loci

[0452] Positive clone cells are injected into blastocysts of BALB / c mice by microinjection. Embryo microinjection is performed according to the methods described in, for example, A. Nagy et al., "Manipulating the Mouse Embryo: A Laboratory Manual (3rd Edition)," Cold Spring Harbor Laboratory Press, 2003. The injected zygotes are then transferred to culture medium for a short time and then transplanted into the oviduct of a recipient mouse to generate genetically modified humanized mice (F0 generation). The mice are then mated with mice of C57BL / 6 background. Figure 22 ) The DNA obtained from the tail of the mice is subjected to PCR analysis. The mice are further crossed with mice of BALB / c background several times (e.g., at least 5 times) to obtain heterozygous mice of BALB / c background with humanized heavy chain immunoglobulin loci.

[0453] To confirm that the mice express human antibody heavy chains, blood is collected from the chimeric mice (F0 generation) and black mice (F1 generation). RNA is extracted and reverse transcribed to obtain cDNA. The sequences are amplified using the following PCR primers and further sequenced.

[0454] Table 13. PCR primers

[0455]

[0456] Example 6: Modification of mouse light chain immunoglobulin loci

[0457] The light chain immunoglobulin loci are located on mouse chromosome 6. Figure 23 is a schematic diagram showing the mouse light chain immunoglobulin loci. Two recombination sites are introduced on both sides of the variable region of the light chain immunoglobulin loci, and the resulting modified chromosome is as shown in Figures 24A-24B . The detailed targeting strategy is as shown in Figure 25 .

[0458] The modification was made in mouse embryonic stem cells. The vector (V3901) has, from 5' to 3': a DNA homology arm sequence upstream of the insertion site, a Flp recognition target (FRT), a mammalian expression promoter from human elongation factor 1 alpha (EF-1a), a hygromycin resistance gene (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), 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 partial sequence of the puromycin resistance gene (3'PuroR), an FMDV self-cleaving peptide (2A), a DT receptor (DTR), a LoxP recognition sequence for Cre recombinase (1102), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0459] The vector (V3901) was introduced into mouse embryonic stem cells. Cells were selected by the respective antibiotic resistance genes or a combination thereof. Integration of the vector V3901 into the correct site of the mouse genome was confirmed by PCR. The results are shown in Figures 26-27

[0460] PCR assays were performed using the following primers:

[0461] IGKV-005-C-5G-L-GT-F: 5'-TCACACACTACAGCTTCCACCACAA-3' (SEQ ID NO: 34);

[0462] IGKV-005-C-5G-L-GT-R2: 5'-CGGGGAAAAGTCGACTCTAGAACGG-3' (SEQ ID NO: 35);

[0463] IGKV-005-C-5G-R-GT-F1: 5'-ACTGCATTCTAGTTGTGGTTTGTCCA-3' (SEQ ID NO: 36);

[0464] IGKV-005-C-5G-R-GT-R: 5'-GGCCTGGAAAACTCAGCTATCCTTT-3' (SEQ ID NO: 37).

[0465] ​Of these primers, IGKV-005-C-5G-L-GT-F and IGKV-005-C-5G-R-GT-R are located on the mouse chromosome, and IGKV-005-C-5G-L-GT-R2 and IGKV-005-C-5G-R-GT-F1 are located on the vector V3901.

[0466] Accordingly, two recombination sites were introduced into the mouse chromosome 6 in mouse embryonic stem cells.

[0467] Example 7: Modification of human chromosome 2

[0468] The human light chain immunoglobulin locus is located in human chromosome 2. Figure 28 is a schematic diagram of human chromosome 2 highlighting the light chain immunoglobulin locus.

[0469] Two recombination sites were introduced flanking the variable region of the light chain immunoglobulin locus. The V HK region between the centromere was deleted to obtain a shorter artificial chromosome for subsequent experiments. Similar recombination sites were introduced in the variable region of the mouse immunoglobulin locus on chromosome 6. The human chromosome was then introduced into mouse recipient cells to obtain a humanized light chain immunoglobulin locus.

[0470] 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), a FMDV self-cleaving peptide (2A), a Blasticidin S deaminase from Aspergillus terreus (Bsr) from Aspergillus terreus, a transcription termination / polyadenylation signal sequence (Poly A; "PA"), a LoxP recognition sequence 2601, a hygromycin resistance gene (hygromycin phosphotransferase portion sequence; "3'HygR", a Flp recognition target (FRT), a DNA homology arm sequence downstream of the insertion site, and a diphtheria toxin A subunit (DTA).

[0471] The vector (V2702) has, from 5' to 3': a DNA homology arm sequence upstream of the insertion site, a LoxP recognition sequence 2602, a PGK promoter, a portion of a puromycin resistance gene sequence (5' PuroR), EF-1a, PBase, IRES, a kanamycin resistance gene sequence (Neo), a transcription termination / polyadenylation signal sequence (Poly A; "PA"), a DNA homology arm sequence downstream of the insertion site, and a DTA.

[0472] The sequence of the vector (V2702) was verified by sequencing. The vector was introduced into human H9 cells by transfection. Cells were then selected by G418 and ubenimex resistance. Integration of the gene into the human genome was confirmed by PCR. The results are shown in Figure 31 Clones numbered 01, 02, 03, and 04 were confirmed as positive clones.

[0473] PCR assays were performed using the following primers: 3’L-L-GT-F: 5’- AAGGTGACTCTGCAATCAGCCTCTG-3’ (SEQ ID NO: 38), 3’L-L-GT-R1: 5’- TCATCTACAGCCACAACGTGAGCAG-3’ (SEQ ID NO: 39); 3’L-R-GT-F1: 5’- CCCATGTACAGGTTCCGCATGAACT-3’ (SEQ ID NO: 40), 3’L-R-GT-R: 5’- CTCCGTCCGCTTTTATTTCCCCTGT-3’ (SEQ ID NO: 41).

[0474] Cells with modified chromosomes suitable for further experimentation were selected. The modified human chromosomes were introduced into recipient cells by chromosome technology. Recipient cells A9 cells were selected by G418 resistance. Cells containing only one human chromosome were selected for further gene editing.

[0475] During the selection process, Figure 31 clone numbered 03 shown in FIG. 3B had the correct recombination and was labeled by human chromosome 2 enumeration probe (CCP2 FISH probe) (CytoTest Inc., Rockville, MD, catalog number CT-CCP002). The results confirmed the presence of the modified human chromosome 2 in the cells Figure 32 ).

[0476] The vector (V2701) was then further introduced into cells. Cells were selected by G418 and blasticidin S resistance. Recombination was confirmed by PCR. Primers are shown in the following table. The results indicated that 5-C3, 1-H2, 1-H9, and 1-G5 were positive clones Figure 33 ).

[0477] Table 14

[0478]

[0479] Example 8: Obtaining mice that can produce humanized antibodies

[0480] Mouse embryonic stem cells were fused with the cells obtained in Example 7 and the modified human chromosome 2 was introduced into mouse embryonic stem cells obtained in Example 6. Mouse ES cells containing only one copy of the modified human chromosome 2 were selected and the cells were screened after Cre-mediated recombination (Cre-mediated recombination) as shown in Figure 34 The cells after Cre recombination were tested to confirm that the human gene sequences had integrated into the mouse genome. Mouse chromosome painting probe (Cytocell Ltd, Cambridge, UK; catalogue number AMP06G) and human specific IGK break apart probe (Cytocell Ltd, Cambridge, UK; catalogue number LPH 034) were used to verify the PCR confirmed positive clones by fluorescence in situ hybridization. The results are shown in Figure 35 which confirmed the presence of the human chromosome fragment in the mouse chromosome. The cells were injected into blastocysts. Mice containing humanized light chain immunoglobulin loci can be obtained.

[0481] Chimeric mice were selected and mated with C57BL / 6 mice to produce grey and black offspring (F1 generation). DNA obtained from the tail of black mice was subjected to PCR analysis and positive F1 generation mice were selected and mated with Flp tool mice. Figure 36 A schematic of Flp-mediated recombination is shown. Mice prepared by this method contain a C57BL / 6 background. Mice with different backgrounds can have different advantageous aspects and heterozygous or homozygous mice prepared by the methods herein can be used to produce mice with some other background by backcrossing (e.g. BALB / c mice have advantageous aspects for humoral immunity) for several generations to obtain mice with the desired background.

[0482] Several mice were selected and crossed with BALB / c mice several times to obtain heterozygous mice with a BALB / c background. The heterozygous mice were then crossed with each other to obtain homozygous mice.

[0483] Mice with humanized light chain immunoglobulin loci and mice with humanized heavy chain immunoglobulin loci were crossed with each other to obtain mice with both humanized heavy chain immunoglobulin loci and humanized light chain immunoglobulin loci.

[0484] Example 9: B cell development in transgenic mice

[0485] Experiments were performed to compare the immune systems of humanized mice and wild-type mice. Three 9-10 week old wild-type (WT), three mice with a heterozygous humanized heavy chain immunoglobulin locus, and three mice with a homozygous humanized heavy chain immunoglobulin locus were selected. Among them, the heterozygous mice and the homozygous mice had similar body weight, appearance, and activity 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 were no observable differences in the size, shape, and weight of the spleen of the three groups of mice). Flow cytometry was performed to analyze the lymphocyte populations and distributions in the peripheral blood, spleen, and lymph nodes of the mice Figures 45-47 ) and the B cell populations in the spleen, lymph nodes, and bone marrow Figures 48-50 ). In the results, white blood cells included: B cells (e.g., characterized as CD45+, CD19+, TCR-), T cells, and natural killer (NK) cells (e.g., characterized as CD45+, TCR-, and NK1.1+). T cells were characterized as CD45+, CD19-, TCR+. CD4+ T cells (CD4) were characterized as CD45+, CD19-, TCR+, CD4+, CD8-. CD8+ T cells (CD8) were characterized as CD45+, CD19-, TCR+, CD4-, CD8+. Only intact single live white blood cells were included in the flow cytometry analysis.

[0486] The developmental stages of B cells in the lymph nodes and spleen were divided into T1 (transitional type 1 B cells, characterized as B220 + IgM + IgD - ), T2 (transitional type 2 B cells, characterized as B220 + IgM + IgD + ), and mature B cells (characterized as B220 + IgM 低 IgD + ). Figures 45-47 The percentages of white blood cells in different tissue samples are shown. Figures 48-49 The percentages of B cells at different developmental stages are shown.

[0487] In addition, the development of B cells was also evaluated in the marginal zone of the spleen (marginal zone B cells, MZ-B, characterized as B220 + CD21 + CD23 - ) and the follicular zone (follicular B cells, referred to as FO-B, characterized as B220 + CD21 低 CD23 + ). Figure 50Percentages of splenic B cells showing marginal zone (MZ-B) and follicular zone (FO-B).

[0488] According to different developmental stages, B cells in bone marrow can be divided into pro-B cells (characterized by B220 低 CD43 高 IgM 低 ), pre-B cells (characterized by B220 低 CD43 中 IgM 低 ) and immature B cells (characterized by B220 高 CD43 低 IgM 高 ). Figures 51A-51C Percentages of B cells at different developmental stages in bone marrow are shown.

[0489] Compared with wild-type mice, the percentages of immune cells and B cells in humanized mice were similar, and there was no statistical difference between different groups. No significant B cell differentiation defects were observed in heavy chain humanized hybrid mice (hybrids) or homozygous mice (homozygotes).

[0490] Example 10: Serum immunoglobulin isotype analysis

[0491] In addition, the levels of various immunoglobulins in the serum of mice not immunized in the above examples were analyzed. The mice included WT mice, mice with hybrid humanized heavy chain immunoglobulin loci, and mice with homozygous humanized heavy chain immunoglobulin loci.

[0492] The experiment was performed using a 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 10 ug / mL with PBS (Solarbio, Catalog No. P1020). Then, 0.1 mL of diluted antibody was added to each well of an enzyme-linked immunosorbent assay (ELISA) plate and incubated at 37°C for 2 hours. Next, the plate was washed and blocked with 1% BSA (Cell Signaling, Catalog No. 9998) gradient dilution at 4°C for 12 hours. 0.1 mL of diluted sample was added to each well, and then incubated at 37°C for 1 hour. 1% BSA was added to the wells as a blank control.

[0493] Next, the plates were washed with PBS containing 0.05% Tween-20 (Amresco, Cat. No. M147). Secondary antibodies conjugated with horseradish peroxidase (goat anti-mouse IgA, IgG (1, 2b, 2c, 3), IgM) were added (diluted 300-fold with 1% BSA, 0.1 mL per well) and incubated with the samples at 37°C for 1 hour. Next, the plates were washed and developed by adding 0.1 mL of TMB chromogen solution (Beyotime Biotechnology, Cat. No. P0209) to each well. After incubation at room temperature in the dark for 8 minutes, 0.1 mL of reaction solution (Beijing Dingguo Changsheng Biotechnology Co. LTD., Cat. No. EIA-0032) was added to each well. The light absorption 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.

[0494] The results showed that mice with humanized heavy chain immunoglobulin loci were able to produce IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM antibody isotypes, and the mice had similar expression levels for each isotype compared to wild-type mice Figures 52-57 ). This indicates that humanization of the heavy chain variable region gene segments did not have a significant adverse effect on antibody class switching, expression, or secretion of various antibody isotypes.

[0495] Example 11: V(D)J recombination of human variable region gene segments in mice

[0496] The gene sequences of the heavy chain variable regions and light chain variable regions in the mice were analyzed by next-generation sequencing.

[0497] One humanized heavy chain homozygous mouse that was not immunized (not exposed to a specific antigen) was selected. Spleen cells were collected from the mouse for RNA extraction. Reverse transcription was performed using a 5’ RACE kit (SMARTer RACE 5’ / 3’ Kit, Takara Bio USA, Inc., Cat. No. 634858) to obtain cDNA. The obtained cDNA was subjected to PCR amplification using an IgM constant region-specific primer and a UPM primer of the 5’ RACE kit to obtain a heavy chain variable region sequence fragment, which was then sequenced. The sequence of the IgM constant region-specific primer was 5’-ccaagcttacgagggggaagacatttgggaa-3’ (SEQ ID NO: 50).

[0498] In another experiment, 11 light chain humanized hybrid mice were selected and blood was collected from the orbital plexus for RNA extraction. After reverse transcription by the method described above, primers VKF1 and IgKC-tag were used to amplify VKI family light chain genes, which were then sequenced. The following primer sequences were used:

[0499] VKF1 sequence: 5'-cataagatctcgmcatccrgwtgacccagt-3' (SEQ ID NO: 51);

[0500] IgKC-tag primer sequence: 5'-ctaacactcattcctgttgaagctcttgac-3' (SEQ ID NO: 52).

[0501] Sequencing results were compared to human immunoglobulin sequences by the NCBI Ig Blast tool to identify expression of human V H , D H , J H , and VK, JK genes after V(D)J recombination. In 135 clones analyzed, preliminary results detected expression of certain V H , D H gene segments and all J H gene segments (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 indicates that after replacement of the endogenous chromosomal fragments with human immunoglobulin heavy chain sequences, human V H , D H , and J H genes on the human chromosome fragments integrated into the mouse can be recombined to express human heavy chains.

[0502] Most VKI family light chain genes were detected in 441 clones derived from humanized light chain hybrid mice (Table 16). Similar to the results for heavy chains, some of these genes on the humanized fragment were located in close proximity to the modification site and some were located far from the modification site. This indicates that after replacement of the endogenous light chain immunoglobulin variable region locus with the human light chain immunoglobulin variable region locus, human VK and JK genes integrated into the mouse genome can be recombined to express light chains with human light chain variable regions. Further analysis of the results in 11 mice indicated that the distribution of IGKV genes detected did not differ significantly among the mice tested Figure 58 .

[0503] Table 15. List of IGHV genes, IGHD genes, and IGHJ detected to be expressed after VDJ recombination

[0504]

[0505] Table 16. List of IGKV genes expressed after detection of VJ recombination

[0506] Gene Name Observed (counts) IGKV1D-43 9 IGKV1D-13 4 IGKV1D-16 9 IGKV1D-12 11 IGKV1-39 17 IGKV1-37 2 IGKV1-33 107 IGKV1-27 14 IGKV1-17 55 IGKV1-16 26 IGKV1-13 1 IGKV1-12 50 IGKV1-9 42 IGKV1-8 24 IGKV1-6 31 IGKV1-5 31 IGKV4-1 8

[0507] Example 12: Immunization and antibody production in humanized mice

[0508] Five wild-type (WT) mice and five humanized heavy chain homozygous mice (9-10 weeks old) were randomly selected and immunized with foreign antigens. Mice were immunized repeatedly every two weeks for a total of three immunizations. Orbital bleeds were performed after the second and third immunizations. Serum was collected and then serum titers were determined by ELISA or FACS to determine and analyze antigen-specific antibody responses. Three antigens were used in the study, hBTLA, dPD1, and OVA Figures 59-63 ). The results showed that after the second immunization, most wild-type (WT) and humanized heavy chain homozygous mice produced antigen-specific antibodies. After the third immunization, antibody titers increased to 1 x 105 4 to 1 x 105 5 . The results of the immunization efficacy test in humanized mice were essentially the same as compared to wild-type mice, indicating that the humanized immunoglobulin variable region loci in the mice were functional and could produce antigen-specific antibodies.

[0509] Example 13: B cell development in hVH / hVL mice

[0510] 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 intercrossed to obtain mice with homozygous humanized heavy chain immunoglobulin loci and homozygous humanized light chain immunoglobulin loci (humanized VH / VL mice, or hVH / hVL mice). The hVH / hVL mice can be used to produce humanized monoclonal antibodies in vivo.

[0511] Experiments were performed to compare the immune systems of unimmunized humanized VH / VL mice with unimmunized wild-type mice. Body weight and spleen weight Figures 65A-65B were measured in wild-type and hVH / hVL mice. No significant differences in mean body weight and spleen weight were detected between wild-type mice and hVH / hVL mice.

[0512] Flow cytometry was performed to analyze lymphocyte populations and distribution in the spleen of mice Figure 66 and B cell populations in the spleen and bone marrow Figures 67A-67B , Figures 68A-68C). The results show that the percentages of B cells, T cells, NK cells, CD4+ T cells, and CD8+ T cells in the spleen are almost identical in hVH / hVL mice as in wild-type mice. In the results, white blood cells include: B cells (e.g., characterized as CD45+, CD19+, TCR-), T cells, and natural killer (NK) cells (e.g., characterized as CD45+, TCR-, and NK1.1+). T cells are characterized as CD45+, CD19-, TCR+. CD4+ T cells (CD4) are characterized as CD45+, CD19-, TCR+, CD4+, CD8-. CD8+ T cells (CD8) are characterized as CD45+, CD19-, TCR+, CD4-, CD8+. Only intact single live white blood cells are included in the flow cytometry analysis.

[0513] Figure 67A The percentages of B cells at different developmental stages are shown. The developmental stages of B cells in the spleen are divided into T1 (transitional 1 type B cells, characterized as B220 + IgM + IgD - ), T2 (transitional 2 type B cells, characterized as B220 + IgM + IgD + ), and mature B cells (characterized as B220 + IgM 低 IgD + ). In addition, the development of B cells was also evaluated in the splenic marginal zone (marginal zone B cells, MZ-B, characterized as B220 + CD21 + CD23 - ) and follicular zone (follicular B cells, referred to as FO-B, characterized as B220 + CD21 低 CD23 + ). Figure 67B The percentages of splenic B cells at the splenic marginal zone (MZ-B) and follicular zone (FO-B) are shown. No significant difference was observed between wild-type mice and hVH / hVL mice.

[0514] Figure 68A The percentages of B cells at different developmental stages in the bone marrow are shown. B cell progenitors in the bone marrow were analyzed by flow cytometry. According to the expression levels of B220 and CD43, B cell progenitors in the bone marrow can be divided into 3 cell populations: pro-B cells (characterized as B220 低 CD43 高 IgM 低 ), pre-B cells (characterized as B220 低 CD43 中 IgM低 ) and immature B cells (characterized by B220) 高 CD43 低 IgM 高 No significant differences were observed between wild-type mice and hVH / hVL mice.

[0515] In addition, flow cytometry was used to assess B cell development in the bone marrow or spleen to evaluate plasma cells (B220). 低 IgM - IgD - CD138 - ) and memory B cells (B220) + IgM + IgD - CD38 + Selective staining () Figures 68B-68C No significant differences were observed between wild-type mice and hVH / hVL mice.

[0516] Different immunoglobulin (Ig) subtypes in the serum of hVH / hVL mice and wild-type mice were quantitatively measured by ELISA. Six mice were selected in each group. No significant differences in the levels of IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM were observed. Figure 69 ).

[0517] These experiments demonstrate that the immune system of hVH / hVL mice is functional, and that the humanized immunoglobulin loci in hVH / hVL mice can interact appropriately with the mouse immunoglobulin homeostasis region.

[0518] Example 14: Germline usage analysis in hVH / hVL mice

[0519] The utilization rates of heavy chain IGHV, IGHD, and IGHJ in unimmunized hVH / hVL mice (without antigen stimulation) were analyzed. The results are shown in Figures 70A-70D. Additionally, the utilization rates of κ chain IGKV and IGKJ were also analyzed. The results are shown in Figures 71A-71C.

[0520] Germ utilization in non-immunized hVH / hVL mice was determined using next-generation sequencing (NGS). For example, as Figure 71C As shown, IGKJ1, IGKJ2, and IGKJ4 were frequently used in unimmunized hVH / hVL mice, while IGKJ3 and IGKJ5 were less frequently observed. This germline usage pattern of IGKJs is consistent with the reported use of human IGKJs in the literature.

[0521] Heavy chain CDR3 length distribution was determined by NGS sequencing of the immunoglobulin repertoire from splenocytes of unimmunized hVH / hVL mice (n=2). As shown in Figure 72 FIG. 6, the median length of CDR3 is 14 amino acids. This result is consistent with the median length of human heavy chain CDR3 in the human immune system.

[0522] The amino acid types at each position of heavy chain CDR3 (HCDR3) were analyzed. Figure 73 ) Multiple patterns were observed, including an increase in the frequency of tyrosine usage and an increase in the usage of the DH2 (IGHD2) germline family. These patterns are similar to the amino acid composition in human HCDR3.

[0523] Cysteine residues can form disulfide bonds. Human HCDR3 can contain one cysteine residue or two cysteine residues, while mouse HCDR3 generally does not contain cysteine. Figure 74 The results in FIG. 7 show the frequency of HCDR3 of hVH / hVL mice containing cysteine residues, and this frequency increases with the length of HCDR3. This result is consistent with the diversity of HCDR3 in human peripheral blood mononuclear cells (PBMC).

[0524] Example 15: Lymphoid organ histological analysis

[0525] Spleen, inguinal lymph node, and Peyer’s patch from unimmunized wild-type mice or unimmunized hVH / hVL mice were stained with hematoxylin-eosin. Representative sections are shown in Figures 75A-75F Wild-type (C57BL / 6) mice and hVH / hVL mice exhibited normal structure of follicles with distinct boundaries, and no significant difference in tissue morphology was observed.

[0526] Example 16: Antibody production in hVH / hVL mice

[0527] After the second and third immunization with BCMA, IL4R, PD-1, Siglec15, and SIRPa antigens, blood was collected and analyzed for antigen-specific antibody titers of wild-type (C57BL / 6) mice and hVH / hVL mice by ELISA Figures 76A-76E The results show that hVH / hVL mice can produce antibodies that specifically bind to antigens, and wild-type mice have similar immune responses to hVH / hVL mice.

[0528] Example 17: B cell development in hVH / hVL mice

[0529] Experiments were performed to compare the immune systems of humanized VH / VL mice and wild-type mice after immunization. Body weight and spleen weight were measured in wild-type mice and hVH / hVL miceFigures 77A-77B No significant differences in mean body weight and spleen weight were detected between wild-type mice and hVH / hVL mice.

[0530] Flow cytometry was performed to analyze the lymphocyte populations and distribution in mouse spleens. Figure 78 ) and B cell populations in the spleen and bone marrow ( Figures 79A-79B , Figures 80A-80C ).

[0531] Figure 79A The percentage of B cells at different developmental stages in the spleen was displayed. Additionally, B cell development was assessed in the marginal zone and follicular zone of the spleen. Figure 79B The percentage of splenic B cells is shown in the marginal zone (MZ-B) and follicular zone (FO-B). No significant difference was observed between wild-type mice and hVH / hVL mice.

[0532] Figure 80A The percentage of B cells at different developmental stages in the bone marrow was shown. B cell progenitor cells in the bone marrow were analyzed by flow cytometry. No significant differences were observed between wild-type mice and hVH / hVL mice.

[0533] In addition, B cell development in the bone marrow or spleen was assessed by flow cytometry to evaluate plasma cell (B220) development. 低 IgM - IgD - CD138 - ) and memory B cells (B220) + IgM + IgD - CD38 + Selective staining () Figures 80B-80C No significant differences were observed between wild-type mice and hVH / hVL mice.

[0534] Different immunoglobulin (Ig) subtypes in the serum of hVH / hVL mice and wild-type mice were quantitatively determined by ELISA. Six mice were selected for each group. No significant differences in the levels of IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM were observed. Figure 81 In addition, the total serum IgG levels in hVH / hVL mice and wild-type mice were quantitatively measured by ELISA. No significant differences were observed. Figure 82 ).

[0535] Other implementation plans

[0536] It is to be understood that while the application has been described in conjunction with the detailed description thereof, the foregoing description is meant to illustrate and not to limit the scope of the application, which is defined by the scope of the appended claims.

[0537] Other aspects, advantageous aspects and modifications are within the scope of the following claims.

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Claims

1. A method for producing genetically modified rodents, the method comprising: The human chromosome is modified to produce a modified human chromosome containing a first exogenous recombination site and a second exogenous recombination site located on both sides of the variable region of the heavy chain immunoglobulin locus; said human chromosome 14. Modify the endogenous chromosomes of rodent cells to produce modified endogenous chromosomes containing a first exogenous recombination site and a second exogenous recombination site; The modified human chromosome was introduced into the cells of the animal; as well as Inducing site-specific recombination between the modified human chromosome and the modified endogenous chromosome, thereby replacing the endogenous sequence in the modified endogenous chromosome with a human sequence from the modified human chromosome; The resulting rodent contains a continuous human sequence of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes from the human chromosome 14 heavy chain immunoglobulin locus, wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked and capable of VDJ rearrangement. The animal in question is a mouse.

2. The method of claim 1, wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin locus on human chromosome 14.

3. The method of claim 1, wherein the animal comprises disruption of its endogenous heavy chain immunoglobulin locus, the disruption comprising a deletion of at least 500 kb of endogenous sequence.

4. The method of claim 3, wherein disruption of the endogenous heavy chain immunoglobulin locus in the animal comprises the deletion of a continuous sequence from the mouse IGHV1-85 gene to the mouse IGHJ4 gene.

5. The method of claim 1, wherein the animal comprises one or more endogenous IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.

6. The method of claim 1, wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus, wherein the unmodified human sequence is at least 500 kb.

7. The method of claim 1, wherein the animal is homozygous for the heavy chain immunoglobulin locus.

8. The method of claim 1, wherein the animal is heterozygous for the heavy chain immunoglobulin locus.

9. The method of claim 1, wherein the animal further comprises one or more human IGKV genes and one or more human IGKJ genes at the endogenous light chain immunoglobulin locus.

10. The method of claim 1, wherein the animal comprises the disruption of its endogenous λ light chain immunoglobulin gene locus.

11. A method for producing genetically modified rodents, the method comprising: The human chromosome is modified to produce a modified human chromosome containing a first exogenous recombination site and a second exogenous recombination site located on both sides of the variable region of the light chain immunoglobulin gene locus, said human chromosome 2; Modify the endogenous chromosomes of rodent cells to produce modified endogenous chromosomes containing a first exogenous recombination site and a second exogenous recombination site; The modified human chromosome was introduced into the cells of the animal; as well as Inducing site-specific recombination between the modified human chromosome and the modified endogenous chromosome, thereby replacing the endogenous sequence in the modified endogenous chromosome with a human sequence from the modified human chromosome; The resulting rodents contain consecutive human sequences of one or more human IGKV genes and one or more human IGKJ genes from the human chromosome 2 κ chain immunoglobulin locus at the endogenous light chain immunoglobulin locus. The animal in question is a mouse.

12. The method of claim 11, wherein the animal comprises all human IGKV genes and all human IGKJ genes at the human chromosome 2 κ chain immunoglobulin gene locus.

13. The method of claim 11, wherein the animal comprises an unmodified human sequence derived from a human light chain immunoglobulin locus, wherein the unmodified human sequence is at least 500 kb.

14. The method of claim 11, wherein the animal comprises disruption of its endogenous light chain immunoglobulin locus, the disruption comprising a deletion of at least 500 kb of endogenous sequence.

15. The method of claim 11, wherein the disruption of the endogenous light chain immunoglobulin locus in the animal comprises a deletion of the sequence from mouse IGKV2-137 to mouse IGKJ5.

16. The method of claim 11, wherein the animal comprises endogenous IGKC.

17. The method of claim 11, wherein the animal is homozygous for the light chain immunoglobulin locus.

18. The method of claim 11, wherein the animal is heterozygous for the light chain immunoglobulin locus.

19. The method of claim 11, wherein 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.

20. The method of claim 11, wherein the animal comprises disruption of its endogenous λ light chain immunoglobulin locus.

21. A method for producing genetically modified mice, the method comprising: The human chromosome is modified to produce a modified human chromosome containing a first exogenous recombination site and a second exogenous recombination site located on both sides of the variable region of the heavy chain immunoglobulin locus; said human chromosome 14. The endogenous chromosomes of mouse cells were modified to produce modified endogenous chromosomes containing a first exogenous recombination site and a second exogenous recombination site. The modified human chromosome was introduced into the cells of the mouse; as well as Inducing site-specific recombination between the modified human chromosome and the modified endogenous chromosome, thereby replacing the endogenous sequence in the modified endogenous chromosome with a human sequence from the modified human chromosome; The resulting mice contain, at the endogenous heavy chain immunoglobulin locus, a sequence of continuous sequences from the mouse IGHV1-85 gene to the mouse IGHJ4 gene, replaced by a sequence of one or more human IGHV, human IGHD, and human IGHJ genes at the human chromosome 14 heavy chain immunoglobulin locus, wherein the human IGHV, human IGHD, and human IGHJ genes are operatively linked to one or more genes selected from the endogenous IGHM, IGHδ, IGHG, IGHE, and IGHA genes.

22. A method for producing genetically modified mice, the method comprising: The human chromosome is modified to produce a modified human chromosome containing a first exogenous recombination site and a second exogenous recombination site located on both sides of the variable region of the light chain immunoglobulin gene locus, said human chromosome 2; The endogenous chromosomes of mouse cells were modified to produce modified endogenous chromosomes containing a first exogenous recombination site and a second exogenous recombination site. The modified human chromosome was introduced into the cells of the mouse; as well as Inducing site-specific recombination between the modified human chromosome and the modified endogenous chromosome, thereby replacing the endogenous sequence in the modified endogenous chromosome with a human sequence from the modified human chromosome; The resulting mice contain, at the endogenous light chain immunoglobulin locus, a sequence of consecutive sequences from mouse IGKV2-137 to mouse IGKJ5, replaced by consecutive human sequences of one or more human IGKV and human IGKJ genes at the human chromosome 2 κ chain immunoglobulin locus, wherein the human IGKV and human IGKJ genes are operatively linked to the endogenous IGKC gene.

23. The method of any one of claims 1-22, wherein the animal lacks an endogenous immunoglobulin heavy chain variable domain locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain.

24. The method of any one of claims 1-22, wherein the animal lacks an endogenous immunoglobulin light chain variable domain locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain.

25. The method of any one of claims 1-22, wherein the animal is capable of producing humanized antibodies.

26. A cell of an animal obtained by the method of any one of claims 1-25, said cell being incapable of developing into an individual.

27. A method for preparing an antibody that specifically binds to an antigen, the method comprising: Nucleic acid sequences encoding variable regions of human heavy chain and light chain immunoglobulins are obtained in cells, said cells expressing chimeric antibodies that specifically bind to said antigen, said cells being obtained by exposing an animal produced by the method of any one of claims 1-25 to said antigen; the nucleic acid encoding the variable region of human heavy chain immunoglobulin is operatively linked with a nucleic acid encoding the constant region of human heavy chain immunoglobulin, and the nucleic acid encoding the variable region of human light chain immunoglobulin is linked with a nucleic acid encoding the constant region of human light chain immunoglobulin; as well as The nucleic acid is expressed in cells to obtain the antibody.

28. A method for obtaining a sample, the method comprising: Animals produced by the method according to any one of claims 1-25 are exposed to the antigen; as well as The sample was collected from the animal.

29. The method of claim 28, wherein the sample is spleen tissue, spleen cells, or B cells.

30. A method for producing genetically modified non-human animals, the method comprising: Modify human chromosomes to produce modified human chromosomes containing a first exogenous recombination site and a second exogenous recombination site located on both sides of the variable region of the immunoglobulin locus; Modify the endogenous chromosomes of non-human animal cells to produce modified endogenous chromosomes containing a first exogenous recombination site and a second exogenous recombination site; The modified human chromosome was introduced into the animal cells; as well as Inducing site-specific recombination between the modified human chromosome and the modified endogenous chromosome, thereby replacing the endogenous sequence in the modified endogenous chromosome with a human sequence from the modified human chromosome; The animal in question is a mouse.

31. The method of claim 30, wherein, The human sequence is integrated into the endogenous chromosome in a one-step replacement via the recombination, and the human sequence is at least 500 kb.

32. A method for producing genetically modified non-human animals, the method comprising: The human chromosome is modified to produce a modified human chromosome containing a first exogenous recombination site and a second exogenous recombination site located on both sides of the variable region of the heavy chain immunoglobulin locus; said human chromosome 14. Modify the endogenous chromosomes of rodent cells to produce modified endogenous chromosomes containing a first exogenous recombination site and a second exogenous recombination site; The modified human chromosome was introduced into the cells of the animal; as well as Inducing site-specific recombination between the modified human chromosome and the modified endogenous chromosome, thereby replacing the endogenous sequence in the modified endogenous chromosome with a human sequence from the modified human chromosome; The endogenous heavy chain immunoglobulin locus of the animal comprises one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked and capable of VDJ rearrangement; wherein the endogenous heavy chain immunoglobulin locus comprises 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; The animal in question is a mouse.

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