Genetically modified non-human animals with humanized immunoglobulin loci

Abstract

The present disclosure relates to genetically modified animals and cells having a humanized lambda light chain immunoglobulin locus and / or a humanized heavy chain immunoglobulin locus. The disclosure also relates to genetically modified animals and cells having a modified locus comprising a human lambda light chain immunoglobulin gene and / or a humanized heavy chain immunoglobulin locus. In some embodiments, the animal also has a humanized kappa light chain immunoglobulin locus.

Description

Genetically modified non-human animals with humanized immunoglobulin loci Technical Field

[0001] This disclosure relates to genetically modified animals and cells having humanized light chain immunoglobulin loci and / or humanized heavy chain immunoglobulin loci. Background Technology

[0002] Therapeutic antibodies are among the fastest-growing classes of therapeutic compounds, developing faster than small molecule drugs. These therapeutic antibodies are typically human or humanized antibodies. Human or humanized antibodies can be generated by humanizing rodent antibodies (e.g., mouse antibodies) or by using phage libraries. Antibodies generated through these methods often have suboptimal binding affinity 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 often exhibit limited diversity and non-natural pairings of immunoglobulin heavy and light chains. Iterative and time-consuming experiments are usually required to improve these properties. Furthermore, in some cases, these antibodies may also induce immunogenicity in patients, causing their efficacy to diminish over time.

[0003] One possible method for generating fully human antibodies is to use transgenic animals engineered to express a human antibody library. The creation of transgenic animals (such as mice with different immunoglobulin loci) has enabled their use in a variety of research and development applications, such as drug discovery and basic research in various biological systems. Many early transgenic animals had incomplete human antibody libraries, resulting in lower-than-normal antibody production due to low V(D)J recombination efficiency, the potential introduction of immunogenic epitopes by endogenous antibody libraries, and various other problems.

[0004] Furthermore, the antibody light chain is encoded by one of two separate loci: κ and λ. Most mouse antibody light chains are κ-type. The ratio of κ to λ light chains used in humans is approximately 60:40. However, in mice, this ratio is approximately 95:5. It has been reported that the preferential use of the κ light chain in mice is preserved in genetically modified mice capable of fully or partially expressing human antibodies. Therefore, mice fully or partially expressing human antibodies show limited utilization of the λ variable region.

[0005] There is a need for efficient and cost-effective methods for generating human antibodies, and for non-human animals containing humanized immunoglobulin loci (including the humanized λ light chain immunoglobulin locus). These non-human animals should have the ability to respond to antigens to efficiently generate humanized antibodies. Summary of the Invention

[0006] This disclosure relates to genetically modified animals and / or cells having a humanized heavy chain immunoglobulin locus and a λ light chain immunoglobulin locus. This disclosure also relates to genetically modified animals and / or cells having a modified locus containing a human λ light chain immunoglobulin gene and / or a humanized heavy chain immunoglobulin locus. In some embodiments, the animals and / or cells also have a humanized κ light chain immunoglobulin locus. Animals can be immunized to produce fully human antibodies having a human heavy chain variable region and a κ or λ light chain variable region.

[0007] In one aspect, this disclosure relates to a genetically modified non-human animal that contains one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes at an endogenous λ light chain immunoglobulin locus. In some embodiments, the animal includes disruption of its endogenous λ light chain immunoglobulin locus. In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous λ light chain immunoglobulin locus includes the deletion of one or more mouse IGLV genes from Table 4, one or more IGLJ genes from Table 5, and / or one or more mouse IGLC genes from Table 6. In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous λ light chain immunoglobulin locus includes the deletion of a continuous sequence from mouse IGLV2 to mouse IGLC1.

[0008] In one aspect, this disclosure relates to a genetically modified non-human animal that contains one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes at an endogenous safe harbor locus, optionally wherein the animal contains disruption of its endogenous λ light chain immunoglobulin locus. In some embodiments, the endogenous safe harbor locus is an endogenous Hipp11 locus or an endogenous Rosa26 locus. In some embodiments, the animal is a mouse, and the one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes are inserted at the mouse Hipp11 locus.

[0009] In some embodiments, these human IGLV, IGLJ, and IGLC genes are operatively linked and can undergo rearrangement. In some embodiments, the animal comprises at least 50 human IGLV genes from Table 1, at least 5 human IGLJ genes from Table 2, and at least 5 human IGLC genes from Table 3. In some embodiments, the animal comprises all human IGLV, all human IGLJ, and all human IGLC genes from the endogenous λ light chain immunoglobulin locus on human chromosome 22 of a human subject. In some embodiments, the animal comprises all human IGLV, all human IGLJ, and all human IGLC genes from the endogenous λ light chain immunoglobulin locus on human chromosome 22 of human cells.

[0010] In some embodiments, the animal comprises an unmodified human sequence derived from the human λ light chain immunoglobulin locus, wherein the unmodified human sequence is at least 800 kb, at least 850 kb, at least 855 kb, at least 895 kb, at least 900 kb, at least 950 kb, or at least 1000 kb. In some embodiments, the animal comprises an unmodified sequence derived from the human λ light chain immunoglobulin locus from human IGLV(I)-70 to human IGLV3-1. In some embodiments, the animal comprises an unmodified sequence derived from the human λ light chain immunoglobulin locus from human IGLV(I)-70 to human IGLC7. In some embodiments, the animal is homozygous for the λ light chain immunoglobulin locus and / or the safe harbor locus. In some embodiments, the animal is heterozygous for the λ light chain immunoglobulin locus and / or the safe harbor locus. In some embodiments, the animal is a rodent (e.g., a mouse).

[0011] In some embodiments, the animal further comprises one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at the endogenous heavy chain immunoglobulin locus. In some embodiments, these human IGHV genes, human IGHD genes, and human IGHJ genes are operatively linked and capable of VDJ rearrangement. In some embodiments, the animal comprises at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9. In some embodiments, 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 in a human subject or cell. In some embodiments, the animal comprises disruption of its endogenous heavy chain immunoglobulin locus. In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous heavy chain immunoglobulin locus comprises the deletion of one or more mouse IGHV genes from Table 4, one or more mouse IGHD genes from Table 5, and / or one or more mouse IGHJ genes from Table 6. In some embodiments, the animal is a mouse, and the disruption of the endogenous heavy chain immunoglobulin locus in the animal comprises the deletion of a continuous sequence from mouse IGHV1-85 to mouse IGHJ4. In some embodiments, the animal comprises one or more endogenous IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus, and in some embodiments, the unmodified human sequence is at least 800 kb. In some embodiments, the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus, which in some embodiments has one of the following characteristics: (1) from human IGHV(III)-82 to human IGHV1-2; (2) from human IGHV(III)-82 to human IGHV6-1; (3) from human IGHD1-1 to human IGHJ6; and (4) from human IGHV(III)-82 to human IGHJ6. In some embodiments, the animal is homozygous or heterozygous for the heavy chain immunoglobulin locus.

[0012] In some embodiments, the animal further includes one or more human IGKV genes and one or more human IGKJ genes at the endogenous κ light chain immunoglobulin locus. In some embodiments, the animal includes all human IGKV genes listed in Table 13 and all human IGKJ genes listed in Table 14. In some embodiments, the animal includes all human IGKV genes and all human IGKJ genes at the endogenous κ light chain immunoglobulin locus on human chromosome 2 in a human subject or cell. In some embodiments, the animal includes disruption of its endogenous κ light chain immunoglobulin locus. In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous light chain immunoglobulin locus includes the deletion of one or more mouse IGKV genes listed in Table 15 and one or more mouse IGKJ genes listed in Table 16. In some embodiments, the animal is a mouse, and the disruption of the animal's endogenous κ light chain immunoglobulin locus includes the deletion of the sequence from mouse IGKV2-137 to mouse IGKJ5. In some embodiments, the animal comprises an unmodified sequence derived from the human κ light chain immunoglobulin locus, from human IGKV3D-7 to human IGKJ5. In some embodiments, the animal comprises endogenous IGKC. In some embodiments, the animal is homozygous or heterozygous for the κ light chain immunoglobulin locus.

[0013] In one aspect, this disclosure relates to a genetically modified non-human animal whose genome includes an endogenous light chain immunoglobulin locus comprising: replacing one or more endogenous IGLV, IGLJ, and IGLC genes with one or more human IGLV, IGLJ, and IGLC genes, wherein in some embodiments, the human IGLV, IGLJ, and IGLC genes are operatively linked. In some embodiments, one or more endogenous IGLV, IGLJ, and IGLC genes are replaced by all human IGLV genes in Table 1, all human IGLJ genes in Table 2, and all human IGLC genes in Table 3. In some embodiments, the animal is a mouse, and all mouse IGLV genes in Table 4, all mouse IGLJ genes in Table 5, and all mouse IGLC genes in Table 6 are replaced.

[0014] In one aspect, this disclosure relates to a genetically modified non-human animal whose genome includes an endogenous safe harbor locus comprising the insertion of one or more human IGLV, human IGLJ, and human IGLC genes, wherein the human IGLV, human IGLJ, and human IGLC genes are operatively linked, optionally wherein the animal includes disruption of its endogenous λ light chain immunoglobulin locus. In some embodiments, all human IGLV genes in Table 1, all human IGLJ genes in Table 2, and all human IGLC genes in Table 3 are inserted at the endogenous safe harbor locus. In some embodiments, the endogenous safe harbor locus is an endogenous Hipp11 locus or an endogenous Rosa26 locus.

[0015] In some embodiments, the animal lacks an endogenous immunoglobulin λ light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous λ light chain variable domain (e.g., mouse λ light chain variable domain). In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain (e.g., mouse heavy chain variable domain). In some embodiments, the animal lacks an endogenous immunoglobulin κ light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous κ light chain variable domain (e.g., mouse κ light chain variable domain).

[0016] In some implementations, the animal can produce humanized antibodies.

[0017] In one aspect, this disclosure relates to cells derived from animals as described herein. In some embodiments, the cell is a B cell expressing an immunoglobulin λ light chain derived from rearrangements of one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes. In some embodiments, the cell is a B cell expressing a chimeric immunoglobulin heavy chain comprising a variable domain of an immunoglobulin heavy chain derived from rearrangements of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein in some embodiments, the variable domain of the immunoglobulin heavy chain is operatively linked to a non-human heavy chain constant region. In some embodiments, the cell is a B cell expressing a chimeric immunoglobulin κ light chain comprising a variable domain of an immunoglobulin κ light chain derived from rearrangements of one or more human IGKV genes and one or more human IGKJ genes, wherein in some embodiments, the variable domain of the immunoglobulin κ light chain is operatively linked to a non-human κ light chain constant region. In some embodiments, the cell is an embryonic stem (ES) cell.

[0018] In one aspect, this disclosure relates to a method for preparing chimeric antibodies that specifically bind to an antigen, the method comprising exposing an animal described herein to the antigen; generating a hybridoma from cells collected from the animal; and collecting the chimeric antibodies generated from the hybridoma. In some embodiments, the method further includes sequencing the genome of the hybridoma.

[0019] In one aspect, this disclosure relates to a method for modifying the genome of a cell, the method comprising modifying one or more human chromosomes; introducing the modified one or more human chromosomes into cells of an animal; and inducing recombination between the modified one or more human chromosomes and one or more endogenous chromosomes, wherein in some embodiments, at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9 are integrated into one or more endogenous chromosomes (e.g., mouse chromosome 12) by recombination; and / or at least 50 human IGLV genes from Table 1, at least 5 human IGLJ genes from Table 2, and at least 5 human IGLC genes from Table 3 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 16 or 11) by recombination.

[0020] In one aspect, this disclosure relates to a method of modifying the genome of a cell, the method comprising modifying one or more human chromosomes; introducing the modified one or more human chromosomes into cells of an animal; and inducing recombination between the modified one or more human chromosomes and one or more endogenous chromosomes, wherein in some embodiments, at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9 are integrated into one or more endogenous chromosomes (e.g., mouse chromosome 12) by recombination; at least 50 human IGKV genes from Table 13 and at least 3 human IGKJ genes from Table 14 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 6); and / or at least 50 human IGLV genes from Table 1, at least 5 human IGLJ genes from Table 2, and at least 5 human IGLC genes from Table 3 are integrated into the one or more endogenous chromosomes (e.g., mouse chromosome 16 or 11) by recombination.

[0021] In one aspect, this disclosure relates to a method for preparing an antibody that specifically binds to an antigen, the method comprising: exposing the animal described herein to the antigen; sequencing nucleic acids encoding variable regions of human heavy chain and light chain immunoglobulins in cells expressing chimeric antibodies that specifically bind to the antigen; and expressing nucleic acids encoding variable regions and constant regions of human heavy chain immunoglobulins and human heavy chain immunoglobulins, as well as nucleic acids encoding variable regions and constant regions of human light chain immunoglobulins, in cells.

[0022] In one aspect, this disclosure relates to a method for preparing an antibody that specifically binds to an antigen, the method comprising obtaining a nucleic acid sequence encoding variable regions of human heavy chain and light chain immunoglobulins in cells expressing a chimeric antibody that specifically binds to an antigen, wherein in some embodiments the cells are obtained by exposing the animal described herein to the antigen; preparing a first nucleic acid, wherein in some embodiments the nucleic acid encoding the variable region of human heavy chain immunoglobulin is operatively linked in the first nucleic acid to a nucleic acid encoding a constant region of human heavy chain immunoglobulin; and preparing a second nucleic acid, wherein in some embodiments the nucleic acid encoding the variable region of human light chain immunoglobulin is operatively linked in the second nucleic acid to a nucleic acid encoding a constant region of human light chain immunoglobulin; and expressing the first and second nucleic acids in the cells to obtain the antibody.

[0023] In one aspect, this disclosure relates to a method for obtaining a nucleic acid encoding an antibody-binding domain that specifically binds to an antigen, the method comprising: exposing an animal described herein to an antigen; and sequencing a nucleic acid encoding variable regions of human heavy and light chain immunoglobulins in a cell expressing a chimeric antibody that specifically binds to the antigen.

[0024] In one aspect, this disclosure relates to a method for obtaining a sample, the method comprising: exposing an animal, as described herein, to an antigen; and collecting the sample from the animal. In some embodiments, the sample is spleen tissue, spleen cells, or B cells.

[0025] This disclosure also relates to the offspring of non-human mammals.

[0026] In some implementations, the non-human mammal is a rodent. In some implementations, the non-human mammal is a mouse.

[0027] This disclosure also provides cells comprising the targeting vectors described herein.

[0028] This disclosure also relates to cells (e.g., stem cells, embryonic stem cells, immune cells, B cells, T cells, or hybridomas) or cell lines, or primary cell cultures thereof, derived from non-human mammals or their offspring. This disclosure also relates to tissues, organs, or cultures thereof derived from non-human mammals or their offspring.

[0029] This disclosure also relates to the use of non-human mammals or their offspring, and animal models generated by the methods described herein, in the development of products related to immune processes, the preparation of human antibodies, or model systems for pharmacological, immunological, microbiological, and medical research.

[0030] Unless otherwise stated, 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 invention pertains. The methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0031] Other features and advantages of the invention will become clear from the detailed description, accompanying drawings, and claims below. Attached Figure Description

[0032] Figure 1 is a schematic diagram showing the human λ light chain immunoglobulin gene locus on chromosome 22.

[0033] Figure 2 is a schematic diagram showing the mouse λ light chain immunoglobulin gene locus on chromosome 16.

[0034] Figure 3 is a flowchart of the method for introducing immunoglobulin genes into the mouse genome.

[0035] Figure 4A illustrates a targeting strategy for modifying mouse heavy chain immunoglobulin loci.

[0036] Figure 4B illustrates a targeting strategy for modifying the mouse κ light chain immunoglobulin locus.

[0037] Figure 5 is a schematic diagram showing the mouse λ light chain immunoglobulin gene locus (not drawn to scale).

[0038] Figure 6A is a schematic diagram showing the mouse λ light chain immunoglobulin locus after two recombination sites were introduced into the genome.

[0039] Figure 6B is a schematic diagram showing the mouse Hipp11 locus after two recombination sites were introduced into the locus.

[0040] Figure 7A illustrates a targeting strategy using a targeting vector at the mouse λ light chain immunoglobulin gene locus on mouse chromosome 16. The targeting vector does not contain the mouse IGLV, IGLJ, or IGLC genes.

[0041] Figure 7B illustrates the targeting strategy using a targeting vector at the mouse Hipp11 locus on mouse chromosome 11. The mouse Hipp11 locus is located in chromosome A1, approximately 3 cM between the Eif4enif1 and Drg1 genes.

[0042] Figure 8 is a schematic diagram of human chromosome 22, highlighting the λ light chain immunoglobulin locus (not drawn to scale).

[0043] Figure 9 is a schematic diagram showing the modified human chromosome 22.

[0044] Figure 10 illustrates a targeting strategy using two targeting vectors at the human λ light chain immunoglobulin locus on human chromosome 22.

[0045] Figure 11A is a schematic diagram illustrating Cre-mediated recombination, in which a human IGL genomic DNA sequence is used to replace the corresponding genomic DNA sequence at a mouse locus.

[0046] Figure 11B is a schematic diagram illustrating Cre-mediated recombination, in which a human IGL genomic DNA sequence is used for insertion into a mouse locus.

[0047] Figures 12A to 12F show the PCR results of the recombinant cells. NC is the negative control. H2O is the water control.

[0048] Figure 13A is a fluorescence in situ hybridization (FISH) image. The arrow next to “2” indicates mouse chromosome 16. The arrow next to “1” indicates a human chromosome segment labeled with a human-specific IGL probe.

[0049] Figure 13B is a fluorescence in situ hybridization (FISH) image. The arrows indicate human chromosome segments labeled with human-specific IGL probes.

[0050] Figure 14 is a schematic diagram illustrating Flp-mediated recombination.

[0051] Figure 15 shows the Southern blot results of cells recombined with IGL-5', IGL-3', IGK, and IGH probes, respectively. WT is the wild-type control. hVH / hVK is the positive control.

[0052] Figure 16 shows hVH H / + / hVK H / + / hVL H / + PCR results for mice.

[0053] Figures 17A and 17B show the percentage of leukocyte subtypes in the spleen of hVH / hVK homozygous mice, hVH / hVK / hVL homozygous mice, and wild-type mice as determined by flow cytometry.

[0054] Figures 18A and 18B show the percentage of leukocyte subtypes in the lymph nodes of hVH / hVK homozygous mice, hVH / hVK / hVL homozygous mice, and wild-type mice, as determined by flow cytometry.

[0055] Figures 19A and 19B show the percentage of T cell subtypes in the peripheral blood of hVH / hVK homozygous mice, hVH / hVK / hVL homozygous mice, and wild-type mice as determined by flow cytometry. Detailed Implementation

[0056] Healthy mice tend to utilize the immunoglobulin κ light chain, while the utilization rate of the λ light chain is only 5%. In healthy humans, the utilization rate of the λ light chain is approximately 40%, significantly higher than in mice. In practice, λ light chain antibodies have also been found to have significant clinical value. For example, reports have concluded that antigens with limited epitopes often produce antibodies with limited light chain utilization, and that in most individuals, antibodies containing the λ light chain exhibit specificity that differs from and is complementary to that of κ-containing antibodies. It is estimated that approximately 24% of antibody drugs on the market contain the λ light chain.

[0057] This disclosure relates to genetically modified animals and / or cells, particularly those possessing a humanized λ light chain immunoglobulin locus or a modified locus containing a human λ light chain immunoglobulin gene. The animals may be immunized to produce antibodies containing human or humanized λ light chains.

[0058] Genetically modified animals can be prepared by introducing human immunoglobulin genes into the genome of non-human animals to produce animals that can express humanized or chimeric antibodies. Figure 3 illustrates a method for preparing humanized mice. In some embodiments, these methods first involve modifying the human immunoglobulin region on a human chromosome. The modified human chromosome is then introduced into mouse recipient cells. The human immunoglobulin variable region is then introduced into the corresponding region of the mouse genome by direct substitution (e.g., in a one-step substitution) or insertion into an endogenous safe harbor locus. Recipient cells are then screened, preferably cells that do not contain human chromosomes. The cells are then injected into blastocysts to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing the complete humanized immunoglobulin gene locus.

[0059] The genetically modified animals described herein possess various advantages. For example, in some cases, these genetically modified animals possess a complete human antibody library, particularly including a human antibody λ chain library. Therefore, the variable domains generated by these animals can exhibit a diversity very similar to that of human variable domains. Furthermore, because the entire sequence at the human immunoglobulin locus is introduced into the animal genome (unmodified or with limited modification), these genes can undergo V(D)J recombination in a manner very similar to that occurring in humans. Moreover, due to efficient V(D)J recombination, antibody production efficiency can be very high, exhibiting a rate similar to that of normal production. Furthermore, because V(D)J recombination can occur between endogenous IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, and / or IGLJ genes and human genes, if these genes are incorporated into rearranged heavy chain VDJ fragments or rearranged light chain VJ fragments, antibodies generated from the antibody library are likely to possess immunogenic epitopes in humans. Immunogenicity can lead to the production of anti-drug antibodies and may weaken therapeutic efficacy. Here, the endogenous IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, IGLJ, and / or IGLC genes have been effectively deleted. Antibodies generated from the antibody library are less likely to be immunogenic in humans. Therefore, these antibodies are more suitable for use as human therapeutic agents. Furthermore, since the human genome contains all IGLJ and IGLC genes located within the JC cluster (see Figure 1), replacing all endogenous IGLV, IGLJ, and IGLC genes with human IGLV, IGLJ, and IGLC genes, or inserting human IGLV, IGLJ, and IGLC genes at endogenous safe harbor loci in modified animals, ensures appropriate recombination to generate functional human λ light chains and avoids potential mismatches during recombination with endogenous IGLV, IGLJ, or IGLC genes.

[0060] Therefore, genetically modified animals provide a favorable platform for the production of humanized antibodies.

[0061] Compared to animals with humanized heavy chain immunoglobulin loci and humanized κ light chain immunoglobulin loci (e.g., hVH / hVK mice described herein), animals with humanized heavy chain immunoglobulin loci, humanized κ light chain immunoglobulin loci, and humanized λ chain immunoglobulin loci (e.g., hVH / hVK / hVL mice described herein), or animals with modified loci containing the human λ chain immunoglobulin gene (e.g., hVH / hVK / hVL mice(v) described herein), can produce fully human antibodies with both human heavy chain variable regions and λ light chain variable regions. Therefore, such animals can produce a greater diversity of antibodies for drug screening.

[0062] In some embodiments, this disclosure relates to animals (e.g., the hVH / hVL mice described herein) having a humanized heavy chain immunoglobulin locus and a humanized λ chain immunoglobulin locus, wherein the entire sequence of the immunoglobulin κ light chain variable region locus is knocked out. In some embodiments, this disclosure relates to animals (e.g., the hVH / hVL mice(v) described herein) having a humanized heavy chain immunoglobulin locus and a modified locus containing the human λ chain immunoglobulin gene, wherein the entire sequence of the immunoglobulin κ light chain variable region locus is knocked out. Such animals can more efficiently produce fully human antibodies with both the human heavy chain variable region and the λ light chain variable region.

[0063] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) linked together by disulfide bonds. Each heavy chain contains a heavy chain variable (VH) domain and a heavy chain constant region (CH). Each light chain contains a light chain variable (VL) domain and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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 with approximately 10-1 of affinity relative to its target epitope. -9 M or smaller (e.g., about or less than 1×10⁻⁶) -9 M, 1×10 -10 M, 1×10 -11 M or 1×10 -12 M) of K D Antibodies. In some implementations, K D It can be measured by surface plasmon resonance (e.g., BIACORE™ or ELISA).

[0064] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0065] As used herein, the term "human antibody" refers to an antibody encoded by nucleic acids present in humans (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies are collected from humans or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., mice) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0066] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). Non-limiting examples of chimeric antibodies are antibodies containing all or part of a variable domain sequence of a human antibody (e.g., all or part of a light chain and / or heavy chain variable domain sequence) and a constant domain of a non-human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0067] As used herein, the term "humanized antibody" refers to a non-human antibody containing a sequence derived from a non-human (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) capable of specifically binding to an antigen.

[0068] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals (human or non-human). This disclosure is intended for both veterinary and non-veterinary applications. A human patient can be an adult or adolescent (e.g., a person 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. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0069] As used herein, when referring to antibodies, the phrase "specific binding" means that, compared to other molecules, an antibody preferably interacts with its target molecule because the interaction depends on the presence of a specific structure on the target molecule (i.e., an antigenic determinant or epitope); in other words, the reagent recognizes and binds to molecules that include this specific structure, rather than all molecules as is typically the case. Antibodies that specifically bind to a target molecule can be called target-specific antibodies.

[0070] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to an amino acid polymer of any length containing at least two amino acids.

[0071] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and refer to a nucleotide polymer of any length consisting of at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modifications.

[0072] As used herein, the term "unmodified human sequence" refers to a sequence derived from a human subject, human cell, cultured human cell, or human cell line that is identical to the gene sequence of the human subject, human cell, cultured human cell, or human cell line.

[0073] Genetically modified mice possessing a humanized λ light chain immunoglobulin gene locus or a modified gene locus containing the human λ light chain immunoglobulin gene.

[0074] The λ light chain immunoglobulin locus (also known as IGL or immunoglobulin λ locus) is a region on a chromosome (e.g., human chromosome 22) containing the light chain gene for human antibodies (or immunoglobulins). Similarly, the immunoglobulin light chain gene can undergo a series of rearrangements, resulting in the production of mature immunoglobulin light chain nucleic acids (e.g., the λ light chain).

[0075] In healthy individuals, the total κ ​​to λ ratio in serum is approximately 2:1 (when measuring intact antibodies), or 1:1.5 if measuring free light chains. In mice, the total κ ​​to λ ratio is approximately 9:1. In some embodiments, the animal comprises a human or humanized λ light chain immunoglobulin locus. In some embodiments, the animal comprises a modified locus containing the human λ light chain immunoglobulin gene (e.g., a modified safe harbor locus).

[0076] The λ light chain immunoglobulin locus (also known as the IGL locus or immunoglobulin λ locus) is located on human chromosome 22 (Chr22q11.2). Table 1 lists the IGLV genes at this locus and their relative sequences. These genes and their sequences are also shown in Figure 1. These IGL genes on human chromosome 22 form three clusters: cluster A, cluster B, and cluster C (from proximal to distal). Specifically, in Figure 1, cluster A begins at ZNF280B and ends at an enhancer downstream of IGLC7; cluster B begins at IGLV5-52 and ends at IGLV7-35; and cluster C begins at BCRL2 and ends at VPREB1.

[0077] Table 1. List of IGLV genes on human chromosome 22

[0078] Table 2 lists all IGLJ genes on human chromosome 22 and their relative sequences. These genes and their sequences are also shown in Figure 1.

[0079] Table 2. List of IGLJ genes on human chromosome 22

[0080] Table 3 lists all immunoglobulin λ constant (IGLC) genes. These genes and their order are also shown in Figure 1. An enhancer is present downstream of IGLC7 at the human λ light chain immunoglobulin locus, and the relative order of the enhancers is 8 in Table 3. In some embodiments, the genetically modified non-human animals described herein contain this human enhancer at the endogenous λ light chain immunoglobulin locus or a modified locus described herein (e.g., a modified safe harbor locus).

[0081] Table 3. List of IGLC genes on human chromosome 22

[0082] The λ light chain immunoglobulin locus is located on mouse chromosome 16. The IGLV, IGLJ, and IGLC genes, along with their relative orders at this locus, are listed in Tables 4 through 6. These genes and their orders are also shown in Figure 2.

[0083] Table 4. List of IGLV genes on mouse chromosome 16

[0084] Table 5. List of IGLJ genes on mouse chromosome 16

[0085] Table 6. List of IGLC genes on mouse chromosome 16

[0086] Enhancers 2-4 and 3-1 are also located at the mouse λ light chain immunoglobulin locus. Further enhancers are present downstream of IGLC1 at the mouse λ light chain immunoglobulin locus. The relative order of these enhancers is shown in Figure 2. In some embodiments, the genetically modified non-human animals described herein do not contain one or more enhancers selected from the following: mouse enhancers downstream of IGLC1, enhancers 2-4, and enhancer 3-1.

[0087] This disclosure provides genetically modified nonhuman animals comprising one or more human IGLV genes, one or more human IGLJ genes, and / or one or more human IGLC genes. In some embodiments, these human IGLV genes, human IGLJ genes, and human IGLC genes are operatively linked together. In some embodiments, the human IGLV genes and human IGLJ genes may undergo VJ rearrangement. In some embodiments, these human IGLV genes, human IGLJ genes, and human IGLC genes are located at an endogenous λ light chain immunoglobulin locus. In some embodiments, these human IGLV genes, human IGLJ genes, and human IGLC genes are located at an endogenous safe harbor locus (e.g., an endogenous Hipp11 locus). In some embodiments, the animals described herein also include disruption of their endogenous λ light chain immunoglobulin locus.

[0088] In some embodiments, the animal contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 human IGLV genes (e.g., genes as shown in Table 1).

[0089] In some implementations, the animal contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes selected from IGLV(I)-70, IGLV4-69, IGLV(I)-68, IGLV10-67, IGLV(IV)-66-1, IGLV(V)-66, IGLV(IV)-65, IGLV(IV)-64, IGLV(I)-63 and IGLV1-62.

[0090] In some implementations, the animal contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes selected from IGLV3-10, IGLV3-9, IGLV2-8, IGLV3-7, IGLV3-6, IGLV2-5, IGLV3-4, IGLV4-3, IGLV3-2 and IGLV3-1.

[0091] In some embodiments, the animal contains about or at least 1, 2, 3, 4, 5, 6, or 7 human IGLJ genes (e.g., genes as shown in Table 2). In some embodiments, the animal contains 1, 2, 3, 4, 5, 6, or 7 human IGLJ genes selected from IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, and IGLJ7. In some embodiments, the animal contains human IGLJ4, IGLJ5, and IGLJ6 at the endogenous λ light chain immunoglobulin gene locus.

[0092] In some embodiments, the animal contains about or at least 1, 2, 3, 4, 5, 6, or 7 human IGLC genes (e.g., genes as shown in Table 3). In some embodiments, the animal contains 1, 2, 3, 4, 5, 6, or 7 human IGLC genes selected from IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, and IGLC7.

[0093] In some embodiments, the animal comprises an unmodified human sequence comprising a sequence that begins with a gene selected from IGLV(I)-70, IGLV4-69, IGLV(I)-68, IGLV10-67, IGLV(IV)-66-1, IGLV(V)-66, IGLV(IV)-65, IGLV(IV)-64, IGLV(I)-63, and IGLV1-62 and ends at a gene selected from IGLV3-10, IGLV3-9, IGLV2-8, IGLV3-7, IGLV3-6, IGLV2-5, IGLV3-4, IGLV4-3, IGLV3-2, and IGLV3-1. In some embodiments, the animal comprises an unmodified human sequence comprising a sequence that begins with a gene selected from IGLV(I)-70, IGLV4-69, IGLV(I)-68, IGLV10-67, IGLV(IV)-66-1, IGLV(V)-66, IGLV(IV)-65, IGLV(IV)-64, IGLV(I)-63, and IGLV1-62 and ends with a gene selected from IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, and IGLJ7. In some embodiments, the animal comprises an unmodified human sequence comprising a sequence that begins with a gene selected from IGLV(I)-70, IGLV4-69, IGLV(I)-68, IGLV10-67, IGLV(IV)-66-1, IGLV(V)-66, IGLV(IV)-65, IGLV(IV)-64, IGLV(I)-63, and IGLV1-62 and ends with a gene selected from IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, and IGLC7.

[0094] In some embodiments, the unmodified human sequence is derived from the human λ light chain immunoglobulin locus from human IGLV(I)-70 to human IGLC7. In some embodiments, the unmodified human sequence is derived from the human λ light chain immunoglobulin locus from human IGLV(I)-70 to human IGLC7. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGLV(I)-70 to the human enhancer immediately downstream of human IGLC7.

[0095] In some embodiments, the animal may have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences derived from the human λ chain immunoglobulin gene locus. In some embodiments, the unmodified human sequence has 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, 810 kb, 820 kb, 830 kb, 840 kb, or 850 kb. Lengths of 855kb, 860kb, 870kb, 880kb, 890kb, 895kb, 896kb, 900kb, 910kb, 920kb, 930kb, 940kb, 950kb, 960kb, 970kb, 980kb, 990kb, 1000kb, 1010kb, 1020kb, 1030kb, 1040kb, or 1050kb.

[0096] In some embodiments, the IGLV gene and / or the IGLJ gene are operatively linked together. VJ recombination can occur in these genes and produce functional antibodies. In some embodiments, these genes are arranged in a sequence similar to that in the human λ light chain immunoglobulin gene locus. This arrangement provides several advantages; for example, the arrangement of these genes allows for the generation of light chain variable domains with a diversity very similar to that of the λ light chain variable domains in humans. In some embodiments, the IGLV gene and / or the IGLJ gene are operatively linked together with an IGLC gene (e.g., the human IGLC gene).

[0097] In some embodiments, the animal is a mouse that does not contain enhancers 2-4 at the endogenous λ light chain immunoglobulin locus. In some embodiments, the animal is a mouse that contains enhancers 3-1 at the endogenous λ light chain immunoglobulin locus.

[0098] In some embodiments, the animal includes disruption of its endogenous λ light chain immunoglobulin gene locus. In some embodiments, disruption of the endogenous light chain immunoglobulin gene locus in the animal includes the deletion of one or more endogenous IGLV genes, one or more endogenous IGLJ genes, and / or one or more immunoglobulin λ constant (IGLC) genes.

[0099] In some embodiments, the animal is a mouse. Disruption of the endogenous λ light chain immunoglobulin locus in the animal includes the deletion of at least or about one, two, or three mouse IGLV genes (e.g., genes shown in Table 4). In some embodiments, the disruption includes the deletion of about or at least one, two, or three mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3. In some embodiments, the mouse also contains about or at least one, two, or three mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3.

[0100] In some embodiments, the disruption comprises the deletion of about or at least 1, 2, 3, 4, 5, or 6 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ2P, IGLJ3, IGLJ3P, and IGLJ4. In some embodiments, the disruption further comprises about or at least 1, 2, 3, 4, 5, or 6 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ2P, IGLJ3, IGLJ3P, and IGLJ4.

[0101] In some embodiments, the disruption comprises the deletion of about or at least one, two, three, or four mouse IGLC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption also comprises about or at least one, two, three, or four mouse IGLC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4.

[0102] In some embodiments, disruption of the endogenous λ light chain immunoglobulin locus in the animal includes deletions of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mouse IGLV, IGLJ, and IGLC genes. In some embodiments, the deletion includes about or at least 1, 2, 3, or 4 mouse IGKC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption includes deletions of about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3. In some embodiments, the disruption includes deletions of about or at least 1, 2, 3, 4, 5, or 6 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ2P, IGLJ3, IGLJ3P, and IGLJ4.

[0103] In some implementations, disruption of the animal's endogenous λ light chain immunoglobulin locus includes the 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 implementations, the endogenous λ light chain immunoglobulin gene in the animal is not disrupted.

[0104] In some implementations, the missing sequence begins from IGLV2 to IGLC1. In some implementations, the missing sequence begins from IGLV3 to IGLC1. In some implementations, the missing sequence begins from IGLJ2 ​​to IGLC1.

[0105] These human IGLV, IGLJ, and IGLC genes are operatively linked together and can undergo rearrangement. In some embodiments, the modified mice have a complete library of human IGLV, IGLJ, and IGLC genes (e.g., including all non-pseudohuman IGLV, IGLJ, and IGLC genes). Therefore, the modified mice can produce antibodies with a human λ light chain. In some embodiments, after recombination, a human IGLV gene (e.g., IGLV4-69, IGLV8-61, IGLV4-60, or IGLV6-57) and a human IGLJ gene (e.g., IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, or IGLJ7) constitute the sequence encoding the variable region of the antibody light chain. In some embodiments, after recombination, a human IGLC gene (e.g., IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, or IGLC7) constitutes the sequence encoding the constant region of the antibody light chain. In addition, in some cases, the entire mouse IGLV, IGLJ, and IGLC genes (all non-pseudogenes) are knocked out, so that the light chain does not contain any sequences encoded by mouse-derived sequences, thereby minimizing immunogenicity in humans.

[0106] In some embodiments, the human cluster A IGL gene is included in the modified chromosome. In some embodiments, the human cluster B IGL gene is included in the modified chromosome. In some embodiments, the human cluster C IGL gene is included in the modified chromosome. In some embodiments, at least one, at least two, or at least three of the human clusters A to C genes are included in the modified chromosome.

[0107] In some implementations, the human IGLV gene, human IGLJ gene, and human IGLC gene are inserted into one or more endogenous "safe harbor" loci (e.g., endogenous Hipp11 locus or endogenous Rosa26 locus).

[0108] Genetically modified heavy chain immunoglobulin locus

[0109] The heavy chain immunoglobulin locus (also known as IGH or immunoglobulin heavy chain locus) is a region on a chromosome (e.g., human chromosome 14) that contains the heavy chain gene for human antibodies (or immunoglobulins).

[0110] This region represents the germline organization of the heavy chain locus. This locus includes V (variable), D (diversity), J (connection), and C (constant) segments. Genes in the V region form the V gene cluster (also known as the IGHV gene cluster). Genes in the D region form the D gene cluster (also known as the IGHD gene cluster). Genes in the J region form the J gene cluster (also known as the IGHJ gene cluster).

[0111] During B cell development, DNA-level recombination events link a single D fragment (also known as the IGHD gene) to a J fragment (also known as the IGHJ gene); then, the fused DJ exon of this partially rearranged DJ region is linked to a V fragment (also known as the IGHV gene). The rearranged VDJ region containing the fused VDJ exon is then transcribed at the RNA level and fused into the IGHM constant region; this transcript encodes the μ heavy chain. Later in development, B cells produce VDJ-Cμ-Cδ premessenger RNA, which is selectively spliced ​​to encode either the μ or δ heavy chain. Mature B cells in lymph nodes undergo conversion recombination, resulting in a fused VDJ gene fragment adjacent to one of the IGHG, IGHA, or IGHE gene fragments, with each cell expressing the γ, α, or ε heavy chain. The potential recombination of many different IGHV genes with several IGHJ genes provides broad antigen recognition. Additional diversity is gained through the linker diversity resulting from the random addition of nucleotides by terminal deoxynucleotidyl transferase and somatic hypermutations occurring during B cell maturation in the spleen and lymph nodes. Several V, D, J, and C fragments are known to not encode proteins and are considered pseudogene fragments (often simply referred to as pseudogenes).

[0112] The human heavy chain immunoglobulin locus is located on human chromosome 14. Table 7 lists the IGHV genes at this locus and their relative sequences.

[0113] Table 7. List of IGHV genes on human chromosome 14

[0114] RPS8P1, ADAM6, and KIAA0125 are also located at this locus. The relative sequence of RPS8P1 is 160, that of ADAM6 is 161, and that of KIAA0125 is 164. Table 8 lists all IGHD genes on human chromosome 14 and their relative sequences. Table 9 lists all IGHJ genes on human chromosome 14 and their relative sequences. Genes containing the immunoglobulin constant domain are located after the IGHV, IGHD, and IGHJ genes. These genes include (in the following order): immunoglobulin weight constant μ (IGHM), immunoglobulin weight constant δ (IGHδ), immunoglobulin weight constant γ3 (IGHG3), immunoglobulin weight constant γ1 (IGHG1), immunoglobulin weight constant εP1 (pseudogene) (IGHEP1), immunoglobulin weight constant α1 (IGHA1), immunoglobulin weight constant γP (nonfunctional) (IGHGP), immunoglobulin weight constant γ2 (IGHG2), immunoglobulin weight constant γ4 (IGHG4), immunoglobulin weight constant ε (IGHE), and immunoglobulin weight constant α2 (IGHA2).

[0115] Table 8. List of IGHD genes on human chromosome 14

[0116] Table 9. List of IGHJ genes on human chromosome 14

[0117] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12. Table 10 lists the IGHV genes at this locus and their relative sequences.

[0118] Table 10. List of IGHV genes on mouse chromosome 12

[0119] Table 11 lists all IGHD genes on mouse chromosome 12 and their relative order. Table 12 lists all IGHJ genes on mouse chromosome 12 and their relative order. Genes of the immunoglobulin constant domain follow the IGHV, IGHD, and IGHJ genes. These genes include (in the following order): immunoglobulin weight constant μ (IGHM), immunoglobulin weight constant δ (IGHδ), immunoglobulin weight constant γ3 (IGHG3), immunoglobulin weight constant γ1 (IGHG1), immunoglobulin weight constant γ2b (IGHG2b), immunoglobulin weight constant γ2a (IGHG2a), immunoglobulin weight constant ε (IGHE), and immunoglobulin weight constant α (IGHA) genes.

[0120] Table 11. List of IGHD genes on mouse chromosome 12

[0121] Table 12. List of IGHJ genes on mouse chromosome 12

[0122] This disclosure provides genetically modified non-human animals comprising one or more human IGHV genes, one or more human IGHD genes, and / or one or more human IGHJ genes. In some embodiments, these human IGHV genes, human IGHD genes, and human IGHJ genes are operatively linked together and are capable of VDJ rearrangement. In some embodiments, these human IGHV genes, human IGHD genes, and human IGHJ genes are located at endogenous heavy chain immunoglobulin loci.

[0123] In some embodiments, the animal contains 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, or 161 individual IGHV genes (e.g., genes as shown in Table 7).

[0124] In some implementations, the animal contains 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.

[0125] In some implementations, the animal contains 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.

[0126] In some embodiments, the animal comprises an unmodified human sequence comprising a sequence that begins with a gene selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75 and ends with a gene selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV1-2. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV(II)-1-1. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV-6-1.

[0127] In some embodiments, the animal contains about 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 individual IGHD genes (e.g., genes as shown in Table 8). In some embodiments, the animal contains 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.

[0128] In some embodiments, the animal contains about or at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes (e.g., genes as shown in Table 9). In some embodiments, the animal contains 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.

[0129] In some embodiments, the animal comprises an unmodified human sequence comprising a sequence beginning with a gene selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27 and ending at a gene selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6. In some embodiments, the unmodified human sequence originates from the human heavy chain immunoglobulin locus from human IGHD1-1 to human IGHJ6.

[0130] In some implementations, the unmodified human sequence is derived from the human heavy chain immunoglobulin loci from human IGHD1-1 to human IGHD7-27.

[0131] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin loci from human IGHJ1P to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin loci from human IGHJ1 to human IGHJ6.

[0132] In some implementations, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHJ6.

[0133] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV1-2 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(II)-1-1 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV6-1 to human IGHJ6.

[0134] In some embodiments, the animal may have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the unmodified human sequence has a length of about 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.

[0135] In some embodiments, the animal contains one or more endogenous genes selected from the group consisting of: immunoglobulin weight constant μ (IGHM), immunoglobulin weight constant δ (IGHδ), immunoglobulin weight constant γ3 (IGHG3), immunoglobulin weight constant γ1 (IGHG1), immunoglobulin weight constant γ2b (IGHG2b), immunoglobulin weight constant γ2a (IGHG2a), immunoglobulin weight constant ε (IGHE), and immunoglobulin weight constant α (IGHA) genes. In some embodiments, these endogenous genes are operatively linked together. In some embodiments, these endogenous genes have the same sequence as those in wild-type animals. In some embodiments, isotype switching (immunoglobulin class switching) may occur in the animal.

[0136] In some embodiments, the IGHV, IGHD, and / or IGHJ genes are operatively linked together. VDJ recombination can occur in these genes and produce functional antibodies. In some embodiments, these genes are arranged in a sequence similar to that in human heavy chain immunoglobulin loci. This arrangement provides several advantages; for example, the arrangement of these genes allows for the generation of heavy chain variable domains with a diversity very similar to that of heavy chain variable domains in humans. Because some random sequences may be inserted into the sequence during VDJ recombination, in some embodiments, a complete human antibody library with no or minimal modifications reduces the likelihood of non-human sequences being inserted during VDJ recombination.

[0137] In some implementations, the IGHV gene, IGHD gene, and / or IGHJ gene are operatively linked together with one or more genes (e.g., all genes) selected from the IGHM gene, IGHδ gene, IGHG3 gene, IGHG1 gene, IGHG2b gene, IGHG2a gene, IGHE gene, and IGHA gene.

[0138] In some embodiments, the animal includes disruption of its endogenous heavy chain immunoglobulin gene loci. In some embodiments, the disruption of the animal's endogenous heavy chain immunoglobulin gene loci includes the deletion of one or more endogenous IGHV genes, one or more endogenous IGHD genes, and one or more endogenous IGHJ genes.

[0139] In some implementations, the animal is a mouse. Disruption of the endogenous heavy chain immunoglobulin loci in the animal includes the 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 shown in Table 10). In some implementations, the disruption includes the 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 also contains 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).

[0140] In some embodiments, the disruption comprises the 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 also comprises the 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.

[0141] In some embodiments, disruption of the endogenous heavy chain immunoglobulin loci in the animal includes the deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mouse IGHD genes (e.g., genes shown in Table 11). In some embodiments, the disruption includes the deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1. In some embodiments, the mouse also contains about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2 and IGHD4-1.

[0142] In some embodiments, the disruption comprises the deletion of about or at least one, two, three, or four mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4. In some embodiments, the mouse also contains about or at least one, two, three, or four mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.

[0143] In some implementations, disruption of the animal's endogenous heavy chain immunoglobulin locus includes the deletion of approximately 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.

[0144] In some implementations, the missing sequence begins from IGHV1-86 to IGHJ4, from IGHV1-85 to IGHJ4, from IGHV1-84 to IGHJ4, from IGHV1-83 to IGHJ4, or from IGHV1-82 to IGHJ4 (e.g., from IGHV1-85 to IGHJ4).

[0145] In some embodiments, the animal contains 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 sequences in the human heavy chain immunoglobulin locus. In some embodiments, the 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, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb. In some embodiments, the sequence begins with human IGHV(III)-82 to IGHV1-2. In some embodiments, the sequence begins with human IGHV7-81 to IGHV1-2. In some embodiments, the sequence begins with human IGHV(II)-1-1 to IGHVJ6. In some implementations, the sequence begins with human IGHV6-1 and extends to IGHVJ6.

[0146] These human IGHV, IGHD, and IGHJ genes are operatively linked together and can undergo VDJ rearrangement. In some embodiments, the modified mice possess a complete library of human IGHV, IGHD, and IGHJ genes (e.g., including all non-pseudohuman IGHV, IGHD, and IGHJ genes). Therefore, the modified mice can generate a complete human antibody library. In some embodiments, after VDJ recombination, one IGHV gene (e.g., IGHV3-21 or IGHV3-74) constitutes the sequence encoding the variable region of the antibody heavy chain. One IGHD gene constitutes the sequence encoding the variable region of the antibody heavy chain. And one IGHJ gene constitutes the sequence encoding the variable region of the antibody heavy chain. In some embodiments, the IGHV gene is IGHV3-21 or IGHV3-74.

[0147] In some embodiments, an IGHV gene (e.g., IGHV3-30, IGHV3-33, IGHV4-39, or IGHV4-34) constitutes the sequence encoding the variable region of the antibody heavy chain. An IGHD gene (e.g., IGHD6-19) constitutes the sequence encoding the variable region of the antibody heavy chain. And an IGHJ gene (e.g., IGHJ4 or IGHJ6) constitutes the sequence encoding the variable region of the antibody heavy chain. In some embodiments, an IGKV gene (e.g., IGKV4-1, IGKV1-33, IGKV2-30) constitutes the sequence encoding the variable region of the antibody light chain. An IGKJ gene (e.g., IGKJ1, IGKJ2, or IGKJ4) constitutes the sequence encoding the variable region of the antibody light chain.

[0148] In addition, in some cases, all mouse IGHV, IGHD, and IGHJ genes (e.g., including all non-pseudogenes) are knocked out, so that the heavy chain variable region does not contain any sequence encoded by mouse-derived sequences, thereby minimizing human immunogenicity.

[0149] Genetically modified κ light chain immunoglobulin locus

[0150] The κ chain immunoglobulin locus (also known as IGK or immunoglobulin κ locus) is a region on a chromosome (e.g., human chromosome 2) containing the human antibody (or immunoglobulin) κ light chain gene. Similarly, the immunoglobulin κ light chain gene can undergo a series of rearrangements to produce mature immunoglobulin κ light chain nucleic acid (e.g., the κ chain).

[0151] The joining of the V segment (also known as the IGKV gene) and the J segment (also known as the IGKJ gene) produces a continuous exon encoding the entire κ light chain variable domain. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far from the C region. The J gene segment (or IGKJ gene cluster) is located close to the C region. The joining of the V and J gene segments also brings the V gene closer to the C region sequence. The rearranged V region J gene segment is separated from the C region sequence only by introns. To prepare the complete immunoglobulin κ light chain messenger RNA, the V region exons are joined to the C region sequence via RNA splicing post-transcriptionally.

[0152] The human κ light chain immunoglobulin locus is located on human chromosome 2. Table 13 lists the IGKV genes at this locus and their relative sequences. There are several distinct groups of human IGKV genes, including IGKV1 genes (including all IGKV genes starting with IGKV1, also known as VκI), IGKV2 genes (including all IGKV genes starting with IGKV2, also known as VκII), IGKV3 genes (including all IGKV genes starting with IGKV3, also known as VκIII), IGKV4 genes (including all IGKV genes starting with IGKV4, also known as VκIV), IGKV5 genes (including all IGKV genes starting with IGKV5, also known as VκV), IGKV6 genes (including all IGKV genes starting with IGKV6, also known as VκVI), and IGKV7 genes (including all IGKV genes starting with IGKV7, also known as VκVII).

[0153] These IGKV genes on human chromosome 2 also form two clusters: the proximal Vκ cluster and the distal Vκ cluster. The sequences in the two clusters are similar but not identical. Large repetitions of these sequences have occurred since the divergence of the human lineage from its most recent common ancestor with other apes.

[0154] Table 13. List of IGKV genes on human chromosome 2

[0155] Table 14 lists all IGKJ genes on human chromosome 2 and their relative sequences. The immunoglobulin κ constant (IGKC) gene, which encodes the κ light chain immunoglobulin constant domain, is located after the IGKV and IGKJ genes.

[0156] Table 14. List of IGKJ genes on human chromosome 2

[0157] The mouse κ light chain immunoglobulin locus is located on mouse chromosome 6. Table 15 lists the IGKV genes at this locus and their relative sequences.

[0158] Table 15. List of IGKV genes on mouse chromosome 6

[0159] Gm9728 and Amd-ps2 are also located at this locus. The relative sequence of Gm9728 is 4, and the relative sequence of Amd-ps2 is 134. Table 16 lists all IGKJ genes on mouse chromosome 6 and their relative sequences. The IGKC gene, encoding the constant domain of the κ light chain immunoglobulin, is located after the IGKV and IGKJ genes.

[0160] Table 16. List of IGKJ genes on mouse chromosome 6

[0161] This disclosure provides genetically modified non-human animals comprising one or more human IGKV genes and / or one or more human IGKJ genes. In some embodiments, the human IGKV and human IGKJ genes are operatively linked together and are capable of undergoing VJ rearrangement. In some embodiments, the human IGKV and human IGKJ genes are located at the endogenous κ light chain immunoglobulin locus.

[0162] In some implementations, the animal contains about 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 13).

[0163] In some implementations, the animal contains 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.

[0164] In some implementations, the animal contains 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.

[0165] In some embodiments, the animal contains about or at least 1, 2, 3, 4, or 5 human IGKJ genes (e.g., genes shown in Table 14). In some embodiments, the animal contains 1, 2, 3, 4, or 5 human IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.

[0166] In some embodiments, the animal contains endogenous IGKC. In some embodiments, the IGKV and / or IGKJ genes are operatively linked together. VJ recombination can occur in these genes and produce functional antibodies. In some embodiments, these genes are arranged in a sequence similar to that in the human κ light chain immunoglobulin gene locus. This arrangement provides several advantages; for example, the arrangement of these genes allows for the generation of κ light chain variable domains with a diversity very similar to that of the κ light chain variable domains in humans.

[0167] In some implementations, the IGKV gene and / or IGKJ gene are operatively linked together with the IGKC gene (e.g., the endogenous IGKC gene).

[0168] In some embodiments, the animal includes disruption of its endogenous κ light chain immunoglobulin gene locus. In some embodiments, disruption of the endogenous κ light chain immunoglobulin gene locus in the animal includes the deletion of one or more endogenous IGKV genes and one or more endogenous IGKJ genes.

[0169] In some implementations, the animal is a mouse. Disruption of the endogenous heavy chain immunoglobulin loci in the animal includes the 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 shown in Table 15). In some implementations, the disruption includes the 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 also contains 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.

[0170] In some embodiments, the disruption comprises the 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 also 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.

[0171] In some embodiments, the disruption comprises the deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5. In some embodiments, the mouse also contains about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5).

[0172] In some implementations, disruption of the animal's endogenous κ light chain immunoglobulin locus includes the deletion of an endogenous sequence 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.

[0173] In some implementations, the missing sequence begins from IGKV2-137 to IGKJ4, from IGKV1-136 to IGKJ4, from IGKV1-135 to IGKJ4, from IGKV2-137 to IGKJ5, from IGKV1-136 to IGKJ5, or from IGKV1-135 to IGKJ5 (e.g., from IGKV2-137 to IGKJ5).

[0174] In some embodiments, the animal contains 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 sequences in the human κ light chain immunoglobulin locus. In some implementations, the sequence has a length of about or at least 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 200kb, 300kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1000kb, 1500kb, 2000kb, 2500kb, 3000kb, or 3500kb.

[0175] In some embodiments, the animal may have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the unmodified human sequence has a length of about 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.

[0176] In some embodiments, the sequence begins with human IGKV3D-7 to IGKJ5. In some embodiments, the sequence begins with human IGKV3D-7 to IGKJ4. In some embodiments, the sequence begins with human IGKV1D-8 to IGKJ5. In some embodiments, the sequence begins with human IGKV1D-8 to IGKJ4.

[0177] The human IGKV gene and the human IGKJ gene are operatively linked together and can undergo VJ rearrangement. In some embodiments, the modified mouse has a complete human IGKV and IGKJ gene library (e.g., including all non-pseudohuman IGKV and IGKJ genes). Therefore, the modified mouse can generate a complete human antibody library. In some embodiments, after VJ recombination, one IGKV gene (e.g., IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12) constitutes the sequence encoding the antibody κ light chain variable region. A human IGKJ gene constitutes the sequence encoding the antibody κ light chain variable region. In some embodiments, the IGKV gene is IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12. Furthermore, in some cases, the entire mouse IGKV and IGKJ genes (all non-pseudogenes) are knocked out, so that the heavy chain variable region does not contain any sequences encoded by mouse-derived sequences, thereby minimizing immunogenicity in humans.

[0178] In some embodiments, the human proximal Vκ cluster IGKV gene is included in the modified chromosome. In some embodiments, the human distal Vκ cluster IGKV gene is included in the modified chromosome. In some embodiments, both the human proximal Vκ cluster IGKV gene and the human distal Vκ cluster IGKV gene are included in the modified chromosome.

[0179] Genetically modified animals

[0180] In one aspect, this disclosure provides genetically modified non-human animals comprising a humanized heavy chain immunoglobulin locus, a humanized λ light chain immunoglobulin locus, and / or a humanized κ light chain immunoglobulin locus. In another aspect, this disclosure provides genetically modified non-human animals comprising a humanized heavy chain immunoglobulin locus, a modified locus containing a human λ light chain immunoglobulin gene, and / or a humanized κ light chain immunoglobulin locus. In some embodiments, the animal comprises one or more human IGHV genes, one or more human IGHD genes, one or more human IGHJ genes, one or more human IGLV genes, one or more human IGLJ genes, and / or one or more human IGLC genes. In some embodiments, the animal also comprises one or more human IGKV genes and one or more human IGKJ genes. In some embodiments, these genes are located at endogenous immunoglobulin loci. In some embodiments, the animal comprises one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes at a safe harbor locus (e.g., an endogenous Hipp11 locus or an endogenous Rosa26 locus).

[0181] In some embodiments, the animal contains a human or humanized λ light chain immunoglobulin locus. In some embodiments, the animal contains disruption of its endogenous λ light chain immunoglobulin locus. In some embodiments, the animal does not have disruption of its endogenous λ light chain immunoglobulin locus. In some embodiments, the animal contains a human or humanized κ light chain immunoglobulin locus. In some embodiments, the animal contains disruption of its endogenous κ light chain immunoglobulin locus. In some embodiments, the animal does not have disruption of its endogenous κ light chain immunoglobulin locus. In some embodiments, the animal contains one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes at an endogenous safe harbor locus (e.g., endogenous Hipp11 locus, endogenous Rosa26 locus, or endogenous COL1A1 locus). In some embodiments, the animal contains disruption of its safe harbor locus. In some embodiments, the animal does not have disruption of its safe harbor locus.

[0182] Theoretically, the safe harbor loci described in this paper (which are intragenic or extragenic regions of the mouse genome) can adapt to predictable expression of newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbor allows sufficient transgene expression to produce the desired level of encoded protein. A safe harbor may also not predispose cells to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it ideally needs to meet the following criteria, including but not limited to: the absence of regulatory elements or gene disruption as determined by sequence annotation; being an intergenetic region within a densely gene-rich area, or a convergence point between two genes transcribed in opposite directions; maintaining distance to minimize the possibility of long-distance interactions between the encoded transcriptional activator and the promoters of adjacent genes (particularly cancer-related genes and microRNA genes); and exhibiting clearly ubiquitous transcriptional activity, as reflected by a broad spatial and temporal expression sequence tag (EST) pattern. Within regions suitable for exogenous insertion, the precise locus selected for insertion should be free of repetitive elements and conserved sequences, and primers for amplifying homologous arms should be readily designable.

[0183] Suitable sites for genome editing or specific targeted integration include, but are not limited to, the Hipp11 locus, Rosa26 locus, COL1A1 locus, Porr2a locus, and AAVS1 locus. Details of safe harbor loci can be found, for example, Ma, X. et al., “Validation of reliable safe harbor locus for efficient porcine transgenesis.” Functional & Integrative Genomics 22.4 (2022): 553-563, the full text of which is incorporated herein by reference.

[0184] The Hipp11 (H11) locus, located between the DRG1 and EIF4ENIF1 genes on mouse chromosome 11, offers significant potential for stable gene knock-in and high-level expression. The robust and ubiquitous function of H11 has been demonstrated in mice, pigs, human embryonic stem cells (hES), and induced pluripotent stem cells (iPS). Details of the Hipp11 locus can be found, for example, in Zhu, F. et al., “DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.” Nucleic Acids Research 42.5 (2014): e34-e34, which is incorporated herein by reference in its entirety.

[0185] The Rosa26 locus is a non-coding gene consisting of three exons on mouse chromosome 6, a region readily fertile for gene insertion. No functional proteins are known to encode Rosa26. Furthermore, the Rosa26 locus facilitates homologous recombination (HR), maintaining protein expression levels of gene constructs inserted into this region without affecting the expression or function of other endogenous genes. Given the widespread expression of the Rosa26 region across all cell types and developmental stages, it is commonly used as a safe site for gene targeting in mouse models. Details of the Rosa26 locus can be found, for example, Friedrich, G. et al., “Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.” Genes & Development 5.9 (1991): 1513-1523, the full text of which is incorporated herein by reference.

[0186] Genetically modified non-human animals can also be various animals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals, where suitable genetically modified embryonic stem (ES) cells are not readily available, other methods can be used to prepare non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent cells) and transferring the modified genome into suitable cells (e.g., oocytes) using nuclear transfer, and gestating the modified cells (e.g., modified oocytes) in the non-human animal under suitable conditions to form an embryo. These methods are known in the art and described, for example, in A. Nagy et al., “Manipulating the MouseEmbryo: A Laboratory Manual (Third Edition)”, Cold Spring Harbor Laboratory Press, 2006, the full text of which is incorporated herein by reference. Therefore, in various embodiments, the human V, D, and / or J fragments (e.g., any corresponding IGH, IGK, or IGL gene fragments described herein) can be operatively linked to non-human animal (e.g., rodent, mouse, rat, hamster) constant region gene sequences. In some embodiments, the human V, D, and / or J fragments (e.g., any corresponding IGH, IGK, or IGL gene fragments described herein) can be operatively linked to human constant region gene sequences. In some embodiments, the human IGLV, IGLJ, and IGLC fragments are operatively linked, which allows for rearrangement of the IGLV and IGLJ fragments. During B cell development, these rearranged human V, D, and / or J fragments can be linked to non-human animal immunoglobulin constant regions or human immunoglobulin constant regions.

[0187] In one embodiment, the animal is a mammal, such as a mammal belonging to the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent may be selected from mice, rats, and hamsters. In some implementations, the genetically modified animals are selected from the following families: Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rats and mice, vole), Muridae (true mice and rat, gerbil, spiny mouse, crested rat), Nesomyidae (climbing mice, rock mice, with-tailed rat, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rate, bamboo rat, and zokor). In some embodiments, the genetically modified rodent is selected from true mice or rats (Muspidae), gerbils, spiny mice, and crested rats. In some embodiments, the non-human animal is a mouse.

[0188] In some implementations, the animal is a C57 background mouse (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 implementations, the mouse is a 129 strain selected from the group consisting of the following strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); and Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference. In some embodiments, the genetically modified mouse is a mixture of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a mixture of the 129 strain or a mixture of the BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a mixture of the BALB strain and another strain. In some implementations, the mouse is derived from a heterozygous line (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129).

[0189] In some embodiments, the animal is a rat. The rat may be selected from Wistar rats, the LEA strain, the Sprague Dawley strain, the Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0190] The animal may have one or more other genetic modifications and / or other modifications suitable for the specific purpose of producing a humanized animal.

[0191] Genetically modified nonhuman animals comprising modifications to endogenous nonhuman immunoglobulin loci. In some embodiments, the modification may comprise a human nucleic acid sequence encoding at least a portion of a human protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human heavy chain variable domain or light chain variable domain sequence). Although genetically modified cells (e.g., ES cells, somatic cells) that may comprise the modifications described herein are also provided, in many embodiments, the genetically modified nonhuman animals comprise modifications to endogenous loci in an animal strain.

[0192] Genetically modified animals can express humanized antibodies and / or chimeric antibodies derived 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, IGKJ, IGLV, IGLJ, and / or IGLC genes). In various embodiments, the endogenous non-human immunoglobulin loci are modified, in whole or in part, to contain human nucleic acid sequences. In some embodiments, the human nucleic acid sequence is linked to the endogenous IGL locus via an upstream linker site (having a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5) and a downstream linker site (having a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6). In some embodiments, integration of a human nucleic acid sequence (e.g., any human nucleic acid sequence described herein) can be verified by sequencing using PCR primer pairs SEQ ID NO: 1 and SEQ ID NO: 2; or SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, integration of a human nucleic acid sequence (e.g., any human nucleic acid sequence described herein) can be verified by Southern blotting using an IGL-5' probe (synthesized from primer pairs SEQ ID NO: 7 and SEQ ID NO: 8) or an IGL-3' probe (synthesized from primer pairs SEQ ID NO: 9 and SEQ ID NO: 10).

[0193] Genetically modified animals can express humanized antibodies and / or chimeric antibodies derived from an endogenous mouse locus, 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 gene, IGHD gene, IGHJ gene, IGKV gene, and / or IGKJ gene), and one or more human λ light chain 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 λ light chain immunoglobulin gene sequences (e.g., IGLV gene, IGLJ gene, and / or IGLC gene) are inserted into the endogenous locus. In some embodiments, the endogenous locus is a safe harbor locus (e.g., any safe harbor locus described herein). In some embodiments, a human nucleic acid sequence is linked to an endogenous locus (e.g., any safe harbor locus described herein) via an upstream linker site (having at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 24) and a downstream linker site (having at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 25). In some embodiments, integration of the human nucleic acid sequence (e.g., any human nucleic acid sequence described herein) can be verified by sequencing (e.g., by PCR). In some embodiments, integration of the human nucleic acid sequence (e.g., any human nucleic acid sequence described herein) can be verified by Southern blotting.

[0194] Genetic, molecular, and behavioral analyses can be performed on the aforementioned non-human mammals. This disclosure also relates to offspring produced by mating a non-human mammal provided herein with a genotype of the same or other. 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.

[0195] This disclosure also provides cell lines or primary cell cultures derived from non-human mammals or their offspring. For example, cell culture-based models can be prepared by methods such as: Cell cultures can be obtained by isolating cells from non-human mammals; alternatively, cells can be obtained from cell cultures established using the same constructs and standard cell transfection techniques. Integration of genetic constructs containing DNA sequences encoding human or humanized immunoglobulins can be detected by various methods.

[0196] Numerous analytical methods are available for detecting modifications on exogenous DNA or genomic DNA, including nucleic acid-level methods (such as mRNA quantification using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, as well as in situ hybridization) and protein-level methods (such as histochemistry, immunoblotting, and in vitro binding studies). Furthermore, the expression levels of genes of interest can be quantified using ELISA techniques well-known to those skilled in the art. Many standard analytical methods are available for performing quantitative measurements. For example, transcriptional levels can be measured using RT-PCR and hybridization methods (including RNase protection, Southern blotting, and RNA dot analysis). Immunohistochemical staining, flow cytometry, and Western blotting can also be used to assess the presence of human or humanized proteins.

[0197] Antibody and antigen binding fragment

[0198] This disclosure provides antibodies and antigen-binding fragments thereof (e.g., humanized antibodies or chimeric antibodies) generated by the methods described herein.

[0199] Typically, antibodies (also known as immunoglobulins) consist of two types of polypeptide chains: a light chain and a heavy chain. The non-limiting antibody disclosed herein can be a complete tetraimmunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype (including IgM, IgG, IgE, IgA, or IgD) or subtype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain can be a κ light chain or a λ light chain. The antibody may comprise two identical copies of the light chain and two identical copies of the heavy chain. Each contains a variable domain (or variable region, V...). H The heavy chains, consisting of multiple constant domains (or constant regions), are linked together by disulfide bonds within their constant domains to form the "backbone" of the antibody. Each chain contains a variable domain (or variable region, V...). L A light chain and a constant structural domain (or constant region) are each bonded to a heavy chain via disulfide bonds. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bonded. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conservative framework regions (FRs).

[0200] These hypervariable regions (called complementarity-determining regions (CDRs)) form loops that comprise the main antigen-binding surface of the antibody. The four framework regions are predominantly in a beta-sheet conformation, while the CDRs form loops that connect the beta-sheet structures and, in some cases, form part of them. The CDRs in each chain are held tightly together by the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding region.

[0201] Identifying the CDR region of an antibody by analyzing its amino acid sequence is a well-known method, and many definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in, for example, Martin, “Protein sequence and structure analysis of antibody variable domains”, Antibody Engineering, Springer Berlin Heidelberg, 2001, 422-439; Abhinandan et al., “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”, Molecular Immunology 45.14 (2008):3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132:211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan. 1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), all of which are cited in full and incorporated herein by reference.

[0202] CDR is crucial for recognizing epitopes of antigens. As used herein, an "epitaph" is the smallest part of a target molecule that can be specifically bound by the 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 do not necessarily have to be in a continuous linear sequence of the primary antigen structure, as epitopes can depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0203] In some implementations, antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, but their constant regions differ, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are known in the art and described in, for example, 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 and Farouk (eds.), “The human IgG subclasses: molecular analysis of structure, function and regulation.”, Elsevier, 2016. All of the above references are incorporated herein by reference in their entirety.

[0204] Antibodies can also be immunoglobulin molecules derived from any species, such as humans, rodents, mice, rats, or camels. The 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" refers to a portion of the antibody that retains the specific binding activity of the intact antibody; that is, any portion of the antibody capable of specifically binding to an epitope on a target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, biantibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide comprising an antibody-binding domain or a binding domain homologous to it. Non-limiting examples of antigen-binding domains include, for example, heavy chain and / or light chain CDRs of an intact antibody, variable regions of heavy chain and / or light chain of an intact antibody, full-length heavy chain or light chain of an intact antibody, or a single CDR from the heavy chain or light chain of an intact antibody.

[0205] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-stranded variable fragment (scFv) as described herein with a CD3-ζ transmembrane domain and an intracellular domain.

[0206] 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, and these two antigen-binding regions can bind to their respective target antigens.

[0207] Antibodies and their antigen-binding fragments (e.g., humanized or chimeric antibodies) generated by the methods described herein offer several advantages. In some embodiments, desired properties (e.g., binding affinity, thermal stability, and / or limited aggregation) can be obtained without further optimization.

[0208] In some specific implementations, the antibody (or its antigen-binding fragment) is delivered in less than 0.1 seconds. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Or less than 0.00001s -1 The dissociation rate (koff) is related to target-specific binding. In some implementations, the dissociation rate (koff) is greater than 0.01 s. -1Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 or greater than 0.000001s -1 .

[0209] In some implementations, the kinetic binding rate (kon) is greater than 1 × 10⁻⁶. 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some implementations, the kinetic binding rate (kon) is less than 1 × 10⁻⁶. 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0210] The affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some implementations, KD is less than 1 × 10⁻⁶. -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -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 × 10⁻⁶. -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M. In some implementations, the antibody binds to the target at a KD concentration 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.

[0211] In some embodiments, thermal stability is determined. The antibody or antigen-binding fragment as described herein may have a Tm greater 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.

[0212] Since IgG can be described as a multidomain protein, its melt profile sometimes shows two or three denaturation temperatures, with the first denaturation temperature being TmD1, the second being TmD2, and optionally the third being TmD3.

[0213] In some embodiments, the antibody or antigen-binding fragment as described herein has a Tm D1 greater 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 antibody or antigen-binding fragment as described herein has a Tm D2 greater 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 antibody or antigen-binding fragment as described herein has a Tm D3 greater 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 implementations, Tm, TmD1, TmD2, and TmD3 are less than 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, or 95℃. In some implementations, when the temperature is below 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, the antibody or antigen-binding fragments described herein do not aggregate.

[0214] Methods for preparing genetically modified animals

[0215] Genetically modified animals can be prepared by introducing human immunoglobulin genes into the genome of non-human animals to produce animals that can express humanized or chimeric antibodies. Figure 3 illustrates a method for preparing humanized animals. In some embodiments, these methods first involve modifying the human immunoglobulin gene locus on a human chromosome. The modified human chromosome is then introduced into mouse recipient cells. The human immunoglobulin variable region is then introduced into the corresponding region of the mouse genome by direct replacement or insertion into an endogenous safe harbor locus. The recipient cells are then screened. In some embodiments, these cells do not contain human chromosomes. These cells are then injected into blastocysts to prepare chimeric mice. Subsequent breeding can be performed to obtain mice containing the complete humanized immunoglobulin gene locus.

[0216] Several other techniques can be used to produce genetically modified animals, including, for example, non-homologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and the CRISPR-Cas system of clustered regularly spaced short palindromic repeats. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to produce 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, the full text of which is incorporated herein by reference. Many other methods for genome editing are also provided, such as microinjection of genetically modified cell nuclei into enucleated oocytes and fusion of enucleated oocytes with another genetically modified cell.

[0217] Gene modification processes may involve replacing endogenous sequences with human sequences or inserting human sequences via homologous recombination. In some implementations, upstream and downstream cleavage at the target site (e.g., via zinc finger nucleases, TALEN, or CRISPR) may result in DNA double-strand breaks, and homologous recombination may be used to replace endogenous sequences with human sequences or insert human sequences.

[0218] In some embodiments, methods for preparing genetically modified humanized animals may include the step of replacing nucleic acids (e.g., V, D, J regions; V, J regions; or V, J, C regions) at an endogenous gene locus (or site) with a corresponding region of a human sequence. This sequence may include regions (e.g., partial or complete regions) of the IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, IGLJ, and / or IGLC genes. In some embodiments, the replacement is mediated by homologous recombination. In some embodiments, the replacement is mediated by Cre recombinase.

[0219] In some embodiments, methods for preparing genetically modified humanized animals may include the step of inserting a nucleic acid (e.g., V, D, J region; V, J region; or V, J, C region) into an endogenous locus (e.g., any safe harbor locus described herein). This sequence may include regions (e.g., partial or complete regions) of the IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, IGLJ, and / or IGLC genes. In some embodiments, the insertion is mediated by homologous recombination. In some embodiments, the insertion is mediated by Cre recombinase.

[0220] Human chromosomes can be obtained from human cell lines, cancer cells, primary cell cultures, and / or human fibroblasts. In some embodiments, a human cell is introduced using a first vector and then fused with a recipient cell. The modified chromosome is then isolated and introduced into another suitable recipient cell. Cells with desired resistance are selected to obtain cells containing only one human chromosome. A second vector is then introduced into the cell, and cells are selected by resistance. A third and / or fourth vector may then be introduced if desired. The recipient cell can be a mammalian cell, a human cell, or a mouse cell. In some embodiments, the recipient cell is a CHO cell, or preferably an A9 cell. In some embodiments, the modified chromosome is fluorescently labeled and isolated. The modified chromosome is then injected into the recipient cell via chromosome microinjection. In some embodiments, donor cells are induced to multinucleate their chromosomes. These nuclei are then forced across the cell membrane to generate microcells that can fuse with the recipient cell. In some embodiments, microcell-mediated chromosome transfer may also be used. Chromosome manipulation techniques are described, for example, in 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 germlinea transmission of a human chromosome fragment in chimaeric mice.” Nature Genetics 16.2 (1997): 133-143; Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284; The full text of the above references is incorporated herein by reference.

[0221] Figure 10 illustrates a similar targeting strategy for the λ light chain immunoglobulin gene locus. Two vectors can be integrated into the human chromosome first. The first vector contains the following upstream DNA homologous arm sequences, a PGK promoter, a red fluorescent protein reporter gene (tdTomato), a transcription termination / polyadenylation signal sequence (PolyA; "PA"), a blastcinin sequence (BSD), an EF-1a promoter, LoxP (Rec01), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "3'HygR"), an Flp recognition target ("FRT"), downstream DNA homologous arm sequences, and the diphtheria toxin subunit A (DTA) gene.

[0222] The second vector contains the DNA homologous arm sequence upstream of the insertion site, Lox (Rec02), a partial sequence of the puromycin resistance gene (5'PuroR), the PGK promoter, the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the neomycin resistance gene sequence (Neo), the internal ribosome entry site (IRES), the piggyBac transposase gene sequence (PBase), the EF-1a promoter, the DNA homologous arm sequence downstream of the insertion site, and the DTA gene.

[0223] The LoxP recognition sequence can also be added to a human chromosome (e.g., human chromosome 22). Cells can also be treated with Cre enzymes to induce recombination at the LoxP sites, thereby removing the genomic DNA sequence. In some implementations, spontaneous chromosome breakage can also be used to remove the genomic DNA sequence.

[0224] The mouse light chain immunoglobulin gene locus can be directly modified, as shown in Figure 7A. In some embodiments, the vector is used directly to replace the entire variable region of the mouse light chain immunoglobulin. In some embodiments, the vector has the following from the 5' to 3' ends: a DNA homologous arm sequence upstream of the insertion site, an Flp recognition target (FRT), a mammalian expression promoter from human elongation factor 1α (EF-1a) (EF-1a promoter), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "5'HygR"), LoxP (Rec01), a 5'PB (piggyBac) transposon sequence (PB5'), a first core insulator, a ubiquitin C promoter, and a sequence encoding the DT receptor (DTR). The sequence encoding the FMDV self-cleaving peptide (2A), the neomycin resistance gene sequence (Neo), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the second core insulator, the 3'PB transposon sequence (PB3'), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the sequence encoding the DT receptor (DTR), the puromycin resistance gene partial sequence (3'PuroR), Lox (Rec02), the downstream DNA homologous arm sequence, and the diphtheria toxin subunit A (DTA) gene.

[0225] Alternatively, the endogenous locus (e.g., any safe harbor locus described herein) may be modified to insert nucleic acids, as shown in Figure 7B. In some embodiments, the vector is used to insert nucleic acids at the endogenous locus. In some embodiments, the vector has the following from the 5' to 3' ends: a DNA homologous arm sequence upstream of the insertion site, an Flp recognition target (FRT), a mammalian expression promoter from human elongation factor 1α (EF-1a) (EF-1a promoter), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "5'HygR"), LoxP (Rec01), a 5'PB (piggyBac) transposon sequence (PB5'), a first core insulator, a ubiquitin C promoter, and a sequence encoding the DT receptor (DTR). The sequence encoding the FMDV self-cleaving peptide (2A), the neomycin resistance gene sequence (Neo), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the second core insulator, the 3'PB transposon sequence (PB3'), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the sequence encoding the DT receptor (DTR), the puromycin resistance gene partial sequence (3'PuroR), Lox (Rec02), the downstream DNA homologous arm sequence, and the diphtheria toxin subunit A (DTA) gene.

[0226] In some embodiments, sequences encoding the variable and / or constant regions of mouse immunoglobulins can be replaced by sequences encoding the variable and / or constant regions of human immunoglobulins via substitution (e.g., homologous recombination or Cre-mediated recombination). In some embodiments, sequences encoding the variable and / or constant regions of human immunoglobulins can be inserted into endogenous loci (e.g., any safe harbor loci described herein), for example via homologous recombination or Cre-mediated recombination. In some embodiments, Cre recombination can be used to mediate substitution. In some embodiments, the vector can add LoxP recognition sequences to human chromosomes. Similar modifications can be made to mouse chromosomes, wherein two LoxP recognition sequences can be added to the chromosome. For example, Cre recombinase can then mediate the substitution of regions V, J, C on mouse chromosomes with regions V, J, C on human chromosomes.

[0227] Cells lacking human chromosomes can be further screened (e.g., by DT). In some cases, cells not screened by DT may contain recombinant human chromosome fragments, but these fragments are small and unstable in mouse cells (e.g., Shinohara et al., (2000) Chromosome Research, 8: 713-725) and will disappear naturally during cell proliferation. In some embodiments, large segments of modified human chromosomes are deleted, for example, through Cre-mediated deletion or through spontaneous chromosome breakage.

[0228] The 5' end homologous arm and / or the 3' end homologous arm may have a desired length to facilitate homologous recombination. In some embodiments, the homologous 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 homologous 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.

[0229] In some embodiments, the vector may optionally include a reporter protein, such as a luciferase (e.g., Gluc) or a fluorescent protein (e.g., EGFP, BFP, etc.).

[0230] These modifications can be performed in various cells. In some implementations, the cells are stem cells, embryonic stem cells, or fertilized egg cells.

[0231] This disclosure also provides a method for establishing a humanized animal model, which involves the following steps:

[0232] (a) Providing cells (e.g., fertilized egg cells) based on the methods described herein;

[0233] (b) Culture the cells in a liquid culture medium;

[0234] (c) Transplanting the cultured cells into the fallopian tubes or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal.

[0235] (d) Identify the phylogenetic transmission in the offspring of pregnant females (i.e., genetically modified humanized non-human mammals) in step (c).

[0236] In some implementations, the non-human mammal in the aforementioned method is a mouse (e.g., a C57 mouse, a BALB / c mouse, or a C57BL / 6 mouse).

[0237] In some implementations, the non-human mammal in step (c) is a female with a pseudopregnancy (or pseudo-pregnancy).

[0238] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used as described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0239] The fertilized egg can be derived from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. Genetic constructs can be introduced into the fertilized egg via 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, and then the non-human mammal can be born, thereby producing the non-human mammal mentioned in the above methods.

[0240] Cells, tissues, and animals (e.g., mice) containing nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mice) expressing humanized or chimeric antibodies derived from endogenous nonhuman loci, are also provided.

[0241] This disclosure also provides various targeting vectors (e.g., vectors for preparing genetically modified animals). In some embodiments, the vector may comprise: a) a DNA fragment homologous to the 5' end of the region to be modified (5' homologous arm); b) a sequence containing 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 fragment homologous to the 3' end of the region to be modified (3' homologous arm). This disclosure also relates to cells comprising the targeting vectors described herein.

[0242] In some implementations, the genes in the cell are heterozygous. In some implementations, the genes in the cell are homozygous.

[0243] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

[0244] This disclosure also relates to methods for generating genetically modified animal models having two or more human or chimeric genes. The animal may comprise one or more human or humanized immunoglobulin loci and sequences encoding additional human or chimeric proteins. In some embodiments, the additional human or chimeric protein may be programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), B and T lymphocyte-associated protein (BTLA), programmed cell death 1 ligand 1 (PD-L1), CD27, CD28, CD47, CD137, CD154, T-cell immune receptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin and mucin-containing domain-3 (TIM-3), glucocorticoid-induced TNFR-associated protein (GITR), or TNF receptor superfamily member 4 (TNFRSF4 or OX40).

[0245] Methods for generating genetically modified animal models with additional human or chimeric genes (e.g., humanized genes) may include the following steps:

[0246] (a) Using the methods described herein to obtain genetically modified non-human animals;

[0247] (b) Mating a 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 with two or more human genes or chimeric genes.

[0248] In some embodiments, in step (b) of the method, the genetically modified animal may mate with a genetically modified non-human animal that possesses human or chimeric PD-1, CTLA-4, OX40, TIGIT, PD-L1, BTLA, TIM-3, LAG-3, CD137, CD47, SIRPa, CD27, CD28, CD154, TIGIT, or GITR. Some of these genetically modified non-human animals are described in, for example, 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, and PCT / CN2018 / 081629; the full text of each of these patents is incorporated herein by reference.

[0249] In some embodiments, the genetically modified animal may possess the human ADAM6 gene, the endogenous ADAM6 gene, or a modified ADAM6 gene. The ADAM6 protein is a member of the ADAM protein family, where ADAM is an acronym for Disintegrin and Metalloprotease. The human ADAM6 gene, typically found between the human IGHV genes IGHV1-2 and IGHV6-1, is a pseudogene. In mice, two ADAM6 genes exist (ADAM6a and ADAM6b). They are located in the intergenic region between the mouse IGHV gene cluster and the IGHD gene cluster. Mouse ADAM6a is located between mouse IGHV5-1 and mouse IGHD5-1. Mouse ADAM6b is located between mouse IGHD3-1 and mouse IGHD1-1. Therefore, in some embodiments, the genetically modified animal may possess the human ADAM6 gene. In some embodiments, the genetically modified animal does not possess the endogenous ADAM6 gene.

[0250] In some embodiments, the genetically modified animal is a mouse. In some embodiments, the mouse is modified to contain a nucleotide sequence encoding an ADAM6 protein (e.g., ADAM6a or ADAM6b). In some embodiments, this sequence is placed at any suitable location. The sequence may be placed in an intergenic region or at any suitable location in the genome. In some embodiments, the nucleic acid encodes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the mouse ADAM6a gene (e.g., 113539230-113547024 of NC_000078.6; SEQ ID NO: 53) or the mouse ADAM6b gene (e.g., 113486188-113492125 of NC_000078.6; SEQ ID NO: 54). In some implementations, the nucleic acid additionally contains regulatory elements (e.g., promoters) of the ADAM6a and ADAM6b genes.

[0251] In some embodiments, the functional mouse ADAM6 locus may be positioned in the middle of the human IGHV gene cluster. In some embodiments, the mouse ADAM6 locus is located between two human IGHVH genes. In some embodiments, the human ADAM6 pseudogene between human VH1-2 and human VH(II)-1-1 is replaced by the mouse ADAM6 locus. In some embodiments, the ADAM6a and ADAM6b genes are located in the animal genome between human IGHV1-2 and human VH(II)-1-1. In some embodiments, the position of the mouse ADAM6 sequence within the human gene sequence may approximate the position of the human ADAM6 pseudogene or the position of the mouse ADAM6 sequence (e.g., within the VD gene region). In some embodiments, the genetically modified mouse has a humanized heavy chain immunoglobulin locus. In some embodiments, mouse ADAM6a and mouse ADAM6b are located between the human IGHV1-2 and IGHV6-1 genes. Placing mouse ADAM6a and mouse ADAM6b between the human IGHV1-2 and IGHV6-1 genes offers various advantages. For example, because these genes replace the human ADAM6 gene at the same locus, it is possible that the replacement of the human ADAM6 gene will have a limited impact on VDJ recombination, and the mouse ADAM6a and mouse ADAM6b genes can also function appropriately (e.g., at positions similar to endogenous loci).

[0252] Therefore, in one aspect, this disclosure provides a genetically modified animal comprising a first sequence, a second sequence, and a third sequence at an endogenous heavy chain immunoglobulin locus, wherein the first sequence comprises one or more human IGHV genes, the second sequence comprises an ADAM6 gene, and the third sequence comprises one or more human IGHD genes and one or more human IGHJ genes. In some embodiments, the first, second, and third sequences are operatively linked.

[0253] In some embodiments, the first sequence comprises all human IGHV genes listed in Table 7 except for IGHV2-10, IGHV3-9, IGHV1-8, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the first sequence comprises all human IGHV genes listed in Table 7 except for IGHV5-10-1, 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.

[0254] In some embodiments, the second sequence comprises one or both of the mouse ADAM6a and mouse ADAM6b genes. In some embodiments, the animal is a fertile male mouse. In some embodiments, the second sequence does not contain the mouse ADAM6a or mouse ADAM6b genes.

[0255] In some embodiments, the third sequence comprises all human IGHD genes in Table 8 and all human IGHJ genes in Table 9. In some embodiments, the third sequence comprises human IGHV6-1. In some embodiments, the third sequence comprises human IGHV(II)-1-1. In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus.

[0256] In some embodiments, AMAM6a and / or ADAM6b are endogenous sequences. In some embodiments, AMAM6a and / or ADAM6b are not substituted, and / or are located at their endogenous or native positions. In some embodiments, the mouse IGHV gene preceding mouse IGHV1-2 at the heavy chain variable region locus is replaced by the human IGHV gene. In some embodiments, the mouse IGHV gene, IGHD gene, and IGHJ gene following mouse IGHV6-1 at the heavy chain variable region locus are replaced by one or more human IGHV genes, IGHD genes, and / or IGHJ genes.

[0257] Therefore, in some embodiments, the mouse IGHV, IGHD, and IGHJ genes can be replaced with human IGHV, IGHD, and IGHJ genes more than once. In the first step, a selected number of mouse IGHV genes on the 5' side of ADAM6a are replaced with human IGHV genes (e.g., all mouse IGHV genes in Table 10). In the second step, a selected number of mouse IGHD and IGHJ genes on the 3' side of ADAM6b are replaced with human IGHD and human IGHJ genes (e.g., all mouse IGHD genes except IGHD5-1 and IGHD3-1 in Table 11 and all IGHJ genes in Table 12). The replacement can be performed via homologous recombination or Cre-mediated recombination.

[0258] In some embodiments, the mice do not possess the mouse ADAM6a or ADAM6b gene. In some embodiments, the mice possess the human ADAM6 gene.

[0259] Various methods can be used to increase fertility in mice. In some embodiments, superovulating female mice can be used for mating. In some embodiments, in vitro fertilization can be used. Superovulation can be induced by injecting serum gonadotropins and human chorionic gonadotropin (hCG) into mature female mice. Mature male mice can be euthanized and their epididymal tails isolated. The ducts of the epididymal tails are cut to release sperm. Next, superovulating mature female mice can be euthanized and their oviducts isolated. The cumulus-oocyte complex (COC) can be released from the oviduct. A sperm suspension can then be added to the COC and incubated for fertilization. Pathogenic oocytes containing only one pronucleus can be retrieved. After incubation, the 2-cell stage embryos can be transferred to a recipient female. Methods for increasing mouse fertility are known in the art.

[0260] This disclosure also provides nucleic acid sequences having 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%, and 99% identity with any nucleotide sequence as described herein, and amino acid sequences having 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%, and 99% identity with any amino acid sequence as described herein.

[0261] In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is fewer 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 has fewer 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.

[0262] In some embodiments, the amino acid sequence (i) includes an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0263] In some implementations, the nucleic acid sequence (i) includes a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0264] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are considered identical at that position 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 (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). Considering the number of vacancies that need to be introduced to achieve optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences is a function of the number of common positions shared by the sequences. For example, the comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0265] The percentage of conserved residues with similar physicochemical properties (homology percentage), such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage. Therefore, this disclosure also provides amino acid sequences, or nucleic acids encoding such amino acid sequences, that have 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%, or 99% homology with any amino acid sequence described herein.

[0266] Using genetically modified animals

[0267] Genetically modified animals can be used to generate humanized or chimeric antibodies that specifically bind to a target. In some embodiments, the target (e.g., a protein or a fragment of a protein) can be used as an immunogen to generate antibodies in these animals using standard techniques for preparing polyclonal and monoclonal antibodies. In some embodiments, the genetically modified animals are exposed to a selected antigen for a period of time, allowing the animals to produce antibodies specific to the antigen.

[0268] Polyclonal antibodies can be generated in an animal through multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. Animals may be injected with the antigenic peptide or protein more than once (e.g., twice, three times, or four times).

[0269] Full-length polypeptides or proteins, or fragments of their antigenic peptides, can be used as immunogens. The antigenic peptide of a protein contains at least eight (e.g., at least 10, 15, 20, or 30) amino acid residues of an amino acid sequence and covers an epitope of the protein, such that antibodies generated against the peptide form specific immune complexes with the protein.

[0270] Immunogens are commonly used to generate antibodies by immunizing suitable subjects (e.g., genetically modified animals as described herein). Appropriate immunogenic formulations may contain, for example, recombinantly expressed or chemically synthesized peptides (e.g., fragments of proteins). The formulation may also include adjuvants (such as Freund's complete or incomplete adjuvants) or similar immunostimulants.

[0271] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide or its antigenic peptide (e.g., a portion of a protein) as an immunogen. Antibody titers in immunized subjects can be monitored over time using immobilized polypeptides or peptides via standard techniques, such as enzyme-linked immunosorbent assay (ELISA). If necessary, antibody molecules can be isolated from mammals (e.g., from blood) and further purified using well-known techniques such as protein A or protein G chromatography to obtain IgG fractions. At an appropriate time following immunization, for example when the specific antibody titer is at its highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies using standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature, Vol. 256: pp. 495–497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today, Vol. 4: p. 72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77–96, 1985), or the trioma technique. Techniques for generating hybridomas are well-known (see generally "Current Protocols in Immunology," 1994, edited by Coligan et al., John Wiley & Sons, Inc., New York, NY). Hybridoma cells that produce monoclonal antibodies are detected by screening for antibodies that bind to the target peptide or epitope in the supernatant of hybridoma culture, for example, using a standard ELISA assay.

[0272] In one aspect, this disclosure provides a mouse comprising a modification of an endogenous immunoglobulin heavy chain gene locus, wherein the mouse produces B cells comprising a rearranged immunoglobulin sequence operatively linked to a heavy chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operatively linked to the heavy chain constant region gene sequence comprises human heavy chain V, D, and / or J sequences. In some embodiments, the heavy chain constant region gene sequence comprises a human or mouse heavy chain sequence selected from the group consisting of CH1, hinge, CH2, CH3, and combinations thereof.

[0273] In one aspect, this disclosure provides mice comprising modifications to endogenous immunoglobulin light chain (e.g., κ or λ) loci, and / or loci containing modifications to the human λ light chain immunoglobulin gene (e.g., any safe harbor loci described herein), wherein the mice produce B cells comprising rearranged immunoglobulin sequences operatively linked to light chain constant region gene sequences. In some embodiments, the rearranged immunoglobulin sequences operatively linked to the light chain constant region gene sequences comprise human light chain V and / or J sequences. In some embodiments, the light chain constant region gene sequences comprise human or mouse light chain constant regions.

[0274] Mouse B cells or spleen cells may contain rearranged non-mouse immunoglobulin variable gene sequences, for example, operatively linked to mouse immunoglobulin constant region genes. Sequences encoding human heavy chain variable regions and human light chain variable regions have been identified. These sequences can be determined, for example, by sequencing the hybridoma or B cells of interest. In some embodiments, single-B-cell screening is used. It allows screening of a natural antibody library without requiring hybridoma fusion and combinatorial display. For example, B cells may be mixed with a set of DNA barcode antigens, allowing the antigen barcodes and B-cell receptor (BCR) sequences of individual B cells to be recovered via a single-cell sequencing protocol.

[0275] Antibodies can be further modified to obtain humanized antibodies or human antibodies, for example by operatively linking a sequence encoding the variable region of the human heavy chain to a sequence encoding the constant region of the human heavy chain, and / or operatively linking a sequence encoding the variable region of the human light chain to a sequence encoding the constant region of the human light chain.

[0276] In some implementations, it may be difficult to elicit an immune response in mice if they express proteins that are very similar to the antigen of interest. This is because B cells and T cells that recognize MHC molecules that bind to peptides of their own origin are absent from the immune cell pool during immune cell development. In those cases, humanized mice can be further modified. The corresponding gene can be knocked out in the mouse, and then the mouse is exposed to the antigen of interest. Since the mouse does not undergo negative selection for the gene product, it can produce antibodies that can readily bind specifically to the target.

[0277] This disclosure also provides methods for preparing antibodies, nucleic acids, cells, and tissues (e.g., spleen tissue). In some embodiments, these methods involve exposing an animal, as described herein, to an antigen. Antibodies (e.g., chimeric antibodies), nucleic acids encoding the antibodies, cells, and / or tissues (e.g., spleen tissue) may be obtained from the animal. In some embodiments, nucleic acids encoding the variable regions of human heavy and light chain immunoglobulins are determined, for example, by sequencing. In some embodiments, the nucleic acid encoding the variable region of human heavy chain immunoglobulins may be operably linked to a nucleic acid encoding the constant region of human heavy chain immunoglobulins. In some embodiments, the nucleic acid encoding the variable region of human light chain immunoglobulins may be operably linked to a nucleic acid encoding the constant region of human light chain immunoglobulins. In some embodiments, cells containing nucleic acids as described herein are cultured and antibodies are collected.

[0278] In some embodiments, the mouse immunoglobulin V, D, and J genes (e.g., the mouse IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, and / or IGLJ genes) are absent from the heavy and / or light chain variable region sequences. In some embodiments, the heavy and / or light chain variable region sequences generated by the animal are fully humanized and consist entirely of human immunoglobulin V, D, and J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, and / or IGLJ genes).

[0279] Variants of the antibody or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized, or chimeric antibody or its antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence constituting the antigen-binding domain of the antibody or antigen-binding domain. Within this group of variants, some antibody or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and / or combinations can be made to obtain an antibody or its antigen-binding fragment with increased binding affinity to the target. Introducing amino acid changes into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are linked by enzymes present in the cell), or introducing new glycosylation sites.

[0280] The antibodies disclosed herein may be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include those derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camels (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits, including genetically engineered rodents to produce human antibodies).

[0281] Human and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequence as antibodies derived from human immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDR.

[0282] Further modifications can be made to the antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in that region. The resulting homodimeric antibody can have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional cross-linking agents, as described, for example, by Wolff et al. (Cancer Res., Vol. 53: pp. 2560-2565, 1993). Alternatively, the antibody can be engineered to have a dual Fc region (see, for example, Stevenson et al., Anti-CancerDrug Design, Vol. 3: pp. 219-230, 1989).

[0283] In some embodiments, the antibody or its antigen-binding fragment may be covalently modified. These covalent modifications can be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications to the antibody or antibody fragment can be introduced into the molecule by reacting the target amino acid residues of the antibody or fragment with an organic derivatizer capable of reacting with selected side chain or N-terminal or C-terminal residues.

[0284] Example

[0285] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0286] Example 1: Overview

[0287] Experiments were conducted to introduce immunoglobulin genes into the mouse genome. Figure 3 illustrates the methods used to create humanized mice. These methods first involve modifying the human immunoglobulin region on a human chromosome. The modified human chromosome is then introduced into mouse recipient cells.

[0288] The mouse immunoglobulin region is replaced by the human immunoglobulin region through direct substitution (e.g., homologous recombination or Cre-mediated recombination). In some cases, the human immunoglobulin region can be introduced into the mouse genome via a stepwise approach. Recipient cells with the correct substitution are then screened. These cells are then injected into blastocysts to generate chimeric mice. Subsequent breeding yields mice containing the complete human immunoglobulin region.

[0289] Example 2: Modification of mouse heavy chain immunoglobulin loci and κ light chain immunoglobulin loci

[0290] hVH / hVK mice possess humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci. The heavy chain immunoglobulin locus is a region of the chromosome containing antibody heavy chain genes. This locus includes IGHV (variable), IGHD (diversity), IGHJ (linking), and heavy chain constant domain genes (Figure 4A). The κ chain immunoglobulin locus is a region of the chromosome containing antibody light chain (κ chain) genes. The κ chain immunoglobulin locus includes IGKV (variable), IGKJ (linking), and light chain constant domain genes (Figure 4B). A detailed description of hVH / hVK mice can be found in PCT / CN2020 / 075698, the full text of which is incorporated herein by reference.

[0291] κ light chain locus knockout mice

[0292] The immunoglobulin κ light chain locus is located on mouse chromosome 6. Mouse chromosome 6 was modified by knocking out the entire sequence of the immunoglobulin κ light chain variable region locus. Detailed knockout methods can be found, for example, in WO2020169022A1 and US20200390073A1; Zou, YR et al., "Gene targeting in the Ig kappa locus: efficient generation of lambda chain-expressing B cells, independent of gene rearrangements in Ig kappa." The EMBOJournal 12.3 (1993): 811-820; and Takeda, S. et al., "Deletion of the immunoglobulinkappa chain intron enhancer abolishes kappa chain gene rearrangement in cisbut not lambda chain gene rearrangement in trans." The EMBO Journal 12.6(1993): References 2329-2336; The full text of all the above references is incorporated herein by reference.

[0293] λ light chain locus knockout mice

[0294] The immunoglobulin λ light chain is located on mouse chromosome 16. Mouse chromosome 16 was modified by knocking out the entire sequence of the immunoglobulin λ light chain variable region locus. Detailed knockout methods can be found, for example, in Zou, X. et al., “Block indevelopment at the pre-B-II to immature B cell stage in mice without Igκ and Igλ light chain.” The Journal of Immunology 170.3 (2003): 1354-1361, which is incorporated herein by reference in its entirety.

[0295] The mice described in this article can be bred together to obtain mice with humanized heavy chain immunoglobulin loci but lacking all or part of the mouse immunoglobulin light chain loci.

[0296] Example 3: Preparation of mice containing a modified locus containing the human λ light chain immunoglobulin gene

[0297] Modification of mouse λ light chain immunoglobulin locus

[0298] The λ light chain immunoglobulin locus (also known as the immunoglobulin λ locus or IGL) is located on mouse chromosome 16. Figure 5 is a schematic diagram of the mouse λ light chain immunoglobulin locus. Two recombination sites (Rec01 and Rec02) were introduced flanking the variable region of the λ light chain immunoglobulin locus, and the resulting modified chromosome is shown in Figure 6A. One of these recombination sites is the wild-type loxP site, and the other is an xenogeneic mutant lox site (lox2272). Recombination cannot occur between the wild-type loxP site and the xenogeneic mutant lox site. The modification was performed in mouse embryonic stem cells. An overview of the targeting strategy is shown in Figure 7A.

[0299] The vector (V01) contains the following elements from the 5' to 3' ends: a DNA homologous arm sequence upstream of the insertion site, an Flp recognition target (FRT), a mammalian expression promoter derived from human elongation factor 1α (EF-1a) (EF-1a promoter), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "5'HygR"), LoxP (Rec01), a 5'PB (piggyBac) transposon sequence (PB5'), a first core insulator, a ubiquitin C promoter, and a sequence encoding the DT receptor (DTR). The sequence includes: the FMDV self-cleaving peptide (2A), the neomycin resistance gene sequence (Neo), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the second core insulator, the 3'PB transposon sequence (PB3'), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the sequence encoding the DT receptor (DTR), the puromycin resistance gene partial sequence (3'PuroR), Lox (Rec02), the downstream DNA homologous arm sequence, and the diphtheria toxin subunit A (DTA) gene. In some cases, the first core insulator and / or the second core insulator are optional elements.

[0300] The vector (V01) was introduced into cells, and cells were then screened using G418. Recombination was confirmed by PCR. Positive clones were selected after screening.

[0301] Modification of the mouse Hipp11 locus

[0302] The mouse Hipp11 locus on mouse chromosome 11 was modified using a method similar to that described above. Specifically, two recombination sites (Rec01 and Rec02) were introduced at the mouse Hipp11 locus, and the resulting modified chromosome is shown in Figure 6B. One of these recombination sites is a wild-type loxP site, and the other is a xenogeneic mutant lox site (lox2272). Recombination cannot occur between the wild-type loxP site and the xenogeneic mutant lox site. The modification was performed in mouse embryonic stem cells. An overview of the targeting strategy is shown in Figure 7B. As shown in Figure 7B, two recombination sites were introduced at the mouse Hipp11 locus, located between exon 19 of the Eif4enif1 gene and exon 7 of the Drg1 gene. The Hipp11 locus is also described, for example, in Hippenmeyer, S. et al., “Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration.” Neuron 68.4 (2010): 695-709, the full text of which is incorporated herein by reference.

[0303] The vector (V04) contains the following elements from the 5' to 3' ends: a DNA homologous arm sequence upstream of the insertion site, an Flp recognition target (FRT), a mammalian expression promoter derived from human elongation factor 1α (EF-1a) (EF-1a promoter), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "5'HygR"), LoxP (Rec01), a 5'PB (piggyBac) transposon sequence (PB5'), a first core insulator, a ubiquitin C promoter, and a sequence encoding the DT receptor (DTR). The sequence includes: the FMDV self-cleaving peptide (2A), the neomycin resistance gene sequence (Neo), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the second core insulator, the 3'PB transposon sequence (PB3'), the transcription termination / polyadenylation signal sequence (PolyA; "PA"), the sequence encoding the DT receptor (DTR), the puromycin resistance gene partial sequence (3'PuroR), Lox (Rec02), the downstream DNA homologous arm sequence, and the diphtheria toxin subunit A (DTA) gene. In some cases, the first core insulator and / or the second core insulator are optional elements.

[0304] The vector (V04) was introduced into cells, and cells were then screened using G418. Recombination was confirmed by PCR. Positive clones were selected after screening.

[0305] Modification of human λ light chain immunoglobulin locus

[0306] The goal of the experiment was to generate modified human chromosomes with at least two recombination sites. Two recombination sites were introduced flanking the λ light chain immunoglobulin gene locus.

[0307] The λ light chain immunoglobulin locus is located on human chromosome 22. Figure 8 is a schematic diagram of human chromosome 22, highlighting the λ light chain immunoglobulin locus. The modified human λ light chain immunoglobulin locus (IGL) is shown in Figure 9. An overview of the targeting strategy is shown in Figure 10, where Rec01 and Rec02 are recombination sites.

[0308] Experiments were conducted to insert the vector into human chromosome 22. The first targeting vector (V02) has the following elements from the 5' to 3' ends: a DNA homologous arm sequence upstream of the insertion site, a PGK promoter, a red fluorescent protein reporter gene (tdTomato), a transcription termination / polyadenylation signal sequence (PolyA; "PA"), a blastcinin sequence (BSD), an EF-1a promoter, LoxP (Rec01), a hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; "3'HygR"), an Flp recognition target ("FRT"), a downstream DNA homologous arm sequence, and a diphtheria toxin subunit A (DTA) gene.

[0309] The second vector (V03) has the following elements from the 5' to 3' ends: DNA homologous arm sequence upstream of the insertion site, Lox (Rec02), partial sequence of puromycin resistance gene (5'PuroR), PGK promoter, transcription termination / polyadenylation signal sequence (PolyA; "PA"), neomycin resistance gene sequence (Neo), internal ribosome entry site (IRES), piggyBac transposase gene sequence (PBase), EF-1a promoter, DNA homologous arm sequence downstream of the insertion site, and DTA gene.

[0310] In some experiments, vectors (VO2 and VO3) are introduced into cells, and cells are selected by appropriate resistance markers or combinations thereof (e.g., blastomycin and / or G418). There are numerous methods for introducing vectors of interest into human chromosomes. Human chromosomes can be obtained from human cell lines, cancer cells, primary cell cultures, and / or human fibroblasts.

[0311] In one experiment, a first vector is introduced into a chromosome. The modified chromosome can then be added to a recipient cell, and a second vector can be inserted into the modified chromosome.

[0312] In one experiment, human fibroblasts were selected and transduced using vector V02. 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 containing only one human chromosome were then selected for resistance to blast fungicide. Vector V03 was then introduced into the cells, and cells were selected for resistance to G148. Positive clones with Rec01 and Rec02 were selected after screening. Chromosome manipulation techniques are described in, for example, by 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, Vol. 13, No. 3, 1987, pp. 279-284; the full text of all the above references are incorporated herein by reference.

[0313] Introducing human chromosomes or fragments into mouse ES cells

[0314] The modified chromosome was introduced into embryonic stem (ES) cells using the previously described method. Cells were then screened for antibiotic resistance. Cells containing only one human chromosome were selected. Figure 11A shows the modified mouse chromosome 16. Recombinase-mediated recombination was then used to obtain the humanized mouse IGL locus, in which the IGLV, IGLJ, and IGLC genes on the mouse chromosome were replaced by the IGLV, IGLJ, and IGLC genes on the human chromosome.

[0315] Human chromosomal DNA sequences were introduced into the chromosomes of mouse ES cells by replacing the sequence between recombination sites Rec01 and Rec02. Hygromycin and puromycin were used to screen for positive cells. The cells were further screened by DTA to obtain mouse cells that did not contain human chromosomes, and these cells were then injected into mouse blastocysts.

[0316] Cells recombined with Cre were tested to confirm the integration of the human gene sequence into mouse chromosome 16. Clones identified by PCR were then validated using Southern blotting to confirm the presence of random insertions. The results are shown in Figures 12A to 12C. The results showed that cells numbered 1-E02, 1-F01, 1-A01, and 1-B01 were verified as positive clones without random insertions. These primers are shown in the table below.

[0317] Table 17. PCR primer sequences and target fragment sizes

[0318] In addition, mouse chromosome 16-specific BACs (e.g., RP23-15M16 and RP23-155M1) labeled with a dye conjugated with PromoFluor 488P (PromoKine, catalog number: PK-PF488P-8-100); and human chromosome 22 IGL-specific BACs (e.g., CH17-117C7, CH17-238D3, CH17-303C15, and CH17-95F2) labeled with a dye conjugated with PromoFluor 555P (PromoKine, catalog number: PK-PF555P-8-100) were detected by fluorescence in situ hybridization (FISH) to integrate human IGL fragments into mouse chromosome 16. The preparation methods of the BAC probe are known to those skilled in the art (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987)), all of which are incorporated herein by reference. The results are shown in Figure 13A. The arrow next to “1” indicates the human IGL fragment. The arrow next to “2” indicates mouse chromosome 16. The results show that the human IGL fragment was successfully integrated into mouse chromosome 16.

[0319] In another experiment, Figure 11B shows the modified mouse chromosome 11. Cre recombinase then mediated recombination to obtain the humanized mouse Hipp11 locus, in which the IGLV, IGLJ, and IGLC genes from the human chromosome were inserted at the Hipp11 locus on the mouse chromosome.

[0320] Cells recombined with Cre were tested to confirm the integration of the human gene sequence into mouse chromosome 11. Clones identified by PCR were then validated using Southern blotting to confirm the presence of random insertions. The results are shown in Figures 12D to 12F. The results showed that cells numbered 1-B12, 1-D12, 1-E10, 1-G11, and 1-H01 were validated as positive clones without random insertions. These primers are shown in the table below.

[0321] Table 18. PCR primer sequences and target fragment sizes

[0322] Furthermore, using the previously described method, the integration of the human IGL fragment into mouse chromosome 11 was detected by fluorescence in situ hybridization (FISH). The results are shown in Figure 13B. The arrows indicate the human IGL fragment. The results show that the human IGL fragment was successfully integrated into the mouse chromosome.

[0323] Preparation of mice containing the humanized λ light chain immunoglobulin locus

[0324] Positive clonal cells (e.g., 1-E02, 1-F01, 1-A01, and 1-B01) are injected via microinjection into the blastocysts of wild-type mice (e.g., C57BL / 6 mice). Embryo microinjection is performed according to methods described, for example, in A. Nagy et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006. The injected fertilized eggs are then transferred to a culture medium for a short period of culture before being transplanted into the oviducts of recipient mice to produce genetically modified humanized mice (F0 generation). F1 generation mice are obtained by backcrossing F0 generation chimeric mice with hVH / hVK mice, and then F2 generation homozygous mice are bred together. Alternatively, the obtained F0 generation mice can also be used for in vitro fertilization (IVF) with hVH / hVK mice. The mice obtained with humanized heavy chain immunoglobulin loci, humanized κ light chain immunoglobulin loci, and humanized λ light chain immunoglobulin loci were named "hVH / hVK / hVL mice".

[0325] hVH / hVK / hVL mice were bred with mice possessing humanized heavy chain immunoglobulin loci but lacking all or part of the mouse immunoglobulin κ light chain locus to obtain "hVH / hVL mice" that possess both humanized heavy chain and humanized λ light chain immunoglobulin loci, but lack all or part of the mouse immunoglobulin κ light chain locus. Positive mice were also bred with Flp transgenic mice to remove the positive selection marker gene (illustrated in Figure 14). These mice were further crossbred several times (e.g., at least 5 times) with mice having a BALB / c background to obtain heterozygous mice with both humanized heavy chain and humanized light chain immunoglobulin loci and a BALB / c background.

[0326] Humanized mice were engineered to express the humanized IGL molecule in vivo, which contains the complete human IGLV, IGLJ, and IGLC genes. The 5' end of the human IGL gene was linked to the mouse sequence as follows:

[0327]

[0328]

[0329] The 5' italicized sequence is the mouse sequence, the bold sequence is the restriction site sequence or additional sequence, the double underlined sequence is the FRT site, the underlined and italicized sequence is the lox site, the underlined sequence is the EF1a+HYG+PA sequence, and the 3' sequence is the human sequence.

[0330] The linker between the 3' end of the human IGL gene and the mouse sequence was designed as follows:

[0331]

[0332] The 3' italicized sequence is the mouse sequence, the bold sequence is the restriction site sequence or additional sequence, the underlined sequence is the lox site, and the 5' anterior sequence is the human sequence.

[0333] Humanized VH with heterozygous humanized heavy chain immunoglobulin loci and heterozygous humanized light chain immunoglobulin loci can be identified by PCR analysis and Southern blotting. H / + / hVK H / + / / hVL H / + Mice.

[0334] Mice identified as positive by PCR were then verified by Southern blotting to confirm whether the clones had random insertions (cell DNA was digested with AseI, BglII, Ndel, or Sspl and then hybridized with the corresponding probe). Restriction enzymes, probes, and target fragment sizes are shown in the table below.

[0335] Table 19. Enzymes and probes used in Southern blotting

[0336] The results of the Southern blot are shown in Figure 15. The results show that mice numbered F1-1, F1-2, F1-3, F1-4, F1-5, F1-6, F1-7, F1-8, F1-9, F1-10 and F1-11 were verified as positive mice without random insertion.

[0337] The following primers are used for probe synthesis in Southern blotting:

[0338] IGL-5' probe:

[0339] IGL-5' probe-F: 5'-AACTCAGTGATGCCAAACAGCTAGG-3' (SEQ ID NO: 7)

[0340] IGL-5' probe-R: 5'-TGCATTCTTTGGGTTCAATGTTTCCA-3' (SEQ ID NO: 8);

[0341] IGL-3' probe:

[0342] IGL-3' probe-F: 5'-GCTTCCTCCCTTGAGTTCAGTCCTC-3' (SEQ ID NO: 9)

[0343] IGL-3' probe-R: 5'-TAGGCAGAGGATCCACAGGAACCAT-3' (SEQ ID NO: 10);

[0344] IGK probe:

[0345] IGK probe-F: 5'-TGATGGGTCAACCATGTTCCTGTGG-3' (SEQ ID NO: 11)

[0346] IGK probe-R: 5'-TCAGCCATTGCTTCTGCTTTCTCCT-3' (SEQ ID NO: 12);

[0347] IGH probe:

[0348] IGH probe-F: 5'-CTTAGACCACCTTGCACCTTCCCTG-3' (SEQ ID NO: 13)

[0349] IGH probe-R: 5'-CAGATCTGAAGCTGGGTCTGTCACG-3' (SEQ ID NO: 14).

[0350] In addition, in order to verify hVH H / + / hVK H / + / hVL H / +The integrity of the human IGL sequence in mice was determined using a PCR-based method to detect the integrity of the human IGLV, IGLJ, and IGLC genes. The PCR results are shown in Figure 16. This method detected 22 IGLV genes, 5 IGLJ genes, and 5 IGLC genes. Furthermore, the PCR-based method can be used to design primers to detect all human IGLV, IGLJ, and IGLC genes based on their nucleotide sequences, and to test the continuity and integrity of all IGLV, IGLJ, and IGLC genes.

[0351] Expression of humanized IGL protein in positive mice can also be confirmed, for example, by flow cytometry. Specifically, one 6-week-old C57BL / 6 wild-type mouse and one hVH mouse were selected. H / H / hVK - / - / hVL H / H Mice (hVH / hVL homozygous mice). Mouse spleen cells were collected. Cells were first stained with PE anti-human Ig light chain λ antibody, FITC anti-mouse Ig light chain κ antibody, and Pacific Blue™ anti-mouse / human CD45R / B220 antibody, followed by flow cytometry analysis. The results of the flow cytometry analysis are shown in the table below. The results showed the detection of humanized IGL protein in hVH / hVL homozygous mice.

[0352] Table 20. Flow cytometry detection in C57BL / 6 mice and hVH / hVL homozygous mice

[0353] Mice containing the modified Hipp11 locus were generated.

[0354] Positive clonal cells (e.g., 1-B12, 1-D12, 1-E10, 1-G11, and 1-H01) were injected via microinjection into the blastocysts of wild-type mice (e.g., C57BL / 6 mice). The injected fertilized eggs were then transferred to a culture medium for short-term culture before being transplanted into the oviducts of recipient mice to generate genetically modified humanized mice (F0 generation). Heterozygous mice were produced by crossing F0 generation chimeric mice with hVH... H / H / hVK H / H / hVL - / - The mice (hVH / hVK mice lacking all or part of the mouse immunoglobulin λ light chain locus) were bred, and then heterozygous mice were bred together to produce homozygous mice. Alternatively, the resulting F0 generation mice could also be used with hVH mice. H / H / hVK H / H / hVL - / -Mice underwent in vitro fertilization (IVF). The mice obtained with humanized heavy chain immunoglobulin loci, humanized κ light chain immunoglobulin loci, and humanized λ light chain immunoglobulin loci were named "hVH / hVK / hVL mice (v)".

[0355] hVH / hVK / hVL mice (v) were bred with mice possessing humanized heavy chain immunoglobulin loci but lacking all or part of the mouse immunoglobulin κ light chain loci and λ light chain loci to obtain "hVH / hVL mice (v)" that possess humanized heavy chain immunoglobulin loci and a modified Hipp11 locus with inserted human IGLV, IGLJ, and IGLC genes, but lack all or part of the mouse immunoglobulin κ light chain loci and λ light chain loci. Positive mice were also bred with Flp transgenic mice to remove the positive selection marker genes. These mice were further crossbred several times (e.g., at least 5 times) with mice with a BALB / c background to obtain heterozygous mice with a BALB / c background possessing both humanized heavy chain immunoglobulin loci and humanized light chain immunoglobulin loci.

[0356] Humanized mice were engineered to express the humanized IGL molecule in vivo, which contains the complete human IGLV, IGLJ, and IGLC genes. The 5' end of the human IGL gene was linked to the mouse sequence as follows:

[0357]

[0358]

[0359] The 5' italicized sequence is the mouse sequence, the bold sequence is the restriction site sequence or additional sequence, the double underlined sequence is the FRT site, the underlined and italicized sequence is the lox site, the underlined sequence is the EF1a+HYG+PA sequence, and the 3' sequence is the human sequence.

[0360] The linker between the 3' end of the human IGL gene and the mouse sequence was designed as follows:

[0361]

[0362] The 3' italicized sequence is the mouse sequence, the bold sequence is the restriction site sequence or additional sequence, the underlined sequence is the lox site, and the 5' normal anterior sequence is the human sequence.

[0363] Humanized hVH / hVK / hVL mice (v) and hVH / hVL mice (v) were identified by PCR analysis and Southern blotting. Mice that tested positive by PCR were then verified by Southern blotting to confirm whether the clones had random insertions.

[0364] In addition, in order to verify hVH H / + / hVK H / + / hVL H / + Mouse (v) and hVH H / + / hVL H / + The integrity of the human IGL sequence in mice (v) was determined using a PCR-based method to detect the integrity of the human IGLV, IGLJ, and IGLC genes. The results were consistent with Figure 16.

[0365] Example 4: Human IGL region gene fragment in mice

[0366] Next-generation sequencing was used to analyze the mRNA sequence of the IGL region in mice. Specifically, two non-immunized (not exposed to the specific antigen) hVH mice were selected. H / H / hVK H / H / hVL H / H Mice and two unimmunized hVH H / H / hVL H / H Mice. Spleen cells were collected from mice for RNA extraction. A 5' RACE kit (SMARTer) was used. ® Reverse transcription was performed using the RACE 5' / 3' kit (Takara Bio USA, Inc., catalog number 634858) to obtain cDNA. The obtained IGH cDNA was amplified by PCR using mouse IGH constant region-specific primers and the upstream primer of the 5'RACE kit to obtain the IGH region sequence fragment, which was then sequenced. The mouse IGH constant region-specific primer sequence is shown as 5'-CCCAAGCTTACGAGGGGGAAGACATTTGGGAA-3' (SEQ ID NO: 15). The obtained IGK cDNA was amplified by PCR using mouse IGK region-specific primers and the upstream primer of the 5'RACE kit to obtain the IGK region sequence fragment, which was then sequenced. The mouse IGK region-specific primer sequence is shown as 5'-CTAACACTCATTCCTGTTGAAGCTCTTGAC-3' (SEQ ID NO: 16). The obtained IGL cDNA was amplified by PCR using human IGL region-specific primers and the upstream primer of the 5'RACE kit to obtain the IGL region sequence fragment, which was then sequenced. The human IGL region-specific primer sequence is shown as 5'-GCTCCCGGGTAGAAGTCAC-3' (SEQ ID NO: 17).

[0367] The sequencing results were compared with the NCBI IgBlast tool used for human immunoglobulin sequencing to identify the expression of the V(D)J recombinant human IHGV, IGHD, IGHJ, IGKV, IGKJ, IGLV, and IGLJ genes. The results are shown in the table below.

[0368] Table 21. In hVH H / H / hVL H / H List of IGHV, IGHD, and IGHJ genes detected in mice after V(D)J recombination.

[0369]

[0370] Table 22. In hVH H / H / hVL H / H List of detected IGLV and IGLJ genes expressed after V(D)J recombination in mice

[0371]

[0372] Table 23. In hVH H / H / hVK H / H / hVL H / H List of IGHV, IGHD, and IGHJ genes detected in mice after V(D)J recombination.

[0373]

[0374] Table 24. In hVH H / H / hVK H / H / hVL H / H List of detected IGKV and IGKJ genes expressed after V(D)J recombination in mice.

[0375]

[0376] Table 25. In hVH H / H / hVK H / H / hVL H / H List of detected IGLV and IGLJ genes expressed after V(D)J recombination in mice

[0377]

[0378] In addition, hVH was analyzed using next-generation sequencing. H / H / hVK H / H / hVL H / H Mouse (v) and hVH H / H / hVL H / H mRNA sequence of the IGL region in mice (v). The results are consistent with those described above for hVH. H / H / hVK H / H / hVL H / H Mice and hVH H / H / hVL H / H The results obtained in mice were consistent.

[0379] Example 5: B cell development in hVH / hVK / hVL mice

[0380] hVH, which uses a homozygous humanized heavy chain immunoglobulin locus and a homozygous humanized κ light chain immunoglobulin locus. H / H / hVK H / H Mice (hVH / hVK homozygous mice) and hVH mice possessing homozygous humanized heavy chain immunoglobulin loci, homozygous humanized κ light chain immunoglobulin loci, and homozygous humanized λ chain immunoglobulin loci H / H / hVK H / H / hVL H / H B cell development was tested in mice (hVH / hVK / hVL homozygous mice). Six mice were used, including three females and three males. Experiments were performed to compare the immune systems of pre-immune hVH / hVK homozygous mice, hVH / hVK / hVL homozygous mice, and wild-type mice. Body weight and the weight of several organs (e.g., spleen, thymus, liver, heart, lungs, and kidneys) were measured in hVH / hVK homozygous, hVH / hVK / hVL homozygous, and wild-type mice. No significant differences in mean body weight and organ (e.g., spleen, thymus, liver, heart, lungs, and kidneys) weight were detected between humanized and wild-type mice.

[0381] Flow cytometry was performed to analyze the lymphocyte populations and distribution in the spleen (Fig. 17A–17B), lymph nodes (Fig. 18A–18B), and peripheral blood (Fig. 19A–19B) of hVH / hVK homozygous mice, hVH / hVK / hVL homozygous mice, and wild-type mice. The results showed that the percentages of B cells, T cells, NK cells, CD4+ T cells, and CD8+ T cells in the peripheral blood, spleen, and lymph nodes of VH / hVK / hVL homozygous mice were almost identical to those in wild-type and hVH / hVK homozygous mice. In the results, leukocytes included: B cells (e.g., characterized by CD45+, CD19+, TCRβ-), T cells, macrophages (e.g., characterized by CD45+, CD11b+, F4 / 80+), monocytes (e.g., characterized by CD45+, CD11b+, F4 / 80-, high Ly-6C, Ly6G-), neutrophils (e.g., characterized by CD45+, CD11b+, F4 / 80-, Ly-6C+, Ly6G+), dendritic cells (e.g., characterized by CD45+, CD11b+, F4 / 80-, Ly-6C+, Ly6G+), and natural killer (NK) cells (e.g., characterized by CD45+, TCRβ-, CD19-, NK1.1+). T cells were further characterized by CD45+, CD19-, and TCR+. CD4+ T cells (CD4) were characterized by CD45+, CD19-, TCR+, CD4+, and CD8-; and CD8+ T cells (CD8) were characterized by CD45+, CD19-, TCR+, CD4-, and CD8+. Flow cytometry analysis included only intact, single, and live leukocytes.

[0382] Flow cytometry was performed to analyze B cell populations in the spleen (Table 26), lymph nodes (Table 27), and bone marrow (Table 28) of hVH / hVK homozygous mice and hVH / hVK / hVL homozygous mice, respectively. B cell progenitors in the bone marrow could be divided into three cell populations: progenitor 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 ).

[0383] The developmental stages of B cells in the spleen are divided into T1 (transitional type 1 B cells, characterized by B220) and T220. + IgM + IgD -T2 (transitional type 2 B cell, characterized by B220) + IgM + IgD + ) and mature B cells (characterized by B220) + IgM 低 IgD + B cell development in the spleen was also assessed by flow cytometry to selectively stain plasma cells (B220). 低 IgM - IgD - CD138 + ) and memory B cells (B220) + IgM + IgD - CD38 + Furthermore, in the spleen marginal zone (marginal zone B cells, referred to as MZ-B, characterized by B220...), + CD21 + CD23 - ) and follicular regions (follicular B cells, referred to as FO-B, characterized by B220 + CD21 低 CD23 + B cell development was assessed.

[0384] The developmental stages of B cells in lymph nodes are divided into T1 (transitional type 1 B cells, characterized by B220) and T220. + IgM + IgD - T2 (transitional type 2 B cell, characterized by B220) + IgM + IgD + ) and mature B cells (characterized by B220) + IgM 低 IgD + B cell development in lymph nodes was also assessed by flow cytometry to selectively stain plasma cells (B220). 低 IgM - IgD - CD138 + ) and memory B cells (B220) + IgM + IgD - CD38 + The results are shown in the table below.

[0385] Table 26. Flow cytometry analysis of B cell development in the spleen

[0386] Table 27. Flow cytometry analysis of B cell development in lymph nodes

[0387] Table 28. Flow cytometry analysis of B cell development in bone marrow

[0388] Example 6: Antibody production in hVH / hVK / hVL mice

[0389] Antigen A is a member of the tumor necrosis factor receptor superfamily. Five hVH mice were co-immunized with antigen A. H / H / hVK H / H / hVL H / H Mice and 5 hVH H / H / hVK - / - / hVL H / H Mice were immunized five times. The first immunization used Freund's complete adjuvant (CFA) and 20 μg of antigen A. The second, third, fourth, and fifth immunizations used Freund's incomplete adjuvant (IFA), CpG oligonucleotides (CpG), and 20 μg of antigen A. A total of five immunizations were administered. The third and fourth immunizations were 6 weeks apart, and the remaining immunizations were 2 weeks apart. Three weeks after the third immunization, retro-orbital blood was collected, and serum antibody titers were detected by fluorescence-activated cell sorting (FACS). One week later, mice with high antibody titers were further immunized with a pulse immunization of 20 μg of human antigen A via intraperitoneal injection.

[0390] Using a Biacore™ (Biacore, Inc., Piscataway, NJ, USA) 8K biosensor equipped with a pre-immobilized protein A sensor chip, the binding affinity of anti-antigen A antibodies to His-tagged human antigen A protein (hA-His) was measured using surface plasmon resonance (SPR). Data analysis was performed using a standard 1:1 binding model in Biacore™ 8K evaluation software 3.0. Affinity values ​​were derived from the quotient of the kinetic rate constant (KD = koff / kon).

[0391] As those skilled in the art will understand, the same method was used to adjust appropriate parameters (e.g., antibody concentration) for each test antibody. The binding affinity of all antibodies reached or even exceeded 10. -8 M indicates that this method can successfully prepare antibodies with high binding affinity to antigens.

[0392] Table 29. Binding affinity of anti-antigen A antibodies

[0393] Antigen B is a sialic acid adhesin molecule and a member of the immunoglobulin superfamily. Immunization with antigen B against hVH... H / H / hVK - / - / hVL H / H Mice. The affinity of eight human common light chain antibodies against antigen B was then detected using Biacore™.

[0394] Table 30. Binding affinity of anti-antigen B antibody

[0395] Other implementation plans

[0396] It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A genetically modified non-human animal that contains one or more human IGLV genes, one or more human IGLJ genes and one or more human IGLC genes at the endogenous λ light chain immunoglobulin gene locus.

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

3. The animal of claim 2, wherein the animal is a mouse, and the disruption of the endogenous λ light chain immunoglobulin gene locus of the animal includes the deletion of one or more mouse IGLV genes in Table 4, one or more IGLJ genes in Table 5, and / or one or more mouse IGLC genes in Table 6.

4. The animal according to claim 2 or 3, wherein the animal is a mouse, and the disruption of the endogenous λ light chain immunoglobulin locus of the animal comprises a deletion of a continuous sequence from mouse IGLV2 to mouse IGLC1.

5. A genetically modified nonhuman animal comprising one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes at an endogenous safe harbor locus, optionally wherein the animal comprises disruption of its endogenous λ light chain immunoglobulin locus.

6. The animal according to claim 5, wherein the endogenous safe harbor locus is the endogenous Hipp11 locus or the endogenous Rosa26 locus.

7. The animal according to claim 5 or 6, wherein the animal is a mouse, and the one or more human IGLV genes, one or more human IGLJ genes and one or more human IGLC genes are inserted at the mouse Hipp11 locus.

8. The animal according to any one of claims 1 to 7, wherein the human IGLV gene, human IGLJ gene and human IGLC gene are operatively linked and rearrangeable.

9. The animal according to any one of claims 1 to 8, wherein the animal comprises at least 50 human IGLV genes from Table 1, at least 5 human IGLJ genes from Table 2, and at least 5 human IGLC genes from Table 3.

10. The animal according to any one of claims 1 to 8, wherein the animal comprises all human IGLV genes, all human IGLJ genes and all human IGLC genes at the endogenous λ light chain immunoglobulin locus on human chromosome 22 in a human subject.

11. The animal according to any one of claims 1 to 8, wherein the animal comprises all human IGLV genes, all human IGLJ genes and all human IGLC genes at the endogenous λ light chain immunoglobulin locus on human chromosome 22 in human cells.

12. The animal according to any one of claims 1 to 11, wherein the animal comprises an unmodified human sequence derived from the human λ light chain immunoglobulin locus, wherein the unmodified human sequence is at least 800 kb, at least 850 kb, at least 855 kb, at least 895 kb, at least 900 kb, at least 950 kb, or at least 1000 kb.

13. The animal according to any one of claims 1 to 12, wherein the animal comprises an unmodified sequence derived from the human globulin locus from human IGLV(I)-70 to human IGLV3-1.

14. The animal according to any one of claims 1 to 12, wherein the animal comprises an unmodified sequence derived from the human λ light chain immunoglobulin locus, from human IGLV(I)-70 to human IGLC7.

15. The animal according to any one of claims 1 to 14, wherein the animal is homozygous for the λ light chain immunoglobulin locus and / or the safe harbor locus.

16. The animal according to any one of claims 1 to 14, wherein the animal is heterozygous for the λ light chain immunoglobulin locus and / or the safe harbor locus.

17. The animal according to any one of claims 1 to 16, wherein the animal is a rodent (e.g., a mouse).

18. The animal according to any one of claims 1 to 17, wherein the animal further comprises one or more human IGHV genes, one or more human IGHD genes and one or more human IGHJ genes in the endogenous heavy chain immunoglobulin locus.

19. The animal of claim 18, wherein the human IGHV gene, human IGHD gene, and human IGHJ gene are operatively linked and capable of VDJ rearrangement.

20. The animal of claim 18 or 19, wherein the animal comprises at least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9.

21. The animal according to claim 18 or 19, wherein the animal contains all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus on human chromosome 14 in a human subject or cell.

22. The animal according to any one of claims 18 to 21, wherein the animal comprises the disruption of its endogenous heavy chain immunoglobulin locus.

23. The animal of claim 22, wherein the animal is a mouse, and the disruption of the endogenous heavy chain immunoglobulin locus of the animal comprises the 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.

24. The animal of claim 22, wherein the animal is a mouse, and the disruption of the endogenous heavy chain immunoglobulin locus of the animal comprises a deletion of a continuous sequence from mouse IGHV1-85 to mouse IGHJ4.

25. The animal according to any one of claims 18 to 24, wherein the animal comprises one or more endogenous IGHM genes, IGHδ genes, IGHG3 genes, IGHG1 genes, IGHG2b genes, IGHG2a genes, IGHE genes, and IGHA genes.

26. The animal according to any one of claims 18 to 25, wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus, wherein the unmodified human sequence is at least 800 kb.

27. The animal according to any one of claims 18 to 26, wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus, wherein the unmodified human sequence has one of the following characteristics: (1) from human IGHV(III)-82 to human IGHV1-2; (2) from human IGHV(III)-82 to human IGHV6-1; (3) from human IGHD1-1 to human IGHJ6; and (4) from human IGHV(III)-82 to human IGHJ6.

28. The animal according to any one of claims 18 to 27, wherein the animal is homozygous or heterozygous for the heavy chain immunoglobulin locus.

29. The animal according to any one of claims 1 to 28, wherein the animal further comprises one or more human IGKV genes and one or more human IGKJ genes at the endogenous κ light chain immunoglobulin gene locus.

30. The animal of claim 29, wherein the animal comprises all human IGKV genes in Table 13 and all human IGKJ genes in Table 14.

31. The animal according to claim 29 or 30, wherein the animal comprises all human IGKV genes and all human IGKJ genes at the endogenous κ light chain immunoglobulin locus on human chromosome 2 in a human subject or cell.

32. The animal according to any one of claims 29 to 31, wherein the animal comprises the disruption of its endogenous κ light chain immunoglobulin locus.

33. The animal of claim 32, wherein the animal is a mouse, and the disruption of the endogenous light chain immunoglobulin locus of the animal comprises the deletion of one or more mouse IGKV genes in Table 15 and one or more mouse IGKJ genes in Table 16.

34. The animal of claim 32 or 33, wherein the animal is a mouse, and the disruption of the endogenous κ light chain immunoglobulin locus of the animal comprises a deletion of the sequence from mouse IGKV2-137 to mouse IGKJ5.

35. The animal according to any one of claims 29 to 34, wherein the animal comprises an unmodified sequence derived from the human κ light chain immunoglobulin locus from human IGKV3D-7 to human IGKJ5.

36. The animal according to any one of claims 29 to 35, wherein the animal comprises endogenous IGKC.

37. The animal according to any one of claims 29 to 35, wherein the animal is homozygous or heterozygous for the κ light chain immunoglobulin locus.

38. A genetically modified non-human animal, the genome of which comprises an endogenous light chain immunoglobulin locus, the endogenous light chain immunoglobulin locus comprising: replacing one or more endogenous IGLV genes, endogenous IGLJ genes, and endogenous IGLC genes with one or more human IGLV genes, human IGLJ genes, and human IGLC genes, wherein, The human IGLV gene, human IGLJ gene, and human IGLC gene are operatively linked.

39. The animal of claim 38, wherein one or more endogenous IGLV genes, endogenous IGLJ genes and endogenous IGLC genes are replaced by all human IGLV genes in Table 1, all human IGLJ genes in Table 2 and all human IGLC genes in Table 3.

40. The animal of claim 38 or 39, wherein the animal is a mouse, and all mouse IGLV genes in Table 4, all mouse IGLJ genes in Table 5, and all mouse IGLC genes in Table 6 are replaced.

41. A genetically modified nonhuman animal whose genome includes an endogenous safe harbor locus, the endogenous safe harbor locus including: an insertion of one or more human IGLV, human IGLJ and human IGLC genes, wherein the human IGLV, human IGLJ and human IGLC genes are operatively linked, optionally, wherein the animal includes disruption of its endogenous λ light chain immunoglobulin locus.

42. The animal of claim 41, wherein all human IGLV genes in Table 1, all human IGLJ genes in Table 2, and all human IGLC genes in Table 3 are inserted at the endogenous safe harbor locus.

43. The animal according to claim 41 or 42, wherein the endogenous safe harbor locus is the endogenous Hipp11 locus or the endogenous Rosa26 locus.

44. The animal according to any one of claims 1 to 43, wherein the animal lacks an endogenous immunoglobulin λ light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous λ light chain variable domain (e.g., mouse λ light chain variable domain).

45. The animal according to any one of claims 1 to 44, wherein the animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain (e.g., mouse heavy chain variable domain).

46. ​​The animal according to any one of claims 1 to 45, wherein the animal lacks an endogenous immunoglobulin κ light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous κ light chain variable domain (e.g., mouse κ light chain variable domain).

47. The animal according to any one of claims 1 to 46, wherein the animal is capable of producing humanized antibodies.

48. A cell obtained from any one of claims 1 to 47.

49. The cell of claim 48, wherein the cell is a B cell expressing an immunoglobulin λ light chain, the immunoglobulin λ light chain being derived from rearrangements of one or more human IGLV genes, one or more human IGLJ genes, and one or more human IGLC genes.

50. The cell of claim 48 or 49, wherein the cell is a B cell expressing a chimeric immunoglobulin heavy chain, the chimeric immunoglobulin heavy chain comprising a rearranged immunoglobulin heavy chain variable domain derived from one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the immunoglobulin heavy chain variable domain is operatively linked to a nonhuman heavy chain constant region.

51. The cell according to any one of claims 48 to 50, wherein the cell is a B cell expressing a chimeric immunoglobulin κ light chain, the chimeric immunoglobulin κ light chain comprising a rearranged immunoglobulin κ light chain variable domain derived from one or more human IGKV genes and one or more human IGKJ genes, wherein the immunoglobulin κ light chain variable domain is operatively linked to a non-human κ light chain constant region.

52. The cell of claim 48, wherein the cell is an embryonic stem (ES) cell.

53. A method for preparing a chimeric antibody that specifically binds to an antigen, the method comprising exposing an animal according to any one of claims 1 to 47 to the antigen; generating a hybridoma from cells collected from the animal; and collecting the chimeric antibody generated from the hybridoma.

54. The method of claim 53, wherein the method further comprises sequencing the genome of the hybridoma.

55. A method for modifying the genome of a cell, the method comprising modifying one or more human chromosomes; introducing the modified one or more human chromosomes into the cells of the animal; and inducing recombination between the modified one or more human chromosomes and one or more endogenous chromosomes, wherein, At least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9 are integrated into one or more endogenous chromosomes (e.g., mouse chromosome 12) through recombination; and / or at least 50 human IGLV genes from Table 1, at least 5 human IGLJ genes from Table 2, and at least 5 human IGLC genes from Table 3 are integrated into one or more endogenous chromosomes (e.g., mouse chromosome 16 or 11) through recombination.

56. A method for modifying the genome of a cell, the method comprising modifying one or more human chromosomes; introducing the modified one or more human chromosomes into the cells of the animal; and inducing recombination between the modified one or more human chromosomes and one or more endogenous chromosomes, wherein, At least 150 human IGHV genes selected from Table 7, at least 20 human IGHD genes selected from Table 8, and at least 5 human IGHJ genes selected from Table 9 were integrated into one or more endogenous chromosomes (e.g., mouse chromosome 12) through recombination; at least 50 human IGKV genes selected from Table 13 and at least 3 human IGKJ genes selected from Table 14 were integrated into one or more endogenous chromosomes (e.g., mouse chromosome 6) through recombination; and / or at least 50 human IGLV genes selected from Table 1, at least 5 human IGLJ genes selected from Table 2, and at least 5 human IGLC genes selected from Table 3 were integrated into one or more endogenous chromosomes (e.g., mouse chromosome 16 or 11) through recombination.

57. A method for preparing an antibody that specifically binds to an antigen, the method comprising exposing an animal according to any one of claims 1 to 47 to the antigen; sequencing nucleic acids encoding variable regions of human heavy chain and light chain immunoglobulins in cells, said cells expressing chimeric antibodies that specifically bind to the antigen; and expressing nucleic acids encoding the variable regions and constant regions of said human heavy chain immunoglobulins and human heavy chain immunoglobulins, as well as nucleic acids encoding the variable regions and constant regions of said human light chain immunoglobulins, in the cells.

58. A method for preparing an antibody that specifically binds to an antigen, the method comprising obtaining a nucleic acid sequence encoding the variable regions of human heavy and light chain immunoglobulins in a cell expressing a chimeric antibody that specifically binds to said antigen, wherein, The cells are obtained by exposing the animal of any one of claims 1 to 47 to the antigen; preparing a first nucleic acid, wherein the nucleic acid encoding the variable region of the human heavy chain immunoglobulin is operatively linked in the first nucleic acid to a nucleic acid encoding the constant region of the human heavy chain immunoglobulin; preparing a second nucleic acid, wherein the nucleic acid encoding the variable region of the human light chain immunoglobulin is operatively linked in the second nucleic acid to a nucleic acid encoding the constant region of the human light chain immunoglobulin; and expressing the first nucleic acid and the second nucleic acid in the cells to obtain the antibody.

59. A method for obtaining nucleic acid, said nucleic acid encoding an antibody-binding domain that specifically binds to an antigen, said method comprising exposing an animal according to any one of claims 1 to 47 to said antigen; and sequencing a nucleic acid in a cell encoding variable regions of human heavy and light chain immunoglobulins, said cell expressing a chimeric antibody that specifically binds to said antigen.

60. A method of obtaining a sample, the method comprising exposing an animal according to any one of claims 1 to 47 to the antigen; and collecting the sample from the animal.

61. The method of claim 60, wherein the sample is spleen tissue, spleen cells, or B cells.

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