Genetically modified non-human animals with humanized immunoglobulin locus

Abstract

To provide genetically modified animals and cells with a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus.SOLUTION: A genetically modified, non-human animal comprises, at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes and one or more human IGHJ genes, where the human IGHV genes, the human IGHD genes and the human IGHJ genes are operably linked and can undergo VDJ rearrangement.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Claim of Priority This application claims priority to PCT / CN2019 / 075406, filed on February 18, 2019, and PCT / CN2019 / 106320, filed on September 18, 2019. The entire contents of the above patent applications are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to genetically modified animals and cells having a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus.

Background Art

[0003] Therapeutic antibodies are one of the most rapidly growing classes of therapeutic compounds and are overtaking the growth of small molecule drugs at a remarkable pace. Such therapeutic antibodies are typically human or humanized antibodies. Human or humanized antibodies can be created by humanizing rodent antibodies (e.g., mouse antibodies) or by using phage libraries. Antibodies created by these methods often do not optimally meet the binding affinity and biophysical properties, leading to manufacturing difficulties and poor pharmacokinetics. In particular, the humanization process can negatively affect the binding affinity and may introduce immunogenic epitopes into the antibody, and antibodies discovered using phage libraries exhibit unnatural pairing of immunoglobulin heavy and light chains with limited diversity. In many cases, it is necessary to repeat experiments and spend time to improve the characteristics. Also, in some cases, such antibodies can be immunogenic in patients, leading to a decrease in their effectiveness over time.

Summary of the Invention

Problems to be Solved by the Invention

[0004] One possible approach to generating fully human antibodies is to use transgenic animals engineered to express a human antibody repertoire. The creation of transgenic animals, such as mice with altered immunoglobulin loci, enables the use of such transgenic animals in a variety of research and development applications, for example in drug discovery and basic research regarding various biological systems. Many of the initial generations of transgenic animals had an incomplete human antibody repertoire, had antibody production rates below normal due to inefficient V(D)J recombination, had an endogenous antibody repertoire that could introduce immunogenic epitopes, and had various other problems. There is a need for an efficient and cost-effective method of producing human antibodies, and there is a need for non-human animals that have a humanized immunoglobulin locus capable of generating humanized antibodies in response to an antigen.

Means for Solving the Problems

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

[0006] In some embodiments, the present disclosure relates to genetically modified non-human animals that include one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes in an endogenous heavy chain immunoglobulin locus. In some embodiments, the human IGHV gene, the human IGHD gene, and the human IGHJ gene are operably linked and capable of undergoing VDJ recombination.

[0007] In some embodiments, the animal comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 human IGHV genes selected from Table 1, about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2, and about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3. In some embodiments, the animal comprises all human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, and IGHV1-8, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes in the endogenous heavy-chain immunoglobulin locus of human chromosome 14 of a human subject. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes in the endogenous heavy-chain immunoglobulin locus of human chromosome 14 of human cells (e.g., somatic cells, cultured cells, non-immune cells, cells without any V(D)J rearrangement).

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

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

[0010] In some embodiments, the animal is a mouse, and disruption of the endogenous heavy-chain immunoglobulin locus of the animal comprises deletion of a contiguous sequence starting from the mouse IGHV1-85 gene to the mouse IGHJ4 gene.

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

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

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

[0014] In some embodiments, the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus beginning at human IGHV(III)-82 and extending to human IGHV1-2. In some embodiments, the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus beginning at human IGHV(III)-82 and extending to human IGHV6-1. In some embodiments, the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus beginning at human IGHD1-1 and extending to human IGHJ6.

[0015] In some embodiments, the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus beginning at human IGHV(III)-82 and extending to human IGHJ6.

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

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

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

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

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

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

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

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

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

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

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

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

[0028] In some embodiments, the animal is a mouse, and disruption of the animal's endogenous heavy chain immunoglobulin locus comprises deletion of the sequence starting from mouse IGHV1-85 to mouse IGHJ4.

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

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

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

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

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

[0034] In some aspects, the disclosure relates to a genetically modified non-human animal comprising one or more human IGKV genes and one or more human IGKJ genes in the endogenous light chain immunoglobulin locus.

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

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

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

[0038] In some embodiments, the animal is a mouse, and disruption of the animal's endogenous light chain immunoglobulin locus comprises deletion of one or more mouse IGKV genes in Table 9 and one or more mouse IGKJ genes in Table 10. In some embodiments, the animal comprises all human IGKV genes and all human IGKJ genes at the endogenous κ chain immunoglobulin locus of human chromosome 2 of a human subject. In some embodiments, the animal comprises all human IGKV genes and all human IGKJ genes at the endogenous κ immunoglobulin locus of human chromosome 2 of human cells (e.g., somatic cells, cultured cells, non-immune cells, cells without any V(D)J recombination).

[0039] In some embodiments, the animal is a mouse, and disruption of the animal's endogenous light chain immunoglobulin locus comprises deletion of the sequence starting from mouse IGKV2-137 to mouse IGKJ5.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0053] In some embodiments, the animal is capable of producing humanized antibodies.

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

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

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

[0057] In some embodiments, the cell is an embryonic stem (ES) cell.

[0058] In some aspects, the disclosure relates to a method of making a chimeric antibody that specifically binds an antigen, the method comprising exposing an animal as described herein to the antigen; generating hybridomas from cells recovered from the animal; and recovering the chimeric antibody produced by the hybridomas. In some embodiments, the cells of interest are isolated and the sequences of the rearranged heavy and light chain variable regions are determined by performing sequencing.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Brief Description of the Drawings

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Mode for Carrying Out the Invention

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

[0079] A genetically modified animal can be produced by introducing a human immunoglobulin gene into the genome of a non-human animal to produce an animal capable of expressing a humanized antibody or a chimeric antibody. Figure 1A shows a method for producing a humanized mouse. In some embodiments, the method first involves modifying the human immunoglobulin region on a human chromosome. Next, the modified human chromosome is introduced into mouse recipient cells. Next, the human immunoglobulin variable region is introduced by direct replacement (e.g., in a one-step replacement) into the corresponding region of the mouse genome. Next, the recipient cells are screened, preferably with respect to cells that do not contain the human chromosome. Next, the cells are injected into blastocysts to prepare chimeric animals (e.g., mice). Subsequently, breeding is carried out, and animals containing an intact humanized immunoglobulin locus can be obtained.

[0080] The genetically modified animals described in this specification can have various advantages. For example, in some cases, the genetically modified animals described in this specification have a complete human antibody repertoire. Therefore, the variable domains created by such animals can have a diversity very similar to the diversity of human variable domains. Furthermore, since the entire sequence of the human immunoglobulin locus (either unmodified or with limited modifications) is introduced into the animal genome, these genes can undergo V(D)J recombination very similar to that which occurs in humans. In addition, antibody production can be extremely efficient, and because V(D)J recombination is efficient, the production rate is comparable to the normal production rate. In addition, since V(D)J recombination can occur between the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes and the human genes, when the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes are incorporated into the rearranged heavy chain VDJ segment or the rearranged light chain VJ segment, the antibodies created by the antibody repertoire are likely to have immunogenic epitopes in humans. This immunogenicity can lead to the production of anti-drug antibodies and can include efficacy. Here, the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes have been substantially deleted. The antibodies created by this antibody repertoire are less likely to be immunogenic in humans. Therefore, this antibody is more suitable for use as a therapeutic agent in humans. Therefore, the genetically modified animals provide an advantageous platform for the production of humanized antibodies.

[0081] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, linked to each other by disulfide bonds. Each heavy chain comprises a heavy chain variable (VH) domain and a heavy chain constant region (CH). Each light chain comprises a light chain variable (VL) domain and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with regions called more conserved framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy 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 having a K D of about 10 -9 M or less (e.g., about 1×10 -9 M, 1×10 -10 M, 1×10 -11 M or 1×10 -12 M or less) for its target epitope. In some embodiments, K D can be measured by surface plasmon resonance, e.g., BIACORE™ or ELISA.

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

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

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

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

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

[0087] As used herein, the terms "subject" and "patient" are used interchangeably throughout this specification and refer to an animal, human or non-human. Depending on the disclosure, veterinary and non-veterinary applications are contemplated. A human patient can be an adult human or a juvenile human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, mini-pigs), equids, canids, felids, bovids and other domesticated animals, farm animals and zoo animals are included.

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

[0089] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to an amino acid polymer of any length that is at least 2 amino acids.

[0090] As used herein, the terms "polynucleotide", "nucleic acid molecule" and "nucleic acid sequence" are used interchangeably herein to refer to a nucleotide polymer of any length that is at least 2 nucleotides and includes, without limitation, DNA, RNA, DNA / RNA hybrids and modifications thereof.

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

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

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

[0094] During B cell development, a single D segment (also known as the IGHD gene) is joined to a J segment (also known as the IGHJ gene) by a recombination event at the DNA level. Next, the fused D-J exon of this partially rearranged D-J region is joined to a V segment (also known as the IGHV gene). Next, the rearranged V-D-J region containing the fused V-D-J exon is transcribed and fused to the IGHM constant region at the RNA level. This transcript encodes the μ heavy chain. In late development, B cells produce V-D-J-Cμ-Cδ pre-messenger RNA, which, upon undergoing alternative splicing, comes to encode either the μ heavy chain or the δ heavy chain. Mature B cells in the lymph nodes undergo switch recombination, whereupon the fused V-D-J gene segment comes into proximity with one of the IGHC, IGHA, or IGHE gene segments, and each cell expresses either the γ, α, or ε heavy chain. Recombination of many different IGHV genes with several IGHJ genes can occur, conferring broad antigen recognition. Additional diversity is achieved by junctional diversity resulting from random nucleotide addition by terminal deoxynucleotidyl transferase and by somatic hypermutation that occurs during B cell maturation in the spleen and lymph nodes. Several V, D, J, and C segments are known to lack the ability to encode a protein and are considered pseudogene segments (often simply referred to as pseudogenes).

[0095] The human heavy chain immunoglobulin locus is located on human chromosome 14. Table 1 shows a list of the IGHV genes in this locus and their relative order.

[0096]

Table 1

[0097] RPS8P1, ADAM6, and KIAA0125 are also located at this locus. The relative order of RPS8P1 is 160, the relative order of ADAM6 is 161, and the relative order of KIAA0125 is 164. Table 2 shows a list of all IGHD genes on human chromosome 14 and their relative orders. Table 3 shows a list of all IGHJ genes on human chromosome 14 and their relative orders. Genes for immunoglobulin constant domains are located behind the IGHV, IGHD, and IGHJ genes. These genes include the following (in the order shown below): immunoglobulin heavy chain constant mu (IGHM), immunoglobulin heavy chain constant delta (IGHδ), immunoglobulin heavy chain constant gamma 3 (IGHG3), immunoglobulin heavy chain constant gamma 1 (IGHG1), immunoglobulin heavy chain constant epsilon P1 (pseudogene) (IGHEP1), immunoglobulin heavy chain constant alpha 1 (IGHA1), immunoglobulin heavy chain constant gamma P (non-functional) (IGHGP), immunoglobulin heavy chain constant gamma 2 (IGHG2), immunoglobulin heavy chain constant gamma 4 (IGHG4), immunoglobulin heavy chain constant epsilon (IGHE), and immunoglobulin heavy chain constant alpha 2 (IGHA2). These genes and their order are also shown in FIGS. 37, 41A, 41B, 41C, 41D, 41E, and 41F.

[0098]

Table 2

[0099]

Table 3

[0100] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12. Table 4 shows a list of IGHV genes at this locus and their relative orders.

[0101]

Table 4

[0102] Table 5 shows a list of all the IGHD genes on mouse chromosome 12 and their relative order. Table 6 shows a list of all the IGHJ genes on mouse chromosome 12 and their relative order. There are genes encoding immunoglobulin constant domains following the IGHV, IGHD, and IGHJ genes. These genes include the following (in the order shown): immunoglobulin heavy chain constant μ (IGHM), immunoglobulin heavy chain constant δ (IGHδ), immunoglobulin heavy chain constant γ3 (IGHG3), immunoglobulin heavy chain constant γ1 (IGHG1), immunoglobulin heavy chain constant γ2b (IGHG2b), immunoglobulin heavy chain constant γ2a (IGHG2a), immunoglobulin heavy chain constant ε (IGHE), and immunoglobulin heavy chain constant α (IGHA) genes. These genes and their order are also shown in FIGS. 38A, 38B, 42A, 42B, 42C, 42D, 42E, 42F, 42G, 42H, and 42I.

[0103]

Table 5

[0104]

Table 6

[0105] The present disclosure provides a genetically modified non-human animal comprising one or more human IGHV genes, one or more human IGHD genes, and / or one or more human IGHJ genes. In some embodiments, the human IGHV gene, the human IGHD gene, and the human IGHJ gene are operably linked to each other and are capable of undergoing VDJ recombination. In some embodiments, the human IGHV gene, the human IGHD gene, and the human IGHJ gene are at the endogenous heavy chain immunoglobulin locus.

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

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

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

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

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

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

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

[0113] In some embodiments, it is an unmodified human sequence derived from the human heavy chain immunoglobulin locus starting from human IGHD1-1 and ending at human IGHD7-27.

[0114] In some embodiments, it is an unmodified human sequence derived from the human heavy chain immunoglobulin locus starting from human IGHJ1P and ending at human IGHJ6. In some embodiments, it is an unmodified human sequence derived from the human heavy chain immunoglobulin locus starting from human IGHJ1 and ending at human IGHJ6.

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

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

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

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

[0119] In some embodiments, the IGHV gene, the IGHD gene, and / or the IGHJ gene are operably linked to each other. VDJ recombination occurs between these genes, and a functional antibody can be produced. In some embodiments, these genes are arranged in an order similar to the order of the human heavy-chain immunoglobulin locus. This arrangement provides various advantages. For example, because these genes are arranged in this way, it is possible to produce a heavy-chain variable domain having a diversity very similar to the diversity of the heavy-chain variable domain in humans. Since some random sequences may be inserted into the sequence during VDJ recombination, in some embodiments, in a fully human antibody repertoire with no or minimal modifications, the possibility of non-human sequences being inserted during VDJ recombination can be reduced.

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

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

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

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

[0124] In some embodiments, disruption of the endogenous heavy chain immunoglobulin locus in an animal comprises 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 murine IGHD genes (e.g., genes as shown in Table 5). In some embodiments, the disruption comprises deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 murine IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 murine IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1.

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

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

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

[0128] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that are about or 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 sequences are about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, or 3500 kb in length. In some embodiments, the sequences are from human IGHV(III)-82 to IGHV1-2. In some embodiments, the sequences are from human IGHV7-81 to IGHV1-2. In some embodiments, the sequences are from human IGHV(II)-1-1 to IGHVJ6. In some embodiments, the sequences are from human IGHV6-1 to IGHVJ6.

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

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

[0131] Furthermore, in some cases, the entire mouse IGHV gene, IGHD gene and IGHJ gene (e.g., including all non-pseudogenes) are knocked out, and there is no sequence encoded by a mouse-derived sequence in the variable region of the heavy chain, thus minimizing immunogenicity in humans.

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

[0133] When the V segment (also known as the IGKV gene) and the J segment (also known as the IGKJ gene) are joined, a continuous exon encoding the entire light chain variable domain is created. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far from the C region. The J gene segment (or IGKJ gene cluster) is located close to the C region. When the V segment joins the J gene segment, the V gene also approaches the C region sequence. The J gene segment of the rearranged V region is separated from the C region sequence by only one intron. To generate a full-length immunoglobulin light chain messenger RNA, the V region exon is joined to the C region sequence by RNA splicing after transcription.

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

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

[0136]

Table 7

[0137] Table 8 shows a list of all the IGKJ genes on human chromosome 2 and their relative order. Behind the IGKV and IGKJ genes, the immunoglobulin kappa constant (IGKC) gene encoding the light chain immunoglobulin constant domain is located. These genes and their order are also shown in FIGS. 39, 43A, 43B, 43C, and 43D.

[0138]

Table 8

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

[0140]

Table 9

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

[0142]

Table 10

[0143] The present disclosure provides a genetically modified non-human animal comprising one or more human IGKV genes and / or one or more human IGKJ genes. In some embodiments, the human IGKV gene and the human IGKJ gene are operably linked to each other and can undergo VJ recombination. In some embodiments, the human IGKV gene and the human IGKJ gene are at an endogenous light chain immunoglobulin locus.

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

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

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

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

[0148] In some embodiments, the animal comprises endogenous IGKC. In some embodiments, the IGKV gene and / or the IGKJ gene are operably linked to each other. VJ recombination can occur between these genes, and a functional antibody can be produced. In some embodiments, these genes are arranged in an order similar to the order of the human light chain immunoglobulin locus. This arrangement provides various advantages. For example, because these genes are arranged in this way, it is possible to produce light chain variable domains with a diversity very similar to the diversity of light chain variable domains in humans.

[0149] In some embodiments, the IGKV gene and / or the IGKJ gene are operably linked to the IGKC gene (e.g., the endogenous IGKC gene).

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

[0151] In some embodiments, the animal is a mouse. The disruption of the animal's endogenous heavy chain immunoglobulin locus comprises 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 as shown in Table 9). 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 IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130 and IGKV9-129. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130 and IGKV9-129.

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

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

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

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

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

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

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

[0159] The human IGKV gene and the human IGKJ gene are operably linked to each other and can undergo VJ recombination. In some embodiments, the modified mouse has a complete human IGKV and IGKJ gene repertoire (e.g., including all non-pseudo human IGKV and IGKJ genes). Thus, the modified mouse can produce a complete human antibody repertoire. In some embodiments, after VJ recombination, one of the IGKV genes in Table 16 (e.g., IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12) contributes to the sequence encoding the variable region of the antibody light chain. One human IGKJ gene contributes to the sequence encoding the variable region of the antibody light chain. In some embodiments, the IGKV gene is IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12. Further, in some cases, the entire mouse IGKV gene and IGKJ gene (all non-pseudo genes) are knocked out, and there is no sequence encoded by a mouse-derived sequence in the variable region of the light chain, thus minimizing immunogenicity in humans.

[0160] In some embodiments, the modified chromosome contains the human proximal Vκ cluster IGKV genes. In some embodiments, the modified chromosome contains the human distal Vκ cluster IGKV genes. In some embodiments, the modified chromosome contains both the human proximal Vκ cluster IGKV genes and the human distal Vκ cluster IGKV genes.

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

[0162] In some embodiments, the animal contains the human λ light chain immunoglobulin locus.

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

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

[0165]

Table 11

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

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

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

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

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

[0171] Genetically modified non-human animals can be various animals, such as mice, rats, rabbits, pigs, cattle (e.g., female cattle, male cattle, calves), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modifiable embryonic stem (ES) cells cannot be easily obtained, other methods are used for the production of non-human animals containing genetic modifications. Such methods include, for example, modification of non-ES cell genomes (e.g., fibroblasts or induced pluripotent cells) and transfer of the modified genome to suitable cells, such as oocytes, using nuclear transfer, and formation of embryos by pregnancy of the modified cells (e.g., modified oocytes) in non-human animals under suitable conditions. These methods are known in the art and are described, for example, in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)”, Cold Spring Harbor Laboratory Press, 2003 (which is hereby incorporated by reference in its entirety). In this way, in various embodiments, human V, D, and / or J segments can be operably linked to non-human animal (e.g., rodent, mouse, rat, hamster) constant region gene sequences. During B cell development, these rearranged human V, D, and / or J segments are linked to non-human animal immunoglobulin constant regions.

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

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

[0174] In some embodiments, the animal is a rat. The rat can be selected from Wistar rats, LEA strain, Sprague-Dawley strain, Fisher strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a hybrid of two or more strains selected from the group consisting of Wistar, LEA, Sprague-Dawley, Fisher, F344, F6, and Dark Agouti.

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

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

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

[0178] For the non-human mammals described above, genetic analysis, molecular analysis, and behavioral analysis can be performed. This disclosure also relates to progeny produced by mating the non-human mammals provided by this disclosure with the same or other genotypes. The non-human mammals 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.

[0179] This disclosure also provides cell lines or primary cell cultures derived from non-human mammals or their progeny. Cell culture-based models can be prepared, for example, by the following methods. Cell cultures can be obtained through isolation from non-human mammals. Alternatively, cells can be obtained from cell cultures established using the same construct and standard cell transfection techniques. The integration of a gene construct containing a DNA sequence encoding a human or humanized immunoglobulin can be detected by various methods.

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

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

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

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

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

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

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

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

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

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

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

[0191] In some implementations, the antibody (or its antigen-binding fragment) specifically binds to the target with a dissociation rate (koff) of less than 0.1 s -1 less than 0.01 s -1 less than 0.001 s -1 less than 0.0001 s -1 less than or 0.00001 s -1 less than. In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 greater than 0.001 s -1 greater than 0.0001 s -1 greater than 0.00001 s -1 greater than or 0.000001 s -1 greater than.

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

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

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

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

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

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

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

[0199] Method for producing a genetically modified animal Genetically modified animals can be produced by introducing human immunoglobulin genes into the genome of non-human animals to produce animals that can express humanized antibodies or chimeric antibodies. Figure 1A shows a method for producing humanized animals. In some embodiments, the method initially involves modifying the human immunoglobulin locus on the human chromosome. Next, the modified human chromosome is introduced into mouse recipient cells. Next, the human immunoglobulin variable region is introduced by direct replacement into the corresponding region of the mouse genome. Next, the recipient cells are screened. In some embodiments, the cells do not contain the human chromosome. Next, the cells are injected into blastocysts to prepare chimeric mice. Subsequently, breeding is carried out, and mice containing intact humanized immunoglobulin loci can be obtained.

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

[0201] The gene modification process can include replacing an endogenous sequence with a human sequence by homologous recombination. In some embodiments, cleavage (e.g., by zinc finger nucleases, TALENs or CRISPR) upstream and downstream of the target site can result in a DNA double-strand break, and the endogenous sequence is replaced with a human sequence using homologous recombination.

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

[0203] FIG. 9 shows a targeting strategy for adding a functional genetic element to a human chromosome. These vectors can be inserted between the upstream of the V region and between the J region and the C region.

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

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

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

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

[0208] Human chromosomes can be obtained from human cell lines, cancer cells, primary cell cultures and / or human fibroblasts. In some embodiments, a first vector is introduced into a human cell and then fused with a recipient cell. Next, the modified chromosome is isolated and introduced into another suitable recipient cell. By selecting cells with the desired resistance, cells containing only one human chromosome are obtained. Next, a second vector is introduced into the cells and the cells are selected by resistance. Next, a third vector and / or a fourth vector can be introduced as needed. The recipient cell can be a mammalian cell, a human cell or a mouse cell. In some embodiments, the recipient cell is a CHO cell or preferably an A9 cell. In some embodiments, the modified chromosome is labeled by fluorescence and isolated. Also, the modified chromosome is injected into the recipient cell by chromosome microinjection. In some embodiments, donor cells are induced to multinucleate their chromosomes. Next, when these nuclei are pushed through the cell membrane, microcells are created, which can be fused with recipient cells. In some embodiments, microcell-mediated chromosome transfer can also be used.Regarding chromosomal manipulation techniques, for example, see the specifications of Chinese Patent Application Publication No. 1200014A; Chinese Patent Application Publication No. 109837307A; US Patent Application Publication No. 20120093785A1; and US Patent Application Publication No. 2009253902; Kuroiwa et al., “Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts,” Nature Biotechnology 18, no. 10 (2000): 1086-1090; the specification of Chinese Patent Application Publication No. 1717483A; Paulis, Marianna, “Chromosome Transfer Via Cell Fusion,” Methods in Molecular Biology 738 (2011): 57; Genes, Chromosomes & Cancer 14: 126-127 (1995); Tomizuka et al., “Functional expression and germline transmission of a human chromosome fragment in chimaeric mice,” Nature Genetics 16, no. 2 (1997): 133-143; Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284 (each of which is incorporated herein by reference in its entirety).

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

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

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

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

[0213] LoxP recognition sequences can also be added to the human chromosome (e.g., human chromosomes 2, 14, 22). The cells can also be treated with Cre enzyme, leading to recombination of the loxP sites and thus removal of the genomic DNA sequence. In some embodiments, genomic DNA sequences can be removed using natural chromosomal breaks as well.

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

[0215] The mouse immunoglobulin variable region can be replaced with a human immunoglobulin variable region by substitution (e.g., homologous recombination or Cre-mediated recombination). In some embodiments, the substitution can be mediated using Cre recombination. In some embodiments, the vector can add a LoxP recognition sequence to the human chromosome. Similar modifications can be made to the mouse chromosome, where two LoxP recognition sequences can be added to the chromosome. For example, next, Cre recombinase can mediate the substitution from the V, J regions on the mouse chromosome to the V, J regions on the human chromosome or from the V, D, J regions on the mouse chromosome to the V, D, J regions on the human chromosome.

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

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

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

[0219] These modifications can be carried out in various cells. In some embodiments, the cells are stem cells, embryonic stem cells, or fertilized egg cells.

[0220] The present disclosure is a method for establishing a humanized animal model, comprising the following steps: (a) providing a cell (e.g., a fertilized egg cell) based on the method described herein; (b) culturing the cell in a liquid culture medium; (c) transplanting the cultured cell into the fallopian tube or uterus of a recipient female non-human mammal, thereby enabling the cell to develop in the uterus of the female non-human mammal; (d) Identifying germline transmission in the genetically modified humanized non-human mammalian offspring of the pregnant female of step (c). The present invention further provides a method comprising the same.

[0221] In some embodiments, 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).

[0222] In some embodiments, the non-human mammal of step (c) is a female in pseudopregnancy (or false pregnancy).

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

[0224] The fertilized egg can be from any non-human animal, for example, any non-human animal as described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection of DNA. For example, after culturing the fertilized egg after microinjection, the cultured fertilized egg can be transplanted into a pseudopregnant non-human animal, and then when it gives birth to a non-human mammal, the non-human mammal mentioned in the method described above will be created.

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

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

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

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

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

[0230] A method for creating a genetically modified animal model comprising an additional human or chimeric gene (e.g., humanized gene) includes the following steps: (a) The step of obtaining a genetically modified non-human animal using the method as described in this specification; (b) The step of obtaining a genetically modified non-human animal containing two or more human or chimeric genes by mating a genetically modified non-human animal with another genetically modified non-human animal and then screening the offspring can be included.

[0231] In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal having human or chimeric PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa or OX40. Some of these genetically modified non-human animals are described, for example, in PCT / CN2017 / 090320, PCT / CN2017 / 099577, PCT / CN2017 / 099575, PCT / CN2017 / 099576, PCT / CN2017 / 099574, PCT / CN2017 / 106024, PCT / CN2017 / 110494, PCT / CN2017 / 110435, PCT / CN2017 / 120388, PCT / CN2018 / 081628, PCT / CN2018 / 081629 (each of which is incorporated herein by reference in its entirety).

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

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

[0234] In some embodiments, a functional mouse ADAM6 locus can be placed at the center of the human IGHV gene cluster. In some embodiments, the mouse ADAM6 locus is between two human IGHV genes. In some embodiments, the human ADAM6 pseudogene between human VH1-2 and human VH(II)-1-1 is replaced with the mouse ADAM6 locus. In some embodiments, the ADAM6a gene and the ADAM6b gene are located between human IGHV1-2 and human VH(II)-1-1 in the genome of the animal. In some embodiments, the location of the mouse ADAM6 sequence within the human gene sequence can approximate the position of the human ADAM6 pseudogene (e.g., within the V-D intergenic region) or can approximate the position of the mouse ADAM6 sequence. 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 gene and the IGHV6-1 gene. Placing mouse ADAM6a and mouse ADAM6b between the human IGHV1-2 gene and the IGHV6-1 gene can have various advantages. For example, since these genes replace the human ADAM6 gene at the same locus, the effect of the replacement of the human ADAM6 gene on VDJ recombination is limited, and the mouse ADAM6a and mouse ADAM6b genes are also likely to function normally (due to being at a similar location as the endogenous locus).

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

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

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

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

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

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

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

[0242] The fertility of the mouse can be increased using various methods. In some embodiments, superovulated female mice can be used for mating. In some embodiments, in vitro fertilization can be used. Superovulation can be induced by injecting a mature female mouse with serum gonadotropin and chorionic gonadotropin (e.g., human or mouse CG). A mature male mouse can be sacrificed and the caudal epididymis can be excised. The ducts of the caudal epididymis can be incised to release sperm. Next, a mature female mouse during superovulation can be sacrificed and the oviducts can be excised. Cumulus-oocyte complexes (COCs) can be released from the oviducts. Next, a sperm suspension can be added to the COCs and incubated for fertilization. Abnormal oocytes containing only one pronucleus can be removed. After incubation, two-cell stage embryos can be transferred to a recipient female. Methods for increasing mouse fertility are known in the art.

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

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

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

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

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

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

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

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

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

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

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

[0254] In one aspect, the present disclosure provides a mouse comprising a modification of the endogenous immunoglobulin heavy chain locus, wherein the mouse produces B cells comprising a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises 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.

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

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

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

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

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

[0260] In some embodiments, none of the murine immunoglobulin V, D, J genes (e.g., none of the murine IGHV, IGHD, IGHJ, IGKV or IGKJ genes) contribute to the heavy and / or light chain variable region sequences. In some embodiments, the heavy and / or light chain variable region sequences produced by the animal are fully human and are fully contributed to by human immunoglobulin V, D, J genes (e.g., human IGHV, IGHD, IGHJ, IGKV and IGKJ genes).

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

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

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

[0264] Further modifications can be made to the present antibody or antigen-binding fragment. For example, one or more cysteine residues can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced can have a somewhat 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, for example, as described in Wolff et al. (Cancer Res. 53:2560-2565, 1993), using a heterobifunctional cross-linking agent. Alternatively, antibodies having a double Fc region can be engineered (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0265] In some embodiments, covalent modifications can be made to the present antibody or its antigen-binding fragment. Such covalent modifications can be made by chemical or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting a targeted amino acid residue of the antibody or fragment with an organic derivatizing agent having the ability to react with a selected side chain or N-terminal or C-terminal residue.

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

Examples

[0267] Example 1: Overview An experiment was conducted to introduce human immunoglobulin genes into the mouse genome to produce mice that express humanized antibodies. Figure 1A shows the method for producing humanized mice. This method initially involves modifying the human immunoglobulin region on human chromosomes. Next, the modified human chromosomes were introduced into mouse recipient cells.

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

[0269] Since the mouse heavy chain gene and the two light chain genes are located on chromosomes 12, 6, and 16, respectively, mice containing the human heavy chain variable region or the human light chain variable region can be prepared separately (Figures 1B and 1C). Next, when these mice are mated with each other, mice that can express both the human heavy chain variable domain and the human light chain variable domain can be obtained.

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

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

[0272] Vectors (V1401 and V1402) were introduced into mouse embryonic stem cells. Next, the cells were screened with hygromycin B and puromycin. Integration of the exogenous gene into the mouse genome was confirmed by PCR. The results are shown in FIGS. 5A - 5B and FIGS. 6A - 6B. Clones No. 030, 035, 036 and 037 were confirmed to be positive.

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

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

[0275] Example 3: Modification of Human Chromosome 14 The purpose of this experiment is to create a modified human chromosome containing at least two recombination sites. Two recombination sites were introduced on both sides of the variable region of the heavy-chain immunoglobulin locus.

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

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

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

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

[0280] In some experiments, the vectors (V401, V402) were introduced into cells, and the cells were selected using appropriate drug resistance markers or combinations thereof (zeocin, G418).

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

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

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

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

[0285] In one experiment, human fibroblasts were selected and vector 402 was introduced. Next, the human fibroblasts were fused with recipient cells (A9 cells or CHO cells). The modified chromosomes were isolated and introduced into another suitable recipient cell. Next, the cells were selected by G418 resistance to obtain cells containing only one human chromosome. Next, vector 401 was introduced into the cells and the cells were selected by zeocin resistance. Thereafter, vector 403 was introduced into the cells and the cells were selected by puromycin resistance. The positive clones selected after screening were treated with Cre enzyme. For chromosome techniques, see, for example, Kuroiwa et al. “Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts”. Nature Biotechnology 18.10 (2000): 1086-1090; Chinese Patent Application Publication No. 1200014A; Chinese Patent Application Publication No. 109837307A; US Patent Application Publication No. 20120093785A1; US Patent Application Publication No. 2009253902; Chinese Patent Application Publication No. 1717483A; Paulis, Marianna. “Chromosome Transfer Via Cell Fusion”. Methods in Molecular Biology 738 (2011): 57; Genes, Chromosomes & Cancer 14:126 - 127 (1995); Tomizuka et al. “Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice”. Nature Genetics 16.2 (1997): 133-143; and Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284 (each of which is hereby incorporated by reference in its entirety herein).

[0286] PCR was performed to confirm the presence of 5'-terminal recombination site 301 and 3'-terminal recombination site 302 on the chromosome. Southern blot was used to confirm the absence of random insertions in the cells, and fluorescence in situ hybridization (FISH) was used for analysis.

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

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

[0289] FISH analysis was performed using the CCP14 FISH probe (CytoTest Inc., Rockville, MD, catalog number CT-CCP014) with the positive clone. Representative FISH images of clone 1-D10 are shown in FIGS. 13 and 14. In FIG. 13, the white arrow indicates the full-length human chromosome 14 (before modification). In FIG. 14, the white arrow indicates the modified human chromosome 14 fragment.

[0290] Example 4: Introduction of Human Chromosomes or Fragments into Mouse ES Cells The modified chromosome obtained in Example 3 was introduced into the cells obtained in Example 2 by the method described previously. Next, the cells were screened with G418. Only the cells containing only one human chromosome were selected. FIG. 15 shows the modified mouse chromosome 12.

[0291] Next, by the mediation of Cre recombinase, the V, D, J regions on mouse chromosome mChr12-mut2 were replaced with the V, D, J regions on human chromosome hChr14-mut3 (FIG. 16). The human chromosome DNA sequence replaced the sequence between recombination sites 1301 and 1302. Hygromycin and puromycin were used for screening of positive cells. After the cells were further screened with DT to obtain mouse cells without human chromosomes, they were injected into mouse blastocysts. Optionally, the cells were directly injected into blastocysts without DT screening.

[0292] The cells after Cre recombination were tested to confirm that the human gene sequence was integrated into the mouse genome. The PCR results are shown in Figures 17, 18, 19 and 20. All PCR results showed correct recombination in cells 1-B4, 1-B10 and 2-A7, and the absence of human chromosomes in 1-B10 cells. 1-B10 cells were tested by FISH using mouse whole chromosome painting probes (Cytocell Ltd, Cambridge, UK; catalog number AMP12R) and human-specific IGH break-apart probes (Cytocell Ltd, Cambridge, UK; catalog number LPH 014). The results are shown in Figure 21, and it was confirmed that human chromosome fragments were present in the mouse chromosomes. These primers are shown in the following table.

[0293]

Table 12

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

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

[0296]

Table 13

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

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

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

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

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

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

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

[0304] Two recombination sites were introduced on both sides of the variable region of the light chain immunoglobulin locus. V HK The region between and the centromere (kinetochore) was deleted to obtain a shorter artificial chromosome for subsequent experiments. Recombination sites similar to those in the variable region of the mouse immunoglobulin locus on chromosome 6 were introduced. Next, human chromosomes were introduced into mouse recipient cells to obtain a humanized light chain immunoglobulin locus.

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

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

[0307] The sequence of the vector (V2702) was confirmed by sequencing. This vector was introduced into human H9 cells by transfection. Next, the cells were screened by G418 and ouabain resistance. It was confirmed by PCR that the gene was integrated into the human genome. The results are shown in FIG. 31. Clones 01, 02, 03, and 04 were confirmed to be positive clones.

[0308] The PCR assay was performed using the following primers: 3’L-L-GT-F: 5’-AAGGTGACTCTGCAATCAGCCTCTG-3’ (SEQ ID NO: 38), 3’L-L-GT-R1: 5’-TCATCTACAGCCACAACGTGAGCAG-3’ (SEQ ID NO: 39); 3’L-R-GT-F1: 5’-CCCATGTACAGGTTCCGCATGAACT-3’ (SEQ ID NO: 40), 3’L-R-GT-R: 5’-CTCCGTCCGCTTTTATTTCCCCTGT-3’ (SEQ ID NO: 41).

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

[0310] During screening, clone No. 03 shown in FIG. 31 had the correct recombination, and this was labeled with a human chromosome 2 counting probe (CCP2 FISH probe) (CytoTest Inc., Rockville, MD, catalog number CT-CCP002). From these results, it was confirmed that the modified human chromosome 2 was present in the cells (FIG. 32).

[0311] Next, a vector (V2701) was further introduced into the cells. These cells were screened for G418 and blasticidin S resistance. Recombination was confirmed by PCR. The primers are shown in the following table. From these results, it was shown that 5-C3, 1-H2, 1-H9, and 1-G5 were positive clones (FIG. 33).

[0312] [Table 14]

[0313] Example 8: Generation of a mouse capable of producing a humanized antibody Mouse embryonic stem cells were fused with the cells obtained in Example 7, and the modified human chromosome 2 was introduced into the mouse embryonic stem cells obtained in Example 6. Mouse ES cells containing only one modified human chromosome 2 were selected, and the cells were screened after Cre-mediated recombination (Cre-mediated recombination) as shown in FIG. 34. The cells after Cre recombination were tested to confirm that the human gene sequence was integrated into the mouse genome. Positive clones confirmed by PCR were verified by fluorescence in situ hybridization (FISH) using a mouse whole chromosome painting probe (Cytocell Ltd, Cambridge, UK; catalog number AMP06G) and a human-specific IGK break-apart probe (Cytocell Ltd, Cambridge, UK; catalog number LPH 034). The results are shown in FIG. 35, and it was confirmed that human chromosome fragments were present in the mouse chromosomes. These cells were injected into blastocysts. A mouse containing the humanized light chain immunoglobulin locus can be obtained.

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

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

[0316] A mouse having a humanized light chain immunoglobulin locus and a mouse having a humanized heavy chain immunoglobulin locus were crossed with each other to obtain a mouse having both a humanized heavy chain immunoglobulin locus and a humanized light chain immunoglobulin locus.

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

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

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

[0320] Based on different developmental stages, bone marrow B cells were classified into pro - B cells (B220 low CD43 high IgM low (characterized by), pre - B cells (B220 low CD43 int IgM low (characterized by) and immature B cells (B220 high CD43 low IgM high (characterized by). Figures 51A to 51C show the percentages of B cells by developmental stage in the bone marrow.

[0321] Compared with wild - type mice, the proportions of immune cells and B cells in humanized mice were similar, and there was no statistical difference between different groups. No significant defects in B cell differentiation were observed in either heavy - chain humanized heterozygous mice (heterozygotes) or homozygous mice (homozygotes).

[0322] Example 10: Serum Immunoglobulin Isotype Analysis Furthermore, various immunoglobulin levels in the sera of non-immunized mice in the above examples were analyzed. The mice included WT mice, mice having a heterozygous humanized heavy chain immunoglobulin locus, and mice having a homozygous humanized heavy chain immunoglobulin locus.

[0323] The experiment was carried out using the Clonotyping System-B6 / C57J-HRP (Southern Biotech, catalog number 5300-05B) kit. First, the capture antibody goat anti-mouse Ig, human ads-UNLB was diluted to 10 μg / mL with PBS (Solarbio, catalog number P1020). Next, 0.1 mL of the antibody dilution was added to each well of an enzyme-linked immunosorbent assay (ELISA) plate and incubated at 37°C for 2 hours. Next, the plate was washed and blocked at 4°C for 12 hours. Serum samples were serially diluted with 1% BSA (Cell Signaling, catalog number 9998). 0.1 mL of the sample dilution was added to each well, followed by incubation at 37°C for 1 hour. As a blank control, 1% BSA was added to one well.

[0324] Next, the plates were washed with PBS containing 0.05% Tween-20 (Amresco, catalog number M147). HRP-conjugated secondary antibody (goat anti-mouse IgA, IgG (1, 2b, 2c, 3), IgM) (diluted 300-fold with 1% BSA, 0.1 mL per well) was added and incubated with the samples at 37 °C for 1 hour. Next, the plates were washed and developed by adding 0.1 mL of TMB chromogen solution (Beyotime Biotechnology, catalog number P0209) to each well. After incubation at room temperature in the dark for 8 minutes, 0.1 mL of reaction solution (Beijing Dingguo Changsheng Biotechnology Co., LTD., catalog number EIA-0032) was added to each well. The absorbance at 450 nm and 570 nm was measured using a microplate reader (Thermo MULTISKAN GO, Thermo Fisher Scientific), and the standard OD value was calculated.

[0325] From these results, it was shown that mice having a humanized heavy-chain immunoglobulin locus had the ability to produce IgA, IgG1, IgG2b, IgG2c, IgG3, and IgM antibody isotypes, and that these mice had comparable expression levels for each isotype compared to wild-type mice (Figs. 52 - 57). This indicates that humanization of the heavy-chain variable region gene segments did not have a significant detrimental effect on antibody class switching, expression, or secretion of various antibody isotypes.

[0326] Example 11: V(D)J Recombination of Human Variable Region Gene Segments in Mice The mRNA sequences of the heavy-chain variable region and light-chain variable region in mice were analyzed by next-generation sequencing.

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

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

[0329] By comparing the sequencing results with the NCBI Ig Blast tool for human immunoglobulin sequences, the expression of human V H , D H , J H and Vκ, Jκ genes after V(D)J recombination was identified. Among the 135 clones analyzed, as preliminary results, specific V H , D H gene segments and all J HExpression of gene segments was detected (Table 15). Some of these gene segments were located near the modification sites on the humanized fragments, and some were away from the modification sites. From this, after replacing the endogenous chromosomal fragment with the human immunoglobulin heavy chain sequence, it was pointed out that human heavy chains could be expressed by recombination of human V H , D H and J H genes.

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

[0331]

Table 15

[0332]

Table 16

[0333] Example 12: Immunization and Antibody Production in Humanized Mice Five wild-type (WT) mice and five humanized heavy-chain homozygous mice (9 - 10 weeks old) were randomly selected and immunized with exogenous antigens. The mice were immunized repeatedly once every two weeks for a total of three times. Retro-orbital blood was collected after the second immunization and after the third immunization. Serum was recovered, and then the serum titer was measured by ELISA or FACS to determine and analyze the antigen-specific antibody response. In this study, three antigens, hBTLA, dPD1, and OVA, were used (Figs. 59 - 63). From these results, it was shown that most wild-type (WT) and humanized heavy-chain homozygous mice produced antigen-specific antibodies after the second immunization. After the third immunization, the antibody titer increased to 1×10 4 ~1×10 5 . Since the results of the immune titer test were essentially the same in humanized mice compared to wild-type mice, it was pointed out that the humanized immunoglobulin variable region locus of the mice was functional and could produce antigen-specific antibodies.

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

[0335] An experiment was conducted to compare the immune systems of naive humanized VH / VL mice and naive wild-type mice. The body weights and spleen weights of wild-type and hVH / hVL mice were measured (Figs. 65A - 65B). No significant difference was detected between wild-type mice and hVH / hVL mice in terms of average body weight and spleen weight.

[0336] Flow cytometry was performed to analyze the lymphocyte population and distribution in the spleen of mice (Figure 66) and the B cell population in the spleen and bone marrow (Figures 67A-67B, 68A-68C). From these results, it was shown that in hVH / hVL mice, the proportions of B cells, T cells, NK cells, CD4+ T cells, and CD8+ T cells in the spleen were almost the same as those in wild-type mice. In these results, leukocytes included B cells (characterized by, for example, CD45+, CD19+, TCR-), T cells, and natural killer (NK) cells (characterized by, for example, CD45+, TCR-, NK1.1+). T cells were characterized by CD45+, CD19-, TCR+. CD4+ T cells (CD4) were characterized by CD45+, CD19-, TCR+, CD4+, CD8-. Also, CD8+ T cells (CD8) were characterized by CD45+, CD19-, TCR+, CD4-, CD8+. Flow cytometry analysis included only intact single viable leukocytes.

[0337] Figure 67A shows the proportion of B cells at different developmental stages. The developmental stages of B cells in the spleen were classified into T1 (transitional type 1 B cells, characterized by B220 + IgM + IgD - , T2 (transitional type 2 B cells, characterized by B220 + IgM + IgD + , and mature B cells (characterized by B220 + IgM low IgD + . In addition, B cell development was classified into the spleen marginal zone (marginal zone B cells, MZ-B, characterized by B220 + CD21 + CD23 - and the follicular zone (follicular B cells, called FO-B, characterized by B220 + CD21 low CD23 +It was also evaluated in (characterized by). Figure 67B shows the percentages of splenic B cells in the marginal zone of the spleen (MZ-B) and the follicular zone (FO-B). No significant difference was observed between wild-type mice and hVH / hVL mice.

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

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

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

[0341] From these experiments, it was shown that the immune system of hVH / hVL mice is functional and that the humanized immunoglobulin locus of hVH / hVL mice can interact appropriately with the mouse immunoglobulin constant region.

[0342] Example 14: Analysis of Germline Usage in hVH / hVL Mice The heavy chain IGHV, IGHD, and IGHJ usage of naive hVH / hVL mice (without antigen stimulation) was analyzed. The results are shown in FIGS. 70A to 70D. In addition, the κ chain IGKV and IGKJ usage was also analyzed. The results are shown in FIGS. 71A to 71C.

[0343] The germline usage of naive hVH / hVL mice was determined by next-generation sequencing (NGS). For example, as shown in FIG. 71C, IGKJ1, IGKJ2, and IGKJ4 were used frequently in naive hVH / hVL mice, while the frequencies of IGKJ3 and IGKJ5 observed were low. Such a pattern of IGKJ germline usage is consistent with the literature reports on human IGKJ germline usage.

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

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

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

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

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

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

[0350] Flow cytometry was performed to analyze the lymphocyte populations and distributions in the spleens of mice (Figure 78) and the B cell populations in the spleens and bone marrows (Figures 79A - 79B, 80A - 80C).

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

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

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

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

[0355] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A genetically modified non-human animal comprising at least 150 human IGHV genes selected from Table 1 below, at least 20 human IGHD genes selected from Table 2 below, and at least 5 human IGHJ genes selected from Table 3 below at its endogenous heavy chain immunoglobulin locus, wherein the human IGHJ genes are operably linked and capable of undergoing VDJ rearrangement. 【Table 1】 【Table 2】 【Table 3】

2. the animal comprises a contiguous human sequence comprising one or more human IGHV genes, one or more human IGHD genes and one or more human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14, the contiguous human sequence having a length of at least 300 kb; 2. The animal of claim 1, wherein the animal preferably comprises all human IGHV genes, all human IGHD genes and all human IGHJ genes at the endogenous heavy chain immunoglobulin locus of human chromosome 14 of the human subject.

3. the animal does not express an endogenous immunoglobulin heavy chain variable domain; Preferably, 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; 10. The animal of claim 1, wherein the animal preferably comprises a disruption of the animal's endogenous heavy chain immunoglobulin locus.

4. The animal of claim 1 , wherein the human immunoglobulin variable region is introduced into the genome of the animal by a stepwise procedure or one-step replacement.

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

6. A genetically modified non-human animal comprising at least 50 human IGKV genes in Table 7 below and at least 3 human IGKJ genes in Table 8 below at its endogenous light chain immunoglobulin locus. 【Table 7】 【Table 8】

7. the animal comprises a contiguous human sequence comprising one or more human IGKV genes and one or more human IGKJ genes at an endogenous kappa chain immunoglobulin locus on human chromosome 2, the contiguous human sequence having a length of at least 300 kb; 7. The animal of claim 6, wherein the animal preferably comprises all human IGKV genes and all human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2.

8. the animal does not express an endogenous immunoglobulin light chain variable domain; Preferably, the animal lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain; 7. The animal of claim 6, wherein the animal preferably comprises a disruption of the animal's endogenous light chain immunoglobulin locus.

9. The animal of claim 6 , wherein the human immunoglobulin variable region is introduced into the genome of the animal by a stepwise procedure or one-step replacement.

10. 7. The animal of claim 6, wherein the animal further comprises one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at the endogenous heavy chain immunoglobulin locus.

11. The animal according to any one of claims 1 to 10, wherein the animal is a mouse.

12. A cell obtained from an animal according to any one of claims 1 to 11.

13. 1. A method for producing a genetically modified non-human animal, comprising: Modifying human chromosomes; introducing the modified human chromosome into cells of the animal; and Inducing recombination between the modified human chromosome and an endogenous chromosome, thereby integrating a human sequence comprising one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. A method comprising:

14. 14. The method of claim 13, characterized by one or more of the following: (i) the modified human chromosome comprises two or more exogenous recombination sites; (ii) the endogenous chromosome contains two or more exogenous recombination sites; (iii) at least 150 human IGHV genes selected from Table 1 below, at least 20 human IGHD genes selected from Table 2 below, and at least 5 human IGHJ genes selected from Table 3 below are integrated into the endogenous chromosome by recombination; Preferably, all of the human IGHV genes, all of the human IGHD genes and all of the human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14 are integrated; (iv) the animal does not express an endogenous immunoglobulin heavy chain variable domain; Preferably, 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; Preferably, the animal comprises a disruption of the animal's endogenous heavy chain immunoglobulin locus; (v) the human immunoglobulin variable region is introduced into the genome of the animal by a stepwise procedure or by one-step replacement; and / or (vi) The animal is a mouse. 【Table 1】 【Table 2】 【Table 3】

15. (a) modifying a first human chromosome, wherein said modified first human chromosome comprises two or more exogenous recombination sites; (b) introducing the modified first human chromosome into cells of the animal; and (c) inducing recombination between the modified first human chromosome and an endogenous chromosome, wherein the endogenous chromosome comprises two or more exogenous recombination sites, thereby integrating a human sequence comprising one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14.

14. The method of claim 13, comprising:

16. one or more human IGKV genes and one or more human IGKJ genes are further incorporated, 14. The method of claim 13, wherein a human sequence is integrated, preferably comprising all human IGKV genes and all human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2.

17. 1. A method for producing a genetically modified non-human animal, comprising: Modifying human chromosomes; introducing the modified human chromosome into cells of the animal; and Inducing recombination between the modified human chromosome and an endogenous chromosome, thereby integrating a human sequence comprising one or more human IGKV genes and one or more human IGKJ genes. A method comprising:

18. 18. The method of claim 17, characterized by one or more of the following: (i) the modified human chromosome comprises two or more exogenous recombination sites; (ii) the endogenous chromosome contains two or more exogenous recombination sites; (iii) at least 50 human IGKV genes in Table 7 below and at least three human IGKJ genes in Table 8 below are integrated into the endogenous chromosome by recombination; Preferably, all human IGKV genes and all human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2 are integrated; (iv) the animal does not express an endogenous immunoglobulin light chain variable domain; Preferably, the animal lacks an endogenous immunoglobulin light chain variable region locus capable of rearranging and forming a nucleic acid sequence encoding an endogenous light chain variable domain; Preferably, the animal comprises a disruption of the animal's endogenous light chain immunoglobulin locus. (v) the human immunoglobulin variable region is introduced into the genome of the animal by a stepwise procedure or by one-step replacement; and / or (vi) The animal is a mouse. 【Table 7】 【Table 8】

19. (a) modifying a first human chromosome, wherein the modified first human chromosome comprises two or more exogenous recombination sites; (b) introducing the modified first human chromosome into cells of the animal; and (c) inducing recombination between the modified first human chromosome and an endogenous chromosome, wherein the endogenous chromosome comprises two or more exogenous recombination sites, thereby integrating a human sequence comprising one or more human IGKV genes and one or more human IGKJ genes at the endogenous kappa chain immunoglobulin locus of human chromosome 2.

20. The method of claim 17, comprising:

20. further incorporating one or more human IGHV genes, one or more human IGHD genes and one or more human IGHJ genes at the heavy chain immunoglobulin locus of human chromosome 14; 18. The method of claim 17, wherein a human sequence is incorporated, preferably comprising all human IGHV genes, all human IGHD genes and all human IGHJ genes in the heavy chain immunoglobulin locus of human chromosome 14.

21. 1. A method for producing an antibody that specifically binds to an antigen, comprising: obtaining nucleic acid sequences encoding human heavy and light chain immunoglobulin variable regions in cells that express a hybrid antibody that specifically binds to said antigen, said cells being obtained by exposing an animal of any one of claims 1 to 11 to said antigen; operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region with a nucleic acid encoding a human heavy chain immunoglobulin constant region, and the nucleic acid encoding the human light chain immunoglobulin variable region with a nucleic acid encoding a human light chain immunoglobulin constant region; and expressing said nucleic acid in a cell, thereby obtaining said antibody. A method comprising:

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