Genetically modified non-human animals possessing a common light chain immunoglobulin locus.

Abstract

Provided are genetically modified animals with a humanized light chain immunoglobulin locus, wherein the endogenous light chain immunoglobulin locus comprises a finite number of human IGKV and IGKJ genes, cells obtained from the animals, and methods for exposing the animals to antigens.

Description

[Technical Field]

[0001] [Claiming priority] This application claims the rights to PCT application number PCT / CN2020 / 094000, filed on 2 June 2020, and PCT application number PCT / CN2021 / 085839, filed on 7 April 2021. The above is incorporated herein by reference in its entirety.

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

[0003] Antibodies are immune proteins that bind to specific antigens. Typically, antibodies possess specificity to their target, the ability to mediate immune effector mechanisms, and a long half-life in serum. These properties make antibodies potent therapeutic agents. Monoclonal antibodies are used in the treatment of a variety of diseases, including cancer, infectious diseases, autoimmune diseases, and inflammatory diseases. Many novel antibody forms have already been proposed for various therapeutic purposes. For example, bispecific antibodies can bind to two different targets or two different epitopes within a target, producing additive or synergistic effects superior to those of monoclonal antibodies. However, the production of bispecific antibodies is challenging due to mismatch problems.

[0004] These therapeutic antibodies are typically human antibodies or humanized antibodies. Human antibodies or humanized antibodies can be produced by humanizing rodent antibodies (e.g., mouse antibodies) or by using phage libraries. Antibodies produced by these methods usually have suboptimal binding affinity and biophysical properties, leading to manufacturing difficulties and poor pharmacokinetics. In particular, the humanization process can negatively affect binding affinity and may introduce immunogenic epitopes into the antibody, while antibodies discovered using phage libraries exhibit unnatural pairing with limited diversity in immunoglobulin heavy and light chains. Often, repeated experiments and considerable time are required to improve the properties. Furthermore, in some cases, these antibodies can induce immunogenicity in patients.

[0005] There is a need for an effective and cost-effective method for producing humanized antibodies, and in particular, a platform for producing humanized bispecific antibodies for various therapeutic purposes. [Overview of the project]

[0006] This disclosure relates to genetically modified animals and cells having humanized heavy chain and light chain immunoglobulin loci. In some embodiments, the genetically modified animals have a finite number of human IGKV and IGKJ genes at the endogenous light chain immunoglobulin loci. In one embodiment, the genetically modified animals described herein can produce immunoglobulin light chain variable domains, which can be paired with a fairly diverse family of heavy chain variable domains, including, for example, affinity maturation or somatic mutation variable domains.

[0007] In one embodiment, the disclosure provides a genetically modified non-human animal in which an exogenous light chain variable region gene sequence is located at an endogenous light chain immunoglobulin locus. In some embodiments, the exogenous light chain variable region gene sequence includes three or fewer human IGKV genes and two or fewer human IGKJ genes. In some embodiments, the three or fewer human IGKV genes and the two or fewer human IGKJ genes are all operably linked to an endogenous light chain constant domain gene.

[0008] In some embodiments, three or fewer human IGKV genes are selected from Table 1, and two or fewer human IGKJ genes are selected from Table 2.

[0009] In some embodiments, the exogenous light chain variable region gene sequence includes one human IGKV gene and one human IGKJ gene.

[0010] In some embodiments, the exogenous light chain variable region gene sequence further comprises a human IGKJ 3'-UTR sequence. In some embodiments, somatic hypermutation can occur in the exogenous light chain variable region gene sequence in one or more cells of the animal.

[0011] In some embodiments, the somatic hypermutation can cause one, two, or three amino acid changes in the light chain variable region of one or more cells of the animal.

[0012] In some embodiments, the exogenous light chain variable region gene sequence includes one human IGKV gene and one human IGKJ gene. In some embodiments, the human IGKV gene is selected from the group consisting of IGKV3-20, IGKV3-11, and IGKV1-39. In some embodiments, the human IGKV gene and the human IGKJ gene are operably linked. In some embodiments, the human IGKV gene is IGKV3-11. In some embodiments, the human IGKJ gene is selected from the group consisting of IGKJ1 and IGKJ4. In some embodiments, the human IGKV gene is IGKV1-39 and the human IGKJ gene is IGKJ4. In some embodiments, the human IGKV gene is IGKV3-11 and the human IGKJ gene is IGKJ1. In some embodiments, the human IGKV gene is IGKV3-20 and the human IGKJ gene is IGKJ1. In some embodiments, the animal further includes a promoter sequence operably linked to the human IGKV gene. In some embodiments, the promoter sequence is located within 2500 bp or 3000 bp of the human IGKV gene. In some embodiments, the promoter is the IGKV3-20 promoter, the IGKV3-11 promoter, or the IGKV1-39 promoter.

[0013] In some embodiments, the animal includes a disruption at the animal's endogenous light chain immunoglobulin locus. In some embodiments, the animal is a mouse, and the disruption at the animal's endogenous light chain immunoglobulin locus includes deletions of one or more mouse IGKV genes in Table 3 and one or more mouse IGKJ genes in Table 4. In some embodiments, the animal is a mouse, and the disruption at the animal's endogenous light chain immunoglobulin locus includes deletions of sequences from mouse IGKV2-137 to mouse IGKJ5.

[0014] In some embodiments, the animal comprises endogenous IGKC. In some embodiments, the animal further comprises a κ-intron type enhancer 5' and / or κ-3' enhancer for the endogenous IGKC.

[0015] In some embodiments, the human light chain variable region is a reconfigured sequence.

[0016] In some embodiments, the animal is homozygous for the light chain immunoglobulin locus. In some embodiments, the animal is heterozygous for the light chain immunoglobulin locus. In some embodiments, the animal includes disruption of the animal's endogenous λ light chain immunoglobulin locus.

[0017] In some embodiments, the animal is a rodent (e.g., a mouse).

[0018] 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 an endogenous heavy chain immunoglobulin locus. In some embodiments, the human IGHV gene, human IGHD gene, and human IGHJ gene can be operably ligated and undergo VDJ rearrangement.

[0019] In some embodiments, the animal comprises at least 150 human IGHV genes selected from Table 5, at least 20 human IGHD genes selected from Table 6, and at least 5 human IGHJ genes selected from Table 7. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin locus on human chromosome 14 of a human subject. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin locus on human chromosome 14 of a human cell. In some embodiments, the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus. In some embodiments, the unmodified human sequence is at least 800 kb.

[0020] In some embodiments, the animal contains unmodified human sequences from human IGHV(III)-82 to human IGHV1-2, derived from the human heavy chain immunoglobulin locus. In some embodiments, the animal contains unmodified human sequences from human IGHV(III)-82 to human IGHV6-1, derived from the human heavy chain immunoglobulin locus. In some embodiments, the animal contains unmodified human sequences from human IGHD1-1 to human IGHJ6, derived from the human heavy chain immunoglobulin locus. In some embodiments, the animal contains unmodified human sequences from human IGHV(III)-82 to human IGHJ6, derived from the human heavy chain immunoglobulin locus. In some embodiments, the animals include IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, IGHV3-75, and IGHV(II)-74-1. In some embodiments, the animals include IGHV5-10-1 and IGHV3-64D.

[0021] In some embodiments, the animal further includes, at the endogenous heavy chain immunoglobulin locus, a first sequence containing one or more human IGHV genes, a second sequence containing an endogenous sequence, and a third sequence containing 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.

[0022] In some embodiments, the first sequence comprises at least 150 human IGHV genes selected from Table 5. In some embodiments, the first sequence comprises at least 20 human IGHD genes selected from Table 6. 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 at least 800 kb. In some embodiments, the second sequence comprises an endogenous sequence of at least 3 kb. In some embodiments, the third sequence comprises at least 20 human IGHD genes selected from Table 6 and at least 5 human IGHJ genes selected from Table 7.

[0023] In some embodiments, the third sequence includes all human IGHD genes in Table 6 and all human IGHJ genes in Table 7. In some embodiments, the third sequence is an unmodified sequence derived from the human heavy chain immunoglobulin locus. In some embodiments, the third sequence is at least 50 kb. In some embodiments, the animal includes a disruption at the animal's endogenous heavy chain immunoglobulin locus.

[0024] In some embodiments, the animal is a mouse, and the disruption at the animal's endogenous heavy chain immunoglobulin locus includes deletions of one or more mouse IGHV genes in Table 8, one or more mouse IGHD genes in Table 9, and one or more mouse IGHJ genes in Table 10. In some embodiments, the animal is a mouse, and the disruption at the animal's endogenous heavy chain immunoglobulin locus includes deletions of sequences from mouse IGHV1-85 to mouse IGHJ4. In some embodiments, the animal includes one or more endogenous IGHM, IGHδ, IHG3, IHG1, IHG2b, IHG2a, IGHE, and IGHA genes.

[0025] In some embodiments, the animal is homozygous for the heavy chain immunoglobulin locus. In some embodiments, the animal is heterozygous for the heavy chain immunoglobulin locus.

[0026] In one embodiment, the Disclosure provides a genetically modified non-human animal whose genome includes an endogenous light chain immunoglobulin locus, the endogenous light chain immunoglobulin locus including one or more endogenous IGKV genes substituted by one or more human IGKV genes selected from Table 1, and one or more endogenous IGKJ genes substituted by one or more human IGKJ genes selected from Table 2. In some embodiments, the human IGKV genes and the human IGKJ genes are operably linked to an endogenous IGKC gene.

[0027] In some embodiments, the one or more human IGKV genes are selected from the group consisting of IGKV3-20, IGKV3-11, and IGKV1-39. In some embodiments, the human IGKV gene is IGKV3-11. In some embodiments, the one or more human IGKJ genes are selected from the group consisting of IGKJ1 and IGKJ4. In some embodiments, the animal further includes an insertion of a human IGKJ 3'-UTR sequence. In some embodiments, the human IGKV gene is IGKV1-39 and the human IGKJ gene is IGKJ4. In some embodiments, the human IGKV gene is IGKV3-11 and the human IGKJ gene is IGKJ1. In some embodiments, the human IGKV gene is IGKV3-20 and the human IGKJ gene is IGKJ1. In some embodiments, all endogenous IGKV genes are replaced by the one or more human IGKV genes. In some embodiments, all endogenous IGKJ genes are replaced by one or more of the aforementioned human IGKJ genes.

[0028] In some embodiments, the animal further comprises a promoter sequence preceding the human IGKV gene. In some embodiments, the promoter sequence is located within 3000 bp of the human IGKV gene. In some embodiments, the animal further comprises a κ intron-type enhancer at 5' of the endogenous IGKC. In some embodiments, the animal further comprises a κ 3' enhancer.

[0029] In some embodiments, the animal genome further comprises an endogenous heavy chain immunoglobulin locus, the endogenous heavy chain immunoglobulin locus comprising one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes, which are replaced by one or more human IGHV, human IGHD, and human IGHJ genes. In some embodiments, the one or more human IGHV, human IGHD, and human IGHJ genes are operably linked to one or more endogenous IGHM, IGHδ, IHG, IGHE, and IGHA genes. In some embodiments, the one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes are replaced by at least 150 human IGHV genes in Table 5, at least 20 human IGHD genes in Table 6, and at least 5 human IGHJ genes in Table 7.

[0030] In some embodiments, the animal is a mouse, and at least 180 mouse IGHV genes in Table 8, all mouse IGHD genes in Table 9, and all mouse IGHJ genes in Table 10 are replaced.

[0031] In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable region locus that can reconstruct and form a nucleic acid sequence encoding an endogenous heavy chain variable domain (e.g., mouse heavy chain variable domain). In some embodiments, the animal lacks an endogenous immunoglobulin light chain variable region locus that can reconstruct and form a nucleic acid sequence encoding an endogenous light chain variable domain (e.g., mouse light chain variable domain).

[0032] In some embodiments, the animal can produce a humanized antibody. In some embodiments, the antibody includes a light chain variable region encoded by the IGKV gene and the IGKJ gene.

[0033] In one embodiment, the present disclosure provides cells obtained from animals described herein. In some embodiments, the cells are B cells expressing a chimeric immunoglobulin light chain comprising an immunoglobulin light chain variable domain encoded by a human IGKV gene and a human IGKJ gene, wherein the human IGKV gene is selected from the group consisting of IGKV3-20, IGKV3-11, and IGKV1-39, and the human IGKJ gene is selected from the group consisting of IGKJ1 and IGKJ4. In some embodiments, the immunoglobulin light chain variable domain is operably linked to a non-human light chain constant region.

[0034] In some embodiments, B cells express a chimeric immunoglobulin heavy chain containing an immunoglobulin heavy chain variable domain, the immunoglobulin heavy chain variable domain being derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. In some embodiments, the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region.

[0035] In some embodiments, the cells are embryonic stem (ES) cells.

[0036] In one embodiment, the present disclosure provides a method for producing a chimeric antibody that specifically binds to an antigen, the method comprising exposing an animal described herein to an antigen, producing a hybridoma from cells recovered from the animal, and recovering the chimeric antibody produced from the hybridoma.

[0037] In some embodiments, the method further includes sequencing the genome of the hybridoma.

[0038] In one embodiment, the present disclosure provides a method for producing an antibody that specifically binds to an antigen, the method comprising: exposing an animal described herein to an antigen; sequencing a nucleic acid encoding a human heavy chain and light chain immunoglobulin variable region in cells expressing a hybrid antibody that specifically binds to the antigen; and operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region to a nucleic acid encoding the human heavy chain immunoglobulin constant region in the cells, and operably linking the nucleic acid encoding the human light chain immunoglobulin variable region to a nucleic acid encoding the human light chain immunoglobulin constant region.

[0039] In one embodiment, the present disclosure provides a method for producing an antibody that specifically binds to an antigen, the method for obtaining a nucleic acid sequence encoding a human heavy chain and light chain immunoglobulin variable region from cells expressing a hybrid antibody that specifically binds to the antigen, the method comprising: obtaining the cells by exposing an animal described herein to the antigen; operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region to a nucleic acid encoding the human heavy chain immunoglobulin constant region; operably linking the nucleic acid encoding the human light chain immunoglobulin variable region to a nucleic acid encoding the human light chain immunoglobulin constant region; and obtaining the antibody by expressing the nucleic acids in the cells.

[0040] In one embodiment, the present disclosure provides a method for obtaining a nucleic acid encoding an antibody-binding domain that specifically binds to an antigen, the method comprising exposing an animal described herein to an antigen and sequencing a nucleic acid encoding a human heavy-chain and light-chain immunoglobulin variable region in cells expressing a hybrid antibody that specifically binds to the antigen.

[0041] In one embodiment, the present disclosure provides a method for obtaining a sample, the method comprising exposing an animal described herein to the antigen and recovering the sample from the animal. In some embodiments, the sample is spleen tissue, spleen cells, or B cells.

[0042] In one embodiment, the present disclosure provides a method for producing a bispecific antibody, the method comprising expressing in cells a nucleic acid sequence encoding a light chain polypeptide containing human VL, a nucleic acid sequence encoding a first heavy chain polypeptide containing a first human VH, and a nucleic acid sequence encoding a second heavy chain polypeptide containing a second human VH, wherein the sequence encoding the first VH is obtained after exposing an animal described herein to a first antigen, and the sequence encoding the second VH is obtained after exposing the animal or a different animal to a second antigen.

[0043] In some embodiments, the first VH and VL form a first antigen-binding site that specifically binds to the first antigen. In some embodiments, the second VH and VL form a second antigen-binding site that specifically binds to the second antigen.

[0044] In one embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a human light chain variable region having a sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 38, 39, or 40.

[0045] In one embodiment, the disclosure provides a plurality of antibodies or antigen-binding fragments thereof, each antibody or antigen-binding fragment comprising a human light chain variable region having a sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 38, 39, or 40.

[0046] In one embodiment, the present disclosure provides a method for producing an antibody that specifically binds to a protein of interest, the method comprising exposing an animal that does not express an endogenous protein homologous to the protein of interest as described herein to the protein of interest, and sequencing nucleic acids encoding human heavy chain and light chain immunoglobulin variable regions in cells that express an antibody that specifically binds to the protein of interest.

[0047] In some embodiments, the gene encoding the endogenous protein is disrupted in vivo in the animal. In some embodiments, the gene encoding the endogenous protein is knocked out.

[0048] In some embodiments, the endogenous protein is at least 80%, 90%, or 95% homologous to the target protein.

[0049] In some embodiments, the target protein is a human protein. In some embodiments, the target protein is PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art may also be used. Materials, methods and examples are illustrative and not intended to limit the scope. All publications, patent applications, patents, sequences, database entries and other references referenced herein are incorporated herein by reference in their entirety. In case of any inconsistency, the provisions of this specification (including definitions) shall prevail.

[0051] Other features and advantages of the present invention will become apparent from the embodiments, drawings, and claims for which the invention is to be carried out. [Brief explanation of the drawing]

[0052] [Figure 1A] This is a flowchart showing how to introduce human immunoglobulin genes into the mouse genome. [Figure 1B]This is an overview of substituting the variable region of a mouse immunoglobulin heavy chain with the variable region of a human immunoglobulin heavy chain. [Figure 2A] This shows the human κ chain V region gene used at the top. [Figure 2B] This shows the human κ chain J region gene used at the top. [Figure 3A] This is a schematic diagram showing the mouse light chain immunoglobulin gene locus. [Figure 3B] This is an overview of replacing mouse immunoglobulin light chain variable region genes with rearranged human IGKV and IGKJ genes. [Figure 4] This is a schematic diagram illustrating the targeting strategy for humanizing the κ-light chain gene. [Figure 5A] This is a histogram showing the body weight of wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 5B] This histogram shows the liver weight of wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 5C] This is a histogram showing the thymic weight of wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 5D] This is a histogram showing the spleen weight of wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 5E] This is a histogram showing lung weights in wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 5F] This is a histogram showing the heart weight of wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 5G] This histogram shows the kidney weights of wild-type (WT) and hVH / hcVL heterozygous mice. [Figure 6A] This histogram shows the percentage of blood immune cells that underwent flow cytometry. The blood immune cells are from wild-type or hVH / hcVL mice. [Figure 6B] This histogram shows the percentage of splenic immune cells that underwent flow cytometry. The immune cells are from wild-type or hVH / hcVL mice. [Figure 7] This histogram shows the percentage of lymph node immune cells that underwent flow cytometry. The lymph node immune cells are from wild-type or hVH / hcVL mice. [Figure 8A] This shows the proportion of pro-B cells (low B220, high CD43, low IgM), pre-B cells (low B220, medium CD43, low IgM), and immature B cells (high B220, low CD43, high IgM) in bone marrow B cells. The bone marrow B cells are from wild-type or hVH / hcVL mice. [Figure 8B] This shows the proportion of plasma cells (B220 low IgM-IgD-CD138+) and memory B cells (B220+IgM+IgD-CD38+) in bone marrow B cells. The bone marrow B cells are from wild-type or hVH / hcVL mice. [Figure 9] This shows the proportions of type 1 transitional (T1, B220+IgM+IgD-), type 2 transitional (T2, B220+IgM+IgD+), and mature B cell (M, B220+IgM-lowIgD+) populations in spleen B cells. The spleen B cells are from wild-type or hVH / hcVL mice. [Figure 10A] This shows the proportion of plasma cells (B220 low IgM-IgD-CD138+) in spleen B cells. Spleen B cells are from wild-type or hVH / hcVL mice. [Figure 10B] This shows the proportion of memory B cells (B220+IgM+IgD-CD38+) in the spleen B cell population. The spleen B cells are from wild-type or hVH / hcVL mice. [Figure 10C] This shows the proportion of marginal zone B cells (MZ, B220+CD21+CD23-) and follicular B cells (F0, B220+CD21-lowCD23+) in spleen B cells. Spleen B cells are from wild-type or hVH / hcVL mice. [Figure 11A] The flow cytometry results of mB220-labeled spleen B cells in wild-type female mice are shown. [Figure 11B] The flow cytometry results of mB220-labeled spleen B cells in hVH / hcVL female mice are shown. [Figure 11C]The flow cytometry results of mB220-labeled spleen B cells in wild-type male mice are shown. [Figure 11D] Flow cytometry results of mB220-labeled spleen B cells in hVH / hcVL male mice are shown. [Figure 11E] Flow cytometry results of spleen B cells labeled with mIgG κ-FITC and mIgG λ-PE in wild-type female mice are shown. [Figure 11F] Flow cytometry results of spleen B cells labeled with mIgG κ-FITC and mIgG λ-PE in hVH / hcVL female mice are shown. [Figure 11G] Flow cytometry results of spleen B cells labeled with mIgG κ-FITC and mIgG λ-PE in wild-type male mice are shown. [Figure 11H] Flow cytometry results of spleen B cells labeled with mIgG κ-FITC and mIgG λ-PE in hVH / hcVL male mice are shown. [Figure 11I] This histogram shows the proportion of B cells labeled with mIgG λ-PE (mIgG λ) and spleen B cells labeled with mIgG κ-FITC (mIgG κ). [Figure 12A] This shows the proportions of type 1 transitional (T1, B220+IgM+IgD-), type 2 transitional (T2, B220+IgM+IgD+), and mature B cell (M, B220+IgM-lowIgD+) populations in lymph node B cells. The lymph node B cells are from wild-type or hVH / hcVL mice. [Figure 12B] This shows the proportion of plasma cells (B220 low IgM-IgD-CD138+) and memory B cells (B220+IgM+IgD-CD38+) in lymph node B cells. Lymph node B cells are from wild-type or hVH / hcVL mice. [Figure 13A] The flow cytometry results of mB220-labeled lymph node B cells in wild-type female mice are shown. [Figure 13B]The flow cytometry results of mB220-labeled lymph node B cells in hVH / hcVL female mice are shown. [Figure 13C] The flow cytometry results of mB220-labeled lymph node B cells in wild-type male mice are shown. [Figure 13D] The flow cytometry results of mB220-labeled lymph node B cells in hVH / hcVL male mice are shown. [Figure 13E] The flow cytometry results of lymph node B cells labeled with mIgG κ-FITC and mIgG λ-PE in wild-type female mice are shown. [Figure 13F] Flow cytometry results of lymph node B cells labeled with mIgG κ-FITC and mIgG λ-PE in hVH / hcVL female mice are shown. [Figure 13G] Flow cytometry results of lymph node B cells labeled with mIgG κ-FITC and mIgG λ-PE in wild-type male mice are shown. [Figure 13H] Flow cytometry results of lymph node B cells labeled with mIgG κ-FITC and mIgG λ-PE in hVH / hcVL male mice are shown. [Figure 13I] This histogram shows the proportion of B cells labeled with mIgG λ-PE (mIgG λ) and lymph node B cells labeled with mIgG κ-FITC (mIgG κ). [Figure 13J] This shows the serum immunoglobulin (Ig) subtype concentrations in wild-type (WT) or pre-immunization hVH / hcVL mice. Ig subtype concentrations are assayed by ELISA. [Figure 13K] The results of the Ig subtype analysis before and after immunization in hVHH / H / hcVLK / K mice and wild-type mice are shown. [Figure 14]This is a schematic diagram highlighting the light chain immunoglobulin locus on human chromosome 2 (the scale of the diagram is not constant). VHK represents the IGKV gene cluster segment, JHK represents the IGKJ gene cluster segment, and CHK represents the IGKC gene. [Figure 15] This document lists the human distal Vκ cluster IGKV genes and the human proximal Vκ cluster IGKV genes. [Figure 16] This is a schematic diagram showing the human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.33). [Figure 17-1] This is a schematic diagram showing the mouse (Mus musculus) IGH gene locus on chromosome 12 (12F2) (strain C57BL / 6). [Figure 17-2] This is a schematic diagram showing the mouse (Mus musculus) IGH gene locus on chromosome 12 (12F2) (strain C57BL / 6). [Figure 18] This is a schematic diagram showing the human immunoglobulin κ chain (IGK) gene locus on chromosome 2 (2p11.2). [Figure 19-1] This is a schematic diagram showing the mouse (Mus musculus) IGK gene locus on chromosome 6 (6C1). [Figure 19-2] This is a schematic diagram showing the mouse (Mus musculus) IGK gene locus on chromosome 6 (6C1). [Figure 20-1] This shows a list of IMGT repertoire for the human heavy chain immunoglobulin (IGH) locus. [Figure 20-2] This shows a list of IMGT repertoire for the human heavy chain immunoglobulin (IGH) locus. [Figure 20-3] This shows a list of IMGT repertoire for the human heavy chain immunoglobulin (IGH) locus. [Figure 20-4] This shows a list of IMGT repertoire for the human heavy chain immunoglobulin (IGH) locus. [Figure 20-5] This shows a list of IMGT repertoire for the human heavy chain immunoglobulin (IGH) locus. [Figure 20-6]This shows a list of IMGT repertoire for the human heavy chain immunoglobulin (IGH) locus. [Figure 21-1] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-2] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-3] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-4] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-5] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-6] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-7] This shows a list of IMGT repertoire for mouse IGH. [Figure 21-8] This shows a list of IMGT repertoire for mouse IGH. [Figure 22-1] This shows a list of IMGT repertoires for the human κ-chain immunoglobulin locus (IGK). [Figure 22-2] This shows a list of IMGT repertoires for the human κ-chain immunoglobulin locus (IGK). [Figure 22-3] This shows a list of IMGT repertoires for the human κ-chain immunoglobulin locus (IGK). [Figure 23-1] This shows a list of IMGT repertoire for mouse IGK. [Figure 23-2] This shows a list of IMGT repertoire for mouse IGK. [Figure 23-3] This shows a list of IMGT repertoire for mouse IGK. [Figure 23-4] This shows a list of IMGT repertoire for mouse IGK. [Figure 23-5] This shows a list of IMGT repertoire for mouse IGK. [Figure 23-6] This shows a list of IMGT repertoire for mouse IGK. [Figure 24]This shows antigen-specific antibody titers after three immunizations with the first test antigen in wild-type and hVHH / H / hcVLK / + mice. [Figure 25] This shows antigen-specific antibody titers after three immunizations with a second test antigen in wild-type and hVHH / H / hcVLK / + mice. [Figure 26A] This shows the IGHV utilization rate (frequency > 1%) in unimmunized hVHH / H / hcVLK / + mice. [Figure 26B] This shows the IGHV utilization rate (frequency <1%) in unimmunized hVHH / H / hcVLK / + mice. [Figure 26C] This shows the IGHD utilization rate in unimmunized hVHH / H / hcVLK / + mice. [Figure 26D] This shows the IGHJ utilization rate in unimmunized hVHH / H / hcVLK / + mice. [Figure 26E] This shows the IGHV utilization rate (frequency > 1%) in unimmunized hVHH / H / hcVLK / K mice. [Figure 26F] This shows the IGHV utilization rate (frequency <1%) in unimmunized hVHH / H / hcVLK / K mice. [Figure 26G] This shows the IGHJ utilization rate in unimmunized hVHH / H / hcVLK / K mice. [Figure 27A] This is a histogram showing the heavy chain CDR3 amino acid length distribution from unimmunized hVHH / H / hcVLK / + mice. [Figure 27B] This is a histogram showing the heavy chain CDR3 amino acid length distribution from unimmunized hVHH / H / hcVLK / K mice. [Figure 28A] This shows the amino acid frequencies on the heavy chain CDR3 (where the length of CDR3 is equal to 17 amino acids) in unimmunized hVHH / H / hcVLK / + mice. [Figure 28B] This shows the amino acid frequencies on the heavy chain CDR3 (where the length of CDR3 is equal to 19 amino acids) in unimmunized hVHH / H / hcVLK / + mice. [Figure 28C] This shows the amino acid frequencies on the heavy chain CDR3 (where the length of CDR3 is equal to 21 amino acids) in unimmunized hVHH / H / hcVLK / + mice. [Figure 28D] This shows the amino acid frequencies on the heavy chain CDR3 (where the length of CDR3 is equal to 17 amino acids) in unimmunized hVHH / H / hcVLK / K mice. [Figure 29] This shows the frequencies of heavy chain CDR3s containing multiple cysteine ​​residues, one cysteine ​​residue, and two cysteine ​​residues on CDR3s of different lengths in unimmunized hVHH / H / hcVLK / + mice. [Figure 30A] The sequence exhibits somatic hypermutability of the amino acid (AA) encoded in hVHH / H / hcVLK / + mice, as seen in the reconstituted human IGKV3-11 / J1 sequence. [Figure 30B] This shows the DNA somatic hypermutation rate in the reconstituted human IGKV3-11 / J1 sequence of hVHH / H / hcVLK / + mice. [Figure 30C] This shows the DNA somatic hypermutation rate in the reconstituted human IGKV3-11 / J1 sequence of hVHH / H / hcVLK / K mice. [Figure 31] The epitope clustering results for 15 human anti-antigen A antibodies are shown. [Figure 32-1] A list of sequences described in this disclosure is provided below. [Figure 32-2] A list of sequences described in this disclosure is provided below. [Figure 32-3] A list of sequences described in this disclosure is provided below. [Figure 32-4] A list of sequences described in this disclosure is provided below. [Figure 32-5] A list of sequences described in this disclosure is provided below. [Figure 32-6] A list of sequences described in this disclosure is provided below. [Figure 33A] This shows the heavy chain diversity of the VDJ recombinant assay of antibody-targeted antigen D performed in hVHH / H / hcVLK / K mice. [Figure 33B]This shows the heavy chain diversity of the VDJ recombinant assay of antibody-targeted antigen E performed in hVHH / H / hcVLK / K mice. [Figure 34A] This shows the distribution of the association rate (kon) and dissociation rate (koff) of antibody target antigen D. [Figure 34B] This shows the distribution of the association rate (kon) and dissociation rate (koff) of antibody target antigen D. [Figure 34C] This shows the distribution of the association rate (kon) and dissociation rate (koff) of antibody target antigen E. [Figure 34D] This shows the distribution of the association rate (kon) and dissociation rate (koff) of antibody target antigen E. [Figure 35A] This shows the distribution of binding affinity (KD) for antibody target antigen D. [Figure 35B] This shows the distribution of binding affinity (KD) for antibody target antigen E. [Modes for carrying out the invention]

[0053] Monoclonal antibodies typically contain two heavy chains, where each heavy chain monomer is associated with the same light chain. Due to their target specificity, monoclonal antibodies can usually bind to a single target. However, in drug development, there is a need for antibodies that can bind to two different antigens or epitopes. For example, CD3-specific antibodies typically pair with various tumor-associated antigen-specific antibodies to produce bispecific antibodies that treat cancer. The ability of bispecific antibodies to bind to two different antigens or epitopes offers a wide range of clinical applications. To date, at least two bispecific antibodies are commercially available, and numerous bispecific antibodies are in clinical trials.

[0054] Bispecific antibodies typically have two different heavy chains and two different light chains. Random pairing of the two heavy chains and two light chains results in the expression of 10 different IgG species, of which only one is in the desired format, making their production in a single host cell challenging. Knob-into-hole structures have already been proposed to modify the heavy chains. Several other modifications have also been proposed for the light chains. However, these additional modifications can negatively impact biochemical and / or biophysical properties, serum half-life, and / or stability, potentially leading to low therapeutic efficacy, instability, and high immunogenicity. A method that does not overly rely on antibody engineering is needed to produce bispecific antibodies.

[0055] This disclosure relates to genetically modified animals and cells having a humanized light chain immunoglobulin locus (e.g., a κ chain locus) and / or a humanized heavy chain immunoglobulin locus. In one embodiment, the humanized light chain immunoglobulin locus has a set of finite numbers of IGKV and IGKJ genes. The genetically engineered animal undergoes a long and complex antibody selection process in vivo to make a biologically appropriate selection when pairing a diverse set of human heavy chain variable domains with a finite number of human light chain variable domain options. The animal is engineered to exhibit a combination of a finite number of human light chain variable domain options and a broad range of human heavy chain variable domain options. In the immunogen challenge, the animal develops antibodies against the immunogen, which are limited, primarily or entirely, by the number of light chain options in its repertoire.

[0056] In various embodiments, antibodies produced in genetically modified animals have a heavy chain that can bind to the same or substantially the same light chain. This is particularly useful when producing bispecific antibodies. For example, such an animal can be immunized with a first antigen to produce B cells that express an antibody that specifically binds to the first antigen. The animal (or an animal having the same modification) can be immunized with a second antigen to express B cells that express an antibody that specifically binds to the second antigen. VH can be cloned from the first B cell and the second B cell. The two VHs can be paired with the same light chain VL to produce a bispecific antibody. Therefore, it is not necessary to associate one light chain with one specific heavy chain by antibody engineering (e.g., introducing a modification to the sequence). This can significantly improve the success rate of bispecific antibody development. In fact, the antibodies or sequences described herein can be further bound to each other to produce multispecific antibodies.

[0057] To obtain a finite repertoire of light chain variable domain options, the animals are engineered to limit their ability to produce animal light chain variable domains with natural diversity. The endogenous animal locus may then be modified with a selected exogenous appropriate human light chain variable region gene sequence and operably ligated to an endogenous animal light chain constant domain, thereby reconstructing the exogenous human variable region gene sequence, which can encode a reconstructed chimeric light chain (with human variable endogenous being constant), or can reconstruct and recombine an unreconstructed exogenous human variable region gene sequence with finite diversity to encode a reconstructed chimeric light chain.

[0058] The genetically modified animals described herein may have several other advantages. For example, in some cases, the genetically modified animals described herein have a complete human heavy chain variable region gene and a reconstituted human light chain variable region gene (e.g., human IGKV1-39 / IGKJ4). Since the entire heavy chain variable region (unmodified or with limited modification) on the human immunoglobulin locus is introduced into the animal genome, these genes can undergo VDJ recombination in a manner quite similar to that which occurs in humans. Furthermore, due to effective VDJ recombination, antibody production can be quite effective and at a rate similar to that of normal.

[0059] 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 one another by disulfide bonds. Each heavy chain contains a heavy chain variable (VH) domain and a heavy chain constant region (CH). Each light chain contains a light chain variable (VL) domain and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with relatively conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" refers to an antibody that has K against its target epitope. D about 10 -8 M or less (for example, approximately 1 × 10) -8 M, 1×10 -9 M, 1×10 -10 M or 1 x 10 -11 This refers to antibodies that are M or less. D This is surface plasmon resonance, for example, BIACORE TM Alternatively, it can be measured by ELISA.

[0060] As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, where the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment comprises at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0061] As used herein, the term “human antibody” refers to an antibody encoded by a nucleic acid present in humans (e.g., a reconstituted human immunoglobulin heavy chain or light chain locus). In some embodiments, human antibodies are recovered from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., mice) containing unreconstituted or reconstituted human immunoglobulin loci (e.g., heavy chain or light chain human immunoglobulin loci).

[0062] As used herein, the term “chimeric antibody” refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence from a human antibody (e.g., all or part of the light chain and / or heavy chain variable domain sequence) and a constant domain from a non-human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0063] As used herein, the term “humanized antibody” refers to a non-human antibody that contains sequences derived from non-human (e.g., mouse) immunoglobulins as well as sequences derived from human immunoglobulins.

[0064] Where used herein, the terms “subject” and “patient” are used synonymously throughout this specification and are intended to represent animals, humans, or non-humans. This disclosure encompasses both veterinary and non-veterinary applications. Human patients may be adult humans or young humans (e.g., humans 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, miniature pigs), equids, canids, felines, bovines, and other domestic animals, farm animals, and zoo animals.

[0065] As used herein, when referring to an antibody, the expressions "specifically binds to..." and "specifically binds to..." mean that the antibody preferentially interacts with that target molecule compared to other molecules, because the interaction depends on the presence of a specific structure on the target molecule (i.e., the antigen determines the cluster or epitope). In other words, the reagent does not recognize and bind to all molecules in general, but rather recognizes and binds to molecules containing a specific structure. An antibody that specifically binds to a target molecule may also be called a target-specific antibody.

[0066] As used herein, the terms “polypeptide,” “peptide,” and “protein” may be used synonymously and refer to amino acid polymers of any length having at least two amino acids.

[0067] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” may be used synonymously herein and refer to nucleotide polymers of any length having at least two nucleotides, and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and their modifications.

[0068] As used herein, the term “unmodified human sequence” means a sequence derived from a human subject, human cell, cultured human cell, or human cell line, wherein the sequence is identical to the gene sequence of the human subject, human cell, cultured human cell, or human cell line.

[0069] As used herein, the term “bispecific antibody” includes antibodies that can selectively bind to two or more epitopes. A bispecific antibody typically comprises two distinct heavy chains, each specifically binding to a different epitope, either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule (e.g., different epitopes on the same immunogen). The epitopes specifically bound by a bispecific antibody may be on the same or different targets (e.g., the same or different proteins). For example, a bispecific antibody can be made by binding heavy chains that recognize different epitopes on the same immunogen. For example, a nucleic acid sequence encoding a heavy chain variable sequence may be fused to a nucleic acid sequence encoding the same or different heavy chain constant region, where the heavy chain variable sequence recognizes different epitopes on the same immunogen and such a sequence can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each heavy chain having three heavy chain CDRs, followed (from the N end to the C end) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain, the immunoglobulin light chain not conferring epitope binding specificity but may be associated with each heavy chain, or may be associated with each heavy chain and may bind to one or more epitopes bound by the heavy chain epitope binding region, or may be associated with each heavy chain phase and may bind one or two heavy chains to one or two epitopes.

[0070] As used herein, the term “common light chain antibody” refers to an antibody having two or more identical light chains. In some embodiments, the common light chain antibody is a common light chain bispecific antibody.

[0071] Restricted 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 human antibody (or immunoglobulin) light chain genes. 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).

[0072] The ligation of the V segment (also known as the IGKV gene) and the J segment (also known as the IGKJ gene) produces a continuous exon that codes for the entire light chain variable domain. In unreconstructed DNA, the V gene segment (or IGKV gene cluster) is located far from the C region. The J gene segment (or IGKJ gene cluster) is located near the C region. The ligation of the V and J gene segments also brings the V gene closer to the C region sequence. In the reconstructed V region, the J gene segment is separated from the C region sequence by a single intron. After transcription, RNA splicing ligates the V region exon to the C region sequence to produce fully immunoglobulin light chain messenger RNA.

[0073] The human light chain immunoglobulin locus is located on human chromosome 2. Table 1 lists the IGKV genes located at this locus and their relative order. The human IGKV genes belong to several different groups, including the IGKV1 gene (containing all IGKV genes from IGKV1, also known as VκI), the IGKV2 gene (containing all IGKV genes from IGKV2, also known as VκII), the IGKV3 gene (containing all IGKV genes from IGKV3, also known as VκIII), the IGKV4 gene (containing all IGKV genes from IGKV4, also known as VκIV), the IGKV5 gene (containing all IGKV genes from IGKV5, also known as VκV), the IGKV6 gene (containing all IGKV genes from IGKV6, also known as VκVI), and the IGKV7 gene (containing all IGKV genes from IGKV7, also known as VκVII).

[0074] These IGKV genes on human chromosome 2 also form two clusters: a proximal Vκ cluster and a distal Vκ cluster (Figure 14). The sequences in the two clusters are similar but not identical. This large segmental copy of the sequence occurred when the human repertoire began to diverge from the most recent common ancestor with other great apes. Figure 15 shows an overview of the related IGKV genes in the clusters.

[0075] [Table 1]

[0076] Table 2 lists all IGKJ genes on human chromosome 2 and their relative order. The immunoglobulin κ constant region (IGKC) gene, which encodes the light chain immunoglobulin constant domain, is located after the IGKV and IGKJ genes. These genes and their order are also shown in Figures 18 and 22.

[0077] [Table 2]

[0078] The mouse light chain immunoglobulin locus is located on mouse chromosome 6. Table 3 lists the IGKV genes located at this locus and their relative order.

[0079] [Table 3-1] [Table 3-2] [Table 3-3]

[0080] 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 4 lists all IGKJ genes on mouse chromosome 6 and their relative order. The IGKC gene, which encodes the constant domain of light chain immunoglobulin, is located after the IGKV and IGKJ genes. These genes and their order are also shown in Figures 19 and 23.

[0081] [Table 4]

[0082] This disclosure provides genetically modified non-human animals comprising one, two, three or fewer human IGKV genes and one, two, three or fewer human IGKJ genes. In some embodiments, the human IGKV and human IGKJ genes are located at endogenous light chain immunoglobulin loci. In some embodiments, the human IGKV and human IGKJ genes are reconstituted sequences. In some embodiments, they are non-reconstituted sequences.

[0083] In some embodiments, the human IGKV gene is selected from any one of the IGKV genes in Table 1. In some embodiments, the human IGKJ gene is selected from any one of the IGKJ genes in Table 2.

[0084] In some embodiments, the animal comprises only one human IGKV gene and one human IGKJ gene. In some embodiments, the IGKV gene is selected from any one of the IGKV genes in Table 1. In some embodiments, the IGKJ gene is selected from any one of the IGKJ genes in Table 2.

[0085] In some embodiments, the animal comprises a human IGKV gene selected from IGKV3-20, IGKV1-39, IGKV1D-39, IGKV3-11, IGKV3-15, and IGKV4-1. In some embodiments, the animal comprises a human IGKJ gene selected from IGKJ1, IGKJ2, and IGKJ4.

[0086] In some embodiments, the animal comprises a human IGKV gene selected from IGKV3-20, IGKV3-11, IGKV3-15, IGKV1-39, IGKV1D-39, and IGKV1-12, IGKV1D-12. In some embodiments, the animal comprises a human IGKJ gene selected from IGKJ1, IGKJ4, and IGKJ2.

[0087] In some embodiments, the animal comprises a human IGKV gene selected from IGKV3-20, IGKV3-11, and IGKV1-39. In some embodiments, the animal comprises a human IGKJ gene selected from IGKJ1 and IGKJ4.

[0088] In some embodiments, the animal includes a promoter sequence preceding the first nucleotide of the human IGKV gene. In some embodiments, the promoter sequence is within or approximately within 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3500, 4000, 4500, or 5000 bp preceding the human IGKV gene. In some embodiments, the promoter is the human IGKV3-20 promoter, the human IGKV1-39 promoter, or the human IGKV3-11 promoter. In some embodiments, the promoter sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleotides 1-2000 of SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0089] In some embodiments, the animal includes an auxiliary sequence after the last nucleotide of the human IGKJ gene. In some embodiments, the auxiliary sequence includes a mouse IGKJ 3'-UTR sequence or a human IGKJ 3'UTR sequence. In some embodiments, the sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7.

[0090] In various embodiments, one or more suitable enhancers may be retained within the animal. For example, when the κ locus is modified and the endogenous κ variable region gene segment is replaced with a human κ variable region gene segment, the κ intron type enhancer and the κ 3' enhancer are functionally maintained or not interfered with. In some embodiments, the modified κ locus can undergo somatic hypermutation. In some embodiments, the degree of somatic hypermutation is approximately the same as or similar to that of the wild-type κ locus. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the human light chain variable region in the antibody (before or after immunization with the antigen) has at least one somatic mutation. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the human light chain variable region in the antibody (before or after immunization with the antigen) has at least two somatic mutations. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the human light chain variable region in the antibody (before or after immunization with the antigen) has at least three somatic mutations.

[0091] In some embodiments, the animal contains endogenous IGKC. In some embodiments, the IGKV gene and / or IGKJ gene are operably ligated to the IGKC gene (e.g., endogenous IGKC gene).

[0092] In some embodiments, the IGKV gene is operably ligated to the IGKJ gene.

[0093] In some embodiments, the animal contains endogenous IGKC. In some embodiments, the IGKV gene and / or IGKJ gene are operably ligated. VJ recombination can occur between these genes, and functional antibodies can also be produced. In some embodiments, the IGKV and IGKJ genes are rearranged at the endogenous κ-chain immunoglobulin locus.

[0094] In some embodiments, the animal includes a disruption at the animal's endogenous light chain immunoglobulin locus. In some embodiments, the disruption at the animal's endogenous light chain immunoglobulin locus includes the deletion of one or more endogenous IGKV genes and one or more endogenous IGKJ genes.

[0095] In some embodiments, the animal is a mouse. The disruption of the endogenous light chain immunoglobulin gene locus of the animal includes deletion of at least one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred In some embodiments, the disruption includes deletions of approximately or at least one, two, three, four, five, six, seven, eight, nine, or ten 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 contains about or at least one, two, three, four, five, six, seven, eight, nine, or ten 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.

[0096] In some embodiments, the disruption is selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1 and comprises deletions of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes. In some embodiments, the mouse still contains 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.

[0097] In some embodiments, the disruption involves the deletion of about or at least one, two, three, four, or five mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5. In some embodiments, the mouse still contains about or at least one, two, three, four, or five mouse IGKJ genes (e.g., IGKJ5) selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.

[0098] In some embodiments, disruption of the endogenous κ-light chain immunoglobulin locus in the animal includes a deletion of an endogenous sequence of about or at least 500kb, 600kb, 700kb, 800kb, 900kb, 1000kb, 1500kb, 2000kb, 2500kb, 3000kb, or 3500kb.

[0099] In some embodiments, the deleted sequences are IGKV2-137 to IGKJ4, IGKV1-136 to IGKJ4, IGKV1-135 to IGKJ4, IGKV2-137 to IGKJ5, IGKV1-136 to IGKJ5, or IGKV1-135 to IGKJ5 (for example, IGKV2-137 to IGKJ5).

[0100] In some embodiments, the animal contains approximately or at least one, two, three, four, five, six, seven, eight, nine, or ten 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 the sequence at the human light chain immunoglobulin locus. In some embodiments, the length of the sequence is approximately or at least 2kb or 3kb. In some embodiments, the length of the sequence is 4kb or less.

[0101] In some embodiments, the animal contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reconstructed human IGKV / IGKJ sequences. In some embodiments, the reconstructed human IGKV / IGKJ sequence 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 SEQ ID NO:35 (e.g., nucleotides 2001-2512 of SEQ ID NO:35). In some embodiments, the reconstituted human IGKV / IGKJ sequence 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 SEQ ID NO:36 (e.g., nucleotides 2001-2551 of SEQ ID NO:36). In some embodiments, the reconstituted human IGKV / IGKJ sequence 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 SEQ ID NO:37 (e.g., nucleotides 2001-2572 of SEQ ID NO:37).

[0102] In some embodiments, the length of the reconstituted human IGKV / IGKJ sequence is approximately or at least 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, or 5000 bp. In some embodiments, the reconstituted human IGKV / IGKJ sequence is smaller than 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, or 3100 bp.

[0103] In some embodiments, animals can produce immunoglobulins (e.g., IgG) containing a light chain variable region 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 SEQ ID NO:38. In some embodiments, animals can produce immunoglobulins (e.g., IgG) containing a light chain variable region 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 SEQ ID NO:39. In some embodiments, animals can produce immunoglobulins (e.g., IgG) containing a light chain variable region 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 SEQ ID NO: 40. In some embodiments, the light chain variable region may have 1, 2, 3, 4, or 5 mutations compared to SEQ ID NO: 38, 39, or 40. In some embodiments, the light chain constant domain has 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 SEQ ID NO:41.

[0104] In some embodiments, the animal may have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the length of the unmodified human sequence is about or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kb.

[0105] In some embodiments, the light chain variable region has a SEQ ID NO:38 sequence having 0, 1, or 2 mutations. In some embodiments, the light chain variable region has a SEQ ID NO:39 sequence having 0, 1, or 2 mutations. In some embodiments, the light chain variable region has a SEQ ID NO:40 sequence having 0, 1, or 2 mutations.

[0106] In some cases, the entire mouse IGKV and IGKJ genes (both non-pseudogenes) are knocked out, and the light chain variable region does not contain any sequences encoded by mouse-derived sequences, thereby minimizing immunogenicity in humans. In various embodiments, the light chain variable region is capable of somatic mutation.

[0107] 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 human antibody (or immunoglobulin) heavy chain genes.

[0108] Each region represents the germline tissue of a heavy chain locus. A locus contains V (variable), D (variable), J (linked), and C (stationary) segments. Genes in the V region form the V gene cluster (also known as the IGHV gene cluster). Genes in the D region form the D gene cluster (also known as the IGHD gene cluster). Genes in the J region form the J gene cluster (also known as the IGHJ gene cluster).

[0109] During B cell development, recombination at the DNA level ligates a single D segment (also known as the IGHD gene) to a J segment (also known as the IGHJ gene), and the fused DJ exon of this partially reconstituted DJ region is ligated to the V segment (also known as the IGHV gene). The reconstituted VDJ region, including the fused VDJ exon, is then transcribed and fused to the IGHM constant region at the RNA level, with this transcript encoding a μ heavy chain. In late development, B cells produce VDJ-Cμ-Cδ premessenger RNA, which is alternatively spliced ​​to encode a μ or δ heavy chain. In mature B cells in lymph nodes, switch recombination occurs, causing the fused VDJ gene segment to approach one of the IHG, IGHA, or IGHE gene segments, and each cell expresses a γ, α, or ε heavy chain. Potential recombination between many different IGHV genes and some IGHJ genes provides broad antigen recognition. Further diversity is obtained from ligation diversity produced by random nucleotide addition by terminal deoxyribonucleotide convertases and somatic hypermutation occurring during the B-cell maturation process in the spleen and lymph nodes. Several V, D, J, and C segments are known to be non-coding and are considered pseudogene segments (commonly abbreviated as pseudogenes).

[0110] The human heavy chain immunoglobulin locus is located on human chromosome 14. Table 5 lists the IGHV genes located at this locus and their relative order.

[0111] [Table 5-1] [Table 5-2] [Table 5-3]

[0112] 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 6 lists all IGHD genes on human chromosome 14 and their relative order. Table 7 lists all IGHJ genes on human chromosome 14 and their relative order. The immunoglobulin constant domain genes are located after the IGHV, IGHD, and IGHJ genes. These genes include (as shown in the following order) immunoglobulin heavy chain constant μ (IGHM), immunoglobulin heavy chain constant δ (IGHδ), immunoglobulin heavy chain constant γ3 (IGHG3), immunoglobulin heavy chain constant γ1 (IGHG1), immunoglobulin heavy chain constant εP1 (pseudogene) (IGHEP1), immunoglobulin heavy chain constant α1 (IGHA1), immunoglobulin heavy chain constant γP (non-functional) (IGHGP), immunoglobulin heavy chain constant γ2 (IGHG2), immunoglobulin heavy chain constant γ4 (IGHG4), immunoglobulin heavy chain constant ε (IGHE), and immunoglobulin heavy chain constant α2 (IGHA2). These genes and their order are also shown in Figures 16 and 20.

[0113] [Table 6]

[0114] [Table 7]

[0115] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12. Table 8 lists the IGHV genes located at this locus and their relative order.

[0116] [Table 8-1] [Table 8-2] [Table 8-3]

[0117] Table 9 lists all IGHD genes on mouse chromosome 12 and their relative order. Table 10 lists all IGHJ genes on mouse chromosome 12 and their relative order. Immunoglobulin constant domain genes are located after the IGHV, IGHD, and IGHJ genes. These genes include (as shown in the following order) the 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 Figures 17 and 21.

[0118] [Table 9]

[0119] [Table 10]

[0120] This 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.

[0121] The genetically modified animals can be produced by introducing human immunoglobulin genes into the genome of non-human animals to produce animals capable of expressing humanized antibodies or chimeric antibodies. Figure 1A shows a method for producing humanized mice. In some embodiments, the method first relates to the modification of the human immunoglobulin region on the human chromosome. The modified human chromosome is then introduced into mouse receptor cells. The human immunoglobulin variable region is then introduced into the corresponding region of the mouse genome by direct substitution (e.g., one-step substitution). The receptor cells, preferably cells that do not contain human chromosomes, are then screened. The cells are then injected into blastulas to produce chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing the fully humanized immunoglobulin gene locus.

[0122] In some embodiments, the human IGHV gene, human IGHD gene, and human IGHJ gene are operablely ligated and can undergo VDJ rearrangement. In some embodiments, the human IGHV gene, human IGHD gene, and human IGHJ gene are located at the endogenous heavy chain immunoglobulin locus.

[0123] In some embodiments, the animal contains 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 (for example, the genes shown in Table 5).

[0124] In some embodiments, the animal contains one, two, three, four, five, six, seven, eight, nine, or ten genes selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75.

[0125] In some embodiments, the animal contains one, two, three, four, five, six, seven, eight, nine, or ten genes selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1.

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

[0127] In some embodiments, the animal contains 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., the genes shown in Table 6). In some embodiments, the animal contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27.

[0128] In some embodiments, the animal contains about or at least one, two, three, four, five, six, seven, eight, or nine human IGHJ genes (for example, the genes shown in Table 7). In some embodiments, the animal contains one, two, three, four, five, six, seven, eight, or nine human IGHJ genes selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6.

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

[0130] In some embodiments, the unmodified human sequence is derived from the portion of the human heavy chain immunoglobulin locus from IGHD1-1 to human IGHD7-27.

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

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

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

[0134] In some embodiments, the animal may have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the length of the unmodified human sequence is about or at least 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 200kb, 300kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, or 1000kb.

[0135] In some embodiments, the animal contains one or more endogenous genes selected from the group consisting of immunoglobulin heavy chain constant μ (IGHM), immunoglobulin heavy chain constant δ (IGHδ), immunoglobulin heavy chain constant 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. In some embodiments, these endogenous genes are operably linked. In some embodiments, these endogenous genes have the same order as in wild-type animals. In some embodiments, isotype switches (immunoglobulin class switches) may occur in the animal.

[0136] In some embodiments, the IGHV, IGHD, and / or IGHJ genes are operably linked. VDJ recombination can occur between these genes, producing functional antibodies. In some embodiments, these genes are aligned in an order similar to the order at the human heavy chain immunoglobulin locus. This alignment offers several advantages; for example, it allows for the production of heavy chain variable domains with diversity quite similar to that of heavy chain variable domains in humans. Because some random sequences can be inserted into the sequence during the VDJ recombination process, in some embodiments, a fully human antibody repertoire with no or minimal modifications can reduce the likelihood of non-human sequences being inserted during the VDJ recombination process.

[0137] In some embodiments, the IGHV gene, IGHD gene, and / or IGHJ gene are operably linked to one or more genes selected from the IGHM, IGHδ, IHG3, IHG1, IHG2b, IHG2a, IGHE, and IGHA genes (e.g., all genes).

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

[0139] In some embodiments, the animal is a mouse. The disruption of the endogenous heavy chain immunoglobulin gene locus in the animal includes the deletion of at least one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred In some embodiments, the disruption includes deletions of approximately or at least one, two, three, four, five, six, seven, eight, nine, or ten 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 contains about or at least one, two, three, four, five, six, seven, eight, nine, or ten mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77 (e.g., IGHV1-86).

[0140] In some embodiments, the disruption includes deletions of approximately or at least one, two, three, four, five, six, seven, eight, nine, or ten 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 includes deletions of approximately or at least one, two, three, four, five, six, seven, eight, nine, or ten mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1.

[0141] In some embodiments, disruption at the endogenous heavy chain immunoglobulin locus of the animal includes deletions of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mouse IGHD genes (e.g., the genes shown in Table 9). In some embodiments, the disruption includes deletions of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1. In some embodiments, the mouse still contains about or at least one, two, three, four, five, six, seven, eight, nine, or ten mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1.

[0142] In some embodiments, the disruption comprises deletion of about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4. In some embodiments, the mouse still comprises about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.

[0143] In some embodiments, the disruption in the endogenous heavy chain immunoglobulin locus of the 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.

[0144] 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).

[0145] 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 the sequences in the human heavy chain immunoglobulin locus. In some embodiments, the length of the sequences is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb. In 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.

[0146] The human IGHV gene, human IGHD gene, and human IGHJ gene are operably linked and can undergo VDJ recombination. In some embodiments, the modified mouse has a complete human IGHV, IGHD, and IGHJ gene repertoire (e.g., including non-pseudogenes in all human IGHV, IGHD, and IGHJ genes). Thus, the modified mouse can produce a complete human antibody repertoire.

[0147] In some embodiments, the use of IGHV3-15, IGHV1-18, IGHV3-21, IGHV5-51, IGHV3-74, IGHV3-30-3, IGHV3-43, IGHV1-24, IGHV3-7, IGHV4-4, IGHV3-53, IGHV4-59, IGHV6-1, IGHV3-23, IGHV3-33, IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-34, or IGHV3-66 may be detected (for example, at a frequency of >1% among reconstituted sequences). In some embodiments, the use of IGHV3-38-3, IGHV1-58, IGHV4-38-2, IGHV4-61, IGHV3-NL1, IGHV2-26, IGHV1-2, IGHV7-4-1, IGHV4-28, IGHV3-64, IGHV3-49, IGHV5-10-1, IGHV3-72, IGHV2-5, IGHV2-70, IGHV1-46, IGHV1-3, IGHV3-11, IGHV3-13, IGHV3-20, IGHV3-64D, IGHV1-69, IGHV3-73, IGHV4-30-2, IGHV4-31, or IGHV4-30-4 may be detected (for example, at a frequency of <1% among reconstituted sequences).

[0148] In some embodiments, the use of IGHD5-24, IGHD2-8, IGHD6-25, IGHD1-14, IGHD4-23, IGHD3-16, IGHD1-20, IGHD2-15, IGHD2-21, IGHD1-1, IGHD5-12, IGHD3-22, IGHD7-27, IGHD4-11, IGHD3-9, IGHD3-3, IGHD2-2, IGHD5-18, IGHD4-17, IGHD3-10, IGHD6-6, IGHD1-26, IGHD1-7, IGHD6-19, or IGHD6-13 may be detected (for example, at a frequency of >1% among reconstituted sequences).

[0149] In some embodiments, the use of IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, or IGHJ6 may be detected (for example, at a frequency of >1% among reconstructed sequences).

[0150] In some embodiments, the use of IGHV1-24, IGHV4-30-2, IGHV1-18, IGHV3-43, IGHV4-30-4, IGHV5-51, IGHV3-21, IGHV4-31, IGHV3-7, IGHV3-30-3, IGHV3-53, IGHV4-4, IGHV3-74, IGHV3-66, IGHV3-33, IGHV3-23, IGHV6-1, IGHV3-30, IGHV4-34, IGHV3-48, IGHV4-59, or IGHV4-39 may be detected (for example, at a frequency of >1% among reconstructed sequences).

[0151] In some embodiments, the use of IGHV3-25, IGHV4-38-2, IGHV7-4-1, IGHV3-NL1, IGHV4-61, IGHV1-58, IGHV2-26, IGHV3-72, IGHV5-10-1, IGHV1-46, IGHV3-49, IGHV2-70, IGHV1-2, IGHV3-64, IGHV4-28, IGHV3-20, IGHV1-3, IGHV3-13, IGHV3-73, IGHV3-11, IGHV3-64D, IGHV1-69, IGHV2-5, or IGHV3-15 may be detected (for example, at a frequency of <1% among reconstituted sequences).

[0152] Furthermore, since V(D)J recombination can occur between the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes and human genes, when the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes are incorporated into a reconstituted heavy chain VDJ segment or a reconstituted light chain VJ segment, the antibodies produced by the antibody repertoire are likely to have immunogenic epitopes in humans. This immunogenicity can lead to the production of anti-drug antibodies, which may be active. Here, in some embodiments, the endogenous IGHV, IGHD, IGHJ, IGKV, and IGKJ genes are effectively deleted. In some cases, the entire mouse IGHV, IGHD, and IGHJ genes (including, for example, all non-pseudogenes) are knocked out, and the heavy chain variable region does not contain any sequences encoded by mouse-derived sequences.

[0153] Antibodies produced from the aforementioned antibody repertoire are unlikely to exhibit immunogenicity in humans. Therefore, these antibodies are more suitable for use as therapeutic agents in humans. For this reason, genetically modified animals provide a favorable platform for the production of humanized antibodies.

[0154] Genetically modified λ light chain immunoglobulin locus The lambda chain immunoglobulin locus (also known as IGL or immunoglobulin λ locus) is a region on a chromosome (e.g., human chromosome 22) that contains human antibody (or immunoglobulin) light chain genes. Similarly, immunoglobulin light chain genes can undergo a series of rearrangements that lead to the production of mature immunoglobulin light chain nucleic acids (e.g., lambda chains). In healthy human individuals, the overall κ to λ ratio in serum is approximately 2:1 (measuring complete total antibodies) or 1:1.5 when measuring free light chains. In mice, the overall κ to λ ratio is approximately 9:1.

[0155] In some embodiments, the animal includes the human λ-chain immunoglobulin locus.

[0156] In some embodiments, the animal includes a disruption at the animal's endogenous λ light chain immunoglobulin locus. In some embodiments, the disruption at 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).

[0157] The mouse λ light chain immunoglobulin (IGL) locus is located on mouse chromosome 16. Table 11 lists the IGLV, IGLJ, and IGLC genes located at this locus and their relative order.

[0158] [Table 11]

[0159] The disruption at the endogenous λ light chain immunoglobulin locus of the animal comprises deletions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mouse IGLV, IGLJ, and IGLC genes (e.g., the genes shown in Table 11). In some embodiments, the deletions comprise at least 1, 2, 3, or 4 mouse IGLC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption comprises deletions of at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3. In some embodiments, the disruption comprises deletions of at least 1, 2, 3, 4, or 5 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ3, IGLJ3P, and IGLJ4.

[0160] In some embodiments, disruption of the endogenous λ light chain immunoglobulin locus in the animal includes deletions of nucleotides of approximately or at least 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 110kb, 120kb, 130kb, 140kb, 150kb, 160kb, 170kb, 180kb, 190kb, 200kb, 210kb, 220kb, 230kb, 240kb, 250kb, 260kb, 270kb, 280kb, 290kb, 300kb, 350kb, 400kb, 450kb, 500kb, or 1000kb. In some embodiments, there is no disruption to the endogenous λ light chain immunoglobulin gene in the animal.

[0161] In some embodiments, the deletion sequence is from IGLV2 to IGLC1, from IGLV3 to IGLC1, or from IGLJ2 ​​to IGLC1.

[0162] For a detailed description of genetically modified animals and genetically modified heavy chain immunoglobulin loci, see, for example, PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety.

[0163] Genetically modified animals In one embodiment, the present disclosure provides a genetically modified non-human animal comprising a humanized light chain immunoglobulin locus and / or a humanized heavy chain immunoglobulin locus. The humanized light chain immunoglobulin locus comprises a set of finite numbers of human IGKV genes and / or human IGKJ genes. In some embodiments, these genes are located at an endogenous immunoglobulin locus.

[0164] In some embodiments, the animal includes the human λ-chain immunoglobulin locus. In some embodiments, the animal includes disruption at the animal's endogenous λ-light chain immunoglobulin locus. In some embodiments, the animal does not have disruption at the animal's endogenous λ-light chain immunoglobulin locus.

[0165] The genetically modified non-human animals may be various animals, such as mice, rats, rabbits, pigs, cattle (e.g., female cattle, male cattle, buffalo), 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, non-human animals containing genetic modifications can be produced using other methods. Such methods include, for example, modifying a non-ES cell genome (e.g., fibroblast or induced pluripotent cell), transferring the modified genome into a suitable cell, e.g., an oocyte, using nuclear transfer, and inoculating the modified cell (e.g., modified oocyte) in a non-human animal under appropriate conditions to form an embryo. These methods are known in the art and are described, for example, in Nagy et al., “Manipulating the Mouse Embryo: A Laboratory Manual (3rd Edition),” Cold Spring Harbor Laboratory Press, 2003 (which is hereby incorporated by reference in its entirety). Thus, in various embodiments, the human V, D, and / or J segments may be operably linked to a non-human animal (e.g., rodent, mouse, rat, hamster) constant region gene sequence. In the B cell development process, these rearranged human V, D, and / or J segments are linked to the non-human animal immunoglobulin constant region.

[0166] In one embodiment, the animal is a mammal, for example, a mammal of the superfamily Muroidoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent may be selected from mice, rats and hamsters. In some embodiments, the genetically modified animal is from the families Calomyscidae (e.g., kangaroo hamster), Cricetidae (e.g., hamster, New World rat and mouse, field vole), Muridae (purebred mouse and rat, gerbil, spiny mouse, crested rat), Nesomyidae (climbing mouse, rock mouse, white-tailed hamster, Madagascar rat and mouse), Platacanthomyidae (e.g., spiny dormouse). The genetically modified rodents are selected from families such as dormice and Spalacidae (e.g., mole rats, bamboo rats, and dwarf rats). In some embodiments, the genetically modified rodents are selected from purebred mice or rats (Muridae) such as gerbils, spiny mice, and maned rats. In some embodiments, the non-human animal is a mouse.

[0167] In some embodiments, the animal is a mouse with a C57 background (for example, 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 mice are 129 strains selected from a group consisting of strains 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999), and Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a cross between the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a cross between the 129 strain or the BL / 6 strain. In some embodiments, the mouse is a BALB strain, for example, the BALB / c strain. In some embodiments, the mouse is a cross between the BALB strain and another strain. In some embodiments, the mice are from a crossbreed (e.g., 50% BALB / c-50% 12954 / Sv, or 50% C57BL / 6-50% 129).

[0168] In some embodiments, the animal is a rat. The rat may be selected from Wistar rat, LEA line, Sprague Dawley line, Fischer line, F344, F6, and Dark Agouti. In some embodiments, the rat line is a hybrid of two or more lines selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0169] The animal may have one or more other genetic modifications and / or other modifications suitable for a specific purpose of producing a humanized animal.

[0170] Genetically modified non-human animals comprising modifications of endogenous non-human immunoglobulin loci. In some embodiments, the modifications may include human nucleic acid sequences encoding at least a portion of a human protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human heavy chain variable domain or light chain variable domain sequence). Genetically modified cells (e.g., ES cells, somatic cells) comprising the modifications described herein are also provided, but in many embodiments, the genetically modified non-human animals comprise modifications of endogenous loci in the animal germline.

[0171] Genetically modified animals can express humanized antibodies and / or chimeric antibodies from endogenous mouse loci, where one or more endogenous mouse immunoglobulin genes are replaced with human immunoglobulin genes and / or nucleotide sequences, and the nucleotide sequences and human immunoglobulin gene sequences (e.g., IGHV, IGHD, IGHJ, IGKV, and / or IGKJ genes) are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In various embodiments, endogenous non-human immunoglobulin loci are completely or partially modified to contain human nucleic acid sequences.

[0172] Genetic, molecular, and behavioral analyses can be performed on the above-mentioned non-human mammals. This disclosure also relates to offspring produced by crossing the non-human mammals described herein with the same genotype or other genotypes. The non-human mammal may be any non-human animal known in the art and can be used in the methods described herein. A preferred non-human mammal is a mammal (e.g., a rodent). In some embodiments, the non-human mammal is a mouse.

[0173] This disclosure further provides cell lines or primary cell cultures derived from non-human mammals or their offspring. Cell culture-based models can be prepared, for example, by the following methods: Cell cultures can be obtained by isolation from non-human mammals, or cells can be obtained from cell cultures established using the same construct and standard cell transfection techniques. The incorporation of gene constructs containing DNA sequences encoding human or humanized immunoglobulins can be detected by various methods.

[0174] Numerous analytical methods can be used to detect exogenous DNA or genomic DNA modifications, including nucleic acid-level methods (including mRNA quantification using reverse transcription polymerase chain reaction (RT-PCR) or Southern blotting and in-situ hybridization) and protein-level methods (including histochemistry, immunoblotting, and in vitro binding). Furthermore, the expression level of a target gene can be quantified using ELISA techniques well known to those skilled in the art. Many standard analytical methods can be used to achieve quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods, which include ribonuclease protection, Southern blotting, and RNA dot analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blotting can also be used to assess the presence of human or humanized proteins.

[0175] 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.

[0176] Typically, an antibody (also known as an immunoglobulin) is composed of two polypeptide chains, a light chain and a heavy chain. The non-limiting antibodies of the present disclosure may be four complete immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody may be of any isotype or subclass, including the isotypes IgM, IgG, IgE, IgA or IgD, and the subclasses include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a κ light chain or a λ light chain. The antibody may include two identical copies of the light chain and two identical copies of the heavy chain. Each of these two heavy chains, which includes one variable domain (or variable region, V H ) and multiple constant domains (or constant regions), binds to each other by disulfide bonds within its constant domain to form the "stem" of the antibody. Each of these two light chains, which includes one variable domain (or variable region, V L ) and one constant domain (or constant region), binds to one heavy chain by a disulfide bond respectively. The variable region of each light chain pairs with the variable region of the heavy chain to which it binds. The variable regions of the light and heavy chains both include three hypervariable regions sandwiched between more conserved framework regions (FR).

[0177] These hypervariable regions (referred to as complementarity-determining regions (CDRs)) form loops that include the main antigen-binding surface of the antibody. The four framework regions mainly use a β-sheet conformation, and the CDRs form loops linked to the β-sheet structure, and in some cases, the loops form part of the β-sheet structure. The CDRs in each chain are held in proximity by the framework regions and form an antigen-binding region together with the CDRs from another chain.

[0178] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and many definitions of CDR are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions can be found, 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, TT and Kabat, EA (1970) J. Exp. Med. 132:211-250; Martin et al., Methods Enzymol. 203:121-53(1991); Morea et al., Biophys Chem. 68(1-3):9-16(October 1997); Morea et al., J Mol Biol. 275(2):269-94(January 1998); and Chothia et al., Nature. This is described in 342(6252):877-83 (December 1989) and Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), and each of these references is incorporated herein by reference in its entirety.

[0179] CDRs are important for the recognition of antigen epitopes. As used herein, “epitope” is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids do not need to be located in a continuous linear sequence of the primary structure of the antigen, as the epitope may depend on the three-dimensional arrangement of the antigen based on the secondary and tertiary structures of the antigen.

[0180] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved except for their constant regions, particularly their hinge and upper CH2 domain. The sequences and differences of IgG subclasses are publicly known in this field 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; and Shakib, Farouk (eds.), The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016. Each of these references is incorporated herein by reference in its entirety.

[0181] The antibody may further be an immunoglobulin molecule derived from any species (e.g., human, rodent, mouse, rat, camelid). The antibodies disclosed herein may further include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies containing an immunoglobulin-binding domain that fuses to another polypeptide. The term “antigen-binding domain” or “antigen-binding fragment” refers to any antibody portion that retains the specific binding activity of a complete antibody, i.e., any portion of an antibody that can specifically bind to an epitope on the target molecule of a complete antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment may be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide containing an antibody domain or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, heavy chain and / or light chain CDRs of a complete antibody, heavy chain and / or light chain variable regions of a complete antibody, full-length heavy chain or light chain of a complete antibody, or individual CDRs of heavy chain or light chain from a complete antibody.

[0182] 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 in which the single-chain variable fragment (scFv) described herein fuses with the CD3-ζ transmembrane domain and the internal domain.

[0183] 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 the two antigen-binding regions are capable of binding to their respective target antigens.

[0184] Antibodies and their antigen-binding fragments (e.g., humanized antibodies or chimeric antibodies) produced by the methods described herein have various advantages. In some embodiments, further optimization is not required to achieve desired properties (e.g., binding affinity, thermal stability, and / or limited aggregation).

[0185] In some embodiments, the antibody (or its antigen-binding fragment) specifically binds to the target, where the dissociation rate (koff) is 0.1 s. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 It is less than 0.01s. In some embodiments, the dissociation rate (koff) is 0.01s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Greater than or equal to 0.000001s -1 It is extremely high. In some embodiments, the koff of the majority of these antibodies (e.g., >50%, >60%, >70%, or >80%) is 1 × 10⁻⁶. -2 / S~1×10 -3 It is / S.

[0186] In some embodiments, the association rate (kon) is 1 x 10⁻⁶ 2 / Ms super, 1x10 3 / Ms super, 1x10 4 / Ms super, 1x10 5 / Ms greater than or 1x10 6 It is greater than / Ms. In some embodiments, the association rate (kon) is 1 x 10⁻¹⁰. 5 / Ms less than 1x10 6 / Ms less than or 1x10 7 The kon value is less than / Ms. In some embodiments, the kon value of the majority of these antibodies (e.g., >50%, >60%, >70%, or >80%) is 1 × 10⁻⁶. 5 / Ms~1×10 6 It is / Ms.

[0187] Affinity can be estimated from the quotient of the dynamic rate constant (KD = koff / kon). In some embodiments, KD is 1 x 10⁻¹⁰. -6 Less than M, 1x10 -7 Less than M, 1x10 -8 Less than M, 1x10 -9 Less than M or 1x10 -10 It is less than M. In some embodiments, KD is 50nM, 40nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM. In some embodiments, KD is 1 x 10⁻¹⁶ -7 Super M, 1x10 -8 Super M, 1x10 -9 Super M, 1x10 -10 Super M, 1x10 -11 M or 1x10 -12 The KD is greater than M. In some embodiments, the antibody binds to a target with a KD of approximately 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. In some embodiments, the KD of the majority of these antibodies (e.g., >50%, >60%, >70%, or >80%) is between 0.10 nM and 100.00 nM (e.g., 1 nM to 100 nM).

[0188] In some embodiments, the antibodies produced by the animals described herein have a median heavy chain CDR3 length of 13–15 amino acids. In some embodiments, the majority of these antibodies (e.g., >50%, >60%, >70%, or >80%) have a heavy chain CDR3 length of 9–18 amino acids, or 10–17 amino acids.

[0189] In some embodiments, in antibodies produced by the animals described herein, less than 30%, 20%, or 15% of the heavy chain CDR3 has cysteine ​​residues (e.g., one or two cysteine ​​residues). In some embodiments, the heavy chain CDR3 has 21 or fewer amino acid residues.

[0190] In some embodiments, thermal stability has been established. The antibody or antigen-binding fragments described herein may have a Tm greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0191] Since IgG may be described as a multi-domain protein, the melting curve may show two or three transitions having a first denaturation temperature Tm D1, a second denaturation temperature Tm D2, and an optional third denaturation temperature Tm D3.

[0192] In some embodiments, the antibody or antigen-binding fragment described herein has a Tm D1 greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the antibody or antigen-binding fragment described herein has a Tm D2 greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the antibody or antigen-binding fragment described herein has a Tm D3 greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0193] In some embodiments, Tm, Tm D1, Tm D2, Tm D3 are less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0194] In some embodiments, when the temperature is lower than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C, the antibodies or antigen-binding fragments described herein do not form aggregates.

[0195] Methods for creating genetically modified animals The aforementioned genetically modified animals can be produced by introducing human immunoglobulin genes into the genome of non-human animals to produce animals capable of expressing humanized antibodies or chimeric antibodies. Figure 1A shows a method for producing humanized animals. In some embodiments, the method first relates to the modification of the human immunoglobulin gene locus on the human chromosome. Then, the modified human chromosome is introduced into mouse receptor cells. Then, the human immunoglobulin variable region is introduced into the corresponding region of the mouse genome by direct substitution. Then, the receptor cells are screened. In some embodiments, the cells do not contain human chromosomes. Then, the cells are injected into blastulas to produce chimeric mice. Subsequent breeding can be performed to obtain mice containing the fully humanized immunoglobulin gene locus.

[0196] To produce genetically modified animals, several other techniques can also be used, including, for example, non-homologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), activator-like effector-based nucleases (TALENs), and clustered short palindromic sequence repeats (CRISPR)-Cas systems. Homologous recombination is used in some embodiments. In some embodiments, CRISPR-Cas9 genome editing is used to produce genetically modified animals. Many of these genome editing techniques are publicly 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 herein by reference in its entirety. Many other methods are also provided and can be used for genome editing, for example, by microinjecting genetically modified cells into enucleated oocytes and fusing the enucleated oocytes with other genetically modified cells.

[0197] The gene modification process may also relate to the replacement of endogenous sequences with human sequences by homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., zinc finger nucleases, TALENs, or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace endogenous sequences with human sequences.

[0198] In some embodiments, a method for producing a genetically modified humanized animal may include the step of substituting a nucleic acid (e.g., a V, D, J region or a V, J region) in a corresponding region of a human sequence at an endogenous locus (or site). The sequence may include a region (e.g., a portion or the entirety) of the IGHV, IGHD, IGHJ, IGKV, and / or IGKJ genes. In some embodiments, the substitution is mediated by homologous recombination. In some embodiments, the substitution is mediated by Cre recombinase.

[0199] In some embodiments, modification of the mouse light chain immunoglobulin locus may be performed directly. In some embodiments, the vector may be used to directly replace the entire mouse light chain immunoglobulin variable region. In some embodiments, the vector may be inserted upstream of the V region and between the J and C regions.

[0200] In some embodiments, modification of the mouse light chain immunoglobulin locus can be carried out in more than one step. In some embodiments, the first vector can be used to replace the entire mouse light chain immunoglobulin variable region. In some embodiments, the vector contains one or more selection markers. In some embodiments, the selection marker is a dominant selection marker (e.g., the neomycin resistance gene or Neo). In some embodiments, the selection marker is a negative selection marker (e.g., the diphtheria toxin receptor gene or DTR). In some embodiments, the first vector contains one or more dominant selection markers and / or one or more negative selection markers. As described herein, in some embodiments, the second vector can be used to further replace the region containing one or more selection markers.

[0201] Figure 4 illustrates a targeting strategy for substituting the endogenous mouse light chain immunoglobulin variable region with a human light chain variable region (e.g., a reconstituted or unreconstituted human light chain variable region sequence). For example, the human light chain variable region may include human IGKV genes selected from IGKV3-20, IGKV3-11, and IGKV1-39, and human IGKJ genes selected from IGKJ1 and IGKJ4. In some embodiments, the entire mouse light chain variable region is replaced by a first vector containing Neo and a DTR selection marker gene. In some embodiments, the second vector has one or more of the following from 5' to 3'. The DNA homologous arm sequence upstream of the insertion site (5' homologous arm), the promoter sequence preceding the first nucleotide of the human IGKV gene (e.g., at least or about 2000 bp prior to the human IGKV gene), the human IGKV gene (e.g., IGKV3-20, IGKV3-11, or IGKV1-39), the human IGKJ gene (e.g., IGKJ1 or IGKJ4), auxiliary sequences (e.g., polyA sequence, WPRE sequence, or 3'UTR sequence), and the DNA homologous arm sequence downstream of the insertion site (3' homologous arm).

[0202] These vectors can be incorporated into the cell genome, and cells can be selected using drug resistance markers or combinations thereof (e.g., zeocin, G418, and / or puromycin). In some embodiments, PB transposases may be expressed, and genetic elements between transposase target sequences may be deleted.

[0203] In some embodiments, these vectors are incorporated into a modified human chromosome. The human chromosome can be modified before the first and second vectors are incorporated into the genome. In some embodiments, one or more additional vectors can be added to different locations on the chromosome, as needed. In some embodiments, a vector is added between the C region and the centromere.

[0204] 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 human cells, and the cells are fused with receptor cells. The modified chromosomes are then isolated and introduced into other suitable receptor cells. Cells with the desired resistance are selected to obtain cells containing only one human chromosome. Then, a second vector is introduced into the cells, and cells are selected based on resistance. A third vector and / or a fourth vector may be introduced if necessary. The receptor cells may be mammalian cells, human cells, or mouse cells. In some embodiments, the receptor cells are CHO cells, or preferably A9 cells. In some embodiments, the modified chromosomes are labeled with fluorescence and isolated. The modified chromosomes are then injected into the receptor cells by chromosome microinjection. In some embodiments, donor cells are induced to make their chromosomes multinucleated. These cell nuclei are then extruded through the cell membrane to form microcells that can fuse with receptor cells. In some embodiments, chromosome transfer mediated by microcells may be used.Chromosome manipulation techniques include, for example, CN 1200014 A, CN 109837307 A, US 20120093785 A1 and US 2009253902, Kuroiwa et al. "Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts." Nature Biotechnology 18.10(2000):1086-1090, Chinese Patent CN 1717483 A, Paulis, Marianna. "Chromosome Transfer Via Cell Fusion." Methods in Molecular Biology 738(2011):57, Genes, Chromosomes & Cancer 14:126127(1995), Tomizuka et al. "Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice." Nature Genetics This is described in 16.2(1997):133-143, Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284, where each reference is incorporated herein by reference in its entirety.

[0205] Furthermore, the LoxP recognition sequence can be added to human chromosomes (e.g., human chromosomes 2, 14, and 22). Genomic DNA sequences can also be removed by treating cells with Cre enzymes to induce recombination of the loxP site. In some embodiments, spontaneous chromosome breaks can also be used to remove genomic DNA sequences.

[0206] Mouse immunoglobulin variable regions can be replaced with human immunoglobulin variable regions by substitution (e.g., homologous recombination or Cre-mediated recombination). In some embodiments, Cre recombination can be used to mediate the substitution. In some embodiments, the vector can add a LoxP recognition sequence to the human chromosome. Similar modifications can be made to mouse chromosomes, where two LoxP recognition sequences can be added to the chromosome. For example, Cre recombinase can mediate the substitution of the V,J region on the mouse chromosome with the V,J region on the human chromosome, or the substitution of the V,D,J region on the mouse chromosome with the V,D,J region on the human chromosome.

[0207] Cells that do not possess human chromosomes can be further screened (e.g., by DT). In some cases, cells that have not been DT screened may contain recombinant human chromosome fragments, but these fragments are small, unstable in mouse cells (e.g., Shinohara et al. (2000) Chromosome Research, 8:713-725), and spontaneously disappear during cell proliferation. In some embodiments, large fragments of modified human chromosomes are deleted, for example, by Cre-mediated removal or by being cut by native chromosomes.

[0208] The 5'-end homologous arm and / or 3'-end homologous arm may have a desired length to promote homologous recombination. In some embodiments, the homologous arm is about or at least 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 20kb, 30kb, 40kb, or 50kb (e.g., about 3kb). In some embodiments, the homologous arm is smaller than 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 20kb, 30kb, 40kb, or 50kb.

[0209] In some embodiments, the vector may optionally further include a reporter protein, such as luciferase (e.g., Gluc) or a fluorescent protein (e.g., EGFP, BFP, etc.).

[0210] These modifications can be performed on various cells. In some embodiments, the cells are stem cells, embryonic stem cells, or fertilized egg cells.

[0211] This disclosure further provides a method for establishing a humanized animal model, the method being described below. (a) the step of providing cells (e.g., fertilized egg cells) based on the method described herein, (b) A step of culturing cells in liquid medium, (c) The step of transplanting cultured cells into the fallopian tube or uterus of a female non-human mammal and generating cells within the uterus of the female non-human mammal, (d) The step of identifying germline transmission in genetically modified humanized non-human mammals of the offspring of the female who became pregnant in step (c).

[0212] In some embodiments, the non-human mammal in the method described above is a mouse (e.g., C57 mouse, BALB / c mouse, or C57BL / 6 mouse).

[0213] In some embodiments, the non-human mammal in step (c) is a female animal in a pseudo-pregnancy (or false pregnancy).

[0214] In some embodiments, the embryo used in the above method is a C57BL / 6 embryo. Other embryos that may be used in the method described herein include, but are not limited to, FVB / N embryos, BALB / c embryos, DBA / 1 embryos, and DBA / 2 embryos.

[0215] The fertilized egg may be from any non-human animal, for example, any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. A gene construct can be introduced into the fertilized egg by microinjection of DNA. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred into a pseudopregnant non-human animal, which then gives birth to a non-human mammal, thereby producing the non-human mammal referred to in the above method.

[0216] This specification further provides cells, tissues and animals (e.g., mice) containing the nucleotide sequences described herein, and cells, tissues and animals (e.g., mice) expressing humanized or chimeric antibodies derived from endogenous non-human loci.

[0217] This disclosure further provides various targeting vectors (e.g., vectors for creating genetically modified animals). In some embodiments, the vector may include: a) a DNA fragment homologous to the 5' end of the region to be altered (5' homologous arm); b) a sequence containing the desired genetic element (e.g., a LoxP recognition site, a drug resistance gene, and / or a reporter gene); and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' homologous arm). This disclosure further relates to cells containing the targeting vectors described herein.

[0218] In some embodiments, the genes in the cells are heterozygous. In some embodiments, the genes in the cells are homozygous.

[0219] In some embodiments, the non-human mammalian cells are mouse cells. In some embodiments, the cells are fertilized egg cells.

[0220] This disclosure further relates to a method for producing genetically modified animal models having two or more human or chimeric genes. The animal may include one or more human or humanized immunoglobulin loci and sequences encoding additional human or chimeric proteins. In some embodiments, the additional human or chimeric proteins may 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-associated (BTLA), programmed cell death ligand 1 (PD-L1), T cell immune receptor having CD27, CD28, CD47, CD137, CD154, Ig and ITIM domains (TIGIT), T cell immunoglobulin and mucin-containing domain 3 (TIM-3), glucocorticoid-inducible TNFR-associated protein (GITR), or TNF receptor superfamily member 4 (TNFRSF4 or OX40).

[0221] A method for producing genetically modified animal models having additional human or chimeric genes (e.g., humanization genes) is: (a) the step of obtaining a genetically modified non-human animal by the method described herein, (b) The step may include crossing the genetically modified non-human animal with another genetically modified non-human animal and screening the offspring to obtain a genetically modified non-human animal having two or more human or chimeric genes.

[0222] In some embodiments, in step (b) of the method, the genetically modified animal may be crossed with a human or a genetically modified non-human animal having chimeric PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40. For example, PCT / CN 2017 / 090320, PCT / CN 2017 / 099577, PCT / CN 2017 / 099575, PCT / CN 2017 / 099576, PCT / CN 2017 / 099574, PCT / CN 2017 / 106024, PCT / CN 2017 / 110494, PCT / CN 2017 / 110435, PCT / CN 2017 / 120388, PCT / CN 2018 / 081628, and PCT / CN 2018 / 081629 describe some of these genetically modified non-human animals, and each of these references is incorporated herein by reference in its entirety.

[0223] This disclosure further relates to methods for generating knockout animals. In some embodiments, if an animal (e.g., a mouse) expresses a protein similar to the antigen of interest, it is difficult to induce an immune response in the animal. This is because, during the developmental process of immune cells, B cells and T cells that recognize MHC molecules that bind to self-derived peptides are removed from the repertoire of immune cells. In these cases, the genetically modified animal may be further modified. The relevant gene in the animal may be knocked out, and the animal may be exposed to the antigen of interest. Because the animal does not perform negative selection for the gene product, the animal can produce antibodies that bind specifically to the target. Therefore, in some embodiments, this disclosure further provides methods for knocking out the gene of interest in a genetically modified animal. In some embodiments, the gene can be knocked out by various gene editing techniques, e.g., the CRISPR-Cas system, TALEN, or ZFN. In some embodiments, the genetically modified animal can be crossed with another animal in which the gene of interest has been knocked out. Once an animal having a knockout phenotype is produced, the animal can be exposed to the antigen of interest to produce antibodies (e.g., antibodies having a common light chain). In some embodiments, the antigen of interest is a human protein. In some embodiments, the antigen of interest is PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40.

[0224] In some embodiments, the genetically modified animal may have the human ADAM6 gene, the endogenous ADAM6 gene, or the modified ADAM6 gene (for example, at its endogenous locus). The ADAM6 protein is a member of the ADAM protein family, where ADAM stands for desintegrin and metalloproteinase. The human ADAM6 gene, usually found between the human IGHV genes IGHV 1-2 and IGHV 6-1, is a pseudogene (Figure 16). Mice have two ADAM6 genes, ADAM6a and ADAM6b. They are located in the intergeneric region between the mouse IGHV and IGHD gene clusters. Mouse ADAM6a is located between mouse IGHV 5-1 and mouse IGHD 5-1. Mouse ADAM6b is located between mouse IGHD 3-1 and mouse IGHD 1-1. Therefore, in some embodiments, the genetically modified animal may have the human ADAM6 gene. In some embodiments, the genetically modified animal does not have the endogenous ADAM6 gene.

[0225] In some embodiments, the genetically modified animal is a mouse. In some embodiments, the mouse is modified to include a nucleotide sequence encoding the ADAM6 protein (e.g., ADAM6a or ADAM6b). In some embodiments, the sequence is placed at any suitable location. It may be placed in an intergene region or at any suitable location in the genome. In some embodiments, the nucleic acid encodes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the mouse ADAM6a gene (e.g., NC_000078.6, 113539230-113547024) or the mouse ADAM6b gene (e.g., NC_000078.6, 113486188-113492125). In some embodiments, the nucleic acid further comprises regulatory elements (e.g., promoters) of the ADAM6a and ADAM6b genes.

[0226] In some embodiments, the functional mouse ADAM6 locus may be located in the center of the human IGHV gene cluster. In some embodiments, the mouse ADAM6 locus is located between two human IGHV genes. In some embodiments, the human ADAM6 pseudogene between human VH1-2 and human VH(II)-1-1 is replaced by the mouse ADAM6 locus. In some embodiments, in the animal genome, the ADAM6a and ADAM6b genes are located between human IGHV1-2 and human VH(II)-1-1. In some embodiments, the location of the mouse ADAM6 sequence in the human gene sequence may be close to the location of the human ADAM6 pseudogene, or close to the location of the mouse ADAM6 sequence (e.g., within the VD intergene region). In some embodiments, the genetically modified mouse has a humanized heavy chain immunoglobulin locus. In some embodiments, mouse ADAM6a and mouse ADAM6b are located between human IGHV1-2 and IGHV6-1 genes. Placing mouse ADAM6a and mouse ADAM6b between the human IGHV1-2 and IGHV6-1 genes may offer several advantages. For example, because these genes substitute for the human ADAM6 gene at the same locus, the impact of the human ADAM6 gene substitution on VDJ recombination is limited, and the mouse ADAM6a and mouse ADAM6b genes can also function normally (as if they were located in a position similar to the endogenous locus).

[0227] Therefore, in one embodiment, the present disclosure provides a genetically modified animal, the genetically modified animal, which includes, at an endogenous heavy chain immunoglobulin locus, a first sequence containing one or more human IGHV genes, a second sequence containing an ADAM6 gene, and a third sequence containing 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.

[0228] In some embodiments, the first sequence includes all human IGHV genes in Table 5, except for IGHV2-10, IGHV3-9, IGHV1-8, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the first sequence includes all human IGHV genes in Table 5, except for IGHV5-10-1 and IGHV3-64D, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the first sequence includes IGHV5-10-1 and IGHV3-64D. In some embodiments, the first sequence is an unmodified sequence derived from the human heavy chain immunoglobulin locus.

[0229] In some embodiments, the second sequence includes one or two of the mouse ADAM6a gene and the mouse ADAM6b gene. In some embodiments, the animal is a fertile male mouse. In some embodiments, the second sequence does not contain the mouse ADAM6a gene or the mouse ADAM6b gene.

[0230] In some embodiments, the third sequence includes all human IGHD genes in Table 6 and all human IGHJ genes in Table 7. In some embodiments, the third sequence includes human IGHV6-1. In some embodiments, the third sequence includes human IGHV(II)-1-1. In some embodiments, the third sequence is an unmodified sequence derived from the human heavy chain immunoglobulin locus.

[0231] In some embodiments, AMAM6a and / or ADAM6b are endogenous sequences. In some embodiments, AMAM6a and / or ADAM6b are not substituted and / or are in their endogenous or native positions. In some embodiments, the mouse IGHV gene preceding mouse IGHV1-2 is replaced by a human IGHV gene at the heavy chain variable region locus. In some embodiments, the mouse IGHV, IGHD, and IGHJ genes following mouse IGHV6-1 are replaced by one or more human IGHV, IGHD, and / or IGHJ genes at the heavy chain variable region locus.

[0232] Therefore, in some embodiments, the mouse IGHV, IGHD, and IGHJ genes can be replaced one or more times with the human IGHV, IGHD, and IGHJ genes. In the first step, a selected number of mouse IGHV genes located on the 5' side of ADAM6a (e.g., all mouse IGHV genes in Table 8) are replaced with the human IGHV gene. In the second step, a selected number of mouse IGHD and IGHJ genes located on the 3' side of ADAM6b (e.g., all mouse IGHD genes in Table 9 except IGHD5-1 and IGHD3-1, and all IGHJ genes in Table 10) are replaced with the human IGHD and human IGHJ genes. The replacement can be carried out by homologous recombination or Cre-mediated recombination.

[0233] In some embodiments, the mouse does not have the mouse ADAM6a or ADAM6b gene. In some embodiments, the mouse has the human ADAM6 gene.

[0234] The reproductive capacity of mice can be increased by various methods. In some embodiments, female mice with superovulation may be used for mating. In some embodiments, in vitro fertilization may be used. Superovulation can be induced by injecting serum gonadotropins and chorionic gonadotropins (e.g., human or mouse CG) into mature female mice. A mature male mouse can be sacrificed, and its epididymal tail can be isolated. The tubules of the epididymal tail are incised, and sperm are released. Next, the fallopian tubes can be isolated by sacrificing a mature female mouse undergoing superovulation. The cumulus-oocyte complex (COC) can be released from the fallopian tube. Next, a sperm suspension can be added to the COC and incubated for fertilization. Pathogenic oocytes containing only one pronucleus can be removed. After incubation, two-cell stage embryos can be transferred into female receptors. Methods for increasing mouse reproductive capacity are known in the art.

[0235] This disclosure 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%, and 99% identical to any nucleotide sequence described herein. In addition, it provides an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to any amino acid sequence described herein.

[0236] In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or to any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is smaller 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 smaller 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.

[0237] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence, or (ii) consists of an amino acid sequence, wherein the amino acid sequence is one of the sequences described herein.

[0238] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence, or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is one of the sequences described herein.

[0239] To determine the degree of identity between two amino acid sequences or two nucleic acid sequences, pairing is performed on the sequences for optimal comparison (for example, gaps may be introduced between the first and second amino acids or between the first and second nucleic acid sequences, or both, for optimal alignment, and non-homologous sequences may be ignored for comparison). Then, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are the same at that position. Considering the number of gaps and the length of each gap, the degree of identity between the two sequences is a function of the number of identical positions shared by the sequences, and the introduction of gaps is necessary to achieve optimal alignment of the two sequences. For illustrative purposes, the comparison of two sequences and the determination of the degree of identity between them can be achieved, for example, by a Blosum 62 scoring matrix with a gap penalty 12, a gap extension penalty 4, and a frameshift gap penalty 5.

[0240] The proportion of conserved residues with similar physicochemical properties (homology ratio), such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties are known in this art. These families include, for example, amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged 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). In many cases, the homology ratio is higher than the identity ratio. Therefore, this disclosure further provides amino acid sequences having a homology percentage of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with any amino acid sequence described herein, or nucleic acids encoding such amino acid sequences.

[0241] Methods for using genetically modified animals The genetically modified animals can be used to produce humanized antibodies or chimeric antibodies that can specifically bind to a target. In some embodiments, using standard techniques for polyclonal and monoclonal antibody preparations, antibodies can be produced in these animals using a target (e.g., a protein or protein fragment) as an immunogen. In some embodiments, the genetically modified animals are exposed to the antigen for a certain period of time under conditions that allow the animals to produce antibodies specific to the selected antigen.

[0242] Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of an antigen peptide or protein. In some embodiments, the antigen peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigen peptide or protein may be conjugated with an immunogenic agent in the species to be immunized. The antigen peptide or protein may be injected into the animal more than once (e.g., two, three, or four times).

[0243] A full-length polypeptide or protein may be used, or alternatively, a fragment of the antigen peptide may be used as an immunogen. The antigen peptide of the protein contains at least eight amino acid residues (e.g., at least 10, 15, 20, or 30) of the amino acid sequence and covers an epitope of the protein such that an antibody produced against the peptide forms a specific immune complex with the protein.

[0244] Immunogens are typically used to produce antibodies by immunizing a suitable target (e.g., genetically modified animals as described herein). A suitable immunogenic preparation may include, for example, recombinant expression polypeptides or chemically synthesized polypeptides (e.g., protein fragments). The preparation may further include, for example, an adjuvant such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0245] As described above, polyclonal antibodies can be produced by immunizing a suitable target with a polypeptide or its antigenic peptide (e.g., a part of a protein) as an immunogen. The change in antibody titer over time of the immunized target can be monitored by standard techniques (e.g., enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptides or peptides). If necessary, the antibody molecule can be isolated from a mammal (e.g., blood) and further purified by well-known techniques (e.g., protein A or protein G chromatography) to obtain the IgG portion. At an appropriate point after immunization, for example, when the titer of the specific antibody is highest, antibody-producing cells can be obtained from the target and used to produce monoclonal antibodies using standard techniques such as the hybridoma technique first described by Kohler et al. (Nature 256:495-497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells that produce monoclonal antibodies can be detected, for example, by screening the hybridoma culture supernatant for antibodies that bind to the target polypeptide or epitope using a standard ELISA assay.

[0246] In one embodiment, the disclosure provides a mouse comprising a modification of the endogenous immunoglobulin heavy chain locus, wherein the mouse produces B cells comprising a reconstituted immunoglobulin sequence operably ligated to a heavy chain constant region gene sequence. In some embodiments, the reconstituted immunoglobulin sequence operably ligated 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.

[0247] In one embodiment, the disclosure provides a mouse comprising a modification of an endogenous immunoglobulin light chain (e.g., κ or λ) locus, wherein the mouse produces B cells comprising a reconstituted immunoglobulin sequence operably ligated to a light chain constant region gene sequence. In some embodiments, the reconstituted immunoglobulin sequence operably ligated 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.

[0248] The mouse B cells or spleen cells may include, for example, a reconstituted 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 cells of interest. In some embodiments, screening is performed using a single B cell. This allows screening of the native antibody repertoire without requiring hybridoma fusion and combinatorial display. For example, B cells may be mixed with a set of antigens to which DNA barcodes have been added, so that one or more antigen barcodes and B cell receptor (BCR) sequences of a single B cell can be recovered by a single-cell sequencing protocol.

[0249] Humanized antibodies or human antibodies can be obtained by further modifying the antibody, for example, by operably linking a sequence encoding the human heavy chain variable region to a sequence encoding the human heavy chain constant region, and / or by operably linking a sequence encoding the human light chain variable region to a sequence encoding the human light chain constant region.

[0250] This disclosure further provides methods for producing antibodies, nucleic acids, cells, and tissues (e.g., spleen tissue). In some embodiments, the methods include exposing animals described herein to an antigen. Antibodies (e.g., hybrid antibodies), nucleic acids encoding antibodies, cells, and / or tissues (e.g., spleen tissue) can be obtained from animals. In some embodiments, nucleic acids encoding the variable regions of human heavy and light immunoglobulins can be determined, for example, by sequencing. In some embodiments, nucleic acids encoding the variable regions of human heavy immunoglobulins can be operably linked to nucleic acids encoding the constant region of human heavy immunoglobulins. In some embodiments, nucleic acids encoding the variable regions of human light immunoglobulins can be operably linked to nucleic acids encoding the constant region of human light immunoglobulins. In some embodiments, cells containing the nucleic acids described herein are cultured to recover antibodies.

[0251] In some embodiments, the mouse immunoglobulin V, D, and J genes (e.g., mouse IGHV, IGHD, IGHJ, IGKV, or IGKJ genes) do not contribute to the heavy chain and / or light chain variable region sequences. In some embodiments, the heavy chain and / or light chain variable region sequences produced by the animal are entirely human and entirely contributed to by the human immunoglobulin V, D, and J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, and IGKJ genes).

[0252] Mutants of the antibodies or antigen-binding fragments described herein can be produced by introducing appropriate nucleotide changes into the DNA encoding the human antibodies, humanized antibodies, or chimeric antibodies, or their antigen-binding fragments, as described herein, or by peptide synthesis. Such mutants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence constituting the antigen-binding site or antigen-binding domain of the antibody. In a population of such mutants, some antibodies or antigen-binding fragments will exhibit increased affinity for the target protein. Deletions, insertions, and / or combinations can be arbitrarily combined to obtain antibodies or their antigen-binding fragments with increased binding affinity to the target. The amino acid changes introduced into the antibody or antigen-binding fragment can further alter or introduce novel post-translational modifications into the antibody or antigen-binding fragment, for example, by changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation site (e.g., altering the amino acid sequence so that enzymes present in cells are linked to different sugars), or introducing novel glycosylation sites.

[0253] The antibodies disclosed herein may be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0254] Human and humanized antibodies include antibodies having a variable region and a constant region derived from a human germline immunoglobulin sequence (or having the same amino acid sequence as the amino acid sequence derived from a human germline immunoglobulin sequence). Human antibodies may, for example, include amino acid residues in the CDR that are not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by in vitro random induction or site-directed induction or in vivo somatic mutation).

[0255] Further modifications may be made to the antibody or antigen-binding fragment. For example, by introducing one or more cysteine ​​residues into the Fc region, the formation of interchain disulfide bonds in this region can be enabled. The homodimer antibody produced thereby may have any increased in vitro and / or in vivo half-life. Furthermore, homodimer antibodies with increased in vitro and / or in vivo half-life can be produced using heterobifunctional crosslinking agents, for example, as described by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies having two Fc regions may be engineered (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0256] In some embodiments, covalent modifications may be made to the antibody or its antigen-binding fragment. These covalent modifications can be carried out 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 the targeted amino acid residue of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues.

[0257] Method for producing bispecific antibodies using common light chains This disclosure provides genetically engineered animals expressing a finite repertoire of light chains that may be associated with various heavy chains. In various embodiments, an endogenous κ light chain variable region gene is deleted and replaced with one, two, three, four, or five human light chain variable region genes, which are operably linked to an endogenous κ constant region gene. In various embodiments, the animals further include a non-functional λ light chain locus, or a deletion thereof, or a deletion that prevents the production of a λ light chain by said locus.

[0258] In some embodiments, the animal lacks an endogenous light chain variable gene and includes a light chain variable region locus containing a reconfigured human V / J sequence operably ligated to an endogenous constant region, wherein the locus expresses a light chain containing the human V / J sequence ligated to an endogenous constant region.

[0259] In various embodiments, when immunized with the antigen of interest, the genetically engineered animal produces B cells exhibiting various rearrangements of the human immunoglobulin heavy chain variable region, the various rearrangements being expressed and functional by a finite number (e.g., 1, 2, 3, 4, 5) of rearranged light chains, the embodiments of which include one or two light chains containing the human light chain variable region, the human light chain variable region containing, for example, 1 to 5 somatic mutations. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the human light chain variable region has at least one somatic mutation. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the human light chain variable region has at least two somatic mutations. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the human light chain variable region has at least three somatic mutations.

[0260] In some embodiments, the somatic hypermutability is relatively low. In some embodiments, 30% or less of the human light chain variable region has one or more somatic mutations. In some embodiments, 20% or less of the human light chain variable region has two or more somatic mutations.

[0261] In some embodiments, the animals described herein can be immunized with a first immunogen to produce B cells expressing antibodies that specifically bind to a first epitope. The animals can be immunized with a second immunogen to produce B cells expressing antibodies that specifically bind to a second epitope. The heavy chain variable region can be cloned from B cells onto a vector for transfecting cells to express a reconstituted human heavy chain variable region fused to a human heavy chain constant region and a common light chain variable region fused to a human light chain constant region.

[0262] In some embodiments, the methods described herein are designed to produce bispecific antibodies. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell cultures. For example, the interface may include at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" equal to or similar in size to one or more of the larger side chains is produced on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in whole by reference.

[0263] In some embodiments, a knob-into-hole (KIH) technique may be used to promote heterodimerization by engineering the CH3 domain to form one "knob" or one "hole" on each heavy chain. The KIH technique is described, for example, in "Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system" by Xu, Yiren et al., MAbs. Vol. 7, No. 1, Taylor and Francis, 2015, which is incorporated herein by reference in its entirety. In some embodiments, one heavy chain has the T366W and / or S354C (knob) substitution (EU number), and another heavy chain has the Y349C, T366S, L368A, and / or Y407V (hole) substitution (EU number). In some embodiments, one heavy chain has one or more of the Y349C and T366W (EU number) substitutions. Another heavy chain may have one or more substitutions of E356C, T366S, L368A, and Y407V (EU number). In some embodiments, one heavy chain has a T366Y (knob) substitution, and another heavy chain has one, two, or three substitutions of T366S, L368A, and Y407V (hole).

[0264] Incidentally, anion exchange chromatography can be used for the purification of bispecific antibodies. Anion exchange chromatography is a method for isolating substances based on the use of an ion exchange resin containing a positively charged group such as a diethyl-aminoethyl group (DEAE) as a charge. In a solution, a positively charged counterion (cation) is coated on the resin. The anion exchange resin binds to negatively charged molecules and replaces the counterion. Anion exchange chromatography can be used for the purification of proteins based on their isoelectric points (pI). The isoelectric point is defined as the pH at which a protein has no net charge. When pH > pI, the protein has a net negative charge, and when pH < pI, the protein has a net positive charge. Therefore, in some embodiments, different amino acid substitutions may be introduced into the two heavy chains, so that the pI of the homodimer containing two Arm A's is different from the pI of the homodimer containing two Arm B's. As used herein, the term "arm" in a bispecific antibody refers to an antigen-binding site that specifically binds to a specific antigen (or epitope) in the bispecific antibody. The pI of a bispecific antibody having Arm A and Arm B is between the two pI's of the homodimer. Therefore, the two homodimers and the bispecific antibody can be released under different pH conditions. The present disclosure shows that several amino acid residue substitutions can be introduced on the heavy chain to adjust the pI. Therefore, in some embodiments, the amino acid residue at Kabat number position 83 in VH is lysine, arginine, or histidine. In some embodiments, the amino acid residue at one or more positions of 1, 6, 43, 81, and 105 (Kabat number) in VH is aspartic acid or glutamic acid. In some embodiments, the amino acid residue at one or more positions of 13 and 105 (Kabat number) is aspartic acid or glutamic acid. In some embodiments, the amino acid residue at one or more positions of 13 and 42 (Kabat number) is lysine, arginine, histidine, or glycine.

Example

[0265] Example 1: Overview We conducted experiments to produce mice that express humanized antibodies by introducing human immunoglobulin genes into the mouse genome. Figure 1A shows the method for creating humanized mice. The method first involves modifying the human immunoglobulin region on the human chromosome. Then, the modified human chromosome is introduced into mouse receptor cells.

[0266] Mouse immunoglobulin variable regions were replaced with human immunoglobulin variable regions by direct substitution (e.g., homologous recombination or Cre-mediated recombination). In some cases, human immunoglobulin variable regions could be introduced into the mouse genome by a stepwise method. Then, receptor cells that were more precisely replaced were screened. These cells were then injected into blastulas to create chimeric mice. Subsequently, they were bred to obtain mice containing human or humanized immunoglobulin variable regions.

[0267] Since the mouse heavy chain gene and two light chain genes are located on chromosomes 12, 6, and 16, respectively, mice containing either the human heavy chain variable region or the human light chain variable region can be generated. These mice can then be crossbred to obtain mice that can express both the human heavy chain variable domain and the human common light chain variable domain.

[0268] Example 2: Modification of the mouse heavy chain immunoglobulin gene locus The heavy chain immunoglobulin locus is located on mouse chromosome 12. Two recombination sites were introduced on either side of the variable region of the heavy chain immunoglobulin locus.

[0269] Furthermore, experiments were conducted to produce modified human chromosomes. Two recombination sites were introduced on both sides of the variable region of the heavy chain immunoglobulin locus. These modified human chromosomes were then introduced into mouse cells. The cells were then screened, and only cells containing a single human chromosome were selected. Cre recombinase then mediated the substitution of the V, D, and J regions on the mouse chromosome with the V, D, and J regions on the human chromosome (Figure 1B).

[0270] Positive cloned cells were injected into BALB / c mouse blastulas by microinjection. For example, embryonic microinjection was performed based on the method described in A. Nagy et al., “Manipulating the Mouse Embryo: A Laboratory Manual (3rd Edition),” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to culture medium and cultured for a short period, and then transplanted into the oviducts of receptor mice to produce genetically modified humanized mice (F0 generation). These mice were then crossed with mice with a C57BL / 6 background. PCR analysis was performed on DNA obtained from the tails of the mice. The mice were crossed with mice with a BALB / c background multiple times (e.g., at least 5 times) to obtain BALB / c background humanized heavy chain immunoglobulin locus heterozygous mice.

[0271] Then, heterozygous mice were crossed with each other to obtain homozygous mice. How homozygous mice for the humanized heavy chain immunoglobulin locus were generated is described in detail in PCT / CN 2020 / 075698, submitted on 18 February 2020, which is incorporated herein by reference in its entirety.

[0272] Example 3: Arrangement design of the light chain VJ region We analyzed the applications of human light chain variable region sequences in various antibodies.

[0273] (1) Data was collected from the IMGT / GeneFrequency database. Statistical analysis of the data revealed that the five most utilized κ light chain V region genes were IGKV3-20, IGKV1-39 / IGKV1D-39, IGKV3-11, IGKV3-15, and IGKV4-1. The three most utilized κ light chain J region genes were IGKJ1, IGKJ2, and IGKJ4.

[0274] [Table 12-1] [Table 12-2]

[0275] 2) The light chain genetic information of 110 antibody molecules was analyzed. Sequence information was collected from the IMGT / mAb-DB database. Statistical analysis revealed that the five most frequently used κ light chain V region genes were IGKV3-20, IGKV3-11, IGKV3-15, IGKV1-39 / IGKV1D-39, and IGKV1-12 / IGKV1D-12, while the three most frequently used κ light chain J region genes were IGKJ1, IGKJ4, and IGKJ2 (Figure 2A-2B).

[0276] After analysis, three human light chain V regions (IGKV3-20, IGKV3-11, and IGKV1-39) and two human light chain J regions (IGKJ1 and IGKJ4) were selected as humanized light chain immunoglobulin loci.

[0277] Example 4: Preparation of reconstituted light chain mice The mouse light chain immunoglobulin locus is located on mouse chromosome 6. Figure 3A is a schematic diagram showing the mouse light chain immunoglobulin locus. The entire mouse VJ region in wild-type mice can be replaced with a reconstructed human light chain VJ region sequence (Figure 3B). Alternatively, this process can be carried out stepwise, i.e., by first knocking out the mouse VJ region (for example, by replacing the VJ region with a sequence containing Neo and DTR), and then inserting a sequence containing the reconstructed human light chain VJ region.

[0278] For example, mouse chromosome 6 was modified by knocking out the entire sequence of the variable region of the κ light chain immunoglobulin locus. Three different reconstructed human light chain VJ sequences were constructed and inserted into the κ chain VJ region of mouse chromosome 6.

[0279] A schematic diagram of the humanization strategy is shown in Figure 4. Three homologous recombinant targeting vectors sharing common sequence features were constructed; that is, each homologous recombinant targeting vector has upstream and downstream homologous arm sequences and a reconstructed human light chain VJ sequence. The reconstructed human VJ sequences were selected distinctly for each vector. The three vectors each contain the reconstituted human IGKV1-39 / J4, IGKV3-11 / J1, and IGKV3-20 / J1 sequences, where IGKV1-39 (SEQ ID NO:1) is the same as the nucleotide sequence 89319625-89320099 of NCBI registry number NC_000002.12, IGKV3-11 (SEQ ID NO:2) is the same as the nucleotide sequence 89027171-89027684 of NCBI registry number NC_000002.12, and IGKV3-20 (SEQ ID NO:3) is the same as the nucleotide sequence 89142574-89143108 of NCBI registry number NC_000002.12. NO:4) is the same as the nucleotide sequence 88861886-88861923 of NCBI registration number NC_000002.12, and IgKJ4 (SEQ ID NO:5) is the same as the nucleotide sequence 88860886-88860923 of NCBI registration number NC_000002.12.

[0280] Each reconstructed human light chain VJ sequence further contains the human V promoter sequence of the light chain V region gene. The promoter sequence is located approximately 2000 bp before the V region gene.

[0281] Furthermore, each reconstructed human VJ sequence may be followed by a single auxiliary sequence, for example, a mouse 3'UTR sequence (SEQ ID NO: 6) or a human 3'UTR sequence (SEQ ID NO: 7) following the human light chain VJ region.

[0282] Three vectors were introduced into cells using gene editing, and cells were selected by DT screening. Positive clones were identified by PCR and Southern blotting. PCR assays were performed using the following primers. [Table 13-1] [Table 13-2] In Table 13, L-GT-F is located on the 5' homologous arm, R-GT-R is located on the 3' homologous arm, L-GT-R and R-GT-F are located on the reconstituted human light chain VJ sequence, and the m-5'loxp-L-GT-F, m-5'loxp-R-GT-R, m-3'lox-L-GT-F, and m-3'lox-R-GT-R primers are located on the humanized heavy chain chromosome.

[0283] The following probes were used to perform the Southern blot assay: [Table 14] In Table 14, the 5' probe is located outside the 5' homologous arm, the 3' probe is located outside the 3' homologous arm, and the IGKV1-39, IGKV3-11, and IGKV3-20 probes are located on the humanized fragment.

[0284] Positive clonal cells were injected into BALB / c mouse blastulas by microinjection. For example, embryo microinjection was performed based on the method described in A. Nagy et al., “Manipulating the Mouse Embryo: A Laboratory Manual (3rd Edition),” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to culture medium and cultured for a short period, and then transplanted into the oviducts of receptor mice to produce genetically modified humanized mice (F0 generation). Chimeric mice were selected and mated with mice homozygous for heavy chain gene humanization to produce F1 generation mice. PCR and southern analysis were performed on DNA obtained from the tails of the F1 generation mice to determine whether or not positive heterozygous mice were obtained.

[0285] Example 5: Common light chain expression The mRNA sequences of the light chain variable region in mice were analyzed by sequencing. Humanized mice (heterozygous humanized heavy chain and heterozygous reconstituted light chain) that were not immunized (not exposed to specific antigens) were selected, and RNA was extracted from orbital blood samples. cDNA was obtained by reverse transcription using a 5'RACE kit (SMARTer RACE 5' / 3' kit, Takara Bio USA, Inc., catalog number 634858). The obtained cDNA was amplified using the IGKC-R primer and UPM primer of the 5'RACE kit. Then, heavy chain variable region sequence fragments were sequenced. The IGKC-R primer sequence is 5'-CTAACACTCATTCCTGTTGAAGCTCTTGAC-3' (SEQ ID NO: 34). The sequencing results were compared with the NCBI Ig Blast tool to identify human immunoglobulin sequences, thereby identifying the usage of human Vκ and Jκ region genes. The results showed that in 577 clones from three randomly selected mice, 89% expressed human Vκ and Jκ, and the ratio of human Vκ to Jκ in each mouse exceeded 60%. This indicates that after the endogenous K chain variable region locus is completely replaced by a rearranged human immunoglobulin light chain sequence, the human Vκ and Jκ genes are expressed with high efficiency and dominance in humanized mice. Conversely, in heterozygous mice, the wild-type κ chain was either not expressed or expressed at low levels.

[0286] [Table 15]

[0287] Example 6: B cell development in hVH / hcVL mice F1 generation mice were crossed with each other. Homozygous humanized heavy chain immunoglobulin locus and heterozygous humanized common light chain immunoglobulin locus (humanized VH H / H / cVL K / + Mouse, abbreviated as hVH H / H / hcVL K / +B cell development was detected using mice. Mice possessing both a humanized VH locus (heterozygous or homozygous) and a common light chain immunoglobulin locus (heterozygous or homozygous) are also called hVH / hcVL mice. Experiments were conducted to detect pre-immunization hVH H / H / hcVL K / + The immune systems of mice and wild-type mice were compared. Body weight and the weight of several organs, including the spleen, thymus, liver, heart, lungs, and kidneys (Figure 5A-5G), were measured for wild-type and hVH / hcVL heterozygous mice. There were no significant differences in mean body weight or organ (spleen, thymus, liver, heart, lungs, and kidneys) weight between wild-type and hVH / hcVL heterozygous mice.

[0288] Flow cytometry was performed to analyze the number and distribution of lymphocytes in the blood (Figure 6A), spleen (Figure 6B), and lymph nodes (Figure 7) of wild-type and hVH / hcVL heterozygous mice. The results showed that the proportions of B cells, T cells, NK cells, CD4+ T cells, and CD8+ T cells in the blood, spleen, and lymph nodes of hVH / hcVL heterozygous mice were almost identical to those of wild-type mice. The results showed that leukocytes included B cells (e.g., characterized by CD45+, CD19+, TCR-), T cells, and natural killer (NK) cells (e.g., characterized by CD45+, TCR-, and NK1.1+). Further characteristics of T cells were CD45+, CD19-, and TCR+. Characteristics of CD4+ T cells (CD4) were CD45+, CD19-, TCR+, CD4+, and CD8-. The characteristics of CD8+ T cells (CD8) are CD45+, CD19-, TCR+, CD4-, and CD8+. Flow cytometry analysis includes only complete, single, viable leukocytes.

[0289] Figure 8A shows the proportion of B cells in the bone marrow at different developmental stages. B cell progenitor cells in the bone marrow were analyzed by flow cytometry. Based on the expression levels of B220 and CD43, B cell progenitor cells in the bone marrow were classified as pro-B cells (B220). 低 CD43 高 IgM 低 Characterized by), pre-B cells (B220)低 CD43 中 IgM 低 Characterized by) and immature B cells (B220) 高 CD43 低 IgM 高 It can be divided into three cell populations (characterized by...). No significant difference was observed between wild-type mice and hVH / hcVL mice. Note that plasma cells (B220 低 IgM - IgD - CD138 + ) and memory B cells (B220 + IgM + IgD - CD38 + B cell development in the bone marrow was evaluated by flow cytometry, selectively staining (Figure 8B). No significant differences were observed between wild-type mice and hVH / hcVL mice.

[0290] Figure 9 shows the proportion of B cells at different developmental stages. The developmental stages of B cells in the spleen are T1 (transitional B cells, B220) + IgM + IgD - Characterized by), T2 (transitional B cell, B220 + IgM + IgD + Characterized by) and mature B cells (B220) + IgM 低 IgD + They can be divided into two categories (characterized by...). No significant differences were observed between wild-type mice and hVH / hcVL mice.

[0291] Plasma cells (B220) 低 IgM - IgD - CD138 + ) and memory B cells (B220 + IgM + IgD - CD38 +Flow cytometry was used to evaluate B cell development in the spleen such that [[ID=]] was selectively stained (Figure 10A-10B). No significant difference was observed between wild-type mice and hVH / hcVL mice. Also, B cell development was evaluated in the splenic marginal zone (characterized by marginal zone B cells, MZ-B, B220 + CD21 + CD23 - and the follicular zone (characterized by follicular B cells, FO-B, B220 + CD21 低 CD23 + Figure 10C shows the percentages of splenic B cells in the splenic marginal zone (MZ-B) and the follicular zone (FO-B). No significant difference was observed between wild-type mice and hVH / hcVL mice.

[0292] Flow cytometry was used to evaluate the light chain utilization rate of B cells in the spleen. First, B cells were selectively labeled with mB220 (Pacific Blue TM Anti-Mouse / Human CD45R / B220 Antibody, BioLegend, Cat#103227), and then B cells were selectively labeled with mIgG κ-FITC (FITC Anti-Mouse Ig Light Chain κ Antibody, BioLegend, Cat#409509) and mIgG lambda-PE (PE Anti-Mouse Ig Light Chain λ Antibody, BioLegend, Cat#407307). No significant difference was observed between wild-type mice and hVH / hcVL mice. From the results, it was found that humanization did not affect the expression levels of κ and λ chains in the spleen of hVH / hcVL heterozygous mice (Figure 11A-11I).

[0293] Figure 12A shows the percentages of B cells at different developmental stages. The developmental stages of B cells in the lymph nodes are T1 (type 1 transitional B cells, B220 + IgM + IgD - characterized by), T2 (type 2 transitional B cells, B220 + IgM + IgD + characterized by), and mature B cells (B220+ IgM 低 IgD + is characterized by) and divided. No significant difference was observed between wild-type mice and hVH / hcVL mice. Furthermore, plasma cells (B220 低 IgM - IgD - CD138 + ) and memory B cells (B220 + IgM + IgD - CD38 + ) were selectively stained (Figure 12B), and the development of B cells in lymph nodes was evaluated by flow cytometry. No significant difference was observed between wild-type mice and hVH / hcVL mice.

[0294] The light chain utilization rate of lymph node B cells was evaluated using flow cytometry. B cells were first selectively labeled with mB220 and then labeled with mIgG κ-FITC and mIgG λ-PE. No significant difference was observed between wild-type mice and hVH / hcVL mice. From the results, it was found that humanization did not affect the expression levels of κ and λ chains in the spleen of hVH / hcVL heterozygous mice (Figures 13A-13I).

[0295] Experiments were conducted to evaluate the B cell development of hVH H / H / hcVL K / K mice. There was no significant difference between wild-type and hVH H / H / hcVL K / K mice in terms of B cell development and average immune organ (e.g., spleen) weight. As shown in Figure 13J, before antigen immunization, different immunoglobulin (Ig) subtypes in the sera of hVH H / H / hcVL K / K and wild-type mice were quantitatively measured by ELISA. No significant difference was observed.

[0296] In another similar experiment, the Ig subtypes before and after immunization of hVH H / H / hcVL K / K mice and wild-type mice were analyzed. No significant difference was observed (Figure 13K).

[0297] Example 7: Antibody production in hVH / hcVL mice We conducted experiments to evaluate whether hVH / hcVL mouse B cells developed normally. We measured antigen-specific antibody titers in the serum of immunized hVH / hcVL mice and wild-type mice.

[0298] hVH H / H / hcVL K / + Mice and wild-type (C57BL / 6) mice were immunized with two different antigens, for a total of three immunization cycles. In the first immunization cycle, Freund's complete adjuvant (CFA) and 20 ug of antigen were injected. Two weeks later, Freund's incomplete adjuvant (IFA) and 20 ug of antigen were injected. Two weeks later, the mice were injected with 20 ug of antigen and IFA. Approximately one week after the third immunization cycle, blood was collected, and antigen-specific antibody titers in wild-type mice and hVH / hcVL mice were analyzed using ELISA. H / H / hcVL K / + The mice primarily expressed antibodies containing the humanized common light chain variable region.

[0299] ELISA was performed as follows: The his-labeled antigen was diluted to 0.5 μg / ml in 1×PBS, added to a 96-well plate at a rate of 0.1 ml / well, and incubated at 37°C for 2 hours. After incubation, each well was washed three times with 300 μl 1×PBST, then closed with 250 μl 1×PBS and 5% skimmed milk, and incubated at 37°C for 1 hour. Each well was then washed twice with 300 μl 1×PBST. Serum samples from hVH / hcVL mice or wild-type mice were first diluted in 1×PBS at a ratio of 1:500, 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, or 1:2048000, and then added to the 96-well plate. Serum samples from unimmunized mice were also added to the plate as a blank control after being diluted 1:500. Diluted serum samples (120 μl / well) were incubated in a 96-well plate at 37°C for 1 hour. After incubation, the plate was washed five times with 300 μl / well of 1×PBST. Then, the plate was incubated with 0.1 ml / well of 1:20000 diluted goat anti-mouse IgG Fc(HRP) at 37°C for 1 hour. After incubation, the plate was washed five times with 300 μl / well of 1×PBST. Next, 0.1 ml of TMB developer was added to each well, and the plate was stored in the dark at room temperature for 10 minutes, and then 0.1 ml of stop solution was added to each well. OD450 and OD570 were measured with a plate reader, and the standard OD values ​​in Figures 24-25 were calculated as standard OD value = OD450 - OD570.

[0300] The results of antigen-specific antibody titers after the third immunization are shown in Figures 24 and 25, respectively. The results indicate that hVH / hcVL mice can produce antibodies that specifically bind to the antigen, and that the immune response between wild-type mice and hVH / hcVL mice is similar. In particular, serum antibody titers are similar to those of wild-type mice.

[0301] Example 8: Antibody production in hVH / hcVL mice hVH H / H / hcVL K / KMice and wild-type mice (3 mice per group) were immunized with TFR1 and Ova, respectively. Plasma cells capable of producing antigen-specific monoclonal antibodies were then isolated using the Beacon™ microfluidic optics system. The number of positive cells is as follows:

[0302] [Table 16]

[0303] Antigen A is a member of the tumor necrosis factor receptor superfamily. Antigen A is hVH H / H / hcVL K / K This study was conducted to immunize mice. Forty-two human common light chain antibodies (IGKV3-11 / J1) were obtained. The VH and VL of these antibodies were added to the constant region of human IgG1 to obtain human antibodies. The affinity of the 42 human common light chain antibodies (IGKV3-11 / J1) produced against antigen A was then determined using Biacore.

[0304] The antibody against purified antigen A was diluted to 1 μg / ml and injected into a Biacore 8K biosensor at 10 μl / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 50 response units (RU)). Then, His-labeled antigen A at a concentration of 200 nM was injected at a rate of 30 μl / min for 120 seconds. Dissociation was monitored for 600 seconds. The tip was regenerated after the last injection of each glycine titration (pH 2.0, 30 μl / min, lasting 30 seconds).

[0305] By fitting the data to a 1:1 Langmuir coupled model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore 8K evaluation software 3.0, the association velocity (kon) and dissociation velocity (koff) were obtained simultaneously. The affinity (KD = koff / kon) was derived from the quotient of the velocity constants.

[0306] As will be understood by those skilled in the art, the same method was performed for each antibody tested, with parameters (e.g., antibody concentration) appropriately adjusted. From the results, it was found that, with the exception of A-1C5-IgG1, A-1D10-IgG1, and A-1E1-IgG1, the binding affinity of these antibodies was 10. -8 It was found that the level reached M, and even exceeded it, suggesting that the method can successfully produce antibodies with high binding affinity to the antigen.

[0307] [Table 17]

[0308] Epitope association analysis The relative positions of epitopes were analyzed by surface plasmon resonance (SPR) competition experiments. The mutual binding inhibition (blocking) effects of A-1B10-IgG1, A-1D7-IgG1, A-1C8-IgG1, A-1F2-IgG1, A-H6L6-IgG1, A-1F9-IgG1, A-1B1-IgG1, A-1D12-IgG1, A-1E7-IgG1, A-1A7-IgG1, A-1B8-IgG1, A-H7L7-IgG1, A-1E12-IgG1, A-H3L3-IgG1, and A-H8L8-IgG1 were studied using a total of 15 monoclonal antibodies. Another antibody against antigen A was also used as a positive control (PC).

[0309] HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) was diluted with HBS-EP+ buffer (10×) and used as a running buffer throughout the experimental process. Anti-His antibodies were immobilized on the surface of the S-series sensor chip CM5 by amino coupling, thereby producing an anti-His chip (i.e., CM5-Anti-His-Channel 1,8-Chip). Then, 1 M ethanolamine (pH 8.5) was injected to block the active carboxyl groups remaining on the chip surface, and the chip was then equilibrated with HBS-EP+ buffer for 2 hours. Recombinant antigen A protein with a His tag (1 μg / ml) was injected into the Biacore 8K biosensor at 10 μl / min for 50 seconds and captured on the anti-His chip to achieve the desired protein density (i.e., 200 RU). A pair of antibodies (200 nM each) were injected into the tip sequentially at 30 μl / min. The binding time for the first injected antibody (analyte 1) was 250 seconds, and the binding time for the second antibody (analyte 2) was also 250 seconds. After injecting antibodies in each analysis cycle, the tip was refilled twice with glycine buffer (pH 1.7, 30 μl / min, lasting 30 seconds). When various monoclonal antibodies paired with other antibodies, the same experimental steps were performed for each pair of monoclonal antibodies to obtain binding inhibition data.

[0310] The binding values ​​of various antibodies were obtained using Biacore Insight evaluation software. To quantify the interference between one antibody binding to another, the binding rate was calculated and each pair of antibodies was compared. The binding rate is defined as dividing the binding value of the second antibody (analyte 2) by the binding value of the first antibody (analyte 1). Statistical software was also used for cluster analysis. Epitope associations were analyzed, and 15 types of human anti-antigen A antibodies were divided into four epitope clusters (Figure 31). As described above, A-1B10-IgG1, A-1D7-IgG1, A-1C8-IgG1, and A-1F2-IgG1 have the same or overlapping epitopes, A-1B1-IgG1 and A-1D12-IgG1 have the same or overlapping epitopes, A-1B8-IgG1, A-H7L7-IgG1, and A-1E12-IgG1 have the same or overlapping epitopes, and A-H3L3-IgG1 and A-H8L8-IgG1 have the same or overlapping epitopes. From the results, hVH H / H / hcVL K / K It was found that mice can produce antibodies that target various epitopes.

[0311] Thermal stability measurement QuantStudio TM 5. Using a real-time PCR system, Protein Thermal Shift TM The thermal stability of 17 types of human anti-antigen A antibodies was measured using a staining kit.

[0312] The experiment was conducted according to the manufacturer's protocol. The reaction was carried out sequentially in two steps. Specifically, the first step was carried out at 25°C at a rate of 1.6°C per second for 2 minutes, and the second step was carried out at 99°C at a rate of 0.05°C per second for 2 minutes. The melting temperature (Tm) of each antibody against antigen A was determined and is shown in the table below.

[0313] [Table 18]

[0314] The results showed that the Tm values ​​of human anti-antigen A antibodies were 70°C or higher, or approximately 70°C, and that these antibodies were thermally stable.

[0315] Example 9: Antibody production in hVH / hcVL mice Antigen B is a type II transmembrane serine protease. Antigen B is hVH H / H / hcVL K / K This study involved immunizing mice (IGKV3-11 / J1). Using Biacore, we detected the affinity of eight human common light chain antibodies (IGKV3-11 / J1) produced against antigen B.

[0316] [Table 19]

[0317] Example 10. Blocking the binding of antigen C ligand to antigen C. Antigen C is an inhibitory receptor on antigen-activated T cells and plays a crucial role in inducing and maintaining immune tolerance to itself. A blocking assay was performed to determine whether 129 antibodies against human antigen C could block binding between human antigen C and its ligand. These antibodies were found to be hVH H / H / hcVL K / K It was produced by mice.

[0318] Antibodies against antigen C were collected from the CHO-S supernatant. 30 μl of CHO cells (approximately 1 × 10⁶) were rapidly transfected with human antigen C ligand. 5 The antibody was added to each well in the plate. The antibody (approximately 20-200 ug / ml) in the supernatant was collected and diluted 10-fold and 100-fold. At 4°C, 30 μl of titration antibody (30 μl per well) and 30 μl of bitoin-labeled human antigen C (30 μl per well, with a final concentration of 1 μg / ml in each well) were added to each well. Cells containing bitoin-labeled human antigen C and antibody were incubated at 4°C for 30 minutes.

[0319] After washing twice with phosphate-buffered saline (PBS), 50 μl of Alexa Fluor® 647-labeled streptavidin (AF647 streptavidin, Jackson Immuno Research, Cat# 016-600-084) was added to each well, diluted 1:1000, incubated at 4°C for 15 minutes, and then washed with PBS. The AF647 signal was confirmed by flow cytometry (Thermo Attune NX). The results of the percentage of test cells containing the streptavidin signal from the antibody in the flow cytometry analysis are summarized in the table below. The blocking rate decreased with decreasing antibody concentration, suggesting that these antibodies can block the binding between antigen C and its ligand.

[0320] [Table 20-1] [Table 20-2] [Table 20-3]

[0321] Twenty-three antibodies were selected, and their cross-reactivity with mouse antigen C, monkey antigen C, and canine antigen C was determined using flow cytometry. The results are shown below.

[0322] [Table 21]

[0323] Example 11: Distribution of antibody diversity and binding affinity in hVH / hcVL mice Antigens D and E are type I transmembrane receptor proteins. Antigens D and E are, respectively, hVH H / H / hcVL K / KThis was used to immunize mice (IGKV3-11 / J1). Then, plasma cells capable of producing antigen-specific monoclonal antibodies were isolated using the Beacon™ microfluidic optical system.

[0324] After extracting RNA from plasma cells, the RNA was reverse transcribed into cDNA and sequenced. A total of 1166 antibody heavy chain variable region sequences were obtained from antigen group D. A total of 1290 heavy chain variable region antibody sequences were obtained from antigen group E. The human germline genes of these antibody heavy chain sequences were identified. Representative results are shown in Figures 33A and 33B. Here, the V region genes of all antibodies relate to a total of 5 subgroups (VH1-VH5, including IGHV1, IGHV2, IGHV3, IGHV4, and IGHV5; see the X axis in Figures 33A-33B). The D region genes relate to a total of 6 subgroups (DH1-DH6, including IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, and IGHD6; see the left Y axis in Figures 33A-33B). The J region genes relate to a total of six subgroups, JH1-JH6 (including IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6; see the right Y-axis in Figures 33A-33B). The results showed high V\D\J diversity in antibody heavy chains produced by anti-antigen D and antigen E. This suggests that antibody heavy chain diversity is not affected in humanized common light mice.

[0325] The affinity of these antibodies was then determined using Biacore. By fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110), the association rate (kon) and dissociation rate (koff) were obtained. The affinity (KD = koff / kon) was derived from the quotient of the velocity constants. KD = 1 × 10⁻⁶ -8Antibodies smaller than M were analyzed. These antibodies included 805 human common light chain antibodies against antigen D and 1026 human common light chain antibodies against antigen E. Antibody binding affinity (KD), kon, and koff values ​​were provided and ranked based on KD. Statistical analysis was performed. The distribution of KD, kon, and koff is shown in Figures 34-35.

[0326] Figures 34A-34B show the kon and koff rate distributions of antibodies against antigen D. Figures 34C-34D show the kon and koff rate distributions of antibodies against antigen E. Figure 35 shows the antibody binding affinity distribution, with the kon rates of these antibodies mainly at 1 × 10⁻⁶. 5 / Ms and 1×10 6 It is located between / Ms and koff is 1×10 -2 / S and 1×10 -3 It was found that the antibody is located between / S and has a binding affinity (KD) mainly between 0.10 nM and 100.00 nM. These results are consistent with antibodies derived from innate immune responses. From the data, hVH H / H / hcVL K / K It was found that mice can produce antibodies with high affinity for various antigens.

[0327] Example 12: hVH H / H / hcVL K / + Analysis of germline use in mice Homozygous humanized heavy chain immunoglobulin locus and heterozygous humanized common light chain immunoglobulin locus (humanized VH H / H / cVL K / + Mouse, or hVH H / H / hcVL K / + Transgenic mice possessing the characteristic (represented as mouse) were selected for use in germline analysis. H / H / hcVL K / +The mice possess a reconstituted human IGKV3-11 / J1 sequence. RNA was extracted from mouse spleen cells prior to antigen immunization. The extracted RNA was reverse transcribed, and the immunoglobulin heavy chain variable region was amplified using PCR. More specifically, PCR amplification was performed using upstream primers, i.e., rapid amplification of the 5' end of cDNA-end (RACE) primers, and downstream primers targeting the downstream region of the IgM-coding gene. The amplified PCR products were purified and analyzed by next-generation sequencing (NGS) to identify unimmunized hVH H / H / hcVL K / + The germline usage status of mice was determined. A total of 131,847 valid readings were obtained.

[0328] Unimmunized hVH H / H / hcVL K / + The usage of heavy chain IGHV, IGHD, and IGHJ in mice was analyzed. The results are shown in Figures 26A-26D. Transcripts of 46 IGHV genes (including 2 pseudogenes) were detected. In addition, transcripts of 25 IGHD genes and 6 IGHJ genes were detected. For example, as shown in Figure 26D, IGHJ3, IGHJ4, IGHJ5, and IGHJ6 are unimmunized hVH H / H / hcVL K / + It was frequently used in mice, but not so much in IGHJ1 and IGHJ2. The aforementioned IGHJ germline usage mode is consistent with the usage of human IGHJ germlines in humans as reported in the literature.

[0329] Homozygous hVH that has not been immunized H / H / hcVL K / K We detected the use of heavy chain IGHV and IGHD in mice. A total of 123,188 valid readings were obtained. The results are shown in Figures 26E-26G. Transcripts of 46 IGHV genes (including two pseudogenes, IGHV1-NL1 and IGHV3-38-3) were detected. The detection results were similar to those of heterozygous mice.

[0330] [Table 22]

[0331] The length distribution of heavy chain CDR3 is found in unimmunized hVH. H / H / hcVL K / + This was determined by NGS sequencing of the immunorepertory derived from mouse (n=1) spleen cells. As shown in Figure 27A, the median length of CDR3 is approximately 13-15 amino acids. Homozygous unimmunized hVH H / H / hcVL K / K The results for mice are shown in Figure 27B. These results are consistent with the median length of human heavy chain CDR3 in the human immune system.

[0332] The amino acid types at each position of the heavy chain CDR3 (HCDR3) (Figures 28A-28C, 28D) were also analyzed. These modes are similar to the amino acid composition of human HCDR3.

[0333] Cysteine ​​residues can form disulfide bonds. Human HCDR3 may contain one or two cysteine ​​residues, but mouse HCDR3 usually does not contain cysteine. The results in Figure 29 show hVH containing cysteine ​​residues. H / H / hcVL K / + The study showed the frequency of HCDR3 in mice, and this frequency increased with increasing HCDR3 length. This result is consistent with the diversity of HCDR3 in human peripheral blood mononuclear cells (PBMCs). The results indicate that VDJ recombination allows hVH / hcVL to function normally in mice.

[0334] Example 13: hVH H / H / hcVL K / + Analysis of somatic hypermutation in mice hVH reconstructed from the human IGKV3-11 / J1 sequence H / H / hcVL K / +Somatic hypermutation analysis was performed on selected mice. RNA was extracted from mouse splenocytes before antigen immunization. The extracted RNA was reverse transcribed into cDNA, and the reconstituted human IGKV3-11 / J1 region was amplified using primer PCR against human IGKV3-11 and IGJ1. Next-generation sequencing (NGS) was performed on the amplified PCR products. 48,326 valid reads were obtained and analyzed using the IMGT / HighV-QUEST tool. As shown in Figures 30A-30B, the results indicated that somatic hypermutation can occur in the reconstituted human light chain region, but the majority of somatic hypermutations were limited to changes of one or two amino acids. (Unimmunized homozygous hVH) H / H / hcVL K / K The results for mice are shown in Figure 30C. 48,541 valid readings were obtained and analyzed using the IMGT / HighV-QUEST tool. The results are similar to those for heterozygous mice, and the majority of somatic mutations are limited to changes of 1-2 amino acids, as is the case with hVH. H / H / hcVL K / K In mice, the somatic hypermutability is low and indicates that the changes are limited to approximately 1-2 nucleotides (or amino acids).

[0335] Other Embodiments It should be understood that while the present invention has been described by linking various forms for carrying it out, this description is intended to illustrate, not limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. A genetically modified non-human animal comprising an exogenous κ light chain variable region gene sequence at the endogenous κ light chain immunoglobulin gene locus, wherein the exogenous κ light chain variable region gene sequence comprises one human IGKV gene and one human IGKJ gene, wherein the one human IGKV gene and the one human IGKJ gene are operably linked to an endogenous light chain constant domain gene. (1) The human IGKV gene is IGKV 1-39, the human IGKJ gene is IGKJ4, or (2) The human IGKV gene is IGKV 3-11, and the human IGKJ gene is IGKJ1, The aforementioned non-human animal is a mouse. Genetically modified non-human animals.

2. The animal according to claim 1, wherein the exogenous κ light chain variable region gene sequence further comprises a human IGKJ 3'-UTR sequence.

3. The animal according to claim 1, wherein somatic hypermutation can occur in the exogenous κ light chain variable region gene in one or more cells of the animal.

4. The animal according to claim 1, further comprising a promoter sequence operably linked to the human IGKV gene, wherein the promoter sequence is located at 2500 bp or 3000 bp of the human IGKV gene.

5. The animal according to claim 1, wherein the animal comprises a disruption at the endogenous light chain immunoglobulin gene locus of the animal.

6. The animal is the animal according to claim 1, comprising endogenous IGKC.

7. The animal according to claim 1, wherein the human light chain variable region is a reconstituted sequence.

8. The animal according to claim 1, wherein the animal is homozygous or heterozygous with respect to the light chain immunoglobulin gene locus.

9. The animal according to claim 1, further comprising one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at an endogenous heavy chain immunoglobulin locus, wherein the human IGHV gene, the human IGHD gene, and the human IGHJ gene are operably linked and capable of undergoing VDJ rearrangement.

10. A genetically modified non-human animal whose genome includes an endogenous κ-light chain immunoglobulin locus, the endogenous κ-light chain immunoglobulin locus is One or more endogenous IGKV genes are replaced by one human IGKV gene, It includes one or more endogenous IGKJ genes that are replaced by one human IGKJ gene, Here, the human IGKV gene and the human IGKJ gene are operably linked to the endogenous IGKC gene, (1) The human IGKV gene is IGKV1-39, the human IGKJ gene is IGKJ4, or (2) The human IGKV gene is IGKV3-11 and the human IGKJ gene is IGKJ1, The aforementioned non-human animal is a mouse. Genetically modified non-human animals.

11. The animal according to claim 10, wherein the genome further comprises an endogenous heavy chain immunoglobulin locus, the endogenous heavy chain immunoglobulin locus comprising one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes which are replaced by one or more human IGHV, human IGHD, and human IGHJ genes, wherein the one or more human IGHV, human IGHD, and human IGHJ genes are operably linked to one or more endogenous IGHM, IGHδ, IGHG, IGHE, and IGHA genes.

12. An animal according to any one of claims 1 to 11, which produces an antibody or antigen-binding fragment thereof comprising a human light chain variable region having the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:

39.

13. The exogenous κ light chain variable region gene sequence is (a) SEQ ID NO: 8 and SEQ ID NO: 9; (b) SEQ ID NO: 10 and SEQ ID NO: 11; (c) SEQ ID NO: 16 and SEQ ID NO: 17; and, (d) SEQ ID NO: 18 and SEQ ID NO: 19 Detection is possible by PCR using one or more primer pairs selected from the group consisting of the following. The animal according to any one of claims 1 to 11.

14. The animal according to any one of claims 1 to 11, wherein the endogenous κ light chain variable immunoglobulin gene locus contains a gene sequence that is at least 95% identical to SEQ ID NO: 35 or SEQ ID NO:

36.

15. The animal according to any one of claims 1 to 11, wherein the endogenous κ light chain variable immunoglobulin gene locus comprises the gene sequence of SEQ ID NO: 35 or SEQ ID NO:

36.

16. The animal is the animal according to any one of claims 1 to 15, which is capable of producing humanized antibodies.

17. Cells obtained from an animal according to any one of claims 1 to 16, which are B cells expressing a chimeric immunoglobulin light chain including an immunoglobulin light chain variable region encoded by the human IGKV gene and the human IGKJ gene, (1) The human IGKV gene is IGKV1-39, and the human IGKJ gene is IGKJ4, or (2) The human IGKV gene is IGKV3-11, and the human IGKJ gene is IGKJ1, The immunoglobulin light chain variable region is operably linked to the non-human light chain constant region in the cell.

18. A method for producing an antibody that specifically binds to an antigen, comprising exposing an animal according to any one of claims 1 to 16 to the antigen, Sequencing nucleic acids encoding the variable regions of human heavy chain and light chain immunoglobulins in cells expressing a hybrid antibody that specifically binds to the aforementioned antigen, A method for producing an antibody that specifically binds to an antigen, comprising: activating the linking of a nucleic acid encoding the variable region of human heavy chain immunoglobulin with a nucleic acid encoding the constant region of human heavy chain immunoglobulin in a cell; and activating the linking of a nucleic acid encoding the variable region of human light chain immunoglobulin with a nucleic acid encoding the constant region of human light chain immunoglobulin.

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