Humanized antibody mouse

Abstract

The present application relates to a genetically modified non-human animal comprising a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus and methods of making the same. The present application also provides methods of producing antibodies by the non-human animal.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a non-human animal model containing humanized fragments and a method for constructing the same. Background Technology

[0002] Therapeutic antibody drugs, as one of the most important components of biomedicine, play a vital role in the treatment of major diseases and biosafety control. Among them, humanized antibodies and fully human antibodies, compared with murine antibodies and chimeric antibodies, have further improved safety and efficacy compared with previous antibody drugs, and are the focus of current and future antibody drug development.

[0003] Mainstream technologies for developing fully human antibodies include phage display. However, due to the lack of in vivo antibody variable region recombination and affinity hypermutation processes, the diversity and affinity of antibodies screened by these methods are limited, often requiring significant costs and time for further optimization and modification. In contrast, human antibody transgenic animal technology, having undergone natural selection within animals, yields antibodies with superior overall performance.

[0004] However, while many animal models can generate fully human antibodies, the sheer size and complexity of the human antibody coding lineage lead to several major drawbacks. These include limited diversity and low affinity of the generated antibody molecules, as well as low success rates, high costs, complex procedures, and lengthy processing times in the preparation of these animal models. Therefore, developing efficient, economical, and cost-effective humanized animal models remains a pressing issue in this field. Summary of the Invention

[0005] This application provides a genetically modified non-human animal comprising a modified heavy chain immunoglobulin locus and / or a modified light chain immunoglobulin locus, and cells derived from the non-human animal. This application also provides a method for preparing the non-human animal or cells, and a method for generating antibodies or related samples using the non-human animal. The non-human animal or cells of this application can be used to prepare an antibody-humanized mouse platform or to prepare animal models.

[0006] The technical solution of this application includes one or more of the following advantages:

[0007] The genetically modified non-human animals provided in this application have advantages in antibody production. For example, the non-human animals contain only partially humanized heavy chain immunoglobulin variable regions and / or partially humanized light chain immunoglobulin variable regions; however,

[0008] (1) It can produce antigen-specific antibodies with high titers and good antigen-binding ability, and can be used to produce humanized monoclonal antibodies in vivo.

[0009] (2) It has a highly diverse antibody library with CDR3 length distribution and similar to human antibodies.

[0010] The genetically modified non-human animals provided in this application have advantages in antibody production. For example, the endogenous immunoglobulin constant region genes of the non-human animals are all preserved, including all host immunoglobulin regulatory sequences, such as promoters, enhancers, transition regions, and other potential expression regulatory sequences. This better ensures the normal function of humanized immunoglobulin genes in the non-human animals, resulting in immune cells, B cell subset ratios, B cell development processes, and similar immune responses to wild-type mice. For example, the endogenous immunoglobulin variable region genes of the non-human animals are all deleted, avoiding interference from genes originating from non-human animals and thus minimizing immunogenicity in humans. The B cell development level, antibody affinity, and specificity of the non-human animals described in this application are consistent with those of wild-type mice.

[0011] The method for preparing genetically modified non-human animals provided in this application has advantages. For example, by introducing only a portion of the variable region of humanized heavy chain immunoglobulin and / or a portion of the variable region of humanized light chain immunoglobulin, the method generates genetically modified non-human animals with variable regions that have a diversity very similar to that of human variable regions, thus providing a more efficient method for constructing humanized animal models.

[0012] On one hand, this application provides a genetically modified non-human animal whose genome contains a modified light chain immunoglobulin locus, wherein the modified light chain immunoglobulin locus contains multiple human IGKV genes and one or more human IGKJ genes, wherein the human IGKV genes are composed of IGKV6D-41, IGKV2D-29, IGKV1D-37, IGKV2D-40, IGKV1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-27, IGKV2-24, IGKV6-21, IGKV3-20, IGKV1-17, IGKV1-16, IGKV3-15, IGKV1-12, IGKV3-11, IGKV1-9, IGKV1-8, IGKV1-6, IGKV1-5, IGKV5-2 and IGKV4-1.

[0013] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0014] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, and IGHV6-1.

[0015] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0016] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0017] In some embodiments, the animal's genome further comprises a modified heavy chain immunoglobulin locus, the modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, and IGHV6. It consists of HV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

[0018] In some embodiments, the animal's genome further comprises a modified heavy chain immunoglobulin locus, the modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3- It consists of 35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

[0019] In some embodiments, the animal contains the entire human IGKJ gene at the human chromosome 2 light chain immunoglobulin locus.

[0020] In some embodiments, the animal comprises all human IGHD and all human IGHJ genes at the human chromosome 14 heavy chain immunoglobulin locus.

[0021] In some embodiments, the animal includes disruption of its endogenous light chain immunoglobulin loci, the disruption comprising the deletion of one or more endogenous Igkv genes and one or more endogenous Igkj genes.

[0022] In some embodiments, the animal includes disruption of its endogenous light chain immunoglobulin gene loci, the disruption including the deletion of all endogenous Igkv genes and all endogenous Igkj genes.

[0023] In some embodiments, the animal includes disruption of its endogenous heavy chain immunoglobulin loci, the disruption including the deletion of one or more endogenous Ighv genes, one or more endogenous Ighd genes, and one or more endogenous Ighj genes.

[0024] In some embodiments, the animal includes disruption of its endogenous heavy chain immunoglobulin gene loci, the disruption including the deletion of all endogenous Ighv genes, all endogenous Ighd genes, and all endogenous Ighj genes.

[0025] In some embodiments, the animal contains an endogenous Igkc gene.

[0026] In some embodiments, the human IGKV and human IGKJ genes are operatively linked to the endogenous Igkc gene.

[0027] In some embodiments, the animal contains endogenous Ighm, Ighδ, Ighg, Ighe, and / or Igha genes.

[0028] In some embodiments, the human IGHV, human IGHD, and human IGHJ genes are operatively linked to one or more genes selected from the endogenous Ighm, Ighδ, Ighg, Ighe, and Igha genes.

[0029] In some embodiments, the animal is homozygous for the modified heavy chain immunoglobulin locus.

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

[0031] In some embodiments, the animal is homozygous for the modified light chain immunoglobulin locus.

[0032] In some embodiments, the animal is heterozygous for the modified light chain immunoglobulin locus.

[0033] In some embodiments, the animal is a rodent.

[0034] In some embodiments, the rodent is a mouse or a rat.

[0035] In some embodiments, disruption of the mouse's endogenous light chain immunoglobulin locus includes the deletion of a continuous sequence from the mouse Igkv2-137 gene to the mouse Igkj5 gene.

[0036] In some embodiments, disruption of the mouse's endogenous light chain immunoglobulin gene locus includes the deletion of the endogenous gene between mouse chromosomal locations chr6:67551638 and 70724291.

[0037] In some embodiments, the mouse contains the human immunoglobulin light chain variable region gene at mouse chromosome location chr6:67551638 to 70724291.

[0038] In some embodiments, disruption of the mouse's endogenous heavy chain immunoglobulin loci comprises the deletion of a continuous sequence from the mouse Ighv1-86 gene to the mouse Ighj4 gene.

[0039] In some embodiments, disruption of the mouse's endogenous heavy chain immunoglobulin gene locus includes the deletion of the endogenous gene between mouse chromosomal locations chr12:116012244 and 113427277.

[0040] In some embodiments, the mouse contains the human immunoglobulin heavy chain variable region gene at mouse chromosome location chr12:116012244 to 113427277.

[0041] On the other hand, this application also provides a method for preparing a non-human animal, wherein the genome of the animal contains a modified light chain immunoglobulin locus, the modified light chain immunoglobulin locus contains a plurality of human IGKV genes and one or more human IGKJ genes, wherein the human IGKV genes are composed of IGKV6D-41, IGKV2D-29, IGKV1D-37, IGKV2D-40, IGKV1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-27, IGKV2-24, IGKV6-21, IGKV3-20, IGKV1-17, IGKV1-16, IGKV3-15, IGKV1-12, IGKV3-11, IGKV1-9, IGKV1-8, IGKV1-6, IGKV1-5, IGKV5-2 and IGKV4-1.

[0042] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0043] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, and IGHV6-1.

[0044] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0045] In some embodiments, the animal's genome further includes a modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0046] In some embodiments, the animal's genome further comprises a modified heavy chain immunoglobulin locus, the modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, and IGHV6. It consists of HV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

[0047] In some embodiments, the animal's genome further comprises a modified heavy chain immunoglobulin locus, the modified heavy chain immunoglobulin locus comprising a plurality of human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3- It consists of 35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

[0048] In some implementations, the method includes:

[0049] (a) Breeding any of the animals described herein as the female parent;

[0050] (b) Breeding any of the animals described herein as the paternal parent;

[0051] (c) Using the female parent described in step (a) and the male parent described in step (b) to mate and reproduce, obtaining F1 generation animals.

[0052] In some embodiments, the animal contains the entire human IGKJ gene at the human chromosome 2 light chain immunoglobulin locus.

[0053] In some embodiments, the animal comprises all human IGHD and all human IGHJ genes at the human chromosome 14 heavy chain immunoglobulin locus.

[0054] In some embodiments, the animal includes disruption of its endogenous light chain immunoglobulin loci, the disruption comprising the deletion of one or more endogenous Igkv genes and one or more endogenous Igkj genes.

[0055] In some embodiments, the animal includes disruption of its endogenous light chain immunoglobulin gene loci, the disruption including the deletion of all endogenous Igkv genes and all endogenous Igkj genes.

[0056] In some embodiments, the animal includes disruption of its endogenous heavy chain immunoglobulin loci, the disruption including the deletion of one or more endogenous Ighv genes, one or more endogenous Ighd genes, and one or more endogenous Ighj genes.

[0057] In some embodiments, the animal includes disruption of its endogenous heavy chain immunoglobulin gene loci, the disruption including the deletion of all endogenous Ighv genes, all endogenous Ighd genes, and all endogenous Ighj genes.

[0058] In some embodiments, the animal contains an endogenous Igkc gene.

[0059] In some embodiments, the human IGKV and human IGKJ genes are operatively linked to the endogenous Igkc gene.

[0060] In some embodiments, the animal contains endogenous Ighm, Ighδ, Ighg, Ighe, and / or Igha genes.

[0061] In some embodiments, the human IGHV, human IGHD, and human IGHJ genes are operatively linked to one or more genes selected from the endogenous Ighm, Ighδ, Ighg, Ighe, and Igha genes.

[0062] In some embodiments, the animal is homozygous for the modified heavy chain immunoglobulin locus.

[0063] In some embodiments, the animal is heterozygous for the modified heavy chain immunoglobulin locus.

[0064] In some embodiments, the animal is homozygous for the modified light chain immunoglobulin locus.

[0065] In some embodiments, the animal is heterozygous for the modified light chain immunoglobulin locus.

[0066] In some embodiments, the animal is a rodent.

[0067] In some embodiments, the rodent is a mouse or a rat.

[0068] In some embodiments, disruption of the mouse's endogenous light chain immunoglobulin locus includes the deletion of a continuous sequence from the mouse Igkv2-137 gene to the mouse Igkj5 gene.

[0069] In some embodiments, disruption of the mouse's endogenous light chain immunoglobulin gene locus includes the deletion of the endogenous gene between mouse chromosomal locations chr6:67551638 and 70724291.

[0070] In some embodiments, the mouse contains the human immunoglobulin light chain variable region gene at mouse chromosome location chr6:67551638 to 70724291.

[0071] In some embodiments, disruption of the mouse's endogenous heavy chain immunoglobulin loci comprises the deletion of a continuous sequence from the mouse Ighv1-86 gene to the mouse Ighj4 gene.

[0072] In some embodiments, disruption of the mouse's endogenous heavy chain immunoglobulin gene locus includes the deletion of the endogenous gene at mouse chromosomal locations chr12:116012244 to 113427277.

[0073] In some embodiments, the mouse contains the human immunoglobulin heavy chain variable region gene at mouse chromosome location chr12:116012244 to 113427277.

[0074] In some embodiments, the method includes introducing a modified human nucleic acid sequence into cells of the animal, such that the cells contain the modified human nucleic acid sequence.

[0075] In some embodiments, the method includes:

[0076] (a) Modifying endogenous loci in non-human animal cells to produce endogenous loci lacking endogenous genes;

[0077] (b) Introducing the modified human nucleic acid sequence into the animal cell described in step (a) such that the cell contains the modified human nucleic acid sequence.

[0078] In some embodiments, the method includes:

[0079] (a) Modifying endogenous loci in non-human animal cells to produce endogenous loci lacking the variable region of endogenous light chain immunoglobulin loci;

[0080] (b) Introducing the modified human nucleic acid sequence into the animal cell described in step (a) such that the cell contains the variable region of the human light chain immunoglobulin locus of the modified human nucleic acid sequence.

[0081] In some embodiments, the method includes:

[0082] (a) Modifying endogenous loci in non-human animal cells to produce endogenous loci lacking the variable region of endogenous heavy chain immunoglobulin loci;

[0083] (b) Introducing the modified human nucleic acid sequence into the animal cell described in step (a) such that the cell contains the variable region of the human heavy chain immunoglobulin locus of the modified human nucleic acid sequence.

[0084] In some embodiments, the method includes:

[0085] (a) Modify endogenous loci in non-human animal cells to generate endogenous loci lacking the variable region of endogenous light chain immunoglobulin loci through site-specific recombination;

[0086] (b) Introducing the modified human nucleic acid sequence into the animal cells described in step (a);

[0087] (c) Inducing site-specific recombination between the endogenous locus and the modified human nucleic acid sequence, thereby enabling the cell to contain the variable region of the human light chain immunoglobulin locus of the modified human nucleic acid sequence.

[0088] In some embodiments, the method includes:

[0089] (a) Modify endogenous loci in non-human animal cells to generate endogenous loci lacking the variable region of endogenous heavy chain immunoglobulin loci through site-specific recombination.

[0090] (b) Introducing the modified human nucleic acid sequence into the animal cells described in step (a);

[0091] (c) Inducing site-specific recombination between the endogenous locus and the modified human nucleic acid sequence, thereby enabling the cell to contain the variable region of the human heavy chain immunoglobulin locus of the modified human nucleic acid sequence.

[0092] In some embodiments, the site-specific recombination is mediated by Cre recombinase and / or Flp recombinase.

[0093] On the other hand, this application also provides the offspring of the aforementioned non-human animal. Such offspring can be the offspring of a non-human animal mating with a species of the same or other genotypes.

[0094] On the other hand, this application also provides a non-human animal cell derived from the cells or tissues of the non-human animal, wherein the cells are immortalized.

[0095] In some embodiments, the cells are immortalized by fusing with tumor cells to provide antibody-producing cells and cell lines, or are made by direct cell immortalization.

[0096] In some embodiments, the cells are ES cells.

[0097] On the other hand, this application also provides a method for generating non-human animal ES cells, the method comprising inserting the genome of the non-human animal into the genome of the non-human animal ES cells.

[0098] On the other hand, this application also provides a method for preparing antibodies that specifically bind to antigens, the method comprising:

[0099] (a) Exposing the non-human animal to the antigen;

[0100] (b) Generating hybridomas from cells collected from said animal; and

[0101] (c) Collect the chimeric antibodies produced by the hybridoma.

[0102] In some embodiments, the method further includes sequencing the genome of the hybridoma.

[0103] On the other hand, this application also provides a method for preparing antibodies that specifically bind to antigens, the method comprising:

[0104] (a) Exposing the non-human animal to the antigen;

[0105] (b) Sequencing the nucleic acids encoding the variable regions of human heavy and light chain immunoglobulins in cells expressing chimeric antibodies that specifically bind to the antigen; and

[0106] (c) In a cell, the nucleic acid encoding the variable region of the human heavy chain immunoglobulin and the nucleic acid encoding the constant region of the human heavy chain immunoglobulin, and the nucleic acid encoding the variable region of the human light chain immunoglobulin and the nucleic acid encoding the constant region of the human light chain immunoglobulin are operatively linked.

[0107] On the other hand, this application also provides a method for obtaining nucleic acid encoding an antibody-binding domain that specifically binds to an antigen, the method comprising exposing a genetically modified non-human animal to the antigen, the animal being produced by the method; and sequencing nucleic acid encoding variable regions of human heavy and light chain immunoglobulins in cells, the cells expressing chimeric antibodies that specifically bind to the antigen.

[0108] On the other hand, this application also provides a method for obtaining a sample, the method comprising: exposing the non-human animal to an antigen; and collecting the sample from the non-human animal.

[0109] In some embodiments, the sample includes immune cells.

[0110] In some embodiments, the sample includes spleen cells, B cells, T cells, or hybridoma cells.

[0111] In some embodiments, the sample is derived from bone marrow, spleen tissue, lymph nodes, spleen cells, or peripheral lymphocytes.

[0112] On the other hand, this application also provides the use of non-human animals or their offspring produced using the method in the preparation of antibody-humanized mouse platforms or animal models.

[0113] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0114] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0115] Figure 1 This diagram shows the κ light chain immunoglobulin gene locus on mouse chromosome 6.

[0116] Figure 2 The diagram shows a humanized mouse κ light chain immunoglobulin gene locus.

[0117] Figure 3 The diagram shows a mouse κ light chain immunoglobulin locus after integration of recombinant target A and recombinant target B.

[0118] Figure 4 The diagram shows a target introduction of recombinant target A and recombinant target B into the mouse κ light chain immunoglobulin locus.

[0119] Figure 5 The results shown are PCR identification results of positive ES cell clones that have homologously integrated with both recombinant target A and recombinant target B.

[0120] Figure 6 The display shows the targeting strategy using the humanized IGK vector C.

[0121] Figure 7 The results shown are positive ES cell clone PCR identification results of the humanized κ light chain immunoglobulin variable region gene.

[0122] Figure 8 The results shown are positive ES cell clone PCR identification results of the variable region gene of humanized κ light chain immunoglobulin after removal of the resistance gene.

[0123] Figure 9 The results shown are PCR identification results of the hIGK F0 generation mice containing the variable region gene of humanized κ light chain immunoglobulin.

[0124] Figure 10The results shown are PCR identification results of hIGK F1 generation mice containing the variable region gene of humanized κ light chain immunoglobulin.

[0125] Figure 11 This diagram shows a heavy chain immunoglobulin gene locus on mouse chromosome 12.

[0126] Figure 12 This diagram shows a humanized mouse heavy chain immunoglobulin gene locus.

[0127] Figure 13 The diagram shows a mouse heavy chain immunoglobulin locus after integration of recombinant target D and recombinant target E.

[0128] Figure 14 The diagram shows a target introduction of recombinant target D and recombinant target E into the mouse heavy chain immunoglobulin locus.

[0129] Figure 15 The results shown are PCR identification results of positive ES cell clones that have homologously integrated with both recombinant target D and recombinant target E.

[0130] Figure 16 The image shows the targeting strategy using the humanized IGH vector F.

[0131] Figure 17 The results shown are PCR identification results of positive ES cell clones from the IGHD and IGHJ regions of the humanized heavy chain immunoglobulin gene locus.

[0132] Figure 18 The results shown are PCR identification results of positive ES cell clones of the IGHD and IGHJ regions of the humanized heavy chain immunoglobulin gene locus after the removal of the resistance gene.

[0133] Figure 19 The image shows the structure of the humanized IGH-Set1 gene after integrating the human heavy chain immunoglobulin IGHV region into Set1.

[0134] Figure 20 The display shows the targeting strategy using the humanized IGH vector G.

[0135] Figure 21 The results shown are positive ES cell clone PCR identification results for humanized heavy chain immunoglobulin IGH-Set1.

[0136] Figure 22 The image shows the structure of the humanized IGH-Set2 gene after integrating the human heavy chain immunoglobulin IGHV region into Set2.

[0137] Figure 23The display shows the targeting strategy using the humanized IGH vector H.

[0138] Figure 24 The results shown are positive ES cell clone PCR identification results for humanized heavy chain immunoglobulin IGH-Set2.

[0139] Figure 25 The image shows the structure of the humanized IGH-Set3 gene after integrating the human heavy chain immunoglobulin IGHV region into Set3.

[0140] Figure 26 The display shows the targeting strategy using the humanized IGH vector I.

[0141] Figure 27 The results shown are positive ES cell clone PCR identification results for humanized heavy chain immunoglobulin IGH-Set3.

[0142] Figure 28 The results shown are PCR identification results of hIGH1 F0 generation mice containing humanized heavy chain immunoglobulin IGH-Set1.

[0143] Figure 29 The results shown are PCR identification results of hIGH1 F1 generation mice containing humanized heavy chain immunoglobulin IGH-Set1.

[0144] Figure 30 The image shown is a PCR electrophoresis result of the genotype identification of fully humanized antibody hIGK / hIGH1 mice.

[0145] Figure 31 The results shown are PCR identification results of hIGH2 F0 generation mice containing humanized heavy chain immunoglobulin IGH-Set2.

[0146] Figure 32 The results shown are PCR identification results of hIGH2 F1 generation mice containing humanized heavy chain immunoglobulin IGH-Set2.

[0147] Figure 33 The image shown is a PCR electrophoresis result of the genotype identification of fully humanized antibody hIGK / hIGH2 mice.

[0148] Figure 34 The results shown are PCR identification results of hIGH3 F0 generation mice containing humanized heavy chain immunoglobulin IGH-Set3.

[0149] Figure 35 The results shown are PCR identification results of hIGH3 F1 generation mice containing humanized heavy chain immunoglobulin IGH-Set3.

[0150] Figure 36The image shown is a PCR electrophoresis result of the genotype identification of fully humanized antibody hIGK / hIGH3 mice.

[0151] Figure 37 The image shown is a PCR electrophoresis result of the fully humanized antibody hIGK / hIGH13 mouse genotype identification.

[0152] Figure 38 The image shown is a PCR electrophoresis result of the fully humanized antibody hIGK / hIGH12 mouse genotype identification.

[0153] Figure 39 The results show flow cytometry analysis of lymphocyte subsets in the spleens of fully humanized antibody hIGK / hIGH13 mice and wild-type WT mice, with no significant differences between the two.

[0154] Figure 40 The image shows flow cytometry analysis of B cell subsets in the spleens of fully humanized antibody hIGK / hIGH13 mice and wild-type WT mice, with no significant differences between the two.

[0155] Figure 41 The image shows flow cytometry analysis of B cell subsets in the spleens of fully humanized antibody hIGK / hIGH13 mice and wild-type WT mice, with no significant differences between the two.

[0156] Figure 42 The image shows the antibody titer in the serum of wild-type mice detected by ELISA after immunization with hTfR1-His as the antigen.

[0157] Figure 43 The image shows the antibody titer in the serum of fully humanized antibody hIGK / hIGH13 mice after immunization with hTfR1-His as the antigen, as detected by ELISA.

[0158] Figure 44 The image shows the antibody titers in the serum of wild-type mice and fully humanized antibody mice as detected by ELISA after four immunizations with recombinant IL-11 protein.

[0159] Figure 45 The image shows the antibody titers in the serum of wild-type mice and fully humanized antibody mice as detected by ELISA after three immunizations with TSLP mRNA-LNP.

[0160] Figure 46 The image shows the antibody titers in the serum of wild-type mice and fully humanized antibody mice as detected by ELISA after four immunizations with IGF1R mRNA-LNP.

[0161] Figure 47The image shows the binding of the Anti-PD-L1 recombinant antibody to the PD-L1 protein by ELISA after immunizing mice with the PD-L1 antigen with the fully humanized antibody; PC-BMK is Atezolizumab.

[0162] Figure 48 The image shows the SPR assay for the affinity between the anti-PD-L1 recombinant antibody and the PD-L1 protein after immunizing mice with the PD-L1 antigen; PC-BMK is Atezolizumab.

[0163] Figure 49 The image shows the binding of the Anti-TSLP recombinant antibody to the TSLP recombinant protein verified by ELISA after immunizing fully humanized antibody mice with TSLP antigen; PC-BMK is Tezepelumab.

[0164] Figure 50 The figure shows the OD450 value of the binding of the anti-TSLP recombinant antibody to the TSLP recombinant protein at a concentration of 10 μg / mL, as verified by SPR, after immunizing fully humanized antibody mice with TSLP antigen; PC-BMK is Tezepelumab.

[0165] Figure 51 The image shows the binding of the Anti-TL1A recombinant antibody to the TL1A recombinant protein verified by ELISA after immunizing fully humanized antibody mice with the TL1A antigen; PC-BMK is Tulisokibart.

[0166] Figure 52 The figure shows the OD450 value of the binding of the anti-TL1A recombinant antibody to the TL1A recombinant protein at a concentration of 10 μg / mL, as verified by SPR, after immunizing mice with the TL1A antigen; PC-BMK is Tulisokibart.

[0167] Figure 53 The image shows the binding of the Anti-IGF1R recombinant antibody to the IGF1R recombinant protein verified by ELISA after immunizing fully humanized antibody mice with IGF1R antigen; PC-BMK is Teprotumumab.

[0168] Figure 54 The figure shows the OD450 value of the binding of the anti-IGF1R recombinant antibody to the recombinant IGF1R protein at a concentration of 10 μg / mL, as verified by SPR, after immunizing fully humanized antibody mice with IGF1R antigen; PC-BMK is Teprotumumab.

[0169] Figure 55The image shows the endocytosis of anti-PD-L1 antibody in the MC38-hPD-L1 cell line as detected by flow cytometry; PC-BMK is Atezolizumab.

[0170] Figure 56 The image shows the flow cytometry analysis of the blocking effect of the anti-PD-L1 antibody on the MC38-hPD-L1 cell line; PC-BMK is Atezolizumab.

[0171] Figure 57 The figure shows the frequency distribution of the BCR V(D)J gene fragment combination in non-immunized fully humanized antibody hIGK / hIGH1 mice.

[0172] Figure 58 The figure shows the frequency distribution of the BCR V(D)J gene fragment combination in non-immunized fully humanized antibody hIGK / hIGH12 mice.

[0173] Figure 59 The figure shows the frequency distribution of the BCR V(D)J gene fragment combination in non-immunized fully humanized antibody hIGK / hIGH13 mice.

[0174] Figure 60 The image shows a histogram of the amino acid length distribution of the CDR3 heavy chain in the fully humanized antibody hIGK / hIGH1 mouse.

[0175] Figure 61 The image shows a histogram of the amino acid length distribution of the CDR3 heavy chain in the fully humanized antibody hIGK / hIGH12 mouse.

[0176] Figure 62 The image shows a histogram of the amino acid length distribution of the CDR3 heavy chain of the fully humanized antibody hIGK / hIGH13 mouse. The CDR3 data for the humanized antibody are from doi.org / 10.1371 / journal.pcbi.1007636.

[0177] Figure 63 The display shows the amino acid frequencies on the CDR3 heavy chain of the fully humanized antibody hIGK / hIGH1 mouse.

[0178] Figure 64 The display shows the amino acid frequencies on the CDR3 of the κ light chain of the fully humanized antibody hIGK / hIGH1 mouse.

[0179] Figure 65 The display shows the amino acid frequencies on the CDR3 of the fully humanized antibody hIGK / hIGH12 mouse heavy chain.

[0180] Figure 66The display shows the amino acid frequencies on the CDR3 of the κ light chain of the fully humanized antibody hIGK / hIGH12 mouse.

[0181] Figure 67 The display shows the amino acid frequencies on the CDR3 heavy chain of the fully humanized antibody hIGK / hIGH13 mouse.

[0182] Figure 68 The display shows the amino acid frequencies on the CDR3 of the κ light chain of the fully humanized antibody hIGK / hIGH13 mouse.

[0183] Figure 69 The figure shows the frequency distribution of the BCR V(D)J gene fragment combination in fully humanized antibody hIGK / hIGH12 mice after immunization.

[0184] Figure 70 The figure shows the frequency distribution of the BCR V(D)J gene fragment combination in fully humanized antibody hIGK / hIGH13 mice after immunization.

[0185] Figure 71 The display shows the amino acid usage frequency on the heavy chain CDR3 of fully humanized antibody hIGK / hIGH12 mice after immunization.

[0186] Figure 72 The display shows the amino acid usage frequency on the CDR3 of the κ light chain in fully humanized antibody hIGK / hIGH12 mice after immunization.

[0187] Figure 73 The display shows the amino acid usage frequency on the heavy chain CDR3 of fully humanized antibody hIGK / hIGH13 mice after immunization.

[0188] Figure 74 The display shows the amino acid usage frequency on the CDR3 of the κ light chain in fully humanized antibody hIGK / hIGH13 mice after immunization. Detailed Implementation

[0189] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0190] Terminology Definition

[0191] In this application, the term "non-human animal" generally refers to all non-human vertebrates, such as mammals and non-mammals, including non-human primates, rodents, rabbits, camels, sheep, dogs, cats, horses, cattle, birds, amphibians, reptiles, etc. For example, a non-human animal can be a rat or a mouse.

[0192] In this application, the term "genetically modified" generally refers to an animal whose germ cells contain exogenous human nucleic acid or human nucleic acid sequences. As a non-limiting example, a genetically modified animal can be a transgenic animal or a knock-in animal, as long as the animal contains a human nucleic acid sequence.

[0193] In this application, the term "modified" generally refers to a gene or gene product that exhibits modifications (i.e. altered characteristics) in sequence and / or functional properties compared to a wild-type gene or gene product.

[0194] In this application, the term "endogenous" generally refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0195] In this application, the term "locus" generally refers to a specific location along a chromosome or DNA sequence. Depending on the context, a locus can be a gene, a marker, a chromosome band, or a specific sequence of one or more nucleotides. In this application, when referring to an immunoglobulin locus, it refers to a genetic element or a group of related genetic elements that contains information that a cell can use to express immunoglobulin peptides. In the case of an unrearranged locus, the genetic element can be assembled by B cell precursors to form a gene encoding an immunoglobulin peptide. In the case of a rearranged locus, the gene encoding an immunoglobulin peptide is contained within the locus.

[0196] In this application, the term "immunoglobulin" generally refers to a protein composed primarily of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. The NH2-terminus (approximately 110 amino acids) of the full-length immunoglobulin "light chain" (approximately 25 kDa and 214 amino acids) is encoded by a variable region gene, and the COOH-terminus is encoded by either a κ or λ constant region gene. Similarly, the full-length immunoglobulin "heavy chain" (approximately 50 kDa and 446 amino acids) is encoded by a variable region gene (approximately 116 amino acids) and one of the other aforementioned constant region genes, such as γ (encoding approximately 330 amino acids). The term "immunoglobulin" includes immunoglobulins having a CDR derived from human or non-human sources. The framework of immunoglobulins can be human, humanized, or non-human, such as a mouse framework modified to reduce antigenicity in the human body, or a synthetic framework such as a shared sequence.

[0197] In this application, the term "operably linked" generally refers to a relationship in which the components are operably linked in a predetermined manner. In some embodiments, the nucleic acid sequence of the immunoglobulin variable region (or V(D)J fragment) is operably linked to the nucleic acid sequence of the immunoglobulin constant region to allow proper recombination between sequences into the immunoglobulin heavy or light chain sequence.

[0198] In this application, the term "variable region" generally refers to the region of an antibody molecule that binds to a specific antigen. It consists of antigen-binding sites on both the heavy and light chains. Variable regions differ between different B-cell immunoglobulins, but are identical across all immunoglobulins produced by the same B cell. Variable region diversity arises from genetic recombination processes that occur during B-cell maturation of the variable region gene. This process, known as rearrangement, produces a high degree of diversity in the ability to bind any given antigen, thereby enabling the immune system to recognize and neutralize the large antigenic burden resulting from exogenous and pathogenic structures. Therefore, the antibody library consists of a rich collection of immunoglobulins with different V regions, but all sharing the same Fc moiety. Information on human heavy chain V, D, and J regions, and light chain V and J regions can be found at IMGTRepertoire: https: / / www.imgt.org / IMGTrepertoire / LocusGenes / .

[0199] In this application, the term "constant region" generally refers to the sum of the antibody's domains excluding the variable region. Constant regions are not directly involved in antigen binding but exhibit different effector functions. Antibodies are classified into the following categories based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into categories such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively. The light chain constant regions, which can be found in all five antibody classes, are referred to as κ (kappa) and λ (lambda). Genes encoding the mouse (Mus musculus) constant regions may include IGHA, IGHδ, IGHE, IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHG3, or IGHM.

[0200] In this application, the term "antibody" generally refers to a scaffold or backbone portion comprising the complete antibody or its antigen-binding fragment, and optionally, a portion that allows the binding of the antigen to adopt a conformation that promotes antibody-antigen binding. Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv and / or di-scFv, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, provided they exhibit the desired antigen-binding activity. The term also includes genetically engineered antibodies such as chimeric antibodies (e.g., humanized mouse antibodies), humanized antibodies, fully human antibodies, and heterocovalent antibodies (e.g., bispecific antibodies).

[0201] In this application, the term "chimeric antibody" generally refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing all or part of a variable domain sequence of a human antibody (e.g., a light chain variable domain and / or a heavy chain variable domain sequence) and a constant domain of a non-human antibody (e.g., a mouse antibody).

[0202] In this application, the term "site-specific recombination" generally refers to recombination between two nucleotide sequences, each containing at least one recognition site. "Site-specific" means a specific nucleotide sequence that may be located at a specific location in the host cell genome. The nucleotide sequence may be endogenous to the host cell, at its natural location in the host genome or at some other location in the genome, or it may be a heterologous nucleotide sequence previously inserted into the host cell genome by any of the various known methods.

[0203] In this application, the term "offspring" generally includes descendant cells and includes differentiated or undifferentiated offspring cells derived from parental cells. In some embodiments, the offspring include offspring cells that are genetically identical to the parent. In some embodiments, the offspring include offspring cells that are genetically identical to the parent and phenotypically identical. In some embodiments, the offspring include offspring cells that have differentiated from parental cells.

[0204] In this application, the term "immortification" generally refers to the characteristic of cells acquiring the ability to grow and proliferate continuously. Immortified cells have unlimited proliferative growth, can be passaged for a long time, and are often accompanied by changes in karyotype.

[0205] In this application, the term "sample" generally refers to a wide variety of sample types obtained from an organism, including but not limited to cells, tissues, organs, or organisms. For example, the sample includes immune cells. For example, the sample includes spleen cells, B cells, T cells, or hybridoma cells. For example, the sample is derived from bone marrow, spleen tissue, lymph nodes, spleen cells, or peripheral lymphocytes.

[0206] In this application, the term "wild type" (WT) generally refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a natural source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily designed as either "normal" or "wild-type" forms of the gene. Invention Details

[0208] 1. Genetically modified non-human animals

[0209] On the one hand, this application provides a genetically modified non-human animal that may possess a modified heavy chain immunoglobulin locus and / or a modified light chain immunoglobulin locus (e.g., a κ chain locus). In this application, the research conducted in mice is merely illustrative; unless otherwise stated, references to mice include all non-human mammals, with mice being the preferred non-human mammal.

[0210] i. Modified κ light chain immunoglobulin locus

[0211] The κ light chain immunoglobulin locus (also known as IGK or immunoglobulin κ locus) is a region on a chromosome (e.g., human chromosome 2) containing genes for antibody (or immunoglobulin) light chains. Similarly, immunoglobulin light chain genes can undergo a series of rearrangements, resulting in the production of mature immunoglobulin light chain nucleic acids (e.g., the κ chain). The connection of the V region (also known as the IGKV gene) and the J region (also known as the IGKJ gene) creates consecutive exons that encode the entire light chain variable domain. In unrearranged DNA, the V gene region (or IGKV gene cluster) is located relatively far from the C region. The J gene region (or IGKJ gene cluster) is located near the C region. The connection of the V and J gene regions also brings the V gene closer to the C region sequence. The rearranged V region J gene region is separated from the C region sequence by only one intron. To produce complete immunoglobulin light chain messenger RNA, the V region exons are ligated to the C region sequence post-transcriptionally via RNA splicing.

[0212] The human light chain immunoglobulin gene locus is located on human chromosome 2. Human IGKV genes consist of several distinct groups, including the IGKV1 gene (comprising all IGKV genes beginning with IGKV1, also known as VκI), the IGKV2 gene (comprising all IGKV genes beginning with IGKV2, also known as VκII), the IGKV3 gene (comprising all IGKV genes beginning with IGKV3, also known as VκIII), the IGKV4 gene (comprising all IGKV genes beginning with IGKV4, also known as VκIV), the IGKV5 gene (comprising all IGKV genes beginning with IGKV5, also known as VκV), the IGKV6 gene (comprising all IGKV genes beginning with IGKV6, also known as VκVI), and the IGKV7 gene (comprising all IGKV genes beginning with IGKV7, also known as VκVII).

[0213] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified light chain immunoglobulin locus, wherein the modified light chain immunoglobulin locus may contain multiple human IGKV genes and one or more human IGKJ genes, wherein the human IGKV gene is composed of IGKV6D-41, IGKV2D-29, IGKV1D-37, IGKV2D-40, IGKV1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-27, IGKV2-24, IGKV6-21, IGKV3-20, IGKV1-17, IGKV1-16, IGKV3-15, IGKV1-12, IGKV3-11, IGKV1-9, IGKV1-8, IGKV1-6, IGKV1-5, IGKV5-2 and IGKV4-1.

[0214] In some embodiments, the animal may contain approximately or at least one, two, three, four, or five human IGKJ genes. In some embodiments, the animal may contain one, two, three, four, or five human IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5. In some embodiments, the animal may contain human IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 genes.

[0215] In some embodiments, the animal may include disruption of its endogenous light chain immunoglobulin gene loci. In some embodiments, disruption of the animal's endogenous light chain immunoglobulin gene loci may include the deletion of one or more endogenous Igkv genes and one or more endogenous Igkj genes. In some embodiments, disruption of the animal's endogenous light chain immunoglobulin gene loci may include the deletion of all endogenous Igkv genes and all endogenous Igkj genes.

[0216] In some embodiments, the animal is a mouse, and disruption of the endogenous light chain immunoglobulin gene locus in the animal may include deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163 mouse Igkv genes.

[0217] In some embodiments, disruption of the endogenous heavy chain immunoglobulin loci in the animal may include the deletion of at least or about one, two, three, four, or five mouse Igkj genes selected from Igkj1, Igkj2, Igkj3, Igkj4, and Igkj5.

[0218] In some embodiments, disruption of the endogenous light chain immunoglobulin locus in the animal may comprise a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb of endogenous sequence.

[0219] In some implementations, the missing sequence may start from Igkv2-137 to Igkj4, from Igkv1-136 to Igkj4, from Igkv1-135 to Igkj4, from Igkv2-137 to Igkj5, from Igkv1-136 to Igkj5, or from Igkv1-135 to Igkj5 (e.g., from Igkv2-137 to Igkj5).

[0220] In some embodiments, disruption of the mouse's endogenous light chain immunoglobulin gene locus may include the deletion of the endogenous gene at mouse chromosomal locations chr6:67551638 to 70724291. In some embodiments, the mouse may contain the human immunoglobulin light chain variable region gene at mouse chromosomal locations chr6:67551638 to 70724291.

[0221] In some embodiments, the mice may lack all endogenous Igkv and Igkj genes, and the light chain variable region will not have any sequences encoded by mouse-derived sequences, thereby minimizing immunogenicity in humans.

[0222] In some embodiments, the Igkv gene and / or the Igkj gene are operatively linked together. VJ recombination can occur between these genes and produce functional antibodies. In some embodiments, these genes are arranged in a sequence similar to that in the human light chain immunoglobulin gene locus. This arrangement provides various advantages; for example, the arrangement of these genes allows for the generation of light chain variable domains with a diversity very similar to that in humans.

[0223] In some implementations, the animal may contain endogenous Igkc.

[0224] In some embodiments, the IGKV gene and / or IGKJ gene are operatively linked to the Igkc gene (e.g., the endogenous Igkc gene).

[0225] In some embodiments, the animal may be homozygous for the modified light chain immunoglobulin locus.

[0226] In some embodiments, the animal may be heterozygous for the modified light chain immunoglobulin locus.

[0227] ii. Modified heavy chain immunoglobulin loci

[0228] Heavy chain immunoglobulin loci (also known as IGH or immunoglobulin heavy chain loci) are regions on chromosomes (e.g., human chromosome 14) that contain genes for antibody (or immunoglobulin) heavy chains. This region represents the germline organization of the heavy chain locus. The locus comprises V (variable), D (diversity), J (connection), and C (constant) regions. 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).

[0229] During B cell development, DNA-level recombination events link a single D region (also known as the IGHD gene) to a J region (also known as the IGHJ gene); the fused DJ exon of this partially rearranged DJ region is then linked to the V region (also known as the IGHV gene). The rearranged VDJ region containing the fused VDJ exon is then transcribed and fused at the RNA level to the IGHM constant region; this transcript encodes the μ heavy chain. Later in development, B cells produce VDJ-Cμ-Cδ premessenger RNA, which is selectively spliced ​​to encode either the μ or δ heavy chain. Mature B cells in lymph nodes undergo conversion recombination, resulting in a fused VDJ gene region adjacent to one of the IGHG, IGHA, or IGHE gene regions, with each cell expressing one of the γ, α, or ε heavy chains. The potential recombination of many different IGHV genes with several IGHJ genes provides broad antigen recognition. In addition to rearrangement diversity, linker diversity resulting from the random addition or deletion of nucleotides by non-homologous end repair processes and high-frequency somatic mutations contribute to antibody diversity.

[0230] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified heavy chain immunoglobulin locus. The modified heavy chain immunoglobulin locus may contain multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. The human IGHV genes are composed of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0231] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified heavy chain immunoglobulin locus. The modified heavy chain immunoglobulin locus may contain multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. The human IGHV genes are composed of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, and IGHV6-1.

[0232] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified heavy chain immunoglobulin locus. The modified heavy chain immunoglobulin locus may contain multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. The human IGHV genes are composed of IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0233] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified heavy chain immunoglobulin locus. The modified heavy chain immunoglobulin locus may contain multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. The human IGHV genes are composed of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1.

[0234] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified heavy chain immunoglobulin locus. The modified heavy chain immunoglobulin locus may contain multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, and IGHV5-51. It consists of HV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

[0235] On one hand, this application provides a genetically modified non-human animal whose genome may contain a modified heavy chain immunoglobulin locus. The modified heavy chain immunoglobulin locus may contain multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes consist of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, and IGHV4-3... 9. Composed of IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

[0236] In some embodiments, the animal may contain 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. In some embodiments, the animals may comprise 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 selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD6-6, IGHD1-7, IGHD2- 8. Human IGHD genes of IGHD3-9, IGHD3-10, IGHD4-11, IGHD5-12, IGHD6-13, IGHD1-14, IGHD2-15, IGHD3-16, IGHD4-17, IGHD5-18, IGHD6-19, IGHD1-20, IGHD2-21, IGHD3-22, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27. In some embodiments, the animal may contain all human IGHD genes at the heavy chain immunoglobulin locus on human chromosome 14.

[0237] In some embodiments, the animal may contain about or at least one, two, three, four, five, six, seven, eight, or nine human IGHJ genes. In some embodiments, the animal may include 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. In some embodiments, the animal may include human IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6 genes.

[0238] In some embodiments, the animal may include disruption of its endogenous heavy chain immunoglobulin gene loci. In some embodiments, disruption of the animal's endogenous heavy chain immunoglobulin gene loci may include the deletion of one or more endogenous Ighv genes, one or more endogenous Ighd genes, and one or more endogenous Ighj genes. In some embodiments, disruption of the animal's endogenous heavy chain immunoglobulin gene loci may include the deletion of all endogenous Ighv genes, all endogenous Ighd genes, and all endogenous Ighj genes.

[0239] In some embodiments, the animal is a mouse. Disruption of the endogenous heavy chain immunoglobulin loci in the animal may include at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182 deletions of the mouse Ighv gene.

[0240] In some embodiments, disruption of the endogenous heavy chain immunoglobulin loci in the animal may include deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mouse Ighd genes.

[0241] In some embodiments, disruption of the endogenous heavy chain immunoglobulin loci in the animal may include the deletion of about or at least one, two, three or four mouse Ighj genes selected from Ighj1, Ighj2, Ighj3 and Ighj4.

[0242] In some embodiments, disruption of the endogenous heavy chain immunoglobulin locus in the animal may include the 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.

[0243] In some implementations, the missing sequence may begin with Ighv1-86 to Ighj4, Ighv1-85 to IGHJ4, Ighv1-84 to Ighj4, Ighv1-83 to Ighj4, or Ighv1-82 to Ighj4 (e.g., Ighv1-86 to Ighj4).

[0244] In some embodiments, disruption of the mouse's endogenous heavy chain immunoglobulin gene locus may include the deletion of the endogenous gene at mouse chromosomal locations chr12:116012244 to 113427277. In some embodiments, the mouse may contain the human immunoglobulin heavy chain variable region gene at mouse chromosomal locations chr12:116012244 to 113427277.

[0245] In some embodiments, the mice may lack all endogenous Ighv, Ighd, and Ighj genes, and the heavy chain variable region will not have any sequence encoded by a mouse-derived sequence, thereby minimizing immunogenicity in humans.

[0246] In some embodiments, the IGHV, IGHD, and / or IGHJ genes are operatively linked together. VDJ recombination can occur between these genes and produce functional antibodies. In some embodiments, these genes are arranged in a sequence similar to that in human heavy chain immunoglobulin loci. This arrangement provides various advantages; for example, the arrangement of these genes allows for the generation of heavy chain variable domains with a diversity very similar to that in humans. Since some random sequences can be inserted into the sequence during VDJ recombination, in some embodiments, unmodified or minimally modified whole human antibody lineages can reduce the likelihood of non-human sequences being inserted during VDJ recombination.

[0247] In some embodiments, the animal may contain one or more endogenous genes selected from 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 γ2c (IGHG2c), immunoglobulin heavy chain constant ε (IGHE), and immunoglobulin heavy chain constant α (IGHA) genes. In some embodiments, these endogenous genes are operatively linked together. In some embodiments, these endogenous genes may have the same sequence as in wild-type animals. In some embodiments, isotype switching (immunoglobulin class switching) may occur in the animal.

[0248] In some embodiments, the IGHV gene, IGHD gene, and / or IGHJ gene may be operatively linked together with one or more genes (e.g., all genes) selected from the IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHG2c, IGHE, and IGHA genes.

[0249] In some embodiments, the animal may be homozygous for the modified heavy chain immunoglobulin locus.

[0250] In some embodiments, the animal may be heterozygous for the modified heavy chain immunoglobulin locus.

[0251] iii. Non-human animals

[0252] In this application, the genetically modified non-human animal can be any genetically modified non-human mammal, such as laboratory animals, livestock, and animals, including species such as rats, rodents, dogs, cats, pigs, horses, cattle, sheep, and non-human primates; for example, mice, rats, rabbits, hamsters, guinea pigs, cattle, pigs, sheep, goats, and other transgenic animal species, especially mammal species, as known in the art. In some embodiments, the genetically modified non-human animal can be a mouse, rat, or rabbit.

[0253] In some embodiments, the genetically modified non-human animal may be a mammal. In some embodiments, the non-human animal may be a small mammal, such as those belonging to the superfamily Dipodoidea or Muroidea. In some embodiments, the non-human animal may be a rodent. In some embodiments, the rodent is selected from mice, rats, and hamsters. In some embodiments, the rodent may be selected from the superfamily Muroidea. In some embodiments, the non-human animal may be selected from the following families: Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats)), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice) and Spalacidae (e.g., mole rates, bamboo rats, and zokors)). In some embodiments, the rodent may be selected from true mice or rats (Muridae), gerbils, spiny mice, and crested rats. In some implementations, the mice may be derived from members of the Muridae family.

[0254] In some embodiments, the genetically modified non-human animal may be a rat. In some embodiments, the rat may be selected from Wistar rats, the LEA strain, the Sprague Dawley strain, the Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain may be a hybrid strain selected from two or more strains of the group consisting of: Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0255] In some embodiments, the genetically modified non-human animal may be a mouse, such as a C57BL strain mouse (e.g., C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, C57BL / Ola, etc.). Mice of the 129 strain (e.g., 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2); mice of the BALB strain (e.g., BALB / c); etc. In some embodiments, the mice may be a hybrid of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In some embodiments, the mice may be a hybrid of the aforementioned 129 strain or a hybrid of the aforementioned BL / 6 strain. In some embodiments, the mice may be a BALB strain, such as the BALB / c strain. In some embodiments, the mice may be a cross between the BALB strain and another of the aforementioned strains. In some embodiments, the mice may be derived from a cross (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129).

[0256] In this application, the animal may have one or more other genetic modifications and / or other modifications suitable for the specific purpose of producing humanized animals.

[0257] In this application, the animal may include offspring produced by mating the genetically modified animal provided in this application with the same genotype or other genotypes.

[0258] 2. Methods for preparing genetically modified non-human animals

[0259] The genetically modified non-human animal can be generated using any method known in the art or as described herein.

[0260] On one hand, this application provides a method for preparing the genetically modified non-human animal, the method comprising introducing a modified human nucleic acid sequence into the cells of the animal, so that the cells contain the modified human nucleic acid sequence.

[0261] In some implementations, the method may include:

[0262] (a) Modifying endogenous loci in non-human animal cells to produce endogenous loci lacking endogenous genes;

[0263] (b) Introducing the modified human nucleic acid sequence into the animal cell described in step (a) such that the cell contains the modified human nucleic acid sequence.

[0264] In some implementations, the method may include:

[0265] (a) Modifying endogenous loci in non-human animal cells to produce endogenous loci lacking the variable region of endogenous light chain immunoglobulin loci;

[0266] (b) Introducing the modified human nucleic acid sequence into the animal cell described in step (a) such that the cell contains the variable region of the human light chain immunoglobulin locus of the modified human nucleic acid sequence.

[0267] In some implementations, the method may include:

[0268] (a) Modifying endogenous loci in non-human animal cells to produce endogenous loci lacking the variable region of endogenous heavy chain immunoglobulin loci;

[0269] (b) Introducing the modified human nucleic acid sequence into the animal cell described in step (a) such that the cell contains the variable region of the human heavy chain immunoglobulin locus of the modified human nucleic acid sequence.

[0270] In some embodiments, any of a variety of methods can be used to introduce human nucleic acid sequences into animal cells to generate genetically modified animals expressing human genes. Such techniques are well known in the art and include, but are not limited to, non-homologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and the CRISPR-Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art. Many other methods are also provided for genome editing, such as microinjecting genetically modified cell nuclei into enucleated oocytes and fusing enucleated oocytes with another genetically modified cell.

[0271] In some implementations, the method may include:

[0272] (a) Modify endogenous loci in non-human animal cells to generate endogenous loci lacking the variable region of endogenous light chain immunoglobulin loci through site-specific recombination;

[0273] (b) Introducing the modified human nucleic acid sequence into the animal cells described in step (a);

[0274] (c) Inducing site-specific recombination between the endogenous locus and the modified human nucleic acid sequence, thereby enabling the cell to contain the variable region of the human light chain immunoglobulin locus of the modified human nucleic acid sequence.

[0275] In some implementations, the method may include:

[0276] (a) Modify endogenous loci in non-human animal cells to generate endogenous loci lacking the variable region of endogenous heavy chain immunoglobulin loci through site-specific recombination.

[0277] (b) Introducing the modified human nucleic acid sequence into the animal cells described in step (a);

[0278] (c) Inducing site-specific recombination between the endogenous locus and the modified human nucleic acid sequence, thereby enabling the cell to contain the variable region of the human heavy chain immunoglobulin locus of the modified human nucleic acid sequence.

[0279] In some embodiments, the site-specific recombination technology may include Cre-LoxP technology, FLP / FRT technology, Cin H / RS2 technology, Par A / MRS technology, and phiC31 technology; in some embodiments, the site-specific recombination may be mediated by Cre recombinase and / or Flp recombinase.

[0280] In some implementations, constructs containing human nucleic acid sequences can be transfected into stem cells (ES cells or iPS cells) using known methods such as electroporation, calcium phosphate precipitation, and lipid transfection. The presence of the introduced nucleic acid can be assessed in the cells by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing) or by protein analysis (e.g., ELISA, Western blotting). Cells that have been determined to have incorporated the expression construct can then be introduced into preimplantation embryos. Such methods are commonly used for the targeted integration of transfected nucleic acid sequences into the genomes of stem cells and, consequently, non-human animals.

[0281] In some implementations, cells identified as having incorporated the expression construct can be introduced into blastocysts, and the injected blastocysts can be transferred into pseudopregnant mice to produce F0 mice. Chimeric mice are selected from littermates and, optionally, F1 heterozygous animals are generated by backcrossing the chimeric mice with wild-type mice, and F2 homozygous animals are generated by hybridizing the chimeric mice with the F1 animals. The presence of the introduced construct can be assessed in the offspring by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blotting, etc.).

[0282] In some embodiments, the genetically modified nonhuman animal can be created by introducing a human nucleic acid sequence into an oocyte (e.g., via microinjection) and allowing the oocyte to develop in a female gestating animal. In a preferred embodiment, a construct containing the human nucleic acid sequence is injected into a fertilized oocyte. Fertilized oocytes can be collected from superovulated females on the day after mating and the expression construct injected. The injected oocytes are cultured overnight or directly transferred into the oviduct of a 0.5-day-old pseudopregnant female. Methods for superovulation, oocyte harvesting, expression construct injection, and embryo transfer are known in the art and described in *Manipulating the Mouse Embryo* (2002, *A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press). The presence of the introduced nucleic acid in the offspring can be assessed by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blotting, etc.). Such methods typically result in the random integration of the injected nucleic acid sequence into the genome of the oocyte and thus the nonhuman animal.

[0283] In some embodiments, the method may include crossbreeding the genetically modified non-human animals described in this application with other genetically modified non-human animals described in this application to breed a new generation of animals carrying genetic modifications. In some embodiments, the method may include further breeding the genetically modified non-human animals described in this application with other genetically modified non-human animals to obtain a new generation of animals. In some embodiments, the method may include further breeding the genetically modified non-human animals described in this application with unmodified non-human animals to obtain a new generation of animals.

[0284] 3. Methods using genetically modified animals

[0285] The genetically modified non-human animals described in this application can be used for a variety of purposes, such as preparing antibodies that specifically bind to antigens, obtaining samples, preparing antibody-humanized mouse platforms, or preparing animal models.

[0286] Recombination of the V(D)J gene is a key mechanism for antibody diversity. The body first randomly selects V, D, and J fragments, introduces double-strand breaks near each fragment, deletes (or reverses) the intermediate DNA, and then joins these fragments together to form diverse gene regions encoding antibody variable regions—the V(D)J regions—thus creating an initial antibody library. When the body is stimulated by antigens, mature B cells undergo further diversification: somatic hypermutation (SHM) in the variable region and class switch recombination (CSR) in the constant region. SHM, through the introduction of point mutations, further diversifies the structure of the antibody variable region, allowing for the acquisition of antibodies with higher antigen affinity through in vivo selection mechanisms.

[0287] On one hand, this application provides a method for preparing antibodies that specifically bind to an antigen, the method comprising exposing the genetically modified animal to the antigen and generating antibodies in the animal using standard techniques for preparing polyclonal or monoclonal antibodies.

[0288] In some implementations, the method may employ hybridoma technology, including but not limited to human B-cell hybridoma technology, EBV-hybridoma technology, or tri-source hybridoma technology.

[0289] In some embodiments, the method may further include sequencing the genome of the hybridoma.

[0290] In some embodiments, the antigen may be at least one of the following: peptide, polypeptide, MHC / peptide complex, mRNA-LNP, DNA, live virus, dead virus or a portion thereof, live bacteria, dead bacteria or a portion thereof, or cancer cell or a portion thereof.

[0291] In some embodiments, the method may expose the animal to the antigen only once, while in other embodiments, the method may expose the animal to the antigen 2, 3, 4, 5, 6, 7, 8 or more times.

[0292] The antigen can be administered to the animal by any suitable means known in the art. In some embodiments, the antigen can be administered to the animal by at least one of the following methods: intrasplenic, intravenous, intraperitoneal, intradermal, intramuscular, and subcutaneous. In some embodiments, the antigen is administered alone, while in other embodiments, it can be administered in combination with a suitable immunomodulator or adjuvant. Examples of adjuvants that can be used in the methods of the present invention include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and alum (Al3(OH)4).

[0293] In this application, the method for separating antibodies can be any method known in the art. Antibodies can be separated from antibody-generating cells, the culture medium in which the antibody-generating cells are cultured, and / or from ascites fluid of an animal, according to methods known in the art. In some embodiments, one or more of the following can be used to separate antibodies: immunoaffinity purification, ammonium sulfate precipitation, protein A / G purification, ion exchange chromatography, and gel filtration.

[0294] On one hand, this application provides a method for obtaining a sample, the method including exposing the non-human animal to an antigen and collecting the sample from the non-human animal.

[0295] In some embodiments, the sample may be isolated from a peripheral lymphoid organ. For example, the peripheral lymphoid organ is a non-splenic organ. For example, any one of the following groups: lymph nodes, tonsils, and mucosa-associated lymphoid tissue (MALT), including gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue (BALT), nose-associated lymphoid tissue (NALT), larynx-associated lymphoid tissue (LALT), skin-associated lymphoid tissue (SALT), vascular-associated lymphoid tissue (VALT), and / or conjunctiva-associated lymphoid tissue (CALT). In some embodiments, the sample may be isolated from the spleen. In some embodiments, the sample may be isolated from bone marrow. In some embodiments, the sample may be immune cells, spleen cells, B cells, T cells, or hybridoma cells. Cell selection and isolation may be performed using flow cytometry or other cell isolation methods. However, those skilled in the art will recognize that production is not limited to these types of cells or methods.

[0296] On the other hand, this application also provides a method for obtaining nucleic acid encoding an antibody-binding domain that specifically binds to an antigen, the method comprising exposing a genetically modified non-human animal to the antigen, the animal being produced by the method; and sequencing nucleic acid encoding variable regions of human heavy and light chain immunoglobulins in cells, the cells expressing chimeric antibodies that specifically bind to the antigen.

[0297] In some embodiments, the nucleic acids encoding the variable regions of human heavy and light chain immunoglobulins can be determined by sequencing. In some embodiments, the nucleic acids encoding the variable regions of human heavy chain immunoglobulins can be operatively linked to the nucleic acids encoding the constant regions of human heavy chain immunoglobulins. In some embodiments, the nucleic acids encoding the variable regions of human light chain immunoglobulins can be operatively linked to the nucleic acids encoding the constant regions of human light chain immunoglobulins. In some embodiments, cells containing said nucleic acids can be cultured and antibodies collected.

[0298] The genetically modified non-human animals described in this application can also be used to construct libraries, such as in vitro display libraries.

[0299] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the non-human animals, preparation methods and uses of this application, and are not intended to limit the scope of the invention.

[0300] Example

[0301] Example 1: Humanization of the variable region of the mouse κ light chain immunoglobulin gene

[0302] The mouse κ light chain immunoglobulin gene is located on chromosome 6. A schematic diagram of the gene structure is shown below. Figure 1 As shown in the figure. The design involved replacing the V and J regions of the mouse κ light chain immunoglobulin gene with the human κ light chain immunoglobulin Igkv and Igkj genes to achieve humanization of the variable region of the mouse κ light chain immunoglobulin gene. The structure of the humanized κ light chain immunoglobulin variable region gene is shown in the figure. Figure 2 As shown.

[0303] The first step involved introducing recombinant target A at the upstream site of the functional V region (Chr6: 67551638) of the mouse κ light chain immunoglobulin Igk gene locus, and introducing recombinant target B at the downstream site of the J region (Chr6: 70724291) of the mouse κ light chain immunoglobulin Igk gene locus. The resulting modified mouse κ light chain immunoglobulin gene is illustrated in the diagram below. Figure 3 As shown. This targeting modification was performed on mouse embryonic stem cells (ES cells) derived from 129 / C57 hybrid blastocysts. The targeting strategy and primer design are as follows. Figure 4 As shown.

[0304] Recombinant vector A includes the upstream recombination site 5' homologous arm, PGK promoter, Cre recombinase mutant-specific recognition site (Lox2272 site), neomycin resistance gene (Neo) expression cassette, Cre recombinase mutant-specific recognition site (Lox2272 site), and upstream recombination site 3' homologous arm, arranged sequentially from 5' to 3'.

[0305] The recombinant vector B includes the following downstream recombination sites arranged sequentially from 5' to 3': 5' homologous arm, Cre recombinase mutant-specific recognition site (Lox2272 site), PGK promoter, Cre recombinase-specific recognition site (Loxp site), hygromycin resistance gene (Hygro) expression cassette, Flp recombinase-specific recognition site (Frt site), and 3' homologous arm.

[0306] Recombinant vectors A and B were introduced into mouse ES cells. Positive ES cell clones with homologous integration of both recombinant target A and recombinant target B were obtained by screening for neomycin resistance genes (Neo) and hygromycin resistance genes (Hygro) and confirmed by PCR. Primers are shown in Table 1, reaction systems in Table 2, and reaction conditions in Table 3.

[0307] Table 1. Primers

[0308]

[0309] Table 2. Reaction System

[0310]

[0311] Table 3. Reaction conditions

[0312]

[0313] Recombinant vector A recombinant-positive ES cell clones simultaneously amplified a 3901 bp band from the Igk Up F1 and Igk Up R1 primer pairs, and a 3811 bp band from the Igk Up F2 and Igk Up R2 primer pairs; recombinant vector B recombinant-positive ES cell clones simultaneously amplified a 3648 bp band from the Igk Down F1 and Igk Down R1 primer pairs, and a 3720 bp band from the Igk Down F2 and Igk Down R2 primer pairs. Results are as follows... Figure 5 As shown. Numbered 1A, 1B, 1C, 1E, 1F, 1H, 2B, 2C, 2E, 2F, 2H, 3A, 3B, 3C, 3D, and 3F are positive ES cell clones that have homologously integrated with both recombinant target A and recombinant target B.

[0314] The second step involves introducing the humanized IGK vector C into the aforementioned positive ES cell clones via Cre recombinase-mediated recombination. The targeting strategy and primer design are as follows: Figure 6 As shown, the V and J regions of the recombinant mouse κ light chain immunoglobulin Igk gene locus were replaced with a combination of the human IGKV region and the complete human IGKJ gene.

[0315] The humanized IGK vector C includes, from 5' to 3', the Cre recombinase mutant-specific recognition site (Lox2272 site), the neomycin resistance gene (Neo) expression cassette, the Cre recombinase mutant-specific recognition site (Lox5171 site), the human IGKV region assembly (containing human κ light chain immunoglobulin V region fragments as shown in Table 4), the complete human IGKJ genome fragment, the Flp recombinase-specific recognition site (Frt site), the PGK promoter, the puro resistance gene (Puro) expression cassette, the Cre recombinase-specific recognition site (Loxp site), the hygromycin resistance gene (Hygro) expression cassette, and the Flp recombinase-specific recognition site (Frt site).

[0316] Table 4. Human IGKV region assemblage in IGK humanized vector C

[0317]

[0318] Following Flp recombinase-mediated recombination, the puromycin resistance gene and the hygromycin resistance gene were removed. PCR identification confirmed the final humanized ES cell clone with the variable region of the κ light chain immunoglobulin. Primers are shown in Table 5, reaction systems in Table 2, and reaction conditions in Table 6.

[0319] Table 5. Primers

[0320]

[0321] Table 6. Reaction conditions

[0322]

[0323] Following Cre recombinase-mediated recombination, the hIGK LAsqF and hIGK LAsqR primer pairs amplified a 464 bp band, a 5' end recombination-positive band; the hIGK RAsqF and hIGK RAsqR primer pairs amplified a 772 bp band, a 3' end recombination-positive band (and the Flp recombinase did not remove the resistance gene), as shown in the results. Figure 7 As shown. Clones 3B and 3F are ES cell clones that are positive for recombination at both the 5' and 3' ends. After removing the puro and hygromycin resistance genes with Flp recombinase, the hIGKFlpF and hIGKFlpR primer pairs amplified a 708bp band, which is a positive band for humanized recombination of the variable region of the κ light chain immunoglobulin gene. The identification results are as follows. Figure 8 As shown; numbers 2F and 4G are positive ES cell clones of humanized recombination of the variable region of the κ light chain immunoglobulin gene.

[0324] Example 2: Obtaining humanized hIGK mice with κ light chain immunoglobulin

[0325] The humanized ES cell clones of the κ light chain immunoglobulin variable region obtained in Example 1 above were expanded and then microinjected into C57BL / 6 mouse blastocysts. After embryo transfer, three positive F0 generation mice were obtained, and the results are as follows. Figure 9 As shown, numbers 1#, 2#, and 4# are F0 generation positive mice.

[0326] Positive F0 generation mice were crossed with C57BL / 6 mice to obtain F1 generation mice. After PCR identification and sequencing confirmation, a total of 10 heterozygous humanized hIGK κ light chain immunoglobulin F1 generation mice were obtained, numbered as: 3#, 4#, 9#, 10#, 16#, 18#, 19#, 21#, 23#, and 24#. The results are as follows: Figure 10 As shown, this indicates that the humanized κ light chain immunoglobulin hIGK mouse was successfully constructed.

[0327] The identification method is as follows: primer pairs hIGK-F and hIGK-R can amplify a 669bp product band for the integrated humanized IGK gene, and primer pairs hIGK-WT-F and hIGK-R can amplify a 998bp product band for the mouse endogenous IgK gene. This allows for the differentiation between wild-type, humanized hIGK heterozygous, and homozygous mice. Primers are shown in Table 7, reaction systems in Table 8, and reaction conditions in Table 6.

[0328] Table 7. Primers

[0329]

[0330] Table 8. Reaction System

[0331]

[0332] Example 3: Humanization of the variable D and J regions of the mouse heavy chain immunoglobulin gene

[0333] The mouse heavy chain immunoglobulin gene is located on chromosome 12, as shown in the diagram. Figure 11 As shown. The design involved replacing the V, D, and J regions of the mouse heavy chain immunoglobulin Igh gene with human heavy chain immunoglobulin IGHD and IGHJ genes, as well as a partial fragment of the human heavy chain immunoglobulin IGHV region, to achieve humanization of the variable regions D and J of the mouse heavy chain immunoglobulin gene. The gene structures of the humanized heavy chain immunoglobulin variable regions D and J are shown below. Figure 12 As shown.

[0334] The first step involved introducing recombinant target D at the upstream site of the functional V region (Chr12: 116012244) of the mouse heavy chain immunoglobulin Igh gene locus, and introducing recombinant target E at the downstream site of the J region (Chr12: 113427277) of the mouse heavy chain immunoglobulin Igh gene. A schematic diagram of the modified mouse heavy chain immunoglobulin gene is shown below. Figure 13 As shown. This targeting modification was performed on mouse embryonic stem cells (ES cells) derived from 129 / C57 hybrid blastocysts. The targeting strategy and primer design are as follows. Figure 14 As shown.

[0335] The recombinant vector D includes the upstream recombination site 5' homologous arm, PGK promoter, Cre recombinase mutant-specific recognition site (Lox2272 site), neomycin resistance gene (Neo) expression cassette, Cre recombinase mutant-specific recognition site (Lox2272 site), and upstream recombination site 3' homologous arm, arranged sequentially from 5' to 3'.

[0336] The recombinant vector E includes the following downstream recombination sites arranged sequentially from 5' to 3': 5' homologous arm, Cre recombinase mutant-specific recognition site (Lox2272 site), PGK promoter, Cre recombinase-specific recognition site (Loxp site), hygromycin resistance gene (Hygro) expression cassette, Flp recombinase-specific recognition site (Frt site), and 3' homologous arm.

[0337] Recombinant vectors D and E were introduced into mouse ES cells. Positive ES cell clones with homologous integration of both recombinant target D and recombinant target E were obtained by screening for neomycin resistance genes (Neo) and hygromycin resistance genes (Hygro) and confirmed by PCR. Primers are shown in Table 9, reaction systems are shown in Table 2, and reaction conditions are shown in Table 3.

[0338] Table 9. Primers

[0339]

[0340] Recombinant vector D recombinant-positive ES cell clones simultaneously amplified a 3635 bp band from the Igh Up F1 and Igh Up R1 primer pairs, and a 3729 bp band from the Igh Up F2 and Igh Up R2 primer pairs; recombinant vector E recombinant-positive ES cell clones simultaneously amplified a 5903 bp band from the Igh Down F1 and Igh Down R1 primer pairs, and a 1831 bp band from the Igh Down F2 and Igh Down R2 primer pairs. Results are as follows... Figure 15As shown. Numbered 1A, 1B, 1C, 1D, 1F, 1G, 2A, 2B, 2C, 2D, 2E, 2H, 3B, and 3G are positive ES cell clones that have homologously integrated with both recombinant target D and recombinant target E.

[0341] The second step involves introducing the IGH humanized vector F into the aforementioned positive ES cell clones via Cre recombinase-mediated recombination. The targeting strategy and primer design are as follows: Figure 16 As shown, the variable regions V, D, and J of the recombinant mouse heavy chain immunoglobulin Igh gene locus were replaced with the complete human IGHD and IGHJ genes, as well as a partial fragment of the human IGHV region.

[0342] The IGH humanized vector F includes, from 5' to 3', the Cre recombinase mutant-specific recognition site (Lox2272 site), the neomycin resistance gene (Neo) expression cassette, the Cre recombinase mutant-specific recognition site (Lox5171 site), the human IGHV6-1 (NCBI Gene ID: 28385) fragment, the complete human IGHD genome fragment, the complete human IGHJ genome fragment, the Flp recombinase-specific recognition site (Frt site), the PGK promoter, the puro resistance gene (Puro) expression cassette, and the Cre recombinase-specific recognition site (Loxp site).

[0343] Following Flp recombinase-mediated recombination, the puromycin resistance gene and the hygromycin resistance gene were removed. PCR identification confirmed the final humanized ES cell clones containing the D and J regions of the heavy chain immunoglobulin gene locus. Primers are shown in Table 10, reaction systems in Table 2, and reaction conditions in Table 6.

[0344] Table 10. Primers

[0345]

[0346] Following Cre recombinase-mediated recombination, the hIGH LAsqF and hIGH LAsqR primer pairs amplified a 464 bp band, a 5' end recombination-positive band; the hIGH RAsqF and hIGH RAsqR primer pairs amplified a 772 bp band, a 3' end recombination-positive band (and the Flp recombinase did not remove the resistance gene), as shown in the results. Figure 17 As shown. Clones 1C and 3B are ES cell clones positive for recombination at both the 5' and 3' ends. After removing the puro and hygromycin resistance genes with Flp recombinase, the hIGHFlpF and hIGH FlpR primer pairs amplified a 940bp band, which is a positive band for humanized recombination of the D and J regions of the heavy chain immunoglobulin gene. The identification results are as follows. Figure 18As shown; numbers 1B, 1E, 2A, 2B, 2H, and 3C are positive ES cell clones for humanized recombination of regions D and J of the heavy chain immunoglobulin gene.

[0347] Example 4: Humanization of the V region of the mouse heavy chain immunoglobulin gene locus

[0348] The design involved inserting three different human heavy chain immunoglobulin IGH gene V region fragments into the upstream regions of the D and J regions of the recombinant humanized heavy chain immunoglobulin gene locus obtained in Example 3 above, thereby achieving the humanization of the mouse heavy chain immunoglobulin gene locus V region and ultimately obtaining a mouse ES cell clone with humanized VDJ region of the heavy chain immunoglobulin IGH gene.

[0349] 4.1 Obtaining humanized hIGH1 mouse ES cells from the Set1 combination of human heavy chain immunoglobulin IGHV region.

[0350] The design involved inserting the human heavy chain immunoglobulin IGHV region combination Set1 fragment upstream of regions D and J of the recombinant humanized heavy chain immunoglobulin gene locus obtained in Example 3 above. The structure of the humanized IGH-Set1 gene after integrating the human heavy chain immunoglobulin IGHV region combination Set1 is shown below. Figure 19 As shown.

[0351] Recombinase-mediated recombination was used to introduce the IGH humanized vector G into the humanized ES cell clones of the heavy chain immunoglobulin gene locus D and J regions obtained in Example 3 above. Targeting strategies and primer design are as follows: Figure 20 As shown.

[0352] The humanized IGH vector G includes, from 5' to 3', the Cre recombinase mutant-specific recognition site (Lox2272), the puromycin resistance gene expression cassette, the inverse hygromycin resistance gene expression cassette, the inverse PGK promoter, the human IGHV region combination Set1 fragment (containing human V region fragments as shown in Table 11), and the Cre recombinase mutant-specific recognition site (Lox5171).

[0353] Table 11. Human IGHV region composition in IGH humanized vector G

[0354]

[0355] Recombinant positive ES cell clones were obtained through screening for resistance to the puromycin resistance gene and the hygromycin resistance gene. PCR identification and sequencing confirmed the correct recombinant humanized IGH-Set1 positive ES cell clones. The hIGH 2nd LAsqF and hIGH 2nd LAsqR primer pairs amplified a 762bp band, indicating a 5' end recombination positive band; the hIGH 2nd RAsqF2 and hIGH 2nd RAsqR1 primer pairs amplified a 1232bp band, indicating a 3' end recombination positive band. Results are as follows... Figure 21 As shown, clones numbered 1A, 3B, and 3D are humanized heavy chain immunoglobulin IGH-Set1 positive ES cells. Primers are shown in Table 12, reaction systems are shown in Table 2, and reaction conditions are shown in Table 13.

[0356] Table 12. Primers

[0357]

[0358] Table 13. Reaction Conditions

[0359]

[0360] 4.2 Obtaining humanized hIGH2 mouse ES cells from the human heavy chain immunoglobulin IGHV region combinatorial Set2

[0361] The design involved inserting the human heavy chain immunoglobulin IGHV region combination Set2 fragment upstream of regions D and J of the recombinant humanized heavy chain immunoglobulin gene locus obtained in Example 3 above. The structure of the humanized IGH-Set2 gene after integrating the human heavy chain immunoglobulin IGHV region combination Set2 is shown below. Figure 22 As shown.

[0362] Recombinase-mediated recombination was used to introduce the IGH humanized vector H into the humanized ES cell clones of the heavy chain immunoglobulin gene locus D and J regions obtained in Example 3 above. Targeting strategies and primer design are as follows: Figure 23 As shown.

[0363] The humanized IGH vector H includes, from 5' to 3', the Cre recombinase mutant-specific recognition site (Lox2272), the puromycin resistance gene expression cassette, the reverse hygromycin resistance gene expression cassette, the reverse PGK promoter, the human IGHV region combination Set2 fragment (containing human V region fragments as shown in Table 14), and the Cre recombinase mutant-specific recognition site (Lox5171).

[0364] Table 14. Human IGHV region assemblage in IGH humanized vector H

[0365]

[0366] Recombinant positive ES cell clones were obtained through screening for resistance to the puromycin resistance gene and the hygromycin resistance gene. PCR identification and sequencing confirmed the correct recombinant humanized IGH-Set2 positive ES cell clones. The hIGH 2nd LAsqF and hIGH 2nd LAsqR primer pairs amplified a 762bp band, indicating a 5' end recombination positive band; the hIGH 2nd RAsqF1 and hIGH 2nd RAsqR1 primer pairs amplified a 717bp band, indicating a 3' end recombination positive band. Results are as follows... Figure 24 As shown, clones numbered 1B, 2C, and 3F are humanized heavy chain immunoglobulin IGH-Set2 positive ES cells. Primers are shown in Table 15, reaction systems are shown in Table 2, and reaction conditions are shown in Table 13.

[0367] Table 15. Primers

[0368]

[0369] 4.3 Obtaining humanized hIGH3 mouse ES cells from the human heavy chain immunoglobulin IGHV region combination Set3

[0370] The design involved inserting the human heavy chain immunoglobulin IGHV region combination Set3 fragment upstream of regions D and J of the recombinant humanized heavy chain immunoglobulin gene locus obtained in Example 3 above. The structure of the humanized IGH-Set3 gene after integrating the human heavy chain immunoglobulin IGHV region combination Set3 is shown below. Figure 25 As shown.

[0371] Recombinase-mediated recombination was used to introduce the IGH humanized vector I into the humanized ES cell clones of the heavy chain immunoglobulin gene locus D and J regions obtained in Example 3 above. Targeting strategies and primer design are as follows: Figure 26 As shown.

[0372] IGH humanized vector I includes, from 5' to 3', the Cre recombinase mutant-specific recognition site (Lox2272 site), the puro resistance gene expression cassette, the reverse hygromycin resistance gene expression cassette, the reverse PGK promoter, the human IGHV region combination Set3 fragment (containing human V region fragments as shown in Table 16), and the Cre recombinase mutant-specific recognition site (Lox5171 site).

[0373] Table 16. Human IGHV region composition in IGH humanized vector I

[0374]

[0375] Recombinant positive ES cell clones were obtained through screening for resistance to the puromycin resistance gene and the hygromycin resistance gene. PCR identification and sequencing confirmed the correct recombinant humanized IGH-Set3 positive ES cell clones. The hIGH 2nd LAsqF and hIGH 2nd LAsqR primer pairs amplified a 762bp band, indicating a 5' end recombination positive band; the hIGH 2nd RAsqF1 and hIGH 2nd RAsqR1 primer pairs amplified a 717bp band, indicating a 3' end recombination positive band. Results are as follows... Figure 27 As shown, clones 2A and 2G are humanized heavy chain immunoglobulin IGH-Set3 positive ES cells. Primers are shown in Table 15, reaction systems are shown in Table 2, and reaction conditions are shown in Table 13.

[0376] Example 5: Obtaining fully humanized antibody hIGK / hIGH mice

[0377] 5.1 Obtaining hIGH1 mice expressing humanized heavy chain immunoglobulin IGH-Set1

[0378] The humanized IGH-Set1 positive ES cell clones obtained in Example 4.1 above were expanded and then microinjected into C57BL / 6 mouse blastocysts. After embryo transfer, four positive F0 generation mice were obtained, numbered 2#, 4#, 5#, and 6#. The results are as follows: Figure 28 As shown in the figure. Positive F0 generation mice were crossed with C57BL / 6 mice to obtain F1 generation mice. Tail DNA was extracted and confirmed by PCR and sequencing. A total of eight humanized heavy chain immunoglobulin IGH-Set1 positive F1 generation hIGH1 mice were obtained, numbered 4#, 5#, 7#, 8#, 9#, 11#, 14#, and 15#. The results are as follows. Figure 29 As shown, this indicates that the humanized heavy chain immunoglobulin hIGH1 mouse model was successfully constructed.

[0379] The identification method is as follows: a 717 bp PCR amplification band was obtained in hIGH1 mice that successfully integrated the humanized reassorted immunoglobulin IGH-Set1 gene. Primers are shown in Table 17, reaction system is shown in Table 2, and reaction conditions are shown in Table 13.

[0380] Table 17. Primers

[0381]

[0382] 5.2 Obtaining fully humanized antibody hIGK / hIGH1 mice

[0383] By mating the humanized κ light chain immunoglobulin hIGK mice obtained in Example 2 with the humanized heavy chain immunoglobulin hIGH1 mice obtained in Example 5.1, fully humanized antibody hIGK / hIGH1 mice with both κ light chain and heavy chain immunoglobulins were obtained. Tail DNA was extracted and confirmed by PCR and sequencing. The hIGK mouse identification method was the same as described in Example 2. The hIGH1 mouse identification method is as follows: primer pairs P-IGH1-1 and P-IGH1-2 amplified a 2131bp product band targeting the mouse endogenous Igh gene, and primer pairs P-IGH1-3 and P-IGH1-2 amplified a 554bp product band targeting the integrated humanized heavy chain IGH-Set1 gene. Primers are shown in Table 18, reaction systems are shown in Table 19, and reaction conditions are shown in Table 20.

[0384] Table 18. Primers

[0385]

[0386] Table 19. Reaction System

[0387]

[0388] Table 20. Reaction Conditions

[0389]

[0390] The results are as follows Figure 30 As shown, mice numbered 1#, 4#, 5#, 7#, 8#, 11#, 12#, and 14# are homozygous for humanized κ light chain hIGK and heterozygous for humanized heavy chain hIGH1; mice numbered 2#, 3#, 6#, 9#, 13#, 15#, and 16# are homozygous for humanized κ light chain hIGK and homozygous for humanized heavy chain hIGH1. This indicates that fully humanized antibody-homozed hIGK / hIGH1 mice were successfully obtained.

[0391] 5.3 Obtaining hIGH2 mice expressing humanized heavy chain immunoglobulin IGH-Set2

[0392] The humanized IGH-Set2 positive ES cell clones obtained in Example 4.1 above were expanded and then microinjected into C57BL / 6 mouse blastocysts. After embryo transfer, five positive F0 generation mice were obtained, numbered 1#, 2#, 4#, 5#, and 6#. The results are as follows: Figure 31As shown in the figure. Positive F0 generation mice were crossed with C57BL / 6 mice to obtain F1 generation mice. Tail DNA was extracted and confirmed by PCR and sequencing. A total of nine humanized heavy chain immunoglobulin IGH-Set2 positive F1 generation mice were obtained, numbered 1#, 3#, 4#, 6#, 8#, 12#, 14#, 15#, and 16#. The results are as follows. Figure 32 As shown, this indicates that the humanized heavy chain immunoglobulin hIGH2 mouse model was successfully constructed.

[0393] The identification method is as follows: a 1232 bp PCR amplification band can be obtained in hIGH2 mice that have successfully integrated the humanized reassorted immunoglobulin IGH-Set2 gene. Primers are shown in Table 21, reaction system is shown in Table 2, and reaction conditions are shown in Table 13.

[0394] Table 21. Primers

[0395]

[0396] 5.4 Obtaining fully humanized antibody hIGK / hIGH2 mice

[0397] By mating the humanized κ light chain immunoglobulin hIGK mice obtained in Example 2 with the hIGH2 mice expressing humanized heavy chain immunoglobulin IGH-Set2 obtained in Example 5.3, fully humanized antibody hIGK / hIGH2 mice with both κ light chain and heavy chain immunoglobulins were obtained. Tail DNA was extracted and confirmed by PCR and sequencing. The identification method for the humanized κ light chain immunoglobulin IGK gene was the same as described in Example 2. The identification method for the humanized heavy chain immunoglobulin hIGH2 mice was as follows: primer pairs P-WT-1 and P-WT-2 amplified a 2131bp product band targeting the mouse endogenous Igh gene; primer pairs P-IGH2-1 and P-IGH2-2 amplified a 213bp product band targeting the integrated humanized heavy chain IGH-Set2 gene; primer pairs P-IPC-1 and P-IPC-2 served as positive control primers for the internal reference gene, producing a 521bp product band. The primers are shown in Table 22, the reaction system 1 and reaction conditions 1 are shown in Table 23 and Table 20, and the reaction system 2 and reaction conditions 2 are shown in Table 24 and Table 25.

[0398] Table 22. Primers

[0399]

[0400] Table 23. Reaction System 1

[0401]

[0402] Table 24. Reaction System 2

[0403]

[0404] Table 25. Reaction Condition 2

[0405]

[0406] The results are as follows Figure 33 As shown, mice numbered D102#, D104#, D105#, D107#, D108#, D112#, D114#, D115#, D116#, D117#, and D118# are homozygous for both the humanized κ light chain hIGK and the humanized heavy chain hIGH2. This indicates that fully humanized antibody-homozed hIGK / hIGH2 mice were successfully obtained.

[0407] 5.5 Obtaining hIGH3 mice expressing humanized heavy chain immunoglobulin IGH-Set3

[0408] The humanized IGH-Set3 positive ES cell clones obtained in Example 4.1 above were expanded and then microinjected into C57BL / 6 mouse blastocysts. After embryo transfer, seven positive F0 generation mice were obtained, numbered 1#, 2#, 3#, 5#, 7#, 8#, and 10#. The results are as follows: Figure 34 As shown in the figure. Positive F0 generation mice were crossed with C57BL / 6 mice to obtain F1 generation mice. Tail DNA was extracted and identified by PCR and sequencing. A total of 11 humanized heavy chain immunoglobulin IGH-Set3 positive F1 generation mice were obtained, numbered as: 2#, 3#, 4#, 6#, 7#, 9#, 12#, 16#, 19#, 22#, and 23#. The results are as follows. Figure 35 As shown, this indicates that the humanized heavy chain immunoglobulin hIGH3 mouse model was successfully constructed.

[0409] The identification method is as follows: a 717 bp PCR amplification band can be obtained in mice that have successfully integrated the humanized reassorted immunoglobulin IGH-Set3 gene. Primers are shown in Table 17, reaction system is shown in Table 2, and reaction conditions are shown in Table 13.

[0410] 5.6 Obtaining fully humanized antibody hIGK / hIGH3 mice

[0411] By mating the humanized κ light chain immunoglobulin hIGK mice obtained in Example 2 with the humanized heavy chain immunoglobulin IGH-Set3 hIGH3 mice obtained in Example 5.5, fully humanized antibody hIGK / hIGH3 mice with both κ light chain and heavy chain immunoglobulins were obtained. Tail DNA was extracted and confirmed by PCR and sequencing. The hIGK mouse identification method was the same as described in Example 2, and the hIGH3 mouse identification method was the same as described in Example 5.4. Primer pairs P-WT-1 and P-WT-2 amplified a 2131bp product band targeting the mouse endogenous Igh gene, primer pairs P-IGH2-1 and P-IGH2-2 amplified a 613bp product band targeting the integrated humanized heavy chain IGH-Set3 gene, and primer pairs P-IPC-1 and P-IPC-2 served as positive control primers for the internal reference gene, producing a 521bp product band.

[0412] The results are as follows Figure 36 As shown, mice numbered E82# and E84# are homozygous for both the humanized κ light chain hIGK and the humanized heavy chain hIGH3. This indicates that fully humanized antibody-homozed hIGK / hIGH3 mice were successfully obtained.

[0413] 5.7 Obtaining fully humanized antibody hIGK / hIGH13 mice

[0414] The humanized hIGK / hIGH1 and hIGK / hIGH3 mice obtained in Examples 5.2 and 5.6 above were mated to obtain fully humanized antibody hIGK / hIGH13 mice. Tail DNA was extracted from the mice and identified by PCR and sequencing. Genotyping electrophoresis results are as follows: Figure 37 As shown. The fully humanized antibody hIGK / hIGH13 mice were numbered as follows: A1#, A3#, A6#, A7#, A11#, A14#, A15#, A17#, A19#, A20#, A24#.

[0415] 5.8 Obtaining fully humanized antibody hIGK / hIGH12 mice

[0416] The humanized hIGK / hIGH1 and hIGK / hIGH2 mice obtained in Examples 5.2 and 5.4 above were mated to obtain fully humanized antibody hIGK / hIGH12 mice. Tail DNA was extracted from the mice and identified by PCR and sequencing. The genotype identification electrophoresis results are as follows: Figure 38 As shown. The fully humanized antibody hIGK / hIGH12 mice were numbered as follows: B194#, B196#, B197#, B199#, B200#, B205#, B209#, B211#, and B214#.

[0417] Example 6: B cell development in fully humanized antibody mice

[0418] Spleen tissues were collected from 8-10 week old wild-type (WT) and fully humanized antibody-homogeneous hIGK / hIGH13 mice, and single-cell suspensions were extracted for flow cytometry analysis of lymphocyte subsets and B cell subsets in the spleen. Results are as follows: Figures 39-41 As shown. Figure 39 The proportions of B cells, NK cells, and T cells in the spleen of wild-type mice and fully humanized antibody mice were similar. Figure 40 The proportions of memory B cells and plasma B cell subsets in the spleen were similar in wild-type mice and fully humanized antibody mice. Figure 41 The development of B cells in wild-type mice and fully humanized antibody mice is similar.

[0419] The results showed that, compared with wild-type mice, fully humanized antibody mice had similar proportions of immune cells and B cell subsets and B cell development processes.

[0420] Example 7: Immunity and Antibody Production in Fully Humanized Antibody Mice

[0421] Six-week-old female fully humanized antibody-modified hIGK / hIGH13 mice and wild-type (WT) mice were used as immunization subjects. Recombinantly expressed hTfR1-His protein from eukaryotic cells was used as the antigen. The first immunization was performed using an emulsion of the antigen protein with Freund's complete adjuvant. Fourteen days later, the second, third, and fourth immunizations were performed using an incomplete Freund's adjuvant and antigen emulsion, with each immunization spaced 14 days apart. Blood samples were collected on day 7 after the third and fourth immunizations to verify the immunization efficacy.

[0422] The results are as follows Figures 42-43 As shown in the figure. The results indicate that the fully humanized antibody mice have normal immune function, can produce antibodies, and have an immune response similar to that of wild-type mice.

[0423] Example 8 Antibody titer in fully humanized antibody mice

[0424] Wild-type C57BL / 6 (WT) mice and fully humanized antibody-modified hIGK / hIGH13 mice were immunized with IL-11, TSLP, and IGF1R antigens, respectively. Immunogens could be recombinant proteins or mRNA-LNPs. Serum was collected after multiple immunizations, and the antigen-specific antibody titers in wild-type and fully humanized antibody-modified mice were detected by ELISA. The results are as follows: Figures 44-46 As shown in the figure. The results indicate that fully humanized antibody mice can produce antibodies that specifically bind to the antigen.

[0425] Example 9 Antibody binding in fully humanized antibody mice

[0426] Mice immunized with the fully humanized antibody hIGK / hIGH13 were immunized with PD-L1, TSLP, TL-1A, and IGF1R antigens, respectively. Spleen cells were then collected and enriched with plasma B cells. Microfluidic cell sorting was used to separate plasma B cells capable of binding to the antigens. The sorted B cells were subjected to 10×genomics high-throughput BCR sequencing to obtain a large number of antibody sequences. Selected sequences were recombinantly expressed, and the antibody binding positivity rate was verified by ELISA. Specific antibodies produced by the fully humanized antibody hIGK / hIGH13 mice against specific antigens were verified for antibody binding ability by ELISA and / or SPR. Results are as follows: Figures 47-54 As shown in Table 26, the results indicate that the antigen-specific antibodies produced by fully humanized antibody mice have good antigen-binding ability.

[0427] Table 26. Affinity test results of Anti-PD-L1 recombinant antibody and PD-L1 protein

[0428]

[0429] Example 10: In vitro functional validation of anti-PD-L1 antibody

[0430] After the anti-PD-L1 antibody binding was verified by ELISA in Example 9, 12 positive binding antibodies were screened out, and the antibody function was further verified by endocytosis and blocking experiments.

[0431] 10.1 Internalization Experiment

[0432] The endocytosis of anti-PD-L1 antibody in the MC38-hPD-L1 cell line was detected using FACS. The anti-PD-L1 antibody was labeled using a pH-sensitive IgG labeling kit; the labeled antibody showed green fluorescence under acidic conditions. After culturing the labeled antibody with MC38-hPD-L1 cells in a 37°C, 5% CO2 incubator for 20 hours, the cells were collected for flow cytometry analysis to detect the antibody endocytosis effect using either the FITC or AF488 channel. Results are as follows: Figure 55 The results showed that eight antibodies exhibited endocytosis effects similar to those of the positive control antibody.

[0433] 10.2 Blocking Experiment

[0434] The blocking effect of anti-PD-L1 antibody on the binding of MC38-hPD-L1 cells to recombinant hPD-1 protein was detected using FACS. MC38-hPD-L1 cells were incubated with different gradient concentrations of anti-PD-L1 antibody at 4°C for 30 min. After incubation, the cells were washed three times with pre-chilled PBS, and biotin-labeled hPD-L1 recombinant protein was added. The cells were incubated at room temperature for 30 min. After incubation, streptavidin-APC was added and the cells were incubated at 4°C for 30 min. After washing three times with pre-chilled PBS, the cells were resuspended in 200 μL of PBS and analyzed. The results are as follows: Figure 56 The results showed that five antibodies exhibited similar blocking effects to the positive control antibody, with the highest blocking rate reaching 53.7%.

[0435] Example 11 Antibody diversity in fully humanized antibody mice

[0436] 11.1 Frequency of use of the V(D)J gene combination in fully humanized antibody mice

[0437] By isolating single B cells from the spleen of unimmunized fully humanized antibody mice and performing high-throughput sequencing, the V, D, and J genes of the BCR heavy and light chains of each cell were precisely annotated using a bioinformatics workflow. The frequency of use of the V, D (heavy chain), and J gene family combinations in unimmunized fully humanized antibody mice was calculated by counting and statistically analyzing all paired sequences.

[0438] Results of variable region gene combination usage frequency in fully humanized antibody hIGK / hIGH1 mice are as follows: Figure 57 As shown, the frequency of use of the variable region gene combination in fully humanized antibody hIGK / hIGH12 mice is as follows: Figure 58 As shown, the frequency of use of the variable region gene combination in fully humanized antibody hIGK / hIGH13 mice is as follows: Figure 59 The diagram shows the usage of the V, D (heavy chain), and J gene families in the heavy chain (top) and κ light chain (bottom) of non-immunized fully humanized antibody mice. The results indicate that the variable region genes in the B cell immune repertoire of fully humanized antibody mice cover the introduced human V, D, and J gene fragments, exhibiting combinatorial diversity.

[0439] 11.2 Distribution of CDR3 length in the mouse heavy chain of fully humanized antibody

[0440] Based on the high-throughput single-cell V(D)J sequencing data, CDR3 length analysis was performed using a bioinformatics workflow. The CDR3 amino acid sequence of each heavy chain variable region was accurately extracted, the number of heavy chain CDR3 amino acid residues was counted, and the frequency of each length value in all valid sequences was calculated.

[0441] The amino acid length distribution of the CDR3 region of the fully humanized antibody hIGK / hIGH1 mouse heavy chain is as follows: Figure 60 As shown, the amino acid length distribution of the CDR3 region of the fully humanized antibody hIGK / hIGH12 mouse heavy chain is as follows: Figure 61 As shown, the amino acid length distribution of the CDR3 region of the fully humanized antibody hIGK / hIGH13 mouse heavy chain is as follows: Figure 62 As shown in the figure. The results show that the amino acid length distribution of the CDR3 region of the fully humanized mouse antibody is highly similar to the known characteristics of the natural human antibody library.

[0442] 11.3 CDR3 amino acid polymorphism in fully humanized antibody mice

[0443] Based on the sequencing data obtained above, sequence alignment and statistical analysis were performed on the CDR3 regions of the heavy and light chains to analyze the compositional variations and polymorphism distribution at different amino acid positions. The amino acid polymorphisms of the CDR3 region of the fully humanized hIGK / hIGH1 mouse antibody are shown below. Figures 63-64 As shown, the CDR3 amino acid polymorphism in fully humanized antibody hIGK / hIGH12 mice is as follows: Figures 65-66 As shown in Figures 67-68, the CDR3 amino acid polymorphism in fully humanized antibody hIGK / hIGH13 mice is illustrated. The results indicate that there are multiple patterns of amino acid mutations in the CDR3 region of fully humanized antibody mice.

[0444] 11.4 Frequency of gene combination and CDR3 amino acid usage in the variable region of the mouse antigen-specific B cell receptor for fully humanized antibodies

[0445] Single cells were isolated from the spleens of fully humanized antibody-treated mice immunized with the target antigen. Using the 10xGenomics Chromium single-cell 5' platform and a human B cell V(D)J enrichment kit, an antibody variable region sequencing library containing individual B cell-specific barcodes was constructed. High-throughput sequencing was then performed, and the V, D, and J genes of the antibody heavy and light chains for each cell were precisely annotated using bioinformatics. Genes in all productive paired sequences were counted and statistically analyzed to determine their specific usage frequency in the post-immunization antibody library. Sequence alignment and statistical analysis were performed on the CDR3 regions of the heavy and light chains to analyze the compositional changes and polymorphic distribution at different amino acid positions.

[0446] The results of the frequency of use of the variable region gene combination in fully humanized antibody hIGK / hIGH12 mice after immunization are as follows: Figure 69 As shown, the frequency of gene usage in the variable region of the fully humanized antibody hIGK / hIGH13 mouse is as follows: Figure 70The figure shows the usage of the V, D (heavy chain), and J gene families of antibody heavy chains (top) and κ light chains (bottom) obtained from B cells of humanized immunoglobulin-derived mice after immunization. The results indicate that, among the thousands of productive antibody pairing sequences obtained, the use of antibody variable region genes covers the introduced human V, D, and J gene fragments.

[0447] Following immunization, the frequency of CDR3 amino acid usage in the CDR3 region of fully humanized antibody hIGK / hIGH12 mice was as follows: Figures 71-72 As shown, the frequency of CDR3 amino acid usage in the CDR3 region of the fully humanized antibody hIGK / hIGH13 mouse is as follows: Figures 73-74 As shown in the figure. The results indicate that there are multiple patterns of amino acid mutations in the CDR3 amino acid mutation of fully humanized antibody-producing mice after immunization.

[0448] The above results indicate that fully humanized antibody mice possess a diverse antibody library, which can be used for antibody discovery, antibody drug or vaccine evaluation.

Claims

1. A method for preparing non-human animals, wherein, The genome of the animal contains a modified light chain immunoglobulin locus, which contains multiple human IGKV genes and one or more human IGKJ genes, wherein the human IGKV genes consist of IGKV6D-41, IGKV2D-29, IGKV1D-37, IGKV2D-40, IGKV1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-27, IGKV2-24, IGKV6-21, IGKV3-20, IGKV1-17, IGKV1-16, IGKV3-15, IGKV1-12, IGKV3-11, IGKV1-9, IGKV1-8, IGKV1-6, IGKV1-5, IGKV5-2, and IGKV4-1.

2. The method according to claim 1, wherein, The animal's genome also includes a modified heavy chain immunoglobulin locus, which further includes multiple human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes are selected from the group consisting of: (1) The human IGHV gene described therein is composed of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1; (2) The human IGHV gene therein is composed of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1; (3) The human IGHV gene therein is composed of IGHV4-59, IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30 and IGHV6-1; (4) The human IGHV gene therein is composed of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1; (5) The human IGHV gene therein is composed of IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2, and IGHV6-1; and (6) The human IGHV gene is composed of IGHV3-53, IGHV5-51, IGHV3-48, IGHV1-46, IGHV4-39, IGHV3-35, IGHV4-34, IGHV4-30-2, IGHV3-33, IGHV3-30, IGHV4-28, IGHV3-23, IGHV3-21, IGHV1-18, IGHV3-15, IGHV3-13, IGHV3-11, IGHV3-9, IGHV1-8, IGHV3-7, IGHV2-5, IGHV4-4, IGHV1-2 and IGHV6-1.

3. The method according to claim 2, wherein the animal comprises all human IGHD genes and all human IGHJ genes at the human chromosome 14 heavy chain immunoglobulin gene locus.

4. The method of claim 3, wherein the animal comprises all human IGKJ genes at the human chromosome 2 light chain immunoglobulin locus.

5. The method of claim 4, wherein the animal comprises disruption of its endogenous heavy chain immunoglobulin loci, the disruption comprising deletion of one or more endogenous Ighv genes, one or more endogenous Ighd genes, and one or more endogenous Ighj genes.

6. The method of claim 5, wherein the animal comprises disruption of its endogenous light chain immunoglobulin gene loci, the disruption comprising deletion of one or more endogenous Igkv genes and one or more endogenous Igkj genes.

7. The method of claim 6, wherein the animal comprises endogenous Ighm, Ighδ, Ighg, Ighe and / or Igha genes, wherein the human IGHV, human IGHD and human IGHJ genes are operatively linked to one or more genes selected from the endogenous Ighm, Ighδ, Ighg, Ighe and Igha genes.

8. The method of claim 7, wherein the animal comprises an endogenous Igkc gene, wherein the human IGKV and human IGKJ genes are operatively linked to the endogenous Igkc gene.

9. The method of claim 8, wherein the animal is a rodent.

10. The method of claim 9, further comprising introducing a modified human nucleic acid sequence into cells of the animal, such that the cells contain the modified human nucleic acid sequence.

11. A non-human animal cell derived from the cells or tissues of a genetically modified non-human animal, said animal being produced by the method of any one of claims 1-10, wherein said cell is immortalized.

12. A method for generating non-human animal ES cells, the method comprising inserting the genome of a genetically modified non-human animal into the genome of a non-human animal ES cell, wherein, The animal is produced by the method according to any one of claims 1-10.

13. A method for preparing an antibody that specifically binds to an antigen, the method comprising: (a) Exposing a genetically modified nonhuman animal to the antigen, said animal being produced by the method of any one of claims 1-10; (b) Collect antibodies targeting the antigen or cells that produce the antibody.

14. A method for obtaining a sample, the method comprising: (a) Exposing a genetically modified nonhuman animal to an antigen, said animal being produced by the method of any one of claims 1-10; (b) Collect the sample from the non-human animal, wherein the sample includes immune cells.

15. Use of non-human animals or their offspring produced by the method according to any one of claims 1-10 in the preparation of antibody humanization animal platforms or animal models.

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