Genetically modified mice for producing antibodies and methods for producing the same

Abstract

Disclosed is a genetically modified mouse in which the immunoglobulin heavy chain locus has been modified to insert a gene segment of a human immunoglobulin heavy chain variable region. The mouse can breed normally and produce human-mouse chimeric antibodies, which contain a human heavy chain variable region and a mouse constant region. Methods for producing the genetically modified mouse and uses of the mouse are also provided.

Description

[Technical Field]

[0001] The present invention relates to genetically modified mice, cells, embryos, and tissues. Specifically, the present invention relates to a mouse whose immunoglobulin heavy chain variable region genome has been humanized and a method for producing the mouse. The present invention also relates to the genome of the modified mouse, cells and tissues containing the genome, and a method for producing a monoclonal antibody using the mouse and its use. The present invention also relates to a mouse having a modified genome. [Background technology]

[0002] Humanized mice possess a fully functional humoral immune system that is essentially indistinguishable from that of wild-type mice. These mice exhibit normal cell populations at all stages of B cell development and normal lymphoid organ morphology. Their antibody sequences exhibit normal V(D)J rearrangements and normal somatic hypermutation frequencies. Their antibody populations reflect the isotype distribution resulting from normal holotype switching (e.g., normal isotype switching). Immunization of these mice results in a robust humoral immune response and the generation of a large and diverse antibody repertoire with human immunoglobulin variable regions suitable for use as therapeutic candidates.

[0003] Genetically modified or transgenic mice can be created by precisely replacing mouse immunoglobulin variable sequences with human immunoglobulin variable sequences. However, due to the divergent evolution of immunoglobulin loci between mice and humans, even when very large segments of human immunoglobulin sequences are sequentially recombined to precisely replace endogenous mouse immunoglobulin sequences at heavy and light chain loci with corresponding human immunoglobulin sequences, certain problems can arise. For example, genetic spacer sequences scattered within the immunoglobulin loci are inconsistent between mice and humans and may not be functionally equivalent in some cases. Differences between mouse and human immunoglobulin loci can still result in abnormalities in humanized mice, especially when specific portions of the endogenous mouse immunoglobulin heavy chain locus are humanized or engineered. Some modifications in the mouse immunoglobulin heavy chain locus are deleterious, such as the loss of the modified mice's ability to mate and produce offspring. Reduced or lost fertility in male mice has been found to be associated with damage to the Adam6 gene.

[0004] The ADAM6 protein is a member of the ADAM protein family, where ADAM is an acronym for A Disintegrin And Metalloprotease. The ADAM protein family is large and diverse, with multiple functions, including cell adhesion. Some members of the ADAM family are involved in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of fertilin, which is involved in sperm-egg interaction. ADAM3 or cyritestin appears to be required for sperm binding to the zona pellucida. Deficiency of either ADAM2 or ADAM3 results in infertility. It has been hypothesized that ADAM2, ADAM3, and ADAM6 form a complex on the surface of mouse spermatids. The human ADAM6 gene is located between the human VH genes VH1-2 and VH6-1. In mice, two ADAM6 genes, ADAM6a and ADAM6b, are present in the intergenic region between the VH and DH gene segments, and in mice, the direction of transcription of the ADAM6a and ADAM6b genes is opposite to that of the surrounding immunoglobulin gene segments.

[0005] CN105861548B discloses an ADAM6 mouse, in which one or more human immunoglobulin gene sequences are inserted into the immunoglobulin heavy chain locus of the germline of a mouse, disrupting the function of the endogenous ADAM6 gene through the insertion, and then a nucleic acid sequence encoding a mouse ADAM6a protein and a nucleic acid sequence encoding a mouse ADAM6b protein are inserted into the germline of the mouse. Here, the mouse ADAM6a protein and the mouse ADAM6b protein are expressed by the nucleic acid sequence, and the mouse ADAM6a protein and the mouse ADAM6b protein are expressed from the nucleic acid sequence and improve or restore fertility when expressed in male mice. Disruption of the endogenous ADAM6 gene is undesirable and may affect the long-term fertility of mice.

[0006] In view of the above, the present invention provides a mouse genome with an altered heavy chain locus that retains the endogenous ADAM6 gene and its functions and is capable of producing human-mouse chimeric antibodies while maintaining reproductive capacity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] CN105861548B Summary of the Invention [Problem to be solved by the invention]

[0008] In a first aspect of the present invention, (a) inserting a first partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of a first mouse, wherein the first partial segment comprises a first partial hIgHV contiguous segment, all hIgHD segments, and all hIgHJ segments, wherein the first partial segment does not comprise a segment between downstream of the hIgHV1-2 gene and upstream of the hIgHV6-1 gene, and wherein the first partial segment comprises a first recombination site upstream of the first partial segment and downstream of the mIgHJ region; (b) inserting a second partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the second mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment comprises a second partial hIgHV contiguous segment, and a second recombination site is included between the downstream of the second partial hIgHV contiguous segment and the upstream of the mIgHC region; (c) crossbreeding the first mouse with the second mouse and screening a third mouse, wherein the second partial segment of the human immunoglobulin heavy chain variable region locus and the first partial segment downstream of the second partial segment are inserted between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the third mouse, and a third recombination site is located between the second partial segment and the first partial segment; and (d) knocking out the mIgHV5-1 gene and all mIgHV segments upstream thereof, the continuous segment between the mIgHD1-1 gene and the mIgHJ4 gene, as well as the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes in the immunoglobulin heavy chain locus of the third mouse, to obtain the genetically modified mouse.

[0009] In the present invention, the first partial hIgHV contiguous segment of the first partial segment may start from any hIgHV segment gene (including a functional gene, pseudogene, or ORF) upstream of the hIgHV1-2 gene and end at the hIgHV1-2 gene. In one embodiment, any gene in the hIgHV segment upstream of the hIgHV1-2 gene may be any functional gene, pseudogene, or ORF between the hIgHV(III)-82 and hIgHV1-2 genes. In one embodiment, any functional gene in the hIgHV segment upstream of the hIgHV1-2 gene, such as hIgHV3-74, hIgHV3-73, hIgHV3-72, hIgHV2-70, hIgHV1-69D, hIgHV1-69-2, hIgHV2-70D, hIgHV1-69, hIgHV3-66, or hIgHV3-6 4, hIgHV3-62, hIgHV4-61, hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgH V3-48, hIgHV1-46, hIgHV1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-3 5, hIgHV4-34, hIgHV3-33, hIgHV4-31, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-3 0-2, hIgHV4-30-1, hIgHV3-30, hIgHV4-28, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-23, hIgHV3-21, hIgHV3-20, hIgHV1-18, hIgHV3-15, hIgHV3-13, hIgHV3-11, hIgHV5-10-1, hIgHV3-9, hIgHV3-64D, hIgHV3-8, hIgHV3-7, hIgHV2-5, hIgHV7-4-1, hIgHV4-4 or hIgHV1-3.

[0010] In one embodiment, the first partial hIgHV contiguous segment of the first partial segment is selected from the group consisting of hIgHV3-74, hIgHV3-73, hIgHV3-72, hIgHV2-70, hIgHV1-69D, hIgHV1-69-2, hIgHV2-70D, hIgHV1-69, hIgHV3-66, hIgHV3-64, hIgHV3-62, hIgHV4-61 , hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgHV3-48, hIgHV1-46, hIgHV1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-35, hIgHV4-34, hIgHV3-33, hIgHV4-3 1, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-30-2, hIgHV4-30-1, hIgHV3-30, hIgHV4-2 8, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-23, hIgHV3-21, hIgHV3-20, hIgHV1-18, hIgHV3-1 In one embodiment, the first partial hIgHV contiguous segment of the first partial segment is a contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0011] In the present invention, the second partial segment is located upstream of the first partial segment, and the second partial hIgHV contiguous segment may start with any gene (functional gene, pseudogene, or ORF) of the hIgHV segment and end with any gene (including a functional gene, pseudogene, or ORF) of the hIgHV segment upstream of the start gene of the first partial hIgHV contiguous segment. In some embodiments, the second partial segment and the first partial segment together constitute all functional genes upstream of hIgHV1-2 and the contiguous segments therebetween, i.e., the contiguous segment between the hIgHV3-74 and hIgHV1-2 genes. For example, in some embodiments, the second partial hIgHV contiguous segment includes or is the contiguous segment between the hIgHV3-74 gene and the hIgHV3-30 gene, and the first partial hIgHV contiguous segment includes or is the contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0012] In some embodiments, the immunoglobulin heavy chain locus of the resulting genetically modified mouse comprises, in this order: (i) the mouse Adam6a gene; (ii) the mouse Adam6b gene; (iii) the contiguous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iv) the contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (v) the contiguous segment between the hIgHV6-1 gene and the hIgHJ6 gene; and (vi) the mIgHC region.

[0013] In some embodiments, in step (a), the first partial hIgHV contiguous segment, all of the hIgHD segments, and all of the hIgHJ segments of the first partial segment are inserted between the mIgHJ region and the mIgHC region by at least two steps, and the at least two steps include: (a1) inserting a first continuous segment between the hIgHV6-1 and hIgHJ6 genes between the mIgHJ region and the mIgHC region; and The method includes a step (a2) of inserting a second continuous segment between the hIgHV4-28 and hIgHV1-2 genes upstream of the first continuous segment.

[0014] In some embodiments, step (a1) specifically comprises: (a11) inserting a continuous segment from hIgHD6-25 to hIgHJ6 genes between the mIgHJ region and the mIgHC region; (a12) inserting a continuous segment between the hIgHD6-13 and hIgHD5-24 genes upstream of the inserted segment of step (a11); a step (a13) of inserting a continuous segment between the hIgHD1-1 and hIgHD5-12 genes upstream of the insertion segment of step (a12); and (a14) inserting a continuous segment between the hIgHV6-1 and hIgHD1-1 genes and loxP and lox2272-PB5' sites upstream of the inserted segment of step (a13).

[0015] In some embodiments, step (a2) specifically comprises: a step (a21) of recombining a BAC vector containing a second continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene, loxP and lox2272 sites located on both ends of the second continuous segment and oriented in the same direction as in step (a14), and Cre recombinase with the genome obtained in step (a1), wherein PB3' is included between the lox2272 site and the second continuous segment; and (a22) contacting the genome to be recombined obtained in step (a21) with PiggyBac transposase and screening for genomes containing the first partial segment and a loxP site upstream of the first partial segment.

[0016] In some embodiments, in step (b), the second partial hIgHV contiguous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps: (b1) inserting a continuous segment between the hIgHV3-74 and hIgHV3-72 genes between the mIgHJ region and the mIgHC region; Step (b2) of inserting a continuous segment between the hIgHV2-70 and hIgHV1-69D genes and PB3'-lox5171 and loxP sites downstream of the inserted segment of step (b1); Step (b3) of recombining a BAC vector containing a continuous segment between the hIgHV1-69-2 gene and the hIgHV3-30 gene and loxP and lox5171 located at both ends and in the same orientation as in step (b2), and Cre recombinase with the genome obtained in step (b2), wherein PB5' is included between the lox5171 site and the continuous segment in step (b3); and (b4) contacting the genome to be recombined obtained in step (b3) with PiggyBac transposase and screening for genomes containing the second partial segment and a loxP site located downstream of the second partial segment.

[0017] In some embodiments, step (c) comprises: (c1) screening Cre-positive mice comprising the first partial segment and the second partial segment; and (c2) crossing the mice obtained in step (c1) with wild-type mice and screening for Cre-free mice comprising a first partial segment and a second partial segment, wherein a loxP site is present between the second partial segment and the first partial segment.

[0018] In some embodiments, step (d) comprises: (d1) crossing the genetically modified mouse with a wild-type mouse and screening for a positive mouse; and (d2) mating the male and female positive mice obtained in step (d1) with each other and screening for homozygous mice.

[0019] Another aspect of the present invention provides a genetically modified mouse genome, wherein the immunoglobulin heavy chain locus of the genetically modified mouse comprises, in this order: (i) a mouse Adam6a gene; (ii) a mouse Adam6b gene; (iii) a second partial segment of a human immunoglobulin heavy chain variable region locus comprising a second partial hIgHV contiguous segment; (iv) a first partial segment of a human immunoglobulin heavy chain variable region locus located downstream of the second partial segment and comprising the first partial hIgHV contiguous segment, all hIgHD segments, and all hIgHJ segments, wherein the first partial segment does not include the segment between downstream of the hIgHV1-2 gene and upstream of the hIgHV6-1 gene; and (v) an mIgHC region.

[0020] In some embodiments, the first partial hIgHV contiguous segment of the first partial segment may start from any gene (including a functional gene, pseudogene, or ORF) of the hIgHV segment upstream of the hIgHV1-2 gene and end at the hIgHV1-2 gene.

[0021] In one embodiment, any gene in the hIgHV segment upstream of the hIgHV1-2 gene may be any functional gene, pseudogene, or ORF between the hIgHV(III)-82 and hIgHV1-2 genes. In one embodiment, any functional gene in the hIgHV segment upstream of the hIgHV1-2 gene, such as hIgHV3-74, hIgHV3-73, hIgHV3-72, hIgHV2-70, hIgHV1-69D, hIgHV1-69-2, hIgHV2-70D, hIgHV1-69, hIgHV3-66, or hIgHV3-6 4, hIgHV3-62, hIgHV4-61, hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgH V3-48, hIgHV1-46, hIgHV1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-3 5, hIgHV4-34, hIgHV3-33, hIgHV4-31, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-3 0-2, hIgHV4-30-1, hIgHV3-30, hIgHV4-28, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-23, hIgHV3-21, hIgHV3-20, hIgHV1-18, hIgHV3-15, hIgHV3-13, hIgHV3-11, hIgHV5-10-1, hIgHV3-9, hIgHV3-64D, hIgHV3-8, hIgHV3-7, hIgHV2-5, hIgHV7-4-1, hIgHV4-4 or hIgHV1-3.

[0022] In one embodiment, the first partial hIgHV contiguous segment of the first partial segment is selected from the group consisting of hIgHV3-74, hIgHV3-73, hIgHV3-72, hIgHV2-70, hIgHV1-69D, hIgHV1-69-2, hIgHV2-70D, hIgHV1-69, hIgHV3-66, hIgHV3-64, hIgHV3-62, hIgHV4-61 , hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgHV3-48, hIgHV1-46, hIgHV1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-35, hIgHV4-34, hIgHV3-33, hIgHV4-3 1, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-30-2, hIgHV4-30-1, hIgHV3-30, hIgHV4-2 8, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-23, hIgHV3-21, hIgHV3-20, hIgHV1-18, hIgHV3-1 In one embodiment, the first partial hIgHV contiguous segment of the first partial segment is a contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0023] In the present invention, the second partial segment is located upstream of the first partial segment, and the second partial hIgHV continuous segment may start with any gene (functional gene, pseudogene, or ORF) of the hIgHV segment and end with any gene (including functional gene, pseudogene, or ORF) of the hIgHV segment upstream of the start gene of the first partial hIgHV continuous segment. In some embodiments, the second partial segment and the first partial segment together constitute all functional genes upstream of hIgHV1-2 and the continuous segments therebetween, i.e., the continuous segment between the hIgHV3-74 and hIgHV1-2 genes. For example, in some embodiments, the second partial hIgHV contiguous segment includes or is the contiguous segment between the hIgHV3-74 gene and the hIgHV3-30 gene, and the first partial hIgHV contiguous segment includes or is the contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0024] In some embodiments, the first partial segment and the second partial segment are unrearranged.

[0025] In some embodiments, the immunoglobulin heavy chain locus of the resulting genetically modified mouse comprises, in this order: (i) the mouse Adam6a gene; (ii) the mouse Adam6b gene; (iii) the contiguous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iv) the contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (v) the contiguous segment between the hIgHV6-1 gene and the hIgHJ6 gene; and (vi) the mIgHC region.

[0026] In some embodiments, the immunoglobulin heavy chain locus of the resulting genetically modified mouse does not include mIgHV5-1 and all mIgHV segments upstream of that gene, the contiguous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes.

[0027] In some embodiments, the fertility of the resulting genetically modified mice is not reduced compared to wild-type mice that are not genetically modified.

[0028] In some embodiments, the resulting genetically modified mouse is capable of producing a human-mouse chimeric antibody, said chimeric antibody comprising a human heavy chain variable region and a mouse constant region.

[0029] Another aspect of the present invention provides a cell, tissue, organ, or mouse comprising the mouse genome described above.

[0030] In some embodiments, the invention provides a cell comprising the mouse genome described above, wherein the cell is an embryonic cell, a B cell, or a hybridoma cell.

[0031] In some embodiments, the present invention provides a tissue comprising the mouse genome described above, wherein said tissue is the white pulp of the spleen or a lymph node thereof.

[0032] In some embodiments, the present invention provides an organ comprising the mouse genome described above, wherein said organ is a spleen.

[0033] In some embodiments, the present invention provides a mouse comprising the mouse genome described above.

[0034] Another aspect of the present invention provides a method for producing a monoclonal antibody, said method comprising: (a) immunizing a mouse having any of the genomes described in the present invention with an antigen; (b) isolating from the mouse cells containing monoclonal antibodies against the antigen; and (c) culturing the cells to obtain the monoclonal antibody.

[0035] In some embodiments, the cells described in step (c) are spleen cells, B cells, or hybridoma cells.

[0036] In some embodiments, the monoclonal antibody has a human heavy chain variable region and no mouse heavy chain variable region, hi some embodiments, the monoclonal antibody comprises a human heavy chain variable region and a mouse constant region.

[0037] Another aspect of the invention provides the use of a cell, tissue, organ, or mouse according to any of the invention in the production of a monoclonal antibody.

[0038] In some embodiments, the monoclonal antibody has a human heavy chain variable region and no mouse heavy chain variable region, hi some embodiments, the monoclonal antibody comprises a human heavy chain variable region and a mouse constant region.

[0039] The present invention provides a mouse genome with an altered heavy chain locus that retains the endogenous ADAM6 gene and its function, and is capable of producing human-mouse chimeric antibodies containing a human heavy chain variable region and a mouse constant region while maintaining fertility. [Brief explanation of the drawings]

[0040] [Figure 1] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 2] The results of PCR identification of each modification step are shown. [Figure 3] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 4] The results of PCR identification of each modification step are shown. [Figure 5] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 6] The results of PCR identification of each modification step are shown. [Figure 7] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 8] The results of PCR identification of each modification step are shown. [Figure 9] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 10-1] The results of PCR identification of each modification step are shown. [Figure 10-2] The results of PCR identification of each modification step are shown. [Figure 10-3] The results of PCR identification of each modification step are shown. [Figure 11] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 12] The results of PCR identification of each modification step are shown. [Figure 13] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 14] The results of PCR identification of each modification step are shown. [Figure 15] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 16] The results of PCR identification of each modification step are shown. [Figure 17] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 18] The results of PCR identification of each modification step are shown. [Figure 19] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 20] The results of PCR identification of each modification step are shown. [Figure 21] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 22-1] The results of PCR identification of each modification step are shown. [Figure 22-2] The results of PCR identification of each modification step are shown. [Figure 22-3] The results of PCR identification of each modification step are shown. [Figure 23] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 24] The results of PCR identification of each modification step are shown. [Figure 25] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 26] The results of PCR identification of each modification step are shown. [Figure 27] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 28] The results of PCR identification of each modification step are shown. [Figure 29] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 30] The results of PCR identification of each modification step are shown. [Figure 31] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 32] The results of PCR identification of each modification step are shown. [Figure 33] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 34] The results of PCR identification of each modification step are shown. [Figure 35] A schematic diagram of the genomic changes associated with each modification step is shown. [Figure 36] The results of PCR identification of each modification step are shown. [Figure 37] The results of PCR identification of each modification step are shown. [Figure 38] The results of PCR identification of each modification step are shown. [Figure 39] The results of PCR identification of each modification step are shown. [Figure 40] A gene usage distribution map at the reads level created based on the obtained gene usage data is shown. [Figure 41] A gene usage distribution map at the reads level created based on the obtained gene usage data is shown. [Figure 42] A gene usage distribution map at the reads level created based on the obtained gene usage data is shown. [Figure 43] A distribution map of CDR3 amino acid lengths at the reads level created based on the obtained CDR3 length frequency data is shown. [Figure 44] The results of Weblog feature analysis at the reads level of the CDR3 amino acid sequence of each sample are shown. [Figure 45] This shows that genetically modified mice and wild-type mice have similar immune characteristics. [Figure 46] Representative examples of positive hybridoma cells detected by screening are shown. [Figure 47] Several dozen positive hybridoma cells were screened, and five of them showed very strong binding ability to antigen-positive cells. [Figure 48] All four genetically modified homozygous mice demonstrate a higher immune response capacity. [Figure 49] This indicates that all antibody sequences obtained from genetically modified mice are human antibody sequences and have abundant sequence diversity. DETAILED DESCRIPTION OF THE INVENTION

[0041] definition

[0042] In the present invention, "hIgHV" refers to the V region of the human immunoglobulin heavy chain variable region locus. When used independently, it refers to the entire V region of the human immunoglobulin heavy chain variable region locus. When a specific gene number is added, such as in "hIgHV3-30," it refers to 3 to 30 genes located in the V region of the human immunoglobulin heavy chain variable region locus. Similarly, in the present invention, "hlgHD" and "hlgHJ" are also used to refer to the D region and J region, respectively, of the human immunoglobulin heavy chain variable region locus.

[0043] In the present invention, "mIgHV" refers to the V region of the mouse immunoglobulin heavy chain variable region locus. When used independently, it refers to the entire V region of the mouse immunoglobulin heavy chain variable region locus. When a specific gene number is added, such as in "mIgHV5-1," it refers to the 5-1 gene located in the V region of the mouse immunoglobulin heavy chain variable region locus. Similarly, in the present invention, "mIgHD" and "mIgHJ" are also used, which refer to the D region and J region, respectively, of the mouse immunoglobulin heavy chain variable region locus.

[0044] A "contiguous segment" refers to an uninterrupted nucleotide fragment between two specified endpoint genes, including functional genes, pseudogenes, ORFs, and other nucleotide sequences (e.g., spacer sequences) located between the two endpoint genes. The term "contiguous segment between gene A and gene B" refers to a segment that includes gene A, gene B, and any contiguous genes between the two. The terms "upstream of gene A" or "downstream of gene A" do not include gene A itself.

[0045] Example

[0046] 1. Construction of mice based on ES cell lines

[0047] 1. Insertion of approximately 20 kb of human gene sequence (including IGHJ1-6, IGHD7-27, IGHD1-26, IGHD6-25 and all gene spacer sequences) between the mouse J and C regions.

[0048] The constructed vector was electroporated into wild-type ES cells. The vector conferred Neo resistance. The cells were screened with the drug G418, and relevant ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0049] Using four pairs of primers (the sequences are shown in Table 1 below, 5' to 3', and so on below; the primer identification positions are also marked on the map (Figure 1)), we identified that the sequences had been inserted at the corresponding positions. Three clones, 1A3, 1A7, and 1B7, simultaneously amplified positive bands using the four pairs of primers (Figure 2). These three clones were used as positive clones in the first step.

[0050] [Table 1]

[0051] 2. Insertion of approximately 20 kb of human D genomic sequence (including IGHD5-24, IGHD4-23, IGHD3-22, IGHD2-21, IGHD1-20, IGHD6-19, IGHD5-18, IGHD4-17, IGHD3-16, IGHD2-15, IGHD1-14, IGHD6-13 and all gene spacer sequences) in front of the cellular genome obtained in the first step.

[0052] The constructed vector was electroporated into the positive 1A7 clone from the first step. The vector conferred Puro resistance, and one end of the homologous arm was placed in the human sequence of the positive clone from the first step, resulting in the loss of Neo resistance by homologous recombination. The cells were screened with puromycin, and related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0053] Using four pairs of primers (the sequences are shown in Table 2 below, and the primer identification positions are also marked on the map (Figure 3)), we identified that the sequences had been inserted at the corresponding positions. Three clones, 1A7-1C2, 1A7-1C3, and 1A7-1B4, simultaneously amplified positive bands using the four pairs of primers (Figure 4). These three clones were used as positive clones in the second step.

[0054] [Table 2]

[0055] 3. Insertion of approximately 20 kb of human D genomic sequence (including IGHD5-12, IGHD4-11, IGHD3-10, IGHD3-9, IGHD2-8, IGHD1-7, IGHD6-6, IGHD5-5, IGHD4-4, IGHD3-3, IGHD2-2, IGHD1-1 and all gene spacer sequences) in front of the cellular genome obtained in the second step.

[0056] The constructed vector was electroporated into the positive cell line 1A7-1C3 clone from the second step. The vector conferred Neo resistance, and one end of the homologous arm was placed in the human sequence of the positive clone from the second step, resulting in the loss of Puro resistance through homologous recombination. The cells were screened with the drug G418, and relevant ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0057] Four pairs of primers (the sequences are shown in Table 3 below, and the primer identification positions are also marked on the map (Figure 5)) were used to identify the insertion of the sequences at the corresponding positions. Four clones, 1A7-1C3-1B1, 1A7-1C3-1C6, 1A7-1C3-1C9, and 1A7-1C3-1F10, simultaneously amplified positive bands using the four pairs of primers (Figure 6). These four clones were used as positive clones in the third step.

[0058] [Table 3]

[0059] 4. Insertion of approximately 20 kb of human V genome sequence (including all spacer sequences between IGHV6-1 and IGHD1-1 upstream) in front of the cellular genome obtained in the third step, and introduction of two lox sites, loxp-lox2272.

[0060] The constructed vector was electroporated into the positive cell line 1A7-1C3-1B1 clone in the third step. The vector conferred Puro resistance, and one end of the homologous arm was engineered to the human sequence of the positive clone in the second step, resulting in the loss of Puro resistance through homologous recombination. The two ends of the Puro resistance arm contained loxP and lox2272-PB5' (5'ITR, 5' inverted terminal repeat) elements, respectively. The cells were screened with puromycin, and related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0061] Four pairs of primers (the sequences are shown in Table 4 below, and the primer identification positions are also marked on the map (Figure 7)) were used to identify the insertion of the sequences at the corresponding positions. Three clones, 1A7-1C3-1B1-2C1, 1A7-1C3-1B1-2C2, and 1A7-1C3-1B1-2D5, simultaneously amplified positive bands using the four pairs of primers (Figure 8). These three clones were used as positive clones in the fourth step.

[0062] [Table 4]

[0063] 5. Insertion of approximately 400 kb of human V genome sequence (including IGHV1-2, IGHV1-3, IGHV4-4, IGHV7-4-1, IGHV2-5, IGHV3-7, IGHV3-64D, IGHV5-10-1, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV1-18, IGHV3-20, IGHV3-21, IGHV3-23, IGHV1-24, IGHV2-26, IGHV4-28 and all gene spacer sequences) in front of the cellular genome obtained in step 4.

[0064] The constructed fusion BAC and Cre were electroporated into the 1A7-1C3-1B1-2D5 clones that were positive for the fourth step. The BAC also contained Neo resistance (Neo antibody has inverted terminal repeats at both ends). The human sequences on the BAC contained loxP and lox2272 elements, oriented in the same direction as the positive clones in the fourth step. A 3' inverted terminal repeat (3'ITR) was located between the lox2272 element and the human genomic sequence. Cre recombinase replaced the loxP and lox2272 sequences in the BAC and the positive clones from the fourth step, replacing the Puro resistance with the human genomic sequence on the BAC and Neo resistance. The cells were screened with the drug G418, and related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0065] Eleven pairs of primers (the sequences are shown in Table 5 below, and the primer identification positions are also marked on the map (Figure 9)) were used to identify sequence substitutions. Two clones, 1A7-1C3-1B1-2D5-2A2 and 1A7-1C3-1B1-2D5-2B3, simultaneously amplified positive bands using the eleven pairs of primers (Figure 10). These two clones were used as positive clones in the fifth step.

[0066] [Table 5] TIFF2026508379000006.tif159166

[0067] 6. Electroporation of pBase (PiggyBac transposase) into cell clones obtained in step 5

[0068] pBase (PiggyBac transposase) was electroporated into the positive cell clones 1A7-1C3-1B1-2D5-2B3 in step 5. The action of PiggyBac transposase excised the two inverted terminal repeats from the genome, eliminating the Neo sequence and lox2272. The positive cell clones contained only the inserted human gene sequence and one loxP element. ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0069] Two pairs of primers (the sequences are shown in Table 6 below, and the primer identification positions are also marked on the map (Figure 11)) were used to identify the deletion sequence. Four clones, 1A7-1C3-1B1-2D5-2B3-1C2, 1A7-1C3-1B1-2D5-2B3-1D1, 1A7-1C3-1B1-2D5-2B3-1D5, and 1A7-1C3-1B1-2D5-2B3-1D6, simultaneously amplified positive bands using the two pairs of primers (Figure 12). These four clones were used as positive clones in the sixth step.

[0070] [Table 6]

[0071] 7. F0 mice derived from ES1

[0072] The positive cell clones obtained in step 6 were injected into blastocysts, which were then implanted into surrogate mother mice. After a gestation period of approximately 20 days, mice were born. The legs of 5-7 day old mice were amputated, DNA was extracted, and PCR typing was performed to identify and confirm the genotype of the mice.

[0073] After injection of the 1A7-1C3-1B1-2D5-2B3-1C2 clone, a total of eight mice were born. Five pairs of primers (Table 7) were used for simultaneous identification, and all eight mice were positive (Figures 13 and 14).

[0074] [Table 7]

[0075] 8. F1 mice derived from ES1

[0076] Mice identified as positive in the F0 stage were crossed with CMV-Cre mice to obtain F1 generation mice. The genotypes of the mice were identified and confirmed by PCR typing of genomic DNA from the tails of the F1 generation mice.

[0077] A total of six F1 mice were born. ES1 was simultaneously identified using five pairs of primers (Table 8), and CMV-Cre was identified using one pair of primers. As a result, two mice (1#, 5#) were double-gene positive (Figures 15 and 16).

[0078] [Table 8]

[0079] II. Construction of mice based on ES cell line II

[0080] 1. Insertion of approximately 20 kb of human gene sequence (including IGHV3-74, IGHV3-73, IGHV3-72, and all gene spacer sequences) between the mouse J and C regions.

[0081] The constructed vector was electroporated into wild-type ES cells. The vector conferred Neo resistance. The cells were screened with the drug G418, and relevant ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0082] Using four pairs of primers (the sequences are shown in Table 9 below, and the primer identification positions are also marked on the map (Figure 17)), we identified that the sequences were inserted at the corresponding positions. There were two clones, 1B2 and 1H5, that simultaneously amplified positive bands using the four pairs of primers (Figure 18). These two clones were used as positive clones in the first step.

[0083] [Table 9]

[0084] 2. Insertion of approximately 20 kb of human V genome sequence (including IGHV2-70, IGHV1-69D, and all gene spacer sequences) downstream of the genome inserted into the cells obtained in the first step, and introduction of two lox sites loxp-lox5171.

[0085] The constructed vector was electroporated into the positive 1H5 clone from the first step. The vector conferred Puro resistance, and one end of the homologous arm was engineered to the human sequence of the positive clone from the first step, resulting in the loss of Neo resistance by homologous recombination. The two ends of the Puro resistance region contained PB3' (3' inverted terminal repeat)-lox5171 and loxP elements, respectively. The cells were screened with puromycin, and related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0086] Using four pairs of primers (the sequences are shown in Table 10 below, and the primer identification positions are also marked on the map (Figure 19)), we identified that the sequences were inserted at the corresponding positions. Two clones, 1H5-1B1 and 1H5-1E3, simultaneously amplified positive bands using the four pairs of primers (Figure 20). These two clones were used as positive clones in the second step.

[0087] [Table 10]

[0088] 3. Insertion of 400 kb of human V genome sequence (including IGHV1-69-2, IGHV2-70D, IGHV1-69, IGHV3-66, IGHV3-64, IGHV4-61, IGHV4-59, IGHV1-58, IGHV3-53, IGHV8-5-51, IGHV5-51, IGHV3-49, IGHV3-48, IGHV1-46, IGHV1-45, IGHV3-43, IGHV4-39, IGHV3-38, IGHV3-35, IGHV4-34, IGHV3-33, IGHV4-31, IGHV3-30 and all gene spacer sequences) downstream of the cellular genome obtained in the second step.

[0089] The constructed fusion BAC and Cre were electroporated into the positive cell clone 1H5-1B1 from the second step. The BAC also contained Neo resistance (both ends of the Neo resistance region contained inverted terminal repeats). The human sequence on the BAC contained loxP and lox5171 elements in the same orientation as the positive clone from the second step, and a 5' inverted terminal repeat (5'ITR) was inserted between the lox5171 element and the human genomic sequence. Cre recombinase replaced the loxP and lox5171 sequences between the BAC and the positive cell clone from the second step, replacing the Puro resistance with the human genomic sequence on the BAC and Neo resistance. The cells were screened with the drug G418, and related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0090] Eleven pairs of primers (the sequences are shown in Table 11 below, and the primer identification positions are also marked on the map (Figure 21)) were used to identify sequence substitutions. Three clones, 1H5-1B1-1A6, 1H5-1B1-1B4, and 1H5-1B1-1E2, simultaneously amplified positive bands using the eleven pairs of primers (Figure 22). These three clones were used as positive clones in the third step.

[0091] [Table 11] TIFF2026508379000013.tif58166

[0092] 4. Electroporation of pBase (PiggyBac transposase) into cell clones obtained in step 3

[0093] In the third step, pBase (PiggyBac transposase) was electroporated into the positive 1H5-1B1-1A6 clone. The action of PiggyBac transposase excised the two inverted terminal repeats from the genome, eliminating the Neo sequence and lox5171. The positive clone contained only the inserted human gene sequence and one loxP element. ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0094] Three pairs of primers (the sequences are shown in Table 12 below, and the primer identification positions are also marked on the map (Figure 23)) were used to identify the deletion of the sequence. Five clones, 1H5-1B1-1A6-1B7, 1H5-1B1-1A6-1C8, 1H5-1B1-1A6-1D4, 1H5-1B1-1A6-1E8, and 1H5-1B1-1A6-1F5, simultaneously amplified positive bands using the three pairs of primers (Figure 24). These five clones were used as positive clones in the third step.

[0095] [Table 12]

[0096] 5. F0 mice obtained from ES2

[0097] The positive cell clones obtained in the fourth step were injected into blastocysts, which were then implanted into surrogate mother mice. After a gestation period of approximately 20 days, mice were born. The legs of 5-7 day-old mice were amputated, DNA was extracted, and PCR typing was performed to identify and confirm the genotype of the mice.

[0098] After injection of the 1H5-1B1-1A6-1C8 clone, a total of eight mice were born, and all eight mice were positive for the 1H5-1B1-1A6-1C8 clone (Figures 25 and 26) as identified using four pairs of primers (Table 13).

[0099] [Table 13]

[0100] 6. F1 mice derived from ES2

[0101] Mice identified as positive in the F0 stage were crossed with wild-type mice to obtain F1 generation mice. The genotypes of the mice were identified and confirmed by PCR typing of genomic DNA from the tails of the F1 generation mice.

[0102] A total of six F1 mice were born, and ES2 was simultaneously identified using four pairs of primers (Table 14). As a result, a total of three mice (2#, 5#, and 6#) were double gene-positive (Figures 27 and 28).

[0103] [Table 14]

[0104] The ES1 double-positive F1 mice were crossed with the F1-positive mice derived from the ES2 gene to generate Cre-positive, ES1-positive, and ES2-positive F2-generation mice. The resulting F2-positive mice were crossed with wild-type mice to generate 3.5-day-old blastocysts, from which ES cell lines were established. Various ES cell lines were detected by PCR and screened for ectopic cells that lacked Cre. These cells encompassed the entire human genome sequence of ES1 and ES2 and contained a single loxP site between IGHV3-30 and IGHV4-28.

[0105] This gene was identified as ectopic using three pairs of primers (Table 15). Four clones were identified as ectopic: 1A3, 1A5, 1A6, and 1B3 (Figures 29 and 30). These four clones were designated ES cell line III.

[0106] [Table 15]

[0107] 3. Gene knockout (KO)

[0108] 1. First homologous recombination knockout (V region) in ES cell line III

[0109] A vector was constructed. This vector was Neo-resistant (recombinase sites existed at both ends of the Neo gene). Its 5'-terminal homologous arm sequence contained a 10-kb contiguous sequence upstream of the mIgHV1-86 gene, and its 3'-terminal homologous arm sequence contained an 8-kb contiguous sequence downstream of the mIgHV5-2 gene. The constructed vector was electroporated into ES cell line III. The cells were screened with the drug G418, and related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0110] Using two pairs of primers (the sequences are shown in Table 16 below, and the primer identification positions are also marked on the map (Figure 31)), we identified V region genes (mIgHV1-86 to mIgHV5-2) that had recombined due to Neo resistance, indicating that the mouse IgH chain V gene had undergone recombination deletion. Eight clones, 1A3-1B3, 1A3-1B4, 1A3-1B5, 1A3-1B7, 1A3-1E1, 1A3-1E2, 1A3-1E3, and 1A3-1E5, simultaneously amplified positive bands using two pairs of primers (Figure 32). These eight clones were used as positive clones in the first step.

[0111] [Table 16]

[0112] 2. Second homologous recombination knockout of the cells obtained in the first step (to knock out all gene sequences between IgHJ4 and IgHD1-1)

[0113] A vector was constructed. The vector also conferred Puro resistance (Puro recombinase sites existed at both ends), and its 5'-terminal homologous arm sequence contained an 8-kb contiguous sequence upstream of the mIgHD1-1 gene, and its 3'-terminal homologous arm sequence contained a 5-kb contiguous sequence downstream of the mIgHJ4 gene. The constructed vector was electroporated into the positive cell clone 1A3-1B3 from the first step, and the cells were screened with the drug puromycin. Related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0114] Using two pairs of primers (the sequences are shown in Table 17 below, and the primer identification positions are also marked on the map (Figure 33)), the gene between mIgHJ4 and mIgHD1-1 was identified as having undergone recombination with Puro resistance, indicating that the gene between mouse IgH chain mIgHJ4 and mIgHD1-1 had undergone recombination deletion. Three clones, 1A3-1B3-1C3, 1A3-1B3-1C6, and 1A3-1B3-1C8, simultaneously amplified positive bands using two pairs of primers (Figure 34). These three clones were used as positive clones in the second step.

[0115] [Table 17]

[0116] 3. Third homologous recombination KO of the cells obtained in the second step (to knock out the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes).

[0117] A vector was constructed. The vector possessed Hygro resistance (Hygro recombinase sites existed at both ends), and its 5'-end homologous arm sequence contained an 8 kb contiguous sequence upstream of the mIgHD1-3 gene, while its 3'-end homologous arm sequence contained a 5 kb contiguous sequence downstream of the mIgHD3-1 gene. The constructed vector was electroporated into the positive clones 1A3-1B3-1C6 from the second step, and the cells were screened with the drug hygromycin B. Related ES clones were selected, cultured, and amplified, followed by PCR typing and identification.

[0118] Using two pairs of primers (the sequences are shown in Table 18 below, and the primer identification positions are also marked on the map (Figure 35)), the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes were identified as recombined with Hygro resistance, indicating that the mouse IgH chain mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes had undergone recombination deletion. Three clones, 1A3-1B3-1C6-1D2, 1A3-1B3-1C6-1D5, and 1A3-1B3-1C6-1D8, simultaneously amplified positive bands using two pairs of primers (Figure 36). These three clones were used as positive clones in the third step.

[0119] [Table 18]

[0120] 4. Obtaining positive F0 mice by injecting the positive cell clones obtained in the third step

[0121] The positive cell clones obtained in the third step were injected into blastocysts, which were then implanted into surrogate mother mice. After a gestation period of approximately 20 days, mice were born. The legs of 5-7 day-old mice were amputated, DNA was extracted, and PCR typing was performed to identify and confirm the genotype of the mice.

[0122] After injection of the 1A3-1B3-1C6-1D8 clone, a total of 18 mice were born. Three pairs of primers (Table 19) were used to simultaneously identify three mice: 3#, 15#, and 18#, as positive mice (Figure 37).

[0123] [Table 19]

[0124] 5. Obtaining Positive F1 Mice

[0125] Mice identified as positive in the F0 stage were crossed with wild-type mice to obtain F1 generation mice. The genotypes of the mice were identified and confirmed by PCR typing of genomic DNA from the tails of the F1 generation mice.

[0126] A total of 16 F1 mice were born, and three pairs of primers (Table 20) were used to identify them simultaneously. As a result, a total of three mice (2#, 6#, 16#) were double gene-positive mice (Figure 38).

[0127] [Table 20]

[0128] 6. Obtaining Homozygous Mice

[0129] Homozygous mice were obtained by mating males of mice identified as positive in the F1 generation with males. The genotypes of the mice were identified and confirmed by PCR typing of genomic tail DNA from the F2 generation mice.

[0130] A total of 12 F2 mice were born and simultaneously identified using six pairs of primers (Table 21). The genotypes of mice 1, 3, and 4# were homozygous, those of mice 2, 5, 6, 11, and 12# were heterozygous, and those of mice 7, 8, 9, and 10# were wild-type (Figure 39).

[0131] [Table 21]

[0132] 4. Detection of breeding performance

[0133] The resulting homozygous mice were further bred and their reproductive performance was statistically analyzed.

[0134] A total of 25 breeding pairs were mated, and the number of mice born one month after mating was counted. There were a total of 25 breeding pairs, of which 20 pairs gave birth to mice one month after mating. The effective breeding pair rate reached 80%. At the same time, the average number of litters was 5.35, which was consistent with the number of litters of wild-type mice. The data are shown in Table 22.

[0135] [Table 22]

[0136] 5. Detection of H chain immunohistochemistry

[0137] The resulting homozygous mice were euthanized, and their spleens were collected by autopsy for RNA extraction. After the total RNA samples passed detection, libraries were constructed and immune repertoire sequencing was performed. (Immuno-Seq is a study of B / T lymphocytes. It uses 5' RACE or multiplex PCR technology to specifically amplify the variable regions (V regions) that determine the diversity of B cell receptors (BCRs) or T cell receptors (TCRs). This is combined with high-throughput sequencing technology to comprehensively evaluate the diversity of the immune system.) The resulting sequences were quality-controlled using quality control software. After filtering for sequencing background, they were compared with the V, D, and J genes in the IMGT immune cell receptor library to search for corresponding gene fragments and identify the exact positions of the V, D, and J gene fragments and sequences. Information such as V, D, and J gene frequencies, clonal frequency distribution, and polypeptide sequence counts were statistically analyzed.

[0138] 40 to 42 show gene usage frequency distribution maps at the reads level, which were created based on the obtained gene usage frequency data. The results in Figure 40 show that unimmunized homozygous mice IGHV43-34 had the highest frequency of usage at the reads level, followed by IGHV6-1, IGHV4-59, IGHV4-39, IGHV3-21, IGHV2-26, IGHV3-23, IGHV2-5, IGHV5-51, IGHV1-18, IGHV3-48, IGHV3-15, IGHV4-4, IGHV3-43, IGHV5-10-1, IGHV3-30, IGHV3-7, IGHV1-3, IGHV4-61, IGHV3-20, IGHV3-74, IGHV1-69, IGHV3-73, IGHV1-2, IGHV7-4-1, IGHV8-1, IGHV9-1, IGHV10-2, IGHV11-2, IGHV12-3, IGHV13-3, IGHV14-4, IGHV15-4, IGHV16-4, IGHV17-4, IGHV18-4, IGHV19-4, IGHV20-4, IGHV21-4, IGHV22-4, IGHV23-4, IGHV24-4, IGHV25-4, IGHV26-4, IGHV27-4, IGHV28-4, IGHV29-4, IGHV29-5, IGHV29-6, IGHV29-7, IGHV21-2, IGHV21-4 ... GHV3-64D, IGHV1-24, IGHV3-53, IGHV1-46, IGHV3-11, IGHV4-28, IGHV3-13, IGHV3-72, IGHV1-58, IGHV3-66, IGHV3-64, IGHV3-69-1, IGHV3-49, IGHV4-38-2, IGHV3-43D, IGHV3-38, IGHV3-33, IGHV2-70, IGHV3-71, IGHV3-NL1, IGHV3-22, IGHV3-30-3, IGHV3-38-3, IGHV3-35, IGHV3-52, IGHV4-31, and IGHV4 / OR15-8 ​​show a decrease in this order. The results in Figure 41 show that the non-immunized homozygous mouse IGHD3-10 had the highest frequency of usage at the reads level, followed by IGHD6-13, IGHD6-19, IGHD3-9, IGHD1-26, IGHD7-27, IGHD5-12, IGHD4-17, IGHD5-18, IGHD2-2, IGHD1-1, IGHD2-15, IGHD3-22, IGHD3-16, IGHD2-21, IGHD4-23, and I The following genes were shown to decrease in this order: GHD1-20, IGHD1-14, IGHD2-8, IGHD3-3, IGHD4-11, IGHD5-24, IGHD1-7, IGHD3 / OR15-3a, IGHD6-6, IGHD6-25, IGHD4 / OR15-4a, IGHD2 / OR15-2a, IGHD5 / OR15-5a, IGHD1 / OR15-1a, IGHD2 / OR15-2b, and IGHD4 / OR15-4b.The results in Figure 42 show that in unimmunized homozygous mice, IGHJ4 was most frequently used at the reads level, followed by IGHJ6, and then IGHJ3, IGHJ5, IGHJ2, and IGHJ1 in decreasing order.

[0139] Figure 43 shows a distribution map of CDR3 amino acid lengths at the reads level, created based on the obtained CDR3 length frequency data. The results showed that the distribution of CDR3 amino acid lengths was close to a normal distribution.

[0140] CDR3 amino acid sequence feature statistics are an important step in immunohistochemical analysis. Quantitative analysis of the abundance of various CDR3 sequences evolved from each germline antibody sequence for each clone is useful for selecting sequences for further antibody expression. Weblog feature analysis of the CDR3 amino acid sequences of each sample at the reads level is shown in Figure 44. The results in Figure 44 show the amino acid sequences with the highest abundance of CDR3 in unimmunized homozygous mice.

[0141] 6. Antibody production

[0142] Three 6- to 8-week-old C57BL / 6N female mice and three 6- to 8-week-old HK homozygous female mice (homozygous mice whose heavy chain variable regions were humanized according to steps 1 to 3 above and whose kappa light chain variable regions were also humanized) were immunized, and the immune cells were the B7-H3-CHO-K1 stably transfected cell line. The immunization schedule consisted of three regular immunizations and one booster immunization. The three regular immunizations were performed once every two weeks, with 2 × 10 7 B7-H3-CHO-K1 cells were injected intraperitoneally into mice. Three days after the third regular immunization, peripheral blood was collected from the mice by retro-orbital bleed, serum was collected, and antibody titers in the mouse serum were detected by flow cytometry. After the titer was determined to be adequate, a booster immunization was performed. The booster immunization was performed 10 days after the third regular immunization, with 2 × 10 7B7-H3-CHO-K1 cells were injected intraperitoneally. Three days after the booster immunization, mouse spleens were harvested and hybridoma fusion was performed to generate hybridoma cells. After hybridoma cell growth, positive hybridoma cells were identified. The supernatants were collected and analyzed by flow cytometry to determine whether they contained B7-H3 antibodies. Experimental results showed that the immune characteristics of HK homozygous mice were comparable to those of wild-type C57BL / 6N mice. After three immunizations, serum antibody titers were comparable between the two groups, with the highest titers exceeding 1:256,000. Spleen cells from HK mice were then fused with myeloma SP20 cells to generate hybridoma cells. After multiple screening rounds, over 70 B7-H3 antibody-positive hybridoma cells were obtained. Of these, antibodies secreted by five hybridoma cells exhibited extremely strong binding ability to antigen-positive cells.

[0143] After the booster immunization, mouse serum was collected to detect antibody titers, and the serum dilution gradient was set at 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. The detection results in Figure 45 showed that HK mice and wild-type mice had similar immune characteristics, and both serum titers were all above 1:256000.

[0144] After hybridoma cell fusion, the culture supernatant of successfully fused cells was selected and co-incubated with antigen-positive cells to identify positive hybridoma cells capable of secreting antibodies. Figure 46 shows a representative example of positive hybridoma cells obtained by screening.

[0145] The mean fluorescence intensity of the positive hybridoma cell supernatants binding to antigen-positive cells was statistically analyzed. The results in Figure 47 show that several dozen positive hybridoma cells were screened, and five of them (5H9, 3A10, 6F12, 325-1G10, and 7D5) had very strong binding ability to antigen-positive cells.

[0146] To generate humoral immune responses against PD-L1 in HK homozygotes (homozygous mice with humanized heavy and kappa light chain variable regions), the first immunization consisted of a subcutaneous injection of 0.05 mg of PD-L1 His (Kactus Biosystems, PDL-HM110) protein in complete Freund's adjuvant (CFA), followed by a subcutaneous injection of 0.025 mg of PD-L1 His protein in incomplete Freund's adjuvant (IFA). These mice were immunized twice weekly for a total of four times to allow antigen-specific antibody production. Serum was collected from HK homozygotes after the third and fourth immunizations, and serum titers were monitored. The mice were coated overnight with 2 μg / ml of antigen in carbonate-buffered saline (CBS), washed with phosphate-buffered saline (PBST), and then blocked with 2% BSA at 37°C for 2 hours. After washing with PBST, serum dilutions (8 serial dilutions starting from 1:2000) were added and blocked for 2 hours at 37°C. After washing with PBST, horseradish peroxidase-conjugated goat anti-mouse secondary antibody (diluted 1:1 in PBS) was added and incubated for 1 hour at 37°C. After washing with PBST, TMB color development solution (Beyotime, P0209-500ml) was added and allowed to develop for 5-10 minutes at 37°C. The reaction was stopped by adding stop solution. The optical density was measured at 450 nm to detect serum titers, and the results are shown in Figure 48. All four HK homozygous mice demonstrated high immune response capabilities, reaching levels of 256K or higher (HK-84, HK-90, HK-106, and HK-110 are HK homozygous mice; WT-C57B6 is a wild-type mouse; NC is a non-immune mouse serum used as a negative control).

[0147] The spleen of the mouse that showed the highest serum titer after four immunizations was harvested and pulverized to collect cells. Total RNA was extracted from the spleen cells using an RNA extraction kit (Fukushima Biosciences, RE-03011). cDNA was synthesized, and the antibody VH and VL sequences were amplified using nested PCR. The vector pComb3XSS (IdiGen, 63890) and the target fragment were digested with SfiI and recovered. The ligated product was electroporated into TG1 competent cells to construct a PD-L1 scFv antibody library. The library capacity was determined to be 1.36 x 109 CFM. Forty-eight clones were randomly selected from the titer plate to measure the number of transformants in the library and sequenced for identification. The results (Figure 49) indicated that all antibody sequences were human and had extensive sequence diversity.

Claims

1. A method for producing a genetically modified mouse, comprising: (a) inserting a first partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of an immunoglobulin heavy chain locus of a first mouse, wherein the first partial segment comprises a first partial hIgHV contiguous segment, all of the hIgHD segments, and all of the hIgHJ segments, wherein the first partial segment does not comprise a segment between downstream of the hIgHV1-2 gene and upstream of the hIgHV6-1 gene, and wherein the first partial segment comprises a first recombination site upstream of the first partial segment and downstream of the mIgHJ region; (b) inserting a second partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the second mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment comprises a second partial hIgHV contiguous segment, and a second recombination site is included between the downstream of the second partial hIgHV contiguous segment and the upstream of the mIgHC region; (c) crossbreeding the first mouse with the second mouse and screening a third mouse, wherein the second partial segment of the human immunoglobulin heavy chain variable region locus and the first partial segment downstream of the second partial segment are inserted between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the third mouse, and a third recombination site is located between the second partial segment and the first partial segment; and (d) knocking out the mIgHV5-1 gene and all of the mIgHV segments upstream thereof, the continuous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes in the immunoglobulin heavy chain locus of the third mouse, to obtain the genetically modified mouse. A method for producing a genetically modified mouse.

2. (i) the first partial hIgHV contiguous segment of the first partial segment comprises a contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (ii) the second partial hIgHV contiguous segment of the second partial segment comprises a contiguous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iii) the first, second, and third recombination sites are loxP sites. The method of claim 1.

3. The immunoglobulin heavy chain locus of the genetically modified mouse comprises, in this order: (i) the mouse Adam6a gene; (ii) the mouse Adam6b gene; (iii) a contiguous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iv) a contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (v) a contiguous segment between the hIgHV6-1 gene and the hIgHJ6 gene; and (vi) an mIgHC region.

3. The method according to claim 1 or 2.

4. In step (a), the first partial hIgHV contiguous segment, all hIgHD segments, and all hIgHJ segments of the first partial segment are inserted between the mIgHJ region and the mIgHC region by at least two steps, and the at least two steps include: (a1) inserting a first continuous segment between the hIgHV6-1 and hIgHJ6 genes between the mIgHJ region and the mIgHC region; and and (a2) inserting a second continuous segment between the hIgHV4-28 and hIgHV1-2 genes upstream of the first continuous segment.

4. The method according to claim 1.

5. The step (a1) (a11) inserting a continuous segment between the hIgHD6-25 and hIgHJ6 genes between the mIgHJ region and the mIgHC region; (a12) inserting a continuous segment between the hIgHD6-13 and hIgHD5-24 genes upstream of the insertion segment of step (a11); (a13) inserting a continuous segment between the hIgHD1-1 and hIgHD5-12 genes upstream of the insertion segment of step (a12); and (a14) inserting a continuous segment between hIgHV6-1 and the upstream of hIgHD1-1 gene and loxP and lox2272-PB5' sites upstream of the inserted segment of step (a13). The method of claim 4.

6. The step (a2) a step (a21) of recombining a BAC vector and Cre recombinase, the BAC vector including a second continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene, and loxP and lox2272 sites located at both ends of the second continuous segment and oriented in the same direction as in step (a14), with the genome obtained in step (a1), wherein PB3' is included between the lox2272 site and the second continuous segment; and (a22) contacting the genome to be recombined obtained in step (a21) with PiggyBac transposase and screening for genomes containing the first partial segment and a loxP site upstream of the first partial segment. The method of claim 5.

7. In step (b), the second partial hIgHV contiguous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps: (b1) inserting a continuous segment between the hIgHV3-74 and hIgHV3-72 genes between the mIgHJ region and the mIgHC region; (b2) inserting a continuous segment between hIgHV2-70 and hIgHV1-69D genes and PB3'-lox5171 and a loxP site downstream of the inserted segment of step (b1); Step (b3) of recombining a BAC vector containing loxP and lox5171 located at both ends of a continuous segment between hIgHV1-69-2 and hIgHV3-30 genes and in the same orientation as in step (b2), and Cre recombinase with the genome obtained in step (b2), wherein PB5' is contained between the lox5171 site and the continuous segment in step (b3); and (b4) contacting the genome to be recombined obtained in step (b3) with PiggyBac transposase and screening for genomes containing the second partial segment and a loxP site located downstream of the second partial segment.

7. The method according to any one of claims 1 to 6.

8. Step (c) (c1) screening Cre-positive mice comprising the first partial segment and the second partial segment; and (c2) crossing the mice obtained in step (c1) with wild-type mice and screening for Cre-free mice comprising a first partial segment and a second partial segment, wherein a loxP site is present between the second partial segment and the first partial segment. The method of claim 7.

9. Step (d) (d1) crossing the genetically modified mouse with a wild-type mouse and screening for a positive mouse; and (d2) crossbreeding the male and female positive mice obtained in step (d1) with each other and screening for homozygous mice. The method of claim 1.

10. 1. A genetically modified mouse genome, comprising: The immunoglobulin heavy chain locus of the genetically modified mouse comprises, in this order: (i) a mouse Adam6a gene; (ii) a mouse Adam6b gene; (iii) a second partial segment of a human immunoglobulin heavy chain variable region locus comprising a second partial hIgHV contiguous segment; (iv) a first partial segment of a human immunoglobulin heavy chain variable region locus located downstream of the second partial segment and comprising the first partial hIgHV contiguous segment, all hIgHD segments, and all hIgHJ segments, wherein the first partial segment does not include a segment between downstream of the hIgHV1-2 gene and upstream of the hIgHV6-1 gene; and (v) an mIgHC region. A genetically modified mouse genome characterized by:

11. (i) the second portion hIgHV contiguous segment comprises or is a contiguous segment between hIgHV3-74 gene and hIgHV3-30 gene; (ii) the first partial segment comprises a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene and a continuous segment between the hIgHV6-1 gene and the hIgHJ6 gene located downstream, or is these two continuous segments; (iii) the immunoglobulin heavy chain locus of the mouse does not include the mIgHV5-1 gene and all mIgHV segments upstream thereof, the contiguous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes; (iv) the mice do not have reduced fertility compared to non-genetically modified wild-type mice; (v) the first sub-segment and the second sub-segment are not rearranged; and (vi) the mouse is capable of producing a human-mouse chimeric antibody, the chimeric antibody having one or more of the following characteristics: a human heavy chain variable region and a mouse constant region. The mouse genome of claim 10.

12. A cell, tissue, organ, or mouse comprising the mouse genome of claim 10 or 11, Preferably, said cell is an embryonic cell, a B cell or a hybridoma cell, preferably said tissue is the white pulp of the spleen or a lymph node thereof, preferably said organ is the spleen, a cell, a tissue, an organ or a mouse.

13. A method for producing a monoclonal antibody, comprising: (a) immunizing a mouse having the genome of claim 10 or 11 with an antigen; (b) isolating from said mouse cells containing monoclonal antibodies against said antigen; and (c) culturing the cells to obtain the monoclonal antibody; Preferably, the cell is a spleen cell, a B cell or a hybridoma cell, and preferably, the monoclonal antibody has a human heavy chain variable region and does not have a mouse heavy chain variable region.

14. Use of the cell, tissue, organ, or mouse according to claim 12 in the production of a monoclonal antibody, Preferably, the monoclonal antibody has a human heavy chain variable region and no mouse heavy chain variable region, and preferably, the monoclonal antibody has a human heavy chain variable region and a mouse constant region.

10. The use characterized by:

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