Genetically modified rodent for preparing quadrivalent fixed light chain and preparation method therefor

Abstract

Disclosed is a genetically modified rodent, in which an immunoglobulin light chain locus is modified to insert a human immunoglobulin light chain gene segment. The rodent is capable of normal reproduction and of producing antibodies containing human light chains. Further provided are a method for preparing the genetically modified rodent and the use.

Description

Genetically modified rodents for making tetra-fixed light chains and methods of making the same TECHNICAL FIELD

[0001] The present invention relates to genetically modified rodents (e.g., mice), cells, embryos, and tissues, and in particular, the present invention relates to the humanization of the immunoglobulin Kappa light chain variable region locus of rodents (e.g., mice) and methods thereof. The present invention also relates to the genome of the modified rodents, cells, tissues comprising the genome, and methods and uses of making monoclonal antibodies using the rodents. The present invention also relates to rodents having a modified genome. BACKGROUND

[0002] Synthesis and expression of bispecific binding proteins has been problematic, in part due to difficulties in identifying appropriate light chains that can associate with two different heavy chains and be expressed together, and in part due to problems with isolation. Mice can be made that express a common light chain, the antibodies expressed by which have heavy chains that can associate with and be expressed with the same or substantially the same light chain. Such mice are particularly useful in making bispecific antibodies. For example, a mouse can be immunized with a first immunogen to produce B cells expressing antibodies that specifically bind to a first epitope. The mouse (or a genetically identical mouse) can be immunized with a second immunogen to produce B cells expressing antibodies that specifically bind to a second epitope. SUMMARY

[0003] One aspect of the present invention provides a method of making a genetically modified rodent, the method comprising (i) knocking out a continuous segment of an endogenous V region to J region of an immunoglobulin Kappa light chain locus of the rodent; and (ii) introducing into the immunoglobulin Kappa light chain locus of the rodent a human immunoglobulin Kappa light chain gene segment operably linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent; wherein the human immunoglobulin Kappa light chain gene segment comprises only four human immunoglobulin Kappa light chain V genes and only one human immunoglobulin Kappa light chain J gene; wherein the four human immunoglobulin Kappa light chain V genes are selected from the group consisting of: hlgKV1-39, hlgKV1-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1; and the one human immunoglobulin Kappa light chain J gene is selected from the group consisting of: hlgKJ1, hlgKJ2, hlgKJ3, hlgKJ4, and hlgKJ5.

[0004] In another aspect, the present application provides a genetically modified rodent genome, wherein the immunoglobulin Kappa light chain locus of the genetically modified rodent genome does not contain a continuous segment of the V region to the J region of the endogenous immunoglobulin Kappa light chain locus of the rodent; and the immunoglobulin Kappa light chain locus of the rodent genome comprises a human immunoglobulin Kappa light chain gene segment operably linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent; wherein the human immunoglobulin Kappa light chain gene segment comprises only four human immunoglobulin Kappa light chain V genes and only one human immunoglobulin Kappa light chain J gene; wherein the four human immunoglobulin Kappa light chain V genes are selected from the group consisting of hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1; and wherein the one human immunoglobulin Kappa light chain J gene is selected from the group consisting of hlgKJl, hlgKJ2, hlgKJ3, hlgKJ4, and hlgKJ5.

[0005] In another aspect, the present application relates to a cell, tissue, organ, or rodent comprising the rodent genome of any one described herein. In another aspect, the present application provides a method of making a monoclonal antibody.

[0006] The present application will construct into 2 models using the most frequent hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1 genes in VH and VL pairing, which is faster to construct, and can be obtained within 1 year, but can cover most of the antibody screening. The present application can be used in common light chain model applications, without immunizing multiple models when immunizing antigens, antibodies can be produced in the same mouse, and light chain diversity is maintained in the same mouse. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 shows the gene orientation and arrangement of the human IgK locus, wherein green indicates functional V genes; yellow indicates V region ORFs; red indicates V region pseudogenes; and blue indicates functional C genes.

[0008] Figure 2 shows the gene orientation and arrangement of the mouse IgK locus, wherein green indicates functional V genes; yellow indicates V region ORFs; red indicates V region pseudogenes; and blue indicates functional C genes.

[0009] Figure 3 shows the Kappa light chain genome of a mouse constructed according to Example 1 of the present application.

[0010] Figure 4 shows the Kappa light chain genome of the mouse constructed according to Example 2 of the present application.

[0011] Figure 5 shows the map of the plasmid vector constructed according to Example 1 shown in Figure 3.

[0012] Figure 6 shows the map of the plasmid vector constructed according to Example 2 shown in Figure 4.

[0013] Figure 7 shows the PCR identification region of Example 1 shown in Figure 3.

[0014] Figures 8 to 11 show the PCR identification results of Example 1.

[0015] Figure 12 shows the PCR identification region of the Fl mice obtained in Example 1.

[0016] Figures 13 to 19 show the PCR identification results of the Fl mice of Example 1.

[0017] Figure 20 shows the PCR identification region of Example 2 shown in Figure 4.

[0018] Figures 21 to 24 show the PCR identification results of Example 1.

[0019] Figure 25 shows the PCR identification region of the Fl mice obtained in Example 2.

[0020] Figures 26 to 31 show the PCR identification results of the Fl mice of Example 2.

[0021] Figure 32 shows the proportion of B cells (CD45+CD19+) in the spleen tissue of the double gene homozygous mice.

[0022] Figure 33 shows the deletion of Lambda type B cells in the spleen tissue of the double gene homozygous mice.

[0023] Figure 34 shows the frequency of four human light chain V genes of the double gene homozygous mice.

[0024] Figures 35 and 36 show whether the antibody sequences of the double gene homozygous mice of the present application are mutated and the mutation positions.

[0025] Figures 37 and 38 show the serum titer detection after immunizing the double gene homozygous mice of the present application. DETAILED DESCRIPTION

[0026] DEFINITIONS

[0027] “hIgKV” refers to the V region of the human (h) immunoglobulin kappa light chain variable region locus in the present invention, and when used independently refers to the entire V region of the human immunoglobulin kappa light chain variable region locus, and when suffixed with a specific gene number, e.g., “hIgKV1-39”, refers to the 1-39 genes located in the V region of the human immunoglobulin kappa light chain variable region locus. Similarly, “hIgKJ” is also used in the present invention to refer to the J region of the human immunoglobulin kappa light chain variable region locus. The human immunoglobulin kappa light chain locus is located on human chromosome 2 (position 2pl l.2), and the orientation and arrangement can be found in IMGT Repertoire (IG and TR), reproduced as Figure 1, and the sequences of each gene and the linking sequences between genes can be found in NCBI Reference Sequence number NC_000002.12. The term “Vk+encoding” is used in the present invention identically to “IgKV+encoding”. The term “Jk+encoding” is used in the present invention identically to “IgKJ+encoding”.

[0028] “mIgKV” refers to the V region of the mouse (m) immunoglobulin kappa light chain variable region locus in the present invention, and when used independently refers to the entire V region of the mouse immunoglobulin kappa light chain variable region locus, and when suffixed with a specific gene number, e.g., “mIgKV2-137”, refers to the 2-137 genes located in the V region of the mouse immunoglobulin kappa light chain variable region locus. Similarly, “mIgKJ” is also used in the present invention to refer to the J region of the mouse immunoglobulin kappa light chain variable region locus; and “mIgKC” refers to the mouse immunoglobulin kappa light chain constant region locus. The mouse immunoglobulin kappa light chain locus is located on mouse chromosome 6 (position 6C1), and the orientation and arrangement can be found in IMGT Repertoire (IG and TR), reproduced as Figure 2, and the sequences of each gene and the linking sequences between genes can be found in NCBI Reference Sequence number NC_000072.7.

[0029] “Contiguous segment” refers to a non-interrupted stretch of nucleotides between two endpoint genes, which includes functional genes, pseudogenes, ORFs, and other nucleotide sequences (e.g., spacer sequences) located between the two endpoint genes. The term “contiguous segment from A to B” refers to a contiguous segment of genes including A, B, and a stretch of genes between the two.

[0030] “Operably linked” includes bringing two or more components (e.g., a V gene and a C gene) into juxtaposition such that each component functions normally. For example, one or more genes of hIgKV operably linked to a gene of hIgKJ to a region of mIgKC refers to each being capable of performing its natural function, including rearrangement to form a diverse antibody light chain variable region, when the components are linked.

[0031] “hIgKV1-39” is human immunoglobulin kappa light chain locus V region 1-39 gene, Gene ID: 28930, its NCBI Reference Sequence number is NC_000002.12: c89320099-89319625, 522 bp, the gene sequence is shown as SEQ ID NO. 60. The nucleotide sequence comprising the gene fragment of hIgKV1-39 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 1.

[0032] “hIgKV1-33” is human immunoglobulin kappa light chain locus V region 1-33 gene, Gene ID: 28933, its NCBI Reference Sequence number is NC_000002.12: c89268475-89268001, 475 bp, the gene sequence is shown as SEQ ID NO. 61. The nucleotide sequence comprising the gene fragment of hIgKV1-33 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 5.

[0033] “hIgKV2-28” is human immunoglobulin kappa light chain locus V region 2-28 gene, Gene ID: 28921, its NCBI Reference Sequence number is NC_000002.12: c89222431-89221698, 734 bp, the gene sequence is shown as SEQ ID NO. 62. The nucleotide sequence comprising the gene fragment of hIgKV2-28 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 2.

[0034] “hIgKV2-30” is human immunoglobulin kappa light chain locus V region 2-30 gene, Gene ID: 28919, its NCBI Reference Sequence number is NC_000002.12: c89245566-89244781, 786 bp, the gene sequence is shown as SEQ ID NO. 63. The nucleotide sequence comprising the gene fragment of hIgKV2-30 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 6.

[0035] “hIgKV3-20” is human immunoglobulin kappa light chain locus V region 3-20 gene, Gene ID: 28912, its NCBI Reference Sequence number is NC_000002.12: c89143108-89142574, 535 bp, the gene sequence is shown as SEQ ID NO. 64. The nucleotide sequence comprising the gene fragment of hIgKV3-20 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 3.

[0036] “hIgKV3-15” is human immunoglobulin kappa light chain locus V region 3-15 gene, Gene ID: 28913, its NCBI Reference Sequence number is NC_000002.12: c89085690-89085177, 514 bp, the gene sequence is shown as SEQ ID NO. 65. The nucleotide sequence comprising the gene fragment of hIgKV3-15 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 7.

[0037] “hIgKV3-11” is human immunoglobulin kappa light chain locus V region 3-11 gene, Gene ID: 28914, its NCBI Reference Sequence number is NC_000002.12: c89027684-89027171, 514 bp, the gene sequence is shown as SEQ ID NO. 66. The nucleotide sequence comprising the gene fragment of hIgKV3-11 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 8.

[0038] “hIgKV4-1” is human immunoglobulin kappa light chain locus V region 4-1 gene, Gene ID: 28908, its NCBI Reference Sequence number is NC_000002.12: 88885572-88886153, 582 bp, the gene sequence is shown as SEQ ID NO. 67. The nucleotide sequence comprising the gene fragment of hIgKV4-1 and its upstream 3 kb and downstream 500 bp is shown as SEQ ID NO. 4.

[0039] “hIgKJ1” is human immunoglobulin kappa light chain locus J region J1 gene, Gene ID: 28950, its NCBI Reference Sequence number is NC_000002.12: c88861923-88861886, 38 bp, the gene sequence is shown as SEQ ID NO. 68. The nucleotide sequence comprising the gene fragment of hIgKJ1 and its upstream 500 bp and downstream 272 bp is shown as SEQ ID NO. 9.

[0040] “hIgKJ2” is human immunoglobulin kappa light chain locus J region J2 gene, Gene ID: 28949, its NCBI Reference Sequence number is NC_000002.12: c88861563-88861525, 39 bp, the gene sequence is shown as SEQ ID NO. 69.

[0041] “hIgKJ3” is the human immunoglobulin Kappa light chain locus J region J3 gene, Gene ID: 28948, with NCBI Reference Sequence number NC_000002.12: c88861258-88861221, 38 bp, with the gene sequence as set forth in SEQ ID NO. 70.

[0042] “hIgKJ4” is the human immunoglobulin Kappa light chain locus J region J4 gene, Gene ID: 28947, with NCBI Reference Sequence number NC_000002.12: c88860923-88860886, 38 bp, with the gene sequence as set forth in SEQ ID NO. 71.

[0043] “hIgKJ5” is the human immunoglobulin Kappa light chain locus J region J5 gene, Gene ID: 28946, with NCBI Reference Sequence number NC_000002.12: c88860605-88860568, 38 bp, with the gene sequence as set forth in SEQ ID NO. 72.

[0044] Methods of making a genetically modified rodent and genomes thereof

[0045] A first aspect of the present application provides a method of making a genetically modified rodent, the method comprising (i) knocking out a continuous segment of endogenous V region to J region of an immunoglobulin Kappa light chain locus of the rodent; and (ii) introducing a human immunoglobulin Kappa light chain gene segment within the immunoglobulin Kappa light chain locus of the rodent, operably linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent; wherein the human immunoglobulin Kappa light chain gene segment comprises only four human immunoglobulin Kappa light chain V genes and only one human immunoglobulin Kappa light chain J gene; wherein the four human immunoglobulin Kappa light chain V genes are selected from the group consisting of: hIgKV1-39, hIgKV1-33, hIgKV2-28, hIgKV2-30, hIgKV3-20, hIgKV3-15, hIgKV3-11, and hIgKV4-1; and the one human immunoglobulin Kappa light chain J gene is selected from the group consisting of: hIgKJ1, hIgKJ2, hIgKJ3, hIgKJ4, and hIgKJ5.

[0046] A second aspect of the application provides a genetically modified rodent genome, wherein the immunoglobulin Kappa light chain locus of the genetically modified rodent genome does not contain a continuous segment of the V region to the J region of the endogenous immunoglobulin Kappa light chain locus of the rodent; and the immunoglobulin Kappa light chain locus of the rodent genome comprises a human immunoglobulin Kappa light chain gene segment operably linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent; wherein the human immunoglobulin Kappa light chain gene segment comprises only four human immunoglobulin Kappa light chain V genes and only one human immunoglobulin Kappa light chain J gene; wherein the four human immunoglobulin Kappa light chain V genes are selected from the group consisting of: hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1; and wherein the one human immunoglobulin Kappa light chain J gene is selected from the group consisting of: hlgKJl, hlgKJ2, hlgKJ3, hlgKJ4, and hlgKJ5.

[0047] In some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are one of each of: hlgKVl-39 and hlgKVl-33; hlgKV2-28 and hlgKV2-30; hlgKV3-20, hlgKV3-15, and hlgKV3-11; and hlgKV4-1.

[0048] In some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are one of each of: hlgKVl-39 and hlgKVl-33; hlgKV2-28 and hlgKV2-30; hlgKV3-20 and hlgKV3-15; and one of hlgKV4-1 and hlgKV3-11.

[0049] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-39, hlgKV2-28, hlgKV3-20, hlgKV4-1, respectively.

[0050] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV3-11, respectively.

[0051] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-39, hlgKV2-30, hlgKV3-20, hlgKV4-1, respectively.

[0052] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-39, hlgKV2-28, hlgKV3-15, hlgKV4-1, respectively.

[0053] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-39, hlgKV2-28, hlgKV3-11, hlgKV4-1, respectively.

[0054] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-39, hlgKV2-30, hlgKV3-15, hlgKV4-1, respectively.

[0055] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-39, hlgKV2-30, hlgKV3-11, hlgKV4-1, respectively.

[0056] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-28, hlgKV3-15, hlgKV3-11, respectively.

[0057] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-28, hlgKV3-20, hlgKV3-11, respectively.

[0058] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV3-11, respectively.

[0059] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-28, hlgKV3-15, hlgKV4-1, respectively.

[0060] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-28, hlgKV3-11, hlgKV4-1, respectively.

[0061] For example, the four human immunoglobulin Kappa light chain V genes are: hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV4-1, respectively.

[0062] For example, the four human immunoglobulin Kappa light chain V genes are, respectively: hlgKVl-33, hlgKV2-30, hlgKV3-11, hlgKV4-1.

[0063] In some embodiments of any of the aspects, for the above described and exemplified embodiments of the four human immunoglobulin Kappa light chain V genes, the one human immunoglobulin Kappa light chain J gene is hlgKJl. In some embodiments of any of the aspects, for the above described and exemplified embodiments of the four human immunoglobulin Kappa light chain V genes, the one human immunoglobulin Kappa light chain J gene is hlgKJ2. In some embodiments of any of the aspects, for the above described and exemplified embodiments of the four human immunoglobulin Kappa light chain V genes, the one human immunoglobulin Kappa light chain J gene is hlgKJ3. In some embodiments of any of the aspects, for the above described and exemplified embodiments of the four human immunoglobulin Kappa light chain V genes, the one human immunoglobulin Kappa light chain J gene is hlgKJ4. In some embodiments of any of the aspects, for the above described and exemplified embodiments of the four human immunoglobulin Kappa light chain V genes, the one human immunoglobulin Kappa light chain J gene is hlgKJ5.

[0064] For example, in some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are, respectively: one of hlgKVl-39 and hlgKVl-33; one of hlgKV2-28 and hlgKV2-30; one of hlgKV3-20, hlgKV3-15, and hlgKV3-11; and hlgKV4-1; and the one human immunoglobulin Kappa light chain J gene is hlgKJl.

[0065] For example, in some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are, respectively: one of hlgKVl-39 and hlgKVl-33; one of hlgKV2-28 and hlgKV2-30; one of hlgKV3-20 and hlgKV3-15; and one of hlgKV4-1 and hlgKV3-11; and the one human immunoglobulin Kappa light chain J gene is hlgKJl.

[0066] For example, in some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are hlgKVl-39, hlgKV2-28, hlgKV3-20, hlgKV4-1, respectively; and the one human immunoglobulin Kappa light chain J gene is hlgKJl.

[0067] For example, in some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV3-11, respectively; and the one human immunoglobulin Kappa light chain J gene is hlgKJl.

[0068] Other combinations of the four human immunoglobulin Kappa light chain V genes and the one human immunoglobulin Kappa light chain J gene can be similarly derived from the above description, and are not repeated here for brevity.

[0069] In the present application, the four human immunoglobulin Kappa light chain V genes can be connected to each other in any order. In some embodiments, the four human immunoglobulin Kappa light chain V genes are connected in the order recited.

[0070] In some embodiments of any of the aspects, the one human immunoglobulin Kappa light chain J gene is downstream of and directly connected to the most downstream one of the four human immunoglobulin Kappa light chain V genes.

[0071] In some embodiments of any of the aspects, the order of connection of the human immunoglobulin Kappa light chain gene segments is: hlgKVl-39, hlgKV2-28, hlgKV3-20, hlgKV4-1, hlgKJl.

[0072] In some embodiments, the order of connection of the human immunoglobulin Kappa light chain gene segments is: hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV3-11, hlgKJl.

[0073] In some embodiments of any of the aspects, the four human immunoglobulin Kappa light chain V genes are connected via the upstream and downstream natural adjacent sequences of the adjacent genes. In some embodiments, the four human immunoglobulin Kappa light chain V genes do not contain rodent endogenous gene fragments.

[0074] In some embodiments of any of the aspects, the upstream natural flanking sequence of each human immunoglobulin Kappa light chain V gene is 500 bp to 3 kb, preferably 1 kb to 3 kb, more preferably 2 kb to 3 kb, upstream of the respective gene.

[0075] In some embodiments of any of the aspects, the downstream natural flanking sequence of each human immunoglobulin Kappa light chain V gene is 50 bp to 500 bp; preferably 100 bp to 500 bp, more preferably 200 bp to 500 bp, downstream of the respective gene.

[0076] In some embodiments of any of the aspects, the human immunoglobulin Kappa light chain J gene is linked to the most downstream one of the four human immunoglobulin Kappa light chain V genes via its upstream natural flanking sequence; preferably, the upstream natural flanking sequence of the human immunoglobulin Kappa light chain J gene is 50 bp to 500 bp, preferably 100 bp to 500 bp, more preferably 200 bp to 500 bp, upstream of the human immunoglobulin Kappa light chain J gene.

[0077] In some embodiments of any of the aspects, the human immunoglobulin Kappa light chain J gene is linked to the most downstream one of the four human immunoglobulin Kappa light chain V genes via its downstream natural flanking sequence; preferably, the downstream natural flanking sequence of the human immunoglobulin Kappa light chain J gene is 50 bp to 280 bp, preferably 100 bp to 280 bp, more preferably 200 bp to 280 bp, downstream of the human immunoglobulin Kappa light chain J gene.

[0078] In some embodiments of any of the aspects, the human immunoglobulin Kappa light chain J gene is linked to the most downstream one of the four human immunoglobulin Kappa light chain V genes via its downstream natural flanking sequence; preferably, the downstream natural flanking sequence of the human immunoglobulin Kappa light chain J gene is 50 bp to 280 bp, preferably 100 bp to 280 bp, more preferably 200 bp to 280 bp, downstream of the human immunoglobulin Kappa light chain J gene.

[0079] For example, in some embodiments of any of the aspects, the human immunoglobulin Kappa light chain gene segments are: [upstream natural flanking sequence]-hlgKVl-39-[downstream natural flanking sequence], [upstream natural flanking sequence]-hlgKV2-28-[downstream natural flanking sequence], [upstream natural flanking sequence]-hlgKV3-20-[downstream natural flanking sequence], [upstream natural flanking sequence]-hlgKV4-l-[downstream natural flanking sequence], [upstream natural flanking sequence]-hlgKJl-[downstream natural flanking sequence]; wherein the upstream and downstream natural flanking sequences have the lengths or preferred lengths described above, respectively.

[0080] For example, in some embodiments of any one of the aspects, the human immunoglobulin Kappa light chain gene segment is: [upstream native flanking sequence]-hIgKV1-33- [downstream native flanking sequence], [upstream native flanking sequence]-hIgKV2-30- [downstream native flanking sequence], [upstream native flanking sequence]-hIgKV3-15- [downstream native flanking sequence], [upstream native flanking sequence]-hIgKV3-11- [downstream native flanking sequence], [upstream native flanking sequence]-hIgKJ1- [downstream native flanking sequence]; wherein the upstream and downstream native flanking sequences have the lengths or preferred lengths described above, respectively.

[0081] It will be appreciated by those skilled in the art that the various V and J gene segments in other embodiments of the human immunoglobulin Kappa light chain gene segments described and exemplified herein can also be linked by analogous means with the upstream and downstream native flanking sequences of adjacent genes to form various embodiments, which for the sake of brevity are not listed individually.

[0082] In some embodiments of any one of the aspects, the rodent can be a mouse, such that the endogenous V region to J region contiguous segment of the immunoglobulin Kappa light chain locus of the mouse refers to a contiguous segment of mIgKV2-137 to mIgKJ5.

[0083] In some embodiments of any one of the aspects, the present application preserves or does not disrupt the endogenous 5'-enhancer and 3'-enhancer of the mouse Kappa light chain constant region gene (mIgKC).

[0084] In some embodiments of any one of the aspects, the immunoglobulin heavy chain variable region locus of the rodent is humanized. Methods of humanizing the immunoglobulin heavy chain variable region locus of a rodent are known in the art, including but not limited to those described in WO2013187953A1, WO2013116609A1, WO2013059230A1, WO2011072204A1, WO2013041844A2, WO2011004192A1, WO2011158009A1, or WO2013079953A1. The present application contemplates all such methods of modifying the heavy chain variable region genome.

[0085] Cells, tissues, animals, and methods of making antibodies

[0086] A third aspect of the present application relates to a cell, tissue, organ, or rodent comprising the rodent genome of any one described herein.

[0087] In some embodiments, the present application provides a cell comprising the rodent genome of any one described herein, the cell being an embryonic cell, a B cell, or a hybridoma cell.

[0088] In some embodiments, the present application provides a tissue comprising the rodent genome of any one described herein, the tissue being white pulp of a spleen or its lymph nodule.

[0089] In some embodiments, the present application provides an organ comprising the rodent genome of any one described herein, the organ being a spleen.

[0090] In some embodiments, the present application provides a rodent comprising the rodent genome of any one described herein, the rodent being a mouse.

[0091] A fourth aspect of the present application provides a method of making a monoclonal antibody, comprising

[0092] (a) immunizing a rodent having any one of the genomes described in the present application with an antigen;

[0093] (b) isolating from the rodent a cell that produces a monoclonal antibody comprising an antibody against the antigen; and

[0094] (c) culturing the cell to obtain the monoclonal antibody.

[0095] In some embodiments, the cell of step (c) is a spleen cell, a B cell, or a hybridoma cell.

[0096] In some embodiments, the method further comprises

[0097] (d) identifying the monoclonal antibody having the same light chain.

[0098] In some embodiments, the monoclonal antibody is a multispecific antibody; preferably a bispecific antibody.

[0099] In some embodiments, the monoclonal antibody has a human heavy chain variable region, a human Kappa light chain variable region, a mouse heavy chain constant region, a mouse Kappa light chain constant region. In some embodiments, the monoclonal antibody does not have a mouse heavy chain variable region and a mouse Kappa light chain variable region.

[0100] A fifth aspect of the present application provides use of any of the cells, tissues, organs or rodents of the present application in the preparation of a monoclonal antibody. In some embodiments, the monoclonal antibody is a multispecific antibody; preferably a bispecific antibody. In some embodiments, the monoclonal antibody has a human heavy chain variable region, a human kappa light chain variable region, a mouse heavy chain constant region, a mouse kappa light chain constant region. In some embodiments, the monoclonal antibody does not have a mouse heavy chain variable region and a mouse kappa light chain variable region.

[0101] The present application will construct the eight genes with the highest frequency of use in VH and VL pairing, hIgKV1-39, hIgKV1-33, hIgKV2-28, hIgKV2-30, hIgKV3-20, hIgKV3-15, hIgKV3-11 and hIgKV4-1 into two models, which can be constructed faster, and can cover most of the antibody screening in one year. The present application can be used in common light chain model application, when immunized with antigen, without immunizing multiple models, antibodies can be produced in the same mouse, and light chain diversity is maintained in the same mouse.

[0102] SEQUENCE LISTING

[0103] Example 1. Construction of four-gene mouse model 1

[0104] The region of mouse Vk2-137 to Igkj5 is replaced with human arrangement genes: Vk1-39, Vk2-28, Vk3-20, Vk4-1 and IgKJ1 gene, and the strategy diagram is shown in Figure 3.

[0105] KI (knock-in) sequence: four different Vk genes (upstream 3 kb sequence to downstream 500 bp gene fragment, synthesized by gene synthesis, sequences are shown in SEQ ID NO. 1 to 4) and one Jk gene (upstream 500 bp to downstream 272 bp, sequence is shown in SEQ ID NO. 9) are synthesized respectively, and the whole KI sequence is about 17 kb in length.

[0106] Homologous arm sequence: 5' arm homologous arm and 3' arm homologous arm were amplified from C57BL / 6 mouse genome by two pairs of primers, the amplification primers and amplification conditions are shown as follows.

[0107] 5F (SEQ ID NO. 10): 5'-GCAAGCAAGATTCAGTCATTGGGTGAG-3'

[0108]

[0109] 5R (SEQ ID NO. 11): 5'-GCAAGCAAGATTCAGTCATTGGGTGAG-3'

[0110]

[0111] 3F (SEQ ID NO. 12): 5'-GCAAGCAAGATTCAGTCATTGGGTGAG-3'

[0112]

[0113] 3R (SEQ ID NO. 13): 5'-GCAAGCAAGATTCAGTCATTGGGTGAG-3'

[0114]

[0115] 5' arm homologous arm was amplified by 5F / 5R, 3' arm homologous arm sequence was amplified by 3F / 3R, and finally 4 Vk genes and 1 Jk gene and Neo (SEQ ID NO. 14), 5' arm and 3' arm homologous arm sequence were connected into pUC57 backbone by infusion to obtain a vector plasmid, and the plasmid map is shown in Figure 5.

[0116] The vector was electroporated into wild-type ES cells, and different clones were obtained after cell culture and Neo resistance screening, and then the clones were selected and cultured, and sent for PCR genotype identification to obtain the final correct positive clones.

[0117] The selected cell clones were subjected to PCR amplification by 4 pairs of primers, and the identification region is shown in Figure 7, and 2F9-1C1, 2F9-1D1, 2F9-1A2, 2F9-1E2, 2F9-1C3, 2F9-1H3, 2F9-1A5, 2F9-1G5, 2F9-1A6, 2F9-1H7, 2F9-1A10, 2F9-1C10, 2F9-1D10, 2F9-1H10, 2F9-1E11, 2F9-1F11, 2F9-1B12, a total of 17 clones were confirmed as positive clones.

[0118] PCR identification primers:

[0119] F1 (SEQ ID NO. 15): 5'-GCAAGCAAGATTCAGTCATTGGGTGAG-3'

[0120] R1 (SEQ ID NO. 16): 5'-CCTACAGATAAGGAACAATGGGGCCA-3'

[0121] Expected PCR Product:

[0122] Wildtype: N.A.

[0123] Targeted: 2227bp

[0124] Results are shown in Figure 8.

[0125] PCR identification primers:

[0126] 1F2 (SEQ ID NO. 17): 5'-TGTCTGCCTGTTGACTGTATCTAA-3'

[0127] 1R2 (SEQ ID NO. 18): 5'-CGGAACCCTTCGAAGTTCCTATTCT-3'

[0128] Expected PCR Product:

[0129] Wildtype: N.A.

[0130] Targeted: 231bp

[0131] Results are shown in Figure 9.

[0132] PCR identification primers:

[0133] 1F3 (SEQ ID NO. 19): 5'-GCTAGCTTGGCTGGACGTA-3'

[0134] 1R3 (SEQ ID NO. 20): 5'-GATGTGCTCTTGCCCTCGAA-3'

[0135] Expected PCR Product:

[0136] Wildtype: N.A.

[0137] Targeted: 356bp

[0138] Results are shown in Figure 10.

[0139] PCR identification primers:

[0140] 1F4 (SEQ ID NO. 21): 5'-TACAAGTCCACCTGAGGAGTCT-3'

[0141] 1R4 (SEQ ID NO. 22): 5'-TGGTTTAGCATTCGCTCTGCT-3'

[0142] Expected PCR Product:

[0143] Wildtype: N.A.

[0144] Targeted: 291bp

[0145] The results are shown in Figure 11.

[0146] The 2F9-1A5 positive cell clones were transplanted into surrogate white B6 mice, and the surrogate mice gave birth to F0 mice after 19-21 days. The F0 mice were black in color, and the chimerism rate was 100%. The mice were confirmed to be positive F0 mice. The F0 mice were bred to sexual maturity and mated with wild-type mice, and the F1 generation was born. At 5-7 days, the F1 mice were identified by PCR, and positive F1 mice were obtained.

[0147] As shown in Figure 12, 7 F1 mice were PCR amplified by 8 pairs of primers, and a total of 89#, 92#, 93#, and 95# mice were confirmed to be heterozygous mice. The F1 mice were subjected to NGS sequencing, and it was confirmed that the human sequence was rearranged in the mouse body. The F1 mice were mated with each other, and Fn generation mice were finally obtained. PCR identification confirmed that the homozygous mice were born. The homozygous mice were immunized, and it was confirmed that the antibody sequence was produced after antigen immunization.

[0148] Primers for KI1 PCR:

[0149] M1F2 (SEQ ID NO. 29): 5'-TACAAGTCCACCTGAGGAGTCT-3'

[0150] M1R2 (SEQ ID NO. 30): 5'-TGGTTTAGCATTCGCTCTGCT-3'

[0151] Internal control PCR primer A1 (SEQ ID NO. 31):

[0152] 5'-CTATCAGGGATACTCCTCTTTGCC-3'

[0153] Internal control PCR primer A2 (SEQ ID NO. 32):

[0154] 5'-GATACAGGAATGACAAGCTCATGGT-3'

[0155] Expected PCR Product:

[0156] Wildtype: N.A.

[0157] Targeted: 291 bp

[0158] Internal control product size: 507 bp

[0159] Results are shown in Figure 13.

[0160] Primers for KI2 PCR:

[0161] M1F3 (SEQ ID NO. 33): 5'-AGTCCCACATGTTAAGGCCC-3'

[0162] M1R3 (SEQ ID NO. 34): 5'-ACTGAGTTGCTCCATGGTGA-3'

[0163] Internal control PCR primer A3 (SEQ ID NO. 35):

[0164] 5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0165] Internal control PCR primer A4 (SEQ ID NO. 36):

[0166] 5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0167] Expected PCR Product:

[0168] Wildtype: N.A.

[0169] Targeted: 504 bp

[0170] Internal control product size: 335 bp

[0171] Results are shown in Figure 14.

[0172] Primers for KI3 PCR:

[0173] M1F4 (SEQ ID NO. 37): 5'-GCACAGTTTCACAGTAATGGC-3'

[0174] M1R4 (SEQ ID NO. 38): 5'-GCATCGCTTTGGTCTGGAAA-3'

[0175] Internal control PCR primer A3 (SEQ ID NO. 35):

[0176] 5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0177] Internal control PCR primer A4 (SEQ ID NO. 36):

[0178] 5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0179] Expected PCR Product:

[0180] Wildtype: N.A.

[0181] Targeted: 510bp

[0182] Internal control product size: 335bp

[0183] Results are shown in Figure 15.

[0184] Primers for KI4 PCR:

[0185] M1F5 (SEQ ID NO. 39): 5'-ACACATCACATGACCGAGCC-3'

[0186] M1R5 (SEQ ID NO. 40): 5'-ACGTCTATGGCCTGATGGTTC-3'

[0187] Internal control PCR primer A3 (SEQ ID NO. 35):

[0188] 5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0189] Internal control PCR primer A4 (SEQ ID NO. 36):

[0190] 5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0191] Expected PCR Product:

[0192] Wildtype: N.A.

[0193] Targeted: 514bp

[0194] Internal control product size: 335bp

[0195] Results are shown in Figure 16.

[0196] Primers for KI5 PCR:

[0197] M1F6 (SEQ ID NO. 41): 5'-CAAGCAAGATTCAGTCATTGGGTG-3'

[0198] M1R6 (SEQ ID NO. 42): 5'-GCTGTTTCATCCTCTGGGTCATTC-3'

[0199] Internal control PCR primer A1 (SEQ ID NO. 31):

[0200] 5'-CTATCAGGGATACTCCTCTTTGCC-3'

[0201] Internal control PCR primer A2 (SEQ ID NO. 32):

[0202] 5'-GATACAGGAATGACAAGCTCATGGT-3'

[0203] Expected PCR Product:

[0204] Wildtype: N.A.

[0205] Targeted: 257bp

[0206] Internal control product size: 507bp

[0207] Results are shown in Figure 17.

[0208] Primers for Neo-del PCR:

[0209] M1F1 (SEQ ID NO. 43): 5'-TGTCTGCCTGTTGACTGTATCTAA-3'

[0210] M1R1 (SEQ ID NO. 44): 5'-GATGTGCTCTTGCCCTCGAA-3'

[0211] Internal control PCR primer A5 (SEQ ID NO. 45):

[0212] 5'-GCAGAAGAGGACAGATACATTCAT-3'

[0213] Internal control PCR primer A6 (SEQ ID NO. 46):

[0214] 5'-CCTACTGAAGAATCTATCCCACAG-3'

[0215] Expected PCR Product:

[0216] Wildtype: N.A.

[0217] Targeted: 378bp

[0218] Internal control product size: 689bp

[0219] Results are shown in Figure 18.

[0220] Primers for wildtype PCR:

[0221] M1F1 (SEQ ID NO. 43): 5'-TGTCTGCCTGTTGACTGTATCTAA-3'

[0222] M1R7 (SEQ ID NO. 47): 5'-GACCCCAATGAAGGAAACTAAATGG-3'

[0223] Internal control PCR primer A1 (SEQ ID NO. 31):

[0224] 5'-CTATCAGGGATACTCCTCTTTGCC-3'

[0225] Internal control PCR primer A2 (SEQ ID NO. 32):

[0226] 5'-GATACAGGAATGACAAGCTCATGGT-3'

[0227] Expected PCR Product:

[0228] Wildtype: 217bp

[0229] Targeted: N.A.

[0230] Internal control product size: 507bp

[0231] Results are shown in Figure 19.

[0232] Example 2. Construction of a four-gene mouse model 2

[0233] The region of mouse Vk2-137 to Igkj5 was replaced with human arrangement genes: Vk1-33, Vk2-30, Vk3-15, Vk3-11 and IgKJ1 gene, the strategy map is shown in Figure 4.

[0234] KI (knock-in) sequence: four different Vk genes (upstream 3 kb sequence to downstream 500 bp gene fragment, synthesized by gene synthesis, sequences are shown in SEQ ID NO. 5 to 8) and one Jk gene (upstream 500 bp to downstream 272 bp, sequence is shown in SEQ ID NO. 9) were synthesized respectively, the whole KI sequence is about 17 kb in length.

[0235] Homologous arm sequence: 5' arm homologous arm and 3' arm homologous arm were amplified from C57BL / 6 mouse genome by two pairs of primers, the amplification primers and amplification conditions are shown below.

[0236] 5F (SEQ ID NO. 10):

[0237]

[0238] 5R (SEQ ID NO. 11):

[0239]

[0240] 3F (SEQ ID NO. 12):

[0241]

[0242] 3R (SEQ ID NO. 13):

[0243]

[0244] The 5' arm homologous arm was amplified by 5F / 5R, the 3' arm homologous arm sequence was amplified by 3F / 3R, and finally the four Vk genes and one Jk gene and Neo, 5' arm and 3' arm homologous arm sequences were connected into the pUC57 backbone by infusion to obtain the vector plasmid, and the plasmid map is shown in Figure 6.

[0245] PCR amplification was performed on the selected cell clones by four pairs of primers, and the identification position is shown in Figure 20, and it was confirmed that 1H9-1C1, 1H9-1G1, 1H9-1H1, 1H9-1F2, 1H9-1G2, 1H9-1B3, 1H9-1C3, 1H9-1C4, 1H9-1C5, 1H9-1B6, 1H9-1E7, 1H9-1A8, 1H9-1E8, 1H9-1B9, 1H9-1C9, 1H9-1E9, a total of 16 clones were positive clones.

[0246] PCR identification primers:

[0247] F1 (SEQ ID NO. 15): 5'-GCAAGCAAGATTCAGTCATTGGGTGAG-3'

[0248] R1 (SEQ ID NO. 16): 5'-CCTACAGATAAGGAACAATGGGGCCA-3'

[0249] Expected PCR Product:

[0250] Wildtype: N.A.

[0251] Targeted: 2517bp

[0252] The results are shown in Figure 21.

[0253] PCR identification primers:

[0254] 2F2 (SEQ ID NO. 23): 5'-TTTGGCATATTGTTCAGAGGACA-3'

[0255] 2R2 (SEQ ID NO. 24): 5'-GTCCAACAAGAGGGAAAGAGACTG-3'

[0256] Expected PCR Product:

[0257] Wildtype: N.A.

[0258] Targeted: 631bp

[0259] Results are shown in Figure 22.

[0260] PCR identification primers:

[0261] 2F3 (SEQ ID NO. 25): 5'-AGGGCTTGAGTTGCAGAATTG-3'

[0262] 2R3 (SEQ ID NO. 26): 5'-CGGAACCCTTCGAAGTTCCTATT-3'

[0263] Expected PCR Product:

[0264] Wildtype: N.A.

[0265] Targeted: 331bp

[0266] Results are shown in Figure 23.

[0267] PCR identification primers:

[0268] 2F4 (SEQ ID NO. 27): 5'-TGGCTGGACGTAAACTCCTCTTC-3'

[0269] 2R4 (SEQ ID NO. 28): 5'-TGGAGCCATATGGGATCTCTCT-3'

[0270] Expected PCR Product:

[0271] Wildtype: N.A.

[0272] Targeted: 253bp

[0273] Results are shown in Figure 24.

[0274] The 1H9-1A8 clone was transplanted into a surrogate white B6 mouse, and the surrogate mouse gave birth to F0 mice after 19-21 days. The F0 mice were black in color, and the chimerism rate was 100%. The mice were confirmed to be positive F0 mice. The F0 mice were raised to sexual maturity and mated with wild-type mice, and the F1 generation was born. The F1 generation mice were 5-7 days old, and PCR identification was performed by cutting the claws. Positive F1 mice were obtained.

[0275] As shown in Figure 25, 15 F1 mice were PCR amplified by 6 pairs of primers, and it was confirmed that a total of 7 mice of 38#, 40#, 44#, 49#, 50#, 51# and 52# were heterozygous mice. The F1 mice were subjected to NGS sequencing to confirm that human sequence rearrangement occurred in mice. At the same time, the F1 mice were mated with each other, and finally Fn generation mice were obtained, which were subjected to PCR identification to confirm the birth of homozygous mice. The homozygous mice were immunized to confirm that antibody sequences were produced after antigen immunization.

[0276] Primers for KI1 PCR:

[0277] M2F3 (SEQ ID NO. 48): 5'-AAGGCCCACTTCATCGTAGC-3'

[0278] M2R3 (SEQ ID NO. 49): 5'-TCCACAGTCTTGTGCTGGAC-3'

[0279] Internal control PCR primer A3 (SEQ ID NO. 35):

[0280] 5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0281] Internal control PCR primer A4 (SEQ ID NO. 36):

[0282] 5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0283] Expected PCR Product:

[0284] Wildtype: N.A.

[0285] Targeted: 585bp

[0286] Internal control product size: 335bp

[0287] The results are shown in Figure 26.

[0288] Primers for KI2 PCR:

[0289] M2F4 (SEQ ID NO. 50): 5'-TTTGGCATATTGTTCAGAGGACA-3'

[0290] M2R4 (SEQ ID NO. 51): 5'-GTCCAACAAGAGGGAAAGAGACTG-3'

[0291] Internal control PCR primer A3 (SEQ ID NO. 35):

[0292] 5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0293] Internal control PCR primer A4 (SEQ ID NO. 36):

[0294] 5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0295] Expected PCR Product:

[0296] Wildtype: N.A.

[0297] Targeted: 631 bp

[0298] Internal control product size: 335 bp

[0299] Results are shown in Figure 27.

[0300] Primers for KI3 PCR:

[0301] M2F5 (SEQ ID NO. 52): 5'-ACGCATTTCAGAAGGCATCTC-3'

[0302] M2R5 (SEQ ID NO. 53): 5'-CATGAGCCATACTACCACCAAGA-3'

[0303] Internal control PCR primer A5 (SEQ ID NO. 45):

[0304] 5'-GCAGAAGAGGACAGATACATTCAT-3'

[0305] Internal control PCR primer A6 (SEQ ID NO. 46):

[0306] 5'-CCTACTGAAGAATCTATCCCACAG-3'

[0307] Expected PCR Product:

[0308] Wildtype: N.A.

[0309] Targeted: 445bp

[0310] Internal control product size: 689bp

[0311] Results are shown in Figure 28.

[0312] Primers for KI4 PCR:

[0313] M2F6 (SEQ ID NO. 54): 5'-TTGCTTCCTCAGTTGTCTGTGTC-3'

[0314] M2R6 (SEQ ID NO. 55): 5'-TTCAAAGTTTGCTCCCACATCC-3'

[0315] Internal control PCR primer A5 (SEQ ID NO. 45):

[0316] 5'-GCAGAAGAGGACAGATACATTCAT-3'

[0317] Internal control PCR primer A6 (SEQ ID NO. 46):

[0318] 5'-CCTACTGAAGAATCTATCCCACAG-3'

[0319] Expected PCR Product:

[0320] Wildtype: N.A.

[0321] Targeted: 378bp

[0322] Internal control product size: 689bp

[0323] Results are shown in Figure 29.

[0324] Primers for Neo-del PCR:

[0325] M2F2 (SEQ ID NO. 56): 5'-GGCTTGAGTTGCAGAATTGTCAT-3'

[0326] M2R2 (SEQ ID NO. 57): 5'-TAACTGGAGCCATATGGGATCTCT-3'

[0327] Internal control PCR primer A5 (SEQ ID NO. 45):

[0328] 5'-GCAGAAGAGGACAGATACATTCAT-3'

[0329] Internal control PCR primer A6 (SEQ ID NO. 46):

[0330] 5'-CCTACTGAAGAATCTATCCCACAG-3'

[0331] Expected PCR Product:

[0332] Wildtype: N.A.

[0333] Targeted: 384bp

[0334] Internal control product size: 689bp

[0335] Results are shown in Figure 30.

[0336] Primers for wildtype PCR:

[0337] M2F1 (SEQ ID NO. 58): 5'-GGGCTTGAGTTGCAGAATTGTCATC-3'

[0338] M2R1 (SEQ ID NO. 59): 5'-CAGCATCAGTGGAGACACAATCTAC-3'

[0339] Internal control PCR primer A5 (SEQ ID NO. 45):

[0340] 5'-GCAGAAGAGGACAGATACATTCAT-3'

[0341] Internal control PCR primer A6 (SEQ ID NO. 46):

[0342] 5'-CCTACTGAAGAATCTATCCCACAG-3'

[0343] Expected PCR Product:

[0344] Wildtype:451bp

[0345] Targeted:N.A.

[0346] Internal control product size:689bp

[0347] Results are shown in Figure 31.

[0348] Example 3. Construction of double gene homozygous mice

[0349] The Kappa chain modified mouse was crossed with the Lambda chain knockout mouse, and double gene heterozygous mice, double gene homozygous mice were obtained in turn, so that the B cells of the double gene homozygous mice only expressed the introduced Kappa light chain sequence, and neither expressed the mouse Kappa light chain sequence nor the mouse Lambda light chain sequence.

[0350] The spleen of the naive state mouse was collected, and the cells were incubated in a solution containing blocking antibodies (such as Fc Block) to prevent non-specific binding. Fluorescently labeled antibodies were added at the recommended concentration according to the antibody instructions, and incubated on ice for 20-30 minutes, avoiding light. The cells were then washed with PBS buffer to remove unbound antibodies. The laser and filter parameters were set to match the antibody fluorescence, and the stained cell sample was loaded into the machine. The fluorescence signal was detected according to the set parameters, and the data was collected using flow cytometry software and saved as a data file for data analysis.

[0351] Representative flow cytometry immunophenotyping analysis and statistical comparison of B cell classification were performed. The detection results are shown in Figures 32 and 33. Figure 32 shows that the B cells (CD45+CD19+) in the spleen tissue of the double gene homozygous mice (mouse 1, mouse 2) developed normally, and the proportion of B cells was comparable to that of wild-type mice (right panel).

[0352] Figure 33 shows that the Lambda type B cells (CD19+Igλ+) in the spleen tissue of the double gene homozygous mice (mouse 1, mouse 2) were absent compared to WT mice (right panel).

[0353] Example 4. Analysis of light chain sequence diversity

[0354] The spleen of a naive mouse is collected, and the spleen RNA is extracted. After the total RNA of the sample is detected and qualified, library construction is performed. In combination with high-throughput sequencing technology, the diversity of the immune system is comprehensively evaluated. The sequences obtained by sequencing are subjected to quality control by using quality control software and filtered from sequencing background. Then, the sequences are compared with the VJ genes in the IMGT immune cell receptor library to search for corresponding gene fragments, find the exact VJ gene fragments and sequence sites, and statistically analyze the VJ gene frequency and other information.

[0355] The results are shown in FIG. 34, which shows that the corresponding antibody sequences can be detected in the inserted four human light chain V genes in mouse 1 and mouse 2. Among them, IGKV4-1 and IGKV3-20 account for the majority in mouse 1, and IGKV2-30, IGKV3-15 and IGKV3-11 account for the majority in mouse 2.

[0356] Further analysis of whether the antibody sequences formed by the parent genes are mutated and the mutation positions is shown in FIG. 35 and FIG. 36. It can be seen that in mouse 1 and mouse 2, mutations mostly occur in the CDR3 region. Each column represents a sequence, and the height of the column can be used to determine the proportion of the sequence in the total sequence. The column from bottom to top represents the proportion of the sequence in the total sequence gradually decreasing, that is, the sequence represented by the lowermost column has the highest proportion.

[0357] Example 5. Immunization of mice and titer determination

[0358] In order to produce a humoral immune response against VEGF165 in the four-gene light chain mouse, two mice (mouse 1 x 2 and mouse 2 x 2) were selected for each of mouse 1 and mouse 2 for immunization, with C57B6 / N wild type (WT) mice as controls. The mice were first immunized with Freund's complete adjuvant (CFA) mixed with 0.1 mg Human VEGF165 His (Baiying Biotechnology) protein, and then subcutaneously injected with Freund's incomplete adjuvant (IFA) mixed with 0.05 mg Human VEGF165 His protein, with an interval of 2 weeks for immunization, a total of 4 times, so as to produce antigen-specific antibodies in light chain mouse 1 and light chain mouse 2.

[0359] Serum titer detection was performed on the serum of the mice after the fourth immunization. The CBS (carbonate buffer solution) was used to coat 1 μg / ml antigen overnight, and 1% BSA was added after PBST (phosphate buffer solution) cleaning, and 37°C blocking for 2h. After PBST cleaning, serum dilution (3 times dilution from 1:100, 11 gradients) was added and reacted at 37°C for 1h. After PBST cleaning, horseradish peroxidase-labeled goat anti-mouse secondary antibody (diluted with PBS at 1:1W) was added and incubated at 37°C for 0.5h. After PBST cleaning, TMB color developing solution (Biyun Tian, P0209-500ml) was added for color development, 25°C, 5-10min, and the reaction was terminated by adding the termination solution, and the serum titer was detected by measuring the optical density at 450nm.

[0360] The results are shown in Figures 37 and 38. The ELISA experiment results showed that the four-gene fixed light chain double-gene homozygous mice ① and ② produced high antibody titers after the fourth immunization, and the antibody titers were all above 656100 (serum dilution fold), wherein HUGO-L (902, 903, 906 and 908) were the four-gene fixed light chain double-gene homozygous mice, WT-C57 was the wild type control, and the negative serum was used as the negative control.

Claims

1. A method of making a genetically modified rodent, comprising: (i) knocking out an endogenous V-to-J segmental stretch of an immunoglobulin Kappa light chain locus of the rodent; and (ii) introducing into the immunoglobulin Kappa light chain locus of the rodent a human immunoglobulin Kappa light chain gene segment operably linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent; wherein the human immunoglobulin Kappa light chain gene segment comprises only four human immunoglobulin Kappa light chain V genes and only one human immunoglobulin Kappa light chain J gene; wherein the four human immunoglobulin Kappa light chain V genes are selected from the group consisting of hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11 and hlgKV4-1; and wherein the one human immunoglobulin Kappa light chain J gene is selected from the group consisting of hlgKJl, hlgKJ2, hlgKJ3, hlgKJ4 and hlgKJ5.

2. The method of claim 1, wherein the four human immunoglobulin Kappa light chain V genes are, respectively: (a) one of hlgKVl-39 and hlgKVl-33; one of hlgKV2-28 and hlgKV2-30; one of hlgKV3-20, hlgKV3-15 and hlgKV3-11; and hlgKV4-1; or (b) one of hlgKVl-39 and hlgKVl-33; one of hlgKV2-28 and hlgKV2-30; one of hlgKV3-20 and hlgKV3-15; and one of hlgKV4-1 and hlgKV3-11.

3. The method of claim 1, wherein the four human immunoglobulin Kappa light chain V genes are, respectively: (a) hlgKVl-39, hlgKV2-28, hlgKV3-20, hlgKV4-1; or (b) hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV3-11.

4. The method of any one of claims 1 to 3, wherein the one human immunoglobulin Kappa light chain J gene is hlgKJl.

5. The method of any one of claims 1 to 4, wherein each human immunoglobulin Kappa light chain V gene is connected to an adjacent gene by an upstream and a downstream natural junction sequence of the respective genes.

6. The method of claim 5, wherein the upstream natural junction sequence of each human immunoglobulin Kappa light chain V gene is between 500 bp and 3 kb, preferably between 1 kb and 3 kb, more preferably between 2 kb and 3 kb, upstream of the respective gene.

7. The method of claim 5 or 6, wherein the downstream natural flanking sequence of each human immunoglobulin Kappa light chain V gene is 50bp to 500bp downstream of the respective gene; preferably 100bp to 500bp, more preferably 200bp to 500bp.

8. The method of any one of claims 1 to 7, wherein the one human immunoglobulin Kappa light chain J gene is linked to the most downstream one of the four human immunoglobulin Kappa light chain V genes by its upstream natural flanking sequence; preferably, the upstream natural flanking sequence of the one human immunoglobulin Kappa light chain J gene is 50bp to 500bp upstream of it, preferably 100bp to 500bp, more preferably 200bp to 500bp.

9. The method of any one of claims 1 to 8, wherein the one human immunoglobulin Kappa light chain J gene is linked to its downstream natural flanking sequence; preferably, the downstream natural flanking sequence of the one human immunoglobulin Kappa light chain J gene is 50bp to 280bp downstream of it, preferably 100bp to 280bp, more preferably 200bp to 280bp.

10. The method of any one of claims 1 to 9, wherein the rodent is a mouse, and the continuous segment of V- to J-regions of the endogenous immunoglobulin Kappa light chain of the mouse is a continuous segment of mlgKV2-137 to mlgKJ5.

11. A genetically modified rodent genome, wherein the immunoglobulin Kappa light chain locus of the genetically modified rodent genome does not contain a continuous segment of V- to J-regions of the endogenous immunoglobulin Kappa light chain locus of the rodent; and the immunoglobulin Kappa light chain locus of the rodent genome comprises human immunoglobulin Kappa light chain gene segments operably linked to the endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent; wherein, the human immunoglobulin Kappa light chain gene segments comprise only four human immunoglobulin Kappa light chain V genes and only one human immunoglobulin Kappa light chain J gene; wherein the four human immunoglobulin Kappa light chain V genes are selected from the group consisting of hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1; and wherein the one human immunoglobulin Kappa light chain J gene is selected from the group consisting of hlgKJl, hlgKJ2, hlgKJ3, hlgKJ4, and hlgKJ5.

12. The rodent genome of claim 11, wherein the four human immunoglobulin Kappa light chain V genes are, respectively: hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKV2-30, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1. (a) one of hlgKVl-39 and hlgKVl-33; one of hlgKV2-28 and hlgKV2-30; one of hlgKV3-20, hlgKV3-15 and hlgKV3-11; and hlgKV4-1; or (b) one of hlgKVl-39 and hlgKVl-33; one of hlgKV2-28 and hlgKV2-30; one of hlgKV3-20 and hlgKV3-15; and one of hlgKV4-1 and hlgKV3-11.

13. The rodent genome of claim 11, wherein the four human immunoglobulin kappa light chain V genes are, respectively: (a) hlgKVl-39, hlgKV2-28, hlgKV3-20, hlgKV4-1; or (b) hlgKVl-33, hlgKV2-30, hlgKV3-15, hlgKV3-11.

14. The rodent genome of any one of claims 11 to 13, wherein the one human immunoglobulin kappa light chain J gene is hlgKJl.

15. The rodent genome of any one of claims 11 to 14, wherein each human immunoglobulin kappa light chain V gene is connected to the adjacent gene by the upstream and downstream natural joining sequences of the adjacent gene.

16. The rodent genome of claim 15, wherein the upstream natural joining sequence of each human immunoglobulin kappa light chain V gene is from 500 bp to 3 kb, preferably from 1 kb to 3 kb, more preferably from 2 kb to 3 kb, upstream of the respective gene.

17. The rodent genome of claim 15 or 16, wherein the downstream natural joining sequence of each human immunoglobulin kappa light chain V gene is from 50 bp to 500 bp, preferably from 100 bp to 500 bp, more preferably from 200 bp to 500 bp, downstream of the respective gene.

18. The rodent genome of any one of claims 11 to 17, wherein the one human immunoglobulin kappa light chain J gene is connected to the most downstream of the four human immunoglobulin kappa light chain V genes by its upstream natural joining sequence; preferably, the upstream natural joining sequence of the one human immunoglobulin kappa light chain J gene is from 50 bp to 500 bp, preferably from 100 bp to 500 bp, more preferably from 200 bp to 500 bp, upstream of the one human immunoglobulin kappa light chain J gene.

19. The rodent genome of any one of claims 11 to 18, wherein the one human immunoglobulin kappa light chain J gene is connected to its downstream natural joining sequence; preferably, the downstream natural joining sequence of the one human immunoglobulin kappa light chain J gene is from 50 bp to 280 bp, preferably from 100 bp to 280 bp, more preferably from 200 bp to 280 bp, downstream of the one human immunoglobulin kappa light chain J gene.

20. The rodent genome of any one of claims 11 to 19, wherein the rodent is a mouse, and the endogenous V to J contiguous segment of the mouse is a contiguous segment of mlgKV2-137 to mlgKJ5.

21. A cell, tissue, organ or rodent comprising the rodent genome of any one of claims 11 to 20; preferably, the cell is an embryonic cell, a B cell or a hybridoma cell; preferably, the tissue is white pulp or its lymph nodule of a spleen; preferably, the organ is a spleen.

22. A method of making a monoclonal antibody, comprising: (a) immunizing a rodent having the rodent genome of any one of claims 11 to 20 with an antigen; (b) isolating from the rodent a cell producing a monoclonal antibody comprising an antibody against the antigen; and (c) culturing the cell to obtain the monoclonal antibody; preferably, the cell is a splenocyte, a B cell or a hybridoma cell.

23. The method of claim 22, wherein the method further comprises: (d) identifying the monoclonal antibodies having the same light chain.

24. The method of claim 23, wherein the monoclonal antibody is a multispecific antibody; preferably a bispecific antibody.

25. Use of the cell, tissue, organ or rodent of claim 21 in the manufacture of a monoclonal antibody; preferably, the monoclonal antibody has the same light chain; preferably, the monoclonal antibody is a multispecific antibody, preferably a bispecific antibody.

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