Genetically modified rodent used for preparing common light chain and preparation method therefor

Abstract

Disclosed is a genetically modified rodent an immunoglobulin light chain locus of which is engineered such that a single rearranged human immunoglobulin light chain gene V / J gene is contained upstream of a constant region of the mouse Kappa light chain locus, and thus a mouse variable region sequence cannot bind to the endogenous constant region to form a complete mouse light chain antibody. The rodent can reproduce normally and produce an antibody containing a human light chain. Also provided are a preparation method for the genetically modified rodent and the use thereof.

Description

Genetically modified rodents for the production of common 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 the rodents for the production of monoclonal antibodies having common light chains. The present invention also relates to rodents having a modified genome. BACKGROUND

[0002] The 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 that express common light chains, whose expressed antibodies have heavy chains that can associate with and be expressed with the same or substantially the same light chain, are particularly useful in the production of bispecific antibodies. For example, such a mouse can be immunized with a first immunogen to produce B cells that express 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 that express 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 inserting a human immunoglobulin kappa light chain single V / J segment upstream of an immunoglobulin kappa light chain constant region locus of the rodent, the single V / J segment directly linked to an endogenous constant region of the immunoglobulin kappa light chain locus of the rodent, and leaving an endogenous V segment and an endogenous J segment of the immunoglobulin kappa light chain locus of the rodent.

[0004] Another aspect of the present invention provides a genetically modified rodent genome, wherein the immunoglobulin kappa light chain locus of the genetically modified rodent genome comprises an endogenous V region of the immunoglobulin kappa light chain locus of the rodent and an endogenous J region of the 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 single V / J segment, the single V / J segment directly linked to an endogenous constant region of the immunoglobulin kappa light chain locus of the rodent.

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

[0006] The present application will construct eight common light chain mouse models using the eight genes of hlgKV1-39, hlgKV1-33, hlgKV2-28, hlgKV1-5, hlgKV3-20, hlgKV3-15, hlgKV3-11 and hlgKV4-1 which are most frequently used in VH and VL pairing, which can be constructed faster and can cover most of the antibody screening within one year. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0009] Figure 3 shows a strategy diagram for constructing eight exemplary mouse models according to the present application.

[0010] Figures 4 to 11 show plasmid vector maps for constructing eight exemplary mouse models of the present application.

[0011] Figure 12 shows a strategy diagram for identifying eight exemplary mouse models.

[0012] Figures 13 to 36 show PCR identification results of positive clones obtained after electroporation of eight plasmid vectors.

[0013] Figure 37 shows a strategy diagram for identifying F1 generation mice.

[0014] Figures 38 to 49 show PCR identification results of F1 generation heterozygous mice.

[0015] Figures 50 to 69 show PCR identification results of F2 generation homozygous mice.

[0016] Figure 70 shows results of flow cytometric immunophenotyping analysis and statistical comparison of B cell classification.

[0017] Figure 71 shows results of flow cytometric immunophenotyping analysis and statistical comparison of immune system.

[0018] Figure 72 shows results of flow cytometric immunophenotyping analysis and statistical comparison of B cell development process.

[0019] Figure 73 shows the analysis results of light chain sequences.

[0020] Figure 74 shows the ability of double-gene homozygous mice to generate immune response after immunization against an antigen.

[0021] Figure 75 shows that the antibody molecules produced by homozygous mice have high binding affinity.

[0022] Figure 76 shows the VDJ gene usage frequency of heavy chain sequences.

[0023] Figure 77 shows the clone frequency distribution of full human common light chain mouse heavy chain antibody variable region. DETAILED DESCRIPTION

[0024] Definitions

[0025] “hIgKV” refers to the V region of the human (h) immunoglobulin Kappa light chain variable region locus in the present application, refers to the entire V region of the human immunoglobulin Kappa light chain variable region locus when used independently, and refers to the 1-39 genes located in the V region of the human immunoglobulin Kappa light chain variable region locus when suffixed with a specific gene number, such as “hIgKV1-39”. Similarly, the present application also uses “hIgKJ” 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 1.2), the orientation and arrangement can be seen in IMGT Repertoire (IG and TR), and the copy is shown in Figure 1, and the sequence of each gene and the connecting sequence between genes can be obtained from NCBI Reference Sequence number NC_000002.12. The term “Vk+ encoding” is used in the present application as equivalent to “IgKV+ encoding”. The term “Jk+ encoding” is used in the present application as equivalent to “IgKJ+ encoding”.

[0026] "mIgKV" refers to the V region of the mouse (m) immunoglobulin Kappa light chain variable region locus, 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 gene located in the V region of the mouse immunoglobulin Kappa light chain variable region locus. Similarly, the present application also uses "mIgKJ" to refer to the J region of the mouse immunoglobulin Kappa light chain variable region locus; and "mIgKC" to refer 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), which is reproduced in Figure 2, and the sequences of each gene and the linking sequences between genes can be obtained from NCBI Reference Sequence number NC_000072.7. The term "Vk+encoding" is used identically to "IgKV+encoding" in the present application. The term "Jk+encoding" is used identically to "IgKJ+encoding" in the present application.

[0027] "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 stretch of contiguous genes including A, B, and the stretch between the two.

[0028] "Operably linked" includes positioning two or more components (e.g., a V gene and a C gene) so that each component functions normally. For example, one hIgKV gene and one hIgKJ gene are operably linked to a mIgKC region refers to the ability of a rodent to form a human light chain variable region when these components are linked.

[0029] "Directly linked" includes positioning two or more components and does not include any spacer sequences between the linked components. For example, when a single human V / J segment is directly linked to the endogenous constant region (C) gene of a mouse Kappa light chain locus, it refers to the linkage of the last nucleotide at the 3' end of the human V / J segment to the first nucleotide at the 5' end of the mouse endogenous C gene.

[0030] “hIgKV1-39” is the human immunoglobulin kappa light chain locus V region 1-39 gene, Gene ID: 28930, with NCBI Reference Sequence number NC_000002.12: c89320099-89319625, 475 bp, with the gene sequence as set forth in SEQ ID NO. 30. For the purposes of the present invention, the coding sequence (CDS) of this gene is as set forth in SEQ ID NO. 43. The leader sequence of this gene is as set forth in SEQ ID NO. 51.

[0031] “hIgKV1-33” is the human immunoglobulin kappa light chain locus V region 1-33 gene, Gene ID: 28933, with NCBI Reference Sequence number NC_000002.12: c89268475-89268001, 475 bp, with the gene sequence as set forth in SEQ ID NO. 31. For the purposes of the present invention, the coding sequence of this gene is as set forth in SEQ ID NO. 46. The leader sequence of this gene is as set forth in SEQ ID NO. 52.

[0032] “hIgKV2-28” is the human immunoglobulin kappa light chain locus V region 2-28 gene, Gene ID: 28921, with NCBI Reference Sequence number NC_000002.12: c89222431-89221698, 734 bp, with the gene sequence as set forth in SEQ ID NO. 32. For the purposes of the present invention, the coding sequence of this gene is as set forth in SEQ ID NO. 47. The leader sequence of this gene is as set forth in SEQ ID NO. 53.

[0033] “hIgKV1-5” is the human immunoglobulin kappa light chain locus V region 1-5 gene, Gene ID: 28299, with NCBI Reference Sequence number NC_000002.12: c88947776-88947301, 476 bp, with the gene sequence as set forth in SEQ ID NO. 33. For the purposes of the present invention, the coding sequence of this gene is as set forth in SEQ ID NO. 45. The leader sequence of this gene is as set forth in SEQ ID NO. 54.

[0034] “hIgKV3-20” is the human immunoglobulin kappa light chain locus V region 3-20 gene, Gene ID: 28912, with NCBI Reference Sequence number NC_000002.12: c89143108-89142574, 535 bp, with the gene sequence as set forth in SEQ ID NO. 34. For the purposes of the present invention, the coding sequence of this gene is as set forth in SEQ ID NO. 44. The leader sequence of this gene is as set forth in SEQ ID NO. 55.

[0035] “hIgKV3-15” is the human immunoglobulin Kappa light chain locus V region 3-15 gene, Gene ID: 28913, with NCBI Reference Sequence number NC_000002.12: c89085690-89085177, 514 bp, with gene sequence as shown in SEQ ID NO. 35. For the purposes of the present invention, the coding sequence of this gene is as shown in SEQ ID NO. 49. The leader sequence of this gene is as shown in SEQ ID NO. 56.

[0036] “hIgKV3-11” is the human immunoglobulin Kappa light chain locus V region 3-11 gene, Gene ID: 28914, with NCBI Reference Sequence number NC_000002.12: c89027684-89027171, 514 bp, with gene sequence as shown in SEQ ID NO. 36. For the purposes of the present invention, the coding sequence of this gene is as shown in SEQ ID NO. 48. The leader sequence of this gene is as shown in SEQ ID NO. 57.

[0037] “hIgKV4-1” is the human immunoglobulin Kappa light chain locus V region 4-1 gene, Gene ID: 28908, with NCBI Reference Sequence number NC_000002.12: 88885572-88886153, 582 bp, with gene sequence as shown in SEQ ID NO. 37. For the purposes of the present invention, the coding sequence of this gene is as shown in SEQ ID NO. 50. The leader sequence of this gene is as shown in SEQ ID NO. 58.

[0038] “hIgKJ1” is the human immunoglobulin Kappa light chain locus J region J1 gene, Gene ID: 28950, with NCBI Reference Sequence number NC_000002.12: c88861923-88861886, 38 bp, with gene sequence as shown in SEQ ID NO. 38.

[0039] “hIgKJ2” is the human immunoglobulin Kappa light chain locus J region J2 gene, Gene ID: 28949, with NCBI Reference Sequence number NC_000002.12: c88861563-88861525, 39 bp, with gene sequence as shown in SEQ ID NO. 39.

[0040] “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 gene sequence as set forth in SEQ ID NO. 40.

[0041] “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 gene sequence as set forth in SEQ ID NO. 41.

[0042] “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 gene sequence as set forth in SEQ ID NO. 42.

[0043] “hIgKV3-15 promoter” is the promoter sequence of the human immunoglobulin Kappa light chain locus V region 3-15 gene, with an exemplary sequence as set forth in SEQ ID NO. 5.

[0044] “mIgKV3-7 leader” is the leader sequence of the mouse immunoglobulin Kappa light chain locus V region 3-7 gene, with an exemplary sequence as set forth in SEQ ID NO. 29.

[0045] Methods of making genetically modified rodents and genomes thereof

[0046] A first aspect of the present application provides a method of making a genetically modified rodent, comprising inserting a human immunoglobulin Kappa light chain single V / J segment upstream of a rodent immunoglobulin Kappa light chain constant region locus, said single V / J segment directly linked to an endogenous constant region of said rodent immunoglobulin Kappa light chain locus, and preserving an endogenous V segment and an endogenous J segment of said rodent immunoglobulin Kappa light chain locus.

[0047] 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 comprises an endogenous V region of the immunoglobulin Kappa light chain locus of the rodent and an endogenous J region of the 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 single V / J segment directly linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent.

[0048] In preferred embodiments of any of the aspects, the single V / J segment comprises, in order, a promoter sequence, a leader sequence, a single human immunoglobulin Kappa light chain V gene or coding sequence (CDS) thereof, and a single human immunoglobulin Kappa light chain J gene.

[0049] In preferred embodiments of any of the aspects, the single human immunoglobulin Kappa light chain V gene is selected from the group consisting of: hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKVl-5, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1.

[0050] In preferred embodiments of any of the aspects, the single human immunoglobulin Kappa light chain J gene is selected from the group consisting of: hlgKJl, hlgKJ2, hlgKJ3, hlgKJ4, and hlgKJ5.

[0051] In preferred embodiments of any of the aspects, the single human immunoglobulin Kappa light chain J gene is hlgKJl or hlgKJ5. Thus, in preferred embodiments, in the single V / J segment, the single human immunoglobulin Kappa light chain V gene is selected from the group consisting of: hlgKVl-39, hlgKVl-33, hlgKV2-28, hlgKVl-5, hlgKV3-20, hlgKV3-15, hlgKV3-11, and hlgKV4-1, and the single human immunoglobulin Kappa light chain J gene is hlgKJl, hlgKJ2, or hlgKJ5.

[0052] In preferred embodiments of any of the aspects, in the single V / J segment, the combination of the single V gene and the single J gene of the single V / J segment is selected from the group consisting of: hlgKVl-39 / hlgKJ5; hlgKV3-20 / hlgKJl; hlgKVl-5 / hlgKJl; hlgKVl-33 / hlgKJl; hlgKV2-28 / hlgKJl; hlgKV3-l l / hlgKJ2; hlgKV3-15 / hlgKJl; and hlgKV4-l / hlgKJl.

[0053] In preferred embodiments of any of the aspects, in the single V / J segment, the promoter sequence is derived from a human immunoglobulin Kappa light chain V gene. The human immunoglobulin Kappa light chain V gene can be any human immunoglobulin Kappa light chain functional V gene. Exemplary human immunoglobulin Kappa light chain functional V genes are hlgKV3D-7, hlgKVlD-8, hlgKVlD-43, hlgKV3D-l l, hlgKV3D-15, hlgKVlD-l 6, hlgKVlD-l 7, hlgKV3D-20, hlgKV6D-21, hlgKV2D-26, hlgKV2D-28, hlgKV2D-29, hlgKV2D-30, hlgKVlD-33, hlgKVlD-39, hlgKV2D-40, hlgKV2-40, hlgKVl-39, hlgKVl-33, hlgKV2-30, hlgKV2-29, hlgKV2-28, hlgKVl-27, hlgKV2-24, hlgKV6-21, hlgKV3-20, hlgKVl-17, hlgKVl-16, hlgKV3-15, hlgKVl-13, hlgKVl-12, hlgKV3-l l, hlgKVl-9, hlgKVl-8, hlgKVl-6, hlgKV5-2, or hlgKV4-l. In preferred embodiments, the promoter sequence is the promoter sequence of hlgKV3-15, e.g., as set forth in SEQ ID NO. 5.

[0054] In preferred embodiments of any of the aspects, in the single V / J segment, the leader sequence is the leader sequence of the single human immunoglobulin Kappa light chain V gene. For example, in the case that the single human immunoglobulin Kappa light chain V gene is hIgKVl-39, the leader sequence is the leader sequence of hIgKVl-39.

[0055] In further preferred embodiments of any of the aspects, in the single V / J segment, the leader sequence is the leader sequence of the single human immunoglobulin Kappa light chain V gene. For example, in the case that the single human immunoglobulin Kappa light chain V gene is hIgKVl-39, the leader sequence is the leader sequence of hIgKVl-39.

[0056] In preferred embodiments of any of the aspects, the rodent is a mouse, the mouse retains the endogenous J segments of its immunoglobulin Kappa light chain locus (contiguous segments of mIgKJl to mIgKJ5) and the endogenous V segments of its immunoglobulin Kappa light chain locus (i.e., contiguous segments of mIgKV2-137 to mIgKV3-l).

[0057] In preferred embodiments of any of the aspects, the rodent is a mouse, and the endogenous enhancers upstream (5') and downstream (3') of mIgKC of the mouse are retained.

[0058] In preferred embodiments of any of the aspects, the endogenous V segments of the immunoglobulin Kappa light chain locus of the rodent are not rearranged with the single human immunoglobulin Kappa light chain J gene.

[0059] In preferred embodiments of any of the aspects, the endogenous V segment and the endogenous J segment of the immunoglobulin Kappa light chain locus of the rodent (e.g., mouse) are not linked to the immunoglobulin Kappa light chain constant region C gene of the rodent. In the present application, the insertion of the single human V / J segment upstream of and direct linkage to the endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent (e.g., mouse) disrupts the splice signal present on the rodent endogenous constant region gene such that the endogenous VJ rearrangement cannot join to the endogenous C gene.

[0060] In preferred embodiments of any of the aspects, the rodent is a mouse, and the endogenous enhancers upstream (5') and downstream (3') of the mIgKC of the mouse are retained.

[0061] In preferred embodiments of any of the aspects, the arrangement of the functional units in the single V / J segment is selected from:

[0062] (a) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV1-39 / CDS]-[hIgKJ5];

[0063] (b) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-20 / CDS]-[hIgKJ1];

[0064] (c) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV1-5 / CDS]-[hIgKJ1];

[0065] (d) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV1-33 / CDS]-[hIgKJ1];

[0066] (e) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV2-28 / CDS]-[hIgKJ1];

[0067] (f) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-11 / CDS]-[hIgKJ2];

[0068] (g) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-15 / CDS]-[hIgKJ1]; and

[0069] (h) [hIgKV3-15 promoter] - [mIgKV3-7 leader] - [hIgKV4-1 / CDS] - [hIgKJ1];

[0070] wherein a pair of square brackets “[]” denotes a functional unit, and “-” between functional units represents a bond or 1-3 nucleotides (e.g., -CC-), and hIgKV gene / CDS denotes the full length of the gene or its CDS.

[0071] In preferred embodiments of any of the aspects, the arrangement of the individual functional units in the single V / J segment is selected from:

[0072] (a) [hIgKV3-15 promoter] - [hIgKV1-39 leader] - [hIgKV1-39 / CDS] - [hIgKJ5];

[0073] (b) [hIgKV3-15 promoter] - [hIgKV3-20 leader] - [hIgKV3-20 / CDS] - [hIgKJ1];

[0074] (c) [hIgKV3-15 promoter] - [hIgKV1-5 leader] - [hIgKV1-5 / CDS] - [hIgKJ1];

[0075] (d) [hIgKV3-15 promoter] - [hIgKV1-33 leader] - [hIgKV1-33 / CDS] - [hIgKJ1];

[0076] (e) [hIgKV3-15 promoter] - [hIgKV2-28 leader] - [hIgKV2-28 / CDS] - [hIgKJ1];

[0077] (f) [hIgKV3-15 promoter] - [hIgKV3-11 leader] - [hIgKV3-11 / CDS] - [hIgKJ1];

[0078] (g) [hIgKV3-15 promoter] - [hIgKV3-15 leader] - [hIgKV3-15 / CDS] - [hIgKJ1]; and

[0079] (h) [hIgKV3-15 promoter] - [hIgKV4-1 leader] - [hIgKV4-1 / CDS] - [hIgKJ1];

[0080] wherein a pair of square brackets “[]” represents a functional unit, and “-” between the functional units represents a bond or 1-3 nucleotides (e.g., -CC-), and hIgKV gene / CDS represents the full length of the gene or its CDS.

[0081] In the above embodiments, an exemplary hIgKV1-39 CDS is set forth in SEQ ID NO. 43. An exemplary hIgKV3-20 CDS is set forth in SEQ ID NO. 44. An exemplary hIgKV1-5 CDS is set forth in SEQ ID NO. 45. An exemplary hIgKV1-33 CDS is set forth in SEQ ID NO. 46. An exemplary hIgKV2-28 CDS is set forth in SEQ ID NO. 47. An exemplary hIgKV3-11 CDS is set forth in SEQ ID NO. 48. An exemplary hIgKV3-15 CDS is set forth in SEQ ID NO. 49. An exemplary hIgKV4-1 CDS is set forth in SEQ ID NO. 50. An exemplary hIgKV3-15 promoter is set forth in SEQ ID NO. 5. An exemplary mIgKV3-7 leader is set forth in SEQ ID NO. 29.

[0082] In the above embodiments, an exemplary hIgKV1-39 leader is set forth in SEQ ID NO. 51. An exemplary hIgKV3-20 leader is set forth in SEQ ID NO. 55. An exemplary hIgKV1-5 leader is set forth in SEQ ID NO. 54. An exemplary hIgKV1-33 leader is set forth in SEQ ID NO. 52. An exemplary hIgKV2-28 leader is set forth in SEQ ID NO. 53. An exemplary hIgKV3-11 leader is set forth in SEQ ID NO. 57. An exemplary hIgKV3-15 leader is set forth in SEQ ID NO. 56. An exemplary hIgKV4-1 leader is set forth in SEQ ID NO. 58.

[0083] In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV1-39 CDS]-[hIgKJ5] is set forth in SEQ ID NO. 6. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV3-20 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 7. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV1-5 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 8. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV1-33 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 9. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV2-28 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 10. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV3-11 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 11. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV3-15 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 12. In exemplary embodiments, the sequence of [mIgKV3-7 leader]-[hIgKV4-1 CDS]-[hIgKJ1] is set forth in SEQ ID NO. 13.

[0084] In some embodiments of any of the aspects, the rodent's immunoglobulin heavy chain variable region locus is humanized. Methods of humanizing a rodent's immunoglobulin heavy chain variable region locus 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 engineering of the heavy chain variable region genome.

[0085] In some embodiments of any of the aspects, the rodent's immunoglobulin Lambda light chain variable region locus is inactivated, e.g., deleted (e.g., knock-out effected).

[0086] Cells, tissues, animals, and methods of antibody production

[0087] A third aspect of the application provides a cell, tissue, organ, or rodent comprising the rodent genome of any one described herein.

[0088] In some embodiments, the 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.

[0089] In some embodiments, the application provides a tissue comprising the rodent genome of any one described herein, the tissue being white pulp of a spleen or a lymph nodule thereof.

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

[0091] In some embodiments, the application provides a rodent comprising the rodent genome of any one described herein, the rodent being a mouse.

[0092] A fourth aspect of the application provides a method of making a monoclonal antibody, comprising

[0093] (a) immunizing a rodent having a genome described herein with an antigen;

[0094] (b) isolating from the rodent a cell that produces a monoclonal antibody comprising an antibody to the antigen; and

[0095] (c) culturing the cell to obtain the monoclonal antibody.

[0096] In some embodiments, the cell of step (c) is a splenocyte, a B cell, or a hybridoma cell.

[0097] In some embodiments, the method further comprises

[0098] (d) identifying the monoclonal antibody as having the same light chain.

[0099] In some embodiments, the monoclonal antibody is a multispecific antibody; preferably a bispecific antibody.

[0100] 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] 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.

[0102] The present application will construct eight common light chain mouse models using the eight genes with the highest frequency in VH and VL pairing, hIgKV1-39, hIgKV1-33, hIgKV2-28, hIgKV1-5, hIgKV3-20, hIgKV3-15, hIgKV3-11 and hIgKV4-1, which can be constructed faster and obtained within one year, but can cover most of the antibody screening.

[0103] SEQUENCE LISTING (PARTIAL)

[0104] Example 1. Construction of eight mouse models

[0105] Eight models were constructed by replacing the mouse Jk1-Jk5 region with Human Vk3-15 Promoter-Mouse Vk3-7 leader-Human Vk-Human Jk sequence, and the strategy diagram is shown in Figure 3. The method is as follows.

[0106] 1. Construction of targeting vector:

[0107] Homologous arm sequence

[0108] The 5' arm sequence corresponds to the region on the mouse genome: GRCm38 / mm10: chr6:70,723,745-70,726,434; and the 3' arm sequence corresponds to the region on the mouse genome: GRCm38 / mm10: chr6:70,726,435-70,729,526.

[0109] The 5' arm homologous arm and the 3' arm homologous arm were amplified from the C57BL / 6 mouse genome by two pairs of primers, and the amplification primers are as follows (all sequences are shown from 5'-3'):

[0110] 5F (SEQ ID NO. 1):

[0111]

[0112] 5R (SEQ ID NO. 2):

[0113]

[0114] 3F (SEQ ID NO. 3):

[0115]

[0116] 3R (SEQ ID NO. 4):

[0117]

[0118] 5F / 5R amplified 5 arm homology arms, 3F / 3R amplified 3 arm homology arms sequences

[0119] Promoter sequence

[0120] Synthetic Human Vk3-15 gene promoter sequence, the synthetic sequence is shown as SEQ ID NO. 5.

[0121] KI (knock-in) sequences: 8 different sequences were synthesized respectively

[0122] Mouse Vk3-7 leader-Human Vk1-39-Human Jk5 (SEQ ID NO. 6)

[0123] Mouse Vk3-7 leader-Human Vk3-20-Human Jk1 (SEQ ID NO. 7)

[0124] Mouse Vk3-7 leader-Human Vk1-5-Human Jk1 (SEQ ID NO. 8)

[0125] Mouse Vk3-7 leader-Human Vk1-33-Human Jk1 (SEQ ID NO. 9)

[0126] Mouse Vk3-7 leader-Human Vk2-28-Human Jk1 (SEQ ID NO. 10)

[0127] Mouse Vk3-7 leader-Human Vk3-11-Human Jk2 (SEQ ID NO. 11)

[0128] Mouse Vk3-7 leader-Human Vk3-15-Human Jk1 (SEQ ID NO. 12)

[0129] Mouse Vk3-7 leader-Human Vk4-1-Human Jk1 (SEQ ID NO. 13)

[0130] Resistance gene Neo (SEQ ID NO. 14)

[0131] The last 5' arm, Neo, promoter sequence, 8 individual Vk genes and 1 Jk gene and 3' arm homology arm sequences were connected into a pUC57 backbone by infusion to obtain 8 different vector plasmids. The plasmid maps are shown in Figures 4 to 11, respectively.

[0132] 2. Electroporation of the vectors into ES cells:

[0133] The linearized 8 vectors were transfected into C57BL / 6 ES cells, respectively. Transfected ES cells were subjected to G418 selection (200 pg / mL) 24 hours after electroporation. G418 resistant colonies were picked and expanded in 96-well plates. Two sets of 96-well plates were made, one was frozen and stored at -80°C, the other set of 96-well plates was used for DNA isolation and subsequent PCR screening for homologous recombination.

[0134] The identification strategy is shown in Figure 12, F1 is outside the 5 arm homology arm on the mouse genome, R1 is on the Neo element, F2 is on the Neo element, R2 is on the promoter, F3 is on the JK gene, and R3 is on the 3' arm homology arm.

[0135] The identification primers are as follows:

[0136] Primers for 5' arm PCR:

[0137] F1 (SEQ ID NO. 15): 5'-tgaatcactgtgattcacgttcg-3'

[0138] R1 (SEQ ID NO. 16): 5'-GACTAGAGCTTGCGGAACCCTT-3'

[0139] Expected PCR Product:

[0140] Wildtype: N.A.

[0141] Targeted: 3053bp

[0142] Primers for Neo-L PCR:

[0143] F2 (SEQ ID NO. 17): 5'-GCTAGCTTGGCTGGACGTA-3'

[0144] R2 (SEQ ID NO. 18): 5'-CCGGGATCTTTCCTGACAAGT-3'

[0145] Expected PCR Product:

[0146] Wildtype: N.A.

[0147] Targeted: 416bp

[0148] Primers for KIPCR:

[0149] F3 (SEQ ID NO. 19): 5'-CCATGGGTTGCAAAAGTTAAACTCA-3'

[0150] R3 (SEQ ID NO. 20): 5'-GAGGCACCTCCAGATGTTAACTGC-3'

[0151] Expected PCR Product:

[0152] Wildtype: N.A.

[0153] Targeted: 367bp

[0154] Cell clones after electroporation of vector 1:

[0155] PCR amplification was performed on selected cell clones by 3 pairs of primers, the identification position was shown in Figure 12, and it was confirmed that 2B1, 2A2, 2B2, 2D2, 2F2, 2G2, 2G4, 2H5, 2G6, 2H8, 2E10, 2F10, 2E11 and 2F11 totally 14 clones were positive clones. The identification results were shown in Figures 13 to 15.

[0156] Cell clones after electroporation of vector 2:

[0157] PCR amplification was performed on selected cell clones by 3 pairs of primers, the identification position was shown in Figure 12, and it was confirmed that 1H8, 1H9 and 2C6 totally 3 clones were positive clones. The identification results were shown in Figures 16 to 18.

[0158] Cell clones after electroporation of vector 3:

[0159] The selected cell clones were amplified by PCR with 3 pairs of primers, and the identification positions are shown in Figure 12. It was confirmed that 2D1, 2H1, 2E2, 2G2, 2D3, 2F4, 2H4, 2G5, 2H5, 2D6, 2F6, 2A7, 2A8, 2E8, 2G8, 2F9, 2C11, 2D12 and 2G12, a total of 19 clones, were positive clones. The identification results are shown in Figures 19 to 21.

[0160] Cell clones after electroporation of vector 4:

[0161] The selected cell clones were amplified by PCR with 3 pairs of primers, and the identification positions are shown in Figure 12. It was confirmed that 1E7, 1G10, 1C11, 1G11, 1E12, a total of 5 clones, were positive clones. The identification results are shown in Figures 22 to 24.

[0162] Cell clones after electroporation of vector 5:

[0163] The selected cell clones were amplified by PCR with 3 pairs of primers, and the identification positions are shown in Figure 12. It was confirmed that 1D3, 1B8, 1D9, 1G11, a total of 4 clones, were positive clones. The identification results are shown in Figures 25 to 27.

[0164] Cell clones after electroporation of vector 6:

[0165] The selected cell clones were amplified by PCR with 3 pairs of primers, and the identification positions are shown in Figure 12. It was confirmed that 1D1, 1H1, 1A3, 1C3, 1G5, 1H5, 1E6, 1H6, 1C7, 1B8, 1F9, 1B10, 1D11, 1A12, a total of 14 clones, were positive clones. The identification results are shown in Figures 28 to 30.

[0166] Cell clones after electroporation of vector 7:

[0167] The selected cell clones were amplified by PCR with 3 pairs of primers, and the identification positions are shown in Figure 12. It was confirmed that 1A1, 1D1, 1H2, 1F3, 1B5, 1C6, 1F6, 1D12, a total of 8 clones, were positive clones. The identification results are shown in Figures 31 to 33.

[0168] Cell clones after electroporation of vector 8:

[0169] The selected cell clones were amplified by PCR using 3 pairs of primers. The positions of the primers are shown in Figure 12. A total of 16 clones, 1C1, 1B2, 1C2, 1F3, 1H3, 1D4, 1A7, 1C7, 1D8, 1E9, 1C10, 1H10, 1A11, 1C11, 1A12, 1G12, were confirmed as positive clones. The results are shown in Figures 34 to 36.

[0170] 3. Eight different targeted ES cell clones were injected into C57BL / 6 albino embryos, which were then reimplanted into CD-1 pseudopregnant females. The founder animals were identified by coat color. The F0 mice were black in color, and the chimerism rate was 100%. The mice were confirmed as positive F0 mice.

[0171] 4. After the eight F0 mice were bred to sexual maturity and mated with wild-type mice, the F1 generation was born. The F1 mice were identified by PCR at 5-7 days of age. The identification strategy for the mice is shown in Figure 37.

[0172] Primers for targeted allele:

[0173] F6 (SEQ ID NO. 21): 5'- ttgtatatatgtgcatcctggccc-3'

[0174] R6 (SEQ ID NO. 22): 5'- CACTGGATGGTGGGAAGATGGATAC-3'

[0175] Wildtype: 201 bp

[0176] F7 (SEQ ID NO. 23): 5'- acactgaaatggagcccttccttg-3'

[0177] R7 (SEQ ID NO. 24): 5'- GGTAGTTGTTGTAGGCACAGGG-3'

[0178] Targeted: 298 bp

[0179] Wildtype: 201 bp

[0180] Homozygotes: 298 bp

[0181] Heterozygotes: 298 bp / 201 bp

[0182] Internal control PCR primer F5 (SEQ ID NO. 25):

[0183] 5'-CTATCAGGGATACTCCTCTTTGCC-3'

[0184] Internal control PCR primer R5 (SEQ ID NO. 26):

[0185] 5'-GATACAGGAATGACAAGCTCATGGT-3'

[0186] Internal control product size: 507 bp

[0187] Internal control PCR primer F4 (SEQ ID NO. 27):

[0188] 5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0189] Internal control PCR primer R4 (SEQ ID NO. 28):

[0190] 5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0191] Internal control product size: 335 bp

[0192] Mouse 1 (Vk1-39):

[0193] PCR amplification was performed on 7 F1 mice by 2 pairs of primers, and it was confirmed that 4 mice of No. 33, 37, 38 and 39 were heterozygous mice in total. The identification results are shown in Figures 38 and 39.

[0194] Mouse 2 (Vk3-20):

[0195] PCR amplification was performed on 1 F1 mouse by 2 pairs of primers, and it was confirmed that this mouse was a heterozygous mouse in total. The identification results are shown in Figures 40 and 41.

[0196] Mouse 3 (Vk1-5):

[0197] PCR amplification was performed on 5 F1 mice by 2 pairs of primers, and it was confirmed that 3 mice of No. 2, 4 and 5 were heterozygous mice in total. The identification results are shown in Figures 42 and 43.

[0198] Mouse 4 (Vk1-33):

[0199] PCR amplification was performed on 5 F1 mice by 2 pairs of primers, and it was confirmed that 5 mice of No. 3, 5, 6, 7 and 8 were hybrid mice in total. The identification results are shown in Figures 44 and 45.

[0200] Mouse 5 (Vk2-28):

[0201] PCR amplification was performed on 9 F1 mice by 2 pairs of primers, and it was confirmed that 9 mice of No. 37, 38, 39, 41, 46, 48, 52, 53 and 54 were hybrid mice in total. The identification results are shown in Figures 46 and 47.

[0202] Mouse 6 (Vk3-11):

[0203] PCR amplification was performed on 3 F1 mice by 2 pairs of primers, and it was confirmed that 3 mice of No. 47, 50 and 51 were hybrid mice in total. The identification results are shown in Figures 48 and 49.

[0204] Mouse 7 (Vk3-15):

[0205] PCR amplification was performed on 2 F1 mice by 2 pairs of primers, and it was confirmed that 2 mice of No. 55 and 58 were hybrid mice in total. The identification results are shown in Figures 50 and 51.

[0206] Mouse 8 (Vk4-1):

[0207] PCR amplification was performed on 5 F1 mice by 2 pairs of primers, and it was confirmed that 5 mice of No. 121, 122, 124, 125 and 127 were hybrid mice in total. The identification results are shown in Figures 52 and 53.

[0208] 5. The F1 mice were mated with each other, and Fn generation mice were finally obtained. PCR identification was performed to confirm the birth of homozygous mice.

[0209] Mouse 1 (Vk1-39):

[0210] PCR amplification was performed on 8 F2 mice by 2 pairs of primers, and it was confirmed that 8 mice of No. 312, 313, 314, 315, 316, 317, 318 and 319 were homozygous mice in total. The identification results are shown in Figures 54 and 55.

[0211] Mouse 2 (Vk3-20):

[0212] PCR amplification was performed on 7 F2 mice by 2 pairs of primers, and it was confirmed that 4 mice of No. 76, 77, 78 and 80 were homozygous mice in total. The identification results are shown in Figures 56 and 57.

[0213] Mouse 3 (Vk1-5):

[0214] The 3 mice of 230#, 231# and 232# were confirmed to be homozygous mice by PCR amplification of 2 pairs of primers on 3 F2 mice. The identification results are shown in Figures 58 and 59.

[0215] Mouse 4 (Vk1-33):

[0216] The 8 mice of 268#, 269#, 270#, 271#, 272#, 273#, 274#, 275# and 276# were confirmed to be homozygous mice by PCR amplification of 2 pairs of primers on 8 F2 mice. The identification results are shown in Figures 60 and 61.

[0217] Mouse 5 (Vk2-28):

[0218] The 5 mice of 287#, 288#, 289#, 290# and 291# were confirmed to be homozygous mice by PCR amplification of 2 pairs of primers on 5 F2 mice. The identification results are shown in Figures 62 and 63.

[0219] Mouse 6 (Vk3-11):

[0220] The 4 mice of 284#, 285#, 286# and 287# were confirmed to be homozygous mice by PCR amplification of 2 pairs of primers on 4 F2 mice. The identification results are shown in Figures 64 and 65.

[0221] Mouse 7 (Vk3-15):

[0222] The 6 mice of 143#, 144#, 146#, 147#, 148# and 149# were confirmed to be homozygous mice by PCR amplification of 2 pairs of primers on 6 F2 mice. The identification results are shown in Figures 66 and 67.

[0223] Mouse 8 (Vk4-1):

[0224] The 4 mice of 155#, 156#, 157# and 158# were confirmed to be homozygous mice by PCR amplification of 2 pairs of primers on 8 F2 mice. The identification results are shown in Figures 68 and 69.

[0225] 6. The Kappa chain modified mouse is crossed with the Lambda chain knockout mouse to obtain double gene heterozygous mice and double gene homozygous mice in sequence, so that the double gene homozygous mouse only expresses the introduced Kappa light chain sequence, neither expresses the mouse Kappa light chain sequence nor the mouse Lambda light chain sequence.

[0226] Collect the spleen of Naive mice, incubate the cells in a solution containing blocking antibodies (such as Fc Block) to prevent non-specific binding, add fluorescently labeled antibodies according to the recommended concentration of the antibody instructions, incubate on ice for 20-30 minutes, avoid light, then wash the cells with PBS buffer to remove unbound antibodies. Set the laser and filter parameters to match the antibody fluorescence, load the stained cell sample into the machine, detect the fluorescence signal according to the set parameters, use the flow cytometry software to collect data, and save the data file for data analysis.

[0227] Representative flow cytometric immunophenotyping analysis and statistical comparison of B cell classification: the results are shown in Figure 70. The results show that the Lambda type B cells are missing in the spleen tissue of double homozygous mice (right panel) compared to WT mice (left panel).

[0228] 7. Detect the immune system in double homozygous mice to confirm that the immune system and B cell development are normal

[0229] Collect the spleen and peripheral blood of Naive mice, incubate the cells in a solution containing blocking antibodies (such as Fc Block) to prevent non-specific binding, add fluorescently labeled antibodies according to the recommended concentration of the antibody instructions, incubate on ice for 20-30 minutes, avoid light, then wash the cells with PBS buffer to remove unbound antibodies. Set the laser and filter parameters to match the antibody fluorescence, load the stained cell sample into the machine, detect the fluorescence signal according to the set parameters, use the flow cytometry software to collect data, and save the data file for data analysis.

[0230] 7.1 Representative flow cytometric immunophenotyping analysis and statistical comparison of the immune system, the results are shown in Figure 71. The results show that there is no significant difference in B cells (CD3-CD19+), T cells (CD3+CD19-), and NK cells (CD3-CD335+) in the spleen tissue of double heterozygous mice compared to WT mice, proving that the immune system of this mouse strain is normal.

[0231] 7.2 Representative flow cytometric immunophenotyping analysis and statistical comparison of B cell development

[0232] The detection results are shown in Figure 72, which shows that the transitional 1 (T1) B cells (CD19+IgM+IgD-), transitional 2 (T2) B cells (CD19+IgM+IgD+) and mature B cells (CD19+IgM-IgD+) in the spleen tissue of double-gene homozygous mice have no obvious difference from WT mice; and in the spleen, T follicular (FO) B cells (CD19+CD21-CD23+) and marginal zone (MZ) B cells (CD19+CD21+CD23+) also have no obvious difference from WT mice.

[0233] 8. Analysis of light chain sequence diversity

[0234] The spleen of a naive state mouse is collected, and spleen RNA is extracted. After the total RNA of the sample is detected to be qualified, library construction is performed. Combined 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 quality control software and filtered from sequencing background, and then compared with the VJ genes of the IMGT immune cell receptor library to search for corresponding gene fragments, find the exact VJ gene fragments and the position of the sequence, and statistically analyze the proportion of VJ sequences without mutation in all light chain sequences.

[0235] The spleen RNA of a naive state mouse is subjected to library construction, and the light chain sequence is analyzed by sequencing. The results are shown in Figure 73. The results show that about 80% of the Kappa light chain sequences in the double-gene homozygous mouse are inserted maternal sequences, and only 10-20% of the sequences are sequences derived from the inserted sequences, i.e. subsequences after high-frequency mutation. There is no other type of light chain sequence.

[0236] 9. Immunizing double-gene homozygous mice with antigens to detect antibody titer levels

[0237] In order to produce a humoral immune response against PD-L1 in double-gene homozygous mice with 8 different light chains, 3 mice were selected for each common light chain mouse for immunization, and C57B6 / NCya wild type (WT) mice were used as controls. The mice were first immunized with Freund's complete adjuvant (CFA) mixed with 0.05 mg of PD-L1 His (Kaiyuan Biotechnology, PDL-HM110) protein, and then subcutaneously injected with Freund's incomplete adjuvant (IFA) mixed with 0.025 mg of PD-L1 His protein, with an interval of 2 weeks for immunization, a total of 3-4 times, so that the fully human antibody mouse produces antigen-specific antibodies.

[0238] After the third and fourth immunization, the mouse serum was taken for serum titer monitoring. The CBS (carbonate buffer solution) was used to coat 2 μg / ml antigen overnight, and 2% BSA was added after PBST (phosphate buffer solution) cleaning. After 2h of 37°C blocking, PBST was cleaned, and serum diluent (from 1:2000, 8 gradients) was added for 2h of 37°C blocking. After PBST cleaning, horseradish peroxidase-labeled goat anti-mouse secondary antibody (diluted with PBS at 1:1W) was added for 1h of 37°C incubation. After PBST cleaning, TMB developing solution (Bi Yun Tian, P0209-500ml) was added for color development, 37°C, 5-10min, and stop solution was added to stop the reaction. The optical density was detected at 450nm to detect the serum titer.

[0239] The results are shown in Figure 74, which shows that the double-gene homozygous mice (numbered starting with L) have the ability to have an immune response equivalent to wild-type mice after immunization against the antigen.

[0240] 10. Further, taking the Vk1-39 double-gene homozygous mouse as an example, after immunization with the VEGF165 antigen according to the above method, the spleen, lymph node and bone marrow cells of the immunized homozygous mouse were taken, enriched by CD138 Beads, and 2x10 6 The 1444 pairs of light and heavy chain paired antibody sequences were obtained by NGS sequencing of the positive B cells after VEGF165 antigen immunization, and the VDJ gene of the heavy chain sequence was statistically analyzed.

[0241] From the 189 pairs of unique sequences obtained by sequencing, 20 molecules (VEGF-1 to VEGF-20) were selected for antibody synthesis and expression, and the purified protein was obtained for ELISA binding activity detection. The VEGF165 antigen was coated, and the candidate antibody was diluted by 5 times gradient at a concentration of 100nM. Anti-Human IgG HRP secondary antibody was added to detect the binding activity of the antibody and the antigen.

[0242] The results are shown in Figure 75, which shows that the antibody molecules produced by the homozygous mouse have high binding affinity.

[0243] Analysis of the sequences obtained after immunization showed that the VDJ gene frequency of the heavy chain sequence (Figure 76) indicated that after immunization of the homozygous mouse with the VEGF165 target, the heavy chain V gene produced was mainly concentrated in the mouse IGHV1 family.

[0244] 11. Fully human mouse

[0245] The double-gene homozygous mice are crossed with the heavy chain variable region fully humanized mice to obtain triple-gene homozygous mice, which are referred to as fully human common light chain mice hereinafter.

[0246] The spleen of the fully human common light chain mice in the Naive state is collected, and the spleen RNA is extracted. After the total RNA of the sample is detected to be 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 quality control software and filtered for sequencing background. Then, the sequences are compared with the VDJ genes of the IMGT immune cell receptor library to search for corresponding gene fragments, find the exact VDJ gene fragments and sequence sites, and statistically analyze the VDJ gene frequency, the clonal frequency distribution, the number of polypeptide sequences, and the CDR3 length distribution, and other information.

[0247] The results are shown in FIG. 77, which indicates that the heavy chain antibody variable region sequence diversity of the fully human common light chain mice is rich, and the use frequency of each family is similar to the use frequency of the heavy chain antibody family in the human body.

Claims

1. A method of making a genetically modified rodent, comprising inserting a single V / J segment of a human immunoglobulin Kappa light chain directly upstream of an endogenous constant region of an immunoglobulin Kappa light chain locus of the rodent, the single V / J segment being linked to the endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent and preserving an endogenous V segment and an endogenous J segment of the immunoglobulin Kappa light chain locus of the rodent.

2. The method of claim 1, wherein the single V / J segment comprises, in order, a promoter sequence, a leader sequence, a single human immunoglobulin Kappa light chain V gene or a coding sequence (CDS) thereof, and a single human immunoglobulin Kappa light chain J gene.

3. The method of claim 2, wherein, the single human immunoglobulin Kappa light chain V gene is selected from the group consisting of hIgKV1-39, hIgKV1-33, hIgKV2-28, hIgKV1-5, hIgKV3-20, hIgKV3-15, hIgKV3-11, and hIgKV4-1.

4. The method of claim 2, wherein, the single human immunoglobulin Kappa light chain J gene is selected from the group consisting of hIgKJ1, hIgKJ2, hIgKJ3, hIgKJ4, and hIgKJ5.

5. The method of claim 4, wherein the single human immunoglobulin Kappa light chain J gene is hIgKJ1, hIgKJ2, or hIgKJ5.

6. The method of claim 2, wherein the combination of the single V gene and the single J gene of the single V / J segment is selected from the group consisting of hIgKV1-39 / hIgKJ5; hIgKV3-20 / hIgKJ1; hIgKV1-5 / hIgKJ1; hIgKV1-33 / hIgKJ1; hIgKV2-28 / hIgKJ1; hIgKV3-11 / hIgKJ2; hIgKV3-15 / hIgKJ1; and hIgKV4-1 / hIgKJ1.

7. The method of claim 2, wherein the promoter sequence is derived from a human immunoglobulin Kappa light chain V gene; preferably, the promoter sequence is the promoter sequence of hIgKV3-15.

8. The method of claim 2 or 7, wherein the leader sequence is derived from a rodent immunoglobulin Kappa light chain V gene, preferably the leader sequence of mIgKV3-7, or the leader sequence is the leader sequence of the single human immunoglobulin Kappa light chain V gene.

9. The method of any one of claims 1 to 8, wherein the rodent is a mouse.

10. The method of any one of claims 1 to 9, wherein the single V / J segment has an arrangement of functional units selected from the group consisting of: (a) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV1-39 / CDS]-[hIgKJ5]; (b) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-20 / CDS]-[hIgKJ1]; (c) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV1-5 / CDS]-[hIgKJ1]; (d) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV1-33 / CDS]-[hIgKJ1]; (e) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV2-28 / CDS]-[hIgKJ1]; (f) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-11 / CDS]-[hIgKJ2]; (g) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-15 / CDS]-[hIgKJ1]; and (h) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV4-1 / CDS]-[hIgKJ1]; wherein a pair of square brackets "[]" represents a functional unit, and the "-" between the functional units represents a bond or 1-3 nucleotides (e.g., -CC-), and hIgKV gene / CDS represents the full length of the gene or its CDS.

11. A genetically modified rodent genome, wherein the immunoglobulin Kappa light chain locus of the genetically modified rodent genome comprises an endogenous V region of the immunoglobulin Kappa light chain locus of the rodent and an endogenous J region of the 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 single V / J segment that is directly linked to an endogenous constant region of the immunoglobulin Kappa light chain locus of the rodent.

12. The rodent genome of claim 11, wherein, the single V / J segment comprises, in order, a promoter sequence, a leader sequence, a single human immunoglobulin Kappa light chain V gene or coding sequence (CDS) thereof, and a single human immunoglobulin Kappa light chain J gene.

13. The rodent genome of claim 12, wherein, the single human immunoglobulin Kappa light chain V gene is selected from the group consisting of: hIgKV1-39, hIgKV1-33, hIgKV2-28, hIgKV1-5, hIgKV3-20, hIgKV3-15, hIgKV3-11, and hIgKV4-1.

14. The rodent genome of claim 12, wherein, the single human immunoglobulin Kappa light chain J gene is selected from the group consisting of: hIgKJ1, hIgKJ2, hIgKJ3, hIgKJ4, and hIgKJ5.

15. The rodent genome of claim 14, wherein the single human immunoglobulin Kappa light chain J gene is hlgKJl, hlgKJ2, or hlgKJ5.

16. The rodent genome of claim 12, wherein the combination of the single V gene and the single J gene of the single V / J segment is selected from the group consisting of: hlgKVl-39 / hlgKJ5; hlgKV3-20 / hlgKJl; hlgKVl-5 / hlgKJl; hlgKVl-33 / hlgKJl; hlgKV2-28 / hlgKJl; hlgKV3-l l / hlgKJ2; hlgKV3-15 / hlgKJl; and hlgKV4-l / hlgKJl.

17. The rodent genome of any one of claims 11 to 16, wherein the promoter sequence is derived from a human immunoglobulin Kappa light chain V gene; preferably, the promoter sequence is the promoter sequence of hlgKV3-15.

18. The rodent genome of any one of claims 11 to 17, wherein the leader sequence is derived from a rodent immunoglobulin Kappa light chain V gene, preferably the leader sequence of mlgKV3-7, or the leader sequence is the leader sequence of the single human immunoglobulin Kappa light chain V gene.

19. The rodent genome of any one of claims 11 to 18, wherein the rodent is a mouse.

20. The rodent genome of any one of claims 11 to 19, wherein the arrangement of the functional units in the single V / J segment is selected from the group consisting of: (a) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKVl-39 / CDS]-[hlgKJ5]; (b) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKV3-20 / CDS]-[hlgKJl]; (c) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKVl-5 / CDS]-[hlgKJl]; (d) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKVl-33 / CDS]-[hlgKJl]; (e) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKV2-28 / CDS]-[hlgKJl]; (f) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKV3-l l / CDS]-[hlgKJ2]; (g) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKV3-15 / CDS]-[hlgKJl]; and (h) [hlgKV3-15 promoter]-[mIgKV3-7 leader]-[hlgKV4-l / CDS]-[hlgKJl]. (g) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV3-15 / CDS]-[hIgKJ1]; and (h) [hIgKV3-15 promoter]-[mIgKV3-7 leader]-[hIgKV4-1 / CDS]-[hIgKJ1]; wherein a pair of square brackets "[]" represents a functional unit, and the "-" between the functional units represents a bond or 1-3 nucleotides (e.g. -CC-), and hIgKV gene / CDS represents the full length of the gene or its CDS.

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 the white pulp of the spleen or its lymph nodule; preferably, the organ is the 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 that produces 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 that have the same light chain.

24. The method of claim 22 or 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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