Recombinant genome, non-human mammalian cells, and methods for their production and uses

Abstract

The objective of the present invention is to provide a recombinant genome, a non-human mammalian cell, and a method for producing the same and its uses. The present invention provides a non-human mammalian cell, a recombinant genome of a non-human mammalian cell, and a method for producing the same. A part or all of an endogenous immunoglobulin variable region gene in the genome is replaced by a human immunoglobulin variable region gene, a part or all of a pseudogene and / or an open reading frame of the human immunoglobulin variable region gene is knocked out, and the human immunoglobulin variable region gene has a human heavy chain function V. H , D H , J H coding and non-coding sequences of human light chain function V L , J L The non-human mammalian cell of the present invention can be used to generate a transgenic animal capable of producing an antibody comprising a human immunoglobulin functional region and having a variable region of fully human origin, and the transgenic animal can be used to efficiently select fully human antibodies, with higher antibody affinity obtained, reduced research and development costs, and a shorter research and development cycle.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 The present invention relates to the field of genetic engineering, and in particular, to genetically modified non-human mammalian cells and their genomes for medical and disease research purposes, a method for obtaining non-human mammals based on such non-human mammalian cells and their genomes, and cells, antibodies, antibody fragments, and derivative pharmaceuticals or pharmaceutical compositions containing such antibody fragments derived from such animals. 〔Background Art〕 Bruggemann et al. 1989a first reported that when non-rearranged human immunoglobulin gene fragments are introduced into an animal body, antibodies derived from the human immunoglobulin gene in the animal serum may be detected, and such attempts have opened the door to directly producing therapeutic antibodies in the animal body with variable regions entirely derived from humans by using genetically modified animals. Many companies have produced transgenic animals having human immunoglobulin genes based on similar principles, and these production methods and examples are described in published documents such as Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, Patent Document 6, Patent Document 7, Patent Document 8, Patent Document 9, Patent Document 10, Patent Document 11, Patent Document 12, Patent Document 13, Patent Document 14, Patent Document 15, Patent Document 16, Non-Patent Document 1, and Non-Patent Document 2.

[0002] These methods relate to the inactivation of the endogenous antibody gene cluster function in the animal body and the recombination and expression of human immunoglobulin genes. The genetic modifications carried out to achieve the two purposes are usually performed in these non-human mammalian embryonic stem cells. For example, part or all of the heavy chain and light chain gene loci of mouse embryonic stem cells are knocked out, and the introduced human heavy chain and light chain gene loci are introduced to compensate for the functional deficiency of this gene, so that the mouse produces antibodies derived from human heavy chain and light chain gene fragments. However, these animal models in the prior art are time-consuming and costly, and usually have some limitations or defects. 1) Since the size of the endogenous immunoglobulin locus of an animal that needs to be knocked out or inactivated is usually on the order of megabases, the gene manipulation efficiency of this knockout or inactivation usually decreases or only partially succeeds. If the knockout or inactivation of the endogenous immunoglobulin locus is incomplete, the resulting animal expresses both human antibodies and endogenous mouse-derived antibodies, increasing the difficulty of antibody selection. 2) The size of the human immunoglobulin locus that needs to be inserted is also on the order of megabases. The size of the vector limits the size of the human DNA fragment introduced at one time, generally requiring more steps and a longer time, and all human immunoglobulin gene fragments can be introduced in several portions. If they cannot be introduced, the resulting animals have only a small V-region library or few constant-region types, and thus can only have a small human antibody diversity or poor B cell development. 3) When successful in inserting a large fragment of DNA of the human immunoglobulin locus, especially when the in situ insertion efficiency is extremely low and multiple insertion steps need to be performed, inefficient factors increase the time cost and risk of failure in obtaining such transgenic animals. When using the random insertion method to introduce a large fragment of DNA of the human immunoglobulin locus into such transgenic animals, due to the deletion of long-range regulatory regions and the uncertain effects caused by random insertion sites, some mouse models have different degrees of inhibition of B cell development, especially the process of developing from T1-B cells to T2-B cells is delayed. It is difficult for the immune response of these animals to antigens to reach the level of non-transgenic animals, and it is also difficult for the affinity of the antibodies produced to reach the affinity of the antibodies produced in non-transgenic animals. 4) Due to restrictions such as the size and quantity of the human immunoglobulin gene fragment introduced, the number of recombinant lines that can be optimally analyzed for expression is limited. The finally obtained transgenic mice have inefficient V(D)J recombination and partial gene complementarity, and generally produce limited antibodies, resulting in a decrease in antibody production efficiency.

[0003] As can be seen from the analysis of the above limitations, there is a need for a method that has a large antibody expression library that can be obtained at low cost and quickly, has a high rearrangement efficiency of human variable region gene fragments, has a high expression efficiency, and in particular, there is a need for a transgenic animal that has a good antigen response ability and can highly express a humanized immunoglobulin with high affinity. 〔Prior Art Documents〕 〔Patent Documents〕 〔Patent Document 1〕International Application Publication No. WO90 / 10077 〔Patent Document 2〕International Application Publication No. WO90 / 04036 〔Patent Document 3〕International Application Publication No. WO2012 / 018610 〔Patent Document 4〕International Application Publication No. WO2010 / 039900 〔Patent Document 5〕International Application Publication No. WO2011 / 004192 〔Patent Document 6〕International Application Publication No. WO2002 / 066630 〔Patent Document 7〕International Application Publication No. WO1994 / 002602 〔Patent Document 8〕International Application Publication No. WO1996 / 030498 〔Patent Document 9〕International Application Publication No. WO1998 / 024893 〔Patent Document 10〕International Application Publication No. WO1994 / 004667 〔Patent Document 11〕International Application Publication No. WO1990 / 006359 〔Patent Document 12〕International Application Publication No. WO1992 / 003917 〔Patent Document 13〕U.S. Application Publication No. US7041871 〔Patent Document 14〕U.S. Application Publication No. US6673986 〔Patent Document 15〕U.S. Application Publication No. US6091001 〔Patent Document 16〕U.S. Application Publication No. US5877397 〔Patent Document 17〕U.S. Patent No. 6,689,610 〔Patent Document 18〕U.S. Patent No. 6,204,061 〔Patent Document 19〕U.S. Patent No. 5,631,153 〔Patent Document 20〕U.S. Patent No. 5,627,059 〔Patent Document 21〕U.S. Patent No. 5,487,992 〔Patent Document 22〕U.S. Patent No. 5,464,764 〔Patent Document 23〕U.S. Patent No. 7,422,889 〔Patent Document 24〕U.S. Patent No. 7,112,715 〔Patent Document 25〕U.S. Patent No. 6,956,146 〔Patent Document 26〕U.S. Patent No. 6,774,279 〔Patent Document 27〕U.S. Patent No. 5,677,177 〔Patent Document 28〕U.S. Patent No. 5,885,836 〔Patent Document 29〕U.S. Patent No. 5,654,182 〔Patent Document 30〕U.S. Patent No. 4,959,317 〔Patent Document 31〕U.S. Patent No. 7,323,618 〔Patent Document 32〕U.S. Patent No. 7,145,057 〔Non-Patent Document〕 〔Non-Patent Document 1〕Nat Biotechnol. 2014 Apr; 32(4): 356 - 63, Proc Natl Acad Sci USA. 2014 Apr 8; 111(14): 5147 - 52 〔Non-Patent Document 2〕Proc Natl Acad Sci USA. 2014 Apr 8; 111(14): 5153 - 8 〔Non-Patent Document 3〕Sambrook and Russell (2001) (Molecular cloning: a laboratory manual 3rd Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press), 〔Non-Patent Document 4〕Nagy, A. (2003). (Manipulating the mouse embryo: a laboratory manual, 3rd Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) [Non-Patent Document 5] Genetic Recombination: Nucleic acid, Homology (biology), Homologous recombination, Non-homologous end joining, DNA repair, Bacteria, Eukaryote, Meiosis, Adaptive immune system, V(D)J recombination (Parallel Edition - December 23, 2009) by Frederic P. Miller, Agnes F. Vandome, and John McBrewster [Non-Patent Document 6] Prosser, H.M., Rzadzinska, A.K., Steel, K.P., and Bradley, A. (2008). Mosaic complementation demonstrates a regulatory role for myosin Vila in actin dynamics of stereocilia. Molecular and Cellular Biology 28, 1702 - 1712; Ramirez-Solis, R., Davis, A.C., and Bradley, A. (1993). Gene targeting in embryonic stem cells. Methods in Enzymology 225, 855 - 878. [Non-Patent Document 7] Evans M.J., Kaufman M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154 - 156. 10.1038 / 292154a0 [Non-Patent Document 8] Datsenko, K.A., B.L. Wanner 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97(12):6640 - 5. [Summary of the Invention] [Problems to be Solved by the Invention] To solve the deficiencies of the prior art, the present invention provides non-human mammalian cells whose genome can contain 41 functional V genes of human immunoglobulin heavy chain variable regions, or 20 functional V genes of human immunoglobulin kappa light chain variable regions, or 31 functional V genes of human immunoglobulin lambda light chain variable regions, and which are used for the selection of fully human therapeutic antibodies and stably inherit. The transgenic animals produced based on the non-human mammalian cells can efficiently and rapidly obtain antibodies with high diversity and strong affinity, and the variable regions are completely derived from humans. 〔Means for Solving the Problems〕 When the inventors of the present application analyzed the human genome database, it was found that the DNA fragment of the human heavy chain variable region is derived from chromosome 14, positions 105863198 to 106879844, the DNA fragment of the human kappa light chain variable region is derived from chromosome 2, positions 88860568 to 90235398, and the DNA fragment of the human lambda light chain variable region is derived from chromosome 22, positions 22023114 to 22922913. All coordinates refer to the human genome database of ENSEMBL GRCh38.p13 version.

[0004] In the human immunoglobulin locus, there may be pseudogenes or open reading frames in which the V-region gene, D-region gene, J-region gene, or constant-region gene does not contribute or contributes inefficiently to the transcriptome of the antibody after rearrangement. For the definitions and characteristics of these three types of genes, refer to the following IMGT database explanation link (http: / / www.imgt.org / IMGTScientificChart / SequenceDescription / IMGTfunctionality.html). Through the http: / / www.imgt.org / genedb / database link, when selecting a species (Homo sapiens for humans), gene type (select variable for V-region genes, diversity for D-region genes, joining for J-region genes, and constant for constant-region genes), and the name of the corresponding immunoglobulin locus (select IGH for the heavy-chain locus, IGK for the Kappa light-chain locus, and IGL for the Lambda light-chain locus), a list of functional, pseudogene, and open reading frame (ORF) gene names within the human immunoglobulin locus can be found. It is particularly noteworthy that all functional genes, pseudogenes, or open reading frame genes reported are included in the IMGT database, and in fact, some genes in the database may exist in only a small number of individuals.

[0005] Gene fragments of the human immunoglobulin heavy chain locus, kappa light chain, and lambda light chain loci can be cloned into a BAC vector or a YAC vector that can replicate in Escherichia coli or yeast by a BAC or YAC library construction method. In the absence of gene editing, pseudo-V genes, open reading frame V genes, and functional V gene fragments are mixed and arranged (Figure 1). These genes may all contain three regions: a gene regulatory region at the 5' end, a gene coding sequence region (including introns and exons), and an antibody gene recombination signal sequence (RSS, Recombination Signal Sequence) at the 3' end. The positions of the functional V genes, pseudo-V genes, and open reading frame gene fragments (5' end regulatory region, gene coding sequence region, and 3' end antibody gene recombination signal sequence (RSS, Recombination Signal Sequence) of the V region gene) derived from humans in the genome are shown in Table 1, and these coordinates refer to the human genome database of the ENSEMBL GRCh38.p13 version.

[0006]

Table 1

[0007] Furthermore, the inventors sequenced the transcriptome cDNA of antibodies after rearrangement of these three variable region gene fragments in the http: / / www.imgt.org / genefrequency / query database and analyzed the gene frequency. They found that functional gene fragments can contribute to the antibody transcriptome at a high frequency through gene rearrangement, while pseudogenes and open reading frames rarely or almost never contribute to the antibody transcriptome.

[0008] Pseudogenes and open reading frames rarely or almost never contribute to the antibody transcriptome. However, these two types of genes can produce ineffective V / D / J or V / J rearrangement products through gene rearrangement. Only B cells with unproductive rearrangement will ultimately disappear by apoptosis, but the existence of a large number of pseudogenes and open reading frames may be an important cause of inefficient unproductive V / D / J or V / J recombination. Therefore, the inventors speculated that when pseudogenes and open reading frames (ORFs) are randomly introduced into the animal genome, the cost of trial and error in the rearrangement of human variable region gene fragments in the animal body will increase and the recombination efficiency will decrease.

[0009] When taking the immunoglobulin variable region locus of humans, mice or rats as an example, functional genes, pseudogenes and open reading frames are interspersed. Even at the gene rearrangement stage, gene transcription stage, and further translation stage, pseudogenes and open reading frames may interfere with effective rearrangement, thereby potentially reducing the effective rearrangement efficiency. After the inventors of the present invention recognized that only functional genes can produce functional immunoglobulins through rearrangement and expression, in order to meet the requirements for efficient production of human antibodies, they attempted to knockout these pseudogenes, the coding sequences and non-coding sequences of open reading frames, and these pseudogenes and open reading frames containing them at the genomic level.

[0010] In one aspect, the present invention provides a nucleic acid construct (or a genetically modified recombinant genome of a non-human mammal), wherein the nucleic acid construct (or recombinant genome) contains variable region gene segments of the human immunoglobulin locus, and one or more or all of the pseudogenes and / or part of the open reading frame genes (i.e., one or more) in the variable region gene segments of the human immunoglobulin locus are deleted.

[0011] In the present application, "one or more or all" means one or more or all.

[0012] Both the coding region and the non-coding region of the variable region gene segment of the human immunoglobulin locus are derived from human immunoglobulins.

[0013] Specifically, the present invention provides a genetically modified recombinant genome (or nucleic acid construct) of a non-human mammal, wherein part or all of the endogenous immunoglobulin variable region genes are replaced by human immunoglobulin variable region genes, and part or all of the pseudogenes and / or open reading frames of the human immunoglobulin variable region genes are knocked out.

[0014] "Human immunoglobulin variable region gene" has the same meaning as "variable region gene (segment) of the human immunoglobulin locus".

[0015] In the present application, optionally, a part or all of other irrelevant genes of the human immunoglobulin variable region gene (for example, the genes marked by the H35 fragment in Table 1) are knocked out.

[0016] Optionally, the variable region of the human immunoglobulin locus is selected from the variable region of the heavy chain of human immunoglobulin, and / or the variable region of the kappa light chain, and / or the variable region of the lambda light chain.

[0017] Optionally, the variable region of the human immunoglobulin locus is selected from any one or a combination of the V, D, and J regions of the variable region of the heavy chain of human immunoglobulin.

[0018] Optionally, the variable region of the human immunoglobulin locus is selected from any one or a combination of the V and J regions of the variable region of the kappa light chain of human immunoglobulin.

[0019] Optionally, the variable region of the human immunoglobulin locus is selected from any one or a combination of the V and J regions of the variable region of the lambda light chain of human immunoglobulin.

[0020] Optionally, the pseudogene and / or open reading frame gene are deleted through gene knockout. Optionally, the gene knockout is carried out in prokaryotic cells or eukaryotic cells such as bacteria, yeast, insect cells, plant cells, Escherichia coli, CHO, Pichia pastoris, etc.

[0021] Note that the definitions of the pseudogene V gene and open reading frame gene refer to the IMGT database.

[0022] In the nucleic acid construct (or genetically modified recombinant genome) of the present invention, both the coding region and the non-coding region of the variable region gene segment of the human immunoglobulin locus are derived from human immunoglobulin. Optionally, the variable region gene segment of the human immunoglobulin locus includes the coding region and the non-coding region of the functional V, D, and J regions of the heavy chain variable region of human immunoglobulin. Optionally, the variable region gene segment of the human immunoglobulin locus includes the coding sequence and the non-coding sequence of the functional V and J regions of the κ light chain variable region of human immunoglobulin, and / or the coding sequence and the non-coding sequence of the functional V and J regions of the λ light chain variable region of human immunoglobulin.

[0023] For example, in one specific embodiment, the nucleic acid construct (or genetically modified recombinant genome) of the present invention comprises the following. (1) Knock out a DNA fragment containing 10 pseudo-V region genes or open reading frame V region genes, namely H2, H4, H9, H11, H13, H15, H19, H21, H23, H17, within the human immunoglobulin heavy chain V region of Table 1, and retain the sequences containing the human immunoglobulin heavy chain D gene region and J gene region derived from human chromosome 14 positions 105863198 to 105939714, or (2) Knock out a DNA fragment containing 14 pseudo-V genes or open reading frame V region genes, namely SEQ ID NOs. H26, H28, H39, H41, H43, H46, H49, H51, H53, H57, H59, H61, H64, H68 in Table 1, and retain the fragment containing 25 functional V region genes, namely SEQ ID NOs. H25, H27, H29, H31, H33, H36, H38, H40, H42, H44, H45, H47, H48, H50, H52, H54, H55, H56, H58, H60, H62, H63, H65, H66, H67 in Table 1, and the fragment containing the pseudo-V region genes SEQ ID NOs. H30, H32, H34, H37 and the H35 gene in Table 1, or (3) Knock out the region containing 10 pseudo-V genes or open reading frames, namely SEQ ID NOs. K3, K6, K9, K12, K16, K19, K21, K24, K26, K28 in Table 1, and retain the region containing 20 functional human Kappa light chain V region genes, namely SEQ ID NOs. K1, K2, K4, K5, K7, K8, K10, K11, K13, K14, K15, K17, K18, K20, K22, K23, K25, K27, K29, K30 in Table 1 and the human immunoglobulin Kappa light chain J gene of human chromosome 2 positions 88861967 to 88860568,Alternatively, knockout the region containing 12 pseudo-V genes or open reading frames with SEQ ID NOs: L2, L4, L10, L12, L14, L17, L19, L21, L23, L25, L28, L30 in Table 1, retain the region containing 19 functional human lambda light chain V region genes with SEQ ID NOs: L1, L3, L5, L6, L7, L8, L9, L11, L13, L15, L16, L18, L20, L22, L24, L26, L27, L29, L31 in Table 1 and the region containing the human immunoglobulin lambda light chain J gene and human C gene at positions 22881432 - 22922913 on human chromosome 22, or (5) knockout the region containing 7 pseudo-V genes or open reading frames with SEQ ID NOs: L36, L38, L41, L43, L45, L48, L50 in Table 1, retain the region containing 12 functional human lambda light chain V region genes with SEQ ID NOs: L32, L33, L34, L35, L37, L39, L40, L42, L44, L46, L47, L49 in Table 1, or include any two or more of (1)-(5), preferably including (1) and (2) or (4) and (5).

[0024] The present invention also provides a method for preparing the nucleic acid construct of the present invention (or the genetically modified recombinant genome of a non-human mammal), the method having the following configuration: (1) Amplify each fragment of the variable region gene segment of the human immunoglobulin locus by PCR, optionally individually insert each fragment obtained by amplification into an appropriate position of a vector, or insert a part or all of the fragments obtained by amplification into an appropriate position of a vector after ligation, or (2) Knock out the pseudo-V genes and / or open reading frame genes in the heavy chain variable region DNA fragment or light chain variable region DNA fragment of human immunoglobulin through a gene knockout method to obtain the variable region gene segment of the human immunoglobulin locus, or (3) A gene synthesis method.

[0025] In the present application, the human immunoglobulin variable region gene is a human heavy chain functional V H, D H , J H The coding and non-coding sequences of, or the human light chain functional V L , J L The coding and non-coding sequences are included, and the light chain is a kappa or lambda light chain.

[0026] Furthermore, the endogenous immunoglobulin variable region gene is the mouse immunoglobulin heavy chain variable region V H , D H , J H or the light chain variable region V L , J L is included, where the light chain refers to a kappa or lambda light chain.

[0027] Furthermore, the coding and non-coding sequences of the human heavy chain functional V H , D H , J H are derived from human chromosome 14, and the coding and non-coding sequences of the human light chain functional V L , J L are derived from human chromosome 2 or human chromosome 22.

[0028] Furthermore, the coding and non-coding sequences of the human heavy chain functional V H , D H , J H include the sequence at nucleotide positions 105863198 - 106879844 derived from human chromosome 14. All coordinates refer to the human genome database of ENSEMBL GRCh38.p13 version, and the sequence numbers H1, H3, H5, H6, H7, H8, H10 (this fragment contains 3 functional V region genes), H12, H14, H16, H18, H20, H22, H24, H25, H27, H29, H31, H33, H36, H38, H40, H42, H44, H45, H47, H48, H50, H52, H54, H55, H56, H58, H60, H62, H63, H65, H66, H67 in Table 1 contain one or more V H region genes in the sequence fragments, preferably 10 - 41 functional V H region genes, more preferably 15 - 41 functional V HRegion gene, more preferably 18 to 41 functional V H Region gene, more preferably 22 to 41 functional V H Region gene, more preferably 25 to 41 functional V H It is a region gene, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 functional V region genes.

[0029] Furthermore, the human kappa light chain functional V L , J L The coding sequence and non-coding sequence of are one or more V in the sequence fragment of nucleotide positions 88860568 to 90235398 derived from human chromosome 2 (the sequence including SEQ ID NOs: K1, K2, K4, K5, K7, K8, K10, K11, K13, K14, K15, K17, K18, K20, K22, K23, K25, K27, K29, K30 in Table 1) L Region genes, or one or more V in the sequence fragment of nucleotide positions 22023114 to 22922913 derived from human chromosome 22 (the sequence including SEQ ID NOs: L1, L3, L5, L6, L7, L8, L9, L11, L13, L15, L16, L18, L20, L22, L24, L26, L27, L29, L31, L32, L33, L34, L35, L37, L39, L40, L42, L44, L46, L47, L49 in Table 1) L Region genes are included, and all coordinates refer to the human genome database of ENSEMBL GRCh38.p13 version.

[0030] Furthermore, the endogenous immunoglobulin variable region gene is partially or completely deleted by homologous recombination, and the human immunoglobulin heavy chain variable region gene is inserted at a position 3KB upstream to 3KB downstream of the deleted endogenous immunoglobulin heavy chain variable region, and the human immunoglobulin light chain variable region gene is inserted at a position 3KB upstream to 3KB downstream of the deleted endogenous immunoglobulin kappa light chain variable region.

[0031] The number of knocked-out (or partially or completely knocked-out) pseudogenes and / or open reading frame genes in the human immunoglobulin variable region gene should be significantly reduced, especially such that the lengths of various genes are shortened. Generally, the lengths of the human immunoglobulin heavy chain and Lambda light chain variable region genes inserted into the non-human mammalian cell genome are 10% to 50% of the full lengths of the human immunoglobulin heavy chain and Lambda light chain variable region genes before knocking out the pseudogenes and / or open reading frame genes, preferably 12% to 47%, preferably 14% to 45%, preferably 15% to 43%, more preferably 16% to 40%, more preferably 16.10%, 18%, 18.50%, 20%, 25%, 30%, 31%, 31.75%, 35%, 38% or 38.06%. Note that the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is 35% to 65% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogenes and / or open reading frame genes, preferably 37% to 63%, preferably 38% to 61%, preferably 40% to 60%, preferably 42% to 58%, preferably 45% to 57%, preferably 47% to 56%, more preferably 50% to 55%, such as 51%, 52%, 53%, 53.08% or 54%.

[0032] The total number of the pseudogenes and / or open reading frame genes is about 75 in the human heavy chain variable region, about 20 in the human Kappa light chain proximal variable region, and about 42 in the human Lambda light chain variable region.

[0033] Preferably, for "partial or complete knockout" or "partial or complete deletion", 10-100% (preferably 15-95%, 20-90%, such as 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 75%, 78%, 80%, 83%, 85% or 88%) of the pseudogenes and / or open reading frame genes are knocked out or deleted, and this ratio is based on the total number of pseudogenes and open reading frame genes of the human immunoglobulin variable region gene.

[0034] Furthermore, the non-human mammalian cell is a mouse embryonic stem cell, the deleted endogenous immunoglobulin heavy chain variable region is located at positions 113428530-116027502 on mouse chromosome 12, the deleted endogenous immunoglobulin kappa light chain variable region is located at positions 67536984-70723924 on mouse chromosome 6, the deleted endogenous immunoglobulin lambda light chain variable region is located at positions 19065021-19260700 on mouse chromosome 16, and the mouse genomic chromosome locus coordinates refer to the C57BL / 6J mouse genomic database positions in the ENSEMBL GRCm38.p6 version. Preferably, the insertion site of the human immunoglobulin heavy chain variable region gene is at position 113428513 on mouse chromosome 12, the insertion site of the human immunoglobulin kappa light chain variable region gene is at position 70723924 on mouse chromosome 6, the insertion site of the human immunoglobulin lambda light chain variable region gene is at position 70726758 on mouse chromosome 6, and the mouse genomic chromosome locus coordinates refer to the C57BL / 6J mouse genomic database positions in the ENSEMBL GRCm38.p6 version.

[0035] Another aspect of the present invention provides a non-human mammalian cell containing the genetically modified recombinant genome. The mammalian cell is a non-human mammalian embryonic stem cell, and more preferably, the embryonic stem cell is a mouse embryonic stem cell, a rat embryonic stem cell or a rabbit embryonic stem cell.

[0036] The present invention also provides a recombinant cell comprising the nucleic acid construct of the present invention, which is prepared by introducing the nucleic acid construct of the present invention into a target cell. Optionally, the cell is an immortalized cell or a non-immortalized cell (e.g., a primary cell, a subculture cell), including a prokaryotic cell or a eukaryotic cell, such as an Escherichia coli cell, a yeast cell, an avian cell, a mammalian cell, an embryonic stem cell of a rat or a mouse, a primordial germ cell of a bird, a C57BL / 6J*129S3 embryonic stem cell.

[0037] The present invention also provides the use of the nucleic acid construct and the recombinant cell of the present invention in the production of transgenic animals.

[0038] In another aspect, the present invention provides a genetically engineered non-human mammalian cell, in which the immunoglobulin locus contained in its genome has a deletion of the endogenous immunoglobulin variable region gene of the host non-human mammalian cell, and both the inserted coding region and non-coding region are derived from gene fragments of the human immunoglobulin variable region, and the gene fragment of the human immunoglobulin variable region has a deletion of some or all of the pseudo-V gene and / or open reading frame.

[0039] Generally, the cell is an immortalized cell or a non-immortalized cell (e.g., a primary cell or a subculture cell).

[0040] In the genetically engineered non-human mammalian cells of the present invention, the inserted gene fragment of the human immunoglobulin variable region contains the coding sequences and non-coding sequences of the heavy chain variable region functional V, D, J regions, and / or the coding sequences and non-coding sequences of the κ or λ light chain variable region functional V, J regions. Optionally, the non-human mammal is a bird such as a mouse, rat, chicken, rabbit, or a rodent. The non-human mammalian cells can be derived from birds, rodents, mice, rats, chickens, rabbits, etc., and can be rat or mouse embryonic stem cells, avian primordial germ cells, C57BL / 6J*129S3 embryonic stem cells, etc. Alternatively, the deleted variable region gene fragment of the endogenous immunoglobulin locus contains the mouse immunoglobulin heavy chain variable region V, D, J regions or the light chain κ or λ variable region V, J regions. Alternatively, the inserted coding sequences and non-coding sequences of the heavy chain variable region functional V, D, J regions contain the sequence from nucleotide position 105863198 to 106879844 on human chromosome 14. Optionally, the inserted coding sequences and non-coding sequences of the light chain κ variable region functional V, J regions contain the sequence from nucleotide position 88860568 to 90235398 on human chromosome 2. Optionally, the inserted coding sequences and non-coding sequences of the light chain λ variable region functional V, J regions contain the sequence from nucleotide position 22023114 to 22922913 on human chromosome 22, and all nucleotide position coordinates refer to the human genome database of ENSEMBL GRCh38.p13 version.

[0041] Another aspect of the present invention provides a method for producing non-human mammalian cells, the method comprising a) introducing, in the same direction and in a compatible manner, recombinase targeting sites upstream and downstream of the variable region gene cluster of the immunoglobulin locus in the genome of a host non-human mammalian cell, respectively; b) subsequently introducing into the cells of step a) a specific recombinase capable of recognizing the recombinase targeting sites, and partially or completely deleting the variable region gene of the endogenous immunoglobulin locus of the host non-human mammalian cell under conditions allowing recombination to occur between the two recombinase targeting sites described in step a) to obtain target cells; c) providing a vector comprising a part or all of the variable region of the human immunoglobulin locus, and knocking out a part or all of the pseudo-V genes and / or open reading frames of the variable region gene of the human immunoglobulin locus contained in the vector to obtain a targeting vector; and d) introducing the targeting vector into the target cells obtained in step b), and replacing the variable region gene of the endogenous immunoglobulin locus that is deleted in the target cells with the variable region of the human immunoglobulin locus contained in the targeting vector to obtain genetically engineered non-human mammalian cells.

[0042] Optionally, the targeting vector of step c) is a heavy chain targeting vector comprising a heavy chain variable region gene, or a light chain targeting vector comprising a κ or λ light chain variable region gene. Note that one or more heavy chain targeting vectors or light chain targeting vectors can be constructed according to the number of variable region genes to be introduced, for example, the targeting vectors shown in FIGS. 10, 13, and 14. When there are multiple heavy chain or light chain targeting vectors, these targeting vectors can be sequentially introduced in step d).

[0043] The targeting vector described in step c) is constructed in Escherichia coli or yeast cells.

[0044] More specifically, another aspect of the present invention provides a method for producing non-human mammalian cells, the method comprising a) deleting or inactivating the light and heavy chain variable regions of the host non-human mammalian Ig locus; b) inserting a human IgH VDJ region comprising a plurality of human IgH V regions, one or more human D regions, and one or more human J regions, in which part or all of the pseudo V gene and / or open reading frame gene is deleted, upstream of the heavy chain constant region of the host non-human mammalian Ig locus; and / or c) inserting a human κ VJ region comprising a plurality of human Ig light chain κ V regions and one or more human Ig light chain κ J regions, in which part or all of the pseudo V gene and / or open reading frame is deleted, upstream of the κ constant region of the host non-human mammalian Ig locus; and / or d) inserting a human λ VJ region comprising a plurality of human Ig light chain λ V regions and one or more human Ig light chain λ J regions, in which part or all of the pseudo V gene and / or open reading frame is deleted, downstream of the κ constant region of the host non-human mammalian Ig locus, wherein steps a) to c) can be carried out in any order, either stepwise or in a single step.

[0045] In addition, another aspect of the present invention provides a method for producing non-human mammalian cells, the method comprising: a) introducing recombinase targeting sites that match in the same direction upstream and downstream of the immunoglobulin variable region gene in the non-human mammalian cell genome; b) introducing a specific recombinase capable of recognizing the recombinase targeting sites described in step a), and partially or completely deleting the endogenous immunoglobulin variable region gene of the non-human mammalian cell under conditions that permit recombination between the two recombinase targeting sites described in step a); c) a targeting vector comprising part or all of the human immunoglobulin variable region (the targeting vector comprises a human functional variable region gene, and part or all of the pseudogene and / or open reading frame is knocked out, and the human functional variable region gene is a human functional V H , D H , J HThe coding and non-coding sequences, or human functional V L , J L comprising the coding and non-coding sequences, wherein the light chain is a kappa or lambda light chain, and the targeting vector is selected from a BAC vector or a YAC vector), d) introducing the targeting vector described in step c) and replacing the endogenous immunoglobulin gene of the non-human mammalian cell lacking the one described in step b) with the human immunoglobulin variable region gene included in the non-human mammalian cell in step c), and e) producing a non-human mammalian cell containing the human immunoglobulin variable region gene in the genome from step d).

[0046] Preferably, the targeting vector described in step c) is constructed in Escherichia coli or yeast cells.

[0047] Another aspect of the present invention provides a targeting vector containing a human immunoglobulin variable region gene, wherein a part or all of the pseudogene and / or open reading frame of the human immunoglobulin variable region gene is knocked out, and the human immunoglobulin variable region gene is human heavy chain functional V H , D H , J H comprising the coding and non-coding sequences, or human light chain functional V L , J L comprising the coding and non-coding sequences, wherein the light chain is a kappa or lambda light chain, and the targeting vector is selected from a BAC vector or a YAC vector.

[0048] Another aspect of the present invention provides a method for producing a non-human mammal that expresses an antibody whose variable region is completely derived from humans, introducing the non-human mammalian cell into the uterus of a female wild-type non-human mammal, and selecting a chimeric non-human mammal of the offspring as a non-human mammal of the F0 generation.

[0049] Furthermore, before introducing non-human mammalian cells into the uterus of a female wild-type non-human mammal, the non-human mammalian cells are selected to obtain non-human mammalian cell clones without chromosomal number increase or decrease. The non-human mammalian cell clones are transplanted into the blastocoel cavity of a wild-type non-human mammalian blastocyst, and the blastocyst is transplanted into the uterus of a pseudo-pregnant female wild-type non-human mammal.

[0050] Furthermore, after mating a non-human mammal of the F0 generation with a wild-type non-human mammal, a non-human mammal of the F1 generation that can stably inherit and insert a human immunoglobulin variable region gene at a predetermined position can be obtained. In addition, when a non-human mammal expressing an antibody with a heavy chain variable region derived entirely from humans and a non-human mammal expressing an antibody with a light chain variable region derived entirely from humans are mated as parents, a non-human mammal expressing an antibody with both heavy and light chain variable regions derived entirely from humans can be obtained.

[0051] Another aspect of the present invention provides a non-human mammal produced by a method for producing a non-human mammal that expresses an antibody with a variable region derived entirely from humans.

[0052] Preferably, the non-human mammal is a mouse, rat, or rabbit, and the non-human mammalian cells are mouse embryonic stem cells, rat embryonic stem cells, or rabbit embryonic stem cells.

[0053] Another aspect of the present invention provides the use of the recombinant genome, the non-human mammalian cells, the targeting vector, or the obtained non-human mammal in the selection of a fully human antibody or the preparation process of a fully human antibody pharmaceutical.

[0054] Another aspect of the present invention provides the use of the recombinant genome, the non-human mammalian cells, the method for producing the non-human mammalian cells, or the targeting vector in the production of a non-human mammal.

[0055] Another aspect of the present invention provides an antibody with a variable region derived entirely from humans, an antibody fragment, or a derivative pharmaceutical or pharmaceutical composition containing the antibody or antibody fragment produced by the non-human mammal.

[0056] The present invention provides the use of the genetically engineered non-human mammalian cells of the present invention in the production of transgenic animals and a method for producing transgenic animals. The method includes injecting the genetically engineered non-human mammalian cells (such as embryonic stem cells) of the present invention into a blastocyst, and then transplanting the chimeric blastocyst into a female animal to produce offspring, and breeding and selecting homozygous recombinants having the desired insertion to obtain transgenic animals. Optionally, the animal can be a rodent such as a rat, mouse, chicken or rabbit.

[0057] In another aspect, the present invention provides a method for generating an antibody or an antigen-binding fragment thereof, which includes immunizing a transgenic animal produced by the present invention with an antigen and recovering the antibody or antibody chain, or recovering the cells producing the antibody or heavy chain or light chain. Optionally, the constant region of the obtained antibody or its antigen-binding fragment is replaced with a human constant region to produce a fully humanized antibody. The present invention also provides the antibody or its antigen-binding fragment prepared in this way, and their use in the preparation of pharmaceutical compositions, and also provides pharmaceutical compositions of these antibodies or their antigen-binding fragments, optionally further including a pharmaceutically acceptable vector. The pharmaceutical composition may be an antibody derivative pharmaceutical which contains an antibody and is a drug formed by binding with other molecules (such as an antibody-toxin conjugate small molecule drug, an antibody-radioimmunoconjugate drug, an antibody-polypeptide conjugate therapeutic drug, a bispecific multispecific antibody, etc.).

[0058] All coordinates of the human immunoglobulin genes in the present invention refer to the human genome database of the ENSEMBL GRCh38.p13 version. The human heavy chain variable region DNA fragment is derived from chromosome 14 at positions 105863198 to 106879844, the human Kappa light chain variable region DNA fragment is derived from chromosome 2 at positions 88860568 to 90235398, and the human Lambda light chain variable region DNA fragment is derived from chromosome 22 at positions 22023114 to 22922913.

[0059] Of the total number of pseudo-V genes and open reading frames in the variable region gene segments of the human immunoglobulin locus, generally 10 to 100% of the pseudo-V genes and / or open reading frames are knocked out, for example 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0060] The method of gene knockout in the present invention can be any known applicable method in the art, such as knockout by homologous recombination, and an exemplary example is to refer to the accompanying drawings.

[0061] The recombinase in the present invention can be any suitable enzyme known in the art, such as Cre, FLP, etc., and the recognition sites can be LoxP, FRT, etc. The constructs, cells, and animals of the present invention can be produced by utilizing a combination of homologous recombination and site-specific recombination. Exemplary homologous recombination methods are described in Patent Documents 17, 18, 19, 20, 21, and 22, which are incorporated herein by reference. Site-specific recombination requires a dedicated recombinase that recognizes the sites and catalyzes recombination at these sites. Many bacteriophage and yeast-derived site-specific recombination systems, such as the tyrosine family bacteriophage P1 Cre / LoxP, yeast FLP-FRT system, and Dre system, each contain a recombinase and a specific homologous site, and the integration of DNA into eukaryotic cells is also suitable for use in the present invention. Such systems and methods of use are described in Patent Documents 23, 24, 25, 26, 27, 28, 29, and 30, which are incorporated herein by reference. The recombinase-mediated cassette exchange (RMCE) procedure is carried out with a combination of wild-type and mutant LoxP (or FRT, etc.) sites and negative selection. Other systems of the tyrosine family, such as bacteriophage λ Int integrase, HK2022 integrase, and systems belonging to the serine family of recombinases, such as bacteriophage phiC31, R4Tp901 integrase, are also suitable for use in the present invention. The introduction of site-specific recombination sites can be achieved through conventional homologous recombination techniques, and such techniques are described in reference documents, such as Non-Patent Documents 3, 4, and 5.

[0062] Gene knockout or knock-in can be identified by any method known in the art, including but not limited to enzyme cleavage identification, PCR identification, hybridization, or selection marker (resistance, nutrition, toxin selection, etc.) identification. Exemplary methods are shown in Figures 11 and 12.

[0063] The targeting vector used in the present invention can be of any known type suitable for use in the present invention. A typical gene targeting vector generally consists of three parts, namely, a targeting gene or an exogenous gene to be inserted into the genome of the recipient cell, DNA sequences flanking the exogenous gene and homologous to the target locus in the cell, and a selection marker. Usually, the neomycin phosphotransferase gene (neo) is used as a positive (+) selection marker, and recipient cells expressing the neomycin phosphotransferase gene can be selected by culturing in a medium containing G418. An exemplary targeting vector is a BAC vector.

[0064] To produce vectors for intracellular homologous recombination, the recombinant engineering methods used in the present invention are described, for example, in WO9929837 and WO0104288, and the techniques are well known in the art. In one aspect, BACs are used as derived from human DNA to perform recombinant engineering of human DNA. Human BAC DNA is isolated using the MN NucleoBond BAC 100 purification kit. The genomic insertion sequences of each human BAC are edited by recombinant engineering so that, when inserted, they form seamless contiguous portions of the human V(D)J genomic region at the mouse IgH or IgK locus. Electroporation transfection and genotyping of BAC DNA can refer to standard protocols (Non-Patent Document 6).

[0065] The genetically engineered non-human mammalian cells of the present invention can be used for the production of transgenic animals because they produce antibodies or antigen-binding fragments thereof of the human immunoglobulin variable region. In one aspect, the host cell in which the endogenous immunoglobulin gene is replaced is an embryonic stem cell, and the embryonic stem cell can then be used for the production of transgenic mammals. Accordingly, the method of the present invention further comprises the steps of isolating embryonic stem cells containing an introduced portion of the human immunoglobulin variable region and producing a transgenic animal containing a partially replaced immunoglobulin locus using the embryonic stem cells. Optionally, the transgenic animal can be a bird, and primordial germ cells are used to produce the transgenic animal. Accordingly, the method of the present invention further comprises the steps of isolating primordial germ cells containing an introduced portion of the human immunoglobulin variable region and producing a transgenic animal containing a partially replaced immunoglobulin locus using the germ cells. Methods for producing such transgenic birds are disclosed in Patent Documents 31 and 32, which are incorporated herein by reference.

[0066] The transgenic animals of the present invention can be used for the production of human antibodies, such as polyclonal antibodies and monoclonal antibodies. These antibodies can be used for conventional applications in the art, including the preparation of compositions (e.g., pharmaceutical compositions), the detection of antigens (e.g., detection reagents or kits), or diagnosis (e.g., diagnostic reagents or kits). Methods and compositions for antigen immunization and antibody preparation, and techniques for preparing detection or diagnostic products are well known in the art. 〔Advantages of the Invention〕 1) By knocking out some or all of the pseudogenes and / or open reading frames, as many functional human antibody variable region gene fragments as possible are collected in the same vector to achieve high-efficiency V H D H J H or V L J L recombination in the mouse body, 2) Since more functional human antibody gene fragments can be introduced into experimental animals by smaller vectors, the construction risk is reduced, the construction time is shortened, and the construction cost is reduced. 3) The knockout operation of the pseudogene or open reading frame of the present invention is completed in Escherichia coli, so the operation is simple, the knockout efficiency is very high, and the time required is short. By reducing the human immunoglobulin variable region gene in Escherichia coli, the gene targeting step in embryonic stem cells can be minimized or even completed in one step. Therefore, as many functional human antibody variable region gene introductions as possible can be completed with very few non-human mammalian cell gene targeting steps, and experimental animals expressing a complete human variable region can be constructed within a very short time. 4) The experimental animals constructed according to the present invention can normally express chimeric antibodies with a variable region derived entirely from humans and a constant region derived from mice, and have a specific immune response to antigens at a level equal to or higher than that of wild-type mice. 5) Compared with conventional antibody selection platforms, the transgenic mice of the present invention can produce a larger antibody library that can be used for high-efficiency selection of antibodies. The affinity of the produced antibodies can reach the nM size and even the pM size, and the affinity is stronger. 6) The ratio of mature B cells to immature B cells in the spleen of the transgenic mice of the present invention is almost the same as that of wild-type mice, ensuring high-efficiency and normal B cell development. 〔Brief Description of the Drawings〕 〔FIG. 1〕Schematic diagram showing DNA fragments of the genomic loci of human immunoglobulin heavy chain, Kappa and Lambda light chain genes inserted into a BAC or YAC vector, and basic structural schematic diagrams of functional variable region V gene fragments, pseudogene variable region V gene fragments and open reading frame V gene fragments. 〔FIG. 2〕Schematic diagram showing recombinant knockout when two recombinase recognition sites are in the same direction on the same chromosome. FIG. 3 is a schematic diagram showing a translocation event when two recombinase recognition sites are in the same direction on two homologous chromosomes. FIG. 4 is a schematic diagram showing a method for efficiently knocking out a long target fragment DNA sequence between recombinase recognition sites by introducing a recombination combination into a complete antibiotic selection gene expression element after stepwise introduction of recombinase recognition sites that match in the same direction and an antibiotic selection gene expression element divided into two on the same chromosome of the genome. FIG. 5 is a schematic diagram showing recombination / knockout of the endogenous immunoglobulin heavy chain variable region (sequence at positions 113428530 to 116027502 on mouse chromosome 12) of the mouse genome (PolyA - hygromycin - LoxP, Puro - CAG, PolyA - Neo - LoxP - PGK, PolyA - hygro - LoxP - PGK). FIG. 6 is a schematic diagram showing recombination / knockout of the endogenous immunoglobulin kappa light chain variable region (sequence at positions 67536984 to 70723924 on mouse chromosome 6) of the mouse genome. FIG. 7 is a schematic diagram showing recombination / knockout of the endogenous immunoglobulin lambda light chain variable region (sequence at positions 19065021 to 19260700 on mouse chromosome 16) of the mouse genome. FIG. 8 is a flowchart showing knockout of a pseudogene or open reading frame in a BAC vector containing a human immunoglobulin region DNA fragment. FIG. 9 is a schematic diagram when the 5' - terminal gene regulatory region, V - region coding sequence (including intron and exon), and 3' antibody gene recombination signal sequence (RSS, Recombination Signal Sequence) of different functional V - region genes are recombined into a new functional V - region gene fragment. FIG. 10 is a schematic diagram showing the sequences and structures of two targeting vectors containing human heavy - chain immunoglobulin variable region gene fragments. 〔Fig. 11〕When taking as an example the bacterial artificial chromosome enzyme cleavage pattern of the heavy chain targeting vector 1 by pulsed field gel electrophoresis, it is a schematic diagram showing a method for identifying the integrity of the DNA fragment of the human immunoglobulin region contained in the BAC vector. 〔Fig. 12〕It is a schematic diagram showing a method for identifying the integrity of the human immunoglobulin gene fragment using the polymerase chain reaction (PCR) method with the bacterial artificial chromosome of the heavy chain targeting vector 1 as a PCR template. 〔Fig. 13〕It is a schematic diagram showing the sequence and structure of a targeting vector containing a human Kappa light chain immunoglobulin variable region gene fragment. 〔Fig. 14〕It is a schematic diagram showing the sequence and structure of two targeting vectors containing a human Lambda light chain immunoglobulin variable region gene fragment. 〔Fig. 15〕It is a schematic diagram showing a protocol for inserting the human immunoglobulin heavy chain variable region gene into the position of the endogenous immunoglobulin heavy chain variable region of the mouse (the original 12th chromosome 113428530 - 116027502 is deleted). 〔Fig. 16〕When stepwise introducing the human heavy chain targeting vectors 1 and 2 into mouse embryonic stem cells lacking the endogenous heavy chain variable region sequence, it is a schematic diagram. 〔Fig. 17〕Taking as an example the in - situ insertion of a gene fragment with the targeting vector of the ACE001 - H2 single homology arm in Fig. 2, it is a schematic diagram showing a PCR identification method for accurately realizing the accurate insertion event of the gene in the in - situ insertion protocol of the gene fragment by the targeting vector through homologous recombination. 〔Fig. 18〕Taking as an example the in - situ insertion of a gene fragment with the targeting vector of the ACE001 - H2 single homology arm in Fig. 2, it is a diagram showing the method for identifying an accurate gene insertion event by 5' - end and 3' - end PCR with primers P1 / P4 and P3 / P2 and the results of electrophoresis. 〔Fig. 19〕It is a schematic diagram showing a protocol for inserting the human immunoglobulin Kappa light chain variable region gene into the position of the endogenous immunoglobulin Kappa light chain variable region of the mouse (the original 6th chromosome 67536984 - 70723924 is deleted). FIG. 20 is a schematic diagram showing a human kappa light chain targeting vector introduced into mouse embryonic stem cells lacking the endogenous kappa light chain variable region sequence of a mouse. FIG. 21 is a schematic diagram showing a protocol for inserting the human immunoglobulin lambda light chain variable region gene downstream of the endogenous immunoglobulin kappa light chain constant region of a mouse (deletion of the original 67536984 to 70723924 on chromosome 6). FIG. 22 is a schematic diagram of the case where human lambda light chain targeting vectors 1 and 2 are sequentially introduced into mouse embryonic stem cells lacking the endogenous kappa light chain sequence of a mouse. FIG. 23 is a diagram showing a comparison of B cell development status between the transgenic mouse of the present invention and a wild-type BALB / c mouse. FIG. 24 is a diagram showing a comparison of OVA-specific antibody serum levels between the transgenic mouse and the wild-type BALB / c mouse after the third booster immunization. FIG. 25 is a diagram showing the affinity level of the antibody obtained from the transgenic mouse of the present invention. FIG. 26 shows SEQ ID NO: 1 in step 2 of Example 4. Term Definition Pseudogene: Also called a pseudo gene or a false gene, it is a residue without a function formed during the evolution of a gene family. A pseudogene resembles a coding gene sequence, is generally not transcribed, and can be regarded as a non-functional genomic DNA copy in the genome without a clear physiological meaning. Pseudogenes have homologous normal genes, and these DNA sequences are very similar. The ancestral gene of a pseudogene was functional, but due to sequence abnormalities caused by mutations, it became unable to be transcribed or the transcript became unable to be translated, so the function of the pseudogene was lost. Pseudogenes are ubiquitous in the mammalian genome and can be regarded as relics of evolution.

[0067] Open reading frame (ORF): An open reading frame is a base sequence on mRNA. An ORF starts from a start codon and ends with a stop codon, and one ORF corresponds to one protein.

[0068] For the specific definitions and characteristics of pseudogenes and open reading frames in the immunoglobulin loci of humans or mice, refer to the description link of the IMGT database (http: / / www.imgt.org / IMGTScientificChart / SequenceDescription / IMGTfunctionality.html).

[0069] Immunoglobulin heavy chain variable region (variable region of heavy chain of Ig, V H ): It is the region with more amino acid sequence changes within the immunoglobulin heavy chain molecule and is a functional region of approximately 115 - 120 residues at the amino terminus. Among these, there are three hypervariable regions with large changes, and the amino acid residues are located at positions 29 - 31, 49 - 58, and 95 - 102 respectively.

[0070] Immunoglobulin light chain variable region (variable region of light chain of Ig, V L ): It is the region with more amino acid sequence changes within the immunoglobulin light chain molecule and is a functional region of approximately 110 amino acid residues. Among them, three parts have large changes and are called hypervariable regions, and the amino acid residues are at positions 28 - 35, 49 - 56, and 91 - 98.

[0071] Immunoglobulin gene rearrangement: Along with the differentiation of B lymphocytes, immunoglobulin genes undergo rearrangement phenomena such as V H / D H / J H 、V L / J L etc., resulting in the generation of immunoglobulin diversity. Coding sequence: The base sequence of DNA that encodes the base sequence of mature RNA in the transcribed region, such as an exon. Less than 2% of the human genome sequence is the coding sequence.

[0072] Non-coding sequence: (1) All sequences excluding the coding sequence in a gene sequence, such as promoters, introns, and enhancers. (2) All sequences in the genomic sequence excluding the gene coding sequence. More than 98% of the human genomic sequence is non-coding sequence.

[0073] Targeting vector: A typical gene targeting vector generally consists of three parts, namely, a gene for targeting or an exogenous gene to be inserted into the genome of the recipient cell, DNA sequences homologous to the target locus in the cell on one or both sides of the exogenous gene, and a selection marker. Usually, the neomycin phosphotransferase gene (neo) is used as a positive (+) selection marker, and recipient cells expressing the neomycin phosphotransferase gene can be selected by culturing in a medium containing G418.

[0074] Derivative pharmaceutical containing an antibody or antibody fragment: A drug formed by binding an antibody or antibody fragment to other molecules (such as antibody-toxin conjugate small molecule drugs, antibody radioimmunoconjugate drugs, antibody-polypeptide conjugate therapeutic drugs, bispecific antibodies, etc.). 〔Mode for Carrying Out the Invention〕 Before further explaining the present invention, it should be understood that the present invention is not limited to the above specific embodiments. Those skilled in the art can change the elements within these embodiments with known techniques.

[0075] Unless otherwise defined specifically, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. All publications mentioned herein are incorporated herein by reference and disclose and explain the methods and / or materials related to the cited publications.

[0076] In the following examples, the coordinates of all mouse genomic chromosomal sites refer to the ENSEMBL GRCm38.p6 version of the C57BL / 6J mouse genomic database, and the coordinates of all human genomic chromosomal sites refer to the ENSEMBL GRCh38.p13 version of the human genomic database.

[0077] As shown in Figure 2, the method for stepwise knockout of the DNA fragment of the present invention can knockout the target fragment in cells through the following steps. 1) Insert a DNA fragment containing a recombinase recognition site (such as LoxP or FRT) through homologous recombination at the 5' end of the target fragment of the cell. 2) Insert a DNA fragment containing a recombinase recognition site (such as LoxP or FRT) that is compatible with the recombinase discrimination site in step 1) and has the same direction through homologous recombination at the 3' end of the target fragment of the same cell clone completed in step 1). 3) Introduce a recombinase (such as Cre or FLP) that recognizes the recombinase recognition site inserted in step 1) or step 2) into the same cell clone completed in step 2). When the two recombinase recognition sites are on the same DNA and in the same direction, the recombinase can effectively cleave the sequence between the two recombinases to achieve knockout of the target fragment (Figure 2).

[0078] In diploid cells, since the target fragment exists on two different chromosomes, the insertion of the recombinase recognition site by homologous recombination in step 1) and the insertion of the recombinase recognition site by homologous recombination in step 2) occur on two different chromosomes with a probability of 50%. In this case, as shown in Figure 3, the recombinase recognition sites in the same direction may still be recognized by the recombinase that may be introduced in step 3), and a translocation event between homologous chromosomes occurs. Even in this case, knockout of the target fragment can be achieved.

[0079] The longer the target fragment is, the lower the knockout efficiency of the target fragment implemented by the method from step 1) to step 3) becomes. In this case, preferably, the same resistance selection gene (promoter, coding region, and polyA transcription termination region) is divided into two parts A and B without resistance selection function, and brought to the 5'-end and 3'-end of the recombinase recognition sites inserted at both ends of the target gene by step 1) and step 2) respectively. After the recombination event occurs, the target fragment between the two recombinases is effectively cleaved, and the two parts A and B are recombined into the resistance selection gene with selection function, so as to efficiently select cell clones that effectively knockout the target gene fragment (Figure 4).

[0080] Mouse embryonic stem cells used for gene knockout or human immunoglobulin variable region gene knock-in can be derived from strains such as 129, C57BL / 6J, C57BL / 6N or hybrid F1 generations, for example, mouse embryonic stem cells of the C57BL / 6J*129 strain. These stem cells are isolated from the inner cell mass of the early mouse embryo (reference: Non-Patent Document 7), or can be purchased from commercial providers such as Cyagen Biosciences (product number MUAES-01001 or MUBES-01001) or Applied Stemcell, Inc. (product number ASE-9005, ASE-9006, ASE-9007, ASE-9008 or ASE-9005). 〔Example 1〕 (Knockout of the endogenous heavy chain immunoglobulin variable region locus in mice) The overall strategy for knocking out the endogenous heavy chain immunoglobulin variable region locus in mice is shown in Figure 5. The specific steps are as follows.

[0081] Step 1: Construct two targeting vectors, Ace001-H1 and Ace001-H2. The construction of the targeting vector is well known to those skilled in the art.

[0082] As shown in Figure 5, the Ace001-H1 vector has the following characteristics: 1) It contains the sequence of mouse chromosome 12 positions 113428529 - 113425469 (HC arm) as a homology arm, and inserts a unique enzyme cleavage site EcoRI linearized at position 113426998. 2) It contains one neomycin selection gene expression element PGK-neo-polyA, where a recombinase recognition site LoxP is inserted before the translation start codon ATG of the neomycin Neo coding gene. As shown in Figure 5, the Ace001-H2 vector has the following characteristics: 1) It contains the sequence of mouse chromosome 12 positions 116032177 - 116027503 (HV arm) as a homology arm, and inserts a unique enzyme cleavage site PmeI linearized at position 116029758. 2) It contains a puromycin resistance gene expression element CAG-puro-polyA with a complete expression function. 3) It contains the coding region and polyA of the hygromycin B resistance gene with a promoter, and has recombinase recognition sites FRT and LoxP at both ends of the element respectively. In the Ace001-H1 and Ace001-H2 vectors, the directions of FRT and LoxP are indicated by arrows.

[0083] Step 2: Sequentially introduce the Ace001-H1 and Ace001-H2 vectors into mouse embryonic stem cells, select with 225 μg / ml neomycin (Neomycin, supplier: Invitrogen (Shanghai) Trading Co., Ltd., product number: 10131027) or 1.25 μg / ml puromycin (Puromycin, supplier: Invitrogen (Shanghai) Trading Co., Ltd., product number A1113803) to obtain surviving embryonic stem cell clones, detect these clones by the conventional PCR method, and obtain embryonic stem cell clones in which the two vectors are knocked into the correct genomic positions of mouse embryonic stem cells. Then, introduce a vector expressing Cre recombinase into these embryonic stem cell clones, select with 50 μg / ml hygromycin B (Hygromycin B, supplier: Invitrogen (Shanghai) Trading Co., Ltd., product number: 10687010) to obtain surviving embryonic stem cell clones, detect these clones by the conventional PCR method, and obtain embryonic stem cell clones in which the knockout of a large fragment mediated by Cre recombinase is successful. Among these mouse embryonic stem cell clones, the endogenous sequence of the mouse on chromosome 12 from 113428530 to 116027502 is deleted on one chromosome. In this step, Ace001-H1 and Ace001-H2 can be introduced into mouse embryonic stem cells in any order. In this step, the homologous recombination targeting vector is knocked into mouse embryonic stem cells by the forward and reverse primer PCR identification method outside the homologous arm region (Figure 17). The specific operation steps for primer design and PCR experiments are well known to those skilled in the art. [Example 2] (Knockout of the endogenous kappa light chain immunoglobulin variable region locus of mice) The overall strategy for knocking out the endogenous kappa light chain immunoglobulin variable region locus of mice is shown in Figure 6. The specific steps are as follows.

[0084] Step 1: Construct two targeting vectors, Ace002-K1 and Ace002-K2.

[0085] As shown in Figure 6, the Ace002-K1 vector has the following characteristics: 1) It contains the sequence of mouse chromosome 6 positions 70718872 - 70723924 (KCL arm) as the left homology arm and the sequence of mouse chromosome 6 positions 70723925 - 70726001 (KCR arm) as the right homology arm. Here, downstream of KCR has a unique linearized enzyme cleavage site NotI. 2) Between the left homology arm and the right homology arm, it contains the neomycin selection gene expression element PGK-neo-polyA. Here, a recombinase recognition site LoxP is inserted before the translation start codon ATG of the neomycin Neo coding gene. As shown in Figure 6, the Ace002-K2 vector has the following characteristics: 1) It contains the sequence of mouse chromosome 6 positions 67532019 - 67536983 as the homology arm, and a unique linearized enzyme cleavage site PmeI is inserted at position 67534443. 2) It contains the puromycin resistance gene expression element CAG-puro-polyA with a complete expression function. 3) It contains the hygromycin B resistance gene coding region with a promoter and polyA, and has recombinase recognition sites FRT and LoxP at both ends of the element respectively. In the Ace002-K1 and Ace002-K2 vectors, the directions of FRT and LoxP are indicated by arrows.

[0086] Step 2: Sequentially introduce the Ace002-K1 and Ace002-K2 vectors into mouse embryonic stem cells, select with 225 μg / ml neomycin or 1.25 μg / ml puromycin to obtain surviving embryonic stem cell clones, detect these clones by the conventional PCR method, and obtain embryonic stem cell clones in which the two vectors are knocked into the correct genomic positions of mouse embryonic stem cells. Then, introduce a vector expressing Cre recombinase into these embryonic stem cell clones, select with 50 μg / ml hygromycin B to obtain surviving embryonic stem cell clones, detect these clones by the conventional PCR method, and obtain embryonic stem cell clones in which the knockout of the large fragment mediated by Cre recombinase was successful. Among these mouse embryonic stem cell clones, the endogenous sequence of the mouse on chromosome 6 from 67536984 to 70723924 was deleted on one chromosome. In this step, Ace002-K1 and Ace002-K2 can be introduced into mouse embryonic stem cells in any order. In this step, the homologous recombination targeting vector is knocked into mouse embryonic stem cells by the forward and reverse primer PCR identification method outside the homologous arm region (Figure 17). The specific operation steps of primer design and PCR experiments are well known to those skilled in the art. Example 3 (Knockout of the endogenous lambda light chain immunoglobulin variable region locus of mice) The overall strategy for knocking out the endogenous lambda light chain immunoglobulin locus of mice is shown in Figure 7. The specific steps are as follows.

[0087] Step 1: Construct two targeting vectors, Ace003-L1 and Ace003-L2. The construction of targeting vectors is well known to those skilled in the art.

[0088] As shown in Fig. 7, the Ace003-L1 vector has the following characteristics: 1) It contains the sequence (LC arm) of mouse chromosome 16 positions 19065020 to 19059018 as a homology arm, and inserts a unique enzyme cleavage site FseI linearized at position 19061523. 2) It contains one neomycin selection gene expression element PGK-neo-polyA. Here, a recombinase recognition site LoxP is inserted before the translation start codon ATG of the neomycin Neo coding gene. As shown in Fig. 7, the Ace003-L2 vector has the following characteristics: 1) It contains the sequence (LV arm) of mouse chromosome 16 positions 19265943 to 19260701 as a homology arm, and inserts a unique enzyme cleavage site NotI linearized at position 19263393. 2) It contains a puromycin resistance gene expression element CAG-puro-polyA with a complete expression function. 3) It contains a hygromycin B resistance gene coding region with a promoter and polyA, and has recombinase recognition sites FRT and LoxP at both ends of the element respectively. In the Ace003-L1 and Ace003-L2 vectors, the directions of FRT and LoxP are indicated by arrows.

[0089] Step 2: Sequentially introduce the Ace003-L1 and Ace003-L2 vectors into mouse embryonic stem cells, select the surviving embryonic stem cell clones with 225 μg / ml neomycin or 1.25 μg / ml puromycin, detect these clones by the conventional PCR method, and obtain embryonic stem cell clones in which the two vectors are knocked into the correct genomic positions of mouse embryonic stem cells. Then, introduce a vector expressing Cre recombinase into these embryonic stem cell clones, select the surviving embryonic stem cell clones with 50 μg / ml hygromycin B, detect these clones by the conventional PCR method, and obtain embryonic stem cell clones in which the knockout of the large fragment mediated by Cre recombinase has been successful. Among these mouse embryonic stem cell clones, one chromosome lacks the endogenous sequence of the mouse at positions 19065021-19260700 on chromosome 16. In this step, Ace003-L1 and Ace003-L2 can be introduced into mouse embryonic stem cells in any order. In this step, the conventional PCR method in which the homologous recombination targeting vector is knocked into mouse embryonic stem cells adopts the forward and reverse primer PCR identification method outside the homology arm region (Figure 17), and the specific operation steps of primer design and PCR experiments are well known to those skilled in the art. [Example 4] (Knockout and identification of pseudogenes and open reading frames in the human immunoglobulin variable region) The human heavy chain variable region DNA fragment is derived from positions 105863198-106879844 on chromosome 14, the human Kappa light chain variable region DNA fragment is derived from positions 88860568-90235398 on chromosome 2, and the human Lambda light chain variable region DNA fragment is derived from positions 22023114-22922913 on chromosome 22. As shown in Figure 1, these DNA fragments derived from the human genome are inserted into a vector containing a non-human DNA fragment, such as a bacterial artificial chromosome (BAC). Suitable BACs can be queried from ENSEMBL, and the BAC vectors used in the present invention were purchased from supplier Source BioScience or Yingjieji (Shanghai) Trading Co., Ltd.

[0090] A process of knocking out unnecessary pseudogenes and open reading frame DNA segments from a BAC containing a human immunoglobulin variable region gene fragment in its original state is carried out in Escherichia coli. An exemplary knockout process is shown in Figure 8, and the specific steps are described below.

[0091] Step 1: 1.1) Prepare a bacterial artificial chromosome BAC1 (with chloramphenicol resistance) containing a human immunoglobulin variable region DNA segment. The BAC1 has been previously transformed into a genetically engineered host Escherichia coli DH10B (supplier: Source BioScience). 1.2) Prepare a recombinase expression vector, such as pKD46 (supplier: HonorGene, product number: HG-VJC0521, reference: Non-Patent Document 8). The vector contains an arabinose-inducible recombinase (such as derived from Escherichia coli λ bacteriophage Redα / Redβ / Redγ proteins) expression element, a temperature-sensitive plasmid vector replicon element, and an ampicillin resistance gene. 1.3) Introduce pKD46 into the DH10B host Escherichia coli containing BAC1 by the conventional electroporation transfection method. Inoculate Escherichia coli on an LB solid medium (supplier: Qingdao Haibo, product number: HB0129) plate containing chloramphenicol and ampicillin, and culture it overnight at 30°C. 2) On the second day, collect a single clone of Escherichia coli and culture it in an LB liquid medium (supplier: Qingdao Haibo, product number: HB0128) containing chloramphenicol (supplier: Shenggong Biology, product number: A100230) and ampicillin (supplier: Shenggong Biology, product number: A100339) under the culture condition of 30°C with shaking for 16 hours. The obtained strain was named Escherichia coli A.

[0092] Step 2: 2.1) For the PCR template rpsL / tetA sequence, referring to SEQ ID NO: 1, forward and reverse primers (supplier: Shenggong Biology) as shown in FIG. 8A can be designed and synthesized. The forward primer contains the 5'-terminal homology arm region HA1 of the 50-bp knockout target region and the 5'-terminal primer region of rpsL / tetA. The reverse primer contains the 3'-terminal homology arm region HA2 of the 50-bp knockout target region and the 3'-terminal primer region of rpsL / tetA. The primer design principles are well known to those skilled in the art. 2.2) Using the above forward and reverse primers, polymerase chain reaction (PCR) technology is adopted to obtain a DNA fragment (FIG. 8A) with 50-bp homology arms and rpsL / tetA expression elements at both ends. 2.3) The bacterial solution of Escherichia coli A obtained in step 1.3) is re-inoculated into 3 ml of liquid LB medium containing chloramphenicol and ampicillin at a final concentration of OD600 = 0.1, and 45 μl of 10% L(+)-arabinose (supplier: Shenggong Biology, product number: A610071) is added, and cultured with shaking at 37°C for 3 - 5 hours to induce the expression of recombinase until the OD600 of the bacterial solution reaches 0.6. 2.4) The rpsL / tetA fragment obtained in step 2.2) is introduced into Escherichia coli A in step 2.3) using the electroporation transfection method. Homologous recombination mediated by recombinase replaces rpsL / tetA with the DNA fragment to be knocked out in situ (FIG. 8B), and cultured overnight at 37°C on an LB solid medium plate containing chloramphenicol and tetracycline (supplier: Shenggong Biology, product number: A100422). 2.5) The colony clones obtained in step 2.4) are collected, cultured at 37°C in LB liquid medium containing chloramphenicol and tetracycline, and Escherichia coli clones containing BAC1 with the knockout target region correctly knocked out are identified by PCR identification and PCR product sequencing methods using selection primers 1 and 2 in FIG. 8C, and named Escherichia coli B. Step 3: 3.1) Introduce the pKD46 vector into E. coli clone C containing BAC1 in which the target knockout region obtained in step 2.5) has been correctly knocked out by the method described in step 1.3), and name it E. coli D. 3.2) Design and synthesize the double-stranded DNA fragment HA1-HA2 of 50bp homology arm HA1 and 50bp homology arm HA2 ligated in sequence. 3.3) Introduce the DNA fragment described in step 3.2) into E. coli D obtained in step 3.1) by the electroporation transfection method. The introduction steps can refer to the methods described in steps 2.3)-2.4). Homologous recombination mediated by recombinase replaces the rpsL / tetA fragment (Figure 8D) inserted when knocking out the target knockout region in BAC1 in situ with the HA1-HA2 fragment, and culture overnight at 37°C on an LB solid medium plate containing chloramphenicol and streptomycin (Supplier: Shenggong Biology, Product Number: A100382). 3.4) Collect the colony clones obtained in step 3.3), culture them at 37°C in an LB liquid medium containing chloramphenicol and streptomycin, and use the selection primers 1 and 2 in Figure 8E to identify the E. coli clone containing BAC1 in which the rpsL / tetA region has been correctly knocked out by PCR identification and PCR product sequencing methods. Step 4: If BAC1 contains a plurality of pseudo-genes or open reading frame regions to be knocked out, steps 1 to 3 are repeated to knock out each target region until all target pseudo-genes or open reading frame regions are knocked out in BAC1. When the segmented human immunoglobulin variable region DNA is inserted into different BAC vectors, the knockout of pseudo-genes and open reading frame regions can be carried out individually with different BAC vectors, and the finally retained DNA fragments can be spliced based on the steps in the published literature "Assisted large fragment insertion by Red / ET-recombination (ALFIRE)-an alternative and enhanced method for large fragment recombineering".

[0093] In a specific embodiment of the present invention, generally for "partial or complete knockout" or "partial or complete deletion", 10 to 100% (preferably 15 to 95%, 20 to 90%, for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 72%, 75%, 78%, 80%, 83%, 85% or 88%) of the pseudo-genes and / or open reading frame genes are knocked out or deleted, and the ratio is based on the total number of pseudo-genes and open reading frame genes of the human immunoglobulin variable region gene.

[0094] For example, in a specific embodiment of the present invention, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the pseudo-genes and / or open reading frames are knocked out.

[0095] For example, in one embodiment, 10 to 25 pseudo-V genes and / or open reading frame genes are knocked out or deleted, and in another embodiment, 10, 15 or 25 pseudo-V genes and / or open reading frame genes are knocked out or deleted.

[0096] In the process of knocking out pseudo-genes and / or open reading frames in the immunoglobulin gene cluster region in humans, the regulatory region, gene coding region, and antibody gene recombination signal sequence of the functional gene fragment to be retained can be derived from continuous fragments of the same immunoglobulin variable region gene, or can also be derived from the recombination of the regulatory region, gene coding region, and antibody gene recombination signal of different immunoglobulin genes. For example, the regulatory region is derived from the 5' terminal regulatory region of the human VA gene, the coding sequence is derived from the VB gene, and the antibody gene recombination signal sequence is derived from the VC gene (Figure 9).

[0097] In one embodiment, the heavy chain targeting vector 1 shown in Figure 10 is constructed, and according to the above steps, 10 pseudo-V H region genes or open reading frames including the H2, H4, H9, H11, H13, H15, H19, H21, H23, H17 of the human immunoglobulin heavy chain V region in Table 1 H are knocked out from the DNA fragment of the V H region gene, and the functional human heavy chain V H region genes IGHV6-1, IGHV1-2, IGHV1-3, IGHV4-4, IGHV7-4-1, IGHV2-5, IGHV3-7, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV1-18, IGHV3-20, IGHV3-21, IGHV3-23 and those derived from human chromosome 14 at positions 105863198 to 105939714, and the human immunoglobulin heavy chain D H gene region and J HRetain the sequence containing the gene region, insert the sequence derived from mouse chromosome 12 positions 113428513 - 113423504 of the unique enzyme cleavage site NotI linearized at the 113426008 site, ligate in order as shown in Figure 10. The heavy chain targeting vector 1 has a neomycin resistance expression element PGK - neo - polyA, a recombinase recognition site FRT, and a pBACe3.6 bacterial artificial chromosome plasmid backbone. After the construction of the vector, two different methods were used to identify whether there were deletions of DNA fragments in the DNA fragments. The first identification method was to determine whether the enzyme cleavage pattern was as expected by pulsed - field electrophoresis after cleavage with the SalI or AgeI restriction enzyme. As shown in Figure 11, after subjecting the heavy chain targeting vector 1 derived from two different colony clones a and b to enzyme cleavage with two different groups of enzymes, the size of each fragment was consistent with the prediction, indicating that the risk of the heavy chain targeting vector 1 derived from these two clones lacking large - fragment DNA was low. The second identification method was, as shown in Figure 12, to randomly design 24 pairs of primers Cargo1 - Cargo24 (Table 2) within the heavy chain targeting vector 1, with the distance between each primer pair within the vector being about 10KB. Then, PCR amplification of these 24 pairs of primers was performed using the heavy chain targeting vector 1 derived from two different colony clones a and b. Through the size of the product and the presence or absence of the product, it was shown that the risk of the heavy chain targeting vector 1 derived from these two clones lacking large - fragment DNA was low.

[0098]

Table 2

[0099] In another embodiment, construct the heavy chain targeting vector 2 shown in Figure 10. The specific characteristics are that in the sequence of human chromosome 14 positions 106879844 - 106268567, according to the above steps, 15 pseudo - Vs of the sequence numbers H26, H28, H35, H39, H41, H43, H46, H49, H51, H53, H57, H59, H61, H64, H68 in Table 1 are in Escherichia coli.H V region gene containing a gene or open reading frame H Knock out the DNA fragment of the V region gene containing the gene, and the 25 functional V regions of SEQ ID NOs. H25, H27, H29, H31, H33, H36, H38, H40, H42, H44, H45, H47, H48, H50, H52, H54, H55, H56, H58, H60, H62, H63, H65, H66, H67 in Table 1 H Fragment of the gene containing the V region and the pseudo-V region gene fragments of SEQ ID NOs. H30, H32, H34, H37 in Table 1 H Retain the DNA fragment of the V region gene fragment and H35 gene fragment, and the sequence of human chromosome 14 from 106276506 to 106268567 as the homology arm. The homology arm inserts a unique enzyme cleavage site PmeI linearized at the 106273423 site. These DNA fragments are ligated in order as shown in Figure 10. The heavy chain targeting vector 2 has a puromycin resistance expression element CAGpuro-polyA, a recombinase recognition site FRT, and a pBACe3.6 bacterial artificial chromosome plasmid backbone. The use of the heavy chain targeting vector 2 is to introduce the sequence of human chromosome 14 positions 106276506 to 106268567 carried in the heavy chain targeting vector 1 into the mouse embryonic stem cell chromosome 12 in a certain direction as the homology arm, and then introduce more V region gene fragments on the heavy chain targeting vector 2 into the mouse embryonic stem cell chromosome 12.

[0100] In another embodiment, a Kappa light chain targeting vector shown in FIG. 13 was constructed. The specific features are as follows: in the sequence at positions 89333431 to 88860568 on human chromosome 2, 10 pseudogenes or open reading frames with SEQ ID NOs. K3, K6, K9, K12, K16, K19, K21, K24, K26, and K28 in Table 1 were knocked out in Escherichia coli according to the above steps, and 20 functional human Kappa light chain V L region genes and the human immunoglobulin Kappa light chain J L gene region at positions 88861967 to 88860568 on human chromosome 2, and the sequence at positions 70723924 to 70729434 on mouse chromosome 6 as a homology arm were retained. A unique enzyme cleavage site NotI linearized at site 70726623 was inserted into the homology arm. The Kappa light chain targeting vector has a neomycin resistance expression element PGK-neo-polyA, a recombinase recognition site FRT, and a pBACe3.6 bacterial artificial chromosome plasmid backbone.

[0101] In another embodiment, Lambda light chain targeting vectors 1 and 2 shown in FIG. 14 were constructed. Lambda light chain targeting vector 1 is derived from positions 22881432 to 22922913 in human chromosome 22 and contains a sequence including the human immunoglobulin Lambda light chain J-C gene region. According to the above steps, 12 pseudogenes or open reading frames with SEQ ID NOs. L2, L4, L10, L12, L14, L17, L19, L21, L23, L25, L28, and L30 in Table 1 were knocked out in Escherichia coli, and 19 functional human Lambda light chain V with SEQ ID NOs. L1, L3, L5, L6, L7, L8, L9, L11, L13, L15, L16, L18, L20, L22, L24, L26, L27, L29, and L31 in Table 1 LRetain the region containing the region gene and the sequence of mouse chromosome 6 positions 70726758 - 70731223 as the homology arm. Insert a unique enzyme cleavage site FseI linearized at the 70729051 site into the homology arm. The Lambda light chain targeting vector 1 has a neomycin resistance expression element PGK-neo-polyA, a recombinase recognition site FRT, and a pBACe3.6 bacterial artificial chromosome plasmid backbone. Lambda light chain targeting vector 2 knocks out the regions containing the 7 pseudogenes or open reading frames with SEQ ID NOs: L36, L38, L41, L43, L45, L48, L50 in E. coli according to the above steps, and the 12 functional human Lambda light chain V L Retain the region containing the region gene and the sequence of human chromosome 22 positions 22381387 - 22387465 as the homology arm. Insert an enzyme cleavage site NotI linearized at the 22383529 site into the homology arm. The Lambda light chain targeting vector 2 has a puromycin resistance expression element CAGpuro-polyA, a recombinase recognition site FRT, and a pBACe3.6 bacterial artificial chromosome plasmid backbone. Its use is to introduce the mouse chromosome 6 positions 70726758 - 70731223 into the Lambda light chain targeting vector 1 in a certain direction, and use the sequence of human chromosome 22 positions 22381387 - 22387465 brought into the Lambda light chain targeting vector 1 as the homology arm to introduce more V region gene fragments on the Lambda light chain targeting vector 2 into mouse embryonic stem cell chromosome 6.

[0102] The method for identifying the integrity of the DNA of the heavy chain targeting vector 2, kappa light chain targeting vector, and lambda light chain targeting vectors 1 and 2 used the pulse field electrophoresis and random fragmentation PCR identification methods after restriction enzyme digestion of vectors similar to the heavy chain targeting vector 1. [Example 5] (The targeting vector is introduced into mouse embryonic stem cells in a certain direction by homologous recombination) In one embodiment, the target cells are mouse embryonic stem cells in the region of mouse chromosome 12, positions 113428530 to 116027502, which were knocked out by the two-step gene targeting and recombinase Cre of Example 1. The targeting vector employed is the heavy chain targeting vector 1 (Figure 10). The process of introducing it in a certain direction can be referred to in Figures 15 and 16. The specific steps are as follows.

[0103] Step 1: 1 - 2×10 7 Individual target mouse embryonic cells were transfected with 50 μg of linearized heavy chain targeting vector 1 by electroporation (240 V, 250 μF, Bio-rad Gene Pulser). The electroporation transfection method is well known to those skilled in the art. According to the resistance gene carried on the targeting vector, the cells after transfection were selected for cell clones that were successfully transfected with 225 μg / ml of neomycin within 24 to 48 hours for 7 days. The antibiotic-resistant cell clones were placed in a 96-well culture plate and subjected to growth culture and genotyping.

[0104] Step 2: A part of the antibiotic-resistant cells obtained in Step 1 was taken, genomic DNA was extracted, and then forward and reverse primers were designed outside the homologous arm region as shown in Fig. 17, and conventional PCR amplification was carried out. 1) Primers P1 and P2 were designed at the 5'-end and 3'-end of the target homologous arm of the wild-type allele, respectively, and the sequences of both primers were outside the homologous arm region. 2) Primers P3 and P4 were designed at the 5'-end and 3'-end of the targeting vector homologous arm, respectively, and the sequences of both primers were outside the homologous arm region. 3) When the linearized targeting vector was inserted into the target homologous arm site of the wild-type allele by homologous recombination, when using primers P1 and P4, P3 and P2, DNA fragments P1P4 and P3P2 with fragment sizes larger than the size of the homologous arm segment could be PCR amplified. However, when using primers P1 and P4, P3 and P2 respectively, DNA fragments with fragment sizes larger than the size of the homologous arm segment could not be amplified in a PCR system having only the wild-type allele template or the targeting vector template. When analyzing the fragment sizes and sequences of the two PCR products P1P4 and P3P2, it is possible to determine whether the targeting vector in the protocol was correctly inserted into the target homologous arm region of the wild-type allele by homologous recombination. As shown in Fig. 18, after introducing the target mouse embryonic stem cells into the heavy-chain targeting vector 1 in a certain direction, the size of the P1P4 product was 5.3 KB, and the size of the P3P2 product was 5.4 KB. This result indicates that multiple cell clones were obtained in which the introduction in this certain direction was successfully introduced into the target region of the mouse embryonic stem cells as expected. The amplification products of P1P4 and P3P2 can be further confirmed by sequencing methods.

[0105] Identification of the integrity of the human immunoglobulin variable region gene in the human heavy chain targeting vector 1 introduced into mouse embryonic stem cells can be identified by PCR using the genomic DNA of these successfully targeted cells as a template. Cargo1 to Cargo24 shown in Table 2 are used as primers for PCR amplification identification. By selecting Cargo1 to 24, PCR products of the expected size can be obtained, indicating that the mouse embryonic stem cell clones have been successfully introduced into the human immunoglobulin variable region locus and there are no clones at risk of major DNA fragment deletion.

[0106] In one embodiment, as shown in FIGS. 15 and 16, 16 human V H functional genes of the human immunoglobulin heavy chain variable region and 27 human D H genes and 6 human J H genes were introduced into mouse embryonic stem cells after completing all the steps shown in Example 1 via the heavy chain targeting vector 1, and the embryonic stem cell clones were further introduced into another 25 human V H functional genes via the heavy chain targeting vector 2.

[0107] In one embodiment, as shown in FIGS. 19 and 20, 20 human kappaV L functional genes of the human immunoglobulin kappa light chain variable region and 5 kappa J L genes were introduced into mouse embryonic stem cells after completing all the steps shown in Example 2 via the kappa light chain targeting vector.

[0108] In one embodiment, as shown in FIGS. 21 and 22, 19 human Lambda V L functional genes of the human immunoglobulin lambda light chain variable region and all 7 lambda J L genes and lambda C LThe gene was introduced into mouse embryonic stem cells after completing all the steps shown in Example 3 via the lambda light chain targeting vector 1, and the embryonic stem cell clones were further introduced into another 12 human Lambda V L functional genes via the lambda targeting vector 2. After the introduction of the lambda light chain targeting vector 1 was completed, FLP expression was introduced into the cells to enable recombination between the two FRT sites, and by knocking out the sequence between the two FRTs, the non-human-derived DNA sequence and the mouse endogenous kappa C L coding sequence derived from the remaining Lambda light chain targeting vector were removed.

[0109] Before introducing the genes containing the human immunoglobulin variable regions in the examples shown in FIGS. 15-16, FIGS. 19-20, and FIGS. 21-22 into mouse embryonic stem cells, some or all of the pseudo-V region genes and / or open reading frame V region genes of these human immunoglobulin V region genes were knocked out. Therefore, the size of the fragment of the V region gene region finally introduced into the mouse embryonic stem cells only occupies a part of the size of the human immunoglobulin V region in the ENSEMBL GRCh38.p13 version of the human genome database, and the ratio is tabulated in Table 3.

[0110]

Table 3

[0111] [Example 6] (Conversion and Propagation of Mouse Embryonic Stem Cells) The technology of converting gene-edited mouse embryonic stem cells into transgenic mice is well known to those skilled in the art. After confirming that the mouse embryonic stem cell clone that passed the genotype identification has no increase or decrease in chromosome number by karyotype detection, the mouse embryonic stem cells of the clone are diluted to the injection concentration and injected into the blastocoel cavity of about 50 blastocysts at 2.5 to 3.5 days old. Next, the microinjected blastocysts are returned to the oviduct or uterus of the surrogate mother mouse. After the mouse is born, using the mouse tail DNA as a template and gene-editing specific primers, the genotype of the progeny chimeras is identified by PCR to confirm whether the gene-edited mouse embryonic stem cells contribute to the somatic cells of the chimeric progeny mice. The grandchild chimeric mice are caged with wild-type mice of different sexes and bred to obtain progeny mice (F1). By identifying the genotype of the F1 generation mice by the same PCR method, it can be determined whether the gene-edited mouse embryonic stem cells can achieve germline transmission. The primer design principles and implementation forms for identifying the mouse genotypes of the F0 generation and F1 generation are well known to those skilled in the art.

[0112] In one example, according to the above method, the mouse embryonic stem cells that successfully introduced the human immunoglobulin heavy chain variable region locus obtained in Example 5 and passed the PCR amplification identification and karyotype detection of Cargo1-24 shown in Table 2 were injected into the blastocoel cavity of 2.5-3.5 day-old mouse blastocysts, and finally F1 generation mice were obtained. In another example, according to the protocols of FIGS. 19 and 20, the Kappa light chain targeting vector shown in FIG. 13 was introduced into mouse embryonic stem cells to obtain F1 generation mice in the same way. These two types of F1 generation mice were bred so that the sites where the heavy chain targeting vector 1 is located and the sites where the Kappa light chain targeting vector is located are both homozygous by the hybridization method. In another example, according to the protocols of FIGS. 21 and 22, the Lambda light chain targeting vectors 1 and 2 shown in FIG. 14 were introduced into mouse embryonic stem cells to obtain F1 generation mice in the same way. Table 4 shows the characteristics of some transgenic mice obtained in the present invention.

[0113]

Table 4

[0114] For the sequence of the variable region V gene related to Table 4, refer to Table 1. 〔Example 7〕 (B cell development test of transgenic mice) In most mammals, the spleen is an organ rich in B cell content. By analyzing the B cells in the spleen, it is possible to determine whether the development of B cells in the animal is normal. (The transgenic mouse spleen obtained in Example 6) The technique of obtaining the spleen, isolating spleen cells, and performing flow cytometry analysis on cells derived from the spleen is well known to those skilled in the art. The spleen of the transgenic mouse group 1 (homozygous transgenic mouse group in Figure 23) in Example 6 was collected without immunization, spleen cells were isolated, and spleen cells derived from transgenic mice and wild-type littermate control mice were compared by flow cytometry analysis. For B220 陽性 / IgM 高 / IgD 低 cells were selected as immature B cells, and B220 陽性 / IgM 低至中等 / IgD 高 as mature B cells. As shown in Figure 23, there was no statistical difference in the ratios of mature and immature B cells among wild-type littermate control mice, heterozygous transgenic mice, and homozygous transgenic mouse groups, indicating that the development of B cells in the transgenic mouse group 1 obtained in the present invention is normal. Similarly, there was no statistical difference in the ratios of mature and immature B cells in transgenic mouse group 2 and transgenic mouse group 3 compared with wild-type littermate control mice and heterozygous transgenic mice, indicating that the development of B cells is normal. 〔Example 8〕 (Immune test of transgenic mice) One of the objectives of the present invention is to obtain a transgenic animal that expresses a human-derived antibody having a gene encoding a variable region, and such a transgenic animal can normally generate an antigen-specific immune response under antigen stimulation. The transgenic mouse groups 1 and 2 obtained in Example 6 and wild-type BALB / c mice in the control group were immunized with ovalbumin (OVA, supplier: Sigma-Aldrich, product number: A5503) at the same dose and the same immunization protocol. After the third booster immunization, mouse sera were taken for OVA-specific enzyme-linked immunosorbent assay (ELISA), and the antigen-specific enzyme-linked immunosorbent assay method is well known to those skilled in the art. The results are shown in Figure 24, and both transgenic mouse group 1 and transgenic mouse group 2 were able to generate an antigen-specific immune response superior to that of wild-type BALB / c mice. [Example 9] (Application example of transgenic mice in the expression of fully human antibody drug candidates) Since the gene encoding the variable region can produce antibodies derived from the rearrangement of human immunoglobulin variable regions, one of the important uses of the mouse model of the present invention is to be used in the expression of fully human antibody drug candidates. The technique of stepwise stimulating transgenic mice with a specific antigen to generate a specific immune response against the specific antigen, collecting the spleen and / or lymph nodes of the mice, and performing cell fusion to obtain hybridomas that can continuously secrete antibodies after immortalization is well known to those skilled in the art.

[0115] Gene editing targeting gene clusters encoding immunoglobulins has a significant adverse effect on the development of mouse B cells. One of the phenomena that can be objectively reflected is that the specific immune response of mice to antigens can only produce low-affinity antibodies. Using the mouse models of transgenic mouse groups 1, 2, and 3 obtained in Example 6 of the present invention, the hybridoma technology was used to obtain 12, 12, and 20 different antigen-specific monoclonal antibodies from three different targets of human BCMA, GALECTIN-10, or TGFb1, respectively. The affinity levels (Figure 25) of these antibodies were measured using BIAcore T200 (GE Healthcare), and it was found that these obtained from the mouse models of the present invention can obtain antibodies with high affinity levels in the pM range for three different targets. The affinity levels were in the ranges of 10 pM to 1 nM, 1 pM to 10 nM, and 1 pM to 10 nM, respectively.

[0116] The transgenic mice produced by the method of the present invention show surprising effects regarding the development level of B cells or the antigen-specific immune response and the affinity level for antibodies, and these effects are unexpected to those skilled in the art.

[0117] The specific embodiments made above are only to clarify the technical content of the present invention, and the scope of the present invention and the scope of the claims should not be limited only to such specific examples, and equivalent embodiments belong to the scope of the present invention. From the above description, in addition to the refinements already described in this specification, those skilled in the art can make various refinements to the present invention, and such refinements are also considered to belong to the scope of the present invention.

Brief Description of the Drawings

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Claims

**Claim 1** A genetically modified recombinant genome of a non-human mammal in which all of the endogenous immunoglobulin variable region genes in the genome have been replaced by human immunoglobulin variable region genes, and a part or all of the pseudogenes and / or open reading frames of the human immunoglobulin variable region genes have been knocked out, and the number of knocked out in the pseudogenes and / or open reading frame genes of the human immunoglobulin variable region genes is such that the lengths of the human immunoglobulin heavy chain variable region gene and the Lambda light chain variable region gene inserted into the non-human mammalian cell genome are 10% to 50% of the full lengths of the human immunoglobulin heavy chain variable region gene and the Lambda light chain variable region gene before knocking out the pseudogenes and / or open reading frame genes, respectively, and / or the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is such that it is 35% to 65% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogenes and / or open reading frame genes, (1) when calculating the full length of the human immunoglobulin heavy chain variable region, calculate according to the positions 105939715 to 106879844 on chromosome 14 of ENSEMBL GRCh38.p13, and the full length is 940130 bp, (2) when calculating the full length of the human Kappa light chain variable region, calculate according to the positions 88861968 to 89333431 on chromosome 2 of ENSEMBL GRCh38.p13, and the full length is 471464 bp, (3) when calculating the full length of the human Lambda light chain variable region, calculate according to the positions 22023114 to 22881431 on chromosome 22 of ENSEMBL GRCh38.p13, and the full length is 858318 bp. A genetically modified recombinant genome of a non-human mammal, characterized in that. **Claim 2** The human immunoglobulin variable region gene is a human heavy chain functional V Ｈ , D Ｈ , J Ｈ coding sequence and non-coding sequence, or a human light chain functional V Ｌ , J Ｌ coding sequence and non-coding sequence, and the light chain is a kappa or lambda light chain, and the genetically modified recombinant genome of the non-human mammal according to claim 1 is characterized in that. **Claim 3** The endogenous immunoglobulin variable region gene is the immunoglobulin heavy chain variable region V of non-human mammalian cells Ｈ , D Ｈ , J Ｈ and / or the light chain variable region V Ｌ , J Ｌ and the light chain refers to the kappa or lambda light chain, and the genetically modified recombinant genome of a non-human mammal according to any one of claims 1 to 2. **Claim 4** The human heavy-chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are derived from human chromosome 14, and the human light-chain functional V Ｌ , J Ｌ The coding sequences and non-coding sequences of are derived from human chromosome 2 or human chromosome 22. The genetically modified recombinant genome of the non-human mammal according to claim 2. **Claim 5** The human heavy chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are sequences from nucleotide positions 105863198 to 106879844 derived from human chromosome 14, and all coordinates refer to the human genome database of the ENSEMBL GRCh38.p13 version. The genetically modified recombinant genome of a non-human mammal according to claim 2 or 4, characterized in that it contains the above sequences. **Claim 6** The human heavy chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are characterized by including a plurality of the V of the sequence numbers shown in the following table Ｈ Genes H1 to H67, and the recombinant genome of the genetically modified non-human mammal according to claim 5. 【Table 1】 **Claim 7** The human heavy chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are 10 to 41 of the V Ｈ genes H1 to H67 described in the table according to claim 6, and the recombinant genome of the non-human mammal with genetically modified genes according to claim 6 is characterized by containing them. **Claim 8** The human heavy chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are the V of the sequence numbers shown in the table according to claim 6 Ｈ The recombinant genome modified with the gene of the non-human mammal according to claim 6, characterized by containing 15 to 41 of the V genes H1 to H67 according to claim 6. **Claim 9** The human heavy chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are the 25 to 41 of the V of the sequence numbers shown in the table according to claim 6 Ｈ A recombinant genome of a non-human mammal modified with a gene according to claim 6, characterized by comprising 25 to 41 of the V genes H1 to H67. **Claim 10** The human heavy-chain functional V Ｈ , D Ｈ , J Ｈ The coding sequences and non-coding sequences of are the V of the sequence numbers shown in the table according to claim 6 Ｈ A recombinant genome modified with a gene of a non-human mammal according to claim 6, characterized by containing 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 of the V genes H1 to H67 according to claim 6 **Claim 11** The human light chain functional V Ｌ , J Ｌ The coding sequence and non-coding sequence of are the sequence of nucleotide positions 88860568 to 90235398 derived from human chromosome 2 or the sequence of nucleotide positions 22023114 to 22922913 derived from human chromosome 22, and all coordinates refer to the human genome database of the ENSEMBL GRCh38.p13 version. The genetically modified recombinant genome of a non-human mammal according to claim 2 or 4, characterized in that it comprises the above. **Claim 12** The human light chain functional V Ｌ , J Ｌ The coding sequence and the non-coding sequence include a plurality of variable region genes K1 to K30 and L1 to L49 of the sequence numbers shown in the following table, and the recombinant genome of the non-human mammal modified by the gene according to claim 11. 【Table 2】 **Claim 13** The endogenous immunoglobulin variable region gene is completely deleted by homologous recombination, the human immunoglobulin heavy chain variable region gene is inserted at a position 3 KB upstream to 3 KB downstream of the deleted endogenous immunoglobulin heavy chain variable region, and the human immunoglobulin light chain variable region gene is inserted at a position 3 KB upstream to 3 KB downstream of the deleted endogenous immunoglobulin kappa light chain variable region. The genetically modified recombinant genome of a non-human mammal according to any one of claims 1, 2, and 4, characterized in that

14. The lengths of the human immunoglobulin heavy chain and Lambda light chain variable region genes inserted into the non-human mammalian cell genome are 12% to 47% of the full lengths of the human immunoglobulin heavy chain and Lambda light chain variable region genes before knocking out the pseudogene and / or open reading frame gene, respectively, and / or the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is 37% to 63% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogene and / or open reading frame gene. The genetically modified recombinant genome of a non-human mammal according to claim 1, characterized in that

15. The lengths of the human immunoglobulin heavy chain and Lambda light chain variable region genes inserted into the non-human mammalian cell genome are 14% to 45% of the full lengths of the human immunoglobulin heavy chain and Lambda light chain variable region genes before knocking out the pseudogene and / or open reading frame gene, respectively, and / or the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is 38% to 61% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogene and / or open reading frame gene. The genetically modified recombinant genome of a non-human mammal according to claim 14, characterized in that

16. The length of the human immunoglobulin heavy chain and Lambda light chain variable region genes inserted into the non-human mammalian cell genome is 16% to 40% of the full length of the human immunoglobulin heavy chain and Lambda light chain variable region genes before knocking out the pseudogene and / or open reading frame gene, respectively, and / or the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is 50% to 55% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogene and / or open reading frame gene. The recombinant genome of the genetically modified non-human mammal according to claim 14, characterized in that.

17. The length of the human immunoglobulin heavy chain and Lambda light chain variable region genes inserted into the non-human mammalian cell genome is 16.10%, 18%, 18.50%, 20%, 25%, 30%, 31%, 31.75%, 35%, 38% or 38.06% of the full length of the human immunoglobulin heavy chain and Lambda light chain variable region genes before knocking out the pseudogene and / or open reading frame gene, respectively, and / or the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is 51%, 52%, 53%, 53.08% or 54% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogene and / or open reading frame gene. The recombinant genome of the genetically modified non-human mammal according to claim 14, characterized in that.

18. The non-human mammalian cell is a mouse embryonic stem cell. The deleted endogenous immunoglobulin heavy chain variable region is located at positions 113428530 to 116027502 on mouse chromosome 12. The deleted endogenous immunoglobulin kappa light chain variable region is located at positions 67536984 to 70723924 on mouse chromosome 6. The deleted endogenous immunoglobulin lambda light chain variable region is located at positions 19065021 to 19260700 on mouse chromosome 16. The mouse genome chromosomal locus coordinates refer to the C57BL / 6J mouse genome database position in the ENSEMBL GRCm38.p6 version. The recombinant genome of the genetically modified non-human mammal according to any one of claims 1, 2, and 4.

19. The insertion site of the human immunoglobulin heavy chain variable region gene is at position 113428513 on chromosome 12 of the mouse genome, the insertion site of the human immunoglobulin kappa light chain variable region gene is at position 70723924 on chromosome 6 of the mouse genome, and the insertion site of the human immunoglobulin lambda light chain variable region gene is at position 70726758 on chromosome 6 of the mouse genome. The recombinant genome of the genetically modified non-human mammal according to claim 18, characterized in that.

20. A non-human mammalian cell comprising the recombinant genome of the non-human mammalian cell according to any one of claims 1 to 19.

21. The non-human mammalian cell according to claim 20, characterized in that the cell is a non-human mammalian embryonic stem cell.

22. The non-human mammalian cell according to claim 21, characterized in that the non-human mammalian embryonic stem cell is a mouse embryonic stem cell, a rat embryonic stem cell or a rabbit embryonic stem cell.

23. A method for producing a non-human mammalian cell according to any one of claims 20 to 22, comprising: a) introducing recombinase targeting sites that are compatible in the same direction upstream and downstream of the immunoglobulin variable region gene of the non-human mammalian cell genome; b) introducing a specific recombinase that can recognize the recombinase targeting site described in step a), and partially or completely deleting the endogenous immunoglobulin variable region gene of the non-human mammalian cell under conditions that allow recombination to occur between the two recombinase targeting sites described in step a); c) A targeting vector comprising part or all of the variable region of human immunoglobulin (the targeting vector contains a human functional variable region gene, and part or all of the pseudogene and / or open reading frame is knocked out, and the human functional variable region gene is the human heavy chain functional V Ｈ , D Ｈ , J Ｈ coding sequence and non-coding sequence, or human functional V Ｌ , J Ｌ coding sequence and non-coding sequence, and the light chain is a kappa or lambda light chain), d) introducing the targeting vector described in step c), and replacing the endogenous immunoglobulin gene of the non-human mammalian cell deleted in step b) with the human immunoglobulin variable region gene included in step c) in the non-human mammalian cell; and e) producing a non-human mammalian cell containing the human immunoglobulin variable region gene in the genome from step d). A method, characterized by comprising.

24. The method according to claim 23, wherein the targeting vector is selected from a BAC vector or a YAC vector.

25. The method according to any one of claims 23 to 24, characterized in that the targeting vector described in step c) is constructed in Escherichia coli or yeast cells.

26. A targeting vector containing a human immunoglobulin variable region gene, wherein a part or all of the pseudogene and / or open reading frame of the human immunoglobulin variable region gene is knocked out, and the human immunoglobulin variable region gene is a human heavy chain functional V Ｈ , D Ｈ , J Ｈ coding sequences and non-coding sequences, or human light chain functional V Ｌ , J Ｌ coding sequences and non-coding sequences, the light chain is a kappa or lambda light chain, and the number of knocked out in the pseudogene and / or open reading frame gene of the human immunoglobulin variable region gene is such that the lengths of the human immunoglobulin heavy chain and lambda light chain variable region genes inserted into the non-human mammalian cell genome are 10% to 50% of the full lengths of the human immunoglobulin heavy chain and lambda light chain variable region genes before knocking out the pseudogene and / or open reading frame gene, respectively, and / or the length of the human immunoglobulin kappa light chain variable region gene inserted into the non-human mammalian cell genome is 35% to 65% of the full length of the human immunoglobulin kappa light chain variable region gene before knocking out the pseudogene and / or open reading frame gene, characterized in that it is a targeting vector.

27. The targeting vector according to claim 26, characterized in that the targeting vector is selected from a BAC vector or a YAC vector.

28. A method for producing a non-human mammalian cell that expresses an antibody whose variable region is completely human-derived, comprising introducing the non-human mammalian cell according to any one of claims 20 to 22, or a non-human mammalian cell obtained by the method for producing the non-human mammalian cell according to any one of claims 23 to 25, into the uterus of a wild-type female non-human mammal, and selecting a chimeric non-human mammalian offspring as a non-human mammal of F0 generation.

29. A method for producing a non-human mammal expressing an antibody whose variable region is completely human-derived, comprising the steps of: before introducing non-human mammalian cells into the uterus of a female wild-type non-human mammal, selecting the non-human mammalian cells to obtain a non-human mammalian cell clone having no increase or decrease in chromosome number, transplanting the non-human mammalian cell clone into the blastocyst cavity of a wild-type non-human mammal, and transplanting the blastocyst into the uterus of a pseudopregnant female wild-type non-human mammal.

30. A method for producing a non-human mammalian animal expressing an antibody in which the variable region is of completely human origin according to any one of claims 28 to 29, characterized in that an F0 generation non-human mammalian animal is mated with a wild-type non-human mammalian animal to obtain an F1 generation non-human mammalian animal which is stably inherited and into which a human immunoglobulin variable region gene can be inserted at a predetermined position.

31. The method for producing a non-human mammal that expresses an antibody whose variable region is of fully human origin, according to any one of claims 28 to 29, characterized in that the non-human mammal is a mouse, a rat, or a rabbit, and the non-human mammal cell is a mouse embryonic stem cell, a rat embryonic stem cell, or a rabbit embryonic stem cell.

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