US-20260123611-A1 - GENETICALLY MODIFIED MOUSE FOR PREPARING ANTIBODY AND METHOD FOR PREPARING GENETICALLY MODIFIED MOUSE

Abstract

Disclosed in the present disclosure is genetically modified mice in which immunoglobulin loci are modified to insert gene segments of a human immunoglobulin variable region. The mice can be bred normally and produce human-mouse chimeric antibodies including a human variable region and a mouse constant region. The present disclosure also provides a method for preparing the genetically modified mice and use of the mice.

Inventors

• Lanqing HAN
• Cui Su
• Xing MOU
• Kang Wang
• Xiaoliang Li
• Liang Zhang
• Xiangyun CHEN
• Shun Zhou
• Kugeng HUO

Assignees

• CYAGEN BIOSCIENCES (SUZHOU) INC.

Dates

Publication Date

20260507

Application Date

20250903

Claims (20)

1. 1 . A method for preparing genetically modified mice, comprising: (i) obtaining heavy chain locus modified mice, comprising (ia) inserting a first partial segment of a human immunoglobulin heavy chain variable region locus between an mIgHJ region and an mIgHC region of an immunoglobulin heavy chain locus of a first mouse, wherein the first partial segment comprises a first partial hIgHV contiguous segment, a whole hIgHD segment and a whole hIgHJ segment, the first partial segment does not comprise a segment between downstream of an hIgHV1-2 gene and upstream of an hIgHV6-1 gene, and first recombination sites are comprised upstream of the first partial segment and downstream of the mIgHJ region; (ib) inserting a second partial segment of the human immunoglobulin heavy chain variable region locus between an mIgHJ region and an mIgHC region of an immunoglobulin heavy chain locus of a second mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment comprises a second partial hIgHV contiguous segment, and second recombination sites are comprised between downstream of the second partial hIgHV contiguous segment and upstream of the mIgHC region; (ic) hybridizing the first mouse with the second mouse, screening a third mouse, and inserting the second partial segment of the human immunoglobulin heavy chain variable region locus and the first partial segment downstream of the second partial segment between an mIgHJ region and an mIgHC region of an immunoglobulin heavy chain locus of the third mouse, wherein third recombination sites are present between the second partial segment and the first partial segment; and (id) respectively knocking out an mIgHV5-1 gene and a whole mIgHV segment upstream of the mIgHV5-1 gene, a contiguous segment between an mIgHD1-1 gene and an mIgHJ4 gene, and mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes of the immunoglobulin heavy chain locus of the third mouse to obtain the heavy chain locus modified mice; (ii) obtaining Kappa light chain locus modified mice, wherein immunoglobulin Kappa light chain loci of the Kappa light chain locus modified mice sequentially comprise a whole hIgKV segment, a whole hIgKJ segment and an mIgKC segment, and a whole mIgKV segment and a whole mIgKJ segment are knocked out; (iii) obtaining Lambda light chain locus modified mice, wherein immunoglobulin Lambda light chain loci of the Lambda light chain locus modified mice sequentially comprise a whole hIgLV segment and a whole hIgLJ-C segment, and a whole mIgLV segment, an mIgLJ segment, and a whole mIgLC segment are knocked out; and (iv) mating the modified mice obtained in the steps (i), (ii) and (iii), screening triple positive mice, mating the obtained male and female triple positive mice with each other, and screening homozygous mice, wherein preferably, the modified mice obtained in the steps (i) and (ii) are mated and screening is performed to obtain first double positive homozygous mice, and the modified mice obtained in the steps (i) and (iii) are mated and screening is performed to obtain second double positive homozygous mice, and the first double positive homozygous mice and the second double positive homozygous mice are mated with each other and screening is performed to obtain triple positive homozygous mice.
2. 2 . The method according to claim 1 , wherein the step (iii) comprises: (iiia) inserting a first partial segment of a human immunoglobulin Lambda light chain locus between mIgLC1 and mIgL Enhancer 3-1 of an immunoglobulin Lambda light chain locus of a seventh mouse, wherein the first partial segment comprises a first partial hIgLV contiguous segment, a whole hIgLJ segment, and a whole hIgLC segment, and seventh recombination sites are comprised upstream of the first partial segment and downstream of mIgLC1; (iiib) inserting a second partial segment of the human immunoglobulin Lambda light chain locus between mIgLC1 and mIgL Enhancer 3-1 of an immunoglobulin Lambda light chain locus of an eighth mouse, wherein the second partial segment is located upstream of the first partial segment on the human immunoglobulin Lambda light chain locus, the second partial segment comprises a second partial hIgLV contiguous segment, the first partial hIgLV contiguous segment and the second partial hIgLV contiguous segment together constitute the whole hIgLV segment, and eighth recombination sites are comprised between downstream of the second partial hIgLV contiguous segment and upstream of mIgL Enhancer 3-1; (iiic) hybridizing the seventh mouse with the eighth mouse, screening a ninth mouse, and inserting the second partial segment of the human immunoglobulin Lambda light chain locus and the first partial segment downstream of the second partial segment between mIgLC1 and mIgL Enhancer 3-1 of an immunoglobulin Lambda light chain locus of the ninth mouse, wherein ninth recombination sites are present between the second partial segment and the first partial segment; and (iiid) knocking out a whole mIgLV segment, a whole mIgLJ segment, and a whole mIgLC segment of the immunoglobulin Lambda light chain locus of the ninth mouse to obtain the Lambda light chain locus modified mice.
3. 3 . The method according to claim 2 , having one or more of the following features: (a) the first partial hIgLV contiguous segment comprises a contiguous segment between an hIgLV3-1 gene and an hIgLV2-34 gene; and the second partial hIgLV contiguous segment comprises a contiguous segment between an hIgLVI-70 gene and an hIgLV7-35 gene; and (b) the seventh recombination sites, the eighth recombination sites, and the ninth recombination sites are loxP sites.
4. 4 . The method according to claim 2 , wherein the step (iiia) comprises: (iiia1) introducing loxP and lox5171-PB5′ between mIgLC1 and mIgL Enhancer 3-1; (iiia2) recombining a BAC vector comprising the first partial segment of the human immunoglobulin Lambda light chain locus, and an loxP site and an lox5171 site which are located at both ends of the first partial segment and in the same direction as that in the step (iiia1), and a Cre recombinase with a genome obtained in the step (iiia1), wherein PB3′ is comprised between the lox5171 site and the first partial segment; and (iiia3) contacting a recombinant genome obtained in the step (iiia2) with a PiggyBac transposase, and screening a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.
5. 5 . The method according to claim 2 , wherein the step (iiib) comprises: (iiib1) inserting a contiguous segment comprised between an hIgLV4-69 gene and an hIgLV10-67 gene between the mIgLC1 and the mIgL Enhancer 3-1; (iiib2) inserting a contiguous segment comprised between an hIGLVIV-66-1 and an hIGLVIV-65 gene, and PB3′-lox2272 and loxP sites downstream of the inserted segment in the step (iiib1); (iiib3) recombining a BAC vector comprising a contiguous segment between an hIGLV7-35 and an hIGLVI-63 gene, and loxP and lox2272 which are located at both ends and in the same direction as that in the step (iiib2), and a Cre recombinase with a genome obtained in the step (iiib2), wherein PB5′ is comprised between the lox2272 site and the contiguous segment described in this step; and (iiib4) contacting a recombinant genome obtained in the step (iiib3) with a PiggyBac transposase, and screening a genome comprising the second partial segment and the loxP site located downstream of the second partial segment.
6. 6 . The method according to claim 2 , wherein the step (iiic) comprises: (iiic1) screening Cre-positive mice comprising the first partial segment and the second partial segment; and (iiic2) mating the mice obtained in the step (iiic1) with wild type mice, and screening mice that do not carry Cre but comprise the first partial segment and the second partial segment, wherein loxP sites are present between the second partial segment and the first partial segment.
7. 7 . The method according to claim 2 , wherein the step (iiid) comprises: (iiid1) mating the ninth mouse with a wild type mouse, and screening positive mice; and (iiid2) mating the male and female positive mice obtained in the step (iiid1) with each other, and screening homozygous mice.
8. 8 . The method according to claim 1 , wherein the step (ii) comprises: (iia) inserting a first partial segment of a human immunoglobulin Kappa light chain variable region locus between an mIgKJ region and an mIgKC region of an immunoglobulin Kappa light chain locus of a fourth mouse, wherein the first partial segment comprises a first partial hIgKV contiguous segment and a whole hIgKJ segment, and fourth recombination sites are comprised upstream of the first partial segment and downstream of the mIgKJ region; (iib) inserting a second partial segment of the human immunoglobulin Kappa light chain variable region locus between an mIgKJ region and an mIgKC region of an immunoglobulin Kappa light chain locus of a fifth mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment comprises a second partial hIgKV contiguous segment, the first partial hIgKV contiguous segment and the second partial hIgKV contiguous segment together constitute the whole hIgKV segment, and fifth recombination sites are comprised between downstream of the second partial hIgKV contiguous segment and upstream of the mIgKC region; (iic) hybridizing the fourth mouse with the fifth mouse, screening a sixth mouse, and inserting the second partial segment of the human immunoglobulin Kappa light chain variable region locus and the first partial segment downstream of the second partial segment between an mIgKJ region and an mIgKC region of an immunoglobulin Kappa light chain locus of the sixth mouse, wherein sixth recombination sites are present between the second partial segment and the first partial segment; and (iid) knocking out a whole mIgKV segment and a whole mIgKJ segment of the immunoglobulin Kappa light chain locus of the sixth mouse to obtain the Kappa light chain locus modified mice.
9. 9 . The method according to claim 8 , having one or more of the following features: (a) the first partial hIgKV contiguous segment of the first partial segment comprises a contiguous segment between an hIgKV4-1 gene and an hIgKV1-37 gene; (b) the second partial hIgKV contiguous segment of the second partial segment comprises a contiguous segment between an hIgKV3D-7 gene and an hIgKV2D-40 gene; and (c) the fourth recombination sites, the fifth recombination sites, and the sixth recombination sites are loxP sites.
10. 10 . The method according to claim 8 , wherein in the step (iia), the first partial hIgKV contiguous segment and the whole hIgKJ segment of the first partial segment are inserted between the mIgKJ region and the mIgKC region by at least two steps comprising: (iia1) inserting a first contiguous segment comprised between an hIgKV1-5 gene and an hIgKJ5 gene between the mIgKJ region and the mIgKC region; and (iia2) inserting a second contiguous segment comprised between an hIgKV1-37 and an hIgKV1-6 gene upstream of the first contiguous segment.
11. 11 . The method according to claim 10 , wherein the step (iia1) specifically comprises: (iia11) inserting a contiguous segment comprised between an hIgKJ1 gene and the hIgKJ5 gene between the mIgKJ region and the mIgKC region; (iia12) inserting a contiguous segment comprised between an hIgKV4-1 gene and an hIgKV5-2 gene upstream of the inserted segment in the step (iia11); (iia13) inserting genes in a contiguous segment comprised between upstream of hIgKV5-2 and downstream of hIgHKV2-4 upstream of the inserted segment in the step (iia12); and (iia14) inserting a contiguous segment comprised between an hIgHKV2-4 gene and an hIgKV1-5 gene, and loxP and lox2272-PB5′ sites upstream of the inserted segment in the step (iia13).
12. 12 . The method according to claim 10 , wherein the step (iia2) specifically comprises: (iia21) recombining a BAC vector comprising the second contiguous segment between the hIgKV1-6 gene and the hIgKV1-37 gene, and an loxP site and an lox2272 site which are located at both ends of the second contiguous segment and in the same direction as that in the step (iia14), and a Cre recombinase with a genome obtained in step (iia1), wherein PB3′ is comprised between the lox2272 site and the second contiguous segment; and (iia22) contacting a recombinant genome obtained in the step (iia21) with a PiggyBac transposase, and screening a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.
13. 13 . The method according to claim 8 , wherein in the step (iib), the second partial hIgKV contiguous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps comprising: (iib1) inserting a contiguous segment comprised between the hIgKV2D-40 gene and an hIgKV2D-38 gene between the mIgKJ region and the mIgKC region; (iib2) inserting a contiguous segment comprised between an hIgKVID-37 and an hIgKVID-35 gene, and PB3′-lox5171 and loxP sites downstream of the inserted segment in the step (iib1); (iib3) recombining a BAC vector comprising a contiguous segment between an hIgKV3D-34 gene and the hIgKV3D-7 gene, and loxP and lox5171 which are located at both ends and in the same direction as that in the step (iib2), and a Cre recombinase with a genome obtained in the step (iib2), wherein PB5′ is comprised between the lox5171 site and the contiguous segment described in this step; and (iib4) contacting a recombinant genome obtained in the step (iib3) with a PiggyBac transposase, and screening a genome comprising the second partial segment and the loxP site located downstream of the second partial segment.
14. 14 . The method according to claim 13 , wherein the step (iic) comprises: (iic1) screening Cre-positive mice comprising the first partial segment and the second partial segment; and (iic2) mating the mice obtained in the step (iic1) with wild type mice, and screening mice that do not carry Cre but comprise the first partial segment and the second partial segment, wherein loxP sites are present between the second partial segment and the first partial segment.
15. 15 . The method according to claim 8 , wherein the step (iid) comprises: (iid1) mating the sixth mouse with a wild type mouse, and screening positive mice; and (iid2) mating the male and female positive mice obtained in the step (iid1) with each other, and screening homozygous mice.
16. 16 . The method according to claim 1 , having one or more of the following features: (a) the first partial hIgHV contiguous segment of the first partial segment comprises a contiguous segment between an hIgHV4-28 gene and the hIgHV1-2 gene; (b) the second partial hIgHV contiguous segment of the second partial segment comprises a contiguous segment between an hIgHV3-74 gene and an hIgHV3-30 gene; and (c) the first recombination sites, the second recombination sites, and the third recombination sites are loxP sites.
17. 17 . The method according to claim 1 , wherein immunoglobulin heavy chain loci of the genetically modified mice sequentially comprise: (i) an Adam6a gene of the mice; (ii) an Adam6b gene of the mice; (iii) a contiguous segment between an hIgHV3-74 gene and an hIgHV3-30 gene; (iv) a contiguous segment between an hIgHV4-28 gene and the hIgHV1-2 gene; (v) a contiguous segment between the hIgHV6-1 gene and an hIgHJ6 gene; and (vi) an mIgHC region.
18. 18 . The method according to claim 1 , wherein in the step (ia), the first partial hIgHV contiguous segment, the whole hIgHD segment and the whole hIgHJ segment of the first partial segment are inserted between the mIgHJ region and the mIgHC region by at least two steps comprising: (ia1) inserting a first contiguous segment comprised between the hIgHV6-1 gene and an hIgHJ6 gene between the mIgHJ region and the mIgHC region; and (ia2) inserting a second contiguous segment comprised between an hIgHV4-28 gene and the hIgHV1-2 gene upstream of the first contiguous segment.
19. 19 . The method according to claim 18 , wherein the step (ia1) specifically comprises: (ia11) inserting a contiguous segment comprised between an hIgHD6-25 gene and the hIgHJ6 gene between the mIgHJ region and the mIgHC region; (ia12) inserting a contiguous segment comprised between an hIgHD6-13 gene and an hIgHD5-24 gene upstream of the inserted segment in the step (ia11); (ia13) inserting a contiguous segment comprised between an hIgHD1-1 gene and an hIgHD5-12 gene upstream of the inserted segment in the step (ia12); and (ia14) inserting a contiguous segment comprised between the hIgHV6-1 gene and the hIgHD1-1 gene, and loxP and lox2272-PB5′ sites upstream of the inserted segment in the step (ia13).
20. 20 . The method according to claim 18 , wherein the step (ia2) specifically comprises: (ia21) recombining a BAC vector comprising the second contiguous segment between the hIgHV4-28 gene and the hIgHV1-2 gene, and an loxP site and an lox2272 site which are located at both ends of the second contiguous segment and in the same direction as that in the step (ia14), and a Cre recombinase with a genome obtained in the step (ia1), wherein PB3′ is comprised between the lox2272 site and the second contiguous segment; and (ia22) contacting a recombinant genome obtained in the step (ia21) with a PiggyBac transposase, and screening a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.

Description

FIELD OF THE INVENTION The present disclosure relates to genetically modified mice, cells, embryos, and tissues. In particular, the present disclosure relates to mice in which an immunoglobulin heavy chain variable region locus, a Kappa light chain variable region locus, and a Lambda light chain locus are all humanized and a method for preparing the mice. The present disclosure also relates to a genome of the modified mice, a cell and tissue including the genome, and a method for preparing a monoclonal antibody by using the mice, and use. The present disclosure also relates to mice having a modified genome. BACKGROUND OF THE INVENTION Humanized mice exhibit a fully functional humoral immune system that is essentially indistinguishable from that of wild type mice. They show normal cell populations at all stages of B cell development. They exhibit normal lymphoid organ morphology. Antibody sequences of the mice exhibit normal V(D)J rearrangements and normal somatic hypermutation frequencies. The antibody populations in these mice reflect the isotype distribution caused by normal isotype switching (such as normal isotype cis-switching). Immunization of mice results in a potent humoral immune response that generates a large and diverse antibody lineage with human immunoglobulin variable regions suitable for use as therapeutic candidates. The precise replacement of mouse immunoglobulin variable sequences with human immunoglobulin variable sequences enables the formation of genetically modified or transgenic mice. However, because of the divergent evolution of immunoglobulin loci between mice and humans, even through sequential recombineering of very large human immunoglobulin sequence segments to precisely replace endogenous mouse immunoglobulin sequences at heavy and light chain loci with corresponding human immunoglobulin sequences, certain problems may arise. For example, intergenic sequences interspersed within immunoglobulin loci are not identical between mice and humans, and in some cases may not be functionally equivalent. Differences between mice and humans in their immunoglobulin loci may still lead to abnormalities in humanized mice, particularly when certain portions of endogenous mouse immunoglobulin heavy chain loci are humanized or otherwise manipulated. Some modifications at mouse immunoglobulin heavy chain loci are deleterious, e.g., a loss of the ability of the modified mice to mate and produce offspring. It has been found that the decrease or disappearance of fertility in male mice is associated with impairment of an Adam6 gene. An ADAM6 protein is a member of an ADAM protein family, where ADAM is an acronym for ADisintegrin And Metalloprotease. The ADAM protein family is large and diverse and has diverse functions including cell adhesion. Some members of the ADAM family are involved in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of a protein fertilin that is involved in sperm-egg interactions. ADAM3 or cyritestin appears to be required for binding of sperms to a zona pellucida. The absence of ADAM2 or ADAM3 will result in infertility. It has been hypothesized that ADAM2, ADAM3 and ADAM6 form a complex on the surfaces of mouse sperm cells. A human ADAM6 gene is located between human VH genes VH1-2 and VH6-1. In mice, there are two Adam6 genes, Adam6a and Adam6b, present in an intergenic region between mouse VH and DH gene segments, and in mice, the direction of transcription of Adam6a and Adam6b genes is opposite to that of surrounding immunoglobulin gene segments. CN105861548B discloses Adam6 mice, wherein one or more human immunoglobulin gene sequences are inserted at an immunoglobulin heavy chain locus in a germline of the mice, wherein the insertion disrupts the function of an endogenous Adam6 gene; then, a nucleic acid sequence encoding a mouse Adam6a protein and a nucleic acid sequence encoding a mouse Adam6b protein are inserted into the germline of the mice, wherein the mouse Adam6a protein and the mouse Adam6b protein are expressed by the nucleic acid sequences, wherein the mouse Adam6a protein and the mouse Adam6b protein are expressed from the nucleic acid sequences and improve or restore fertility when expressed in male mice. Disruption of the endogenous Adam6 gene is undesirable and may have an impact on the long-term reproductive ability of the mice. In view of this, the present disclosure provides a heavy chain locus modified mouse genome that maintains the endogenous Adam6 gene and its function and can produce human-mouse chimeric antibodies while maintaining fertility. SUMMARY OF THE INVENTION In one aspect, the present disclosure provides a method for preparing genetically modified mice, including: (i) obtaining heavy chain locus modified mice, including(ia) inserting a first partial segment of a human immunoglobulin heavy chain variable region locus between an mIgHJ region and an mIgHC region of an immunoglobulin heavy chain locus of a first mouse,