Animal model producing humanized antibodies and method for constructing same

Abstract

The present application relates to a non-human animal and a method for producing the non-human animal, wherein the non-human animal has a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus. The present application also provides a method for producing antibodies by the non-human animal.

Description

[Technical Field]

[0001] The present application relates to the field of biomedicine, and more particularly to antibody-producing non-human animal models and methods for constructing same. [Background technology]

[0002] After more than 30 years of development, therapeutic monoclonal antibody drugs have become one of the most important components of biopharmaceuticals and remain the fastest-growing, most advanced, and most effective drugs. Since the first OKT3 was approved in 1986, therapeutic antibodies have demonstrated rapid and transformative progress in their development, solidifying their position as an important aspect of modern biopharmaceuticals. However, despite this success, challenges remain, including low success rates, high costs, and lengthy development times for antibody-based drugs. In recent years, the issue of anti-drug antibodies (ADAs) due to immunogenicity, especially for therapeutic antibodies classified as macromolecular drugs, has also attracted significant attention. Overcoming the risk of ADA formation during the preclinical stage remains a complex task.

[0003] Currently, the most commonly used methods for discovering and screening therapeutic monoclonal antibodies are "display technologies," including phage display, bacterial display, and yeast display. Although "display technologies" can rapidly screen antibodies, due to the lack of in vivo antibody variable region recombination and affinity hypermutation processes, antibodies screened using these methods have limited diversity and affinity, and require further optimization and modification, which requires significant costs and cycles. Another commonly used method for discovering and screening therapeutic monoclonal antibodies is immune discovery, which is carried out directly using wild-type animal models such as birds, rodents, and non-human primates. Humanization of molecules obtained using the above methods based on experience and computer database predictions is a common method to avoid the ADA effect caused by immunogenicity. However, the final results are often unpredictable and far from the actual clinical effects, resulting in side effects such as hypersensitivity stress during clinical treatment and loss of therapeutic efficacy due to antibody neutralization.

[0004] One possible way to circumvent the above problems and discover fully human antibodies is to use nonhuman animals engineered to contain human antibody genes. While many currently available nonhuman animals are capable of producing fully human antibodies, the vastness and complexity of the human antibody coding system means that many of these models have significant drawbacks and limitations. For example, they often contain only a small or partial representation of the human antibody family. The lack of a significant portion of the human antibody family means a lack of diversity in the antibody molecules ultimately discovered and a reduced likelihood of obtaining high affinity. This, in turn, leads to reduced V(D)J recombination efficiency and sub-normal antibody production. Furthermore, these models face challenges such as immunogenicity issues resulting from the penetration of the animal's endogenous antibody coding system and phenotypic problems resulting from the deletion of large fragments of the animal's endogenous genes. Due to these and several other issues, the industry needs nonhuman animals that contain all human immunoglobulin variable region-encoding genes and have the ability to produce humanized antibodies without altering the immune response. This allows for the effective and efficient production of clinically suitable, fully humanized antibodies with variable regions. Summary of the Invention [Means for solving the problem]

[0005] The present application provides a non-human animal and a method for producing the non-human animal, the genome of which comprises a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus. The non-human animal of the present application avoids interference with the animal's own gene expression and regulation, prevents leakage of endogenous variable region genes, and has a significant advantage in antibody production.

[0006] In one aspect, the present application provides a method of producing a non-human animal, the method comprising operably linking a human immunoglobulin variable region gene downstream of an immunoglobulin locus of the non-human animal.

[0007] In some embodiments, the method comprises operably linking a human immunoglobulin heavy chain variable region gene downstream of a heavy chain locus of the non-human animal. In some embodiments, the method comprises operably linking one or more human heavy chain V, D, or J region, or fragment thereof, genes downstream of a heavy chain constant region locus of the non-human animal. In some embodiments, the multiple human heavy chain V, D, or J region, or fragment thereof, genes can be recombined. In some embodiments, the multiple human heavy chain V, D, or J region, or fragment thereof genes are directly linked.

[0008] In some embodiments, the method comprises operably linking a human immunoglobulin light chain variable region gene downstream of a light chain locus of the non-human animal. In some embodiments, the method comprises operably linking one or more human light chain V or J region, or fragment thereof, genes downstream of a light chain constant region locus of the non-human animal. In some embodiments, the multiple human light chain V or J region, or fragment thereof, genes can be recombined. In some embodiments, the multiple human light chain V or J region, or fragment thereof, genes are directly linked.

[0009] In another aspect, the present application provides a method of producing a non-human animal, the method comprising operably linking a human immunoglobulin heavy chain variable region gene downstream of an immunoglobulin heavy chain locus of the non-human animal.

[0010] In another aspect, the present application provides a method of producing a non-human animal, the method comprising operably linking a human immunoglobulin light chain variable region gene downstream of an immunoglobulin light chain locus of the non-human animal.

[0011] In one aspect, the present application provides a non-human animal or a non-human animal cell, the genome of which comprises a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus, and any of the features contained in the non-human animal referred to below are contained in the non-human animal cell.

[0012] In some embodiments, a human immunoglobulin heavy chain variable region gene is operably linked downstream of the heavy chain locus of the non-human animal. In some embodiments, one or more human heavy chain V, D, or J region, or fragment thereof, genes are operably linked downstream of the heavy chain constant region locus of the non-human animal. In some embodiments, the multiple human heavy chain V, D, or J region, or fragment thereof, genes are recombined. In some embodiments, the multiple human heavy chain V, D, or J region, or fragment thereof, genes are directly linked.

[0013] In some embodiments, a human immunoglobulin light chain variable region gene is operably linked downstream of the light chain locus of the non-human animal. In some embodiments, one or more human light chain V or J region, or fragment thereof, genes are operably linked downstream of the light chain constant region locus of the non-human animal. In some embodiments, the multiple human light chain V or J region, or fragment thereof, genes are recombined. In some embodiments, the multiple human light chain V or J region, or fragment thereof, genes are directly linked.

[0014] In another aspect, the present application provides a non-human animal or a non-human animal cell, wherein a human immunoglobulin heavy chain variable region gene is operably linked downstream of an immunoglobulin heavy chain locus of the non-human animal or the non-human animal cell.

[0015] In another aspect, the present application also provides progeny of the non-human animals described above, which may be progeny produced by mating the non-human animals with the same genotype or with other genotypes.

[0016] In another aspect, the present application also provides cells (e.g., stem cells, embryonic stem cells, immune cells, B cells, T cells, or hybridomas), cell lines, or primary cell cultures thereof derived from the non-human animals or their progeny.

[0017] In another aspect, the present application provides a non-human animal or a non-human animal cell, wherein a human immunoglobulin light chain variable region gene is operably linked downstream of an immunoglobulin light chain locus of the non-human animal or the non-human animal cell.

[0018] In some embodiments, the non-human animal comprises endogenous immunoglobulin variable region genes. In some embodiments, the integrity of the endogenous immunoglobulin variable region genes of the non-human animal is not disrupted. In some embodiments, the function of expression regulatory elements of the endogenous immunoglobulin variable region genes of the non-human animal is not disrupted. In some embodiments, the endogenous immunoglobulin variable region genes of the non-human animal comprise a functional protein-coding gene and a functional microRNA gene. In some embodiments, the non-human animal comprises complete endogenous immunoglobulin variable region expression regulatory elements. In some embodiments, the non-human animal comprises complete endogenous immunoglobulin variable region genes.

[0019] In some embodiments, the non-human animal does not express endogenous immunoglobulin variable regions, hi some embodiments, the non-human animal's endogenous immunoglobulin variable region genes do not express functional proteins.

[0020] In some embodiments, the direction of transcription of the human immunoglobulin variable region genes and the endogenous immunoglobulin variable region genes of the non-human animal is opposite.

[0021] In some embodiments, the direction of transcription of the immunoglobulin constant region genes of the non-human animal and the endogenous immunoglobulin variable region genes is opposite. The immunoglobulin constant region genes of the non-human animal may be endogenous. The non-human animal may not contain exogenous immunoglobulin constant region genes.

[0022] In some embodiments, the direction of transcription of the human immunoglobulin variable region genes and the immunoglobulin constant region genes in the non-human animal is the same.

[0023] In some embodiments, the non-human animal contains a heavy chain constant region gene between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene. In some embodiments, the non-human animal contains one or more heavy chain constant region genes between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene. In some embodiments, the distance between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene in the non-human animal is 169 Kbp to 240 Kbp.

[0024] In some embodiments, the non-human animal contains a light chain constant region gene between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene. In some embodiments, the non-human animal contains one or more light chain constant region genes between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene. In some embodiments, the distance between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene in the non-human animal is 4 Kbp to 42 Kbp.

[0025] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal is a field mouse.

[0026] In some embodiments, the mouse comprises a human immunoglobulin heavy chain variable region gene inserted between the Tmem121 and Igha genes at the locus. In some embodiments, the mouse comprises a human immunoglobulin heavy chain variable region gene inserted between chromosomal locations chr12:113,149,523 to 113,223,857. In some embodiments, the mouse comprises a human immunoglobulin heavy chain variable region gene inserted at chromosomal location chr12:113,190,256.

[0027] In some embodiments, the mouse comprises a human immunoglobulin light chain variable region gene inserted between the Igkc and Rpia genes at the locus. In some embodiments, the mouse comprises a human immunoglobulin light chain variable region gene inserted between chromosomal locations chr6:70,703,738-70,742,704. In some embodiments, the mouse comprises a human immunoglobulin light chain variable region gene inserted at chromosomal location chr6:70,706,267.

[0028] In some embodiments, the method comprises operably linking a human immunoglobulin variable region gene downstream of an immunoglobulin locus of the non-human animal by site-specific recombination. In some embodiments, the site-specific recombination can be one or more times. In some embodiments, the method comprises linking multiple genes for human heavy chain V, D, or J regions, or fragments thereof, using multiple rounds of site-specific recombination. In some embodiments, the method comprises linking multiple genes for human light chain V or J regions, or fragments thereof, using multiple rounds of site-specific recombination. In some embodiments, the site-specific recombination comprises homologous recombination, endonuclease-mediated recombination, or recombination mediated by a site-specific recombinase. In some embodiments, a vector carrying a human genomic fragment is used to insert the human immunoglobulin variable region gene downstream of the immunoglobulin locus of the non-human animal. In some embodiments, the human immunoglobulin variable region gene is inserted downstream of the immunoglobulin locus of the non-human animal using a site-specific integration method mediated by a site-specific recombination system. In some embodiments, the method comprises deleting the resistance marker after inserting the human immunoglobulin variable region gene.

[0029] In some embodiments, the method comprises reversing the direction of transcription of the non-human animal's immunoglobulin constant region genes and endogenous immunoglobulin variable region genes by chromosomal recombination prior to inserting the human variable region genes. In some embodiments, the method comprises reversing the direction of transcription of the non-human animal's immunoglobulin constant region genes by chromosomal recombination prior to inserting the human variable region genes. In some embodiments, the chromosomal recombination comprises genomic fragment reversal mediated by a site-specific recombination system. In some embodiments, the method comprises deleting a resistance marker after chromosomal recombination.

[0030] In another aspect, the present application provides a non-human animal cell whose genome comprises a human immunoglobulin variable region gene downstream of an immunoglobulin locus. In some embodiments, the cell is an embryonic stem (ES) cell. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic cell.

[0031] In some embodiments, the non-human animal cells are capable of developing into a non-human animal.

[0032] In another aspect, the present application provides cells, tissues and organs derived from non-human animals.

[0033] In another aspect, the present application provides a method of producing an antibody that specifically binds to an antigen, the method comprising immunizing the non-human animal of the present application with the antigen.

[0034] In another aspect, the present application provides a method for producing an antibody that specifically binds to an antigen, the method comprising exposing the non-human animal or cells to the antigen and harvesting cells from the animal to produce a hybridoma. The method further comprises harvesting the chimeric antibody produced by the hybridoma. The method can further comprise sequencing the variable region genes of the hybridoma.

[0035] In another aspect, the present application provides a method for producing an antibody that specifically binds to an antigen, the method comprising exposing the non-human animal or cell to the antigen and sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in the animal or cell. The non-human animal or cell is capable of expressing a chimeric antibody that specifically binds to the antigen. In some embodiments, the method comprises operably linking a nucleic acid encoding the human heavy chain immunoglobulin variable region to a nucleic acid encoding a heavy chain immunoglobulin constant region, and expressing an antibody to which the antigen specifically binds. The constant region may be a human heavy chain constant region or a heavy chain constant region of a non-human animal. In some embodiments, the method comprises operably linking a nucleic acid encoding the human light chain immunoglobulin variable region to a nucleic acid encoding a light chain immunoglobulin constant region, and expressing an antibody to which the antigen specifically binds. The constant region may be a human light chain constant region or a light chain constant region of a non-human animal.

[0036] In another aspect, the present application provides a method of producing a sample, the method comprising exposing the non-human animal of the present application to an antigen and collecting the sample from the non-human animal. In some embodiments, the sample comprises immune cells such as B cells. In some embodiments, the sample comprises bone marrow, spleen tissue, lymph nodes, splenocytes, or peripheral lymphocytes.

[0037] The technical solution of the present application includes one or more of the following advantages:

[0038] The non-human animals of the present application have the advantage of avoiding and regulating interference with the animal's own gene expression. For example, by inserting a human variable region gene (e.g., a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region) downstream (or after) the endogenous immunoglobulin locus of a mouse cell, it is possible to avoid inserting a large DNA sequence fragment or deleting a DNA sequence into the endogenous immunoglobulin locus of a non-human animal (e.g., a mouse). The endogenous immunoglobulin locus of a non-human animal (e.g., a mouse) contains a very long "junk sequence" downstream. For example, the recently disclosed complete genome sequence of the GRCm39 mouse contains a non-functional gene report sequence of up to 66 kb downstream (after) the mouse immunoglobulin heavy chain gene and a non-functional gene report sequence of up to 39 kb downstream (after) the mouse immunoglobulin κ light chain gene. By introducing exogenous large fragment genes into these long "Junk sequences", it is possible to avoid interference with the expression and regulation of endogenous genes of non-human animals (e.g., mice) from the genomic layer.

[0039] The non-human animals of the present application also have the advantage of avoiding interference with the expression and regulation of the animal's own genes. For example, modification or deletion of the immunoglobulin gene variable region of a non-human animal (e.g., mouse) is avoided. Because the endogenous immunoglobulin variable region locus of a non-human animal (e.g., mouse) is rich in functional protein-coding genes and functional microRNA genes, deletion or modification of these genes can result in the emergence of unpredictable phenotypes in the animal itself. For example, the immunoglobulin heavy chain locus of a mouse contains the functional protein-coding gene Adam6a, and modification or deletion of the Adam6a gene can cause male infertility in mice.

[0040] The non-human animals of the present application have advantages in antibody production. The non-human animals of the present application retain both the immunoglobulin variable region genes and constant region genes of the host non-human animal. That is, the endogenous immunoglobulin variable region genes and constant region genes of the non-human animal are retained. All host immunoglobulin regulatory sequences, including promoters, enhancers, switch regions, and other potential expression regulatory sequences, are retained. This allows for more optimal immunoglobulin gene recombination, expression, B cell development, affinity maturation, and other processes in the non-human animals.

[0041] The non-human animals of the present application have an advantage in antibody production. For example, in some embodiments, the animal's endogenous immunoglobulin constant region is fully or partially modified (e.g., all or part of the constant region is inverted) to reverse the direction of transcription of the endogenous variable region genes, thereby preventing V(D)J recombination between the endogenous variable region and the endogenous constant region to produce antibodies.

[0042] The non-human animals of the present application have an additional advantage in antibody production. For example, in some embodiments, the inserted exogenous human immunoglobulin variable region is separated from the endogenous variable region and is in the opposite transcriptional direction. By preventing the endogenous variable region from being integrated into the human variable region VDJ or VJ recombination by DNA recombinase correction or recombination, it is highly likely that the production of antibodies bearing epitopes immunogenic in humans, which would be produced by the endogenous variable region lineage, is avoided.

[0043] The non-human animals of the present application have an additional advantage in antibody production. For example, in some embodiments, all fragments of human variable region genes are introduced stepwise and inserted downstream (after) the endogenous immunoglobulin loci of non-human animal (e.g., mouse) cells, allowing the variable domains produced by the resulting non-human animal to have nearly the same diversity as that of human variable domains. This means that the resulting non-human animal has antibody diversity as close as possible to that of the human body, and has the potential to obtain high-affinity, specific antibodies.

[0044] The methods for producing non-human animals described herein also have advantages. For example, in some approaches, all or part of the endogenous immunoglobulin constant region of a non-human animal (e.g., a mouse) is modified so that the transcriptional direction is reversed from that of the endogenous variable region gene. Furthermore, when modified human variable region genes are inserted downstream (behind) of the endogenous immunoglobulin locus in non-human animal (e.g., a mouse) cells, the human variable region genes are split from back to front and can be inserted in the reverse transcriptional direction of the animal's immunoglobulin locus. As a result, cells with modified human variable regions obtained by almost any stage of insertion can be injected into early animal embryos to create chimeric animals (e.g., mice), which can then be produced or bred to produce non-human animals. All of the resulting non-human animals can use the inserted part of the human variable region gene to undergo VDJ or VJ recombination and produce chimeric antibodies with functional human variable regions. These non-human animals with partial human variable regions can serve as a checkpoint for the eventual production of non-human animals with fully human variable regions. The reliability and functionality of the stepwise insertion of human variable regions will be assessed based on antibody production and the diversity of variable region domains, allowing for rapid prediction of success or failure and correction of the route for the creation of non-human animals with fully human variable regions. These non-human animals with partial human variable regions can also be identified using antibodies, allowing for the production of human variable region chimeric antibodies.

[0045] Those skilled in the art can readily discern other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. As those skilled in the art will recognize, the contents of this application will enable those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the invention referred to herein. Correspondingly, the drawings and description in the present application are illustrative and not restrictive. [Brief explanation of the drawings]

[0046] Specific features of the invention referred to herein are set forth in the appended claims. The features and advantages of the invention referred to herein can be better understood through the exemplary embodiments and drawings described in detail below. A brief description of the drawings follows.

[0047] [Figure 1] Figure 1 shows the heavy chain Igh constant region Cpoint, LD targeting and C region modifications. [Figure 2] Figure 2 shows a schematic diagram of heavy chain human variable region insertion, including insertion targeting and deletion of the resistance marker. [Figure 3] Figure 3 shows the light chain Igk constant region Cpoint, LD targeting, and C region modification. [Figure 4] Figure 4 shows a schematic diagram of the light chain human variable region insertion, including insertion targeting and deletion of the resistance marker. [Figure 5] Figure 5 shows the PCR identification results of Cpoint targeting. [Figure 6] Figure 6 shows the PCR identification of LD targeting. [Figure 7] Figure 7 shows the reverse PCR identification results. [Figure 8] Shown in Figure 8 is the PCR confirmation of resistance marker deletion. [Figure 9]Figure 9 shows the PCR identification results of IGH vector1 targeting. [Figure 10] Shown in Figure 10 is PCR confirmation of the integrity of IGH vector1 targeting. [Figure 11] Shown in Figure 11 is PCR identification of the IGH vector1 resistance marker deletion. [Figure 12] Figure 12 shows the utilization test of the IGH insert gene. [Figure 13] Figure 13 shows the utility test of the IGK insert gene. [Figure 14] Shown in Figure 14 are the normal proportions of lymphoid and myeloid cells in the spleens of humanized mice. [Figure 15] Figure 15 shows normal development of B cells in the spleen and bone marrow. [Figure 16] Figure 16 shows the leakage expression status of mouse-derived genes after inversion of different regions of the mouse. [Figure 17] Shown in Figure 17 are ELISA titrations of humanized and control mice. [Figure 18] Figure 18 shows a comparison of the efficacy of the screened 10K4F3 antibody and a commercially available drug in mice. [Figure 19] Figure 19 shows the utilization test of some IGH-V, D, and J inserted genes. [Figure 20] Figure 20 shows the utilization test of some IGK-V,J inserted genes. [Figure 21] Shown in Figure 21 are ELISA titrations of some VDJ humanized and control mice (S0, pre-immune; S1, secondary immunization; S2, tertiary immunization). DETAILED DESCRIPTION OF THE INVENTION

[0048] Although the following describes embodiments of the present invention using specific examples, those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0049] Term definition As used herein, the term "non-human animal" generally refers to all non-human vertebrates, including mammals and non-mammals, such as non-human primates, rodents, rabbits, camelids, sheep, dogs, cats, horses, cows, birds, amphibians, and reptiles. For example, the non-human animal may be a rat or a mouse.

[0050] In this application, the term "constant region" generally refers to the entire domain of an antibody excluding the variable region. The constant region is not directly involved in antigen binding but exhibits different effector functions. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies are classified as IgA, IgD, IgE, IgG, and IgM, and some of these are further classified as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different types of antibodies are called α, δ, ε, γ, and μ, respectively. The light chain constant regions found in all five antibody types are called κ (kappa) and λ (lambda). Genes encoding the constant regions of mice (Mus musculus) can include IGHA, IGHD, IGHE, IGHG1, IGHG2A, IGHG2B, IGHG2C, IGHG3, or IGHM. For information regarding mouse constant region loci, see the IMGT Repertoire: https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=Mus_musculus&group=IGHC.

[0051] As used herein, the term "locus" generally refers to a specific location along a chromosome or DNA sequence. Depending on the context, a locus may be a gene, a marker, a chromosomal band, or a specific sequence of one or more nucleotides. As used herein, an immunoglobulin locus includes a genetic element or set of related genetic elements that a cell can use to express information for an immunoglobulin peptide. In an unrearranged locus, the genetic elements are integrated by B cell precursors to form genes encoding immunoglobulin peptides. In a rearranged locus, genes encoding immunoglobulin peptides are contained within the locus.

[0052] As used herein, the term "antibody" generally refers to an intact antibody or antigen-binding fragment thereof, and optionally includes a framework or scaffold capable of binding to an antigen and partially adopting a structure that promotes antibody-antigen binding. Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFV, dis-scFV, and / or dAb, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody mimetics, or fusion proteins, as long as they exhibit the required antigen-binding activity. The term also includes genetically engineered antibodies, such as chimeric antibodies (e.g., humanized murine antibodies), humanized antibodies, fully human antibodies, and heterocovalently linked antibodies (e.g., bispecific antibodies).

[0053] As used herein, the term "chimeric antibody" generally refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies derived from two different mammalian species, such as human and murine antibodies). Non-limiting examples of chimeric antibodies include antibodies that contain human antibody variable domain sequences (e.g., all or part of the light chain variable domain and / or heavy chain variable domain sequences) and the constant domains of a non-human antibody (e.g., a murine antibody).

[0054] As used herein, the term "immunoglobulin" generally refers to a protein essentially consisting of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The NH2-terminus (approximately 110 amino acids) of a full-length immunoglobulin "light chain" (approximately 25 kD and 214 amino acids) is encoded by a variable region gene, and the COOH-terminus is encoded by a kappa or lambda constant region gene. The full-length immunoglobulin "heavy chain" (approximately 50 kD and 446 amino acids) is similarly encoded by a variable region gene (approximately 116 amino acids) and one of the other constant region genes mentioned above, such as gamma (encoding approximately 330 amino acids). The term "immunoglobulin" includes immunoglobulins with CDRs of human or non-human origin. Immunoglobulins may be human, humanized, or non-human, eg, murine, structures modified to reduce antigenicity in humans, or synthetic structures such as covalent sequences.

[0055] As used herein, the term "variable region" generally refers to the region of an antibody molecule that binds to a specific antigen. The variable region consists of the antigen-binding sites of the heavy and light chains. Variable regions differ among different B cell immunoglobulins but are identical among all immunoglobulins produced by the same B cell. The diversity of variable regions arises from a genetic recombination process that occurs in variable region genes during B cell maturation. This process is a rearrangement process that generates a large diversity of antibodies capable of binding to any given antigen, thereby enabling the immune system to identify and neutralize the large antigen burden posed by exogenous and pathogenic structures. Thus, an antibody library consists of a wealth of immunoglobulins with different V regions, but with the same Fc region.

[0056] As used herein, the term "endogenous" generally refers to any substance that originates and is produced within an organism, cell, tissue, or system. For example, an endogenous gene is a gene that naturally occurs in the body of a non-human animal, as distinguished from an exogenous gene from another animal that has been introduced by genetic recombination.

[0057] As used herein, the terms "upstream" and "downstream" generally describe the relative location of genes within a chromosome, and may refer to either the chromosome or any given gene. To better account for the commonalities between modifications of the two types of gene loci, heavy and light chain, the present application refers to the location of the native non-human animal host immunoglobulin locus and the transcriptional direction of that immunoglobulin (i.e., the public display in the NCBI database); for example, for gene A, if the location of gene A is after the coding end of the immunoglobulin locus relative to the host immunoglobulin locus, then A is considered to be downstream of the host immunoglobulin locus, and the host immunoglobulin locus is considered to be upstream of A.

[0058] As used herein, the term "transcription direction" generally refers to the direction of the genetic code in which a gene ultimately forms a functional protein, i.e., from the sequence encoding the start codon (e.g., ATG) to the sequence encoding the stop codon (e.g., TAG, TAA, TGA).

[0059] Details of the invention This application relates to genetically modified non-human animals and cells with functional human immunoglobulin variable region genes capable of producing chimeric antibodies in which the variable regions are human. In this application, work performed in mice is for illustrative purposes only, and unless otherwise specified, references to mice include all non-human mammals, with mice being the preferred non-human mammal.

[0060] In certain aspects, the non-human animals or cells referred to herein comprise one or more human IGHV regions, one or more human IGHD regions, and / or one or more human IGHJ regions downstream of a heavy chain locus coding region of a host non-human mammal. In some embodiments, the direction of transcription of the coding region after insertion of the human IGHV regions, human IGHD regions, and human IGHJ regions is opposite to the direction of transcription of the host's endogenous heavy chain VDJ primordial gene coding region. The human IGHV regions, human IGHD regions, and human IGHJ regions are operably linked and capable of VDJ recombination.

[0061] In some embodiments, the animal's endogenous heavy chain locus constant region is modified in whole or in part so that all or part of it is in the opposite direction of transcription from the endogenous heavy chain variable region gene.

[0062] In another aspect, the non-human animals or cells referred to herein can have one or more human light chain (e.g., IGK) V regions and / or one or more human light chain (e.g., IGK) J regions downstream of the light chain locus coding region of the host non-human animal. In some embodiments, the direction of transcription of the coding region after insertion of the human light chain (e.g., IGK) V region or human light chain (e.g., IGK) J region is opposite to the direction of transcription of the corresponding endogenous light chain VJ progenitor gene coding region of the host. The human IGK V region and human IGK J region are operably linked and capable of VJ recombination.

[0063] In some embodiments, the animal's endogenous light chain locus constant region is modified in whole or in part so that its transcriptional direction is reversed in whole or in part from that of the corresponding endogenous light chain variable region gene.

[0064] In one aspect, the human immunoglobulin heavy chain VDJ region gene located downstream of the host non-human animal heavy chain locus coding region comprises all of the V, D, and J regions of a heavy chain of human origin arranged in reverse phylogenetic orientation, and some or all of the intervening sequences.

[0065] In another aspect, a human immunoglobulin heavy chain VDJ region gene located downstream of the heavy chain locus coding region of a host non-human animal is arranged in the reverse orientation and operably linked to a modified host constant region, allowing unimpeded VDJ recombination and expression of a functional human variable region chimeric antibody.

[0066] In another aspect, human immunoglobulin genes arranged in reverse orientation downstream of the heavy chain locus coding region of a host non-human animal can be expressed in combination with constant regions of different host antibody subtypes, allowing unimpeded immunoglobulin isotype switching in B cells.

[0067] In one aspect, the human immunoglobulin light chain VJ region gene located downstream of the host non-human animal light chain locus coding region comprises all of the V and J regions of a light chain of human origin arranged in reverse phylogenetic orientation, and some or all of the intervening sequences.

[0068] In another aspect, a human immunoglobulin light chain VJ region gene located downstream of the light chain locus coding region of a host non-human animal is arranged in the reverse orientation and operably linked to a modified host constant region, allowing unimpeded VJ recombination and expression of a functional human variable region chimeric antibody.

[0069] In some aspects, the inserted human immunoglobulin variable region gene DNA comprises 10% to 100% reverse-encoded human heavy chain variable (V) gene coding region sequence, e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%, and even the entire human heavy chain variable region gene coding region sequence.

[0070] In some aspects, the inserted human immunoglobulin variable region gene DNA comprises 50% to 100% of the human heavy chain D region gene coding region encoded in reverse orientation, e.g., more than 60%, more than 70%, more than 80%, more than 90%, and even the entire human D region gene coding region.

[0071] In some aspects, the inserted human immunoglobulin variable region gene DNA comprises 50% to 100% of the human heavy chain J region gene coding region encoded in reverse orientation, e.g., more than 60%, more than 70%, more than 80%, more than 90%, and even the entire human J region gene coding region.

[0072] In certain aspects, the length of the inserted human immunoglobulin heavy chain variable region gene DNA is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb.

[0073] In some aspects, the inserted human immunoglobulin variable region gene DNA comprises 15% to 100% of the human light chain V region gene coding region encoded in the reverse orientation, e.g., more than 60%, more than 70%, more than 80%, more than 90%, and even the entire human V region gene coding region.

[0074] In some aspects, the inserted human immunoglobulin variable region gene DNA comprises 50% to 100% of the human light chain J region gene coding region encoded in the reverse orientation, e.g., more than 60%, more than 70%, more than 80%, more than 90%, and even the entire human light chain J region gene coding region.

[0075] In certain aspects, the length of the inserted human immunoglobulin light chain variable region gene DNA is about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb.

[0076] For information on human heavy chain V, D, and J regions, and light chain V and J regions, see IMGT Repertoire: https: / / www.imgt.org / IMGTrepertoire / LocusGenes / .

[0077] Optionally, the non-human animal may contain only human heavy chain variable region genes located downstream of the heavy chain locus coding region of the host non-human animal, or only human light chain variable region genes located downstream of the light chain locus coding region of the host non-human animal, or both simultaneously.

[0078] Optionally, the endogenous immunoglobulin heavy chain variable region (V region, D region, and J region) genes of the non-human animal are not modified or deleted in any way to prevent the expression or regulation of parts of the host's genes in the variable regions from being affected. At the same time, the endogenous immunoglobulin heavy chain variable region is separated from the inserted human immunoglobulin heavy chain variable region by the coding sequence of the endogenous immunoglobulin heavy chain constant region, with a base length of 169 Kbp to 240 Kbp between them, preferably 202 Kbp, to some extent preventing the endogenous variable region from penetrating into the final chimeric immunoglobulin-encoding gene recombinant.

[0079] Optionally, the endogenous immunoglobulin light chain variable region (V region and J region) genes of the non-human animal are not modified or deleted in any way to prevent the expression or regulation of part of the host's genes in the variable region from being affected. At the same time, the endogenous immunoglobulin light chain variable region is separated from the inserted human immunoglobulin light chain variable region by an endogenous immunoglobulin light chain constant region coding sequence, with a base length of 4 Kbp to 42 Kbp between them, preferably 5 Kbp, to some extent preventing the endogenous variable region from penetrating into the final chimeric immunoglobulin-encoding gene recombinant.

[0080] This application discloses methods for constructing genes with functional human immunoglobulin variable regions in non-human animals (e.g., mice). In this application, research work performed in mice is for illustrative purposes only, and unless otherwise specified, references to mice include all non-human animals, with mice being the preferred non-human animal.

[0081] In one aspect, site-specific recombination is used to target insertion of human immunoglobulin heavy chain variable region gene DNA downstream of the IgH locus on mouse chromosome 12 between the Tmem121 gene and the Igha gene, and in one aspect, insertion is performed between mouse chromosome 12 coordinates 113,149,523 and 113,223,857, preferably at coordinates 113,190,256.

[0082] In one aspect, site-specific recombination is used to target insertion of human immunoglobulin light chain variable region gene DNA, e.g., human light chain κ VJ, downstream of the mouse IgK locus on chromosome 6, between the Igkc and Rpia genes, which in one aspect is inserted between mouse chromosome 6 coordinates 70, 703, 738 and 70, 742, 704, preferably at positions 70, 706, 267, or the equivalent position on chromosome 16 at the mouse λ locus. All coordinates are referenced in the NCBI database GRCm39.

[0083] In some embodiments, the site-specific recombination method includes homologous recombination, endonuclease-mediated recombination (e.g., CRISPR / Cas9), and site-specific recombinase-mediated recombination, and the combined use of one or more of these methods is described.

[0084] In some embodiments, the endogenous heavy chain locus constant region is fully or partially modified to reverse its transcriptional direction in whole or in part relative to that of the corresponding endogenous light chain variable region gene. This modification involves inserting exogenous recombination sites into or at both ends of the constant region of a non-human animal cell or animal, and then inverting all or part of the host's constant region gene sequence by means such as intracellular delivery of a recombinase or animal breeding. The insertion can be achieved by conventional gene editing targeting, such as homologous recombination or endonuclease-mediated recombination (e.g., CRISPR / Cas9). The modification can also be achieved using nucleases, and related techniques are known in the art.

[0085] In some embodiments, the endogenous heavy chain locus constant region is entirely or partially modified to reverse its transcription direction in whole or in part relative to the transcription direction of the corresponding endogenous light chain variable region gene. The modification involves inserting or replacing part or all of the host's constant region gene sequence in the reverse direction using a non-human animal constant region. The insertion or replacement can be achieved by conventional gene editing targeting methods, such as homologous recombination or endonuclease-mediated recombination (e.g., CRISPR / Cas9).

[0086] In some embodiments, modified human variable region genes are first inserted downstream (after) the endogenous immunoglobulin loci of a mouse cell, and then a recombinase is introduced to invert part or all of the endogenous constant region of the mouse cell by introducing a modified short exogenous recombinase binding site into the endogenous constant region of the mouse cell. The resulting modified cells are then screened and identified, and injected into early embryos to generate chimeric animals (e.g., mice). Subsequent breeding can result in animals containing fully humanized immunoglobulin loci.

[0087] In some embodiments, a recombinase is introduced to invert part or all of the endogenous constant region of a mouse cell by first introducing a modified short exogenous recombinase binding site into the endogenous constant region of the mouse cell. The modified human variable region genes are then inserted downstream (after) the endogenous immunoglobulin loci of the mouse cell. The resulting modified cells are then screened and identified, and the cells are injected into early animal embryos to generate chimeric animals (e.g., mice). Subsequent breeding can result in animals containing fully humanized immunoglobulin loci.

[0088] Optionally, in some embodiments, a human heavy chain variable region gene can be introduced downstream of the heavy chain locus coding region of the host non-human animal, a human light chain variable region gene can be introduced downstream of the light chain locus coding region of the host non-human animal, or both can be introduced simultaneously.

[0089] The following examples are merely intended to explain the fusion protein, preparation method, and use of the present invention, and are not intended to limit the scope of the present invention, nor are they limited by any theory. [Example]

[0090] Example 1 The mouse model of the present invention is constructed by inserting a heavy chain gene containing all humanized V, D, and J regions downstream of a mouse immunoglobulin heavy chain-encoding locus and a humanized light chain gene downstream of a mouse immunoglobulin light chain-encoding locus. This insertion is achieved by gene targeting in ES cells using techniques well known in the art. A targeting vector containing human antibody variable region gene sequences is constructed in vitro through a recombination process, and the targeting vector is then inserted downstream of the immunoglobulin locus derived from the mouse to achieve humanized antibody variable region gene modification. Because the human immunoglobulin variable region gene is very large, we have adopted a series of measures to increase the success rate of our work to ensure that we can efficiently and accurately obtain mice containing all human immunoglobulin variable regions without failure. Some of the available options include:

[0091] In the present invention, by inserting a human-derived gene into the Junk sequence downstream of the mouse-derived immunoglobulin locus, the effect on mouse-derived gene expression during multiple gene targeting is avoided, as explained in Example 2 below.

[0092] At the same time, the engineered targeting vectors designed in this invention can be inserted downstream of mouse immunoglobulin loci to produce precisely engineered chimeric antibody molecules by modifying the mouse constant regions, thereby checking the functionality of the gene insertion at each step of the remodeling process, as described in Examples 8 and 9 below.

[0093] When targeting ES cells, not all targeting vectors are fully integrated in the actual procedure. We screened ES cells using high-density gene identification to obtain cells with fully integrated gene insertions. This identification can be performed using high-density PCR or Q-PCR. Although we finally confirmed fully integrated targeting vectors through multiple high-density PCR analyses, the probability of obtaining fully integrated ES cells during the actual procedure is around 10%. In Example 3, Table 6 shows the partial integration array PCR design for the first vector (vector 1) in the heavy chain targeting process.

[0094] Furthermore, due to the karyotypic instability of mouse ES cells in in vitro culture (Gaztelumendi N, Nogues C. Chromosome Instability in Mouse Embryonic Stem Cells [J]. Scientific Reports, Springer Science and Business Media LLC, 2014, 4(1).), only a small number of ES cells with the acquired complete gene insertion retained chromosomal integrity and germline transmission capacity even after long-term in vitro culture and continuous targeting manipulations. In operation, detailed karyotyping methods, such as photomicrographic karyotyping, specifically designed Q-PCR-based karyotyping (D´Hulst C, Parvanova I, Tomoiaga D, et al. Fast Quantitative Real-Time PCR-Based Screening for Common Chromosomal Aneuploidies in Mouse Embryonic Stem Cells[J]. Stem Cell Reports, Elsevier BV, 2013, 1(4):350-359), or polysomy analysis (Barrett MT, Scheffer A, Ben-Dor A, et al. Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA[J]. Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, 2004, 101(51):17765-17770). However, these methods often only partially express the capabilities of the acquired ES, and therefore cannot represent its germline transmission ability or its ability to produce human variable region chimeric antibodies.

[0095] In contrast, one practical approach of the present invention is to generate and verify intermediate animals at appropriate or regular intervals (e.g., every three targeted modifications). The resulting effect is that all partially human variable region-modified ES cells obtained from almost all stepwise insertions can be injected into mouse embryos to produce chimeric mice. All of the resulting chimeric mice can use the inserted partial human variable region genes to perform VDJ or VJ recombination and produce functional chimeric antibodies with human variable regions. This data serves as a checkpoint for ultimately creating non-human animals with fully human variable regions. The reliability and functionality of the obtained process ES cells can be determined from the aspects of animal production, antibody production, and variable region domain diversity, allowing for rapid prediction of success or failure and route correction in the creation of fully human variable regions. These mice with partial human variable regions can also be identified using antibodies, leading to the production of human variable region chimeric antibodies. See Examples 8 and 9 for data.

[0096] Example 2 BALB / c mouse ES cell lines will be independently isolated to serve as the basis for subsequent targeting.

[0097] Using ES targeting techniques well known in the field, vector element 1 (hereinafter referred to as Cpoint, used as an element to induce the insertion of human variable regions) was knocked into ES cells at downstream positions of the mouse IGH and IGK constant region loci (the region between the Igha gene and the Tmem121 gene, specifically IGH chr12:113, 190, 256; the region between the Igkc and Rpia genes, specifically IGK chr6:70, 706, 267; NCBI database GRCm39).

[0098] After obtaining the correct ES cell line, we further used ES targeting technology to knock in vector element 2 (hereinafter referred to as LD, used in conjunction with Cpoint) into the upstream position of the mouse IGH and IGK constant regions (the region between the IgH J4 exon and the Cμ locus, specifically IGH chr12:113, 391, 844; the region between the Igk J5 exon and the Igkc gene, specifically IGK chr6:70, 701, 630; NCBI database GRCm39).

[0099] The above-mentioned positive cell lines were transfected with a Cre expression plasmid to achieve inversion of the mouse-derived C region using the Cre / loxP recombination mechanism (Zheng, B et al., "Engineering a mouse balancer chromosome," Nature genetics vol. 22, 4 (1999): 375-8. doi: 10.1038 / 11949). The positive cell lines were retransfected with a transposase expression plasmid, and the screening marker fragment was deleted via the transposition mechanism (Transposase / Transposon system) (Maragathavally, KJ, et al., "Chimeric Mos1 and piggyBac transposases result in site-directed integration," The FASEB journal 20.11 (2006): 1880-1882; Wilson, Matthew H., Craig J. Coates, and Alfred L. George, "PiggyBac transposon-mediated gene transfer in human cells," Molecular therapy 15.1 (2007): 139-145.).

[0100] Using recombinase-mediated cassette exchange (RMCE) technology (Wallace, Helen AC et al., "Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence," Cell vol. 128, 1 (2007): 197-209. doi:10.1016 / j.cell.2006.11.044; Prosser, Haydn M et al., "Mosaic complementation demonstrates a regulatory role for myosin VIIa in actin dynamics of stereocilia," Molecular and cellular biology vol. 28, 5 (2008): 1702-12. doi:10.1128 / MCB.01282-07), we performed targeting of the vector batch by batch. Each time a vector was inserted, the screening marker fragment was deleted by a transposition mechanism until the last vector was inserted. Finally, we obtained ES cell lines with fully inserted humanized antibody variable region-encoding genes (VDJ).

[0101] ES cells, quality-controlled by PCR and karyotype Q-PCR array analysis, were injected into mouse blastocysts according to the method described in "Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition" (Cold Spring Harbor Laboratory Press, 2014), to obtain chimeric mice. These were then bred with BALB / c mice for genotyping and functional validation, ultimately yielding IGH and IGK gene-humanized mice. One alternative for obtaining functional validation data more quickly is to inject ES cells into mouse blastocysts that are incapable of functional VDJ rearrangement (e.g., Rag1 knockout mice), allowing direct immune system functional validation of the resulting chimeric mice without the need for proliferation.

[0102] The construction diagram and vector design are shown in Figures 1 to 4. Figure 1 shows the Igh constant region C point, LD targeting, and C region modification. Figure 2 shows a schematic diagram of the heavy chain human variable region insertion, illustrating IGH-vector targeting and resistance marker deletion. Figure 3 shows the Igk constant region C point, LD targeting, and C region modification. Figure 4 shows a schematic diagram of the light chain human variable region insertion, illustrating IGK-vector targeting and resistance marker deletion.

[0103] Example 3

[0104] 3.1 Construction of Cpoint targeting vector

[0105] The targeting vector consists of homologous arms (sequences on both sides of the insertion site) at both ends, a screening marker (abbreviated as SM)2, loxP, and a transposon PB fragment. As shown in Figure 1, these fragments are ligated with the commercial vector PMD18T by enzymatic ligation to construct the Cpoint targeting vector.

[0106] Using ES targeting techniques well known in the art, the Cpoint targeting vector was knocked into ES cells at a position downstream of the mouse-derived IGH constant region locus.

[0107] PCR identification confirmed that the IGH-Cpoint targeting of clone 10-24# was correct. The Cpoint identification method is shown in Table 1, and the PCR identification results are shown in Figure 5.

[0108] [Table 1]

[0109] 3.2 Construction of LD targeting vector

[0110] The targeting vector consists of homologous arms (sequences on both sides of the insertion site) at both ends, a screening marker (abbreviated as SM)1, loxP, and a transposon PB fragment. As shown in the figure, these fragments were ligated with the commercial vector PMD18T by enzymatic ligation to construct the LD targeting vector.

[0111] We targeted Cpoint to precise ES cells, and then used ES targeting technology to knock-in the LD targeting fragment into the upstream region of the mouse IGH constant region (the region between the IgH J4 exon and the Cμ locus, specifically IGH chr12:113, 391, 844; the region between the Igk J5 exon and the Igkc gene, specifically IGK chr6:70, 701, 630; NCBI database GRCm39).

[0112] PCR identification confirmed that the IGH-LD targeting of clones 108#, 111#, and 112# was correct. The LD identification method is shown in Table 2, and the PCR identification results are shown in Figure 6.

[0113] [Table 2]

[0114] 3.3 Construction of Cre expression vector

[0115] The Cre expression plasmid consists of a eukaryotic promoter, a Cre coding gene and a terminator, and these fragments were ligated with the commercial vector PMD18T by enzyme ligation to construct the Cre expression vector.

[0116] The C point and LD were precisely targeted into ES cells, transfected with a Cre expression vector, and the mouse C region was inverted using Cre / loxP recombination. PCR identification confirmed that the inversion of clone 14-29# was correct. The inversion identification method is shown in Table 3, and the identification results are shown in Figure 7.

[0117] [Table 3]

[0118] 3.4 Construction of transposase expression vector

[0119] The transposase expression plasmid consisted of a eukaryotic promoter, a transposase-encoding gene, and a terminator, and these fragments were ligated with the commercial vector PMD18T by enzymatic ligation to construct the transposase expression vector.

[0120] The positive cell lines were reversed and retransfected with a transposase expression plasmid, achieving excision of the screening marker fragment by a transposition mechanism.

[0121] PCR identification confirmed that the resistance markers in clones 3-6# and 8-10# had been correctly deleted. The method for identifying the deletion of the resistance markers is shown in Table 4, and the results are shown in Figure 8.

[0122] [Table 4]

[0123] 3.5 Construction of IGH vector1 targeting vector

[0124] As shown in Figure 2, the IGH vector1 targeting vector was constructed by using Red / ET recombination technology (Rivero-Muller, Adolfo et al., "Assisted large fragment insertion by Red / ET-recombination (ALFIRE) -- an alternative and enhanced method for large fragment recombineering," Nucleic Acids Research, Vol. 35, 10 (2007): e78. doi:10.1093 / nar / gkm250) to insert a transposon, promoter, loxP, and the screening marker Neo into the 5'-end of the vector, and loxP, the screening marker Puro, and a transposon element into the 3'-end of the vector.

[0125] Targeting of human IGH vector1 was performed by recombinase-mediated cassette exchange (RMCE) technology.

[0126] PCR identification confirmed that the targeting of IGH vector1 in clone 131# was correct. The targeting identification method is shown in Table 5, the targeting integrity identification method is shown in Table 6, the IGH vector1 targeting PCR identification results are shown in Figure 9, and the IGH vector1 targeting integrity PCR identification results are shown in Figure 10.

[0127] [Table 5]

[0128] [Table 6] TIFF2026508038000007.tif106170

[0129] IGH vector1 targeting-positive cells were retransfected with a transposase expression plasmid to achieve deletion of the screening marker fragment via transposition. PCR identification confirmed that the resistance marker deletion in clone 13-15# was correct. The method for identifying the resistance marker deletion is shown in Table 7, and the PCR identification results for IGH vector1 resistance marker deletion are shown in Figure 11.

[0130] [Table 7]

[0131] IGH vector1-FIAU was targeted to the correct clones for quality control by karyotype Q-PCR array analysis, and the results showed that they met the quality control criteria.

[0132] The ES cells, whose quality was controlled by PCR and karyotype Q-PCR array analysis, were injected into the blastocyst of mice according to the method in the "Manual for Experimental Manipulation of Mouse Embryos," to obtain F0 chimeric mice. These were then bred with BALB / c mice, and after genotyping and functional verification, humanized mice with a partially human IgH gene were finally obtained.

[0133] Example 4 Humanized mice can accurately use human variable region genes

[0134] The spleens of the humanized mice were collected and BCR sequencing was performed to measure the utilization of the human gene insertions in splenic B cells. The results of BCR sequencing are shown in Figures 12 and 13, demonstrating that the humanized mice can accurately utilize the human IGH and IGK variable region genes.

[0135] Example 5 Normal development of humanized mouse immune cells

[0136] The development of immune-related cells, particularly B cells, in the spleen and bone marrow of humanized mice was measured using flow cytometry. The results are shown in Figures 14 and 15, demonstrating that the proportions of lymphocytes and myeloid cells in the spleen were normal, and that B cell development in the spleen and bone marrow was normal.

[0137] Example 6 Constant region inversion reduces leaky expression of mouse-derived genes

[0138] When using humanized mice, the problem of leaky expression of mouse VDJ genes may arise. Previous techniques have focused primarily on inactivating mouse VDJ genes, which can be divided into two main categories: 1. Complete deletion of mouse VDJ genes, resulting in the thorough removal of mouse VDJ genes, but simultaneously deleting a large number of other endogenous mouse genes, disrupting endogenous genes and transcriptional regulatory elements; 2. Inversion of mouse VDJ genes, resulting in the insertion of endogenous mouse VDJ genes adjacent to human VDJ genes. In practice, there is a wide variety of VDJ gene orientations within the mouse Igk locus, including IGKV genes oriented in the same direction as the J and C genes of each IGK and IGKV genes, as well as in the opposite direction (Collins, Andrew M., and Corey T. Watson, “Immunoglobulin light chain gene rearrangements, receptor editing, and the development of a self-tolerant antibody repertoire,” Frontiers in Immunology 9 (2018): 2249). The inverted variable regions are still adjacent to the human variable regions, and both are still spatially upstream (5' end) of the endogenous constant regions. Therefore, based on the principles and direction of genome transcription and translation, there is still a risk of using mouse-derived variable regions during the V(D)J recombination process.

[0139] In this patent, human variable region genes are inserted downstream of mouse-derived immunoglobulin loci, with the endogenous variable region and the inserted human variable region separated by an endogenous constant region. Mice with endogenous variable regions inserted away from the human variable regions disclosed in this patent were compared with mice with endogenous variable regions inserted adjacent to the human variable regions of the prior art. Spleens from both mice were sampled and BCR sequenced to detect the number of variable region sequences with higher homology to the mouse. This confirmed the involvement of mouse-derived variable regions in VDJ rearrangement. As shown in Figure 16, leakage expression was frequently detected in the mouse-derived variable region VDJ rearrangement where the human and mouse variable regions are close to each other, but almost no leakage expression was detected in the mouse-derived variable region gene where the human and mouse variable regions are distant from each other. The effect of inserting human variable region genes downstream of the immunoglobulin genome in this patent is to completely change the relative positions of the human and mouse variable regions, thereby resolving the problem of leakage expression of mouse-derived variable regions involved in VDJ gene rearrangement.

[0140] Example 7 Humanized mice can produce high-affinity antibodies

[0141] Humanized mice were immunized with a commercialized hPD1 recombinant protein and serum antibody ELISA titers were measured. Results showed that the immune responses of humanized mice and BALB / c mice to the same PD1 antigen were similar. Using hybridoma fusion screening technology, a positive antibody, 10K4F3, was isolated. Surface plasmon resonance was used to measure antibody affinity (using the commercially available antibody drug BMK as a positive control). The efficacy of the antibody drug was evaluated in vivo in mice.

[0142] The results are shown in Table 8 and Figures 17 and 18, demonstrating that high-affinity antibodies can be produced using humanized mice, and the corresponding antibodies have relatively good in vivo anti-tumor efficacy data.

[0143] [Table 8]

[0144] Example 8 Generation of partially variable region gene-humanized mice

[0145] Using portions of the human IGH-V, D, and J genes and portions of the human IGK-V and J genes (specific gene names are listed in Figures 19 and 20), partial VDJ gene-humanized mice were obtained using the same method. The spleens of the positive mice were collected, and the utilization of the human gene insertions in splenic B cells was measured by BCR sequencing. The results of BCR sequencing are shown in Figures 19 and 20, demonstrating that even with the insertion of portions of the VDJ genes, the humanized mice were still able to utilize portions of their human variable region genes.

[0146] Example 9 Partially variable region gene-humanized mice can generate normal immune responses

[0147] A portion of VDJ humanized mice was immunized using commercialized OVA protein, and serum antibody ELISA titers were measured.

[0148] The results are shown in Figure 21, and it was found that there was no significant difference in the immune response to the OVA antigen between humanized mice and BALB / c mice.

Claims

1. 1. A method for producing a non-human animal, said method comprising operably linking a human immunoglobulin variable region gene downstream of an immunoglobulin locus of said non-human animal.

2. The method of claim 1, comprising operably linking a human immunoglobulin heavy chain variable region gene downstream of the heavy chain locus of the non-human animal.

3. The method of any one of claims 1 to 2, comprising operably linking genes of one or more human heavy chain variable region V regions, heavy chain variable region D regions, heavy chain variable region J regions, or fragments thereof, downstream of the heavy chain constant region locus of the non-human animal.

4. The method according to claim 3, wherein the genes of the multiple human heavy chain variable region V, heavy chain variable region D, or heavy chain variable region J regions or fragments thereof are directly linked.

5. 2. The method of claim 1, wherein the method comprises operably linking a human immunoglobulin light chain variable region gene downstream of a light chain locus of the non-human animal.

6. The method of claim 5, comprising operably linking one or more human light chain variable region V or J regions or fragments thereof downstream of the light chain constant region locus of the non-human animal.

7. The method of claim 6, wherein the genes of the multiple human light chain variable region V or light chain variable region J regions or fragments thereof are directly linked.

8. The method of any one of claims 1 to 7, wherein the non-human animal comprises endogenous immunoglobulin variable region genes.

9. The method of any one of claims 1 to 8, wherein the genomic integrity of the endogenous immunoglobulin variable regions of the non-human animal is not altered.

10. The method according to any one of claims 1 to 9, wherein the functions of expression regulatory elements of endogenous immunoglobulin variable region genes of the non-human animal are not disrupted.

11. The method according to any one of claims 1 to 10, wherein the non-human animal comprises complete endogenous immunoglobulin variable region expression regulatory elements.

12. The method of any one of claims 1 to 11, wherein the non-human animal comprises complete endogenous immunoglobulin variable region genes.

13. The method of any one of claims 1 to 12, wherein the non-human animal does not express an endogenous immunoglobulin variable region.

14. The method according to any one of claims 1 to 13, wherein the endogenous immunoglobulin variable region genes of the non-human animal do not express functional proteins.

15. The method of any one of claims 1 to 14, comprising reversing the transcriptional direction of the human immunoglobulin variable region gene relative to that of the endogenous immunoglobulin variable region gene.

16. The method of any one of claims 1 to 15, comprising reversing the transcription direction of the immunoglobulin constant region gene of the non-human animal relative to that of the endogenous immunoglobulin variable region gene.

17. The method according to any one of claims 1 to 16, wherein the transcription directions of the human immunoglobulin variable region gene and the immunoglobulin constant region gene of the non-human animal are the same.

18. The method according to any one of claims 1 to 17, wherein the non-human animal comprises a heavy chain constant region gene between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene.

19. The method according to any one of claims 1 to 18, wherein the distance between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene in the non-human animal is 169 Kbp to 240 Kbp.

20. The method according to any one of claims 1 to 19, wherein the non-human animal comprises a light chain constant region gene between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene.

21. The method according to any one of claims 1 to 20, wherein the distance between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene in the non-human animal is 4 Kbp to 42 Kbp.

22. The method of any one of claims 1 to 21, wherein the non-human animal is a rodent.

23. The method according to any one of claims 1 to 22, wherein the non-human animal is a mouse.

24. 24. The method of claim 23, comprising inserting a human immunoglobulin heavy chain variable region gene between the Tmem121 and Igha genes at the mouse locus.

25. The method of any one of claims 23 to 24, comprising inserting a human immunoglobulin heavy chain variable region gene into the mouse chromosome between positions chr12:113,149,523 and 113,223,857.

26. 26. The method of any one of claims 23 to 25, comprising inserting a human immunoglobulin heavy chain variable region gene into the mouse chromosome at location chr12:113,190,256.

27. The method of any one of claims 23 to 26, comprising inserting a human immunoglobulin light chain variable region gene between the Igkc gene and the Rpia gene at the mouse locus.

28. 28. The method of any one of claims 23 to 27, comprising inserting a human immunoglobulin light chain variable region gene into the mouse chromosome between positions chr6:70,703,738 to 70,742,704.

29. 29. The method of any one of claims 23 to 28, comprising inserting a human immunoglobulin light chain variable region gene into the mouse chromosome at location chr6:70,706,267.

30. The method of any one of claims 1 to 29, comprising operably linking a human immunoglobulin variable region gene downstream of the immunoglobulin locus of the non-human animal by site-specific recombination.

31. The method of any one of claims 1 to 30, comprising genomic modification to reverse the transcription direction of the immunoglobulin constant region genes of the non-human animal relative to the transcription direction of the endogenous immunoglobulin variable region genes.

32. A non-human animal, the genome of which comprises human immunoglobulin variable region genes operably linked downstream of an immunoglobulin locus.

33. The non-human animal of claim 32 , comprising a human immunoglobulin heavy chain variable region gene operably linked downstream of the heavy chain locus.

34. The non-human animal according to any one of claims 32 to 33, comprising one or more genes of human heavy chain variable region V region, heavy chain variable region D region, or heavy chain variable region J region, or fragments thereof, operably linked downstream of the heavy chain constant region locus.

35. The non-human animal according to any one of claims 32 to 34, wherein the genes of the multiple human heavy chain variable region V regions, heavy chain variable region D regions, or heavy chain variable region J regions, or fragments thereof, are directly linked.

36. The non-human animal of claim 32 , comprising a human immunoglobulin light chain variable region gene operably linked downstream of the light chain locus.

37. The method of claim 36, comprising operably linking one or more human light chain variable region V or light chain variable region J regions, or fragments thereof, downstream of the light chain constant region locus.

38. The non-human animal according to any one of claims 36 to 37, wherein the genes of the multiple human light chain variable region V or light chain variable region J regions, or fragments thereof, are directly linked.

39. The non-human animal of any one of claims 32 to 38, comprising an endogenous immunoglobulin variable region gene.

40. 40. The non-human animal of any one of claims 32 to 39, wherein the genomic integrity of the endogenous immunoglobulin variable regions is not altered.

41. The non-human animal according to any one of claims 32 to 40, wherein the function of an expression regulatory element of an endogenous immunoglobulin variable region gene is not disrupted.

42. The non-human animal according to any one of claims 32 to 41, comprising expression regulatory elements for a complete endogenous immunoglobulin variable region.

43. The non-human animal of any one of claims 32 to 42, comprising a complete endogenous immunoglobulin variable region gene.

44. The non-human animal according to any one of claims 32 to 43, which does not express an endogenous immunoglobulin variable region.

45. The non-human animal according to any one of claims 32 to 44, wherein the endogenous immunoglobulin variable region gene does not express a functional protein.

46. The non-human animal according to any one of claims 32 to 45, wherein the transcription directions of the human immunoglobulin variable region gene and the endogenous immunoglobulin variable region gene in the genome are opposite to each other.

47. The non-human animal according to any one of claims 32 to 46, wherein the transcription directions of the immunoglobulin constant region gene and the endogenous immunoglobulin variable region gene in the genome are opposite to each other.

48. The non-human animal according to any one of claims 32 to 47, wherein the transcription directions of the human immunoglobulin variable region gene and the immunoglobulin constant region gene of the non-human animal are the same.

49. The non-human animal according to any one of claims 32 to 48, comprising a heavy chain constant region gene between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene.

50. The non-human animal according to any one of claims 32 to 49, wherein the distance between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene is 169 Kbp to 240 Kbp.

51. The non-human animal according to any one of claims 32 to 50, comprising a light chain constant region gene between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene.

52. The non-human animal according to any one of claims 32 to 51, wherein the distance between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene is 4 Kbp to 42 Kbp.

53. The non-human animal according to any one of claims 32 to 52, wherein the non-human animal is a rodent.

54. The non-human animal according to any one of claims 32 to 53, wherein the non-human animal is a mouse.

55. 55. The non-human animal of claim 54, comprising a human immunoglobulin heavy chain variable region gene between the Tmem121 and Igha genes of the locus.

56. 56. The non-human animal of any one of claims 54 to 55, comprising a human immunoglobulin heavy chain variable region gene between chromosomal positions chr12:113,149,523 and 113,223,857.

57. 57. The non-human animal of any of claims 54 to 56, comprising a human immunoglobulin heavy chain variable region gene at chromosomal location chr12:113,190,256.

58. The non-human animal according to any one of claims 54 to 57, wherein a human immunoglobulin light chain variable region gene is inserted between the Igkc and Rpia genes of the gene locus.

59. The non-human animal of any one of claims 54 to 58, wherein a human immunoglobulin light chain variable region gene is inserted between chromosomal positions chr6:70,703,738 to 70,742,704.

60. The non-human animal of any one of claims 54 to 59, wherein a human immunoglobulin light chain variable region gene is inserted at chromosomal position chr6:70,706,267.

61. A non-human animal cell whose genome comprises a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus.

62. A method for producing an antibody that specifically binds to an antigen, comprising immunizing a non-human animal according to any one of claims 32 to 60 with the antigen.

63. A method for preparing a sample, comprising exposing the non-human animal according to any one of claims 32 to 60 to an antigen, and collecting the sample from the non-human animal.

64. 64. The method of claim 63, wherein the sample comprises immune cells.

65. 65. The method of claim 63 or 64, wherein the sample comprises B cells.

66. The method of any of claims 63 to 65, wherein the sample is derived from bone marrow, spleen tissue, lymph nodes, spleen cells or peripheral lymphocytes.

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