Animal model for producing humanized antibody, and construction method therefor

Abstract

The present application relates to a non-human animal and a method for preparing the non-human animal. The non-human animal is operably linked to a human immunoglobulin variable region gene downstream of an immunoglobulin locus. The present application further provides a method for producing an antibody by means of the non-human animal.

Description

Animal model for producing humanized antibodies and method for constructing the same Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a non-human animal model for producing antibodies and a method for constructing the same. Background Art

[0002] After more than three decades of development, therapeutic monoclonal antibody drugs have become one of the most important components of biopharmaceuticals and are currently the fastest-growing, most advanced and effective class of drugs. Since the first approval of OKT3 in 1986, the development of therapeutic antibodies has witnessed rapid and transformative progress, consolidating its position as an important aspect of modern biopharmaceuticals. Despite these successes, challenges remain, including the low success rate, high cost and long time of antibody-based drug development. Recently, the problem of anti-drug antibodies (ADA) caused by immunogenicity has also attracted serious attention, especially for therapeutic antibodies classified as large molecule drugs. Overcoming the risk of ADA formation in the preclinical stage remains a complex task.

[0003] Currently, the most commonly used method for the discovery and screening of therapeutic monoclonal antibodies is "display technology", including phage display, bacterial display, and yeast display. "Display technology" can quickly screen antibodies, but due to the lack of in vivo antibody variable region recombination and affinity hypermutation processes, the diversity and affinity of antibodies screened by these methods are limited, and often require a lot of cost and time for further optimization and modification. Another method commonly used for the discovery and screening of therapeutic monoclonal antibodies is to directly use wild-type animal models for immune discovery, such as poultry, rodents, and non-human primates. Humanization modification of the molecules obtained by the above methods based on experience and computer database predictions is a common method used to avoid the ADA effect caused by immunogenicity, but the final modification results are often unpredictable and divorced from the actual clinical effect, leading to side effects such as allergic stress during clinical treatment and loss of efficacy of anti-antibody neutralization.

[0004] One possible approach to circumventing the above issues and discovering fully human antibodies is to use non-human animals engineered to carry human antibody genes. While many currently available non-human animals are capable of producing fully human antibodies, these models often have major drawbacks or limitations due to the vastness and complexity of the human antibody repertoire. For example, they often contain only a small or partial portion of the human antibody repertoire. The absence of a substantial portion of the human antibody repertoire means a lack of diversity in the resulting antibody molecules and a reduced likelihood of obtaining high-affinity antibodies. This is accompanied by lower V(D)J recombination efficiency and suboptimal antibody production. Furthermore, there are concerns about immunogenicity caused by the infiltration of the animal's own endogenous antibody coding regions and the genetic defect phenotype caused by the deletion of large segments of the animal's own endogenous genes. These and other issues have led to a need for non-human animals that contain all human immunoglobulin variable region coding genes and are capable of producing humanized antibodies without altering the immune response. This would allow for the effective and efficient production of fully humanized antibodies with variable regions suitable for clinical use.

[0005] Summary of the Invention

[0006] The present application provides a non-human animal and a method for preparing the non-human animal, wherein the genome of the non-human animal comprises a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus. The non-human animal of the present application can avoid interference with the animal's own gene expression and regulation, avoid leakage of endogenous variable region genes, and has significant advantages in antibody production.

[0007] In one aspect, the present application provides a method for preparing a non-human animal, comprising operably linking a human immunoglobulin variable region gene downstream of an immunoglobulin locus of the non-human animal.

[0008] In certain embodiments, the method comprises operably connecting a human immunoglobulin heavy chain variable region gene downstream of the heavy chain locus of the non-human animal. In certain embodiments, the method comprises operably connecting one or more human heavy chain V, D or J regions or fragments thereof downstream of the heavy chain constant region locus of the non-human animal. In certain embodiments, the genes of the multiple human heavy chain V, D or J regions or fragments thereof can be recombined. In certain embodiments, the genes of the multiple human heavy chain V, D or J regions or fragments thereof are directly connected.

[0009] In certain embodiments, the method comprises operably connecting a human immunoglobulin light chain variable region gene downstream of the light chain locus of the non-human animal. In certain embodiments, the method comprises operably connecting a gene of one or more people's light chain V or J region or its fragment downstream of the light chain constant region locus of the non-human animal. In certain embodiments, the genes of the multiple people's light chain V or J region or its fragment can be recombined. In certain embodiments, the genes of the multiple people's light chain V or J region or its fragment are directly connected.

[0010] Therefore, on the other hand, the present application provides a method for preparing a non-human animal, which comprises operably linking a human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus of the non-human animal.

[0011] On the other hand, the present application provides a method for preparing a non-human animal, comprising operably linking a human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus of the non-human animal.

[0012] In one aspect, the present application provides a non-human animal or non-human animal cell, wherein the genome comprises a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus. The following features mentioned in the non-human animal are all included in the non-human animal cell.

[0013] In certain embodiments, the heavy chain locus of the non-human animal is operably connected to a human immunoglobulin heavy chain variable region gene downstream. In certain embodiments, the heavy chain constant region locus of the non-human animal is operably connected to one or more human heavy chain V, D or J regions or genes of its fragments downstream. In certain embodiments, the genes of the multiple human heavy chain V, D or J regions or its fragments can be recombined. In certain embodiments, the genes of the multiple human heavy chain V, D or J regions or its fragments are directly connected.

[0014] In certain embodiments, the light chain locus downstream of the non-human animal is operably connected to a human immunoglobulin light chain variable region gene. In certain embodiments, the light chain constant region locus downstream of the non-human animal is operably connected to a gene of one or more human light chain V or J region or its fragment. In certain embodiments, the genes of the multiple human light chain V or J region or its fragment can be recombined. In certain embodiments, the genes of the multiple human light chain V or J region or its fragment are directly connected.

[0015] Therefore, on the other hand, the present application provides a non-human animal or non-human animal cell, wherein the immunoglobulin heavy chain gene locus of the non-human animal or non-human animal cell is operably connected to a human immunoglobulin heavy chain variable region gene downstream.

[0016] On the other hand, the present application also provides offspring of the non-human animal. The offspring can be offspring produced by mating the non-human animal with the same genotype or other genotypes.

[0017] On the other hand, the present application also provides cells (eg, stem cells, embryonic stem cells, immune cells, B cells, T cells or hybridomas) or cell lines or primary cell cultures thereof derived from the non-human animal or its progeny.

[0018] On the other hand, the present application provides a non-human animal or non-human animal cell, wherein the immunoglobulin light chain gene locus of the non-human animal or non-human animal cell is operably connected to a human immunoglobulin light chain variable region gene downstream.

[0019] In certain embodiments, the non-human animal comprises endogenous immunoglobulin variable region genes. In certain embodiments, the integrity of the endogenous immunoglobulin variable region genes of the non-human animal is not destroyed. In certain embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin variable region genes of the non-human animal is not destroyed. In certain embodiments, the endogenous immunoglobulin variable region genes of the non-human animal comprise functional protein-coding genes and functional microRNA genes. In certain embodiments, the non-human animal comprises complete endogenous immunoglobulin variable region expression regulatory elements. In certain embodiments, the non-human animal comprises complete endogenous immunoglobulin variable region genes.

[0020] In certain embodiments, the non-human animal does not express endogenous immunoglobulin variable regions. In certain embodiments, the endogenous immunoglobulin variable region genes of the non-human animal do not express functional proteins.

[0021] In certain embodiments, the human immunoglobulin variable region genes of the non-human animal are transcribed in the opposite direction to the endogenous immunoglobulin variable region genes.

[0022] In certain embodiments, the immunoglobulin constant region genes of the non-human animal are transcribed in the opposite direction to the endogenous immunoglobulin variable region genes. 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.

[0023] In certain embodiments, the transcription direction of the human immunoglobulin variable region genes in the non-human animal is the same as that of the immunoglobulin constant region genes of the non-human animal.

[0024] In certain embodiments, a heavy chain constant region gene is located between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene in the non-human animal. In certain embodiments, one or more heavy chain constant region genes are located between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene in the non-human animal. In certain 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.

[0025] In certain embodiments, a light chain constant region gene is located between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene in the non-human animal. In certain embodiments, one or more light chain constant region genes are located between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene in the non-human animal. In certain 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.

[0026] In certain embodiments, the non-human animal is a mammal. In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse. In certain embodiments, the non-human animal is a voles.

[0027] In certain embodiments, the mouse has a human immunoglobulin heavy chain variable region gene inserted between the Tmem121 and Igha genes at the locus. In certain embodiments, the mouse has a human immunoglobulin heavy chain variable region gene inserted between chromosomal positions chr12: 113,149,523 to 113,223,857. In certain embodiments, the mouse has a human immunoglobulin heavy chain variable region gene inserted at chromosomal position chr12: 113,190,256.

[0028] In certain embodiments, the mouse has a human immunoglobulin light chain variable region gene inserted between the lgkc and Rpia genes at the locus. In certain embodiments, the mouse has a human immunoglobulin light chain variable region gene inserted between chromosomal positions chr6:70,703,738 to 70,742,704. In certain embodiments, the mouse has a human immunoglobulin light chain variable region gene inserted at chromosomal position chr6:70,706,267.

[0029] In certain embodiments, the method comprises operably connecting a human immunoglobulin variable region gene downstream of the immunoglobulin locus of the non-human animal by site-directed recombination. In certain embodiments, the site-directed recombination can be one or more times. In certain embodiments, the method comprises using multiple site-directed recombination to connect multiple human heavy chain V, D or J regions or their fragment genes. In certain embodiments, the method comprises using multiple site-directed recombination to connect multiple human light chain V or J regions or their fragment genes. In certain embodiments, the site-directed recombination comprises: homologous recombination, nucleic acid cutting enzyme-mediated recombination, and site-specific recombinase-mediated recombination. In certain 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 certain embodiments, a site-directed integration method mediated by a site-specific recombination system is used to insert the human immunoglobulin variable region gene downstream of the immunoglobulin locus of the non-human animal. In certain embodiments, the method comprises deleting the resistance marker after inserting the human immunoglobulin variable region gene.

[0030] In certain embodiments, the method comprises, prior to inserting the human variable region genes, causing the transcription direction of the immunoglobulin constant region genes of the non-human animal to be opposite to that of the endogenous immunoglobulin variable region genes by chromosomal recombination. In certain embodiments, the method comprises, prior to inserting the human variable region genes, reversing the transcription direction of the immunoglobulin constant region genes of the non-human animal by chromosomal recombination. In certain embodiments, the chromosomal recombination comprises inversion of genomic segments mediated by a site-specific recombination system. In certain embodiments, the method comprises, after the chromosomal recombination, deleting the resistance marker.

[0031] On the other hand, the application provides a kind of non-human animal cell, it comprises immunoglobulin locus downstream in genome and is operably connected to human immunoglobulin variable region gene.In some embodiments, described cell is embryonic stem (ES) cell.In some embodiments, described cell is germ cell.In some embodiments, described cell is fertilized egg cell.In some embodiments, described cell is embryonic cell.

[0032] In certain embodiments, the non-human animal cell can develop into a non-human animal.

[0033] In another aspect, the present application provides cells, tissues and organs derived from non-human animals.

[0034] In another aspect, the present application provides a method for preparing an antibody that specifically binds to an antigen, the method comprising immunizing the non-human animal described herein with the antigen.

[0035] In another aspect, the present application provides a method for preparing an antibody that specifically binds to an antigen, the method comprising: exposing the non-human animal or cell to the antigen, and generating a hybridoma from cells collected from the animal. The method may further comprise collecting the chimeric antibody produced by the hybridoma. The method may further comprise sequencing the variable region gene of the hybridoma.

[0036] On the other hand, the present application provides a method for preparing an antibody that specifically binds to an antigen, the method comprising: exposing the non-human animal or cell to the antigen, and sequencing the nucleic acid encoding the human heavy chain and light chain immunoglobulin variable regions in the animal or cell. The non-human animal or cell can express a chimeric antibody that specifically binds to the antigen. In one embodiment, the method may comprise operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region with the nucleic acid encoding the heavy chain immunoglobulin constant region, and expressing the antibody that specifically binds to the antigen. The constant region may be a human heavy chain constant region or a heavy chain constant region of a non-human animal. In one embodiment, the method may comprise operably linking the nucleic acid encoding the human light chain immunoglobulin variable region with the nucleic acid encoding the light chain immunoglobulin constant region, and expressing the antibody that specifically binds to the antigen. The constant region may be a human light chain constant region or a light chain constant region of a non-human animal.

[0037] In another aspect, the present application provides a method for preparing a sample, comprising: exposing a non-human animal described herein to an antigen; and collecting the sample from the non-human animal. In certain embodiments, the sample comprises immune cells, such as B cells. In certain embodiments, the sample comprises bone marrow, spleen tissue, lymph nodes, splenocytes, or peripheral lymphocytes.

[0038] The technical solution of this application includes one or more of the following advantages:

[0039] Non-human animal described in the application has advantage in avoiding interfering with animal's own gene expression and regulation.For example, people's variable region gene (for example, light chain variable region, heavy chain variable region, or light chain variable region and heavy chain variable region) is inserted in the endogenous immunoglobulin locus downstream (or back) of mouse cell, avoid inserting large fragment DNA sequence or deleting DNA sequence in non-human animal (for example, mouse) endogenous immunoglobulin locus.Due to there is very long " Junk sequence " in non-human animal (for example, mouse) endogenous immunoglobulin locus downstream, as reference according to the GRCm39 mouse full genome sequence of recent announcement, mouse immunoglobulin heavy chain gene downstream (rear) has the sequence of the non-functional gene report of up to 66kb base, mouse immunoglobulin kappa light chain gene downstream (back) has the sequence of the non-functional gene report of up to 39kb base.In these long " Junk sequences ", introducing exogenous large fragment gene can avoid interfering with expression and the regulation of non-human animal (as mouse) endogenous gene from genome level.

[0040] The non-human animals described in this application also have advantages in avoiding interference with the gene expression and regulation of the animals themselves. For example, modification or deletion of the variable regions of the immunoglobulin genes of non-human animals (e.g., mice) is avoided. Since there is no shortage of functional protein-coding genes and functional microRNA genes within the endogenous immunoglobulin variable region loci of non-human animals (e.g., mice), deletion or modification of these genes may cause unpredictable phenotypes in the animals themselves. For example, the mouse immunoglobulin heavy chain locus contains the functional protein-coding gene Adam6a, and modification or deletion of the Adam6a gene can lead to male infertility in mice.

[0041] The non-human animals described herein have advantages in antibody production. In the non-human animals described herein, both the host non-human animal immunoglobulin variable region genes and constant region genes are retained, i.e., both the non-human animal's endogenous immunoglobulin variable region genes and constant region genes are retained. All host immunoglobulin regulatory sequences, including promoters, enhancers, switch regions, and other potential expression regulatory sequences, are retained. This can better ensure immunoglobulin gene recombination and expression, B cell development, affinity maturation, and the like in the non-human animals.

[0042] The non-human animals described herein have advantages in antibody production. For example, in some embodiments, the animal's endogenous immunoglobulin constant region is fully or partially modified (e.g., the constant region is fully or partially inverted) so that it is transcribed in the opposite direction to the endogenous variable region gene. This prevents V(D)J recombination between the endogenous variable region and the endogenous constant region to produce antibodies.

[0043] The non-human animals described herein also have advantages in antibody production. For example, in some embodiments, the inserted exogenous human immunoglobulin variable regions are located distally from the endogenous variable regions and transcribed in the opposite direction. This prevents the endogenous variable regions from being modified and recombined by DNA recombinases and integrated into human variable regions during VDJ or VJ recombination, thereby significantly preventing the production of antibodies with immunogenic epitopes in humans that are derived from the endogenous variable region repertoire.

[0044] The non-human animals described herein also have advantages in producing antibodies. For example, in some embodiments, all fragments of human variable region genes are gradually introduced into the downstream (behind) endogenous immunoglobulin loci of non-human animal (e.g., mouse) cells in batches. The variable domains produced by the resulting non-human animals can have a diversity that is almost the same as the diversity of variable domains in humans. This also means that the resulting non-human animals have an antibody diversity that is as close as possible to that of the human body itself and the possibility of obtaining high-affinity specific antibodies.

[0045] The method for preparing non-human animals described in the present application also has advantages. For example, in some embodiments, the endogenous immunoglobulin constant region of a non-human animal (e.g., a mouse) is modified in whole or in part so that it is transcribed in the opposite direction to the endogenous variable region gene. The modified human variable region gene is then inserted downstream (behind) the endogenous immunoglobulin locus of the non-human animal (e.g., a mouse) cell. The human variable region gene can be segmented from back to front and inserted in the opposite direction of transcription to the animal immunoglobulin locus. The effect obtained is that almost every segment of the modified cell with the obtained partial human variable region can be injected into an early animal embryo to prepare a chimeric animal (e.g., a mouse), or a non-human animal obtained through breeding. The obtained non-human animals can all use the inserted partial human variable region gene to perform VDJ or VJ recombination and produce chimeric antibodies with functional human variable regions. These non-human animals carrying partial human variable regions can serve as checkpoints for the production of non-human animals with complete human variable regions. The reliability and functionality of human variable region segment insertions can be assessed based on antibody production and variable region structural diversity, allowing for timely prediction of success and correction of routes for the production of non-human animals with complete human variable regions. These non-human animals carrying partial human variable regions can also be used for antibody discovery to produce chimeric antibodies containing human variable regions.

[0046] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0048] Figure 1 shows the heavy chain Igh constant region Cpoint and LD targeting as well as C region modification.

[0049] Figure 2 shows a schematic diagram of the heavy chain human variable region insertion, including insertion targeting and resistance marker deletion.

[0050] Figure 3 shows the Cpoint and LD targeting of the light chain Igk constant region and the C region modification.

[0051] Figure 4 shows a schematic diagram of the insertion of the light chain human variable region, including insertion targeting and resistance marker deletion.

[0052] Figure 5 shows the Cpoint targeted PCR identification results.

[0053] FIG6 shows the results of LD targeting PCR identification.

[0054] FIG7 shows the results of inversion PCR identification.

[0055] FIG8 shows the results of PCR identification of resistance marker deletion.

[0056] Figure 9 shows the results of IGH vector1 targeted PCR identification.

[0057] Figure 10 shows the PCR verification of IGH vector1 targeting integrity.

[0058] FIG11 shows PCR identification of the IGH vector1 resistance marker deletion.

[0059] FIG12 shows the utilization test of the IGH inserted gene.

[0060] FIG13 shows the utilization test of the IGK insertion gene.

[0061] FIG14 shows that the ratio of lymphocytes to myeloid cells in the spleen of humanized mice is normal.

[0062] Figure 15 shows normal B cell development in the spleen and bone marrow.

[0063] Figure 16 shows the leakage expression of mouse genes after inversion of different mouse regions.

[0064] FIG17 shows the ELISA titer detection of humanized mice and control mice.

[0065] Figure 18 shows the comparison of the efficacy of the screened 10K4F3 antibody and marketed drugs in mice

[0066] Figure 19 shows the utilization test of some IGH-V, D, and J inserted genes.

[0067] FIG20 shows the utilization test of some IGK-V and J inserted genes.

[0068] Figure 21 shows the ELISA titer detection of VDJ partially humanized mice and control mice (S0, before immunization; S1, second immunization; S2, third immunization) DETAILED DESCRIPTION

[0069] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0070] Definition of terms

[0071] In this application, the term "non-human animal" generally refers to all non-human vertebrates, such as mammals and non-mammals, such as non-human primates, rodents, rabbits, camels, sheep, dogs, cats, horses, cows, birds, amphibians, reptiles, etc. For example, the non-human animal can be a rat or a mouse.

[0072] In this application, the term "constant region" generally refers to the sum of the domains of an antibody excluding the variable region. The constant region is not directly involved in the binding of antigens, but exhibits different effector functions. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies are divided into the following categories: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into categories such as IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different types of antibodies are referred to as α, δ, ε, γ and μ, respectively. The light chain constant regions that can be found in all five antibody classes are referred to as κ (kappa) and λ (lambda). Genes encoding mouse (Mus musculus) constant regions can include IGHA, IGHD, IGHE, IGHG1, IGHG2A, IGHG2B, IGHG2C, IGHG3 or IGHM. Information about mouse constant region loci can be found in the IMGT Repertoire:

[0073] https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=Mus_musculus&group=IGHC

[0074] In this application, the term "locus" generally refers to a specific location along a chromosome or DNA sequence. Depending on the context, a locus can be a gene, a marker, a chromosome band, or a specific sequence of one or more nucleotides. In this application, when referring to an immunoglobulin locus, a genetic element or a group of related genetic elements that contain information that a cell can use to express immunoglobulin peptides. With regard to a locus that is not rearranged, the genetic element can be assembled by B cell precursors to form genes encoding immunoglobulin peptides. With regard to a rearranged locus, the gene encoding immunoglobulin peptides is contained in the locus.

[0075] In this application, the term "antibody" generally refers to a scaffold or backbone portion comprising a complete antibody or an antigen-binding fragment thereof, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes antibody binding to the antigen. Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, as long as they exhibit the desired antigen-binding activity. The term also includes genetically engineered antibodies such as chimeric antibodies (e.g., humanized mouse antibodies), humanized antibodies, fully human antibodies, and heterologous covalent antibodies (e.g., bispecific antibodies).

[0076] In this application, the term "chimeric antibody" generally refers to an antibody comprising sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody comprising a variable domain sequence (e.g., all or part of a light chain variable domain and / or a heavy chain variable domain sequence) of a human antibody and a constant domain of a non-human antibody (e.g., a mouse antibody).

[0077] In this application, "immunoglobulin" generally refers to a protein consisting of one or more polypeptides that are substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The NH2-terminus (about 110 amino acids) of the full-length immunoglobulin "light chain" (about 25KD and 214 amino acids) is encoded by the variable region gene, and the COOH-terminus is encoded by the kappa or lambda constant region gene. The full-length immunoglobulin "heavy chain" (about 50KD and 446 amino acids) is similarly encoded by the variable region gene (about 116 amino acids) and one of the other constant region genes mentioned above, such as gamma (encoding about 330 amino acids). The term "immunoglobulin" includes immunoglobulins with CDRs from humans or non-human sources. The immunoglobulin framework can be human, humanized, or non-human, such as a murine framework modified to reduce antigenicity in humans, or a synthetic framework such as a consensus sequence.

[0078] In this application, the term "variable region" generally refers to the region of an antibody molecule that binds to a specific antigen. It is composed of the antigen binding sites of the heavy chain and the light chain. The variable region is different between different B cell immunoglobulins, but is identical between all immunoglobulins produced by the same B cell. The diversity of the variable region is produced by the genetic recombination process that occurs during the maturation of the B cell through the variable region genes. This process is a rearrangement process that produces a large amount of diversity that can bind to any given antigen, thereby enabling the immune system to recognize and neutralize a large amount of antigenic burden caused by foreign and pathogenic structures. Therefore, the antibody repertoire is composed of abundant immunoglobulins with different V regions, but these immunoglobulins have the same Fc part.

[0079] In this application, the term "endogenous" generally refers to any substance originating from or produced within an organism, cell, tissue, or system. For example, an endogenous gene is a gene naturally present in a non-human animal organism, as distinguished from an exogenous gene introduced by transgenesis from another animal.

[0080] In this application, the terms "upstream" and "downstream" are generally used to describe the relative positions between genes in a chromosome, and can be based on the chromosome or a given gene. In order to better summarize the common points of modification of the heavy chain and light chain loci in this application, the position of the natural non-human animal host immunoglobulin locus and its immunoglobulin transcription direction (i.e., publicly displayed in the NCBI database) are used as a reference. For example, for gene A, if the position of gene A is behind 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 upstream of A.

[0081] In this application, the term "transcription direction" generally refers to the gene coding direction in which the gene ultimately forms a functional protein, that is, the direction from the sequence encoding the start codon (such as ATG) to the sequence encoding the stop codon (such as TAG, TAA, TGA).

[0082] Detailed Description of the Invention

[0083] This application relates to genetically modified non-human animals and cells containing functional human immunoglobulin variable region genes capable of producing chimeric antibodies with human variable regions. Research conducted in mice is provided herein for illustrative purposes only. Unless otherwise specified, references to mice include all non-human mammals, with mice being the preferred non-human mammal.

[0084] In one aspect, the present application relates to non-human animals or cells comprising one or more human IGHV regions, one or more human IGHD regions, and / or one or more human IGHJ regions downstream of the coding region of the host non-human mammal's heavy chain locus. In some embodiments, after insertion, the transcription direction of the coding regions of the human IGHV, human IGHD, and human IGHJ regions is opposite to the transcription direction of the coding region of the host's endogenous heavy chain VDJ progene. The human IGHV, human IGHD, and human IGHJ regions are operably linked together and capable of undergoing VDJ recombination.

[0085] In some embodiments, all or part of the constant region of the animal's endogenous heavy chain locus is modified so that all or part of it is transcribed in the opposite direction to the endogenous heavy chain variable region gene.

[0086] On the other hand, the non-human animals or cells involved in the present application may 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 host non-human animal light chain locus coding region. In some embodiments, the transcription direction of the coding region after insertion of the human light chain (e.g., IGK) V region and the human light chain (e.g., IGK) J region is opposite to the transcription direction of the coding region of the corresponding endogenous light chain VJ progene of the host. The human IGKV region and the human IGKJ region are operably linked together and can undergo VJ recombination.

[0087] In some embodiments, the constant region of the animal's endogenous light chain locus is modified in whole or in part so that its transcription direction is wholly or partially opposite to the transcription direction of the corresponding endogenous light chain variable region gene.

[0088] On the one hand, the human immunoglobulin heavy chain VDJ region gene located downstream of the coding region of the host non-human animal heavy chain locus comprises all the V regions, D regions and J regions of the human heavy chain and part or all of the intervening sequences arranged in reverse in a germline manner.

[0089] On the other hand, the human immunoglobulin heavy chain VDJ region gene located downstream of the coding region of the host non-human animal heavy chain locus is arranged in reverse and can be operably linked to the modified host constant region, unhindered VDJ recombination and expression of functional human variable region chimeric antibodies.

[0090] On the other hand, the reverse-arranged human immunoglobulin genes located downstream of the heavy chain locus coding region of the host non-human animal can be expressed in combination with the constant regions of different antibody subtypes of the host, allowing for unimpeded immunoglobulin class switching (isotype switching) of B cells.

[0091] On the one hand, the human immunoglobulin light chain VJ region gene located downstream of the host non-human animal light chain locus coding region comprises all the V regions and J regions of the light chain from humans and part or all of the intervening sequences arranged in reverse in a germline manner.

[0092] On the other hand, the human immunoglobulin light chain VJ region gene located downstream of the light chain locus coding region of the host non-human animal is arranged in reverse and can be operably linked to the modified host constant region, so as to carry out VJ recombination without hindrance and express functional human variable region chimeric antibodies.

[0093] On the one hand, the inserted human immunoglobulin variable region gene DNA contains 10%-100% of the reverse-encoding human heavy chain variable (V) gene coding region sequence, such as more than 60%, more than 70%, more than 80%, more than 90% and all human heavy chain variable region gene coding regions.

[0094] On the one hand, the inserted human immunoglobulin variable region gene DNA contains 50%-100% of the reverse-encoding human heavy chain D region gene coding region, such as more than 60%, more than 70%, more than 80%, more than 90% and all human D region gene coding regions.

[0095] On the one hand, the inserted human immunoglobulin variable region gene DNA contains 50%-100% of the reverse-encoding human heavy chain J region gene coding region, such as more than 60%, more than 70%, more than 80%, more than 90% and all human J region gene coding regions.

[0096] In one aspect, 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.

[0097] On the one hand, the inserted human immunoglobulin variable region gene DNA contains 15%-100% of the reverse-encoding human light chain V region gene coding region, such as more than 60%, more than 70%, more than 80%, more than 90% and all human V region gene coding regions.

[0098] On the one hand, the inserted human immunoglobulin variable region gene DNA contains 50%-100% of the reverse-encoding human light chain J region gene coding region, such as more than 60%, more than 70%, more than 80%, more than 90% and all of the human light chain J region gene coding region.

[0099] In one aspect, 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.

[0100] Information on human heavy chain V, D, and J regions, and light chain V and J regions can be found in the IMGT Repertoire: https: / / www.imgt.org / IMGTrepertoire / LocusGenes / .

[0101] Optionally, the non-human animal may contain only the human heavy chain variable region gene located downstream of the coding region of the host non-human animal heavy chain locus, or may contain only the human light chain variable region gene located downstream of the coding region of the host non-human animal light chain locus, or may contain both of the aforementioned.

[0102] Optionally, the non-human animal endogenous immunoglobulin heavy chain variable region (V region, D region, and J region) gene is not modified or deleted in any way to prevent the expression or regulation of the host part of the gene in the variable region 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, and the endogenous immunoglobulin heavy chain constant region coding sequence is separated therebetween. The base length between the two is between 169Kbp and 240Kbp, and the preferred distance is 202Kbp. This prevents the endogenous variable region from infiltrating into the final chimeric immunoglobulin coding gene recombination to a certain extent.

[0103] Optionally, the non-human animal's endogenous immunoglobulin light chain variable region (V region and J region) gene is not modified or deleted in any way to prevent the expression or regulation of the host's partial genes within the variable region from being affected. At the same time, the endogenous immunoglobulin light chain variable region is spaced apart from the inserted human immunoglobulin light chain variable region, with the endogenous immunoglobulin light chain constant region coding sequence separating the two. The base distance between the two is between 4Kbp and 42Kbp, preferably 5Kbp. This prevents the endogenous variable region from infiltrating the final chimeric immunoglobulin coding gene recombination to a certain extent.

[0104] The present application discloses a method for constructing functional human immunoglobulin variable region genes in non-human animals (e.g., mice). In this application, the research work carried out in mice is only illustrative, and unless otherwise specified, reference to mice also includes all non-human animals, with mice being the preferred non-human animal.

[0105] On the one hand, the insertion of human immunoglobulin heavy chain variable region gene DNA is targeted between the gene Tmem121 and the Igha gene downstream of the IgH locus on mouse chromosome 12 by site-directed recombination; in one aspect, the insertion is between coordinates 113,149,523 and 113,223,857 on mouse chromosome 12, suitably at coordinate 113,190,256.

[0106] In one aspect, human immunoglobulin light chain variable region gene DNA, such as human light chain kappa VJ, is inserted into the mouse chromosome 6 downstream of the IgK locus between the lgkc and Rpia genes by site-directed recombination. In another aspect, insertion is performed between coordinates 70,703,738 and 70,742,704 on mouse chromosome 6, preferably at position 70,706,267, or at an equivalent position on the mouse lambda locus on chromosome 16. All coordinates are referenced to the NCBI database GRCm39.

[0107] In some embodiments, the site-directed recombination method includes homologous recombination, nucleic acid cleavage enzyme-mediated recombination (such as CRISPR / Cas9, etc.), and site-specific recombinase-mediated recombination, and involves the combined use of one or more of these methods.

[0108] In some embodiments, the endogenous heavy chain locus constant region is modified in whole or in part so that its transcription direction is wholly or partially opposite to the transcription direction of the corresponding endogenous light chain variable region gene. The modification is performed by inserting exogenous recombination sites inside or at both ends of the constant region of non-human animal cells or animals, and then by means such as intracellular delivery of recombinases or animal breeding, so that the host constant region gene sequence is completely or partially reversed. The insertion can be obtained by commonly used gene editing targeting means, such as homologous recombination or nucleic acid cutting enzyme-mediated recombination (such as CRISPR / Cas9, etc.). The modification can also be achieved by nucleases, and related technologies are known in the prior art.

[0109] In some embodiments, the endogenous heavy chain locus constant region is modified in whole or in part so that its transcription direction is completely or partially opposite to the transcription direction of the corresponding endogenous light chain variable region gene. The modification is achieved by using a non-human animal constant region to reversely insert or replace part or all of the host's constant region gene sequence. The insertion or replacement can be obtained by commonly used gene editing targeting methods, such as homologous recombination or nuclease-mediated recombination (such as CRISPR / Cas9, etc.).

[0110] In some embodiments, by first inserting the modified human variable region gene into the downstream (backward) of the endogenous immunoglobulin locus of the mouse cell, and then by introducing a modified short exogenous recombinase binding site in the endogenous constant region of the mouse cell, the recombinase is introduced to partially or completely invert the endogenous constant region of the mouse cell. After screening and identification to obtain the modified cells, the cells are injected into early embryos to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing complete humanized immunoglobulin loci.

[0111] In some embodiments, a modified short exogenous recombinase binding site can be first introduced into the endogenous constant region of the mouse cell, and the recombinase is introduced to partially or completely invert the endogenous constant region of the mouse cell. The modified human variable region gene is then inserted downstream (behind) of the endogenous immunoglobulin locus of the mouse cell. After screening and identification to obtain the modified cells, the cells are injected into early animal embryos to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing complete humanized immunoglobulin loci.

[0112] Optionally, in some embodiments, the human heavy chain variable region gene can be introduced only downstream of the coding region of the heavy chain locus of the host non-human animal, or the human light chain variable region gene can be introduced only downstream of the coding region of the light chain locus of the host non-human animal, or both of the aforementioned can be introduced at the same time.

[0113] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0114] Example

[0115] Example 1

[0116] The mouse model of the present invention can be established by inserting a heavy chain gene containing all human V, D and J regions into the downstream of the mouse immunoglobulin heavy chain encoding locus, and inserting a human light chain gene into the downstream of the 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 a human antibody variable region gene sequence is constructed in vitro by recombinant engineering, and the targeting vector is inserted downstream of the above-mentioned mouse immunoglobulin locus to achieve the transformation of the human antibody variable region gene. Since the human immunoglobulin variable region gene is very large, in order to ensure that mice containing all human immunoglobulin variable regions can be successfully, effectively and correctly obtained, we have adopted a series of schemes to increase the probability of success of the work. Some optional schemes include the following.

[0117] The present invention inserts the human gene into the Junk sequence downstream of the mouse immunoglobulin locus to avoid affecting the expression of the mouse gene during multiple gene targetings, as described in Example 2 below.

[0118] At the same time, the operable insertion of the targeting vector designed by the present invention downstream of the mouse immunoglobulin locus can produce correctly recombined chimeric antibody molecules by modifying the mouse constant region, thereby checking the function of the inserted gene at each step in the transformation process, as described in Examples 8 and 9 below.

[0119] When targeting ES cells, not all targeting vectors can be fully integrated in practice. We screen ES cells by high-density gene identification to obtain those cells with complete gene insertion. This identification can be performed by high-density PCR or Q-PCR. The complete integration of the targeting vector is finally confirmed by multiple high-density PCR analysis. The probability of obtaining fully integrated ES cells during the actual operation is about 10%. In Example 3, Table 6 represents the partial integrity array PCR design of the first vector (vector1) in the heavy chain targeting process.

[0120] In addition, due to the inherent karyotype instability of mouse ES cells in in vitro culture (Gaztelumendi N, Nogués C. Chromosome Instability in mouse Embryonic Stem Cells[J]. Scientific Reports, Springer Science and Business Media LLC, 2014, 4(1).), only a few of the ES cells with complete gene insertion retained chromosome integrity and germline transmission capacity after prolonged in vitro culture and continuous targeting operations. During the operation, the screened ES cells can be identified using a detailed karyotype analysis scheme, such as karyotype examination based on general microscopy (photomicrographic), karyotype detection based on specially designed Q-PCR (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 based on multiple chromosomes (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 schemes often only reflect part of the capabilities of the obtained ES and cannot represent its germline transmission ability and the production capacity of human variable region chimeric antibodies.

[0121] In this regard, in one feasible operational scheme of the present invention, a timely or scheduled (e.g., three times per cell targeting modification) method is used to produce intermediate animals and verify the results. The effect achieved is that almost every segment of modified ES cells inserted with the obtained partial human variable region can be injected into mouse embryos to produce chimeric mice. The obtained chimeric mice can use the inserted partial human variable region gene to undergo VDJ or VJ recombination and produce chimeric antibodies with functional human variable regions. This data serves as a checkpoint for the production of non-human animals with complete human variable regions. The reliability and functionality of the obtained process ES cells are judged from the aspects of animal production, antibody production, and variable region domain diversity, and the success or failure of the production route with complete human variable regions can be predicted and the route corrected in a timely manner. These mice carrying partial human variable regions can also be used for antibody discovery to produce chimeric antibodies with human variable regions. Refer to the data in Examples 8 and 9.

[0122] Example 2

[0123] The ES cell line of BALB / c mice was obtained through independent isolation and used as the basic material for subsequent targeting.

[0124] Using ES targeting technology well known in the art, vector element 1 (hereinafter referred to as Cpoint, an element for introducing human variable region insertion) was knocked into the 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) in ES cells.

[0125] After obtaining the correct ES cell line, the vector element 2 (hereinafter referred to as LD, used for collaboration with Cpoint) was knocked 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) using ES targeting technology.

[0126] The above positive cell lines were transfected with Cre expression plasmids, and the mouse C region was inverted by 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 above-mentioned positive cell line is then transfected with a transposase expression plasmid, and the deletion of the selection marker fragment is achieved through 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.).

[0127] 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), vector targeting was performed in batches. After each vector insertion, the selection marker fragment was deleted through the transposition mechanism until the last vector was inserted, ultimately obtaining an ES cell line with complete insertion of the human antibody variable region encoding gene (VDJ).

[0128] ES cells, quality-controlled by PCR and karyotype Q-PCR array analysis, were injected into mouse blastocysts according to the methods described in "Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition (Cold Spring Harbor Laboratory Press, 2014)" to generate chimeric mice. These chimeric mice were then bred with BALB / c mice for genotyping and functional validation to obtain humanized mice harboring IGH and IGK genes. To obtain functional validation data more quickly, an alternative approach is to inject ES cells into mouse blastocysts that are incapable of functional VDJ rearrangement (e.g., Rag1 gene-deficient mice). The resulting chimeric mice can then be directly tested for immune system function without breeding.

[0129] The preparation diagrams and vector designs are shown in Figures 1 to 4, wherein Figure 1 shows the Cpoint and LD targeting of the Igh constant region and the modification of the C region; Figure 2 shows a schematic diagram of the insertion of the heavy chain human variable region, showing IGH-vector targeting and deletion of the resistance marker; Figure 3 shows the Cpoint and LD targeting of the Igk constant region and the modification of the C region, and Figure 4 shows a schematic diagram of the insertion of the light chain human variable region, showing IGK-vector targeting and deletion of the resistance marker.

[0130] Example 3

[0131] 3.1 Construction of Cpoint targeting vector

[0132] The targeting vector consists of homology arms at both ends (sequences on both sides of the insertion site), a selection marker (SM) 2, loxP, and a transposon PB fragment. As shown in Figure 1, these fragments were linked to the commercial vector PMD18T by enzyme ligation to construct the Cpoint targeting vector.

[0133] The ES targeting technology well known in the art was used to knock the Cpoint targeting vector into the downstream position of the mouse IGH constant region locus in ES cells.

[0134] PCR identification confirmed that the IGH-Cpoint of clone 10-24 was correctly targeted. The Cpoint identification protocol is shown in Table 1, and the PCR identification results are shown in Figure 5.

[0135] Table 1 Cpoint identification scheme

[0136] 3.2 Construction of LD targeting vector

[0137] The targeting vector consists of homology arms at both ends (sequences on both sides of the insertion site), a selection marker (SM) 1, loxP, and a transposon PB fragment. As shown in the figure, these fragments are connected to the commercial vector PMD18T by enzyme ligation to construct the LD targeting vector.

[0138] The ES cells with correct Cpoint targeting were then injected with the LD targeting fragments using ES targeting technology into the upstream positions 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).

[0139] PCR identification confirmed that the IGH-LD of clones 108#, 111#, and 112# were correctly targeted. The LD identification scheme is shown in Table 2, and the PCR identification results are shown in Figure 6.

[0140] Table 2 LD identification scheme

[0141] 3.3 Construction of Cre expression vector

[0142] The Cre expression plasmid consists of a eukaryotic promoter, a Cre encoding gene, and a terminator. These fragments are connected to the commercial vector PMD18T by enzyme ligation to construct a Cre expression vector.

[0143] ES cells with correctly targeted Cpoint and LD were transfected with a Cre expression vector, achieving inversion of the murine C region via Cre / loxP recombination. PCR confirmed the correct inversion in clone 14-29. The inversion identification protocol is shown in Table 3, and the results are shown in Figure 7.

[0144] Table 3 Inversion identification scheme

[0145] 3.4 Construction of transposase expression vector

[0146] The transposase expression plasmid consists of a eukaryotic promoter, a transposase encoding gene, and a terminator. These fragments are connected to the commercial vector PMD18T by enzyme ligation to construct a transposase expression vector.

[0147] The inversion-positive cell line is then transfected with a transposase expression plasmid to achieve deletion of the selection marker fragment through the transposition mechanism.

[0148] PCR identification confirmed that the resistance marker deletion of clones 3-6# and 8-10# was correct. The resistance marker deletion identification scheme is shown in Table 4, and the identification results are shown in Figure 8.

[0149] Table 4 Resistance marker deletion identification scheme

[0150] 3.5 Construction of IGH vector1 targeting vector

[0151] As shown in Figure 2, the IGH vector1 targeting vector was constructed by inserting a transposon, a promoter, loxP, and a selection marker Neo at the 5' end of the vector, and loxP, a selection marker Puro, and a transposon element at the 3' end of the vector using Red / ET recombination technology (Rivero-Müller, 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).

[0152] Targeting of human IGH vector1 was performed by recombinase-mediated cassette exchange (RMCE) technology.

[0153] PCR identification confirmed that IGH vector 1 in clone 131 was correctly targeted. The targeting identification scheme is shown in Table 5, and the targeting integrity identification scheme is shown in Table 6. The PCR identification results for IGH vector 1 targeting are shown in Figure 9, and the PCR identification results for IGH vector 1 targeting integrity are shown in Figure 10.

[0154] Table 5 IGH vector1 targeting identification scheme

[0155] Table 6 IGH vector1 targeting integrity assessment scheme

[0156] IGH vector1-targeted cells were then transfected with a transposase-expressing plasmid to delete the selection marker fragment through transposition. PCR analysis confirmed that the resistance marker deletion in clones 13-15 was correct. The resistance marker deletion identification protocol is shown in Table 7, and the PCR results for IGH vector1 resistance marker deletion are shown in Figure 11.

[0157] Table 7 Resistance marker deletion identification scheme

[0158] The clones with correct IGH vector1-FIAU targeting were subjected to karyotype Q-PCR array analysis for quality control. The results showed that they met the quality control standards.

[0159] ES cells that had passed PCR and karyotype Q-PCR array analysis were injected into the mouse blastocyst cavity according to the method in the Mouse Embryo Operation Experiment Manual to obtain F0 chimeric mice. They were bred with BALB / c mice, and after genotypic identification and functional verification, humanized mice with partial human IGH genes were finally obtained.

[0160] Example 4

[0161] Humanized mice can correctly use human variable region genes

[0162] Spleens from humanized mice were collected and BCR sequencing was performed to examine the utilization of the inserted human genes in splenic B cells. The BCR sequencing results are shown in Figures 12 and 13, demonstrating that humanized mice can correctly utilize the human IGH and IGK variable region genes.

[0163] Example 5

[0164] Humanized mouse immune cells develop normally

[0165] The development of immune-related cells, especially B cells, in the spleen and bone marrow of humanized mice was detected by flow cytometry. The results are shown in Figures 14 and 15, demonstrating that the proportion of lymphocytes and myeloid cells in the spleen is normal, and that B cells in the spleen and bone marrow develop normally.

[0166] Example 6

[0167] Constant region inversion reduces leaky expression of mouse genes

[0168] In actual use of humanized mice, leaky expression of mouse VDJ genes may occur. Existing technical solutions primarily target the inactivation of mouse variable region genes, with two main approaches: 1. Complete deletion of the mouse variable region gene, completely eliminating the mouse variable region gene, but also deleting a large number of other endogenous mouse genes, potentially damaging endogenous genes or transcriptional regulatory elements; 2. Inversion of the mouse variable region gene, placing the mouse endogenous variable region gene in the opposite direction, but placing the mouse endogenous variable region gene in close proximity to the inserted human variable region gene. In reality, the orientation of V genes within the mouse Igk locus varies, including IGKV genes that have the same and opposite orientations as their respective IGK J and C genes (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 region is still adjacent to the human variable region, and both are still spatially located upstream (5' end) of the endogenous constant region. From the perspective of the principles and directions of genome transcription and translation, there is still a risk of mouse variable regions being involved in the V(D)J recombination process.

[0169] In this patent, human variable region genes are inserted downstream of the mouse immunoglobulin locus, with the endogenous constant region separating the endogenous variable region from the inserted human variable region. The mice disclosed in this patent, whose endogenous variable regions are distal to the inserted human variable regions, were compared with mice in the prior art, whose endogenous variable regions are immediately adjacent to the inserted human variable regions. Spleens from the two mice were subjected to BCR sequencing, and the abundance of mouse variable regions involved in VDJ rearrangement was confirmed by detecting the number of variable region sequences with higher homology to mice. As shown in Figure 16, leaky expression of mouse variable region VDJ rearrangement was detected multiple times in the scenario where the human-mouse variable regions were immediately adjacent, while leaky expression of mouse variable region genes was almost undetectable in the scenario where the human-mouse variable regions were distal. By inserting human variable region genes downstream of the immunoglobulin genome, this patent achieves the effect of completely changing the relative positions of the human and mouse variable regions, solving the problem of leaky expression of mouse variable regions involved in VDJ gene rearrangement.

[0170] Example 7

[0171] Humanized mice can produce high-affinity antibodies

[0172] Humanized mice were immunized with commercially available hPD1 recombinant protein, and serum antibody titers were measured by ELISA. Results showed that the immune response of humanized mice and BALB / c mice against the same PD1 antigen was comparable. A positive antibody, 10K4F3, was obtained through hybridoma fusion screening. Antibody affinity was measured by surface plasmon resonance (BMK, a marketed antibody drug, was used as a positive control). In vivo efficacy experiments in mice were also performed to evaluate the antibody's efficacy.

[0173] The results, as shown in Table 8 and Figures 17 and 18, demonstrate that the use of humanized mice can produce high-affinity antibodies, and the corresponding antibodies have good in vivo anti-tumor efficacy data.

[0174] Table 8 Affinity comparison of the screened 10K4F3 antibodies and marketed drugs

[0175] Example 8

[0176] Preparation of partially humanized mice with variable region genes

[0177] Using partially humanized IGH-V, D, and J genes and partially humanized IGK-V and J genes (specific gene names are listed in Figures 19 and 20), mice with partial VDJ gene humanization were obtained using the same method. Spleens from positive mice were collected, and the utilization of the inserted human genes in splenic B cells was tested by BCR sequencing. The BCR sequencing results are shown in Figures 19 and 20, demonstrating that even with the insertion of partial VDJ genes, humanized mice can still utilize these human variable region genes.

[0178] Example 9

[0179] Some humanized mice with variable region genes can produce normal immune responses

[0180] Commercially available OVA protein was used to immunize VDJ partially humanized mice, and the serum antibody titer was detected by ELISA.

[0181] The results are shown in FIG21 , which indicate that the immune responses of humanized mice and BALB / c mice to OVA antigens were comparable.

Claims

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

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

3. The method according to any one of claims 1-2, which comprises operably linking a gene of one or more human heavy chain variable region V regions, heavy chain variable region D regions or 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 one or more 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. The method according to claim 1, the method comprising operably linking a human immunoglobulin light chain variable region gene downstream of the light chain locus of the non-human animal.

6. The method according to claim 5, which comprises operably linking a gene of 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 according to claim 6, the genes of the one or more human light chain variable region V or light chain variable region J regions or fragments thereof being directly linked.

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

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

10. The method according to any one of claims 1-9, wherein the function of the expression regulatory element of the endogenous immunoglobulin variable region gene of the non-human animal is not disrupted.

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

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

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

14. The method according to any one of claims 1-13, wherein the endogenous immunoglobulin variable region gene of the non-human animal does not express a functional protein.

15. The method according to any one of claims 1-14, which comprises making the transcription direction of the human immunoglobulin variable region gene opposite to that of the endogenous immunoglobulin variable region gene.

16. The method according to any one of claims 1-15, which comprises making the transcription direction of the immunoglobulin constant region gene of the non-human animal opposite to that of the endogenous immunoglobulin variable region gene.

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

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

19. The method according to any one of claims 1-18, 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 - 240 Kbp.

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

21. The method according to any one of claims 1-20, 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 - 42 Kbp.

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

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

24. The method according to claim 23, which comprises inserting a human immunoglobulin heavy chain variable region gene between Tmem121 and Igha genes in the mouse locus.

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

26. The method according to any one of claims 23-25, which comprises inserting a human immunoglobulin heavy chain variable region gene at the mouse chromosomal position chr12: 113,190,256.

27. The method according to any one of claims 23-26, which comprises inserting a human immunoglobulin light chain variable region gene between the lgkc and Rpia genes in the mouse locus.

28. The method according to any one of claims 23-27, which comprises inserting a human immunoglobulin light chain variable region gene between the mouse chromosomal positions chr6: 70,703,738 and 70,742,704.

29. The method according to any one of claims 23-28, which comprises inserting a human immunoglobulin light chain variable region gene at the mouse chromosomal position chr6: 70,706,267.

30. The method according to any one of claims 1-29, which comprises making the human immunoglobulin variable region gene operably linked downstream of the immunoglobulin locus in the non-human animal by site-specific recombination.

31. The method according to any one of claims 1-30, which comprises making the transcriptional direction of the immunoglobulin constant region gene in the non-human animal opposite to that of the endogenous immunoglobulin variable region gene by genome modification.

32. A non-human animal, the genome of which contains a human immunoglobulin variable region gene operably linked downstream of the immunoglobulin locus.

33. The non-human animal according to claim 32, which comprises 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 32-33, which comprises a gene of one or more human heavy chain variable region V regions, heavy chain variable region D regions or heavy chain variable region J regions or fragments thereof operably linked downstream of the heavy chain constant region locus.

35. The non-human animal according to any one of claims 32-34, wherein the genes of the one or more 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 according to claim 32, which comprises a human immunoglobulin light chain variable region gene operably linked downstream of the light chain locus.

37. The non-human animal according to claim 36, which comprises a gene of one or more human light chain variable region V or light chain variable region J regions or fragments thereof operably linked downstream of the light chain constant region locus.

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

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

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

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

42. The non-human animal according to any one of claims 32-41, which comprises a complete expression regulatory element of the endogenous immunoglobulin variable region.

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

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

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

46. The non-human animal according to any one of claims 32-45, wherein the human immunoglobulin variable region gene in the genome has a transcription direction opposite to that of the endogenous immunoglobulin variable region gene.

47. The non-human animal according to any one of claims 32-46, wherein the immunoglobulin constant region gene in the genome has a transcription direction opposite to that of the endogenous immunoglobulin variable region gene.

48. The non-human animal according to any one of claims 32-47, wherein the transcription direction of the human immunoglobulin variable region gene is the same as that of the immunoglobulin constant region gene of the non-human animal.

49. The non-human animal according to any one of claims 32-48, wherein a heavy chain constant region gene is included 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 32-49, wherein the distance between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene is 169 Kbp - 240 Kbp.

51. The non-human animal according to any one of claims 32-50, wherein the light chain constant region gene is included 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 32-51, wherein the distance between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene is 4 Kbp - 42 Kbp.

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

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

55. The non-human animal according to claim 54, which includes the human immunoglobulin heavy chain variable region gene between Tmem121 and the Igha gene at the locus.

56. The non-human animal according to any one of claims 54-55, which includes the human immunoglobulin heavy chain variable region gene between chromosome positions chr12: 113,149,523 and 113,223,857.

57. The non-human animal according to any one of claims 54-56, which includes the human immunoglobulin heavy chain variable region gene at chromosome position chr12: 113,190,256.

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

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

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

61. A non-human animal cell, which includes a human immunoglobulin variable region gene operably linked downstream of an immunoglobulin locus in the genome.

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

63. A method for preparing a sample, the method comprising: Exposing the non-human animal according to any one of claims 32-60 to the antigen; And collecting the sample from the non-human animal.

64. The method according to claim 63, wherein the sample includes immune cells.

65. The method according to claim 63 or 64, wherein the sample includes B cells.

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

Images