Animal model and construction method therefor

Abstract

A non-human animal and a method for preparing the non-human animal. In the genome of the non-human animal, the CH1 position of an immunoglobulin heavy-chain constant region gene is changed, and more specifically, the change in the CH1 position enables the transcription direction to be, sequentially, CH2, CH3 and CH1.

Description

Animal model and construction method thereof 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, in particular to a genetically modified non-human animal that retains the gene encoding the immunoglobulin CH1 region to produce heavy chain antibodies. Background Art

[0002] Monoclonal antibodies have revolutionized cancer treatment. However, the large size of traditional antibodies (150 kDa) limits their penetration into solid tumors and their therapeutic efficacy. Nanobodies (15 kDa) and humanized single heavy-chain antibodies (75 kDa) based on nanobodies offer advantages such as small size, high solubility, high stability, and excellent tissue penetration in vivo.

[0003] The discovery of heavy-chain antibodies has opened up unprecedented opportunities for cancer treatment. Conventional antibodies are composed of paired heavy and light chains. Lack of heavy or light chain expression can lead to B cell developmental arrest. Some species, such as camels (dromedaries / llamas) and sharks, are capable of producing heavy chain-only antibodies (HcAbs). These unique camelid-derived antibodies lack complete light chains and the CH1 domain.

[0004] Camels have exploited their ability to produce single-chain heavy-chain antibodies (HCAbs) for screening and identifying HcAbs. However, this approach to screening single-chain antibodies requires further humanization due to immunogenicity concerns. This humanization process can negatively impact binding affinity and introduce immunogenic epitopes into the antibody. Iterative and time-consuming experiments are often required to improve the properties of these antibodies. In some cases, these antibodies can also become immunogenic in patients, leading to diminished efficacy over time. Using transgenic human HcAb rodent models for screening single-chain heavy-chain antibodies offers a promising solution, enabling in vivo screening without the laborious humanization steps required for camelid single-chain heavy-chain antibodies. Furthermore, transgenic HcAb rodent platforms offer several advantages over camelid HcAb discovery platforms, including shorter immunization times, easier access and manipulation compared to larger camelids, easier access to in vivo immune organ material, more consistent immune responses, and more established antibody discovery technologies such as hybridoma fusion or display. There are currently three main strategies for preparing transgenic human HcAb rodent models (mainly mice). The first is to produce single heavy-chain antibodies by silencing or knocking out the mouse light chain to make it defective and unable to bind to the heavy chain. However, in these mice, B cell development is blocked at the immature B cell stage, affecting downstream antibody screening. The second is to construct an expression vector with human VDJ+mouse CH1 constant region deleted for transgenic purposes. This random insertion method has obvious disadvantages. The expression of the target gene is easily affected by the flanking sequences at the random insertion position, and there is a risk of damaging other endogenous genes. At the same time, this strategy generally requires silencing the entire endogenous heavy chain, which will lead to abnormal B cell development.The third type produces single heavy-chain antibodies by deleting the endogenous CH1 sequence. However, the presence of CH1 exons is quite important, such as: a) CH1 has genetic markers of allotypes (Ternant, David et al. "IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding." Journal of immunology (Baltimore, Md.: 1950) vol. 196, 2 (2016): 607-13.); b) the presence of CH1 can screen out improperly folded proteins (Matthias J, Feige, Sandra, Groscurth, Moritz, Marcinowski et al. An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. [J]. Mol Cell, 2009, 34: 0.), so the deletion of CH1 exons has potential adverse effects. The industry needs a non-human animal model that retains the CH1 exon and produces heavy chain antibodies, thereby effectively and efficiently producing humanized or non-humanized antibodies. Summary of the Invention

[0005] The present application provides a non-human animal and a method for preparing a non-human animal, the method changing the CH1 position by editing the immunoglobulin heavy chain constant region gene in the genome of the non-human animal. The non-human animal of the present application can avoid interfering with the animal's own gene expression and regulation, avoid the risk of CH1 deletion destroying other endogenous genes and B cell dysplasia, and has great advantages in antibody production. The non-human animal of the present application (e.g., mouse) has functional humanized single heavy chain antibodies, and using it to screen single heavy chain antibodies is a good solution. The humanized single heavy chain antibody model can avoid the laborious humanization step required for camel single heavy chain antibodies through in vivo screening. In addition, compared with HcAb discovery platforms such as camelids, the humanized single heavy chain antibody mouse model also has other advantages, such as shorter immunization time, easier access and operation compared to large camelids, easier access to in vivo immune organ materials, more consistent immune response, more mature hybridoma fusion or display and other antibody discovery technologies.

[0006] In one aspect, the present application provides a method for preparing a non-human animal, wherein the method changes the CH1 position by editing the immunoglobulin heavy chain constant region gene in the genome of the non-human animal.

[0007] In certain embodiments, the immunoglobulin heavy chain constant region gene includes the following exons: CH2, CH3 and CH1, and the transcription direction is: CH2, CH3 and CH1.

[0008] In certain embodiments, the immunoglobulin heavy chain constant region gene comprises IgM, IgD, IgG, IgA, or IgE. In certain embodiments, the immunoglobulin heavy chain constant region gene is IgG. In certain embodiments, the immunoglobulin heavy chain constant region gene is IgM. In certain embodiments, the immunoglobulin heavy chain constant region gene is IgD. In certain embodiments, the immunoglobulin heavy chain constant region gene is IgA. In certain embodiments, the immunoglobulin heavy chain constant region gene is IgA.

[0009] In certain embodiments, the IgG comprises IgG3, IgG1, IgG2A, IgG2B and / or IgG2C.

[0010] In certain embodiments, the immunoglobulin heavy chain constant region gene further includes the following exons: H, M1 and M2, and a gene L encoding a linker, and the transcription direction is: H, CH2, CH3, M1, M2, L and CH1.

[0011] In certain embodiments, the linker is a self-cleaving peptide, preferably a self-cleaving peptide with a length of 18-22 amino acids.

[0012] In certain embodiments, the linker is a 2A peptide, preferably F2A, E2A, P2A and T2A.

[0013] In certain embodiments, the immunoglobulin heavy chain constant region gene is derived from the non-human animal.

[0014] In certain embodiments, the method comprises constructing a nucleic acid sequence comprising, in order from the 5' to the 3' end, the following exons: CH2, CH3, and CH1. In certain embodiments, the nucleic acid sequence comprises, in order from the 5' to the 3' end, the following exons and a gene encoding a linker: H, CH2, CH3, M1, M2, L, and CH1.

[0015] In certain embodiments, the method comprises inserting the nucleic acid sequence into an immunoglobulin heavy chain constant region locus of the non-human animal.

[0016] In certain embodiments, the method comprises inserting the nucleic acid sequence into the locus of the immunoglobulin heavy chain constant region IGHG of the non-human animal.

[0017] In certain embodiments, the method comprises replacing the endogenous immunoglobulin heavy chain constant region IGHG gene of the non-human animal with the nucleic acid sequence.

[0018] In certain embodiments, the method comprises operably linking a human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus of the non-human animal.

[0019] In certain embodiments, the method comprises operably linking 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. In certain embodiments, the genes of the plurality of 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.

[0020] In certain embodiments, the non-human animal comprises an endogenous immunoglobulin heavy chain variable region gene. In certain embodiments, the genomic integrity of the endogenous immunoglobulin heavy chain variable region gene of the non-human animal is not altered. In certain embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin heavy chain variable region gene of the non-human animal is not impaired. In certain embodiments, the non-human animal comprises intact endogenous immunoglobulin heavy chain variable region expression regulatory elements. In certain embodiments, the non-human animal comprises intact endogenous immunoglobulin heavy chain variable region gene.

[0021] In certain embodiments, the non-human animal does not express endogenous immunoglobulin heavy chain variable regions.

[0022] In certain embodiments, the non-human animal's endogenous immunoglobulin variable region genes are not expressed as antibody heavy chain variable regions.

[0023] In certain embodiments, the human immunoglobulin heavy chain variable region gene is transcribed in the opposite direction to the endogenous immunoglobulin heavy chain variable region gene.

[0024] In certain embodiments, the human immunoglobulin heavy chain variable region gene in the non-human animal comprises an immunoglobulin heavy chain constant region gene of the non-human animal between the endogenous immunoglobulin heavy chain variable region gene.

[0025] In certain embodiments, the immunoglobulin heavy chain constant region gene is transcribed in the opposite direction to the endogenous immunoglobulin heavy chain variable region gene.

[0026] In certain embodiments, the transcription direction of the human immunoglobulin heavy chain variable region gene and the immunoglobulin heavy chain constant region gene in the non-human animal is the same.

[0027] In certain embodiments, the chromosome of the non-human animal comprises, from upstream to downstream, the endogenous immunoglobulin heavy chain variable region gene, the immunoglobulin heavy chain constant region gene, and the human immunoglobulin heavy chain variable region gene.

[0028] In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse.

[0029] In some embodiments, the mouse has the human immunoglobulin heavy chain variable region gene inserted between chromosomal positions chr12: 113,149,523 to 113,223,857. In some embodiments, the mouse has the human immunoglobulin heavy chain variable region gene inserted at chromosomal position chr12: 113,190,256.

[0030] In certain embodiments, the method comprises operably linking the human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus of the non-human animal by site-directed recombination. In certain embodiments, the method comprises modifying the genome so that the transcription direction of the immunoglobulin heavy chain constant region gene of the non-human animal is opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

[0031] In certain embodiments, the method comprises operably linking a human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus of the non-human animal.

[0032] In certain embodiments, the method comprises operably linking one or more human light chain variable region V region or light chain variable region J region or fragment thereof genes downstream of the light chain constant region locus of the non-human animal. In certain embodiments, the one or more human light chain variable region V region or light chain variable region J region or fragment thereof genes are directly linked.

[0033] In certain embodiments, the non-human animal comprises an endogenous immunoglobulin light chain variable region gene. In certain embodiments, the genomic integrity of the endogenous immunoglobulin light chain variable region gene of the non-human animal is not altered. In certain embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin light chain variable region gene of the non-human animal is not impaired. In certain embodiments, the non-human animal comprises a complete endogenous immunoglobulin light chain variable region expression regulatory element. In certain embodiments, the non-human animal comprises a complete endogenous immunoglobulin light chain variable region gene.

[0034] In certain embodiments, the non-human animal does not express endogenous immunoglobulin light chain variable regions.

[0035] In certain embodiments, the non-human animal's endogenous immunoglobulin variable region genes are not expressed as antibody light chain variable regions.

[0036] In certain embodiments, the human immunoglobulin light chain variable region gene is transcribed in the opposite direction to the endogenous immunoglobulin light chain variable region gene.

[0037] In certain embodiments, the human immunoglobulin light chain variable region gene in the non-human animal comprises an immunoglobulin light chain constant region gene of the non-human animal between the endogenous immunoglobulin light chain variable region gene.

[0038] In certain embodiments, the immunoglobulin light chain constant region gene is transcribed in the opposite direction to the endogenous immunoglobulin light chain variable region gene.

[0039] In certain embodiments, the transcription direction of the human immunoglobulin light chain variable region gene and the immunoglobulin light chain constant region gene in the non-human animal is the same.

[0040] In certain embodiments, 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.

[0041] In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse.

[0042] In some embodiments, the mouse has the human immunoglobulin light chain variable region gene inserted between chromosomal positions chr6: 70,703,738 to 70,742,704. In some embodiments, the mouse has the human immunoglobulin light chain variable region gene inserted at chromosomal position chr6: 70,706,267.

[0043] In certain embodiments, the method comprises operably linking the human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus of the non-human animal by site-directed recombination. In certain embodiments, the method comprises modifying the genome so that the transcription direction of the immunoglobulin light chain constant region gene of the non-human animal is opposite to that of the endogenous immunoglobulin light chain variable region gene.

[0044] In another aspect, the present application provides a non-human animal or a non-human animal cell.

[0045] 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.

[0046] 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.

[0047] On the other hand, the present application provides a non-human animal, wherein the immunoglobulin heavy chain constant region gene in the genome of the non-human animal includes the following exons: CH2, CH3 and CH1, and the transcription directions are: CH2, CH3 and CH1.

[0048] On the other hand, the present application provides a non-human animal genome, wherein the immunoglobulin heavy chain constant region gene in the genome of the non-human animal includes the following exons: CH2, CH3 and CH1, and the transcription directions are: CH2, CH3 and CH1.

[0049] In certain embodiments, the CH2, CH3, and CH1 are derived from IgG and / or IgM.

[0050] In certain embodiments, the IgG comprises IgG3, IgG1, IgG2A, IgG2B and / or IgG2C.

[0051] In certain embodiments, the immunoglobulin heavy chain constant region gene further includes the following exons: H, M1 and M2, and a gene L encoding a linker, and the transcription direction is: H, CH2, CH3, M1, M2, L and CH1.

[0052] In certain embodiments, the linker is a self-cleaving peptide, preferably a self-cleaving peptide with a length of 18-22 amino acids.

[0053] In certain embodiments, the linker is a 2A peptide, preferably F2A, E2A, P2A and T2A.

[0054] In certain embodiments, the immunoglobulin heavy chain constant region gene is derived from the non-human animal.

[0055] In certain embodiments, the non-human animal's immunoglobulin heavy chain locus is operably connected downstream to a human immunoglobulin heavy chain variable region gene. In certain embodiments, the non-human animal's heavy chain constant region locus is operably connected downstream to one or more human heavy chain variable region V region, heavy chain variable region D region, or heavy chain variable region J region, or a fragment thereof. In certain embodiments, the genes for the multiple human heavy chain variable region V, heavy chain variable region D, or heavy chain variable region J region, or a fragment thereof, are directly linked.

[0056] In certain embodiments, the non-human animal comprises an endogenous immunoglobulin heavy chain variable region gene. In certain embodiments, the genomic integrity of the endogenous immunoglobulin heavy chain variable region gene of the non-human animal is not altered. In certain embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin heavy chain variable region gene of the non-human animal is not impaired. In certain embodiments, the non-human animal comprises intact endogenous immunoglobulin heavy chain variable region expression regulatory elements. In certain embodiments, the non-human animal comprises intact endogenous immunoglobulin heavy chain variable region gene.

[0057] In certain embodiments, the non-human animal does not express endogenous immunoglobulin heavy chain variable regions. In certain embodiments, the non-human animal's endogenous immunoglobulin variable region genes do not express antibody heavy chain variable regions.

[0058] In certain embodiments, the human immunoglobulin heavy chain variable region gene is transcribed in the opposite direction to the endogenous immunoglobulin heavy chain variable region gene.

[0059] In certain embodiments, the human immunoglobulin heavy chain variable region gene in the non-human animal comprises an immunoglobulin heavy chain constant region gene of the non-human animal between the endogenous immunoglobulin heavy chain variable region gene.

[0060] In certain embodiments, the immunoglobulin heavy chain constant region gene is transcribed in the opposite direction to the endogenous immunoglobulin heavy chain variable region gene.

[0061] In certain embodiments, the transcription direction of the human immunoglobulin heavy chain variable region gene and the immunoglobulin heavy chain constant region gene in the non-human animal is the same.

[0062] In certain embodiments, the chromosome of the non-human animal comprises, from upstream to downstream, the endogenous immunoglobulin heavy chain variable region gene, the immunoglobulin heavy chain constant region gene, and the human immunoglobulin heavy chain variable region gene.

[0063] In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse.

[0064] In certain embodiments, the mouse comprises the human immunoglobulin heavy chain variable region gene at chromosome position chr12: 113,149,523 to 113,223,857. In certain embodiments, the mouse comprises the human immunoglobulin heavy chain variable region gene at chromosome position chr12: 113,190,256.

[0065] In certain embodiments, the non-human animal's immunoglobulin light chain gene locus is operably linked downstream to a human immunoglobulin light chain variable region gene.

[0066] In certain embodiments, the method comprises operably linking one or more human light chain variable region V region or light chain variable region J region or fragment thereof genes downstream of the light chain constant region locus of the non-human animal. In certain embodiments, the one or more human light chain variable region V region or light chain variable region J region or fragment thereof genes are directly linked.

[0067] In certain embodiments, the non-human animal comprises an endogenous immunoglobulin light chain variable region gene. In certain embodiments, the genomic integrity of the endogenous immunoglobulin light chain variable region gene of the non-human animal is not altered. In certain embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin light chain variable region gene of the non-human animal is not impaired. In certain embodiments, the non-human animal comprises a complete endogenous immunoglobulin light chain variable region expression regulatory element. In certain embodiments, the non-human animal comprises a complete endogenous immunoglobulin light chain variable region gene.

[0068] In certain embodiments, the non-human animal does not express endogenous immunoglobulin light chain variable regions.

[0069] In certain embodiments, the non-human animal's endogenous immunoglobulin variable region genes are not expressed as antibody light chain variable regions.

[0070] In certain embodiments, the human immunoglobulin light chain variable region gene is transcribed in the opposite direction to the endogenous immunoglobulin light chain variable region gene.

[0071] In certain embodiments, the chromosome in the non-human animal comprises, from upstream to downstream, the endogenous immunoglobulin light chain variable region gene, the immunoglobulin light chain constant region gene, and the human immunoglobulin light chain variable region gene.

[0072] In certain embodiments, the immunoglobulin light chain constant region gene is transcribed in the opposite direction to the endogenous immunoglobulin light chain variable region gene.

[0073] In certain embodiments, the transcription direction of the human immunoglobulin light chain variable region gene and the immunoglobulin light chain constant region gene in the non-human animal is the same.

[0074] In certain embodiments, 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.

[0075] In certain embodiments, the non-human animal is a mouse and comprises the human immunoglobulin light chain variable region gene at chromosome position chr6: 70,703,738 to 70,742,704. In certain embodiments, the non-human animal is a mouse and comprises the human immunoglobulin light chain variable region gene at chromosome position chr6: 70,706,267.

[0076] On the other hand, the present application provides an engineered chromosome comprising the non-human animal genome described in the present application.

[0077] On the other hand, the present application provides a non-human animal cell, the immunoglobulin heavy chain constant region gene of whose genome includes the following exons: CH2, CH3 and CH, H, M1 and M2, as well as the gene L encoding the linker, and the transcription direction is H, CH2, CH3, M1, M2, L and CH1 in sequence.

[0078] 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.

[0079] In certain embodiments, the antibody is a heavy chain-only antibody (HCAb).

[0080] 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.

[0081] The technical solution of this application includes one or more of the following advantages:

[0082] The non-human animals described herein produce antibodies lacking light chains, i.e., heavy chain antibodies. Because heavy chain antibodies lack light chains, they are smaller and therefore expected to show better tissue penetration than antibodies comprising light chains, yet they contain similar or more favorable pharmacokinetic profiles and still retain similar effector functions compared to conventional antibodies. Because they are smaller, heavy chain antibodies can also be administered at higher doses in a given volume. The commonly used method for administering antibodies is by subcutaneous injection. For a given antibody dose, the reduced administration volume can provide benefits to the patient and avoid the complications and pain caused by large subcutaneous injection volumes. Another advantage of heavy chain antibodies is that bispecific antibodies can be produced by heterodimerizing heavy chains that are specific for two different epitopes in a single therapeutic agent. Because heavy chain antibodies lack light chains, they are particularly suitable for producing bispecific antibodies because they do not require engineering modifications that do not interfere with the binding affinity or specificity of any heavy chain, but can also enable bispecific antibodies to be appropriately expressed.

[0083] The non-human animals described in this application have advantages in avoiding interference with the animal's own gene expression and regulation. For example, the deletion of the CH1 exon of the heavy chain constant region of the immunoglobulin gene in non-human animals (e.g., mice) is avoided. The presence of the CH1 exon in non-human animals plays an important role, such as the genetic marker of allotypes on CH1. The IgG1 isotype affects the pharmacokinetics of therapeutic monoclonal antibodies through FcRn binding. The type of CH1 domain has an impact on BCR assembly and expression as well as antigen sensing. The presence of CH1 can screen out improperly folded proteins through quality control.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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

[0092] 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:

[0093] Figure 1 shows the heavy chain Igh constant region Cpoint and LD targeting as well as C region modification.

[0094] Figure 2 shows a schematic diagram of the heavy chain human variable region insertion, including insertion targeting and resistance marker deletion.

[0095] Figure 3 shows the Cpoint and LD targeting of the light chain Igk constant region and the C region modification.

[0096] Figure 4 shows a schematic diagram of the insertion of the light chain human variable region, including insertion targeting and resistance marker deletion.

[0097] Figure 5 shows a schematic diagram of the insertion of the heavy chain constant region targeting vector, including LD targeting and constant region targeting.

[0098] FIG6 shows the results of LD targeting PCR identification.

[0099] Figure 7 shows the results of vector targeted PCR identification.

[0100] FIG8 shows the results of PCR identification of single heavy chain mice.

[0101] FIG9 shows the sequencing alignment results of CH1 identification PCR products in genomic DNA.

[0102] FIG10 shows the sequencing alignment results of the PCR products for CH1 identification in IgG-mRNA.

[0103] FIG11 shows the results of the utilization test of the IGH inserted gene.

[0104] Figure 12 shows that the ratio of lymphocytes to myeloid cells in the spleen is normal.

[0105] FIG13 shows the ELISA titer detection of humanized single heavy chain mice and control mice.

[0106] FIG14 shows the ELISA activity detection of anti-human CD98hc humanized single heavy chain antibody.

[0107] FIG15 shows an exemplary CH1 translocation modification method of the present application. DETAILED DESCRIPTION

[0108] 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.

[0109] Definition of terms

[0110] 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.

[0111] In this application, the term "CH1" generally refers to the gene encoding the first domain of the immunoglobulin heavy chain constant region (IGHC), located before the hinge region, specifically within the 3 / 4 or 4 / 5 region near the C-terminus of the antibody heavy chain, and bearing the genetic signature of an Ig allotype. This term encompasses introns, exons, the full-length CH1 gene, and functional CH1 fragments. For the location of the CH1 gene within the IGHC, please refer to the IMGT / GENE-DB.

[0112] In this application, the term "CH2" generally refers to the gene encoding the second domain of the immunoglobulin heavy chain constant region. The CH2 domain has a complement C1q binding site and can participate in the activation of the classical complement pathway, thereby enhancing the immune response. This term includes introns, exons, the full-length CH2 gene, and functional CH2 fragments. For the location of the CH2 gene in the IGHC, please refer to IMGT / GENE-DB.

[0113] In this application, the term "CH3" generally refers to the third domain of the antibody heavy chain constant region. For IgG antibodies, the CH3 domain is capable of binding to Fc receptors (FcγRs) on the surface of various immune cells, mediating different biological effects of immune cells, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The term includes introns, exons, the full-length CH3 gene, and functional CH4 fragments. For the location of the CH4 gene in the IGHC, please refer to IMGT / GENE-DB.

[0114] In this application, the term "H" generally refers to the gene encoding the hinge region of an immunoglobulin, located between the CH1 and CH2 domains. This region is aligned with proline, making it easy to stretch and bend, which can change the distance between the two arms of the "Y" shape, facilitating the simultaneous binding of two identical antigenic epitopes by both arms. It also facilitates the exposure of the complement binding site of the immunoglobulin molecule, binding to complement C1q and activating complement. The hinge regions of the five classes of immunoglobulins and their subclasses are not identical. A hinge region exists between the CH1 and CH2 regions of the IgG, IgA, and IgD heavy chains. The hinge region of IgG1, IgG2, IgG4, and IgA is shorter, consisting of only a little over 10 amino acid residues; whereas the hinge region of IgG3 and IgD is longer, containing approximately 60 amino acid residues. IgM and IgE heavy chains lack a hinge region. In addition, the hinge region is sensitive to papain and pepsin, and enzymatic hydrolysis can break the immunoglobulin into several different fragments at this site. The term includes introns, exons, full-length H gene, and functional fragments of H. For the location of the H gene in IGHC, please refer to IMGT / GENE-DB.

[0115] In this application, the term "M1" generally refers to the gene encoding the transmembrane domain of the immunoglobulin constant region, which enables antibodies to be expressed on the cell membrane or secreted outside the cell. M1 is a transmembrane region close to the CH3 domain, which is involved in the transmembrane transport of antibody molecules and their localization on the cell membrane. The M1 domain is crucial for the correct folding, stability, and transport and distribution of antibody molecules in the body. They ensure that antibodies can be effectively transported outside the cell to exert their immune function. The M1 domain generally contains hydrophobic amino acids, which structurally help the antibody molecule pass through the phospholipid bilayer of the cell membrane. The term includes introns, exons, M1 full-length genes, and M1 functional fragments. For the location of the M1 gene in the IGHC, please refer to IMGT / GENE-DB.

[0116] In this application, the term "M2" generally refers to the gene encoding the transmembrane domain of the immunoglobulin constant region, which enables antibody expression on the cell membrane or secretion. M2 is the transmembrane region located downstream of M1 and is also involved in transmembrane trafficking and localization of antibodies. The term includes introns, exons, the full-length M2 gene, and functional M2 fragments. For the location of the M2 gene in the IGHC, please refer to IMGT / GENE-DB.

[0117] In this application, the term "L" generally refers to a gene encoding a linker, which is located between the M2 and CH1 domains and can connect M2 and CH1 in cells.

[0118] In this application, the term "2A peptide" generally refers to a viral oligopeptide of 18-22 amino acids (aa) in length that mediates the "self-cleavage" of the polypeptide during translation in eukaryotic cells. The 2A-mediated "self-cleavage" mechanism is that the ribosome skips the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, thereby forming two proteins upstream and downstream of 2A. "2A" refers to a specific region of the viral genome, and different viral 2As are usually named after the viruses from which they originate. Common 2A peptides include F2A, E2A, P2A, and T2A.

[0119] 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 an antigen, but displays 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). The genes encoding mouse (Mus musculus) constant regions can include IGHA, IGHD, IGHE, IGHG1, IGHG2A, IGHG2B, IGHG2C, IGHG3, or IGHM. Information on mouse constant region loci can be found in the IMGT Repertoire: https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=Mus_musculus&group=IGHC.

[0120] 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.

[0121] 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).

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In this application, the terms "upstream" and "downstream" are generally used to describe the relative positions of 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, 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 natural host immunoglobulin locus, then A is considered to be downstream and the host immunoglobulin locus is upstream.

[0127] 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).

[0128] Detailed Description of the Invention

[0129] The present application relates to genetically modified non-human animals and cells that retain CH1 exons and are capable of producing antibodies lacking the CH1 domain. In this application, research conducted in mice is provided for illustrative purposes only. Unless otherwise specified, reference to mice also includes all non-human mammals, with mice being the preferred non-human mammal.

[0130] The genetically modified non-human animals of the present application can be used to produce a wide variety of heavy chain antibodies. The genetic modification described herein can be produced in, for example, any suitable mouse germline. The mouse germline can have any genetic background suitable for producing the selected heavy chain antibody.

[0131] On the one hand, the non-human animal or cell involved in the present application has an immunoglobulin heavy chain constant region gene in its genome including the following exons: CH2, CH3 and CH1, and the transcription direction is: CH2, CH3 and CH1.

[0132] In some embodiments, the immunoglobulin heavy chain constant region gene includes the following exons: H, CH2, CH3, M1, M2, L and CH1, and the transcription direction is: H, CH2, CH3, M1, M2, L and CH1.

[0133] In some embodiments, L is a protein linker, which generally includes flexible linkers, rigid linkers, and cleavable linkers.

[0134] In some embodiments, L is F2A, E2A, P2A, or T2A.

[0135] In some embodiments, the modified immunoglobulin heavy chain constant region in the non-human animal is IgM, IgD, IgA, IgE, IgG3, IgG1, IgG2A, IgG2B and / or IgG2C.

[0136] In some embodiments, the non-human animal's endogenous immunoglobulin light chain variable region gene is not modified or deleted in any way.

[0137] In some embodiments, the non-human animal's endogenous immunoglobulin heavy chain variable region gene is not modified or deleted in any way.

[0138] In another aspect, the present application relates to a non-human animal or cell in which one or more human IGHV regions, one or more human IGHD regions, and / or one or more human IGHJ regions are operably linked downstream of the heavy chain constant region locus of the non-human animal. In some embodiments, after insertion, the transcription direction of the coding regions of the human IGHV region, human IGHD region, and human IGHJ region is opposite to the transcription direction of the coding regions of the host endogenous heavy chain VDJ progene. The human IGHV region, human IGHD region, and human IGHJ region are operably linked together and can undergo VDJ recombination.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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 / .

[0155] 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.

[0156] 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.

[0157] 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.

[0158] This application discloses methods for constructing immunoglobulin heavy chain constant region genes with the transcriptional orientation of CH2, CH3, and CH1 in non-human animals (e.g., mice). Research conducted in mice is provided herein for illustrative purposes only. Unless otherwise specified, reference to mice includes all non-human animals, with mice being the preferred non-human animal.

[0159] On the one hand, a DNA sequence comprising CH2, CH3 and CH1 in the transcriptional direction is inserted into the region between the IGHJ4 exon and the Tmem121 locus on mouse chromosome 12 by site-directed recombination; in one aspect, the insertion is at coordinates 113,191,291 to 113,391,844 on mouse chromosome 12.

[0160] On the one hand, the endogenous immunoglobulin heavy chain constant region IGHG gene of the non-human animal is replaced with a DNA sequence comprising CH2, CH3 and CH1 in the transcription direction.

[0161] In one aspect, the inserted immunoglobulin heavy chain constant region CH1 gene comprises 50-100% of the immunoglobulin heavy chain constant region CH1 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.

[0162] In some embodiments, the non-human embryo is a mouse embryo or a rat embryo, and the donor cell is a mouse ES cell or a rat ES cell, respectively.

[0163] In some embodiments, the site-directed recombination method includes homologous recombination, nucleic acid cutting enzyme-mediated recombination (such as CRISPR / Cas9, etc.), and site-specific recombinase-mediated recombination. It involves the use of one or more of these methods in combination. The insertion or replacement can be obtained by commonly used gene editing targeting methods, such as homologous recombination or nucleic acid cutting enzyme-mediated recombination (such as CRISPR / Cas9, etc.). The modification can also be achieved by nuclease, and the relevant technology is known in the prior art.

[0164] In some embodiments, the C1 of the endogenous immunoglobulin heavy chain constant region IGHG gene of a non-human animal cell is first cut out, and then L and C1 are inserted after C3, so that the transcription direction is C2, C3, and C1. For example, L+C1 can be inserted after M2. After screening and identification to obtain modified cells, the cells are injected into early embryos to produce animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing non-humanized immunoglobulin loci.

[0165] In some embodiments, the endogenous immunoglobulin heavy chain constant region IGHG gene of non-human animal cells is replaced in its entirety with a DNA sequence comprising CH2, CH3, and CH1 in the transcriptional orientation. After screening and identification to obtain the modified cells, the cells are injected into early embryos to produce animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing non-humanized immunoglobulin loci.

[0166] On the other hand, the present application discloses a method for constructing functional human immunoglobulin variable region genes in non-human animals (eg, mice).

[0167] In some embodiments, the variable region of the non-human animal immunoglobulin gene is first modified or deleted, and then the CH1 of the endogenous immunoglobulin heavy chain constant region IGHG gene of the non-human animal cell is cut out, and L and CH1 are inserted after CH3, so that the transcription direction is CH2, CH3 and CH1. After screening and identification to obtain the modified cells, the cells are injected into early embryos to produce chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing complete humanized immunoglobulin loci.

[0168] In some embodiments, the variable region of the non-human animal immunoglobulin gene is first modified or deleted, and then the endogenous immunoglobulin heavy chain constant region IGHG gene of the non-human animal cell is replaced as a whole with a DNA sequence containing CH2, CH3 and CH1 in the transcription direction. 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.

[0169] In some embodiments, the C1 of the endogenous immunoglobulin heavy chain constant region IGHG gene of the non-human animal cell is first cut out, and L and C1 are inserted after C3 so that the transcription direction is C2, C3 and C1. Then, the variable region of the non-human animal immunoglobulin gene is modified or deleted. 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.

[0170] In some embodiments, the endogenous immunoglobulin heavy chain constant region IGHG gene of the non-human animal cell is first replaced as a whole with a DNA sequence comprising a transcription direction of CH2, CH3 and CH1, and then the variable region of the non-human animal immunoglobulin gene is modified or deleted. 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 a complete humanized immunoglobulin locus.

[0171] In some embodiments, the modified human variable region gene is transcribed in the opposite direction to the endogenous variable region gene of the non-human animal. In some embodiments, the modified endogenous constant region is transcribed in the opposite direction to the endogenous variable region gene.

[0172] In some embodiments, the modified human variable region gene is first inserted downstream (behind) the endogenous immunoglobulin locus of the mouse cell, and then a modified short exogenous recombinase binding site is introduced into 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 that, the CH1 in the endogenous immunoglobulin constant region IGHG gene of the mouse cell is cut out, and L and CH1 are inserted after CH3 so that the transcription direction is CH2, CH3, L and CH1. 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.

[0173] In some embodiments, the modified human variable region gene is first inserted downstream (behind) the endogenous immunoglobulin locus of the mouse cell, and then a modified short exogenous recombinase binding site is introduced into 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, and then the endogenous immunoglobulin heavy chain constant region IGHG gene of the mouse cell is replaced as a whole with a DNA sequence comprising CH2, CH3 and CH1, so that its transcription direction is CH2, CH3 and CH1. 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.

[0174] In some embodiments, a modified short exogenous recombinase binding site is 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 which the CH1 in the endogenous immunoglobulin heavy chain constant region IGHG gene of the mouse cell is cut off, and L and CH1 are inserted after CH3 so that the transcription direction is CH2, CH3, L and CH1. 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.

[0175] In some embodiments, a modified short exogenous recombinase binding site is 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, and then the mouse cell endogenous immunoglobulin heavy chain constant region IGHG gene is replaced as a whole with a DNA sequence comprising CH2, CH3 and CH1, so that its transcription direction is CH2, CH3, L and CH1. 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.

[0176] In some embodiments, the modified human variable region gene is first inserted downstream (behind) the endogenous immunoglobulin locus of the mouse cell, and then the entire endogenous immunoglobulin heavy chain constant region IGHG gene of the mouse cell is replaced with a DNA sequence comprising CH2, CH3, and CH1, so that its transcription direction is CH2, CH3, L, and CH1. 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.

[0177] In some embodiments, the entire endogenous immunoglobulin heavy chain constant region IGHG gene of the mouse cell is first replaced with a DNA sequence comprising CH2, CH3 and CH1, so that its transcription direction is CH2, CH3, L and CH1, and then the modified human variable region gene is inserted downstream (behind) of the endogenous immunoglobulin locus of the mouse cell. For example, the transcription direction of the modified constant region is opposite to that before modification. 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] In one embodiment, the present application uses gene targeting technology to insert the entire human variable region gene into the mouse locus, and obtains a single heavy chain model by changing the position of the IgG-CH1 exon on the original IgG gene, rather than knocking out CH1. The non-human animal model of the present application still has the CH1 functional domain on the gene (Figures 9 and 10).

[0182] The technical effects that can be achieved by the embodiments of the present application are, for example, as follows: the non-human animals of the present application contain all human heavy chain variable region VDJ genes and can express humanized single heavy chain antibodies, reducing the cost and difficulty of screening human single heavy chain antibodies; the non-human animals of the present application retain the CH1 nucleic acid sequence and retain the endogenous heavy chain regulatory elements, greatly reducing the adverse effects caused by gene or exon deletions.

[0183] Without intending to be bound by any theory, the following examples are merely intended to illustrate the non-human animals, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0184] Example

[0185] Example 1 Obtaining Mouse ES Cells Inserted with Human Variable Regions

[0186] The ES cell line of BALB / c mice was obtained through independent isolation and used as the basic material for subsequent targeting.

[0187] 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.

[0188] 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.

[0189] 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 to achieve deletion of the selection marker fragment 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.).

[0190] 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 in which the target human antibody variable region encoding gene (VDJ) was completely inserted.

[0191] ES cells that have been quality-controlled by PCR and karyotype Q-PCR array analysis are injected into the mouse blastocyst cavity 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 chimeric mice are then bred with BALB / c mice. After genotyping and functional verification, mice containing human immunoglobulin heavy chain variable region genes and human immunoglobulin light chain variable region genes are ultimately obtained. To obtain functional verification data more quickly, an alternative approach is to inject ES cells into mouse blastocysts that are incapable of functional VDJ rearrangement (such as Rag1 gene-deficient mice). The resulting chimeric mice can be directly tested for immune system function without breeding.

[0192] 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.

[0193] Then, mouse embryonic stem cells were obtained through embryo manipulation and cell culture.

[0194] Example 2 Construction of LD targeting vector

[0195] Based on the positive cells obtained in Example 1, this patent further adopts the ES targeting technology well known in the art, using a whole-replacement strategy (as shown in FIG5 ) to replace the endogenous Igh constant region as a whole with a vector containing CH2, CH3 and CH1 constructed in vitro, so that its transcription direction is CH2, CH3 and CH1 or CH2, CH3, L and CH1. A step-by-step replacement strategy for the constant region CH1 can also be used here, first deleting CH1 in the endogenous immunoglobulin constant region IGH gene of the mouse cell, and then inserting CH1 after the endogenous CH3 by homologous recombination, so that its transcription direction is CH2, CH3 and CH1 (as shown in FIG15 ).

[0196] This patent adopts a holistic replacement strategy, which first achieves LD targeting through homologous recombination and then inserts the vector with the help of the Cre / loxP system.

[0197] The LD targeting vector consists of two homology arms (sequences on both sides of the insertion site), a selection marker (SM1), loxP and loxP511 fragments. As shown in Figure 5, these fragments are linked to the commercial vector PMD18T by enzyme ligation to construct the LD targeting vector for collaboration with the vector targeting vector.

[0198] Using ES targeting technology well known in the art, the LD targeting fragment was knocked into the mouse IGH constant region location (the region between the Ighj4 exon and the Tmem121 locus, specifically IGH chr12: 113,191,291 to 113,391,844. NCBI database Mus musculus strain C57BL / 6J, GRCm39) in ES cells.

[0199] PCR identification confirmed that the LDs of clones 1, 3, 6, 7, 10, 12, and 17 were correctly targeted. The LD identification scheme is shown in Table 1, and the PCR identification results are shown in Figure 6.

[0200] Table 1 LD identification scheme

[0201] Example 3 Construction of vector targeting vector

[0202] As shown in Figure 5, the endogenous CH1 sequence was recombined and shifted to the end of the gene and replaced the original gene sequence by 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). At the same time, a promoter, loxP, and a selection marker (SM2) were inserted at the 5 end of the vector, and a loxP original was inserted at the 3 end of the vector to construct a vector targeting vector.

[0203] Vector targeting was performed by 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).

[0204] PCR identification confirmed that the vector targeting of clones 1-11# was correct. The targeting identification scheme is shown in Table 2, and the vector targeting PCR identification results are shown in Figure 7.

[0205] Table 2 Vector Targeting Identification Scheme

[0206] The positive ES cells were injected into the mouse blastocyst cavity according to the method in the Mouse Embryo Operation Manual to obtain F0 chimeric mice, which were bred with BALB / c mice. After genotypic identification and functional verification, a single heavy chain mouse model was finally obtained.

[0207] Example 4 Mouse Model Genome Verification

[0208] Genomic DNA from single heavy chain mice was extracted, and PCR and sequencing confirmed that both the single heavy chain mouse genomic sequence and mRNA sequence contained the CH1 exon sequence. The identification scheme is shown in Table 3, the PCR identification results are shown in Figure 8, the sequencing and alignment analysis results of the genomic PCR products are shown in Figure 9, and the sequencing and alignment analysis results of the mRNA identification PCR products are shown in Figure 10. The results showed that the CH1 sequence was detectable in both the genome and mRNA.

[0209] Table 3 CH1 identification scheme

[0210] Example 5 Humanized single heavy chain mice can correctly use human variable region genes

[0211] Spleens from humanized single heavy chain mice were harvested and BCR sequencing was performed to assess the utilization of the inserted human gene in splenic B cells. The BCR sequencing results are shown in Figure 11, demonstrating that humanized single heavy chain mice are able to correctly utilize the human IGH variable region gene.

[0212] Example 6 Humanized single heavy chain mice have normal immune cell development and normal B cell ratio

[0213] The immune-related cells in the spleen of humanized single heavy chain mice were detected by flow cytometry. The results are shown in Figure 12, which proves that the proportions of lymphocytes and myeloid cells in the spleen are normal.

[0214] Example 7 Humanized single heavy chain mice are able to produce high affinity antibodies

[0215] Humanized single-heavy chain mice were immunized with commercially available human CD98hc recombinant protein, and serum antibody titers were measured by ELISA (enzyme-linked immunosorbent assay). Results showed that the immune response of humanized single-heavy chain mice was comparable to that of fully humanized mice against the same antigen. A positive single-heavy chain antibody, 009E5, was obtained through hybridoma fusion screening, and ELISA activity testing confirmed that this single-heavy chain antibody was capable of binding to the human CD98hc protein.

[0216] The results are shown in Figures 13 and 14, demonstrating that high-affinity antibodies can be produced using humanized single heavy chain mice, and the corresponding antibodies have good in vitro binding activity.

Claims

1. A method for preparing a non-human animal, the method comprising editing the immunoglobulin heavy chain constant region gene in the genome of the non-human animal and altering the position of CH1.

2. The method according to claim 1, wherein the immunoglobulin heavy chain constant region gene comprises the following exons: CH2, CH3, and CH1, and the transcriptional directions are in turn: CH2, CH3, and CH1.

3. The method according to any one of claims 1-2, wherein the CH2, CH3, and CH1 can be derived from IgG and / or IgM.

4. The method according to any one of claims 1-3, wherein the IgG comprises IgG3, IgG1, IgG2A, IgG2B, and / or IgG2C.

5. The method according to any one of claims 1-4, wherein the immunoglobulin heavy chain constant region gene further comprises the following exons: H, M1, and M2, and a gene L encoding a linker, and the transcriptional directions are in turn: H, CH2, CH3, M1, M2, L, and CH1.

6. The method according to claim 5, wherein the linker is a self-cleaving peptide, preferably a self-cleaving peptide with a length of 18-22 amino acids.

7. The method according to any one of claims 5-6, wherein the linker is a 2A peptide, preferably F2A, E2A, P2A, and T2A.

8. The method according to any one of claims 1-7, wherein the immunoglobulin heavy chain constant region gene is derived from the non-human animal.

9. The method according to any one of claims 1-8, which comprises constructing a nucleic acid sequence that sequentially comprises the following exons from the 5'-end to the 3'-end: CH2, CH3, and CH1.

10. The method according to any one of claims 1-9, wherein the nucleic acid sequence sequentially comprises the following exons and a gene L encoding a linker from the 5'-end to the 3'-end: H, CH2, CH3, M1, M2, L, and CH1.

11. The method according to any one of claims 9-10, which comprises inserting the nucleic acid sequence into the immunoglobulin heavy chain constant region locus of the non-human animal.

12. The method according to any one of claims 9-11, which comprises inserting the nucleic acid sequence into the locus of immunoglobulin heavy chain constant region IGHG and / or IGHM of the non-human animal.

13. The method according to any one of claims 9-12, which comprises replacing the endogenous immunoglobulin heavy chain constant region IGHG and / or IGHM gene in the non-human animal with the nucleic acid sequence.

14. The method according to any one of claims 1-13, the method comprising operably linking a human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus of the non-human animal.

15. The method according to claim 14, which comprises operably linking 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 downstream of the heavy chain constant region locus of the non-human animal.

16. The method according to claim 15, wherein the genes of the multiple human heavy chain variable regions V, D or J regions or fragments thereof are directly linked.

17. The method according to any one of claims 14-16, wherein the non-human animal comprises an endogenous immunoglobulin heavy chain variable region gene.

18. The method according to any one of claims 16-17, wherein the genomic integrity of the endogenous immunoglobulin heavy chain variable region of the non-human animal is not altered.

19. The method according to any one of claims 16-18, wherein the function of the expression regulatory element of the endogenous immunoglobulin heavy chain variable region gene of the non-human animal is not disrupted.

20. The method according to any one of claims 1-19, wherein the non-human animal comprises the complete expression regulatory element of the endogenous immunoglobulin heavy chain variable region.

21. The method according to any one of claims 1-20, wherein the non-human animal comprises the complete endogenous immunoglobulin heavy chain variable region gene.

22. The method according to any one of claims 1-21, wherein the non-human animal does not express the endogenous immunoglobulin heavy chain variable region.

23. The method according to any one of claims 1-22, wherein the endogenous immunoglobulin variable region gene of the non-human animal is not expressed as an antibody heavy chain variable region.

24. The method according to any one of claims 1-23, which comprises making the transcriptional direction of the human immunoglobulin heavy chain variable region gene opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

25. The method according to any one of claims 1-24, wherein the immunoglobulin heavy chain constant region gene of the non-human animal is comprised between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene in the non-human animal.

26. The method according to any one of claims 1-25, which comprises making the transcriptional direction of the immunoglobulin heavy chain constant region gene opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

27. The method according to any one of claims 1-26, wherein the transcriptional directions of the human immunoglobulin heavy chain variable region gene and the immunoglobulin heavy chain constant region gene in the non-human animal are the same.

28. The method according to any one of claims 1-27, wherein the chromosome of the non-human animal sequentially comprises, from upstream to downstream: the endogenous immunoglobulin heavy chain variable region gene, the immunoglobulin heavy chain constant region gene, and the human immunoglobulin heavy chain variable region gene.

29. The method according to any one of claims 1-28, wherein the non-human animal is a rodent.

30. The method according to any one of claims 1-29, wherein the non-human animal is a mouse.

31. The method according to claim 30, which comprises inserting the human immunoglobulin heavy chain variable region gene between the positions chr12: 113,149,523 and 113,223,857 on the mouse chromosome.

32. The method according to any one of claims 30-31, which comprises inserting the human immunoglobulin heavy chain variable region gene at the mouse chromosomal position chr12:113,190,256.

33. The method according to any one of claims 1-32, which comprises operably linking the human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus of the non-human animal by site-specific recombination.

34. The method according to any one of claims 1-33, which comprises making the transcriptional direction of the immunoglobulin heavy chain constant region gene of the non-human animal opposite to that of the endogenous immunoglobulin heavy chain variable region gene by genomic modification.

35. A non-human animal, wherein the immunoglobulin heavy chain constant region gene in its genome comprises the following exons: CH2, CH3, and CH1, and the transcriptional directions are in turn: CH2, CH3, and CH1.

36. The non-human animal according to claim 35, wherein the CH2, CH3, and CH1 are derived from IgG and / or IgM.

37. The non-human animal according to claim 36, wherein the IgG comprises IgG3, IgG1, IgG2A, IgG2B, and / or IgG2C.

38. The non-human animal according to any one of claims 35-37, wherein the immunoglobulin heavy chain constant region gene further comprises the following exons: H, M1, and M2, and a gene L encoding a linker, and the transcriptional directions are in turn: H, CH2, CH3, M1, M2, L, and CH1.

39. The non-human animal according to claim 38, wherein the linker is a self-cleaving peptide, preferably a self-cleaving peptide having a length of 18-22 amino acids.

40. The non-human animal according to any one of claims 38-39, wherein the linker is a 2A peptide, preferably F2A, E2A, P2A, and T2A.

41. The non-human animal according to any one of claims 35-40, wherein the immunoglobulin heavy chain constant region gene is derived from the non-human animal.

42. The non-human animal according to any one of claims 35-41, which comprises a human immunoglobulin heavy chain variable region gene operably linked downstream of the immunoglobulin heavy chain locus.

43. The non-human animal according to claim 42, 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 immunoglobulin heavy chain constant region locus.

44. The non-human animal according to claim 42, 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.

45. The non-human animal according to any one of claims 34-44, which comprises an endogenous immunoglobulin heavy chain variable region gene.

46. The non-human animal according to any one of claims 34-45, wherein the genomic integrity of the endogenous immunoglobulin heavy chain variable region is not altered.

47. The non-human animal according to any one of claims 34-46, wherein the function of the expression regulatory element of the endogenous immunoglobulin heavy chain variable region gene is not disrupted.

48. The non-human animal according to any one of claims 34-47, which comprises the expression regulatory element of the complete endogenous immunoglobulin heavy chain variable region.

49. The non-human animal according to any one of claims 34-48, which comprises the complete endogenous immunoglobulin heavy chain variable region gene.

50. The non-human animal according to any one of claims 34-49, which does not express the endogenous immunoglobulin heavy chain variable region.

51. The non-human animal according to any one of claims 34-50, wherein the endogenous immunoglobulin variable region gene is not expressed as an antibody heavy chain variable region.

52. The non-human animal according to any one of claims 34-51, wherein the transcriptional direction of the human immunoglobulin heavy chain variable region gene in its genome is opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

53. The non-human animal according to any one of claims 34-52, wherein the immunoglobulin heavy chain constant region gene of the non-human animal is comprised between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene in its genome.

54. The non-human animal according to any one of claims 34-53, wherein the transcriptional direction of the immunoglobulin heavy chain constant region gene in its genome is opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

55. The non-human animal according to any one of claims 34-54, wherein the transcriptional directions of the human immunoglobulin heavy chain variable region gene and the immunoglobulin heavy chain constant region gene in the non-human animal are the same.

56. The non-human animal according to any one of claims 34-55, wherein its chromosome sequentially comprises, from upstream to downstream: the endogenous immunoglobulin heavy chain variable region gene, the immunoglobulin heavy chain constant region gene, and the human immunoglobulin heavy chain variable region gene.

57. The non-human animal according to any one of claims 34-56, which is a rodent.

58. The non-human animal according to any one of claims 34-57, which is a mouse.

59. The non-human animal according to claim 58, which is a mouse and comprises the human immunoglobulin heavy chain variable region gene between chromosome positions chr12: 113,149,523 to 113,223,857.

60. The non-human animal according to any one of claims 58-59, which is a mouse and the human immunoglobulin heavy chain variable region gene is inserted at chromosome position chr12: 113,190,256.

61. The non-human animal according to any one of claims 34-60, wherein its genome comprises an operably linked human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus.

62. The non-human animal according to claim 61, wherein downstream of the genomic immunoglobulin light chain constant region locus thereof, there are genes of one or more human light chain variable region V regions or light chain variable region J regions or fragments thereof operably linked.

63. The non-human animal according to claim 62, 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.

64. The non-human animal according to any one of claims 34-63, which comprises an endogenous immunoglobulin light chain variable region gene.

65. The non-human animal according to any one of claims 34-64, wherein the genomic integrity of the endogenous immunoglobulin light chain variable region is not altered.

66. The non-human animal according to any one of claims 34-65, wherein the function of the expression regulatory element of the endogenous immunoglobulin light chain variable region gene is not disrupted.

67. The non-human animal according to any one of claims 34-66, which comprises the complete expression regulatory element of the endogenous immunoglobulin light chain variable region.

68. The non-human animal according to any one of claims 34-67, which comprises the complete endogenous immunoglobulin light chain variable region gene.

69. The non-human animal according to any one of claims 34-68, which does not express the endogenous immunoglobulin light chain variable region.

70. The non-human animal according to any one of claims 34-69, wherein the endogenous immunoglobulin variable region gene is not expressed as an antibody light chain variable region.

71. The non-human animal according to any one of claims 34-70, wherein the transcriptional direction of the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene in its genome is opposite.

72. The non-human animal according to any one of claims 34-71, wherein its chromosome sequentially contains, from upstream to downstream: the endogenous immunoglobulin light chain variable region gene, the immunoglobulin light chain constant region gene, and the human immunoglobulin light chain variable region gene.

73. The non-human animal according to any one of claims 34-72, wherein the transcriptional direction of the immunoglobulin light chain constant region gene and the endogenous immunoglobulin light chain variable region gene in its genome is opposite.

74. The non-human animal according to any one of claims 34-73, wherein the transcriptional direction of the human immunoglobulin light chain variable region gene and the immunoglobulin light chain constant region gene in its genome is the same.

75. The non-human animal according to any one of claims 34-74, 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.

76. The non-human animal according to any one of claims 34-75, which is a mouse and contains the human immunoglobulin light chain variable region gene at the chromosomal position chr6: between 70,703,738 and 70,742,704.

77. The non-human animal according to any one of claims 34-76, which is a mouse and comprises the human immunoglobulin light chain variable region gene at chromosomal position chr6: 70,706,267.

78. A non-human animal cell, wherein the immunoglobulin heavy chain constant region genes of its genome include the following exons: CH2, CH3, and CH, H, M1, and M2, and the gene L encoding a linker, and the transcriptional directions are in turn H, CH2, CH3, M1, M2, L, and CH1.

79. 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 35-77 with the antigen.

80. The method according to claim 78, wherein the antibody is a heavy chain only antibody (HCAb).

81. A method for preparing a sample, the method comprising: Exposing the non-human animal according to any one of claims 35-77 to an antigen; And collecting the sample from the non-human animal.

82. The method according to claim 81, wherein the sample comprises immune cells.

83. The method according to any one of claims 81-82, wherein the sample comprises B cells.

84. The method according to any one of claims 81-83, wherein the sample is derived from bone marrow, spleen tissue, lymph nodes, spleen cells, or peripheral lymphocytes.

85. A genetically modified non-human animal genome, wherein the immunoglobulin heavy chain constant region genes thereof include the following exons: CH2, CH3, and CH1, and the transcriptional directions are in turn: CH2, CH3, and CH1.

86. The non-human animal genome according to claim 85, wherein the CH2, CH3, and CH1 are derived from IgG and / or IgM.

87. The non-human animal genome according to claim 86, wherein the IgG includes IgG3, IgG1, IgG2A, IgG2B, and / or IgG2C.

88. The non-human animal genome according to any one of claims 85-87, wherein the immunoglobulin heavy chain constant region genes further include the following exons: H, M1, and M2, and the gene L encoding a linker, and the transcriptional directions are in turn: H, CH2, CH3, M1, M2, L, and CH1.

89. The non-human animal genome according to claim 88, wherein the linker is a self-cleaving peptide, preferably a self-cleaving peptide having a length of 18-22 amino acids.

90. The non-human animal genome according to any one of claims 88-89, wherein the linker is a 2A peptide, preferably F2A, E2A, P2A, and T2A.

91. The non-human animal genome according to any one of claims 85-90, wherein the immunoglobulin heavy chain constant region genes are derived from the non-human animal.

92. The non-human animal genome according to any one of claims 85-91, which comprises an operably linked human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus.

93. The non-human animal genome according to claim 92, which comprises 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 immunoglobulin heavy chain constant region locus.

94. The non-human animal genome according to claim 92, 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.

95. The non-human animal genome according to any one of claims 84-94, which comprises an endogenous immunoglobulin heavy chain variable region gene.

96. The non-human animal genome according to any one of claims 84-95, wherein the genomic integrity of the endogenous immunoglobulin heavy chain variable region is not altered.

97. The non-human animal genome according to any one of claims 84-96, wherein the function of the expression regulatory element of the endogenous immunoglobulin heavy chain variable region gene is not disrupted.

98. The non-human animal genome according to any one of claims 84-97, which comprises the complete expression regulatory element of the endogenous immunoglobulin heavy chain variable region.

99. The non-human animal genome according to any one of claims 84-98, which comprises the complete endogenous immunoglobulin heavy chain variable region gene.

100. The non-human animal genome according to any one of claims 84-99, which does not express the endogenous immunoglobulin heavy chain variable region.

101. The non-human animal genome according to any one of claims 84-90, wherein the endogenous immunoglobulin variable region gene is not expressed as an antibody heavy chain variable region.

102. The non-human animal genome according to any one of claims 84-91, wherein the transcription direction of the human immunoglobulin heavy chain variable region gene is opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

103. The non-human animal genome according to any one of claims 84-92, wherein the immunoglobulin heavy chain constant region gene of the non-human animal is comprised between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene.

104. The non-human animal genome according to any one of claims 84-93, wherein the transcription direction of the immunoglobulin heavy chain constant region gene is opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

105. The non-human animal genome according to any one of claims 84-94, wherein the transcription direction of the human immunoglobulin heavy chain variable region gene and the immunoglobulin heavy chain constant region gene in the non-human animal is the same.

106. The non-human animal genome according to any one of claims 84-95, which comprises, from upstream to downstream in sequence: the endogenous immunoglobulin heavy chain variable region gene, the immunoglobulin heavy chain constant region gene, and the human immunoglobulin heavy chain variable region gene.

107. The non-human animal genome according to any one of claims 84-96, wherein the non-human animal is a rodent.

108. The non-human animal genome according to any one of claims 84-97, wherein the non-human animal is a mouse.

109. The non-human animal genome according to claim 98, wherein the non-human animal is a mouse and comprises the human immunoglobulin heavy chain variable region gene between chromosomal positions chr12: 113,149,523 and 113,223,857.

110. The non-human animal genome according to any one of claims 98-99, which is a mouse and into which the human immunoglobulin heavy chain variable region gene is inserted at chromosomal position chr12: 113,190,256.

111. The non-human animal genome according to any one of claims 84-100, which comprises a human immunoglobulin light chain variable region gene operably linked downstream of an immunoglobulin light chain locus.

112. The non-human animal genome according to claim 111, wherein downstream of the immunoglobulin light chain constant region locus comprises a gene or genes of one or more human light chain variable region V regions or light chain variable region J regions or fragments thereof operably linked.

113. The non-human animal genome according to claim 112, wherein the gene or genes of one or more human light chain variable region V or light chain variable region J regions or fragments thereof are directly linked.

114. The non-human animal genome according to any one of claims 84-113, which comprises an endogenous immunoglobulin light chain variable region gene.

115. The non-human animal genome according to any one of claims 84-114, wherein the genomic integrity of the endogenous immunoglobulin light chain variable region is not altered.

116. The non-human animal genome according to any one of claims 84-115, wherein the function of the expression regulatory element of the endogenous immunoglobulin light chain variable region gene is not disrupted.

117. The non-human animal genome according to any one of claims 84-116, which comprises the expression regulatory element of a complete endogenous immunoglobulin light chain variable region.

118. The non-human animal genome according to any one of claims 84-117, which comprises a complete endogenous immunoglobulin light chain variable region gene.

119. The non-human animal genome according to any one of claims 84-118, which does not express an endogenous immunoglobulin light chain variable region.

120. The non-human animal genome according to any one of claims 84-119, wherein the endogenous immunoglobulin variable region gene is not expressed as an antibody light chain variable region.

121. The non-human animal genome according to any one of claims 84-120, wherein the human immunoglobulin light chain variable region gene is in the opposite transcriptional orientation to the endogenous immunoglobulin light chain variable region gene.

122. The non-human animal genome according to any one of claims 84-121, whose chromosome sequentially comprises, from upstream to downstream: the endogenous immunoglobulin light chain variable region gene, the immunoglobulin light chain constant region gene, and the human immunoglobulin light chain variable region gene.

123. The non-human animal genome according to any one of claims 84-122, wherein the transcription direction of the immunoglobulin light chain constant region gene is opposite to that of the endogenous immunoglobulin light chain variable region gene.

124. The non-human animal genome according to any one of claims 84-123, wherein the transcription direction of the human immunoglobulin light chain variable region gene is the same as that of the immunoglobulin light chain constant region gene.

125. The non-human animal genome according to any one of claims 84-124, 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.

126. The non-human animal genome according to any one of claims 84-125, wherein the non-human animal is a mouse, and the human immunoglobulin light chain variable region gene is contained between chromosome positions chr6: 70,703,738 to 70,742,704.

127. The non-human animal genome according to claim 84-126, wherein the non-human animal is a mouse, and the human immunoglobulin light chain variable region gene is contained at chromosome position chr6: 70,706,267.

128. An engineered chromosome comprising the non-human animal genome according to any one of claims 84-127.

129. A cell comprising the engineered chromosome according to claim 128.

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