Animal model for producing humanized antibody and construction method therefor

Abstract

A non-human animal and a method for preparing the non-human animal. The genome of the non-human animal comprises a human immunoglobulin light-chain variable region gene which is operably linked downstream of an immunoglobulin light-chain locus, and the human immunoglobulin light-chain variable region gene contains only one human V region gene. Further provided are a method for producing an antibody using the non-human animal.

Description

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

[0001] The present application relates to the field of biomedicine, and specifically to a humanized antibody animal model using individual human light chain V regions and a method for constructing the same. Background Art

[0002] After more than three decades of development, therapeutic monoclonal antibody drugs have become one of the most important components of biopharmaceuticals and a rapidly growing, most advanced, and most effective class of drugs. With the advancement of this drug class, monoclonal antibodies capable of binding to two different antigens or epitopes have been developed, namely bispecific monoclonal antibodies (BsMAbs or BsAbs). BsAbs are synthetic, antibody-like proteins that can simultaneously bind to two different antigens or two different epitopes on the same antigen. In other words, bispecific antibodies combine the antigen-binding regions (Fab, fragment of antigen binding) of two different antibodies. Compared to conventional antibodies, BsAbs can bind to two targets simultaneously, which not only physiologically interferes with different biologically active epitopes simultaneously but also physically brings the two antigens closer together. BsAbs can be designed for a variety of applications, including increasing target specificity, recruiting and activating immune cells, interfering with receptor signaling and inactivating signaling ligands, and forcing protein complex binding.

[0003] One of the methods that can be used for the discovery and screening of humanized BsAbs is to use non-human animals engineered to carry human antibody genes. Among them, in order to avoid the diversity brought about by VJ recombination of light chains, the use of humanized common light chain mice (Common Light Chain) carrying only a single human light chain for molecular discovery is a low-cost, time-efficient, high-affinity humanized BsAb antibody discovery method. The antibody molecules obtained through the development of common light chain mice have consistent light chain variable regions, which can be used to avoid non-target heavy and light chain pairing when producing conventional IgG-like BsAbs. This method not only increases the acquisition rate from 12.5% ​​to 50%, but also greatly simplifies the BsAb antibody purification process. Combined with commonly used techniques such as Knob-in-hole, the production acquisition rate of BsAb antibodies can be further increased to 100%.

[0004] Currently, the most common approach used in the industry involves inactivating or deleting all light chain variable region coding regions in the non-human animal genome and inserting a single, functional, fully rearranged human light chain variable region at the endogenous light chain coding genomic location. However, this approach often results in genetic defect phenotypes due to the deletion of large fragments of the non-human animal's endogenous genes. Therefore, a new method for constructing humanized common light chain non-human animal models is urgently needed. Summary of the Invention

[0005] The application provides a kind of non-human animal and the method for preparing non-human animal.On the one hand, the genome of described non-human animal comprises 1 people V district gene and limited number of people J district gene (for example 1, 2, 3, 4 or 5 J district gene) in immunoglobulin light chain gene seat downstream.Described non-human animal, by the antibody selection process of complexity in organism, when the diversified set of people's heavy chain variable domain is paired with limited number of people's light chain variable domain option, makes biologically appropriate selection.Described non-human animal is engineered to present limited number of people's light chain variable domain option and is combined with extensive people's heavy chain variable domain option.Under the challenge of immunogen, described non-human animal develops the antibody of immunogen, mainly or completely subject to the quantity restriction of the light chain option of its pedigree.In various embodiments, the antibody produced by described non-human animal has the heavy chain that can be combined with identical or substantially identical light chain.When preparing BsAb, it is not necessary to associate a light chain with a specific heavy chain by antibody engineering (for example, introducing modification to sequence). This can greatly improve the success rate of bispecific antibody development, thereby overcoming the above-mentioned defects caused by a large number of heavy chain VDJ recombination and light chain VJ recombination.In fact, the antibodies or sequences described herein can be further combined with each other to prepare multispecific antibodies.

[0006] On the other hand, in the genome of the non-human animal, the human variable region gene (for example, the light chain variable region, the heavy chain variable region, or the light chain variable region and the heavy chain variable region) is inserted downstream (or behind) the endogenous immunoglobulin locus of the non-human animal cell. At the same time, the endogenous immunoglobulin constant region gene of the non-human animal is inverted (that is, its transcription direction is opposite to that of the endogenous immunoglobulin variable region gene of the non-human animal), thereby avoiding the insertion of large fragments of DNA sequences or the deletion and modification of endogenous DNA sequences in the endogenous immunoglobulin locus of the non-human animal (for example, mouse), thereby eliminating interference with the expression and regulation of the endogenous genes of the non-human animal, and overcoming the above-mentioned defects caused by the inactivation and deletion of large fragments of the endogenous genes of the non-human animal itself.

[0007] In addition, the non-human animals and methods for producing non-human animals disclosed herein have other advantages. For example, in some cases, the inserted exogenous human immunoglobulin variable regions are located away from the endogenous variable regions and are transcribed in the opposite direction to them, thereby preventing the endogenous variable regions from being integrated into the human variable regions during recombination, thereby preventing the generated antibodies from containing endogenous immune epitopes.

[0008] On the one hand, the present application provides a method for preparing a non-human animal, the method comprising operably linking a human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus of the non-human animal, wherein the human immunoglobulin light chain variable region gene comprises only one human V region gene.

[0009] In certain embodiments, the human immunoglobulin light chain variable region gene further comprises one or more human J region genes. In certain embodiments, the human immunoglobulin light chain variable region gene comprises 2, 3, 4, or 5 human J region genes. In certain embodiments, the human V region gene and the one or more human J region genes are operably linked.

[0010] In certain embodiments, the human V region gene is selected from the group consisting of IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, IGKV1D-33, IGKV 1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-9, IGKV2D-29, IGKV1D-12, IGKV1-12, IGKV1-27, and IGKV1-16. In certain embodiments, the human V region gene is selected from the group consisting of IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, and IGKV1D-33. In certain embodiments, a gene rearrangement intron sequence is included between the human V region gene and the J region gene. In certain embodiments, a gene rearrangement intron sequence is not included between the human V region gene and the J region gene.

[0011] In certain embodiments, the human J region gene is selected from the group consisting of IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1. In certain embodiments, the human J region gene comprises a gene rearrangement intron sequence. In certain embodiments, the human J region gene does not comprise a gene rearrangement intron sequence.

[0012] In certain embodiments, the method comprises operably connecting a human immunoglobulin heavy chain variable region gene downstream of the heavy chain locus of the non-human animal. In certain embodiments, the method comprises operably connecting one or more human heavy chain V, D or J regions or fragments thereof downstream of the heavy chain constant region locus of the non-human animal. In certain embodiments, the genes of the multiple human heavy chain V, D or J regions or fragments thereof can be recombined. In certain embodiments, the genes of the multiple human heavy chain V, D or J regions or fragments thereof are directly connected.

[0013] In another aspect, the present application provides a non-human animal or non-human animal cell, wherein the genome of the non-human animal comprises an operably linked human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus, wherein the human immunoglobulin light chain variable region gene comprises only one human V region gene. The following features included in the non-human animal are all included in the non-human animal cell.

[0014] On the other hand, the present application also provides a genetically modified non-human animal genome, which comprises an operably linked human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus, wherein the human immunoglobulin light chain variable region gene comprises only one human V region gene.

[0015] In certain embodiments, the human immunoglobulin light chain variable region gene further comprises one or more human J region genes, preferably 2 to 5. In certain embodiments, the human V region gene and the one or more human J region genes are operably linked.

[0016] In certain embodiments, the human V region gene is selected from the group consisting of IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, IGKV1D-33, IGKV 1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-9, IGKV2D-29, IGKV1D-12, IGKV1-12, IGKV1-27, and IGKV1-16. In certain embodiments, the human V region gene is selected from the group consisting of IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, and IGKV1D-33. In certain embodiments, a gene rearrangement intron sequence is included between the human V region gene and the J region gene. In certain embodiments, a gene rearrangement intron sequence is not included between the human V region gene and the J region gene.

[0017] In certain embodiments, the human J region gene is selected from the group consisting of IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1. In certain embodiments, the human J region gene comprises a gene rearrangement intron sequence. In certain embodiments, the human J region gene does not comprise a gene rearrangement intron sequence.

[0018] In certain embodiments, the genome of the non-human animal further comprises an endogenous immunoglobulin light chain variable region gene. In certain embodiments, the genomic integrity of the endogenous immunoglobulin light chain variable region gene is not altered. In certain embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin light chain variable region gene is not disrupted. In certain embodiments, the genome of the non-human animal comprises a complete set of expression regulatory elements for the endogenous immunoglobulin light chain variable region. In certain embodiments, the genome of the non-human animal comprises a complete set of endogenous immunoglobulin light chain variable region genes.

[0019] In certain embodiments, the non-human animal does not express endogenous immunoglobulin light chain variable regions. In certain embodiments, the non-human animal's endogenous immunoglobulin light chain variable region genes are not expressed as antibody light chain variable regions.

[0020] In certain embodiments, the transcription direction of the human immunoglobulin light chain variable region gene in the non-human animal is opposite to that of the endogenous immunoglobulin light chain variable region gene. In certain embodiments, the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene contain an immunoglobulin light chain constant region gene of the non-human animal.

[0021] In certain embodiments, the immunoglobulin light chain constant region genes in the non-human animal are transcribed in the opposite direction as the endogenous immunoglobulin light chain variable region genes.

[0022] In certain embodiments, the human immunoglobulin light chain variable region gene in the non-human animal is transcribed in the same direction as the immunoglobulin light chain constant region gene of the non-human animal.

[0023] In certain embodiments, the human immunoglobulin light chain variable region gene in the non-human animal comprises a light chain constant region gene between the endogenous immunoglobulin light chain variable region gene. In certain embodiments, the distance between the human immunoglobulin light chain variable region gene in the non-human animal and the endogenous immunoglobulin light chain variable region gene is 4 Kbp-42 Kbp.

[0024] In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse. In certain embodiments, the mouse comprises the human immunoglobulin light chain variable region gene at its locus between the lgkc and Rpia genes. In certain embodiments, the mouse comprises the human immunoglobulin light chain variable region gene at chromosomal position chr6:70,703,738 to 70,742,704. In certain embodiments, the mouse comprises the human immunoglobulin light chain variable region gene at chromosomal position chr6:70,706,267.

[0025] In certain embodiments, the genome of the non-human animal comprises an operably connected human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus. In certain embodiments, the heavy chain constant region locus downstream of the non-human animal comprises an operably connected gene of 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 of the multiple human heavy chain V region, heavy chain variable region D region or heavy chain variable region J region or a fragment thereof can be recombined. In certain embodiments, the genes of the multiple human heavy chain variable region V region, heavy chain variable region D region or heavy chain variable region J region or a fragment thereof are directly connected.

[0026] In certain embodiments, the genome of the non-human animal comprises endogenous immunoglobulin heavy chain variable region genes. In certain embodiments, the genomic integrity of the endogenous immunoglobulin heavy chain variable region genes 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 genes of the non-human animal is not impaired. In certain embodiments, the genome of the non-human animal comprises complete endogenous immunoglobulin heavy chain variable region expression regulatory elements. In certain embodiments, the genome of the non-human animal comprises complete endogenous immunoglobulin heavy chain variable region genes.

[0027] 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.

[0028] In certain embodiments, the human immunoglobulin heavy chain variable region gene of the non-human animal is transcribed in the opposite direction to the endogenous immunoglobulin heavy chain variable region gene. In certain embodiments, the human immunoglobulin heavy chain variable region gene of the non-human animal and the endogenous immunoglobulin heavy chain variable region gene contain an immunoglobulin heavy chain constant region gene of the non-human animal between them.

[0029] In certain embodiments, 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.

[0030] In certain embodiments, the transcription direction of the human immunoglobulin heavy chain variable region gene of the non-human animal is the same as that of the immunoglobulin heavy chain constant region gene of the non-human animal.

[0031] In certain embodiments, the human immunoglobulin heavy chain variable region gene in the non-human animal comprises a heavy chain constant region gene between the endogenous immunoglobulin heavy chain variable region gene. In certain embodiments, the distance between the human immunoglobulin heavy chain variable region gene in the non-human animal and the endogenous immunoglobulin heavy chain variable region gene is 169 Kbp-240 Kbp.

[0032] In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse. In certain embodiments, the mouse comprises the human immunoglobulin heavy chain variable region gene between the Tmem121 and Igha genes at its locus. In certain embodiments, the mouse comprises the human immunoglobulin heavy chain variable region gene between chromosomal locations chr12:113,149,523 and 113,223,857. In certain embodiments, the mouse has the human immunoglobulin heavy chain variable region gene inserted at chromosomal location chr12:113,190,256.

[0033] 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.

[0034] 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.

[0035] On the other hand, the present application provides an engineered chromosome comprising the non-human animal genome described in the present application.

[0036] On the other hand, the application provides a kind of non-human animal cell, the genome of described non-human animal cell comprises the human immunoglobulin light chain variable region gene that is operably connected downstream of immunoglobulin light chain locus, and described human immunoglobulin light chain variable region gene comprises only one human V district gene.In some embodiments, described cell is embryonic stem (ES) cell.In some embodiments, described cell is germ cell.In some embodiments, described cell is fertilized egg cell.In some embodiments, described cell is embryonic cell.

[0037] In certain embodiments, the non-human animal cell can develop into a non-human animal.

[0038] In another aspect, the present application provides cells, tissues and organs derived from non-human animals.

[0039] In another aspect, the present application provides a method for preparing an antibody that specifically binds to an antigen, the method comprising immunizing the non-human animal described herein with the antigen.

[0040] 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.

[0041] On the other hand, the present application provides a method for preparing an antibody that specifically binds to an antigen, the method comprising: exposing the non-human animal or cell to the antigen, and sequencing the nucleic acid encoding the human heavy chain and light chain immunoglobulin variable regions in the animal or cell. The non-human animal or cell can express a chimeric antibody that specifically binds to the antigen. In one embodiment, the method may comprise operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region with the nucleic acid encoding the heavy chain immunoglobulin constant region, and expressing the antibody that specifically binds to the antigen. The constant region may be a human heavy chain constant region or a heavy chain constant region of a non-human animal. In one embodiment, the method may comprise operably linking the nucleic acid encoding the human light chain immunoglobulin variable region with the nucleic acid encoding the light chain immunoglobulin constant region, and expressing the antibody that specifically binds to the antigen. The constant region may be a human light chain constant region or a light chain constant region of a non-human animal.

[0042] On the other hand, the present application provides a method for preparing antibodies with identical or similar light chain variable regions that specifically bind to two different antigens, the method comprising: exposing the non-human animal or cell to the two different antigens, and sequencing the nucleic acids encoding the human heavy and light chain immunoglobulin variable regions in the animal or cell. The non-human animal or cell can express a series of chimeric antibodies with identical or similar light chain variable regions that specifically bind to the different antigens. In one embodiment, the method can comprise operably linking nucleic acids encoding the human heavy chain immunoglobulin variable regions that specifically bind to the two different antigens to nucleic acids encoding heavy chain immunoglobulin constant regions, operably linking nucleic acids encoding the identical or similar human light chain immunoglobulin variable regions to nucleic acids encoding light chain immunoglobulin constant regions, and ultimately expressing bispecific antibodies that specifically bind to the two different antigens. The heavy chain immunoglobulin constant region can be a human heavy chain constant region or a non-human animal heavy chain constant region. The light chain immunoglobulin constant region can be a human light chain constant region or a non-human animal light chain constant region.

[0043] In some embodiments, the antibody is a shared light chain antibody. In some embodiments, the shared light chain antibody is a shared light chain bispecific antibody. On the other hand, the present application provides a method for preparing a sample, the method comprising: exposing the 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 is derived from bone marrow, spleen tissue, lymph nodes, splenocytes, or peripheral lymphocytes.

[0044] The non-human animal described herein can be a rodent, for example, a mouse.

[0045] The technical solution of this application includes one or more of the following advantages:

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] The non-human animals described herein also have advantages in antibody production. For example, in some embodiments, a limited number of human variable region gene segments (comprising the entire human IGK J region and a small number of human IGK V regions) are gradually introduced and inserted downstream (behind) the endogenous immunoglobulin loci in cells of a non-human animal (e.g., a mouse). The resulting variable domains produced by the non-human animal can have limited light chain diversity, thus avoiding the diversity caused by VJ recombination in the light chain.

[0052] The non-human animals described in the present application also have advantages in producing antibodies. For example, in some embodiments, the present application avoids the problem of different V region usage. Under natural conditions, there are differences in the frequency of use of the V region of the light chain variable region of human immunoglobulin. The reason for this situation may be the physical arrangement of the spatial distance between the V region, J region and C region in the human genome. The strategy of the present application uses a single IGKV, which avoids the problem of light chain V region usage, so that all inserted human IGKVs can be used in almost all B cells in the living biological system of the mouse and obtain sufficient expression levels. In some embodiments, the expression of the V region will not be affected by its low frequency of use in nature, and it can be used to develop antibodies using low-frequency IGKV, such as in the development of antibody drugs for some emerging epidemic viruses. It has unique advantages.

[0053] 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.

[0054] The method for preparing non-human animals described in the present application also has advantages. For example, in some embodiments, the method of the present application can be used to quickly prepare a series of non-human animals carrying different human light chain variable region V genes. In some embodiments, the endogenous constant region of the non-human animal is first inverted to obtain an intermediate (cell or animal), and then a different single human IGK V region is inserted downstream of the endogenous immunoglobulin locus of this intermediate to obtain a series of non-human animals carrying different human light chain variable region V genes. By performing antibody discovery in non-human animals carrying different human light chain variable region V genes, the situation in which high-affinity antibodies cannot be obtained for a certain target due to the lack of diversity in the IGK V region is avoided to the greatest extent. At the same time, a variety of different single human IGK V region animals can also be used to perform antibody discovery on some common targets of multi-antibody drugs (such as CD3 and CD16A), and to establish universal shelf-type antibody molecules to match other targets with increasing diversity.

[0055] This application also provides the following implementation scheme:

[0056] The present application provides a method for preparing a non-human animal, the method comprising operably linking a human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus of the non-human animal, wherein the human immunoglobulin light chain variable region gene comprises only one human V region gene.

[0057] In some embodiments, the human immunoglobulin light chain variable region gene further comprises one or more human J region genes.

[0058] In some embodiments, the human immunoglobulin light chain variable region gene comprises 2 to 5 human J region genes.

[0059] In some embodiments, the one human V region gene and one or more human J region genes are operably linked.

[0060] In some embodiments, the human V region gene is selected from the group consisting of IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, and IGKV1D-33.

[0061] In some embodiments, a gene rearrangement intron sequence is contained between the human V region gene and the J region gene.

[0062] In some embodiments, the human V region gene and the J region gene do not contain a gene rearrangement intron sequence.

[0063] In some embodiments, the human J region gene is selected from the group consisting of IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1.

[0064] In some embodiments, the human J region gene comprises a gene rearranged intron sequence.

[0065] In some embodiments, the human J region gene does not comprise a gene rearranged intron sequence.

[0066] In some embodiments, the non-human animal comprises an endogenous immunoglobulin light chain variable region gene.

[0067] In some embodiments, the non-human animal's endogenous immunoglobulin light chain variable region genomic integrity is unaltered.

[0068] In some embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin light chain variable region genes of the non-human animal is not disrupted.

[0069] In some embodiments, the non-human animal comprises expression regulatory elements for a complete endogenous immunoglobulin light chain variable region.

[0070] In some embodiments, the non-human animal comprises a complete endogenous immunoglobulin light chain variable region gene.

[0071] In some embodiments, the non-human animal does not express endogenous immunoglobulin light chain variable regions.

[0072] In some embodiments, the non-human animal's endogenous immunoglobulin light chain variable region genes are not expressed as antibody light chain variable regions.

[0073] In some embodiments, the method includes causing the transcription of the human immunoglobulin light chain variable region gene to be in the opposite direction to that of the endogenous immunoglobulin light chain variable region gene.

[0074] In some embodiments, a non-human animal immunoglobulin light chain constant region gene is contained between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene.

[0075] In some embodiments, the method comprises directing transcription of the immunoglobulin light chain constant region gene of the non-human animal in the opposite direction to that of the endogenous immunoglobulin light chain variable region gene.

[0076] In some embodiments, in the non-human animal, 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 of the non-human animal.

[0077] In certain embodiments, the human immunoglobulin light chain variable region gene in the non-human animal comprises a light chain constant region gene between the endogenous immunoglobulin light chain variable region gene. In some embodiments, the distance between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene is 4 Kbp-42 Kbp.

[0078] In some embodiments, the non-human animal is a rodent.

[0079] In some embodiments, the non-human animal is a mouse.

[0080] In some embodiments, the method comprises inserting the human immunoglobulin light chain variable region gene between the lgkc and Rpia genes of the mouse locus.

[0081] In some embodiments, the method comprises inserting the human immunoglobulin light chain variable region gene into a mouse chromosome between positions chr6:70,703,738-70,742,704.

[0082] In some embodiments, the method comprises inserting a human immunoglobulin light chain variable region gene at mouse chromosome position chr6:70,706,267.

[0083] In some embodiments, the method comprises operably linking the human immunoglobulin light chain variable region gene downstream of the immunoglobulin locus of the non-human animal by site-directed recombination.

[0084] In some embodiments, the method comprises modifying the genome of the non-human animal so that the transcription direction of the immunoglobulin light chain constant region gene is opposite to that of the endogenous immunoglobulin light chain variable region gene.

[0085] In some embodiments, the method further comprises operably linking a human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus of the non-human animal.

[0086] In some embodiments, the method includes operably linking one or more 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.

[0087] In some embodiments, the genes of the plurality of human heavy chain variable region V, heavy chain variable region D or heavy chain variable region J regions or fragments thereof are directly linked.

[0088] In some embodiments, the non-human animal comprises an endogenous immunoglobulin heavy chain variable region gene.

[0089] In some embodiments, the non-human animal's endogenous immunoglobulin heavy chain variable region genomic integrity is unaltered.

[0090] In some embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin heavy chain variable region genes of the non-human animal is not disrupted.

[0091] In some embodiments, the non-human animal comprises expression regulatory elements for a complete endogenous immunoglobulin heavy chain variable region.

[0092] In some embodiments, the non-human animal comprises a complete endogenous immunoglobulin heavy chain variable region gene.

[0093] In some embodiments, the non-human animal does not express endogenous immunoglobulin heavy chain variable regions.

[0094] In some embodiments, the non-human animal's endogenous immunoglobulin variable region genes are not expressed as antibody heavy chain variable regions.

[0095] In some embodiments, it includes causing the transcription direction of the human immunoglobulin heavy chain variable region gene to be opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

[0096] In some 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.

[0097] In some embodiments, it includes causing the transcription direction of the immunoglobulin heavy chain constant region gene of the non-human animal to be opposite to that of the endogenous immunoglobulin heavy chain variable region gene.

[0098] In some embodiments, the transcription direction of the human immunoglobulin heavy chain variable region gene in the non-human animal is the same as that of the immunoglobulin heavy chain constant region gene of the non-human animal.

[0099] In certain embodiments, the human immunoglobulin heavy chain variable region gene in the non-human animal comprises a heavy chain constant region gene between the endogenous immunoglobulin heavy chain variable region gene. In some embodiments, the distance between the human immunoglobulin heavy chain variable region gene in the non-human animal and the endogenous immunoglobulin heavy chain variable region gene is 169 Kbp-240 Kbp.

[0100] In some embodiments, it comprises inserting the human immunoglobulin heavy chain variable region gene between the Tmem121 and Igha genes of the mouse locus.

[0101] In some embodiments, it comprises inserting the human immunoglobulin heavy chain variable region gene into the mouse chromosome position chr12: 113,149,523 to 113,223,857.

[0102] In some embodiments, it comprises inserting the human immunoglobulin heavy chain variable region gene at mouse chromosome position chr12:113,190,256.

[0103] In some embodiments, it comprises operably linking a human immunoglobulin variable region gene downstream of the immunoglobulin locus of the non-human animal by site-directed recombination.

[0104] In some embodiments, it includes modifying the genome so that the transcription direction of the immunoglobulin constant region gene of the non-human animal is opposite to that of the endogenous immunoglobulin variable region gene.

[0105] The present application provides a non-human animal whose genome comprises an operably linked human immunoglobulin light chain variable region gene downstream of an immunoglobulin light chain locus, wherein the human immunoglobulin light chain variable region gene comprises only one human V region gene.

[0106] In some embodiments, the human immunoglobulin light chain variable region gene further includes one or more human J region genes, preferably 2 to 5.

[0107] In some embodiments, the one human V region gene and one or more human J region genes are operably linked.

[0108] In some embodiments, the human V region gene is selected from the group consisting of IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, and IGKV1D-33.

[0109] In some embodiments, the human V region gene and the J region contain a gene rearrangement intron sequence.

[0110] In some embodiments, the human V region gene and the J region do not contain a gene rearrangement intron sequence.

[0111] In some embodiments, the human J region gene is selected from the group consisting of IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1.

[0112] In some embodiments, the human J region gene comprises a gene rearranged intron sequence.

[0113] In some embodiments, the human J region gene does not comprise a gene rearranged intron sequence.

[0114] In some embodiments, the non-human animal genome further comprises an endogenous immunoglobulin light chain variable region gene.

[0115] In some embodiments, the endogenous immunoglobulin light chain variable region genomic integrity is unaltered.

[0116] In some embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin light chain variable region gene is not disrupted.

[0117] In some embodiments, the non-human animal genome comprises a complete endogenous immunoglobulin light chain variable region expression regulatory element.

[0118] In some embodiments, the non-human animal genome comprises a complete endogenous immunoglobulin light chain variable region gene.

[0119] In some embodiments, the non-human animal does not express endogenous immunoglobulin light chain variable regions.

[0120] In some embodiments, the endogenous immunoglobulin light chain variable region genes are not expressed as antibody light chain variable regions.

[0121] In some embodiments, the human immunoglobulin light chain variable region gene is transcribed in the opposite direction to the endogenous immunoglobulin light chain variable region gene.

[0122] In some embodiments, a non-human animal immunoglobulin light chain constant region gene is contained between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene.

[0123] In some embodiments, the immunoglobulin light chain constant region gene is transcribed in the opposite direction to the endogenous immunoglobulin light chain variable region gene.

[0124] In some embodiments, the transcription direction of the human immunoglobulin light chain variable region gene is the same as that of the non-human animal immunoglobulin constant region gene.

[0125] In certain embodiments, the human immunoglobulin light chain variable region gene in the non-human animal comprises a light chain constant region gene between the endogenous immunoglobulin light chain variable region gene. In some embodiments, the distance between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene is 4 Kbp-42 Kbp.

[0126] In some embodiments, the non-human animal is a rodent.

[0127] In some embodiments, the non-human animal is a mouse.

[0128] In some embodiments, the non-human animal comprises the human immunoglobulin light chain variable region gene between the lgkc and Rpia genes at the locus.

[0129] In some embodiments, the non-human animal comprises the human immunoglobulin light chain variable region gene between chromosome positions chr6:70,703,738-70,742,704.

[0130] In some embodiments, the non-human animal comprises a human immunoglobulin light chain variable region gene at chromosome position chr6:70,706,267.

[0131] In some embodiments, the non-human animal genome comprises an operably linked human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus.

[0132] In some embodiments, downstream of the heavy chain constant region locus comprises one or more operably linked human heavy chain variable region V region, heavy chain variable region D region, or heavy chain variable region J region or fragments thereof.

[0133] In some embodiments, the genes of the plurality of human heavy chain variable region V, heavy chain variable region D or heavy chain variable region J regions or fragments thereof are directly linked.

[0134] In some embodiments, the non-human animal genome comprises an endogenous immunoglobulin heavy chain variable region gene.

[0135] In some embodiments, the endogenous immunoglobulin heavy chain variable region genomic integrity is unaltered.

[0136] In some embodiments, the function of the expression regulatory elements of the endogenous immunoglobulin heavy chain variable region gene is not disrupted.

[0137] In some embodiments, the non-human animal genome comprises a complete endogenous immunoglobulin heavy chain variable region expression regulatory element.

[0138] In some embodiments, the non-human animal genome comprises a complete endogenous immunoglobulin heavy chain variable region gene.

[0139] In some embodiments, the non-human animal does not express endogenous immunoglobulin heavy chain variable regions.

[0140] In some embodiments, the endogenous immunoglobulin variable region genes are not expressed as antibody heavy chain variable regions.

[0141] In some embodiments, the human immunoglobulin heavy chain variable region gene is transcribed in the opposite direction to the endogenous immunoglobulin heavy chain variable region gene.

[0142] In some embodiments, the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene contain an immunoglobulin heavy chain constant region gene of a non-human animal.

[0143] In some embodiments, 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.

[0144] In some embodiments, the human immunoglobulin heavy chain variable region gene and the non-human animal immunoglobulin heavy chain constant region gene are transcribed in the same direction.

[0145] In certain embodiments, the human immunoglobulin heavy chain variable region gene in the non-human animal comprises a heavy chain constant region gene between the endogenous immunoglobulin heavy chain variable region gene. In some embodiments, the distance between the human immunoglobulin heavy chain variable region gene and the endogenous immunoglobulin heavy chain variable region gene is 169 Kbp-240 Kbp.

[0146] In some embodiments, the non-human animal is a mouse and comprises the human immunoglobulin heavy chain variable region gene between the Tmem121 and Igha genes at the locus.

[0147] In some embodiments, the non-human animal is a mouse, and comprises the human immunoglobulin heavy chain variable region gene between chromosome positions chr12: 113,149,523 to 113,223,857.

[0148] In some embodiments, the non-human animal is a mouse, and the human immunoglobulin heavy chain variable region gene is inserted at chromosome position chr12:113,190,256.

[0149] 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

[0150] 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:

[0151] FIG1 shows an exemplary IgK constant region Cpoint and LD targeting and C region modification scheme.

[0152] Figure 2 shows a schematic diagram of the insertion of the human variable region of the light chain, including IGK-vector targeting and resistance marker deletion.

[0153] FIG3 shows an exemplary IgH constant region Cpoint and LD targeting and C region modification scheme.

[0154] Figure 4 shows a schematic diagram of the heavy chain human variable region insertion, including IGH-vector targeting and resistance marker deletion.

[0155] FIG5 shows the PCR identification results of the Cpoint modification of the light chain.

[0156] FIG6 shows the PCR identification results of the Cpoint modification of the heavy chain.

[0157] FIG7 shows the results of LD PCR identification of the light chain.

[0158] FIG8 shows the results of LD PCR identification of the heavy chain.

[0159] FIG9 shows the results of inversion PCR identification of the light chain constant region.

[0160] FIG10 shows the results of inversion PCR identification of the heavy chain constant region.

[0161] FIG11 shows the results of light chain resistance marker deletion identification.

[0162] FIG12 shows the results of heavy chain resistance marker deletion identification.

[0163] Figures 13A and 13B show the results of light chain IGK VJ insertion identification.

[0164] Figures 14A and 14B show the results of PCR identification of IGH vector1 targeting and integrity.

[0165] FIG15 shows the results of PCR identification of the IGH vector1 resistance marker deletion.

[0166] FIG16 shows the frequency of V region usage of human immunoglobulin light chain (IGK).

[0167] FIG17 shows the frequency of J region usage of human immunoglobulin light chain (IGK).

[0168] FIG18 shows two feasible strategies for connecting IGK V and IGK J exemplified in the present application.

[0169] FIG19 shows the usage counts of the light chain variable region V and J regions in BCR sequencing of humanized mice.

[0170] FIG20 shows that the human IGK variable region V region is utilized in nearly 100% of all B cells in humanized mice.

[0171] FIG21 shows that the human IGK variable region J region is nearly 100% utilized in all B cells in humanized mice.

[0172] Figure 22 shows the development of immune cells in the spleen of humanized mice.

[0173] Figure 23 shows the development of myeloid immune cells in humanized mice.

[0174] FIG24 shows the affinity analysis results of 12 unique antibody molecules.

[0175] FIG25 shows the Ec50 data of the Unique antibody molecule.

[0176] FIG26 shows the FACS analysis of yeast enrichment of human antigens at different concentrations.

[0177] FIG27 shows the affinity detection of antibodies in the supernatants of different clones at the FACS level.

[0178] Figure 28 shows the BCR sequencing results of humanized mouse IGH.

[0179] Figure 29 shows the BCR sequencing results of humanized mouse IGK.

[0180] FIG30 shows the results of ELISA titer detection of humanized mouse serum antibodies. DETAILED DESCRIPTION

[0181] 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.

[0182] Definition of terms

[0183] 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, sheep, dogs, cats, horses, cows, birds, amphibians, reptiles, etc. For example, the non-human animal can be a rat or a mouse.

[0184] In this application, the term "constant region" generally refers to the sum of the domains of an antibody excluding the variable region. The constant region is not directly involved in the binding of antigens, but exhibits different effector functions. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies are divided into the following categories: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into categories such as IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different types of antibodies are referred to as α, δ, ε, γ and μ, respectively. The light chain constant regions that can be found in all five antibody classes are referred to as κ (kappa) and λ (lambda). Genes encoding mouse (Mus musculus) constant regions can include IGHA, IGHD, IGHE, IGHG1, IGHG2A, IGHG2B, IGHG2C, IGHG3 or IGHM. Information about mouse constant region loci can be found in the IMGT Repertoire:

[0185] https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=Mus_musculus&group=IGHC

[0186] 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 and light chains, divided into the heavy chain variable region and the light chain variable region, respectively. Based on the exons encoded by its gene, the variable region can be further subdivided into the V (variable) region, the D (diversity) region, and the J (joining) region, of which the light chain variable region is composed only of the V (variable) region and the J (joining) region. The variable region is different between different B cell immunoglobulins, but is identical between all immunoglobulins produced by the same B cell. The variable region is produced by somatic cells through a genetic recombination process that occurs during B cell maturation. This process is called rearrangement, which produces a large variety of proteins that can bind to any given antigen, thereby enabling the immune system to recognize and neutralize the large antigenic burden caused by foreign and pathogenic structures. Therefore, the antibody repertoire is composed of a rich variety of immunoglobulins with different V regions, but these immunoglobulins have the same Fc part.

[0187] 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.

[0188] In this application, the term "antibody" generally refers to a whole antibody or an antigen-binding fragment thereof. And optionally, a scaffold or skeleton 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 show 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).

[0189] In this application, the term "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.

[0190] 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.

[0191] In this application, the term "downstream" is generally used to describe the relative position between genes in a chromosome, and can be based on the chromosome or a given gene. In order to better summarize the common points of modification of the light chain and heavy chain loci in this application, the position of the natural non-human animal host immunoglobulin locus and its immunoglobulin transcription direction (i.e., publicly displayed in the NCBI database) are used as a reference. For example, for gene A, if the position of gene A is behind the coding end of the immunoglobulin locus relative to the host immunoglobulin locus, then A is considered to be downstream of the host immunoglobulin locus.

[0192] 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).

[0193] In this application, the term "gene rearrangement intron" generally refers to a DNA sequence that plays a role in the DNA rearrangement of the immunoglobulin variable region and is generally not transcribed into RNA or expressed as a protein or polypeptide. A gene rearrangement intron may include a rearrangement signal sequence (RSS), which is a sequence located at both ends of the V, D, and J segments in the Ig germline gene, including a 7-nucleotide sequence with a palindromic feature (CACAGTG) and a 9-nucleotide sequence rich in A (ACAAAAACC) plus a 12 or 23 bp spacer sequence between the two. For example, when the gene is not rearranged, the intron sequence between the V region and the J region of the light chain variable region contains an RSS sequence, but after gene rearrangement, the V region and the J region of the light chain variable region do not contain the RSS sequence in the VJ intron.

[0194] Detailed Description of the Invention

[0195] Non-human animals or cells

[0196] This application relates to genetically modified non-human animals and cells containing functional human immunoglobulin variable region genes capable of producing chimeric antibodies with human variable regions. Research conducted in mice is provided herein for illustrative purposes only. Unless otherwise specified, references to mice include all non-human mammals, with mice being the preferred non-human mammal.

[0197] In order to obtain a non-human animal that produces antibodies with human variable regions, a human variable region gene (e.g., a light chain variable region, a heavy chain variable region, or a light chain variable region and a heavy chain variable region) is operably linked to the downstream endogenous immunoglobulin locus of the non-human animal cell. At the same time, in some embodiments, the non-human animal's endogenous immunoglobulin constant region is modified in whole or in part (e.g., the constant region is inverted in whole or in part) so that its transcription direction is opposite to that of the endogenous variable region gene. In this way, while retaining the non-human animal's immunoglobulin variable region gene and constant region gene, the expression of the non-human animal's endogenous variable region can be avoided. Furthermore, the transcription direction of the non-human animal's endogenous variable region gene can be made opposite to that of the non-human animal's endogenous constant region and human variable region gene, thereby avoiding V(D)J recombination between the endogenous variable region and the endogenous constant region to produce antibodies, and also avoiding the integration of the endogenous variable region into the human variable region, thereby producing antibodies with immunogenic epitopes in humans brought by the endogenous variable region repertoire.

[0198] In some embodiments, the human variable region genes in the genome of the non-human animal have only one IGK V region gene and a limited number of IGK J region genes, such as 1, 2, 3, 4, or 5 IGK J region genes. Therefore, the diverse antibodies produced in the non-human animal have diverse heavy chains that can bind to the same or substantially the same light chain. This feature is particularly useful in the preparation of bispecific antibodies (BsAbs). For example, such non-human animals can be immunized with a first antigen to induce the production of B cells, and the antibodies expressed by these B cells can specifically bind to the first antigen. Similarly, the non-human animal (or an animal with the same modification) can also be immunized with a second antigen to produce B cells that express antibodies that can specifically bind to the second antigen. Subsequently, the corresponding VH (heavy chain variable region) can be cloned from the first and second B cells respectively. These two VHs can be paired with the same light chain VL (light chain variable region) to prepare bispecific antibodies. Therefore, there is no need to force a certain light chain to be paired with a specific heavy chain through antibody engineering methods (such as modifying the sequence). This method significantly improves the success rate of bispecific antibody development. In addition, the antibodies or related sequences described herein can be further combined with each other to prepare multispecific antibodies, such as bispecific antibodies (BsAbs).

[0199] The kappa chain immunoglobulin locus (also known as IGK or immunoglobulin kappa locus) is a region on a chromosome (e.g., human chromosome 2) that contains human antibody (or immunoglobulin) light chain genes. The immunoglobulin light chain genes can undergo a series of rearrangements, resulting in the production of mature immunoglobulin light chain nucleic acids (e.g., kappa chains).

[0200] The connection of the V region of the kappa chain immunoglobulin locus (also known as the IGKV gene) and the J region of the kappa chain immunoglobulin locus (also known as the IGKJ gene) produces continuous exons, which encode the entire light chain variable domain. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far away from the constant (C) region. The J gene segment (or IGKJ gene cluster) is located near the C region. The connection between the V region and the J region also brings the V gene close to the constant region sequence. The rearranged V and J gene segments are separated from the C region sequence by only one intron. In order to produce a complete immunoglobulin light chain messenger RNA, the V region exons, J region exons, and C region exons are directly connected in sequence by RNA splicing after transcription to remove introns.

[0201] The human light chain immunoglobulin locus is located on human chromosome 2. There are several different groups of human IGKV genes, including IGKV1 genes (including all IGKV genes starting from IGKV1, also known as VκI), IGKV2 genes (including all IGKV genes starting from IGKV2, also known as VκII), IGKV3 genes (including all IGKV genes starting from IGKV3, also known as VκIII), IGKV4 genes (including all IGKV genes starting from IGKV4, also known as VκIV), IGKV5 genes (including all IGKV genes starting from IGKV5, also known as VκV), IGKV6 genes (including all IGKV genes starting from IGKV6, also known as VκVI) and IGKV7 genes (including all IGKV genes starting from IGKV7, also known as VκVII). In addition, the IGKV genes in human chromosome 2 also form two clusters, the proximal Vκ cluster and the distal Vκ cluster, and the sequences in the two clusters are similar but not identical. Human IGKJ genes include IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.

[0202] Information on human light chain V and J regions, and heavy chain V, D, and J regions can be found in the IMGT Repertoire: https: / / www.imgt.org / IMGTrepertoire / LocusGenes / .

[0203] On the one hand, the non-human animal or cell involved in the present application has a human light chain (such as IGK) V region and one or more human light chain (such as IGK) J regions downstream of the host non-human animal light chain locus coding region, such as comprising 1 human light chain (such as IGK) V region and 1, 2, 3, 4 or 5 human light chain (such as IGK) J regions. In some embodiments, the transcription direction of the coding region after the insertion of the human light chain (such as IGK) V region and the human light chain (such as IGK) J region is opposite to the transcription direction of the coding region of the corresponding endogenous light chain VJ original gene of the host. The human IGK V region and the human IGK J region are operably linked together and can undergo VJ recombination.

[0204] 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.

[0205] In some embodiments, the human V region gene is IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, IGKV1D-33, IGKV 1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-9, IGKV2D-29, IGKV1D-12, IGKV1-12, IGKV1-27, or IGKV1-16. In some embodiments, the human V region gene is IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, or IGKV1D-33.

[0206] In some embodiments, the human J region genes are selected from 1 to 5 of the following group: IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1.

[0207] In some embodiments, the human V region gene is selected from one of the following groups: IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, IGKV1D-33, IGKV 1-39, IGKV1-33, IGKV2-30, IGKV2-28, IGKV1-9, IGKV2D-29, IGKV1D-12, IGKV1-12, IGKV1-27 and IGKV1-16, and at the same time, the human J region gene is selected from 1 to 5 of the following groups: IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1. For example, when the human V region gene is IGKV4-1, the human J region gene is IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1. KJ3, IGKJ2 and IGKJ1, 1, 2, 3, 4 or 5; when the human V region gene is IGKV1-5, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1; when the human V region gene is IGKV3-11, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1; when the human V region gene is IGKV3-11, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1; When the human V region gene is IGKV3-15, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1; when the human V region gene is IGKV3-20, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1; when the human V region gene is IGKV1D-39, the human J region gene is IGKJ5, IGKJ4, 1, 2, 3, 4 or 5 of IGKJ3, IGKJ2 and IGKJ1; when the human V region gene is IGKV2D-28, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1; when the human V region gene is IGKV1D-33, the human J region gene is 1, 2, 3, 4 or 5 of IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1.

[0208] In another aspect, the present application relates to non-human animals or cells comprising one or more human IGHV regions, one or more human IGHD regions, and / or one or more human IGHJ regions downstream of the coding region of the host non-human mammal's heavy chain locus. In some embodiments, after insertion, the transcription direction of the coding regions of the human IGHV, human IGHD, and human IGHJ regions is opposite to the transcription direction of the coding region of the host's endogenous heavy chain VDJ progene. The human IGHV, human IGHD, and human IGHJ regions are operably linked together and capable of undergoing VDJ recombination.

[0209] 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.

[0210] Therefore, the present application provides a non-human animal or cell, the genome of the non-human animal cell comprising an operably linked human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus, and an operably linked human immunoglobulin heavy chain variable region gene downstream of the immunoglobulin heavy chain locus, wherein the human immunoglobulin light chain variable region gene comprises one human light chain V region gene and one or more (e.g., 2, 3, 4 or 5) human light chain J region genes, and the human immunoglobulin heavy chain variable region gene comprises one or more human heavy chain V region, heavy chain D region or heavy chain J region or fragment thereof genes, and at the same time, the genome of the non-human animal comprises endogenous immunoglobulin light chain variable region genes and constant region genes, the endogenous immunoglobulin constant region genes are located between the endogenous immunoglobulin variable region and the human immunoglobulin variable region, and the transcription direction of the endogenous immunoglobulin variable region is opposite to that of the endogenous immunoglobulin constant region and the human immunoglobulin light chain variable region.

[0211] 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 a single V region and J region of the light chain from humans and part or all of the intervening sequences arranged in reverse in a germline manner.

[0212] 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.

[0213] 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 heavy chain from humans and part or all of the intervening sequences arranged in reverse in a germline manner.

[0214] 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.

[0215] 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.

[0216] In one aspect, the inserted human immunoglobulin variable region gene DNA comprises the entire gene coding region of a single functional human V region gene.

[0217] On the one hand, the inserted human immunoglobulin variable region gene DNA contains 18%-100% of the reverse-encoding human light chain J region gene coding region, such as more than 18%, more than 38%, more than 58%, more than 78% and all of the human light chain J region gene coding region.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] Optionally, the non-human animal 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 the human light chain variable region gene located downstream of the coding region of the host non-human animal light chain locus and the human heavy chain variable region gene located downstream of the coding region of the host non-human animal heavy chain locus.

[0223] 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.

[0224] 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.

[0225] Preparation method of non-human animals or cells

[0226] The present application discloses a method for constructing functional human immunoglobulin variable region genes in non-human animals (e.g., mice). In this application, the research work carried out in mice is only illustrative, and unless otherwise specified, reference to mice also includes all non-human animals, with mice being the preferred non-human animal.

[0227] 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 one aspect, insertion is performed between coordinates 70,703,738 and 70,742,704 on mouse chromosome 6, preferably at position 70,729,444, or at the equivalent position on the mouse lambda locus on chromosome 16. All coordinates are referenced to the NCBI database GRCm39.

[0228] 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 113,981,732.

[0229] In some embodiments, the site-directed recombination method includes homologous recombination, nucleic acid cleavage enzyme-mediated recombination (such as CRISPR / Cas9, etc.), and site-specific recombinase-mediated recombination, and involves the combined use of one or more of these methods.

[0230] In some embodiments, the endogenous locus constant region is modified in whole or in part so that its transcription direction is wholly or partially opposite to the transcription direction of the corresponding endogenous variable region gene. The modification is performed by inserting exogenous recombination sites inside or at both ends of the constant region of non-human animal cells or animals, and then by means such as intracellular delivery of recombinases or animal breeding, so that the host constant region gene sequence is completely or partially reversed. The insertion can be obtained by commonly used gene editing targeting means, such as homologous recombination or nucleic acid cutting enzyme-mediated recombination (such as CRISPR / Cas9, etc.). The modification can also be achieved by nucleases, and the relevant technology is known in the prior art.

[0231] In some embodiments, the endogenous light chain locus constant region is modified in whole or in part so that its transcription direction is wholly or partially opposite to the transcription direction of the corresponding endogenous light chain variable region gene. The modification is achieved by using a non-human animal constant region to reversely insert or replace part or all of the host's constant region gene sequence. The insertion or replacement can be obtained by commonly used gene editing targeting methods, such as homologous recombination or nuclease-mediated recombination (such as CRISPR / Cas9, etc.).

[0232] In some embodiments, by first inserting the modified human variable region gene into the downstream (backward) of the endogenous immunoglobulin locus of the mouse cell, and then by introducing a modified short exogenous recombinase binding site in the endogenous constant region of the mouse cell, the recombinase is introduced to partially or completely invert the endogenous constant region of the mouse cell. After screening and identification to obtain the modified cells, the cells are injected into early embryos to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing complete humanized immunoglobulin loci.

[0233] In some embodiments, a modified short exogenous recombinase binding site can be first introduced into the endogenous constant region of the mouse cell, and the recombinase is introduced to partially or completely invert the endogenous constant region of the mouse cell. The modified human variable region gene is then inserted downstream (behind) of the endogenous immunoglobulin locus of the mouse cell. After screening and identification to obtain the modified cells, the cells are injected into early animal embryos to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing complete humanized immunoglobulin loci.

[0234] Optionally, in some embodiments, 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 the human heavy chain variable region gene can be introduced downstream of the coding region of the heavy chain locus of the host non-human animal while the human light chain variable region gene is introduced downstream of the coding region of the light chain locus of the host non-human animal.

[0235] Method for preparing common light chain antibodies using non-human animals

[0236] Non-human animals can be used to generate humanized or chimeric antibodies that specifically bind to a target. Antibody production can be induced in non-human animals using standard techniques, using the target (e.g., a protein or fragment thereof) as an immunogen. For example, polyclonal antibodies can be induced by multiple injections of an antigenic peptide or protein (possibly in combination with an adjuvant). Antibody titer can then be monitored using techniques such as immobilized antigen and ELISA, and purified.

[0237] To obtain monoclonal antibodies, cells can be extracted from immunized animals and screened for cells producing the target antibody using hybridoma technology. Alternatively, single-cell screening can be used, without the need for hybridoma fusion, to directly analyze antibody sequences using DNA barcoding and single-cell sequencing.

[0238] In modified mouse models, B cells are genetically engineered to contain human light and / or heavy chain gene sequences. These modifications may include integrating human immunoglobulin variable and constant region genes into the mouse locus. The resulting antibodies may be composed entirely of human V, D, and J genes and can be further humanized or optimized to improve binding affinity, alter glycosylation properties, and so on.

[0239] Through nucleic acid sequencing or peptide synthesis technology, the antigen binding site of the antibody can be modified to optimize its functionality. All antibodies can be derived from mammals, including humans and humanized antibodies. Subsequent modification of antibodies can also include covalent modification or other post-translational modifications to enhance specific properties or functions.

[0240] Method for preparing bispecific antibodies using common light chains

[0241] The present disclosure provides a non-human animal that expresses a light chain with a limited pedigree and can be associated with a variety of heavy chains. In some embodiments, the non-human animal comprises a human light chain variable region locus, which has an IGK V region gene and a small amount (such as 1-5) of IGK J region genes, and is operably connected to the downstream of an endogenous constant region gene. The endogenous constant region can be partially or completely modified (such as inverted) so that its transcription direction is opposite to that of the endogenous variable region gene.

[0242] The animal can be immunized with various immunogens to generate B cells that express specific antibodies. For example, the heavy chain variable region can be cloned from B cells and fused to the human heavy chain constant region; the light chain can be fused to the human light chain constant region for cell transfection and antibody expression.

[0243] In addition, this method can also be used to prepare bispecific antibodies. By modifying the antibody molecular interface (such as the CH3 domain), the proportion of heterodimers recovered from recombinant cell culture can be maximized, thereby achieving efficient production of bispecific antibodies.

[0244] 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.

[0245] Example

[0246] Example 1: Overall Strategy

[0247] The mouse model of the present application can be established by inserting a sequence containing only one functional V region and one or more J regions of the human immunoglobulin light chain variable region into the downstream of the mouse immunoglobulin light chain encoding locus, and can also be simultaneously established by inserting a heavy chain gene containing all human V, D and J regions into the downstream of the mouse immunoglobulin heavy chain encoding locus. This insertion is achieved by gene targeting in ES cells using techniques well known in the art. A targeting vector containing a human antibody variable region gene sequence is constructed in vitro by recombinant engineering, and the targeting vector is inserted downstream of the above-mentioned mouse immunoglobulin locus to achieve the transformation of the human antibody variable region gene. Since the human immunoglobulin variable region gene is very large, in order to ensure that mice containing all human immunoglobulin variable regions can be successfully, effectively and correctly obtained, the present application has adopted a series of schemes to increase the probability of success of the work, and some optional schemes include the following.

[0248] The present application selects a human IGK V / J sequence inserted downstream of the mouse immunoglobulin light chain encoding gene locus that contains only one functional human immunoglobulin light chain variable region V region, and does not limit the number of subsequent human immunoglobulin light chain variable region J regions. As described in Example 4, an alternative embodiment of the present application is to produce a sequence containing only one human IGK V region and all human IGK J regions, and insert it downstream of the mouse IgK constant region to obtain a mouse. An alternative embodiment of the present application is to produce a sequence containing only one human IGK V region and only one human IGK J region, and insert it downstream of the mouse IgK constant region to obtain a mouse. The only human IGK V region included is connected to the sequence of one or more subsequent IGK J regions, and the middle may not contain intron sequences or further do not contain recombination signal sequences (RSS) in VJ introns. That is, the inserted human IGK V region and IGK J region may be rearranged or only retain a limited rearrangeable J.

[0249] The present application inserts the human gene into the Junk sequence downstream of the mouse immunoglobulin locus to avoid affecting the expression of the mouse gene in the final targeted cells or mice, as described in Example 2 below.

[0250] When targeting ES cells, not all targeting vectors are fully integrated in practice. We screen ES cells by high-density gene identification to obtain those with complete gene insertion. This identification can be performed by high-density PCR or Q-PCR. High-density PCR analysis is used to finally confirm the complete integration of the targeting vector. In practice, the probability of obtaining fully integrated ES cells is about 10%. In Example 3, Table 6 represents the partial integrity array PCR design of the first vector (vector1) in the heavy chain targeting process.

[0251] In addition, due to the inherent karyotype instability of mouse ES cells in in vitro culture (Gaztelumendi N, Nogués C. Chromosome Instability in mouse Embryonic Stem Cells. Scientific Reports, Springer Science and Business Media LLC, 2014, 4(1).), only a few of the ES cells with complete gene insertion retained chromosome integrity and germline transmission capacity after prolonged in vitro culture and continuous targeting operations. During the operation, the screened ES cells can be identified using a detailed karyotype analysis scheme, such as karyotype examination based on general microscopy (photomicrographic), or karyotype detection based on specially designed Q-PCR (D'Hulst C, Parvanova I, Tomoiaga D, et al. Fast Quantitative Real-Time PCR-Based Screening for Common Chromosomal Aneuploidies in Mouse Embryonic Stem Cells[J]. Stem Cell Reports, Elsevier BV, 2013, 1(4): 350–359.), or based on multiple chromosomes (Barrett MT, Scheffer A, Ben-Dor A, et al. Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA. Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, 2004, 101(51): 17765–17770.). However, these schemes often only reflect part of the capabilities of the obtained ES and cannot represent its germline transmission ability and the production capacity of human variable region chimeric antibodies.

[0252] In this regard, in a feasible operational scheme of the present application, the intermediate animals are produced and verified in a timely or regular manner (such as 3 times per cell targeting modification). The effect obtained is that almost every modified ES cell with the obtained partial human variable region can be injected into a mouse embryo to produce a chimeric mouse to verify its germline transmission ability. At the same time, due to the presence of the human variable region gene that can be inserted operably downstream of the mouse immunoglobulin locus, the obtained chimeric mice can use the inserted human variable region gene to perform VDJ or VJ recombination and produce chimeric antibodies with functional human variable regions. This data serves as a checkpoint for the production of non-human animals with complete human variable regions. The reliability and functionality of the obtained process ES cells are judged from the animal production level, antibody production level and variable region domain diversity, and the success or failure prediction and route correction of the production of the route with complete human variable regions are made in a timely manner. These mice carrying partial human variable regions can also be used for antibody discovery to produce chimeric antibodies with human variable regions. At the same time, these mice can be used to produce a general scheme for ultimately producing animals with human immunoglobulin light chain variable regions, which can be used to produce a series of animals containing different single human light chain variable region V regions, and can also be used to produce animals containing all or part of the human light chain variable region V regions.

[0253] Example 2: Modification of mouse immunoglobulin loci

[0254] The ES cell line of BALB / c mice was obtained through independent isolation and used as the basic material for subsequent targeting.

[0255] Using ES targeting techniques 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 position of the mouse IGK locus (the region between the Igkc and Rpia genes, specifically IGK chr6:70,706,267. NCBI database GRCm39) in ES cells. After element 1 (Cpoint) was knocked into the correct ES cell line, ES targeting technology was used to knock vector element 2 (hereinafter referred to as LD, used to cooperate with Cpoint) into the upstream position of the mouse IGK constant region (the region between the Igk J5 exon and the Igkc gene, specifically IGK chr6:70,701,630. NCBI database GRCm39).

[0256] This protocol can also be used for modifications near the immunoglobulin heavy chain locus. In ES cells, vector element 1 (referred to as Cpoint, an element for introducing human variable region insertion) is knocked into the downstream position of the mouse IGH locus (the region between the Igha gene and the Tmem121 gene, specifically IGH chr12:113,190,256. NCBI database GRCm39). Similarly, after obtaining element 1 (Cpoint) knocked into the correct ES cell line, ES targeting technology is used to knock vector element 2 (LD, used to cooperate with Cpoint) into the upstream position of the mouse IGH constant region (the region between the IgH J4 exon and the Cμ locus, specifically IGH chr12:113,391,844).

[0257] 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.).

[0258] 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 is performed single or in batches. After each vector insertion, the selection marker fragment is deleted by the transposition mechanism until the last vector is inserted, ultimately obtaining an ES cell line in which the target human antibody variable region encoding gene (VDJ) is completely inserted.

[0259] ES cells, quality-controlled by PCR and karyotype Q-PCR array analysis, are injected into the blastocyst cavity of mice 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 light chain variable region genes and with or without human immunoglobulin heavy chain variable region genes are 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.

[0260] The preparation diagrams and vector designs are shown in Figures 1 to 4, where Figure 1 shows the Cpoint and LD targeting of the IgK constant region and the modification of the C region; Figure 2 shows a schematic diagram of the insertion of the light chain human variable region, showing IGK-vector targeting and deletion of the resistance marker; Figure 3 shows the Cpoint and LD targeting of the IgH constant region and the modification of the C region, and Figure 4 shows a schematic diagram of the insertion of the heavy chain human variable region, showing IGH-vector targeting and deletion of the resistance marker.

[0261] Example 3: Selection of light chain VJ regions: unrearranged VJ and rearranged VJ

[0262] 3.1 Construction of Cpoint targeting vector

[0263] The targeting vector consists of two homology arms (sequences flanking the insertion site), a selection marker (SM) 2, loxP, and a transposon PB fragment. As shown in Figures 1 and 3, these fragments are ligated to the commercial vector pMD18T via enzyme ligation to construct the Cpoint targeting vector. As described in Example 2, in addition to being used for modification near the immunoglobulin light chain locus, the present invention can also be used for modification near the immunoglobulin heavy chain locus.

[0264] (1) Cpoint modification near the light chain

[0265] The ES targeting technology well known in the art was used to knock the Cpoint targeting vector into the downstream position of the mouse IgK constant region locus in ES cells.

[0266] PCR and sequencing confirmed that the IgK-Cpoints of clones C6, E5, G4, D2, and A2 were correctly targeted. The Cpoint identification protocol is shown in Table 1, and the PCR identification results are shown in Figure 5.

[0267] Table 1 Cpoint identification scheme for light chain

[0268] (2) Cpoint transformation near the heavy chain

[0269] The ES targeting technology well known in the art was used to knock the Cpoint targeting vector into the downstream position of the mouse IGH constant region locus in ES cells.

[0270] PCR identification confirmed that the IGH-Cpoint of clone 10-24 was correctly targeted. The Cpoint identification protocol is shown in Table 2, and the PCR identification results are shown in Figure 6.

[0271] Table 2 Cpoint identification scheme for heavy chain

[0272] 3.2 Construction of LD targeting vector

[0273] The targeting vector consists of two homology arms (sequences flanking the insertion site), a selection marker (SM) 1, loxP, and a transposon PB fragment. As shown in the figure, these fragments are enzymatically linked to the commercial vector pMD18T to construct the LD targeting vector. As described in Example 2, in addition to being used for modification near the immunoglobulin light chain locus, this application scheme can also be used for modification near the immunoglobulin heavy chain locus.

[0274] (1) Cpoint modification near the light chain

[0275] The ES cells with the correct Cpoint targeting were then knocked into the upstream position of the mouse IgK constant region (the region between the Igk J5 exon and the Igkc gene, specifically IGK chr6:70,701,630. NCBI database GRCm39) using ES targeting technology.

[0276] PCR identification confirmed that the IGK-LD of clones 5, 6, 8, and 24 were correctly targeted. The LD identification scheme is shown in Table 3, and the PCR identification results are shown in Figure 7.

[0277] Table 3 Light chain LD identification scheme

[0278] (2) Cpoint transformation near the heavy chain

[0279] The ES cells with the correct Cpoint targeting were then injected with the LD targeting fragments into the upstream position of the mouse IgH constant region (the region between the IgH J4 exon and the Cμ locus, specifically IGH chr12:113,391,844. NCBI database GRCm39) using ES targeting technology.

[0280] PCR identification confirmed that the IGH-LD of clones 108#, 111#, and 112# were correctly targeted. The LD identification scheme is shown in Table 4, and the PCR identification results are shown in Figure 8.

[0281] Table 4 Heavy chain LD identification scheme

[0282] 3.3 Construction of Cre expression vector

[0283] The Cre expression plasmid consists of a eukaryotic promoter, a Cre encoding gene, and a terminator. These fragments are connected to the commercial vector PMD18T by enzyme ligation to construct a Cre expression vector.

[0284] ES cells with correct Cpoint and LD targeting were transfected with Cre expression vector, and the mouse C region was inverted through the Cre / loxP recombination mechanism.

[0285] (1) Inversion modification of the light chain constant region

[0286] PCR identification confirmed that the light chain constant region inversion of clone 268# was correct. The light chain constant region inversion identification scheme is shown in Table 5, and the identification results are shown in Figure 9.

[0287] Table 5 Identification scheme for light chain constant region inversion

[0288] (2) Inversion modification of the heavy chain constant region

[0289] PCR identification confirmed that the heavy chain constant region inversion of clone 14-29# was correct. The heavy chain constant region inversion identification protocol is shown in Table 6, and the identification results are shown in Figure 10.

[0290] Table 6 Heavy chain constant region inversion identification scheme

[0291] 3.4 Construction of transposase expression vector

[0292] The transposase expression plasmid consists of a eukaryotic promoter, a transposase encoding gene, and a terminator. These fragments are connected to the commercial vector PMD18T by enzyme ligation to construct a transposase expression vector.

[0293] The inversion-positive cell line is then transfected with a transposase expression plasmid to achieve deletion of the selection marker fragment through the transposition mechanism.

[0294] PCR identification confirmed that the light chain resistance marker was correctly deleted in clones 1, 2, 4, and 6. The light chain resistance marker deletion identification scheme is shown in Table 7, and the identification results are shown in Figure 11.

[0295] Table 7 Light chain resistance marker deletion identification scheme

[0296] PCR identification confirmed that the heavy chain resistance marker deletion in clones 3-6# and 8-10# was correct. The heavy chain resistance marker deletion identification scheme is shown in Table 8, and the identification results are shown in Figure 12.

[0297] Table 8 Heavy chain resistance marker deletion identification scheme

[0298] 3.5 Construction of IGK VJ targeting vector

[0299] As shown in Figure 2, the IGK VJ targeting vector was constructed using Red / ET recombination technology (Rivero-Müller, Adolfo et al. "Assisted large fragment insertion by Red / ET-recombination (ALFIRE)--an alternative and enhanced method for large fragment recombineering." Nucleic acids research vol.35,10(2007):e78.doi:10.1093 / nar / gkm250). It consists of the human IGK V / J sequence and homology arms at both ends (sequences on both sides of the insertion site). It also carries a screening marker (SM) 2 that facilitates homologous recombination screening and transposon resistance deletion, a transposon PB fragment, and the like.

[0300] As described in Example 1, the human IGK V / J sequence consists of a sequence of only one functional human immunoglobulin light chain variable region V region and one or more human immunoglobulin light chain variable region J regions. The selected single human IGK V region is linked to the sequence of one or more subsequent IGK J regions, and may not contain an intron sequence or further does not contain a recombination signal sequence (RSS) in a VJ intron.

[0301] Directly linked IGKV1-5 and IGKJ1 sequences, without the recombination signal sequence (RSS) between them, are inserted downstream of the mouse IgK constant region. The resulting mice are then used for immunization and screening of antibodies with IGKV1-5 / J1 light chains (Sequence 1).

[0302] Directly linked IGKV1-39, IGKJ1, IGKJ2, and IGKJ5 sequences contain a recombination signal sequence (RSS) between them. This sequence is inserted downstream of the mouse IgK constant region. The resulting mice can be used for immunization and screening of antibodies containing IGKV1-39 / J1, IGKV1-39 / J2, and IGKV1-39 / J5 light chains (Sequence 2).

[0303] This application adopts ES targeting technology well known in the art, and knocks the IGK VJ targeting vector into the downstream position of the mouse IgK locus in ES cells, which can be the same as the Cpoint knock-in position. The targeting technology used here is homologous recombination, and other site-directed recombination methods can also be used, including nucleic acid cutting enzyme-mediated recombination (such as CRISPR / Cas9, etc.) and site-specific recombinase-mediated recombination.

[0304] PCR identification confirmed the correct insertion of the light chain IGK V1-39 / J1-J2-J5 ​​in clones 1, 2, 4-7, 9, 13, 14, 19, and 23. The light chain IGK VJ insertion identification scheme is shown in Table 9, and the identification results are shown in Figure 13A.

[0305] Table 9 Light chain IGK VJ insertion identification scheme

[0306] PCR identification confirmed that the light chain IGK V1-5 / J1 insertion of clone #331-360 was correct. The light chain IGK VJ insertion identification scheme is shown in Table 10, and the identification results are shown in Figure 13B.

[0307] Table 10 Light chain IGK VJ insertion identification scheme

[0308] in,

[0309] The sequence of IGKV1-5 / J1 is shown below:

[0310] The sequence of IGKV1-39 / J1-J2-J5 ​​is shown below:

[0311] 3.6 Construction of IGH vector1 targeting vector

[0312] As shown in Figure 4, the IGH vector1 targeting vector was constructed by inserting a transposon, a promoter, loxP, and a selection marker Neo at the 5' end of the vector, and loxP, a selection marker Puro, and a transposon element at the 3' end of the vector using Red / ET recombination technology (Rivero-Müller, Adolfo et al. "Assisted large fragment insertion by Red / ET-recombination (ALFIRE)--an alternative and enhanced method for large fragment recombineering." Nucleic acids research vol. 35, 10(2007): e78. doi: 10.1093 / nar / gkm250).

[0313] Targeting of human IGH vector1 was performed by recombinase-mediated cassette exchange (RMCE) technology.

[0314] PCR identification confirmed that IGH vector 1 of clone 131 was correctly targeted. The targeting identification scheme is shown in Table 11, and the targeting integrity identification scheme is shown in Table 12. The PCR identification results of IGH vector 1 targeting are shown in Figure 14A, and the PCR identification results of IGH vector 1 targeting integrity are shown in Figure 14B.

[0315] Table 11 IGH vector1 targeting identification scheme

[0316] Table 12 IGH vector1 targeting integrity identification scheme

[0317] IGH vector1-targeted cells were then transfected with a transposase-expressing plasmid to delete the selection marker fragment through transposition. PCR analysis confirmed that the resistance marker deletion in clones 13-15 was correct. The resistance marker deletion identification protocol is shown in Table 13, and the PCR results for IGH vector1 resistance marker deletion are shown in Figure 15.

[0318] Table 13 Resistance marker deletion identification scheme

[0319] The clones with correct IGH vector1-FIAU targeting were subjected to karyotype Q-PCR array analysis for quality control. The results showed that they met the quality control standards.

[0320] ES cells that had passed PCR and karyotype Q-PCR array analysis were injected into the mouse blastocyst cavity according to the method in the Mouse Embryo Operation Experiment Manual to obtain F0 chimeric mice. They were bred with BALB / c mice, and after genotypic identification and functional verification, humanized mice with partial human IGH genes were finally obtained.

[0321] Example 4: Selection of light chain VJ regions: unrearranged VJ and rearranged VJ

[0322] By collecting data based on the IMGT / GeneFrequency database published on the Web by LIGM (University of Montpellier, CNRS) in 2003, the V region usage frequency and J region usage frequency of human immunoglobulin light chain (IGK) were statistically analyzed, as shown in Figures 16 and 17, Tables 14 and 15.

[0323] Table 14 Selected human IGK V regions and their usage frequencies published by IMGT (only frequencies greater than 100 are listed)

[0324] Table 15 Selected human IGK J regions and their frequencies of use as published by IMGT

[0325] We selected nine IGK light chain V region genes that showed high utilization rates in IMGT data analysis: IGKV1-39, IGKV1-5, IGKV1D-33, IGKV1D-39, IGKV1-39, IGKV2D-28, IGKV3-11, IGKV3-15, and IGKV3-20. We chose one of them to be inserted downstream of the mouse immunoglobulin constant region to create a series of mouse models.

[0326] Since the J region usage frequency of these mouse models of human immunoglobulin light chain (IGK) is relatively high, and different J region uses have appeared in the currently published antibody drugs, the mouse models produced in this application are not limited to the J region of human IGK. Some of them have all the J regions of human IGK, and some have only one J region of human IGK, as shown in Figure 18. For example, Examples 3 and 5 list the mouse production scheme using IGK1-39 / J1-J2-J5 ​​insertion and the IGK1-39 / J1 mouse B cell light chain variable region usage data. For example, Example 7 selects mice obtained in this application using IGKV4-1 / J1 insertion for antibody discovery. For example, Example 8 selects mice obtained in this application using IGKV4-1 / J1-J2-J3-J4-J5 insertion for antibody discovery.

[0327] Example 5: Sequencing analysis of light chain expression without affecting the diversity of heavy chains

[0328] The spleens of humanized mice using IGK1-39 and IGKJ1 were collected, and the usage of the immunoglobulin light chain variable region of the spleen B cells was detected by BCR sequencing. The results of BCR sequencing are shown in Figures 19-21. The results show that IGK1-39 and IGKJ1 inserted downstream of the mouse immunoglobulin light chain locus can be correctly transcribed and expressed as BCR variable regions, and are unique at the same time. Basically all B cells use the inserted single human light chain variable region V region and J region. This data proves the success of the strategy of inserting only one person's IGKV variable region downstream of the mouse immunoglobulin light chain locus in this application.

[0329] Under natural circumstances, there are differences in the frequency of use of the V region of the variable region of the light chain of human immunoglobulins. This may be due to the physical arrangement of the spatial distance between the V region, J region, and C region in the human genome. The strategy of this application uses a single IGKV, which avoids the problem of light chain V region usage, so that all inserted human IGKVs can be used in the living biological system of the mouse. Adequate expression levels are obtained. That is, the present application scheme will not affect its expression due to the frequency of use of the V region in nature.

[0330] Example 6: Immune cells in the transgenic mice developed normally

[0331] The development of immune-related cells, especially B cells, in the spleen and bone marrow of the humanized mice of the present application containing only a single human light chain variable region V gene, wild-type Balb / c, and humanized mice containing all human light chain V genes were detected by flow cytometry. The results are shown in Figures 22 and 23, demonstrating that the proportion of lymphocytes and myeloid cells in the spleen is normal, and that B cells in the spleen and bone marrow develop normally.

[0332] Example 7: High frequency usage of heavy chains and light chains in phage library construction

[0333] Based on the humanized mice carrying IGK4-1 and IGKJ1 obtained in this application, we used commercial phage display technology to immunize mice with commercial human PD1 (hPD1) recombinant protein for antibody molecule discovery. Table 16 shows the data of the phage library established using this mouse, and its library capacity can reach 10 8. Through multiple rounds of antigen-antibody affinity panning, we picked a total of 518 clones and further screened 183 positive clones that bound to human antigens. After sequence analysis of these 183 positive clones that bound to human antigens, we obtained 50 Unique antibody molecules with heavy chain sequence diversity, Table 17. The light chain variable regions of these molecules are all IGK4-1 and IGKJ1 carried by mice. Furthermore, we measured the antibody affinity of 12 of these molecules by ELISA and BLI, showing that there are high-affinity antibodies with an affinity close to that of BMK (marketed antibody drug Pembrolizumab) targeting PD1 in these common light chain molecules. The results are shown in Figures 24 and 25.

[0334] These results demonstrate that humanized mice can generate high-affinity antibodies with consistent light chain variable region origin.

[0335] Table 16. Phage library data established using humanized mice carrying IGK4-1 and IGKJ1 immunized with hPD1

[0336] Table 17. Heavy chain sequence diversity of 18 screened unique antibody molecules

[0337] Example 8: Yeast library construction to obtain high-affinity molecules

[0338] Based on the humanized mice carrying IGKV4-1 and IGKJ1-J2-J3-J4-J5 obtained in the present application scheme, we used commercial yeast display technology to immunize mice with commercial target A protein for antibody molecule discovery. We used human target A protein (Human-antigen A), monkey target A protein (Cyno-antigen A) and irrelevant antigen chicken ovalbumin (OVA) for flow cytometry (FACS) selection. Table 18 represents the use of different immune organs (spleen and lymph nodes and peripheral blood PBM and bone marrow) of different mice to establish a yeast display library, and the increase in the proportion of positive antibody display yeast after two rounds of selection. Figure 26 shows the enrichment of different positive antibody display yeasts obtained by panning with different concentrations of human target A protein (Human-antigen A). We selected 48 clones for FACS analysis of antigen-binding capacity in cell supernatants. The results (Figure 27) showed that 11 clones exhibited antigen-binding capacity, some even outperforming the control BMK antibody. Sequencing analysis of the top 10 binding clones revealed diverse CDR3 regions and diverse IGH VDJ rearrangement selection (Table 19).

[0339] These results demonstrate that humanized mice carrying IGKV4-1 and IGKJ1-J2-J3-J4-J5 obtained using the method of the present application are capable of producing high-affinity antibodies with diverse heavy chains and constant light chains.

[0340] Table 18. Yeast library construction and selection data

[0341] Table 19. Sequence diversity analysis of clones with higher affinity

[0342] Example 9: Preparation of partially humanized mice with variable region genes

[0343] Using partially humanized IGH-V, D, and J genes and limited humanized IGK-V and J genes (specific gene names are listed in Figures 19 and 20), mice with partial VDJ gene humanization were generated using the same method. Spleens from positive mice were collected, and the utilization of the inserted human genes in splenic B cells was tested by BCR sequencing. The BCR sequencing results are shown in Figures 28 and 29, demonstrating that even with the insertion of partial VDJ genes, humanized mice can still utilize these human variable region genes.

[0344] Example 10: Partially humanized variable region gene mice can produce normal immune responses

[0345] Commercially available OVA protein was used to immunize VDJ partially humanized mice, and the serum antibody titer was detected by ELISA.

[0346] The results are shown in FIG30 , which indicate that the immune responses of humanized mice and BALB / c mice to OVA antigens were comparable.

Claims

1. A method for producing a non-human animal, the method comprising operably linking a human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus of the non-human animal, the human immunoglobulin light chain variable region gene comprising only one human V region gene, preferably, the immunoglobulin light chain constant region gene of the non-human animal is comprised between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene of the non-human animal.

2. The method according to claim 1, wherein the human immunoglobulin light chain variable region gene further comprises one or more human J region genes.

3. The method according to claim 2, wherein the human immunoglobulin light chain variable region gene comprises 2 to 5 human J region genes.

4. The method according to claim 2 or 3, wherein the only one human V region gene and the one or more human J region genes are operably linked.

5. The method according to any one of claims 1-4, wherein the human V region gene is selected from the group consisting of: IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28 and IGKV1D-33.

6. The method according to any one of claims 1-5, wherein a gene rearrangement intron sequence is comprised between the human V region gene and the J region gene.

7. The method according to any one of claims 1-5, wherein no gene rearrangement intron sequence is comprised between the human V region gene and the J region gene.

8. The method according to any one of claims 2-7, wherein the human J region gene is selected from the group consisting of: IGKJ5, IGKJ4, IGKJ3, IGKJ2 and IGKJ1.

9. The method according to any one of claims 2-8, wherein the human J region gene comprises a gene rearrangement intron sequence.

10. The method according to any one of claims 2-8, wherein the human J region gene does not comprise a gene rearrangement intron sequence.

11. The method according to any one of claims 1-10, wherein the genomic integrity of the endogenous immunoglobulin light chain variable region of the non-human animal is not altered.

12. The method according to any one of claims 1-11, wherein the function of the expression regulatory element of the endogenous immunoglobulin light chain variable region gene of the non-human animal is not disrupted.

13. The method according to any one of claims 1-12, wherein the non-human animal comprises a complete expression regulatory element of the endogenous immunoglobulin light chain variable region.

14. The method according to any one of claims 1-13, wherein the non-human animal comprises a complete endogenous immunoglobulin light chain variable region gene.

15. The method according to any one of claims 1-14, wherein the non-human animal does not express the endogenous immunoglobulin light chain variable region.

16. The method according to any one of claims 1-15, wherein the endogenous immunoglobulin light chain variable region gene of the non-human animal is not expressed as an antibody light chain variable region.

17. The method according to any one of claims 1-16, which comprises making the transcriptional direction of the human immunoglobulin light chain variable region gene opposite to that of the endogenous immunoglobulin light chain variable region gene.

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

19. The method according to claim 18, which comprises making the transcriptional direction of the immunoglobulin light chain constant region gene of the non-human animal opposite to that of the endogenous immunoglobulin light chain variable region gene.

20. The method according to any one of claims 1-19, wherein in the non-human animal, the transcriptional direction of the human immunoglobulin light chain variable region gene is the same as that of the immunoglobulin constant region gene of the non-human animal.

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

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

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

24. The method according to claim 23, which comprises inserting the human immunoglobulin light chain variable region gene between the lgkc and Rpia genes of the mouse locus.

25. The method according to any one of claims 23-24, which comprises inserting the human immunoglobulin light chain variable region gene between the mouse chromosomal positions chr6: 70,703,738 - 70,742,704.

26. The method according to any one of claims 23-25, which comprises inserting the human immunoglobulin light chain variable region gene at the mouse chromosomal position chr6: 70,706,267.

27. The method according to any one of claims 1-26, which comprises making the human immunoglobulin light chain variable region gene operably linked downstream of the immunoglobulin locus of the non-human animal by site-specific recombination.

28. The method according to any one of claims 19-27, which comprises making the transcriptional direction of the immunoglobulin light chain constant region gene of the non-human animal opposite to that of the endogenous immunoglobulin light chain variable region gene by genome modification.

29. A non-human animal, whose genome comprises a human immunoglobulin light chain variable region gene operably linked downstream of the immunoglobulin light chain locus, the human immunoglobulin light chain variable region gene comprises only one human V region gene, and preferably, an immunoglobulin light chain constant region gene of the non-human animal is comprised between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene of the non-human animal.

30. The non-human animal according to claim 29, wherein the human immunoglobulin light chain variable region gene further comprises one or more human J region genes, preferably 2 - 5.

31. The non-human animal according to claim 30, wherein the only one human V region gene and the one or more human J region genes are operably linked.

32. The non-human animal according to any one of claims 29-31, wherein the human V region gene is selected from the group consisting of: IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, and IGKV1D-33.

33. The non-human animal according to any one of claims 29-32, wherein a gene rearrangement intron sequence is included between the human V region gene and the J region gene.

34. The non-human animal according to any one of claims 29-33, wherein a gene rearrangement intron sequence is not included between the human V region gene and the J region gene.

35. The non-human animal according to any one of claims 30-34, wherein the human J region gene is selected from the group consisting of: IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1.

36. The non-human animal according to any one of claims 30-35, wherein the human J region gene includes a gene rearrangement intron sequence.

37. The non-human animal according to any one of claims 30-36, wherein the human J region gene does not include a gene rearrangement intron sequence.

38. The non-human animal according to any one of claims 29-37, wherein the genomic integrity of the endogenous immunoglobulin light chain variable region is not altered.

39. The non-human animal according to any one of claims 29-38, wherein the function of the expression regulatory element of the endogenous immunoglobulin light chain variable region gene is not disrupted.

40. The non-human animal according to any one of claims 29-39, whose genome includes the expression regulatory elements of the complete endogenous immunoglobulin light chain variable region.

41. The non-human animal according to any one of claims 29-40, whose genome includes the complete endogenous immunoglobulin light chain variable region gene.

42. The non-human animal according to any one of claims 29-41, which does not express the endogenous immunoglobulin light chain variable region.

43. The non-human animal according to any one of claims 29-42, wherein the endogenous immunoglobulin light chain variable region gene is not expressed as an antibody light chain variable region.

44. The non-human animal according to any one of claims 29-43, wherein the transcription direction of the human immunoglobulin light chain variable region gene is opposite to that of the endogenous immunoglobulin light chain variable region gene.

45. The non-human animal according to any one of claims 29-44, wherein an immunoglobulin light chain constant region gene of the non-human animal is included between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene.

46. The non-human animal according to claims 29-44, 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.

47. A non-human animal according to any one of claims 29-46, wherein the transcriptional orientation of the human immunoglobulin light chain variable region gene is the same as that of the immunoglobulin constant region gene of the non-human animal.

48. A non-human animal according to any one of claims 29-47, 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.

49. A non-human animal according to any one of claims 29-48, which is a rodent.

50. A non-human animal according to any one of claims 29-49, which is a mouse.

51. The non-human animal according to claim 50, which comprises the human immunoglobulin light chain variable region gene between the lgkc and Rpia genes at the locus.

52. The non-human animal according to any one of claims 50-51, which comprises the human immunoglobulin light chain variable region gene at a chromosomal position between chr6: 70,703,738 and 70,742,704.

53. The non-human animal according to any one of claims 50-52, which comprises the human immunoglobulin light chain variable region gene at the chromosomal position chr6: 70,706,267.

54. A non-human animal cell, the genome of which comprises an operably linked human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus, and the human immunoglobulin light chain variable region gene comprises only one human V region gene.

55. A method for preparing an antibody that specifically binds to an antigen, the method comprising immunizing a non-human animal according to any one of claims 29-53 with the antigen.

56. A method for preparing a sample, the method comprising: Exposing a non-human animal according to any one of claims 29-53 to an antigen; And collecting the sample from the non-human animal.

57. The method according to claim 56, wherein the sample comprises immune cells.

58. The method according to any one of claims 56-, wherein the sample comprises B cells.

59. The method according to any one of claims 56-58, wherein the sample is derived from bone marrow, spleen tissue, lymph nodes, spleen cells or peripheral lymphocytes.

60. A genetically modified non-human animal genome, which comprises an operably linked human immunoglobulin light chain variable region gene downstream of the immunoglobulin light chain locus, and the human immunoglobulin light chain variable region gene comprises only one human V region gene. Preferably, the human immunoglobulin light chain variable region gene comprises the immunoglobulin light chain constant region gene of the non-human animal between it and the endogenous immunoglobulin light chain variable region gene of the non-human animal.

61. The non-human animal genome according to claim 60, wherein the human immunoglobulin light chain variable region gene further comprises one or more human J region genes, preferably 2-5.

62. The non-human animal genome according to claim 61, wherein the only one human V region gene and the one or more human J region genes are operably linked.

63. The non-human animal genome according to any one of claims 60-62, wherein the human V region gene is selected from the group consisting of: IGKV4-1, IGKV1-5, IGKV3-11, IGKV3-15, IGKV3-20, IGKV1D-39, IGKV1-39, IGKV2D-28, and IGKV1D-33.

64. The non-human animal genome according to any one of claims 60-63, wherein a gene rearrangement intron sequence is included between the human V region gene and the J region gene.

65. The non-human animal genome according to any one of claims 60-64, wherein a gene rearrangement intron sequence is not included between the human V region gene and the J region gene.

66. The non-human animal genome according to any one of claims 61-65, wherein the human J region gene is selected from the group consisting of: IGKJ5, IGKJ4, IGKJ3, IGKJ2, and IGKJ1.

67. The non-human animal genome according to any one of claims 61-65, wherein the human J region gene includes a gene rearrangement intron sequence.

68. The non-human animal genome according to any one of claims 61-67, wherein the human J region gene does not include a gene rearrangement intron sequence.

69. The non-human animal genome according to any one of claims 61-68, wherein the genomic integrity of the endogenous immunoglobulin light chain variable region is not altered.

70. The non-human animal genome according to any one of claims 60-69, wherein the function of the expression regulatory element of the endogenous immunoglobulin light chain variable region gene is not disrupted.

71. The non-human animal genome according to any one of claims 60-70, which includes the expression regulatory element of the complete endogenous immunoglobulin light chain variable region.

72. The non-human animal genome according to any one of claims 60-71, which includes the complete endogenous immunoglobulin light chain variable region gene.

73. The non-human animal genome according to any one of claims 60-72, which does not express the endogenous immunoglobulin light chain variable region.

74. The non-human animal genome according to any one of claims 60-73, wherein the endogenous immunoglobulin light chain variable region gene is not expressed as an antibody light chain variable region.

75. The non-human animal genome according to any one of claims 60-74, wherein the transcriptional direction of the human immunoglobulin light chain variable region gene is opposite to that of the endogenous immunoglobulin light chain variable region gene.

76. The non-human animal genome according to any one of claims 60-75, wherein an immunoglobulin light chain constant region gene of the non-human animal is included between the human immunoglobulin light chain variable region gene and the endogenous immunoglobulin light chain variable region gene.

77. The non-human animal genome according to claims 60-76, wherein the transcriptional direction of the immunoglobulin light chain constant region gene is opposite to that of the endogenous immunoglobulin light chain variable region gene.

78. The non-human animal genome according to any one of claims 60-77, wherein the transcriptional orientation of the human immunoglobulin light chain variable region gene is the same as that of the immunoglobulin constant region gene of the non-human animal.

79. The non-human animal genome according to any one of claims 60-78, 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.

80. The non-human animal genome according to any one of claims 60-79, wherein the non-human animal is a rodent.

81. The non-human animal genome according to any one of claims 60-80, wherein the non-human animal is a mouse.

82. The non-human animal genome according to claim 81, which contains the human immunoglobulin light chain variable region gene between the lgkc and Rpia genes.

83. The non-human animal genome according to any one of claims 81-82, which contains the human immunoglobulin light chain variable region gene between chromosomal positions chr6: 70,703,738 to 70,742,704.

84. The non-human animal genome according to any one of claims 81-83, which contains the human immunoglobulin light chain variable region gene at chromosomal position chr6: 70,706,267.

85. An engineered chromosome, which contains the non-human animal genome according to any one of claims 60-84.

86. A cell, which contains the engineered chromosome according to claim 85.

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