Method for preparing antibody

Abstract

A method for preparing an antibody and an antibody prepared by using the method. In the method, an antigen is used for immunizing an animal in which an immune system is reconstituted and a stem cell having differentiation potential is transplanted, and an antibody capable of binding to the antigen is obtained, wherein the stem cell having differentiation potential is derived from a donor animal carrying one or more human immunoglobulin variable region gene segments. The problems in the prior art that a living transgenic animal used when preparing a human antibody is restricted by related requirements such as policy and quarantine, a transportation time is long, and costs are high are solved. In the method, when preparing an antibody having relatively high homology between a transgenic animal and a human target antigen, an immunosuppressive animal having a target antigen coding gene knocked out can be used as a transplantation receptor, thereby solving the problem in the prior art that a transgenic animal is difficult to generate an antibody against regions of homology between human and transgenic animal target antigens.

Description

A method for preparing an antibody Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for preparing human antibodies Background Art

[0002] Antibody drugs are a rapidly growing, advanced, and effective class of drugs. The advent of monoclonal antibody technology has made the research and production of antibody drugs a reality. After more than three decades of development, therapeutic monoclonal antibodies have become one of the most important components of biopharmaceuticals, with broad application prospects in disease treatment, including successful treatments for tumors, autoimmune diseases, infectious diseases, and transplant rejection. Since the first therapeutic monoclonal antibody was approved in 1975, over 100 monoclonal antibodies have been approved for marketing, with the number of monoclonal antibodies increasing rapidly in recent years.

[0003] The earliest therapeutic antibodies were entirely animal-derived, and their immunogenicity led to the production of human anti-mouse antibodies (HAMAs) in humans, even causing severe allergic side effects. Humanizing animal-derived antibodies through antibody engineering can reduce immunogenicity, but this process is costly and time-consuming, and it is difficult to completely eliminate immunogenicity while maintaining high affinity. The solution lies in developing fully human antibodies, and four main approaches are currently available: A) display technology; B) transgenic mouse immune screening; C) immortalized human B cells; and D) single-cell screening of human memory B cells. Display technology allows for rapid, high-throughput screening of antibodies. However, because the screening process is a purely artificial in vitro system, the diversity and affinity of the antibodies obtained are limited, and the presence of non-natural structures can still lead to immunogenicity, requiring significant time and cost for optimization and modification. Human B cells can only be isolated from humans who have been exposed to the antigen and have an immune response. Due to limited sources, screening methods using human B cells can yield a limited variety of antibodies, and the probability of identifying high-affinity antibodies is very low. Compared with other methods, genetically modified mouse models that have been transferred with human antibody-encoding genes have obvious advantages. Mice carrying complete human antibody variable region encoding genes (>3Mb) can produce immune responses close to those of humans against antigens, and obtain fully human antibodies with high affinity that are encoded by human genes and undergo natural somatic hypermutation in the body. Because it relies on a completely natural B cell screening and maturation process, there is no need for post-optimization. The antibodies are encoded by human genes and there will be no immunogenicity issues. This method can obtain high-affinity monoclonal antibodies at a much lower time and cost than other methods. For example, the HuMab platform developed by Medarex, the Trianni Mouse developed by Tranni, the VelocImmune developed by Regeneron, and the Kymouse developed by Kymab all belong to this type of model.

[0004] Mice carrying human antibody-encoding genes possess robust immune systems, and immunization with target antigens can produce high-affinity antibodies encoded by human genes. This greatly simplifies the technical process for therapeutic antibody discovery, eliminating the time and cost of antibody engineering modifications (such as humanization and affinity enhancement).

[0005] However, mouse and human proteins share a high degree of homology. For example, the amino acid identity of CLAUDIN-18 between humans and mice is 88%, with some proteins exceeding 95% in human-mouse homology. The B cell maturation process involves a "negative selection" process, which eliminates B cell clones that recognize the body's own proteins. This negative selection mechanism makes it difficult for mice to produce antibodies against the human-mouse homologous regions of proteins, a major obstacle to obtaining antibodies against human targets. To overcome this difficulty, existing techniques involve knocking out the gene encoding the target antigen in mice carrying human antibody-encoding genes, generating homozygous transgenic mice for immunization. While this method can produce antibodies against homologous antigens (sequence conserved regions), it requires the creation of a knockout strain for each target antigen, which is time-consuming. Furthermore, when it is necessary to produce antibodies against targets related to immune system function (target antigens), knocking out the gene encoding the target antigen in the mouse itself may affect the development and function of the mouse immune system, making it impossible to produce the corresponding antibodies through immune response.

[0006] Furthermore, because the immunization process is completed in transgenic (or transgenic + knockout) mice, live animals are required for experiments. Limited by policies and quarantine requirements, the transportation of live animals is time-consuming and expensive, which increases the availability of transgenic live animals and makes it difficult to use transgenic mice for antibody screening.

[0007] Summary of the Invention

[0008] The present invention provides a new method for preparing human antibodies, in which stem cells of a donor transgenic animal carrying one or more human immunoglobulin variable region gene fragments are transplanted into an immunosuppressed animal to rebuild the immune system of the recipient animal. After the recipient animal with a successful immune system reconstruction is immunized with an antigen, the immune cells from the donor are used to produce antibodies, so as to solve the problem that under the existing technology, the living transgenic animals used in the preparation of human antibodies are restricted by policies and quarantine requirements, and the transportation time is long and the cost is expensive. The immune cells from the donor can be transplanted fresh, or frozen and revived, and are convenient for long-distance transportation. The frozen donor immune cells (rather than living animals) can be revived and transplanted into recipient mice to rebuild the immune system, and antibodies can be produced through immunization, thus avoiding the difficulty of transporting living animals.

[0009] When preparing antibodies that have a high degree of homology between transgenic animals and human target antigens, the method described in the present invention can use immunosuppressed animals with target antigen encoding genes knocked out as transplant recipients, thereby solving the problem that transgenic animals have difficulty producing antibodies to homologous regions of human and transgenic animal target antigens under existing technologies.

[0010] The specific technical solutions of the present invention are as follows:

[0011] A method for preparing antibodies comprises immunizing a recipient animal with an antigen and obtaining antibodies that can bind to the antigen. The recipient animal is an animal whose immune system has been reconstructed by transplanting stem cells with differentiation potential, wherein the stem cells with differentiation potential are derived from a donor animal carrying one or more human immunoglobulin variable region gene segments.

[0012] Preferably, the human immunoglobulin is selected from one or more of IgG, IgM, IgA, IgD, and IgE, and the variable region is V H D H J H Recombinant and / or V K J K Recombinant or V L J L One or more of the regions encoded by the recombinant gene segments. Preferably, the variable region encoding segment is composed of J H1 、J H1P 、J H2 、J H2P 、J H3 、J H3P 、J H4 、J H5 or J H6 Preferably, the variable region encoding segment is composed of J K1 、J K2 、J K3 、J K4 or J K5 One of them participates in recombination formation.

[0013] The antibody of the present invention is a human antibody that can specifically recognize and bind to a target antigen.

[0014] The target antigen can be a molecule or compound that is artificially synthesized, recombinantly expressed, or naturally present in nature.

[0015] The target antigen may be a polypeptide chain comprising three or more amino acids.

[0016] The target antigen can be a membrane receptor or a free protein.

[0017] The human antibody can be a diagnostic, detection or therapeutic antibody targeting a specific disease-related therapeutic target, preferably a therapeutic antibody.

[0018] The human antibody may be an antibody whose variable region is encoded by a human gene, and is preferably a therapeutic antibody, such as a therapeutic antibody for treating human diseases such as tumors, autoimmune diseases, metabolic diseases, and nervous system diseases.

[0019] In the above method, the donor and recipient animals can have the same or different genetic backgrounds. The stem cells with differentiation potential are stem cells capable of differentiating into a variety of immune cells, and can further be one or more of bone marrow cells, fetal liver cells, hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells, preferably bone marrow cells or hematopoietic stem cells. The stem cells can be freshly isolated or frozen and revived.

[0020] In the above method, the donor and recipient animals for transplantation are non-human mammals or rodents, selected from rats, mice, rabbits, sheep, and non-human primates. In one embodiment of the present invention, the donor and recipient animals for transplantation are mice.

[0021] In the method of the present invention, the immunosuppressed recipient animal is an immunosuppressed recipient animal obtained by one or more of the following means: bone marrow ablation, immunosuppression and immunoregulation using low-toxic drugs, and gene modification to make the recipient's immune system defective or suppressed.

[0022] The bone marrow ablation is to use sublethal doses of ionizing radiation or chemical means to eliminate hematopoietic stem cells in the animal bone marrow; the use of low-toxic drugs is to use drugs that inhibit stem cell proliferation or block differentiation information transmission (such as imatinib, etc.) to perform immunosuppression and immunoregulation; the genetic modification to make the recipient's immune system defective or suppressed is hematopoietic stem cell developmental defect, including but not limited to c-kit or thrombopoietin gene mutant mice.

[0023] The method for removing hematopoietic stem cells from the bone marrow of an animal by ionizing radiation or chemical means according to the present invention can adopt the types, dosages and operation methods conventionally used in the prior art. For example, ionizing radiation is X-ray or Co60 irradiation, and the irradiation dose is 600cGy-950cGy, or a higher sublethal dose. The specific reference (Eunbee Park, et al., J Vis Exp, 2021.) The chemical means described is to give the animal one or more of busulfan, cyclophosphamide, and melphalan. Taking busulfan as an example, myeloablative preconditioning can be completed by connecting intraperitoneal injections at a dose of 20 mg / kg twice, 24 hours apart (Encarnacion Montecino-Rodriguez, et al., STAR Protocols, 2020.).

[0024] The recipient animal of the present invention can be a wild-type or genetically engineered animal. The genetically engineered animal can be a transgenic animal carrying a human gene, an animal carrying a mutant gene, an animal expressing a reporter gene, or an animal lacking a target antigen encoding gene (e.g., a target antigen encoding gene knockout). Preferably, the animal lacks a target antigen encoding gene.

[0025] The method for screening human antibodies can be a hybridoma, single B cell screening or display library screening method. Those skilled in the art will understand that the B cell collection obtained after immunization of an animal obtained by the transplantation reconstruction method provided by the present invention can be used to obtain a monoclonal clone that produces the target antibody through existing antibody screening technology, and further prepare the target human antibody that meets the requirements.

[0026] The method of the present invention specifically comprises the following steps:

[0027] (1) Subjecting animals to immunosuppressive pretreatment to obtain immunosuppressed recipient animals; constructing or purchasing animals carrying one or more human immunoglobulin variable region gene fragments as donors, collecting their bone marrow cells, embryonic cells or purified hematopoietic stem cells, and transplanting them into the immunosuppressed recipient animals.

[0028] The level of immune cell reconstruction in the recipient animal after transplantation can be detected by flow cytometry, immunofluorescence, quantitative PCR or other corresponding technologies. When the immune cells derived from the donor are stably reconstituted in the recipient, the animal can be immunized with antigens.

[0029] (2) Immunize the recipient animal obtained in step (1) with the antigen.

[0030] (3) Select animals that can produce antibodies that bind to the target antigen, collect their immune cells, and further prepare human antibodies.

[0031] Serum from immunized animals can be collected and the titer of serum antibodies capable of binding to the target antigen can be tested using ELISA or other similar techniques. Animals that produce antibodies that bind to the target antigen are selected, and their immune cells are collected. Using hybridomas, flow cytometry, high-throughput sequencing, and other methods, clones of monoclonal antibodies that meet the required parameters, such as affinity and functional activity, are screened. The antibody coding regions are sequenced to obtain the coding sequence, and the antibody variable region sequences are further combined with the human antibody constant region to produce antibodies through methods such as recombinant expression.

[0032] The order of constructing the transplant donor and recipient in the above method is not particular.

[0033] Another object of the present invention is to provide a humanized antibody prepared by the method of the present invention. Another object of the present invention is to provide a pharmaceutical composition comprising the humanized antibody of the present invention.

[0034] Advantages of the present invention:

[0035] 1. Due to policy and quarantine requirements, the transportation of live animals used in antibody production under existing technologies is time-consuming and expensive. The method described in the present invention allows for the cryopreservation of pre-prepared donor-derived immune cells, facilitating long-distance transportation. The revived donor immune cells (rather than live animals) can be transplanted into local or remote recipient mice to regenerate the immune system, and the target antigen can be used to immunize the mice to produce antibodies, thus avoiding the difficulties of transporting live animals.

[0036] 2. When using transgenic mice to produce human antibodies, prior art directly uses transgenic mice carrying human antibody-encoding genes for immunization, requiring a large number of humanized transgenic animals, which are often expensive. The present invention, through transplantation and reconstruction, can significantly reduce the number of transgenic animals used. Stem cells from a single donor mouse can be used to reconstitute three or more recipient mice, providing more animals for immunization and reducing costs.

[0037] 3. The prior art knocks out the gene encoding the target antigen in transgenic mice carrying human antibody encoding genes, establishes a genetically modified strain that cannot express the target antigen, and then uses it for antibody screening of specific targets. However, this method requires the production of a gene knockout strain for each target antigen, which is time-consuming and costly. In order to overcome the problem that immunocompetent animals cannot produce antibodies against human-animal homologous antigens, the present invention separately constructs transgenic animals carrying human antibody encoding genes (transplant donors) and transgenic animals with target antigen gene knockout (transplant recipients). Through stem cell transplantation, a normal immune cell population derived from the donor animal is reconstructed in the transgenic animal (recipient) with target antigen gene knockout, which contains mature functional T cells, B cells, and antigen-presenting cells and other fully functional immune cells. Stem cells with differentiation potential from the donor differentiate and mature in the transgenic animal (recipient) with target antigen gene knockout, and T cells and B cells are negatively selected in the transplant recipient. For target antigens that are lacking in the recipient after transplantation and reconstruction, the "negative selection" for the target antigen cannot effectively eliminate the B cell clones that produce antibodies that recognize the human / animal homologous region of the target antigen. Thus, transgenic animals with successfully immune reconstructed target antigen gene knockout can use immune cells from animals with humanized antibody encoding genes to produce antibodies encoded by human genes.

[0038] 4. The present invention solves the problem that it is difficult for ordinary animals (mice) to obtain antibodies against homologous antigens (such as conserved regions of amino acid sequences of homologous proteins). Transgenic animals (receptors) with antigen gene knockout are used to develop therapeutic antibodies against corresponding targets. The diversity of the antibody spectrum is improved, the immune response is enhanced, and the immune cycle is shortened, which is of great value for screening therapeutic antibodies.

[0039] 5. The present invention adopts the method of bone marrow (or hematopoietic stem cell) transplantation, and can use the bone marrow (or hematopoietic stem cells) of the same transgenic ordinary animal (mouse) to reconstruct the immune system in different recipient animals (mice), thereby realizing personalized antibody production.

[0040] 6. The recipient animals used in the present invention can be wild-type or genetically modified strains. For example, the recipient animals can be genetically modified models of diseases such as autoimmune diseases, tumors, and metabolic diseases, carrying gene mutations or transgenes. Such models can be used to screen for antibodies that are potential therapeutic or detection targets for specific diseases.

[0041] The recipient animal can also be a genetically modified animal carrying a specific reporter gene expression system. For example, when the animal produces the target antibody, its specific tissues or cells will show phenotypes such as emitting fluorescence and secreting cytokines, which assists in antibody screening.

[0042] 7. The stem cell transplantation and reconstruction adopted in the present invention is a non-genetic method, which does not require target gene knockout on the basis of transgenic mice, eliminating the long and complicated genetic modification and mouse breeding time, and improving efficiency.

[0043] 8. The target gene knockout animals (recipients) used for transplantation in the present invention can be produced simultaneously with the transplant donor animals, or they can be produced in large quantities and with multiple targets. These gene knockout models themselves do not carry human antibody encoding genes and can also be used for other research.

[0044] 9. The hematopoietic stem cells collected from transgenic animals (recipients) with target antigen gene knockout in the present invention can be frozen, revived, and transplanted. The stem cells themselves can be used as a non-living product, which is easy to mass produce, store, and transport. They can also be revived, transplanted, immunized, and antibodies screened according to experimental needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the curve of body weight changes in B6 recipient mice after transplantation of EGFP transgenic mouse bone marrow.

[0046] Figure 2 shows the survival rate of B6 recipient mice after transplantation of EGFP transgenic mouse bone marrow.

[0047] Figure 3 shows the results of immune cell tracing analysis after B6 recipient mice were transplanted with EGFP transgenic mouse bone marrow.

[0048] FIG4 is a curve showing changes in body weight of gene-modified recipient mice and B6 wild-type recipient mice after transplantation of transgenic mouse bone marrow cells carrying human immunoglobulin variable region encoding genes.

[0049] FIG5 shows the survival rate of gene-modified recipient mice and B6 wild-type recipient mice transplanted with bone marrow cells of transgenic mice carrying genes encoding human immunoglobulin variable regions.

[0050] FIG6 shows the results of the determination of immune serum antibody titers in gene-modified recipient mice and B6 wild-type recipient mice transplanted with bone marrow cells of transgenic mice carrying genes encoding human immunoglobulin variable regions.

[0051] Figure 7 shows the binding activity of anti-PD1 antibodies to hPD1 recombinant protein obtained by screening after PD1 KO recipient mice were transplanted with bone marrow cells of transgenic mice carrying human immunoglobulin variable region encoding genes.

[0052] Figure 8 shows the binding activity of anti-PD1 antibodies to Jurkat-PD1 cells obtained by screening PD1 KO recipient mice transplanted with bone marrow cells of transgenic mice carrying genes encoding human immunoglobulin variable regions.

[0053] Figure 9 shows the in vivo efficacy results of anti-PD1 antibodies (9G4C5 and 6F2A11) obtained by screening after PD1 KO recipient mice were transplanted with transgenic mouse bone marrow cells carrying human immunoglobulin variable region encoding genes.

[0054] FIG10 is a curve showing the weight change of mice reconstructed by fetal liver cell transplantation.

[0055] FIG11 shows the survival rate of mice reconstructed by fetal liver cell transplantation.

[0056] Figure 12 shows the results of tracing analysis of immune cells reconstructed in mice by fetal liver cell transplantation.

[0057] Figure 13 shows the results of B cell tracing analysis after cryopreserved and fresh bone marrow cell transplantation and reconstruction.

[0058] Figure 14 shows the results of immune cell tracing analysis after bone marrow transplantation in TIGIT KO mice and LAG3 KO mice.

[0059] FIG15 shows the titer results of immune serum from bone marrow transplantation in TIGIT KO mice and LAG3 KO mice.

[0060] Figure 16 shows the results of immune cell tracing in gene-modified recipient mice (TG), CLDN KO mice, and CLDN KO mice.

[0061] FIG17 shows the results of determination of immune serum antibody titers in gene-modified recipient mice (TG), CLDN KO mice, and CLDN KO recipient mice transplanted with bone marrow cells of transgenic mice carrying genes encoding human immunoglobulin variable regions. DETAILED DESCRIPTION

[0062] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0063] Unless otherwise specified, the terms used in the present invention generally have the meanings commonly understood by those skilled in the art.

[0064] The present invention will be described in further detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0065] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0066] Example 1 Construction of immune reconstitution mice by bone marrow cell transplantation

[0067] Immunosuppressive pretreatment of recipient mice:

[0068] In this example, 4-week-old B6 mice (product of Jiangsu Jicui Yaokang Biotechnology Co., Ltd., strain number: N000013) were selected as recipients and pretreated with irradiation myeloablation. However, those skilled in the art will appreciate that animals of other ages and strains can serve as transplant recipients, and other pretreatment methods (such as chemical myeloablation) that achieve myeloablation can also be used. The inventors have found that 600-900 cGY is an appropriate myeloablative irradiation dose for the selected recipients, so 600-900 cGY was selected as the irradiation dose. To improve the survival rate of mice after irradiation, antibiotics can be fed before irradiation.

[0069] Donor mouse bone marrow cell extraction and transplantation:

[0070] In this study, bone marrow from EGFP transgenic donor mice was transplanted into irradiated C57BL / 6JGpt (B6) mice. These EGFP transgenic donor mice are a fluorescent mouse model (strain number T006163) developed by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The green fluorescent protein (EGFP) is controlled by a CAG promoter and is widely expressed in mouse tissues. This model can be used to obtain various mouse cells labeled with GFP, particularly for cell transplantation research.

[0071] The specific operation steps are as follows: the donor mouse is euthanized, the hind leg bones are separated, and placed in a sterile culture dish containing 4°C PBS. The donor mouse bone marrow cells are obtained after washing the bone marrow with PBS, and the bone marrow cells are collected after resuspending and lysing for later use. The collected bone marrow cells are transplanted into the irradiated recipient mice through tail vein injection. The mice are observed and weighed every week after transplantation, and the body weight and survival rate are calculated. Figure 1 shows the changes in body weight of B6 recipient mice after transplantation of EGFP transgenic mouse bone marrow. The results showed that the body weight of the recipient mice recovered with increasing age after irradiation and transplantation. The trend of weight change showed that the physiological state of the mice after transplantation was normal. Figure 2 shows the survival rate of B6 recipient mice after transplantation of EGFP transgenic mouse bone marrow. The results showed that the survival rate of the recipient mice after irradiation and transplantation was 100%, and no mice died during the observation period of more than 90 days after transplantation. This shows that the transplanted mice (100%) can survive for at least 90 days or more.

[0072] Immune system reconstruction marker detection:

[0073] In this example, cells from the donor's EGFP transgenic mice fluoresce, whereas cells from the recipient's wild-type B6 mice do not. Therefore, flow cytometry was used to examine B cells (mCD19+) in the peripheral blood of reconstituted mice. Detection of green fluorescence indicated that the cells were donor-derived; absence of fluorescence indicated that the cells were recipient-derived.

[0074] Starting from the 4th week after transplantation, the proportion of immune cells of donor origin in the peripheral blood of reconstructed mice was collected, and the proportion of immune cells of donor origin was detected by flow cytometry using EGFP as a fluorescent marker. Figure 3 shows the results of immune cell tracing analysis after reconstruction of B6 recipient mice transplanted with EGFP transgenic mouse bone marrow. The results showed that 60 days after irradiation and transplantation, 94.2% of the white blood cells (mCD45+) and 99.9% of the B cells (mCD19+) of the recipient mice were EGFP+ cells, derived from the donor, proving that the bone marrow cells of donor origin can stably reconstruct immune cells in the recipient mice, and the immune cells of the recipient mice themselves did not reappear after immunosuppression. This example proves that the method of the present invention can reconstruct the immune system by transplanting bone marrow cells from donor mice into recipients pretreated with immunosuppression.

[0075] Example 2 Preparation of specific antibodies using bone marrow transplantation immune reconstitution mice

[0076] Bone marrow cell transplantation and reconstitution animals were constructed using the transplantation and reconstitution method described in Example 1, grouped according to Table 1. G1 and G3 were PD1 knockout recipient mice (PD1 KO) (recipient mice were a product of Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number: T011515) and wild-type B6 mice (recipient mice were a product of Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number: N000013), respectively, transplanted with humanized mouse (TG) bone marrow cells containing human immunoglobulin variable region gene segments. G2 and G4 were PD1 knockout recipient mice (PD1 KO) and wild-type B6 mice, respectively, that were not immunosuppressed prior to transplantation. The donor mice used in this experiment carried the human immunoglobulin heavy chain (IGH) and light chain (IGK) variable region encoding genes in their genomes. Furthermore, the corresponding murine Igh and Igk genes were inactivated (prepared according to the method disclosed in E-Chiang L et al., Nature Biotechnology, 2014). Specifically, referring to the ES targeting method described in the literature, a human IGH variable region gene fragment (including human V H 、D H and J H gene fragment), and a targeting vector carrying a human IGK variable region gene fragment (including human V K and J K The targeting vectors are electroporated into ES cells to obtain ES clones that have successfully integrated the human IGH gene fragment upstream of the mouse Igh gene constant region coding fragment, and ES clones that have successfully integrated the human IGK gene fragment upstream of the mouse Igk gene constant region coding fragment. The above-mentioned targeted clones are injected into mouse blastocysts and transplanted into the uterus of pseudo-pregnant mice, and finally targeted mice developed from the targeted ES cells are obtained. The IGH targeted mice are mated with the IGK targeted mice to obtain homozygous mice with IGH / IGK dual targets, which are used as humanized mice (donors) carrying human immunoglobulin variable region coding genes to provide stem cells with differentiation potential for transplantation.

[0077] After transplantation, mice were observed and weighed weekly to assess weight and survival. Results showed that recipient mice grew steadily after transplantation, with 100% survival for over 90 days (Figures 4 and 5). Following successful reconstruction, recipients were immunized with commercially available hPD-1 recombinant protein and Freund's adjuvant.

[0078] Table 1 Grouping of animal experiments on bone marrow cell transplantation and reconstruction

[0079] Immunization program:

[0080] Primary immunization: 100 μg of antigen was thoroughly mixed with an equal volume of Freund's complete adjuvant using a vortex shaker and injected subcutaneously at multiple points in each mouse. The day of the primary immunization was designated as day 0.

[0081] Second immunization: On the 21st day, 100 μg of antigen was thoroughly mixed with an equal volume of Freund's incomplete adjuvant using a vortex oscillator and injected subcutaneously at multiple points. The dosage, method and route were the same as the primary immunization.

[0082] The third immunization: On the 42nd day, 100 μg of antigen was thoroughly mixed with an equal volume of Freund's incomplete adjuvant using a vortex oscillator and injected subcutaneously at multiple points. The dosage, method and route were the same as the initial immunization (if continued immunization is required, the regimen shall be the same).

[0083] Antibody titer detection:

[0084] a) Two weeks after the third immunization, approximately 100-200 μL of blood was collected from the mouse's orbital cavity and centrifuged at 7000 rpm for 5 min. The upper pale yellow serum was collected as a sample.

[0085] b) Dilute the antigen (hPD1 recombinant protein) in coating buffer to different concentrations (1 μg / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 50 ng / mL). Add 100 μl / well to the ELISA plate, with two replicates for each concentration. Coat the plate overnight at 4°C.

[0086] c) Discard the coating buffer and wash three times with 200 μl PBST per well for 3 minutes each time. Invert the ELISA plate on a piece of flat paper to remove excess PBST. Coat the plate with 200 μl blocking solution per well at 37°C for 2 hours.

[0087] d) Discard the blocking solution and wash three times with 200 μl of PBST per well for 3 minutes each. Dilute the positive and negative sera in the coating solution in two-fold increments (1:200, 1:400, 1:800, and 1:1600). Add 100 μl / well to the ELISA plate. Set up a blank control and incubate at 37°C for 1.5 hours.

[0088] e) Discard the blocking solution and wash three times with 200 μl PBST per well, each wash for 3 minutes. Dilute the goat anti-mouse enzyme-linked secondary antibody 1:5000 in PBST + 1% BSA. Add 100 μl / well to the ELISA plate and incubate at 37°C for 1 hour.

[0089] f) Discard the liquid in the wells and wash three times with 200 μl of PBST per well for 5 minutes each. Add 100 μl of TMB colorimetric primer to the ELISA plate in the dark for 10 minutes at room temperature. Add 100 μl of stop solution and measure the OD450nm value. The results showed that after diluting the mouse serum at 1:250,000, the OD405nm absorbance of the G1-G4 group samples was higher than that of the non-immunized control serum. The results showed that the titers of antigen-specific antibodies in the sera of these four groups reached 250,000, and the serum antibody titers of transplanted and reconstructed recipient mice (G1, G3) were higher than those of the control group (G2, G4) that did not undergo irradiation transplantation ( Figure 6 ).

[0090] Screening and production of anti-hPD1 monoclonal antibodies

[0091] (1) Hybridoma screening

[0092] When the mouse serum titer reached the appropriate range, mice in the G1 group with a significant immune response were selected for intraperitoneal immunization with hPD1 recombinant protein, with 25 μg of hPD1 recombinant protein injected intraperitoneally per mouse. Three to four days later, the spleens were harvested, ground using a 70 μm mesh, and fused with SP2 / 0 cells using an electrofusion instrument. The cells were then plated and cultured for seven days using HT. The supernatant of the fused hybridomas was then plated for hPD1 recombinant protein testing and positive hybridoma screening was performed by ELISA.

[0093] (2) Subclone screening and identification

[0094] After fusion, ELISA-positive parent hybridoma clones were subcloned. Positive cells were aspirated and transferred to a 1.5 ml EP tube. 1000 μl of HT medium was added. A small number of cells were counted and 100 cells were dissolved in 23 ml of HT medium. 200 μl per well was used for limiting dilution in a 96-well plate. After 7 days of culture, the subcloned cell supernatants were plated with hPD1 recombinant protein for screening of ELISA-positive subclones. Simultaneously, supernatants from ELISA-positive subclones were incubated with Jurkat-hPD1 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) and analyzed by flow cytometry to screen for binding to native hPD1 protein. Four double-positive subclones were identified that bound to both the hPD1 recombinant protein and Jurkat-hPD1 cells and were designated 6F2A11, 9G4C5, 19E3C1, and 10E9G7, respectively. The four positive subclone wells were transferred to a 6-well plate for expansion. After confluence, the cells were divided into two parts, one part was frozen in liquid nitrogen for sequencing, and the other part was frozen for future use.

[0095] (3) Positive monoclonal sequencing

[0096] Positive subclones (6F2A11, 9G4C5, 19E3C1, and 10E9G7) were lysed, and mRNA was extracted and reverse-transcribed into cDNA. Using this cDNA as a template, PCR was used to amplify the IgG light and heavy chain variable regions, respectively. The heavy and light chain variable regions were then analyzed by Sanger sequencing.

[0097] Table 2: Heavy chain and light chain variable region amino acid sequences and human heavy chain and light chain constant region amino acid sequences of 4 positive clones (6F2A11, 9G4C5, 19E3C1, 10E9G7)

[0098] (5) Production and preparation of anti-hPD1 antibodies

[0099] Taking 6F2A11 as an example, the 6F2A11 heavy chain antibody sequence (heavy chain variable region sequenced from 6F2A11 positive clones + human IgG1 constant region) and the 6F2A11 light chain antibody sequence (light chain variable region sequenced from 6F2A11 positive clones + human Kappa chain constant region) were genetically synthesized. Corresponding nucleic acid sequences were designed based on the synthesized 6F2A11 heavy chain antibody sequence and light chain antibody, and the resulting sequences were ligated to the PTT5 vector to obtain the 6F2A11 heavy chain PTT5 vector and the 6F2A11 light chain PTT5 vector, respectively. These two vectors were transiently transfected into 293T cells at the same time, and the 293T supernatant protein was harvested. After purification by Protein A affinity chromatography, the anti-hPD1 antibody was obtained and named uw.6F2A11.

[0100] Anti-hPD1 antibodies uw.9G4C5, uw.19E3C1, and uw.10E9G7 of the positive clones 9G4C5, 19E3C1, and 10E9G7 were prepared according to the above method.

[0101] Evaluation of Anti-hPD1 Antibody Activity in Vitro

[0102] The four antibodies purified above (uw.6F2A11, uw.9G4C5, uw.19E3C1, and uw.10E9G7) were selected for in vitro activity evaluation. First, hPD1 recombinant protein was plated, and different gradients of the four antibodies were added to detect the binding of the antibodies to the hPD1 recombinant protein. The results are shown in Figure 7. The results show that uw.6F2A11, uw.9G4C5, uw.19E3C1, and uw.10E9G7 can all bind to the hPD1 recombinant protein, and the binding of uw.6F2A11 and uw.9G4C5 to the hPD1 recombinant protein is better than the positive control Keytruda. Furthermore, the purified antibodies were incubated with Jurkat-hPD1 cells, and then an APC-conjugated anti-IgG1-Fc secondary antibody was added. The binding of the purified antibodies to Jurkat-hPD1 cells was evaluated by the APC mean fluorescence intensity (MFI) of the secondary antibody. The results are shown in Figure 8, showing that uw.6F2A11, uw.9G4C5, uw.19E3C1, and uw.10E9G7 all bound to Jurkat-hPD1 cells. hIgG4 was used as a negative control, and Keytruda was used as a positive control.

[0103] In vivo efficacy evaluation of anti-hPD1 monoclonal antibodies

[0104] Taking the two antibodies uw.9G4C5 and uw.6F2A11 as examples, further in vivo pharmacodynamic activity evaluation was performed. In vivo pharmacodynamic experiment based on the MC38 subcutaneous inoculation model of B6-hPD1 mice. MC38 mouse colon cancer cells in the logarithmic growth phase were inoculated subcutaneously into 6-8 week-old B6-hPD1 (a product of Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.). On the 6th day after inoculation, the average tumor volume reached 82.13 mm 3 32 mice were randomly divided into hIgG4 (negative control), Keytruda (positive control), uw.9G4C5, and uw.6F2A11 treatment groups (n=8) according to tumor volume, and treated with the corresponding drugs (hIgG4, Keytruda, uw.9G4C5, and uw.6F2A11). Specific dosing groups are shown in Table 3 below. The drugs were administered twice a week for a total of 6 times. The results are shown in Figure 9, which show that the antibody uw.9G4C5 has a significant inhibitory effect on tumor growth, and its inhibitory effect on tumor growth is better than that of the positive drug Keytruda.

[0105] Table 3 In vivo efficacy of anti-PD1 antibodies in groups

[0106] Example 3 Construction of immune reconstitution mice by fetal liver cell transplantation

[0107] To test whether stem cells derived from mouse embryos can reconstitute the immune system in transplant recipients, this example isolated fetal liver cells of appropriate gestational age and transplanted them into recipient mice that had been pretreated with immunosuppression (Table 4). The specific procedures were as follows:

[0108] Immunosuppressive Preconditioning of Recipient Mice: Four-week-old B6 recipient mice were pre-irradiated and fed antibiotics for 7 days before irradiation and for 14 consecutive days after irradiation (Table 4). Donor embryos were F1 hybrid embryos (129×BALB / c F1 fetal livers) obtained by mating BALB / cJGpt (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number N000020) with 129S1 / SvImJGpt (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number N000017). Recipient mice were B6 mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number N000013).

[0109] Donor Mouse Fetal Liver Cell Extraction: Euthanize a suitable pregnant mouse, open the uterus, remove the embryo, strip the fetal membranes, and rinse the embryo repeatedly with 4°C PBS. Finely dissect the anterior abdominal wall of the embryo, remove the fetal liver intact, and wash it in a sterile Petri dish containing 4°C PBS. Rinse three times, then coarsely cut the liver, grind it with frosted glass, filter it through a 40μm filter, and carefully pipette it into a 50mL centrifuge tube. RBC Lysis Buffer is used to lyse the red blood cells and collect the cells for transplantation.

[0110] Fetal liver cell transplantation: After irradiation, fetal liver cells were transplanted into irradiated recipient mice via tail vein injection. The day of irradiation was defined as D0. Following transplantation, mice were observed and weighed weekly, and body weight and survival rates were calculated. The results are shown in Figures 10 and 11.

[0111] The results showed that mouse fetal liver cells can reconstitute the immune system in recipient mice. After transplantation, the weight of the recipient mice increased steadily (Figure 10), and the survival rate of the fetal liver transplant group was 90% (Figure 11). Figure 12 shows the results of the B cell (mCD19+) tracing analysis of fetal liver transplant recipient mice. Among them, A is a B6J control mouse that has not been transplanted with fetal liver cells, and B is the B cell tracing analysis of the fetal liver of the recipient mouse. Since the IgM of B6 background mice is type b, and the IgM of 129 and BALB / c background mice is type a, flow cytometry is used to detect different types of IgM for distinction. mIgM-A is a protein expressed on the surface of B cells of 129×BALB / c background mice, and mIgM-B is a protein expressed on the surface of B cells of B6J mice. The results showed that almost all B cells (mCD19+) originated from donor mice, proving that fetal liver transplantation can reconstitute the immune system in recipient mice. In this example, F1 mice, a hybrid of 129 and BALB / c strains, were used as stem cell donors and transplanted into B6J recipient mice. The results demonstrated that the transplantation and reconstruction method proposed in this invention can be performed between mice with different genetic backgrounds.

[0112] Table 4 Grouping of animal experiments with fetal liver cell transplantation and reconstruction

[0113] Note: G, group; N, number of animals.

[0114] Example 4: Construction of immune reconstitution mice using frozen-thawed stem cells

[0115] In this example, bone marrow cells frozen in liquid nitrogen were revived and transplanted into recipient mice that had undergone immunosuppressive pretreatment (referring to the method described in Example 1, with specific groupings shown in Table 5) for immune reconstitution. Flow cytometry analysis, as shown in Figure 13, demonstrated that frozen-thawed bone marrow cells could reconstitute the immune system in the recipient mice.

[0116] Table 5 Frozen-thawed bone marrow cell transplantation and reconstruction experiment

[0117] Example 5 Different target immunization and human antibody screening test

[0118] With reference to the method of Example 2, donor mouse bone marrow cells carrying human immunoglobulin variable region encoding genes were prepared and transplanted into the corresponding target gene knockout recipient mice that had been pretreated with immunosuppression (refer to the method in Example 1, see Table 6 for specific grouping) for immune reconstruction. The successfully reconstructed mice were immunized to screen for antibodies encoded by human genes. The recipient mice with target gene knockout were TIGIT KO mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number: T037162) and LAG3 KO mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number: T002755). The immune cells of TIGIT KO mice and LAG3 KO mice after bone marrow transplantation were traced and analyzed, and the immune serum titer was determined.

[0119] The results, as shown in Figures 14-15, show that bone marrow cells from humanized mouse models encoding antibody genes were able to successfully reconstitute the immune system in mice with different gene knockouts. Following immunization with the hTIGIT recombinant protein antigen, transplanted and reconstructed TIGIT KO mice were able to generate an immune response and produce specific antibodies with a titer exceeding 100,000. Following immunization with the hLAG3 recombinant protein antigen, transplanted and reconstructed LAG3 KO mice were able to generate an immune response and produce specific antibodies with a titer exceeding 100,000. These results demonstrate that the technical solutions described herein are suitable for screening humanized antibodies against different targets.

[0120] Table 6 Screening test of human antibodies with different targets

[0121] Example 6: Breaking B cell immune tolerance and enhancing immune response through bone marrow transplantation

[0122] Bone marrow cells from donor mice carrying human immunoglobulin variable region encoding genes were prepared according to the method of Example 2 and transplanted into recipient mice with corresponding target gene knockout that had been pretreated with immunosuppression (see the method of Example 1) for immune reconstitution. Successfully reconstituted mice, antibody-encoding gene humanized mice (TG), and CLDN KO mice were immunized according to Table 7 to evaluate whether bone marrow transplantation breaks through B cell peripheral immune tolerance and enhances immune response. The target gene knockout recipient mice were CLDN KO mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain number: T014374).

[0123] The results, as shown in Figures 16-17, show that bone marrow cells from humanized antibody-encoding mice successfully reconstituted the immune system in mice with various gene knockouts. Following immunization with commercially available HEK293-hClaudin18.2 cells, transplanted CLDN KO mice were able to generate an immune response that was greater than that of both TG and CLDN KO mice. This suggests that bone marrow transplantation from TG mice with humanized antibody-encoding genes enhances the immune response in CLDN KO mice.

[0124] Table 7 Immune tolerance experiment design and grouping

Claims

1. A method for preparing an antibody, characterized in that An antigen is used to immunize a recipient animal and obtain antibodies that can bind to the antigen. The recipient animal is an animal whose immune system has been reconstructed by transplanting stem cells with differentiation potential. The stem cells with differentiation potential are derived from a donor animal carrying one or more human immunoglobulin variable region gene segments.

2. The method according to claim 1, characterized in that The stem cells with differentiation potential are one or more of bone marrow cells, fetal liver cells, hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells.

3. The method according to claim 1, characterized in that The immune system reconstruction is performed by bone marrow stem cell elimination, immunosuppression and immunoregulation using low-toxic drugs, or gene modification to make the recipient animal's immune system defective or suppressed, and then stem cells with differentiation potential are transplanted into the recipient animal.

4. The method according to claim 3, characterized in that The bone marrow clearance is the use of sublethal doses of ionizing radiation or chemical means to eliminate hematopoietic stem cells in the animal bone marrow; the low-toxicity drug is a drug that inhibits stem cell proliferation or blocks the conduction of differentiation information; the gene modification that causes the recipient's immune system to be defective or suppressed is a defect in the development or differentiation of hematopoietic stem cells or a variety of immune cell development.

5. The method according to claim 4, characterized in that The ionizing radiation is X-ray or Co60 ray irradiation, and the chemical means is to administer one or more of busulfan, cyclophosphamide, and melphalan to the animal.

6. The method according to claim 1, characterized in that The recipient animal is a non-human mammal or a rodent.

7. The method according to claim 1, characterized in that The recipient animal is selected from rats, mice, rabbits, sheep, camels, and non-human primates.

8. The method according to claim 1, characterized in that The recipient animal is a wild type animal or an animal carrying a non-natural endogenous gene.

9. The method according to claim 8, characterized in that The animal carrying the non-natural endogenous gene is an animal in which the target antigen encoding gene is knocked out.

10. The method according to any one of claims 1 to 9, characterized in that The steps include: (1) Subjecting animals to immunosuppressive pretreatment to obtain immunosuppressed recipient animals; constructing or purchasing one or more animals carrying human immunoglobulin variable region gene fragments as donors, collecting stem cells with differentiation potential from the animals and transplanting them into immunosuppressed recipient animals to obtain immune-reconstructed recipient animals whose immune cells are derived from the donors; (2) using the antigen to immunize the recipient animal obtained in step (1); (3) Selecting animals that can produce antibodies that bind to the target antigen, collecting their immune cells, and further preparing human antibodies.

11. A humanized antibody, characterized in that The method is prepared by any one of claims 1 to 10.

12. A pharmaceutical composition, characterized in that Comprising the humanized antibody according to claim 11.

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