A method for preparing an antibody

Abstract

The present invention discloses a method for preparing an antibody and an antibody prepared by using the method. The method uses an antigen to immunize an animal with a reconstituted immune system transplanted with stem cells having differentiation potential, and obtains an antibody capable of binding to the antigen. The stem cells having differentiation potential are derived from a donor animal carrying one or more human immunoglobulin variable region gene segments. The method of the present invention solves the problems that the live transgenic animals used in the prior art for preparing human antibodies are limited by relevant requirements such as policies and quarantine, with a long transportation time and high costs. When preparing an antibody with a high homology between the transgenic animal and the human target antigen, the method of the present invention can use an immunosuppressed animal with the target antigen-encoding gene knocked out as a transplantation recipient, so as to solve the problem that it is difficult for transgenic animals in the prior art to produce antibodies against the homologous region between the human and transgenic animal target antigens.

Description

Technical Field

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

[0002] Antibody drugs are currently a rapidly growing, most advanced, and effective class of drugs. The advent of monoclonal antibody technology has made it possible to research and produce antibody drugs. After more than thirty years of development, therapeutic monoclonal antibody drugs have now become one of the most important components of biomedicine and have broad application prospects in disease treatment. They have been successfully used to treat various diseases such as tumors, autoimmune diseases, infectious diseases, and transplant rejection reactions. Since the first therapeutic monoclonal antibody was approved in 1975, more than 100 monoclonal antibodies have been approved for market, showing a rapid growth trend in recent years

[0003] The earliest therapeutic antibodies were completely of animal origin, and their immunogenicity could cause the human body to produce anti-antibodies (Human Anti-Mouse Antibodies, HAMA), and even cause serious side effects such as allergies. By engineering the humanization of animal-derived antibodies through antibody engineering, the immunogenicity can be reduced, but this process requires huge costs and time, and it is difficult to completely eliminate immunogenicity while maintaining high affinity. The solution is to develop fully human antibodies, and currently there are mainly four methods: A) display technology; B) transgenic mouse immune screening; C) immortalization of human B cells; D) single-cell screening of human memory B cells. Among them, for the "display technology", this technology can screen antibodies quickly and in a high-throughput manner. However, since the screening process is a pure artificial in vitro system, the antibody diversity and affinity obtained are limited, and there are non-natural structures, and there is still immunogenicity, and a large amount of time and cost are still required for optimization and transformation. Human B cells can only be isolated from humans who have been exposed to antigens and have an immune response. Due to source limitations, the screening methods using human B cells can obtain fewer antibody types, and the probability of screening high-affinity antibodies is very low. Compared with other methods, the gene-modified mouse model transfected with human antibody-encoding genes has obvious advantages. Mice carrying the complete human antibody variable region-encoding gene (>3Mb) can produce an immune response similar to that of humans against antigens, and obtain fully human antibodies with high affinity encoded by human genes and matured through natural somatic hypermutation and other processes in vivo. Due to relying on the completely natural B cell screening and maturation process, without the need for later optimization, the antibodies are encoded by human genes and there will be no immunogenicity problems. This method can obtain high-affinity monoclonal antibodies at a time and cost far lower 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 of Kymab all belong to this type of model

[0004] Mice carrying human antibody-encoding genes can have a sound immune system, and high-affinity antibodies encoded by human genes can be produced by immunization with the target antigen. This greatly simplifies the technical process of therapeutic antibody discovery and saves the time and cost of antibody engineering (such as humanization and affinity improvement).

[0005] However, there is a high homology between mouse and human proteins. Taking CLAUDIN-18 as an example, the amino acid homology of CLAUDIN-18 between humans and mice is 88%, and the human-mouse homology of some proteins can reach over 95%. There is a "negative selection" process during B cell maturation, which clones and eliminates B cells 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, becoming a major obstacle to obtaining human target antibodies. To overcome this difficulty, the prior art knocks out the gene encoding the target antigen in mice carrying human antibody-encoding genes to obtain homozygous knockout transgenic mice for immunization. Although this method can obtain antibodies against homologous antigens (sequence conserved regions), it requires the production of a gene knockout strain for each target antigen, consuming a large amount of time. In addition, when antibodies against targets (target antigens) related to the immune system function need to be prepared, knocking out the gene encoding the target antigen in mice may affect the development and function of the mouse immune system, and thus corresponding antibodies cannot be obtained through the immune response.

[0006] In addition, since the immune process is completed in transgenic (or transgenic + knockout) mice, live animals need to be obtained to conduct experiments. Limited by policies and quarantine requirements, the transportation time of live animals is long and the cost is high, which also reduces the accessibility of transgenic live animals and brings difficulties to antibody screening using transgenic mice. Summary of the Invention

[0007] The present invention provides a new method for preparing human antibodies. Stem cells of donor transgenic animals carrying one or more human immunoglobulin variable region gene fragments are transplanted into immunosuppressed animals to reconstruct the immune system of the recipient animals. After the immune system of the recipient animals is successfully reconstructed and immunized with the antigen, antibodies are produced using immune cells from the donor, so as to solve the problem that live transgenic animals used in the prior art for preparing human antibodies are limited by policies and quarantine requirements, with long transportation time and high cost. The immune cells from the donor can be freshly transplanted, or cryopreserved and revived, and are convenient for long-distance transportation. The cryopreserved donor immune cells (instead of live animals) can be revived and transplanted into recipient mice to reconstruct the immune system, and antibodies can be produced through immunization, avoiding the transportation difficulties of live animals.

[0008] When the method of the present invention is used to prepare an antibody with high homology to a human target antigen in a transgenic animal, an immunosuppressed animal with a knockout of the target antigen-encoding gene can be used as a transplantation recipient, solving the problem that it is difficult for a transgenic animal to produce an antibody against the homologous region of the human and transgenic animal target antigens in the prior art.

[0009] The specific technical solution of the present invention is as follows:

[0010] A method for preparing an antibody, which uses an antigen to immunize a recipient animal and obtains an antibody capable of binding to the antigen, wherein the recipient animal is an animal with a reconstituted immune system transplanted with stem cells having differentiation potential, and the stem cells having differentiation potential are derived from a donor animal carrying one or more human immunoglobulin variable region gene fragments.

[0011] 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 recombination or / and V K J K recombination or V L J L one or more of the regions encoded by the gene fragments formed by recombination. Preferably, the variable region coding fragment is formed by the participation of one of J H1 、J H1P 、J H2 、J H2P 、J H3 、J H3P 、J H4 、J H5 or J H6 in recombination. Preferably, the variable region coding fragment is formed by the participation of one of J K1 、J K2 、J K3 、J K4 or J K5 in recombination.

[0012] The antibody of the present invention is a human antibody capable of specifically recognizing and binding to a target antigen.

[0013] The target antigen can be a molecule or compound that is artificially synthesized, recombinantly expressed, or naturally present in nature.

[0014] The target antigen can be a polypeptide chain containing more than 3 amino acids.

[0015] The target antigen can be a membrane receptor or a free protein.

[0016] The human antibody may be a diagnostic, detection or therapeutic antibody targeting a specific disease-related therapeutic target, preferably a therapeutic antibody.

[0017] The human antibody may be an antibody whose variable region is encoded by human genes, preferably a therapeutic antibody, such as a therapeutic antibody for treating human diseases such as tumors, autoimmune diseases, metabolic diseases, and nervous system diseases.

[0018] In the above method, the donor and recipient animals for transplantation may have the same genetic background or different genetic backgrounds. The stem cells with differentiation potential are stem cells capable of differentiating into multiple immune cells, and may 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 may be newly isolated cells or cells revived after freezing.

[0019] 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 example of the present invention, the donor and recipient animals for transplantation are mice.

[0020] In the method of the present invention, the immunosuppressed recipient animal is an immunosuppressed recipient animal obtained by one or more of bone marrow ablation, immunosuppression and immunomodulation using low-toxicity drugs, and genetic modification to make the recipient immune system defective or inhibited.

[0021] The bone marrow ablation is to ablate hematopoietic stem cells in the bone marrow of animals by using a sub-lethal dose of ionizing radiation or chemical means; the use of low-toxicity drugs is to use drugs that inhibit stem cell proliferation or block the transmission of differentiation information (such as imatinib, etc.) for immunosuppression and immunomodulation; the genetic modification to make the recipient immune system defective or inhibited is a hematopoietic stem cell development defect, including but not limited to c-kit or thrombopoietin gene mutation mice.

[0022] The method for clearing hematopoietic stem cells in the bone marrow of animals by ionizing irradiation or chemical means described in the present invention can adopt the types, doses, and operation methods commonly used in the prior art. For example, for ionizing irradiation, X-rays or Co60 rays are used for irradiation, and the irradiation dose is 600 cGy - 950 cGy, or a higher sublethal dose. Specifically, the chemical means described in the reference (Eunbee Park, et al., JVis Exp, 2021.) is to administer one or several of busulfan, cyclophosphamide, and melphalan to the animals. Taking busulfan as an example, the myeloablative pretreatment can be completed by intraperitoneal injection at a dose of 20 mg / kg twice with an interval of 24 hours (Encarnacion Montecino-Rodriguez, et al., STAR Protocols, 2020.).

[0023] The recipient animals described in the present invention can be wild-type or genetically engineered animals. The genetically engineered animals can be transgenic animals carrying human genes, animals carrying mutant genes, animals expressing reporter genes, or animals lacking target antigen-encoding genes (such as knockout of target antigen-encoding genes). Animals lacking target antigen-encoding genes are preferred.

[0024] The method for screening human antibodies can be methods such as hybridoma, single B cell screening, or display library screening, etc. Those skilled in the art understand that monoclonal antibodies producing target antibodies can be obtained from the B cell population obtained after immunizing the animals obtained based on the transplantation and reconstruction method provided by the present invention through existing antibody screening techniques, and further prepare target human antibodies that meet the requirements.

[0025] The method described in the present invention specifically includes the following steps:

[0026] (1) Pretreat the animals with immunosuppression to obtain immunosuppressed recipient animals; construct or purchase animals carrying one or more human immunoglobulin variable region gene fragments as donors, collect their bone marrow cells, embryonic cells, or purified hematopoietic stem cells, and transplant them into the immunosuppressed recipient animals.

[0027] The immune cell reconstruction level in the transplanted recipient animals can be detected by flow cytometry, immunofluorescence, quantitative PCR, or other corresponding techniques. When the immune cells derived from the donor are stably reconstructed in the recipient, the animals can be immunized with the antigen.

[0028] (2) Immunize the recipient animals obtained in step (1) with the antigen.

[0029] (3) Select the animals that can produce antibodies binding to the target antigen, collect their immune cells, and further prepare human antibodies.

[0030] It is possible to collect the sera of immunized animals and detect the titers of serum antibodies that can bind to the target antigen by ELISA or other similar techniques. Select animals that can produce antibodies that bind to the target antigen, collect their immune cells, and screen for clones that can produce monoclonal antibodies with parameters such as affinity and functional activity meeting the requirements through methods such as hybridoma, flow sorting, and high-throughput sequencing. Then sequence the antibody-encoding region to obtain the coding sequence. Further, combine the antibody variable region sequence with the human antibody constant region and prepare the antibody through methods such as recombinant expression.

[0031] The construction order of the donors and recipients in the above method is not limited to a specific sequence.

[0032] Another object of the present invention is to provide a human antibody prepared by the method described in the present invention. Another object of the present invention is to provide a pharmaceutical composition comprising the human antibody described in the present invention.

[0033] Advantages of the present invention:

[0034] 1. Limited by policies and quarantine requirements, etc., the transportation of live animals used in antibody preparation in the prior art takes a long time and is expensive. The method described in the present invention can cryopreserve the immune cells from the donor prepared in advance and is convenient for long-distance transportation. The revived donor immune cells (instead of live animals) can be transplanted into local or remote recipient mice to reconstruct the immune system, and the mice can be immunized with the target antigen to produce antibodies, avoiding the transportation difficulties of live animals.

[0035] 2. When using transgenic mice to prepare human antibodies in the prior art, transgenic mice carrying human antibody-encoding genes are directly used for immunization, and a relatively large number of humanized transgenic animals are required, and such humanized transgenic animals are often expensive. The method of reconstruction by transplantation in the present invention can save the usage of transgenic animals several times. The stem cells from a single donor mouse can reconstruct 3 or more recipient mice, obtaining more animals for immunization and reducing costs.

[0036] 3. In the prior art, the gene encoding the target antigen in transgenic mice carrying the human antibody encoding gene is knocked out to establish a gene-modified strain that cannot express the target antigen, and then it is used 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 constructs a transgenic animal (transplant donor) carrying the human antibody encoding gene and a transgenic animal with the target antigen gene knocked out (transplant recipient) respectively. Through stem cell transplantation, a functionally normal immune cell population derived from the donor animal is reconstituted in the transgenic animal (recipient) with the target antigen gene knocked out, including mature functional T cells, B cells, antigen-presenting cells and other immune cells with complete functions. The stem cells with differentiation potential from the donor differentiate and mature in the transgenic animal (recipient) with the target antigen gene knocked out, and negative selection of T cells and B cells occurs in the transplant recipient. For the target antigen lacking in the recipient after transplantation and reconstruction, "negative selection" against the target antigen cannot effectively eliminate the B cell clones that produce antibodies recognizing the human / animal homologous region of the target antigen. Thus, the transgenic animal with the target antigen gene knocked out with successful immune reconstruction can produce antibodies encoded by human genes using the immune cells of the antibody-encoding gene humanized animal.

[0037] 4. The present invention solves the problem that ordinary animals (mice) are not easily obtained antibodies against homologous antigens (such as the conserved region of the amino acid sequence of homologous proteins), and uses transgenic animals with the antigen gene knocked out (recipients) to develop therapeutic antibodies for corresponding targets. The diversity of its antibody spectrum is increased, the immune response is enhanced, and the immune cycle is shortened, which has extremely important value for screening therapeutic antibodies.

[0038] 5. The present invention adopts the method of bone marrow (or hematopoietic stem cell) transplantation, and the immune system can be reconstituted in different recipient animals (mice) using the bone marrow (or hematopoietic stem cells) of the same transgenic ordinary animal (mice) to achieve the personalization of antibody production.

[0039] 6. The recipient animals used in the present invention can be wild-type or gene-modified strains. For example, the recipient animals can be disease models with genetic modifications such as gene mutations or transgenes, such as autoimmune diseases, tumors and metabolic diseases. Such models can be used to screen antibodies against potential therapeutic or detection targets for specific diseases.

[0040] The recipient animals can also be gene-modified animals carrying a specific reporter gene expression system. For example, when the animal produces the target antibody, specific tissues or cells will show phenotypes such as fluorescence emission and cytokine secretion to assist antibody screening.

[0041] 7. The stem cell transplantation reconstruction adopted by the present invention is a non-genetic method, which does not require target gene knockout on the basis of transgenic mice, saving the long and complex gene modification and mouse breeding time and improving the efficiency.

[0042] 8. The target gene knockout animals (recipients) for transplantation in the present invention can be produced synchronously with the transplantation donor animals, or can be produced in large batches and multiple targets. These gene knockout models do not carry human antibody encoding genes themselves and can also be used for other research.

[0043] 9. The hematopoietic stem cells of the transgenic animals (recipients) with target antigen gene knockout collected in the present invention can be cryopreserved and transplanted after resuscitation. The stem cells themselves can be used as a non-living product, which is easy to produce, preserve, transport in batches, and can be resuscitated, transplanted, immunized and screened for antibodies according to experimental needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the body weight change curve after transplanting the bone marrow of EGFP transgenic mice into B6 recipient mice.

[0045] Figure 2 It is the survival rate after transplanting the bone marrow of EGFP transgenic mice into B6 recipient mice.

[0046] Figure 3 It is the result of immune cell tracing analysis after transplanting the bone marrow of EGFP transgenic mice into B6 recipient mice.

[0047] Figure 4 It is the body weight change curve after transplanting the bone marrow cells of transgenic mice carrying the human immunoglobulin variable region encoding gene into gene-modified recipient mice and B6 wild-type recipient mice.

[0048] Figure 5 It is the survival rate after transplanting the bone marrow cells of transgenic mice carrying the human immunoglobulin variable region encoding gene into gene-modified recipient mice and B6 wild-type recipient mice.

[0049] Figure 6 It is the result of determination of the immune serum antibody titer after transplanting the bone marrow cells of transgenic mice carrying the human immunoglobulin variable region encoding gene into gene-modified recipient mice and B6 wild-type recipient mice.

[0050] Figure 7 It is the binding activity of the anti-PD1 antibody screened after transplanting the bone marrow cells of transgenic mice carrying the human immunoglobulin variable region encoding gene into PD1 KO recipient mice to the hPD1 recombinant protein.

[0051] Figure 8The binding activity of the anti-PD1 antibody obtained by screening the bone marrow cells of transgenic mice carrying the gene encoding the variable region of human immunoglobulin in PD1 KO recipient mice to Jurkat-PD1 cells.

[0052] Figure 9 The in vivo efficacy results of the anti-PD1 antibodies (9G4C5 and 6F2A11) obtained by screening the bone marrow cells of transgenic mice carrying the gene encoding the variable region of human immunoglobulin in PD1 KO recipient mice.

[0053] Figure 10 The weight change curve of mice after fetal liver cell transplantation and reconstitution.

[0054] Figure 11 The survival rate of mice after fetal liver cell transplantation and reconstitution.

[0055] Figure 12 The results of immune cell tracing analysis of mice after fetal liver cell transplantation and reconstitution.

[0056] Figure 13 The results of B cell tracing analysis after cryopreserved and fresh bone marrow cell transplantation and reconstitution.

[0057] Figure 14 The results of immune cell tracing analysis after bone marrow transplantation in TIGIT KO mice and LAG3 KO mice.

[0058] Figure 15 The results of immune serum titer of bone marrow transplantation in TIGIT KO mice and LAG3 KO mice.

[0059] Figure 16 The immune cell tracing results of gene-modified recipient mice (TG), CLDN KO mice and CLDN KO mice.

[0060] Figure 17 The determination results of immune serum antibody titers of gene-modified recipient mice (TG), CLDN KO mice and CLDN KO recipient mice after transplantation of bone marrow cells of transgenic mice carrying the gene encoding the variable region of human immunoglobulin. Detailed implementation manners

[0061] The following examples illustrate the specific steps of the present invention, but are not limited by the examples.

[0062] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0063] The present invention will be further described in detail below with reference to specific examples and data. It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention in any way.

[0064] In the following embodiments, various processes and methods not described in detail are conventional methods well known in the art.

[0065] Example 1 Construction of immune - reconstructed mice by bone marrow cell transplantation

[0066] Immunosuppressive pretreatment of recipient mice:

[0067] In this example, 4 - week - old B6 mice (product of Jiangsu Jicui Yakang Biotechnology Co., Ltd., strain number: N000013) were selected as recipients and pretreated by irradiation - based myeloablation. However, those skilled in the art know that animals of other ages and strains can be used as transplantation recipients, and other pretreatment methods (such as chemical myeloablation) that can achieve the myeloablation effect can also be used. The inventors found through research that 600 - 900 cGY is the 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.

[0068] Extraction and transplantation of donor mouse bone marrow cells:

[0069] In this study, the bone marrow of EGFP - transgenic donor mice was transplanted into the irradiated recipient mice of the background strain C57BL / 6JGpt (B6). The EGFP - transgenic donor mice are a fluorescent mouse model developed by Jiangsu Jicui Yakang Biotechnology Co., Ltd. (strain number T006163), and the green fluorescent protein EGFP is controlled by the CAG promoter and is widely expressed in mouse tissues. This model can be used to obtain various mouse cells labeled with green fluorescent protein, especially in the research of cell transplantation.

[0070] The specific operation steps are as follows: The donor mice were euthanized, the hind leg bones were separated and placed in a sterile culture dish containing PBS at 4°C. After rinsing the bone marrow with PBS, donor mouse bone marrow cells were obtained, resuspended and lysed to remove red blood cells, and then the bone marrow cells were collected for standby. The collected bone marrow cells were transplanted into the irradiated recipient mice through tail vein injection. After transplantation, the mice were observed and weighed weekly, and the body weight and survival rate were statistically analyzed. Figure 1 Body weight change of B6 recipient mice after transplantation of EGFP - transgenic mouse bone marrow. The results showed that the body weight of the irradiated and transplanted recipient mice recovered with the increase of the week age. The trend of body weight change indicated that the physiological state of the transplanted mice was normal. Figure 2 Survival rate of B6 recipient mice after transplantation of EGFP - transgenic mouse bone marrow. The results showed that the survival rate of the irradiated and transplanted recipient mice was 100%. During the observation period of more than 90 days after transplantation, no mouse death occurred. This indicates that the transplanted mice (100%) can survive for at least 90 days and above.

[0071] Detection of immune system reconstruction indicators:

[0072] In this example, the EGFP transgenic mouse cells from the donor can fluoresce, while the B6 wild-type mouse cells of the recipient cannot. Therefore, by detecting B cells (mCD19+) in the peripheral blood of the reconstituted mouse through flow cytometry, if green fluorescence is detected, it indicates that the cells are from the donor; if no fluorescence is detected, the cells are from the recipient mouse.

[0073] Starting from the 4th week after transplantation, the proportion of immune cells from the donor in the peripheral blood of the reconstituted mouse was collected, with EGFP as the fluorescent label, and the proportion of immune cells from the donor was detected by flow cytometry. Figure 3 Results of immune cell tracing analysis after transplanting EGFP transgenic mouse bone marrow into B6 recipient mice for reconstitution. The results showed that 94.2% of white blood cells (mCD45+) and 99.9% of B cells (mCD19+) in the recipient mice were EGFP+ cells 60 days after irradiation and transplantation, which were from the donor, proving that bone marrow cells from the donor could stably reconstitute immune cells in the recipient mouse, and the immune cells of the recipient mouse itself did not reappear after immunosuppression. This example proves that the method described in the present invention can reconstitute the immune system by transplanting bone marrow cells of the donor mouse into the immunosuppressed pre-treated recipient.

[0074] Example 2 Preparation of specific antibodies using bone marrow transplantation immune reconstituted mice

[0075] Using the transplantation and reconstitution method described in Example 1, bone marrow cell transplantation and reconstitution animals were constructed according to the grouping in Table 1. Among them, G1 and G3 were PD1 gene knockout recipient mice (PD1 KO) (the recipient mice were products of Jiangsu Genscript Biotech Co., Ltd., strain number: T011515) and wild-type B6 mice (the recipient mice were products of Jiangsu Genscript Biotech Co., Ltd., strain number: N000013) that were respectively transplanted with bone marrow cells of humanized mice (TG) carrying human immunoglobulin variable region gene fragments, and G2 and G4 were PD1 gene knockout recipient mice (PD1KO) and wild-type B6 mice that were not pretreated with immunosuppression and transplanted. The donor mice used in this experiment carried the genes encoding the variable regions of human immunoglobulin heavy chain (IGH) and light chain (IGK) in their genomes. And the corresponding murine Igh and Igk genes were inactivated (prepared according to the method disclosed in the reference E-Chiang L et al., Nature Biotechnology. 2014). Specifically, referring to the ES targeting method described in the reference, a targeting vector carrying a human IGH variable region gene fragment (including human V H , D H and J H gene fragments), and a targeting vector carrying a human IGK variable region gene fragment (including human V K and J KTargeting vectors of gene fragments). The above targeting vectors were respectively electroporated into ES cells to obtain ES clones in which the human IGH gene fragment was successfully integrated upstream of the coding fragment of the constant region of the mouse Igh gene, and ES clones in which the human IGK gene fragment was successfully integrated upstream of the coding fragment of the constant region of the mouse Igk gene. The above targeted clones were injected into mouse blastocysts and transplanted into the uterus of pseudopregnant mice, and finally targeted mice developed from the targeted ES cells were obtained. The IGH-targeted mice were mated with the IGK-targeted mice to obtain homozygous mice with double targeting of IGH / IGK, which were used as humanized mice (donors) carrying the coding genes of the variable regions of human immunoglobulins, and stem cells with differentiation potential for transplantation were provided.

[0076] After transplantation, the recipients were observed and weighed weekly, and the body weight and survival rate were statistically analyzed. The results showed that after transplantation, the recipient mice grew steadily and 100% survived for more than 90 days ( Figure 4 , Figure 5 ). After successful reconstruction, immunization was carried out with commercial hPD1 recombinant protein and Freund's adjuvant.

[0077] Table 1 Grouping of animal experiments for bone marrow cell transplantation reconstruction

[0078]

[0079]

[0080] Immunization procedure:

[0081] Primary immunization: 100 μg of antigen was thoroughly mixed with an equal volume of Freund's complete adjuvant using a vortex oscillator and injected subcutaneously at multiple points in each mouse. The day of primary immunization was recorded as day 0.

[0082] Second immunization: On day 21, 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 dose, method, and route were the same as those of the primary immunization.

[0083] Third immunization: On day 42, 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 dose, method, and route were the same as those of the primary immunization (if continuous immunization is required, the protocol is the same).

[0084] Antibody titer detection:

[0085] a) Two weeks after the third immunization, about 100 - 200 μL of blood was collected from the mouse's orbit, centrifuged at 7000 rpm for 5 min, and the upper light yellow serum was taken as the sample.

[0086] b) Dilute the antigen (recombinant hPD1 protein) to different concentrations (1 μg / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 50 ng / mL) using the coating buffer, add 100 μl / well to the ELISA plate, perform 2 replicates for each concentration, and coat overnight at 4°C;

[0087] c) Discard the coating buffer, wash each well 3 times with 200 μl of PBST for 3 minutes each time. Invert the ELISA plate on filter paper to absorb the excess PBST, and coat each well with 200 μL of blocking solution at 37°C for 2 hours;

[0088] d) Discard the blocking solution, wash each well 3 times with 200 μl of PBST for 3 minutes each time. Dilute the positive and negative sera two-fold with the coating solution (1:200, 1:400, 1:800, 1:1600), add 100 μL / well to the ELISA plate, and set up a blank control at the same time. Incubate at 37°C for 1.5 hours.

[0089] e) Discard the blocking solution, wash each well 3 times with 200 μl of PBST for 3 minutes each time. Dilute the goat anti-mouse enzyme-labeled secondary antibody at 1:5000, and the dilution solution is PBST + 1% BSA. Add 100 μL / well to the ELISA plate and incubate at 37°C for 1 hour.

[0090] f) Discard the liquid in the wells, wash each well 3 times with 200 μl of PBST for 5 minutes each time. Add the TMB chromogenic substrate solution at 100 μL / well to the ELISA plate under light-proof conditions, react at room temperature in the dark for 10 minutes, add 100 μL of the stopping solution, and measure the OD450nm value. The results show that after diluting the mouse serum at 1:250000, the OD405nm absorption values of the samples in groups G1 - G4 are higher than those of the non-immunized control serum This result shows that the antigen-specific antibody titers in these 4 groups of sera reach 250000, and the serum antibody titers of the transplanted and reconstructed recipient mice (G1, G3) are higher than those of the non-irradiated transplantation control group (G2, G4) ( Figure 6 )

[0091] Screening and Production of Anti-hPD1 Monoclonal Antibody

[0092] (1) Hybridoma Screening

[0093] When the serum titer of the mice reached the appropriate range, mice with obvious immune responses in the G1 group were selected and boosted intraperitoneally with the hPD1 recombinant protein at a dose of 25 μg per mouse. After 3 - 4 days, the spleens of the mice were taken, ground through a 70 - μm sieve, fused with SP2 / 0 cells using an electrofusion apparatus, and then plated. After culturing with HT for 7 days, the supernatant of the fused hybridoma cells was detected by plating the hPD1 recombinant protein for positive screening of hybridoma by ELISA.

[0094] (2) Sub - cloning screening and cell line determination

[0095] The mother - clone hybridomas that were ELISA - positive after fusion were sub - cloned. The cells determined to be positive were blown up and transferred into 1.5 - ml EP tubes, and 1000 μl of HT medium was added. A small amount of cells was counted, and 100 cells were dissolved in 23 ml of HT medium, with 200 μl per well for limited dilution in a 1×96 - well plate. After culturing the sub - cloned cells for 7 days, the supernatant of the sub - cloned cells was detected by plating the hPD1 recombinant protein for positive screening of sub - cloned ELISA. At the same time, the supernatant of the ELISA - positive sub - clones was co - incubated with Jurkat - hPD1 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.), and flow cytometry was performed for screening the binding of the sub - cloned supernatant to the natural hPD1 protein. Finally, 4 double - positive sub - clones that bound both the hPD1 recombinant protein and Jurkat - hPD1 cells were identified, named 6F2A11, 9G4C5, 19E3C1, and 10E9G7 respectively. The 4 positive sub - cloned wells were transferred to a 6 - well plate for expansion culture. After growing to confluence, the cells were divided into two parts, one part was cryopreserved in liquid nitrogen for sequencing, and the other part was cryopreserved for backup.

[0096] (3) Sequencing of positive monoclonal antibodies

[0097] The screened positive sub - cloned cells (6F2A11, 9G4C5, 19E3C1, 10E9G7) were lysed, and mRNA was extracted and reverse - transcribed into cDNA. Using this cDNA as a template, the nucleic acid sequences of the variable regions of the light and heavy chains of the IgG antibody were amplified by PCR respectively, and Sanger sequencing analysis was performed on the variable regions of the heavy and light chains.

[0098] Table 2: Amino acid sequences of the variable regions of the heavy and light chains of 4 positive clones (6F2A11, 9G4C5, 19E3C1, 10E9G7) and amino acid sequences of the human heavy and light chain constant regions

[0099]

[0100]

[0101] (5) Production and preparation of Anti - hPD1 antibody

[0102] Taking 6F2A11 as an example, the heavy chain antibody sequence of 6F2A11 (the variable region of the heavy chain of the 6F2A11 positive clone sequencing + the constant region of human IgG1) and the light chain antibody sequence of 6F2A11 (the variable region of the light chain of the 6F2A11 positive clone sequencing + the constant region of human kappa chain) were synthesized by gene. According to the synthesized heavy chain antibody sequence and light chain antibody of 6F2A11, the corresponding nucleic acid sequences were designed respectively, and ligated to the PTT5 vector respectively to obtain the 6F2A11 heavy chain PTT5 vector and the 6F2A11 light chain PTT5 vector. These two vectors were co-transfected into 293T cells transiently, and the 293T supernatant protein was harvested. After purification by Protein A affinity chromatography, the Anti-hPD1 antibody was obtained and named uw.6F2A11.

[0103] Referring to the above method, the Anti-hPD1 antibodies uw.9G4C5, uw.19E3C1, and uw.10E9G7 of the positive clones 9G4C5, 19E3C1, and 10E9G7 were prepared.

[0104] In vitro activity evaluation of Anti-hPD1 antibody

[0105] Four antibodies purified above (uw.6F2A11, uw.9G4C5, uw.19E3C1, uw.10E9G7) were selected for in vitro activity evaluation. First, the hPD1 recombinant protein was plated, and different gradients of the four antibodies were added respectively to detect the binding of the antibodies to the hPD1 recombinant protein. The results are as Figure 7 shown. The results showed that uw.6F2A11, uw.9G4C5, uw.19E3C1, and uw.10E9G7 could all bind to the hPD1 recombinant protein, and the binding of uw.6F2A11 and uw.9G4C5 to the hPD1 recombinant protein was better than that of the positive control Keytruda. Further, the purified antibodies were co-incubated with Jurkat-hPD1 cells, and then the anti-IgG1-Fc secondary antibody conjugated with APC was added. The binding of the purified antibodies to Jurkat-hPD1 cells was evaluated by the average fluorescence intensity (MFI) of the APC of the secondary antibody. The results are as Figure 8 shown. The results showed that uw.6F2A11, uw.9G4C5, uw.19E3C1, and uw.10E9G7 could all bind to Jurkat-hPD1 cells. Among them, hIgG4 was used as the negative control and Keytruda was used as the positive control.

[0106] In vivo efficacy evaluation of Anti-hPD1 monoclonal antibody

[0107] Taking two antibodies, uw.9G4C5 and uw.6F2A11, as examples, the in vivo pharmacodynamic activity was further evaluated. Based on the in vivo pharmacodynamic experiment of the subcutaneous inoculation of MC38 model in B6-hPD1 mice. The murine colon cancer cell line MC38 in the logarithmic growth phase was inoculated subcutaneously into 6-8-week-old B6-hPD1 mice (products of Jiangsu GICC Biotech Co., Ltd.). On the 6th day after inoculation, the average tumor volume reached 82.13 mm 3 When it reached, 32 mice were randomly grouped into hIgG4 (negative control), Keytruda (positive control), uw.9G4C5, and uw.6F2A11 administration groups (n = 8) according to the tumor volume, and the corresponding drugs (hIgG4, Keytruda, uw.9G4C5, uw.6F2A11) were used for treatment respectively. The specific administration grouping is shown in Table 3 below. Administer the drugs 2 times a week for a total of 6 times. The results are as Figure 9 shown. The results showed that the antibody uw.9G4C5 had a significant effect on inhibiting tumor growth, and the effect of inhibiting tumor growth was better than that of the positive drug Keytruda.

[0108] Table 3 Administration grouping of anti-PD1 antibody in vivo pharmacodynamics

[0109] Group Number of animals (each) Dosing dose (mg / kg) Route of administration Dosing frequency and cycle hIgG4 6 5 i.p. BIW×3week Keytruda 6 5 i.p. BIW×3week uw.6F2A11 6 5 i.p. BIW×3week uw.9G4C5 6 5 i.p. BIW×3week

[0110] Example 3 Construction of immune-reconstituted mice by fetal liver cell transplantation

[0111] To test whether stem cells derived from mouse embryos can reconstruct the immune system in transplanted recipients, in this example, fetal mouse liver (fetal liver) cells of appropriate gestational age were isolated and transplanted into recipient mice pretreated with immunosuppression (Table 4). The specific operations are as follows:

[0112] Recipient mouse immunosuppressive pretreatment: Select 4-week-old B6 recipient mice for irradiation pretreatment. Antibiotics were fed 7 days before irradiation, and antibiotics were continuously fed for 14 days after irradiation (Table 4). The donor embryos were F1 hybrid embryos (129×BALB / c fetal liver) obtained by mating BALB / cJGpt (Jiangsu GICC Biotech Co., Ltd., strain number N000020) with 129S1 / SvImJGpt (Jiangsu GICC Biotech Co., Ltd., strain number N000017). The recipient mice were B6 mice (products of Jiangsu GICC Biotech Co., Ltd., strain number: N000013).

[0113] Extraction of fetal liver cells from donor mice: Select appropriate pregnant mice. After euthanasia, cut open the uterus, take out the embryos, strip the fetal membranes, and repeatedly rinse the embryos with PBS at 4°C. Carefully separate the anterior abdominal wall of the embryo, completely remove the fetal liver, place it in a sterile petri dish containing PBS at 4°C for washing. After rinsing 3 times, coarsely cut the fetal liver, grind it with frosted glass, filter it through a 40μm filter, and then carefully aspirate it into a 50mL centrifuge tube with a sterile pipette. After lysing red blood cells with RBC Lysis Buffer, collect the cells for transplantation.

[0114] Transplantation of fetal liver cells: After irradiation, transplant the fetal liver cells into the irradiated recipient mice via tail vein injection. The day of irradiation is defined as D0. Observe and weigh the mice weekly after transplantation, and count the body weight and survival rate. The results are as Figure 10 , 11 shown.

[0115] The results show that fetal liver cells of mice can reconstruct the immune system in recipient mice. After transplantation, the body weights of the recipient mice increased steadily ( Figure 10 ), and the survival rate of the fetal liver transplantation group was 90% ( Figure 11 ). Figure 12 This is the tracing analysis result of B cells (mCD19+) in recipient mice of fetal liver transplantation. Among them, A is a B6J control mouse that has not received fetal liver cell transplantation, and B is the tracing analysis of B cells in the recipient mouse of fetal liver. Since the IgM of B6 background mice is of type b, while the IgM of 129 and BALB / c background mice is of type a, different types of IgM are distinguished by flow cytometry. mIgM-A is the protein expressed on the surface of B cells of 129×BALB / c background mice, and mIgM-B is the protein expressed on the surface of B cells of B6J mice. The results show that almost all B cells (mCD19+) are derived from donor mice, proving that fetal liver transplantation can reconstruct the immune system in recipient mice. In this example, the F1 generation mice hybridized from 129 and BALB / c strains were used as stem cell donors and transplanted into B6J recipient mice. The results prove that the transplantation and reconstruction method proposed by the present invention can be carried out between different genetic backgrounds.

[0116] Table 4 Grouping of animal experiments for transplantation and reconstruction of fetal liver cells

[0117] Group Quantity Recipient mice Donor cell source G5 10 B6 129×BALB / c F1 fetal liver

[0118] Remarks: G, group, group number; N, number of animals.

[0119] Example 4 Construction of immune-reconstituted mice with cryopreserved and thawed stem cells

[0120] In this example, bone marrow cells cryopreserved in liquid nitrogen were thawed and transplanted into recipient mice that had been pre-treated with immunosuppression (refer to the method in Example 1, and the specific grouping is shown in Table 5) for immune reconstitution. Tracing analysis was performed by flow cytometry, and the results are as Figure 13 shown. The cryopreserved and thawed bone marrow cells can reconstitute the immune system in recipient mice.

[0121] Table 5 Transplantation and reconstitution test of cryopreserved and thawed bone marrow cells

[0122] Group Quantity Recipient mice Number of transplanted cells Donor cell source G6 10 B6 <![CDATA[1×10 7 cells / per]]> Cryopreserved and thawed bone marrow cells G7 10 B6 <![CDATA[1×10 7 cells / per]]> Freshly collected bone marrow cells

[0123] Example 5 Immune response and screening of human antibodies against different targets

[0124] Donor mouse bone marrow cells carrying the human immunoglobulin variable region-encoding gene were prepared according to the method of Example 2 and transplanted into recipient mice with corresponding target gene knockout that had been pre-treated with immunosuppression (refer to the method in Example 1, and the specific grouping is shown in Table 6) for immune reconstitution. The successfully reconstituted mice were immunized to screen for antibodies encoded by human genes. The recipient mice with target gene knockout were TIGIT KO mice (Jiangsu GICC Biopharma Co., Ltd., strain number: T037162) and LAG3 KO mice (Jiangsu GICC Biopharma Co., Ltd., strain number: T002755). Tracing analysis was performed on immune cells after bone marrow transplantation in TIGIT KO mice and LAG3 KO mice, and the immune serum titer was measured.

[0125] The results are as Figure 14-15 shown. The bone marrow cells of antibody-encoding gene humanized mice can successfully reconstitute the immune system in mice with different gene knockouts. After immunization with hTIGIT recombinant protein antigen, the transplanted and reconstituted TIGIT KO mice can produce an immune response and produce specific antibodies with a titer exceeding 100,000. After immunization with hLAG3 recombinant protein antigen, the transplanted and reconstituted LAG3 KO mice can produce an immune response and produce specific antibodies with a titer exceeding 100,000. The results prove that the technical solution of the present invention is applicable to the screening of human antibodies against different targets.

[0126] Table 6 Screening test of human antibodies against different targets

[0127] Group Quantity Recipient mice Number of transplanted cells Donor bone marrow cell source G8 5 TIGIT KO mice <![CDATA[1×10 7 cells / per unit]]> Antibody-encoding gene humanized mice G9 5 LAG3 KO mice <![CDATA[1×10 7 cells / per]]> Antibody-encoding gene humanized mice

[0128] Example 6 Breaking B cell immune tolerance and enhancing immune response through bone marrow transplantation reconstitution

[0129] The donor mouse bone marrow cells carrying the human immunoglobulin variable region encoding gene were prepared by referring to the method of Example 2 and transplanted into the corresponding target gene knockout recipient mice pretreated with immunosuppression (referring to the method in Example 1) for immune reconstitution. And the successfully reconstituted mice, antibody-encoding gene humanized mice (TG), and CLDN KO mice were immunized according to Table 7 to evaluate whether the peripheral immune tolerance of B cells was broken and the immune response was enhanced after bone marrow transplantation. The recipient mice with target gene knockout were CLDN KO mice (Jiangsu Jicui Yakang Biotech Co., Ltd., strain number: T014374).

[0130] The results are as Figure 16-17 shown. The bone marrow cells of antibody-encoding gene humanized mice could successfully reconstitute the immune system in mice with different gene knockouts. After immunization with HEK293-hClaudin18.2 cells (commercialized), the transplanted and reconstituted CLDN KO mice could produce an immune response, and the immune response was higher than that of antibody-encoding gene humanized mice (TG) and CLDN KO mice, indicating that the immune response was enhanced after the bone marrow transplantation of antibody-encoding gene humanized mice (TG) into CLDN KO mice.

[0131] Table 7 Experimental design and grouping of immune tolerance

[0132]

Claims

1. A method for preparing an antibody, characterized in that Immunize a receptor animal with an antigen and obtain an antibody capable of binding to the antigen. The receptor animal is an animal with a reconstituted immune system into which stem cells with differentiation potential have been transplanted. The stem cells with differentiation potential are derived from a donor animal carrying one or more human immunoglobulin variable region gene segments. The receptor and donor animals are non-human mammalian animals of the same species. The human immunoglobulin is selected from one or more of IgG, IgM, IgA, IgD, and IgE. The donor animal is a rodent. The reconstitution of the immune system is achieved by one or more of the following means: clearing bone marrow stem cells, immunosuppression and immunomodulation using low-toxicity drugs, and making the immune system of the receptor animal defective or suppressed through gene modification, and then transplanting stem cells with differentiation potential into the receptor animal.

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

3. The method according to claim 1, wherein The clearing of bone marrow stem cells is achieved by using a sub-lethal dose of ionizing radiation or chemical means to remove hematopoietic stem cells in the bone marrow of the animal. The low-toxicity drug is a drug that inhibits stem cell proliferation or blocks the conduction of differentiation information. Making the receptor immune system defective or suppressed through gene modification means defects in the development and differentiation of hematopoietic stem cells or defects in the development of multiple immune cells.

4. The method according to claim 3, characterized in that The ionizing radiation is irradiation with X-rays or Co60 rays, and the chemical means is administering one or more of busulfan, cyclophosphamide, and melphalan to the animal.

5. The method according to claim 1, wherein The receptor animal is selected from rats and mice.

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

7. The method according to claim 6, characterized in that The animal carrying a non-natural endogenous gene is an animal with a knockout of the target antigen-encoding gene.

8. The method according to any one of claims 1-7, characterized in that It includes the following steps: (1) Pretreat the animal with immunosuppression to obtain an immunosuppressed receptor animal; construct or purchase one or more animals carrying human immunoglobulin variable region gene segments as donors, collect their stem cells with differentiation potential and transplant them into the immunosuppressed receptor animal to obtain an immune-reconstituted receptor animal with immune cells derived from the donor. (2) Immunize the receptor animal obtained in step (1) with the antigen. (3) Select an animal that can produce an antibody capable of binding to the target antigen, collect its immune cells, and further prepare a human antibody. The donor animal and the receptor animal are rodents.

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