Screening method and application of human-mouse protein high homologous target antibody based on fully humanized antibody mouse

Abstract

The invention belongs to the field of antibody development, and discloses a screening method and application of a human-mouse protein high homologous target antibody based on a fully humanized antibody mouse. Aiming at the problem of weak antibody response caused by immune tolerance of human-mouse high homologous targets (protein homology is greater than or equal to 95%), the following scheme is provided: in embryonic stem cells (ES cells) of HUGO-Mabfully humanized antibody transgenic mice, a mouse target gene (such as ACVR2A) is knocked out through a Turbo Knockout technology, and homozygous knockout ES clones are screened; carrying out microinjection on the clones to the whitened B6 mouse blastocyst, and transplanting a pregnant mouse to obtain a Founder mouse; the Founder mouse is subjected to target antigen immunization for more than or equal to 4 times (the Freund's complete adjuvant is used for the first time), and the titer of the serum antibody is detected. According to the invention, 100% homozygous knockout chimeric efficiency is realized in the Founder stage, the mouse construction period is shortened from traditional 8-10 months to 3-4 months, and the diversity and affinity of the antibody are significantly improved (titer reaches 1: 729,000). The obtained antibody can be used for preparing medicines for treating tumors or autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of antibody development, and specifically discloses a method and application for screening antibodies targeting human and mouse proteins with high homology based on fully humanized antibody mice. Background Technology

[0002] Therapeutic antibodies have become the leading drugs for treating cancer and other human diseases. With the continuous development and improvement of gene editing technology, the successful establishment of mouse model technology platforms expressing human antibody genes is not only a revolutionary innovation in the development of therapeutic antibody drugs, but also promotes the research and development of therapeutic antibodies as drugs and their widespread clinical application.

[0003] Therapeutic antibodies have become the best-selling drugs in the pharmaceutical market. As of 2022, more than 160 antibody therapies had been approved by regulatory agencies, and the proportion of fully human antibodies is increasing. In 2022, fully human antibodies accounted for 45% of the top 50 best-selling antibody drugs, and 70% of these approved fully human antibodies were derived from transgenic mice. This demonstrates that the feasibility and advantages of using transgenic mouse platforms for fully human antibody development have been fully validated.

[0004] Currently, there are only a handful of mature humanized antibody transgenic mouse platforms internationally. Kymab (now acquired by Sanofi) in the UK and Regeneron in the US are globally recognized as having the best platforms in terms of technology and drug discovery effectiveness. Kymab created the Kymouse™ mouse for research and development of therapeutic antibodies by inserting human antibody genes into mice. Regeneron created the VelocImmune mouse by replacing variable region segments of mouse antibody genes in situ with human antibody heavy chain and light chain variable region genes, respectively. In addition, Medarex (now acquired by Bristol-Myers Squibb) developed the HuMAb-Mouse™ technology platform and collaborated with Kyowa Kirin in Japan to establish the KM mouse technology. Abgenix (now acquired by Amgen) integrated the 1020kb VH and 800kb Vκ genes of human antibodies to create the XenoMouse mouse. OMT created the first fully humanized monoclonal antibody platform based on transgenic rats—OmniRat™. OMT also developed OmniMouse™ to compensate for the shortcomings of OmniRat™ and further expand epitope coverage in the development of humanized antibodies.

[0005] Among domestic pharmaceutical companies, Harbour BioMed has developed the Harbour Mice® transgenic mouse platform, which includes the H2L2 transgenic mouse capable of producing conventional fully human antibodies containing two light chains and two heavy chains, and the HCAbz transgenic mouse capable of producing novel fully human heavy chain antibodies. Biocytogen, based on the RenMice® (RenMab®, RenLite®, and RenNano®) platform, has established six major fully human antibody technology platforms for the discovery of fully human therapeutic monoclonal antibodies, bispecific / multispecific antibodies, bispecific antibody ADCs, nanobodies, and TCR-like antibodies. Biocytogen is currently conducting large-scale drug development on over 1000 potential druggable targets (the "Thousand Mice, Ten Thousand Antibodies™" program). NeoMab™, a fully human antibody transgenic mouse model independently developed by Yaokang Biotech, retains the constant region sequence encoding the mouse antibody gene in the BALB / c genetic background. Through gene editing, the gene sequence encoding the variable region of the human antibody is replaced in situ with the mouse sequence. Jinmaibo independently developed the fully human antibody mouse strain (CAMouseHG) and the fully human single-domain antibody mouse strain (CAMouseH). CAMouse is the first fully human antibody mouse independently bred in China for the development of fully human antibody drugs. ImmuMab® mice are humanized immunoglobulin genes created using the MASIRT® ultra-large fragment cross-species in situ substitution technology.

[0006] Because the mouse and human genome sequences are highly homologous (approximately 99%), when antigens from humans and mice share high homology, the antibody response produced by B cells in immunized mice may be weak. This phenomenon is known as "immune tolerance" or "immune neglect." This is primarily because highly homologous antigens may be recognized by the mouse's immune system as "self" or "self-like" substances. The immune system then eliminates B cells with high affinity for the self-antigen through a negative selection process to prevent autoimmune diseases. This results in a weaker immune response.

[0007] The conventional technique involves knocking out mouse-derived genes in fertilized eggs and then transplanting them into surrogate mice to obtain positive Founder mice. Since these positive Founder mice are chimeric, they need to be mated with wild-type mice to produce F1 offspring with germline inheritance. These F1 mice are typically heterozygous for gene knockout (except for sex chromosome genes). To ensure complete gene knockout, F1 mice still need to be mated to produce F2 homozygous gene knockout mice.

[0008] Defects and shortcomings of existing technology: Typically, the industry's overall cycle for obtaining homozygous knockout mice of a certain gene is 8-10 months, and the age of the mice obtained may vary. Summary of the Invention

[0009] To address the aforementioned problems in the prior art, this invention provides a method and application for screening antibodies targeting human and mouse proteins with high homology based on fully humanized antibody mice.

[0010] This invention includes the following technical solutions: A method for screening antibodies targeting highly homologous human-mouse proteins based on fully humanized antibody mice, such as... Figure 1 As shown, it includes the following steps: (a) 3.5-day blastocysts were generated by mating female and male HUGO-Mab™ mice, and HUGO-Mab™ homozygous ES cells were isolated. (b) The mouse-derived target gene was knocked out in HUGO-Mab™ mouse embryonic stem cells (ES cells) to obtain homozygous knockout ES clones; (c) The homozygous knockout ES clone was microinjected into a recipient embryo and transplanted into a surrogate mouse to obtain a Founder mouse. (d) Immunize the Founder mice with the target antigen and detect the serum antibody titer.

[0011] Furthermore, in the above screening method, the knockout of the mouse target gene in step (a) is achieved through TurboKnockout technology.

[0012] Furthermore, in the above screening method, the protein homology of the human-mouse high homology target is ≥95%.

[0013] Furthermore, in the above screening method, the HUGO-Mab™ mouse is a fully humanized antibody transgenic mouse.

[0014] Furthermore, in the above screening method, step (a) involves screening homozygous gene knockout ES clones by PCR.

[0015] Furthermore, in the above screening method, the recipient embryo in step (b) is an albino B6 mouse blastocyst.

[0016] Furthermore, in the above screening method, the number of antigen immunizations in step (c) is ≥4, and the first immunization uses Freund's complete adjuvant.

[0017] Furthermore, in the above screening method, the target gene is ACVR2A.

[0018] The present invention also discloses high-affinity antibodies obtained by the above screening method.

[0019] The present invention also discloses the use of the above-mentioned antibody in the preparation of drugs for treating tumors or autoimmune diseases.

[0020] Compared with the prior art, the present invention has the following outstanding advantages: This invention utilizes Cyagen Biosciences' proprietary TurboKnockout technology to construct knockout mice targeting specific immune targets for immunization. It achieves 100% chimeric efficiency in homozygous gene knockout at the Founder stage, with the overall mouse construction cycle taking only 3-4 months. Compared to traditional methods, the gene knockout mouse construction cycle is shortened by more than half. Theoretically, knocking out the mouse gene significantly increases the diversity of antibodies produced after immunization, providing the possibility of screening antibodies with different functions. Based on the application potential of humanized antibody mice and the huge demand in the new drug development market, this invention can meet the animal model requirements of the "target discovery" process in new drug development, accelerating new drug development. Attached Figure Description

[0021] Figure 1. Technical flow of TurboKnockout® ES shooting technology; Figure 2. Main technical flow of the present invention; Figure 3. Acvr2a gene knockout strategy diagram; Figure 4. Detection of mRNA expression levels in ES clones after mouse Acvr2a gene knockout; Figure 5. Detection of Acvr2a mRNA expression in 4-week-old Founder mice injected with ES clones; Figure 6. ELISA detection of serum titer of ACVR2A protein in mice after 4 immunizations; Figure 7 shows the serum binding of HEK293-ACVR2A to ACVR2A protein after four immunizations, as detected by FACS. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 A Brief Overview of the Production of Cyagen HUGO-Mab™ Mice Using TurboKnockout® ES targeting technology, the variable region sequences of the mouse antibody heavy chain and Kappa light chain are replaced in situ with human variable region gene sequences, without expressing the mouse V(D)J sequence, but retaining the mouse constant region coding genes and regulatory elements to support antibody type conversion. Additionally, the variable and constant region sequences of the mouse Lambda light chain are completely replaced with human gene sequences, without expressing the mouse Lambda light chain sequence. HUGO-Mab™ mice contain all human antibody variable region sequences, exhibiting rich diversity. Upon antigen stimulation, they produce a strong immune response at the same level as wild-type mice, generating high-titer and diverse antibodies.

[0024] Establishment of HUGO-Mab™ mouse ES cell lines, see Figure 2 .

[0025] 1. Preparation of HUGO-Mab™ mice (1) PMSG injection: On the evening of the first day, after 18:00, HUGO-Mab™ female mice (Cygaen, HUGO-Mab-C106) were injected with PMSG.

[0026] (2) HCG injection: HCG is usually injected 24 hours after PMSG injection. The HCG injection is performed after 18:00 on the second day.

[0027] (3) Mice co-breeding and plug detection: After HCG injection, HUGO-Mab™ female mice were co-breeded with HUGO-Mab™ male mice (Cygaen, HUGO-Mab-C106). At 8:00 AM the next day, plugs were detected in the co-breeding female mice. Female mice with obvious plugs were separated from those without plugs, and were given sufficient nutrition and special care. The day the plug was detected was recorded as day 0.5 of embryonic development at 12:00 PM.

[0028] 2. Feeder preparation: Note the number of days of embryonic development. When the embryos develop to 2.5 days, prepare the feeders by placing them in 48-well plates, culturing them in MEF medium, and incubating them in a 37℃, 5% CO2 incubator.

[0029] 3. Treatment of laboratory mice: (1) Sacrifice the mice: When the female HUGO-Mab™ mice reach 3.5 days of development, the mice are euthanized by cervical dislocation. The euthanized mice are placed on the operating table with their abdomen facing up and covered with plastic wrap. Immediately spray 75% alcohol on the abdomen and around the mice to prevent the mice's hair from contaminating the subsequent operations.

[0030] (2) Opening the abdominal cavity of the mouse: Hold the mouse's abdomen with dissecting forceps in the left hand, and make a 1-1.5cm horizontal incision in the middle of the mouse's abdomen with dissecting scissors in the right hand. Hold the two ends of the incision with both hands and pull them towards the head and tail to fully open the abdominal cavity. At this time, the mouse's fur will also be completely away from the abdominal cavity. Then, hold ophthalmic forceps in the left hand and cut the peritoneum with a scalpel in the right hand. Use the scalpel in the right hand to push the intestine covering the uterus to the top, exposing the uterus, fallopian tubes and ovaries.

[0031] (3) Obtaining the mouse uterus: First, cut the connection between the uterine body and the cervix with scissors. Then, use pointed forceps with your left hand to firmly grasp the uterus, lift it upwards, and separate the uterus from the mesentery with scissors. Next, cut the connection between the fallopian tube and the uterine horn to separate the uterus and place it in preheated M2 in vitro manipulation solution.

[0032] 4. Establishment of mouse ES: (1) Obtaining mouse E3.5 blastocysts: Using pointed forceps, make a "V" shaped opening at the uterine horn, insert a 1mL syringe filled with sufficient M2 processing solution into the cervix, and flush out the embryos from both sides of the uterus in turn. Pick out the embryos with a pipette, wash them twice with M2, and place them in M2 for later use.

[0033] (2) Inoculation of mouse E3.5d blastocysts: Two hours in advance, replace the culture medium in the 48-well plate feeder with ES establishment medium, adding 500 μL of culture medium to each well; inoculate the treated E3.5 blastocysts one by one into the 48-well plate with feeder using an oral pipette. After all the embryos have been inoculated into the 48-well plate, immediately and gently place it in an incubator for culture, recording the number of embryos, date, and other information. Do not move the cell culture plate within 48 hours from the time of inoculation. In this experiment, observation generally begins on day 4-5 to observe embryo growth and change the medium.

[0034] (3) Establishment of mouse ES: 6-7 days after embryo inoculation, observe the adherence and growth of the embryos. When a distinct cell cluster is observed to grow around the embryo, digest it with 0.25% trypsin and inoculate it onto a new feeder culture plate, and change the ES medium as usual.

[0035] Example 2 Obtaining human and mouse KO ES cells with high homology to protein targets: 1. The activin-activin receptor (ActR) signaling pathway is an important component of the transforming growth factor-β (TGF-β) superfamily, participating in a series of physiological processes such as cell growth, differentiation, apoptosis, and development. Activin / ActR exhibits typical TGF-β signaling pathway characteristics, namely: the dimeric ligand exerts its function by forming a hexameric signaling complex containing two type I and two type II TGF-β family receptors.

[0036] Type II ActRs: Five type II receptors are currently known, including ACVR2A, ACVR2B, MPR2, TBRII, and AMHR2. Among them, ACVR2A and ACVR2B are the main initial binding sites for ligands such as activin and myostatin. Their biological functions are inseparable from ActRIs; after binding to ligands, they must recruit and phosphorylate ActRIs before transmitting signals downstream. Activin receptor type-2A (ACVR2A) forms a receptor complex upon binding to its ligand, consisting of two type II and two type I transmembrane serine / threonine kinases. Type II receptors phosphorylate and activate type I receptors, which then autophosphorylate and bind to and activate SMAD transcriptional regulators. It also contains receptors for activin A, activin B, and inhibin A.

[0037] ACVR2A shows 99.6% amino acid homology between human and mouse proteins. Figure 4 Furthermore, homozygous knockout mice targeting this target can survive, making it an ideal target for eKO validation.

[0038] 2. Design the ACVR2A KO carrier for the mouse, such as... Figure 3 As shown, the Acvr2a gene is located on mouse chromosome 2 and consists of 11 exons, with the ATG start codon in exon 1 and the TGA stop codon in exon 11. Homologous arms of the KO vector were constructed by PCR amplification of the BAC clone RP23-33K4, with exons 2-4 forming the KO region. Deletion of this region leads to the loss of function of the mouse Acvr2a gene, thereby triggering a series of biological effects.

[0039] 3. Electroporate the mouse ACVR2A KO vector obtained in the previous step into the HUGO-Mab™ ES cells established in Example 1, and screen for homozygous mouse ACVR2A KO ES clones by PCR identification through single clone selection; 4. The homozygous KO ES clones of mouse ACVR2A obtained in the previous step were subjected to mouse ACVR2A mRNA detection to verify that ES clones that do not express mouse ACVR2A mRNA were obtained. Figure 4 ).

[0040] Example 3 Acquisition and expression verification of mouse ACVR2A homozygous KO Founder 1. The homozygous KO ES clone of mouse ACVR2A obtained in Example 2 was microinjected into the preblastocyst embryo of albino B6, and then transferred to the uterus of a surrogate mouse the next day. Founder mice were born 19 days later.

[0041] 2. Four weeks after the birth of the Founder mice, samples were taken from the cerebral cortex, lungs, and cerebellum. RT-qPCR was used to detect the expression of mRNA in the three tissues with high ACVR2A expression. The results showed that none of the KO mice expressed the mouse ACVR2A gene. Figure 5 ).

[0042] Example 4 ACVR2A antigen protein immunoassay and serum titer detection 1. After the Founder mice obtained in Example 3 were 6 weeks old, the mice that underwent ACVR2A KO and the mice that did not undergo KO were immunized with protein antigens. 2. For the first immunization, the ACVR2A-FC antigen was emulsified with Freund's complete adjuvant and injected subcutaneously into each mouse at a dose of 100 μL / 50 μg. 3. On day 14, the second immunization was performed using Freund's incomplete adjuvant emulsified ACVR2A-FC antigen, with 100 μL / 25 μg injected subcutaneously into each mouse; 4. On day 28, the third immunization was performed using Freund's incomplete adjuvant emulsified ACVR2A-FC antigen, with 100 μL / 25 μg injected subcutaneously into each mouse; 5. Blood was collected on day 35. The plate was coated with ACVR2A-His protein at 1 μg / mL. The initial concentration was 1000 times that of mouse serum. The concentration was then serially diluted 3 times and 7 times to measure the titer. 6. On day 42, the antigen was mixed with water-soluble manganese adjuvant, 60 μg / 300 μl per mouse, 100 μl subcutaneously and 200 μl intraperitoneally; 7. Blood was collected on day 49. The plate was coated with ACVR2A-His protein at 1 μg / mL. The initial concentration was 1000 times that of mouse serum. The concentration was then serially diluted 3 times and 7 times to measure the titer. 8. ELISA experiments showed that the immunogenicity of ACVR2A knockout mice was significantly higher than that of humanized mice with non-cVR2A gene knockout. For example... Figure 6 As shown, the highest molecular binding titer of ACVR2A in mice after KO was 1:729000, while the highest molecular binding titer in mice before KO was 1:27000. 9. FACS experiments showed that the serum of mice after ACVR2A knockout (KO) had a significantly higher binding capacity to HEK293-ACVR2A cells (HEK293 cells overexpressing ACVR2A) than that of humanized mice with non-knockout ACVR2A genes. Figure 7 As shown, when the serum was diluted 1000 times, the highest average fluorescence intensity of HEK293-ACVR2A MFI-647 in the serum of mice after ACVR2A KO reached 568104.58, while the highest average fluorescence intensity of HEK293-ACVR2A MFI-647 in the serum of mice before ACVR2A KO was only 49026.22.

[0043] In summary, this invention innovatively applies the independently developed TurboKnockout technology to the proprietary fully humanized antibody mouse HUGO-Mab™ to construct knockout mice with high homology to specific human and mouse immune target proteins. This allows for immunization with mice, achieving 100% chimeric efficiency of homozygous gene knockout at the Founder stage, with the overall mouse construction cycle taking only 3-4 months. Compared to traditional methods, the gene knockout mouse construction cycle is shortened by more than half. Theoretically, knocking out the mouse gene significantly increases the diversity of antibodies produced after immunization, providing the possibility of screening antibodies with different functional types.

[0044] It is worth noting that the above description of the embodiments focuses on illustrating the technical solution of the present invention, rather than precisely defining its scope of protection. Those skilled in the art should understand that appropriate adjustments and optimizations can be made based on the technical details disclosed in the embodiments of the present invention, or equivalent substitutions can be implemented for individual or even all technical elements. Such adjustments and substitutions will not deviate from the core essence of the technical solution of the present invention and should be included within the technical protection scope of the embodiments of the present invention. In short, the protection of the present invention should not be limited to the concrete presentation of the above embodiments, but broadly covers all equivalent changes and improvements that do not depart from its basic concept. In summary, the protection definition of the present invention should be based on the statement of the claims, and the above embodiments are only used as a reference guide for understanding the present invention.

Claims

1. A method for screening antibodies targeting highly homologous human-mouse proteins based on fully humanized antibody mice, characterized in that, Includes the following steps: (a) 3.5-day blastocysts were generated by mating female and male HUGO-Mab™ mice, and HUGO-Mab™ homozygous ES cells were isolated. (b) The mouse-derived target gene was knocked out in HUGO-Mab™ mouse embryonic stem cells (ES cells) to obtain homozygous knockout ES clones; (c) The homozygous knockout ES clone was microinjected into a recipient embryo and transplanted into a surrogate mouse to obtain a Founder mouse. (d) Immunize the Founder mice with the target antigen and detect the serum antibody titer.

2. The method as described in claim 1, characterized in that, In step (a), the knockout of the mouse target gene is achieved using TurboKnockout technology.

3. The method as described in claim 1, characterized in that, The proteins of the high homology targets between humans and mice have a homology of ≥90%.

4. The method as described in claim 1, characterized in that, The HUGO-Mab™ mice are fully humanized antibody transgenic mice.

5. The method as described in claim 1, characterized in that, In step (a), homozygous gene knockout ES clones are screened by PCR.

6. The method as described in claim 1, characterized in that, In step (b), the recipient embryo is an albino B6 mouse blastocyst.

7. The method as described in claim 1, characterized in that, In step (c), the number of antigen immunizations is ≥4, and the first immunization uses Freund's complete adjuvant.

8. The method as described in claim 1, characterized in that, The target gene is ACVR2A.

9. A high-affinity antibody obtained by any of the methods described in claims 1-8.

10. The use of an antibody as described in claim 9 in the preparation of a drug for treating tumors or autoimmune diseases.

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