An antibody that can cross-bind human, mouse and cynomolgus monkey AMHR2 protein, and a preparation method and application thereof

Abstract

The present application relates to an antibody capable of cross-binding human, mouse and cynomolgus monkey AMHR2 proteins and a preparation method and application thereof, the antibody comprising one of anti-AMHR2 monoclonal antibodies Ab11, Ab12, Ab13, Ab15, Ab16, Ab18, Ab24, Ab25, Ab26, Ab29 and Ab30. The present application identifies a conserved epitope region by sequence alignment of the extracellular domains of human, mouse and cynomolgus monkey AMHR2 proteins, and obtains anti-AMHR2 monoclonal antibodies with cross-species binding activity based on hybridoma technology and B cell sorting technology, and constructs a bispecific T cell engager (TCE) targeting AMHR2xCD3, thereby solving the problems of strong species specificity, low affinity, great difficulty in clinical transformation and insufficient safety of bispecific antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to an antibody that can cross-bind to human, mouse and cynomolgus monkey AMHR2 protein, its preparation method and application. Background Technology

[0002] Anti-Müllerian hormone type II receptor (AMHR2) is a member of the transforming growth factor-β (TGF-β) superfamily and a type I transmembrane serine / threonine kinase receptor. Upon binding to its ligand AMH, it activates the Smad signaling pathway, regulating cell proliferation, differentiation, and apoptosis. In a recent study, researchers developed an immunocompatible in vivo gene screening method using the StealthyCRISPR platform, bypassing the immunogenicity of Cas9. This immunocamouflage strategy helps identify metastasis drivers, revealing the AMH-AMHR2 axis as a clinically actionable pathway for inhibiting cancer metastasis. While AMHR2 possesses conserved extracellular domain sequences in humans, mice, and monkeys, certain amino acid differences exist between species. This means that most existing AMHR2 antibodies can only recognize AMHR2 proteins from a single species, failing to meet the needs of cross-species translational research from animal experiments to clinical applications.

[0003] In research on AMHR2-related diseases (such as polycystic ovary syndrome, ovarian cancer, and colorectal cancer), there is an urgent need for an antibody tool that can simultaneously recognize AMHR2 in humans, mice, and monkeys. This would enable cross-species validation of disease mechanisms, evaluation of animal models for drug screening, and consistency comparison of clinical sample testing. While some existing AMHR2 antibodies exhibit some species cross-reactivity, they suffer from low affinity, poor specificity, or insufficient signaling pathway antagonistic activity, limiting their application in translational medicine. Therefore, developing an AMHR2 antibody with high affinity, high specificity, and strong species cross-reactivity has significant clinical value and research implications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an antibody that can cross-bind to human, mouse and cynomolgus monkey AMHR2 protein, as well as its preparation method and application, to solve the problems of existing AMHR2 antibodies having high species specificity, low affinity, difficulty in clinical translation and insufficient safety of dual antibody killing.

[0005] The present invention provides an antibody that can cross-bind to the AMHR2 protein of humans, mice and cynomolgus monkeys, said antibody comprising one of the following anti-AMHR2 monoclonal antibodies: Ab11, Ab12, Ab13, Ab15, Ab16, Ab18, Ab24, Ab25, Ab26, Ab29 and Ab30.

[0006] Furthermore, the heavy chain variable region of the monoclonal antibody Ab11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.26-SEQ ID NO.28, respectively; the light chain variable region of Ab11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.29-SEQ ID NO.31, respectively. The heavy chain variable region of the monoclonal antibody Ab12 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.32-SEQ ID NO.34, respectively; the light chain variable region of Ab12 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.35-SEQ ID NO.37, respectively. The heavy chain variable region of the monoclonal antibody Ab13 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.38-SEQ ID NO.40, respectively; the light chain variable region of Ab13 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.41-SEQ ID NO.43, respectively. The heavy chain variable region of the monoclonal antibody Ab15 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.44-SEQ ID NO.46, respectively; the light chain variable region of Ab15 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.47-SEQ ID NO.49, respectively. The heavy chain variable region of the monoclonal antibody Ab16 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.50-SEQ ID NO.52, respectively; the light chain variable region of Ab16 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.53-SEQ ID NO.55, respectively. The heavy chain variable region of the monoclonal antibody Ab18 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.56-SEQ ID NO.58, respectively; the light chain variable region of Ab18 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.59-SEQ ID NO.61, respectively. The heavy chain variable region of the monoclonal antibody Ab24 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.62-SEQ ID NO.64, respectively; the light chain variable region of Ab24 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.65-SEQ ID NO.67, respectively. The heavy chain variable region of the monoclonal antibody Ab25 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.68-SEQ ID NO.70, respectively; the light chain variable region of Ab25 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.71-SEQ ID NO.73, respectively. The heavy chain variable region of the monoclonal antibody Ab26 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.74-SEQ ID NO.76, respectively; the light chain variable region of Ab26 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.77-SEQ ID NO.79, respectively. The heavy chain variable region of the monoclonal antibody Ab29 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.80-SEQ ID NO.82, respectively; the light chain variable region of Ab29 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.83-SEQ ID NO.85, respectively. The heavy chain variable region of the monoclonal antibody Ab30 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.86-SEQ ID NO.88, respectively; the light chain variable region of Ab30 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.89-SEQ ID NO.91, respectively.

[0007] Further, the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab11 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab11 is shown in SEQ ID NO.5; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab12 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab12 is shown in SEQ ID NO.7; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab13 is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab13 is shown in SEQ ID NO.9; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab15 is shown in SEQ ID NO.10, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab15 is shown in SEQ ID NO.11; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab16 is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab16 is shown in SEQ ID NO.13; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab18 is shown in SEQ ID NO. As shown in NO.14, the amino acid sequence of the light chain variable region of monoclonal antibody Ab18 is shown in SEQ ID NO.15; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab24 is shown in SEQ ID NO.16, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab24 is shown in SEQ ID NO.17; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab25 is shown in SEQ ID NO.18, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab25 is shown in SEQ ID NO.19; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab26 is shown in SEQ ID NO.20, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab26 is shown in SEQ ID NO.21; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab29 is shown in SEQ ID NO.22, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab29 is shown in SEQ ID NO.23; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab30 is shown in SEQ ID NO. As shown in NO.24, the amino acid sequence of the light chain variable region of the monoclonal antibody Ab30 is shown in SEQ ID NO.25.

[0008] Furthermore, the present invention allows for conserved amino acid substitutions in the antibody frame region (FR), wherein the identity of VH with the corresponding sequence in SEQ ID NO. 4-25 is at least 85%, preferably 90%, more preferably 95%; and the identity of VK with the corresponding sequence in SEQ ID NO. 4-25 is at least 85%, preferably 90%, more preferably 95%, but the CDR sequence must remain completely unchanged to ensure the cross-binding activity and specificity of the antibody.

[0009] This invention also provides a method for preparing antibodies that can cross-bind to human, mouse, and cynomolgus monkey AMHR2 proteins, comprising the following steps: Positive clones were obtained by cross-immunization with BALB / c mice, and the antibody sequences were obtained by sequencing. The variable region sequence of the antibody was fused with the constant region of mouse IgG2a or human IgG1 to construct a recombinant expression vector, which was transfected into 293 cells and purified to obtain high-purity antibody (purity > 95%).

[0010] The present invention also provides a bispecific T cell adaptor targeting AMHR2×CD3, comprising a combination of the antibody that cross-binds to human, mouse and cynomolgus monkey AMHR2 protein and CD3 antibody OKT3.

[0011] This invention also provides a method for preparing a bispecific T-cell connector targeting AMHR2×CD3, comprising the following steps: The selected antibody (preferably Ab26 antibody) was combined with the CD3 antibody OKT3, and a bispecific antibody expression vector was constructed by designing a dual-end scFv structure, combined with KIH heterodimer technology and Fc mutation (L234A / L235A / P329G) modification. The vector was transfected into 293 cells, purified by Protein G affinity chromatography, and concentrated by dialysis to obtain a bispecific antibody (BAM26T3) with a purity >95%.

[0012] This invention also provides an antibody that can cross-bind to human, mouse and cynomolgus monkey AMHR2 proteins and its application in the detection of human, mouse and monkey AMHR2 proteins and cross-species research.

[0013] This invention also provides the application of an antibody that can cross-bind to human, mouse and cynomolgus monkey AMHR2 proteins in the preparation of diagnostic and therapeutic products for AMHR2-positive diseases.

[0014] This invention also provides the application of a bispecific T-cell connector targeting AMHR2×CD3 in the preparation of diagnostic and therapeutic products for AMHR2-positive diseases.

[0015] The biological characteristics of the monoclonal antibody of this invention are as follows: 1. Cross-species binding activity: Among the 11 anti-AMHR2 antibodies, Ab12, Ab16, Ab18, Ab26, Ab29, and Ab30 can specifically bind to the extracellular domains of AMHR2 proteins in humans, mice, and cynomolgus monkeys. ELISA detection showed that the EC50 values ​​for AMHR2 in all three species were ≤5 nM. The remaining antibodies can bind to AMHR2 proteins in humans and monkeys, with EC50 values ​​<0.3 nM.

[0016] 2. Signaling pathway antagonistic activity: Some antibodies (AMBM6, AMBM7, AMBM8, etc.) can be completely blocked by AMH protein, while antibodies such as Ab12 can be partially blocked by AMH protein, competitively inhibiting the binding of AMH to AMHR2.

[0017] 3. Competitive binding characteristics: AMBM2 antibody can compete with AMBM7 and AMBM7 antibody for binding to human AMHR2 protein, but does not compete with other candidate antibodies, indicating that candidate antibodies and AMBM2 recognize different epitopes.

[0018] 4. Bispecific antibody killing activity: AMHR2×CD3 bispecific TCE (BAM26T3) can redirect human PBMC cells to specifically kill AMHR2-overexpressing 293-Luc cells, with a significant dose-dependent killing effect, and can achieve a clear EC50 and maximum killing rate.

[0019] The application scenarios of the monoclonal antibody of this invention are as follows: 1. Research tool applications: It can be used as a detection reagent for experiments such as Western blot, ELISA, flow cytometry, and immunohistochemistry for the qualitative and quantitative detection of AMHR2 protein in human, mouse, and monkey samples; it can be used for cross-species research on AMHR2-related signaling pathway mechanisms, providing a reliable tool for drug screening.

[0020] 2. Diagnostic applications: It can be prepared into diagnostic reagents for clinical sample testing and prognostic assessment of AMHR2-positive diseases (such as ovarian cancer, colorectal cancer, polycystic ovary syndrome, etc.).

[0021] 3. Therapeutic applications: Anti-AMHR2 monoclonal antibodies can be used directly as therapeutic drugs or prepared into antibody-drug conjugates (ADCs) for the treatment of AMHR2 expression-related diseases; AMHR2×CD3 bispecific TCEs can be used for the immunotherapy of AMHR2-positive tumors, killing tumor cells by redirecting T cells, with high safety and significant killing effect.

[0022] 4. Animal model applications: Since some antibodies have good cross-binding activity between mice and monkeys, they can be directly used for in vivo efficacy evaluation in mouse tumor xenograft models and cynomolgus monkey disease models without the need to construct additional species-specific antibodies, thus accelerating the clinical translation research process.

[0023] Beneficial effects This invention identifies conserved epitope regions by sequence alignment of the extracellular domains of AMHR2 proteins in humans, mice, and cynomolgus monkeys, and obtains anti-AMHR2 monoclonal antibodies with cross-species binding activity based on hybridoma technology and B-cell sorting technology. At the same time, it constructs a bispecific T-cell adaptor (TCE) targeting AMHR2×CD3, which solves the problems of existing AMHR2 antibodies, such as high species specificity, low affinity, difficulty in clinical translation, and insufficient safety of bispecific antibodies. Attached Figure Description

[0024] Figure 1 SDS-PAGE gel image of AMHR2 protein.

[0025] Figure 2 The results show the detection of AMHR2 control antibody binding to different tag proteins of human AMHR2; where a is the his tag recombinant protein and b is the Fc tag recombinant protein.

[0026] Figure 3 The results of AMHR2-Luc 293 cells overexpressing AMHR2 were validated using AMHR2 control antibodies; where a is human AMHR2-overexpressing 293 cells and b is mouse AMHR2-overexpressing 293 cells.

[0027] Figure 4 The results are for AMHR2 immune serum titer detection.

[0028] Figure 5 shows the AMHR2 antibody binding to human (a), monkey (b), and mouse (c) AMHR2-His protein.

[0029] Figure 6 The AMHR2 antibody blocks the binding of human AMHR2 protein to AMH protein.

[0030] Figure 7 The AMBM2 antibody competes with other AMHR2 antibodies for binding to the human AMHR2 protein.

[0031] Figure 8 This study investigated the killing effect of the AMHR2×CD3 bispecific antibody TCE on AMHR2-Luc 293 cells. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Example 1

[0033] Preparation of AMHR2 antigen and control antibody: The truncated AMHR2 extracellular domain genes (amino acid sequences Pro18-Leu149) from humans, mice, and cynomolgus monkeys were obtained from the uniprot database and synthesized after codon optimization. The amino acid sequences of the human, mouse, and cynomolgus monkey AMHR2 proteins are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, as shown in Table 1 below. The control antibody sequences AMBM1-AMBM8 are derived from patented sequences, as shown in Table 2 below. They were constructed into expression vectors for subsequent preparation of recombinant AMHR2 extracellular domain proteins and control antibodies from humans, mice, and cynomolgus monkeys. The above plasmids were transfected into 293 cells. After 5-7 days, the cell culture supernatant was collected, filtered through a 0.22 μm filter, and the Fc-tagged fusion protein and antibody were purified by Protein G chromatography. After elution, the protein was immediately neutralized with Tris-HCl buffer and dialyzed against 1×PBS. The His-tagged fusion protein was purified by nickel column chromatography, eluted with imidazole solutions of different concentrations, and dialyzed against 1×PBS. The purified protein was analyzed by SDS-PAGE, and the results are as follows: Figure 1 The results showed that the purity of the AMHR2 recombinant protein was >90%, which met the requirements for mouse immunization; the purity of the AMHR2 control antibodies was >95%, which met the standard requirements for antibody application.

[0034] Table 1. Amino acid sequences of the AMHR2 extracellular domain in humans, mice, and cynomolgus monkeys. .

[0035] Table 2. Sources of control antibody sequences . Example 2

[0036] AMHR2 antigen and AMHR2-293 cell function verification: First, coating was performed. The AMHR2 fusion proteins of each tag were diluted to a concentration of 1 μg / mL with coating buffer, and 100 μL was added to each well. The plate was incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plate was washed three times with washing buffer, allowing it to stand for 1 minute each time before discarding the waste liquid. Blocking buffer was then added to each well, and the plate was blocked at 37°C for 1 hour. After blocking, the washing steps were repeated, and serially diluted AMHR2 control antibodies AMBM1 and AMBM2 were added to each well. The plate was incubated at 37°C for 1 hour. After incubation, the plate was washed again, and HRP-labeled secondary antibody was added. The plate was incubated at 37°C in the dark for 30 minutes. After incubation, the plate was washed thoroughly, and substrate solution was added to each well. The plate was incubated in the dark for 10-15 minutes. Once a clear color change was observed, stop solution was added to each well to terminate the reaction. The absorbance at OD450 nm was immediately measured using a microplate reader. Based on the absorbance results, the binding ability of the different AMHR2 fusion proteins to the control antibodies was analyzed to verify the integrity of their antigenic function. The test results showed that both control antibodies could bind to AMHR2 fusion proteins with different tags, as shown in the results below. Figure 2 As shown, both the AMHR2 fusion protein and the control antibody function normally and can be used for subsequent immunization and control experiments.

[0037] AMHR2 expression in AMHR2-Luc 293 cells was verified by flow cytometry. The specific procedure was as follows: AMHR2-Luc 293 cells in logarithmic growth phase were collected, washed twice with PBS, centrifuged, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL were divided into experimental and blank control groups. The experimental group received the AMHR2 control antibody used for validation, diluted 3-fold at a maximum concentration of 10 μg / mL. The blank control group received an equal volume of PBS and incubated at room temperature in the dark for 1 hour. After incubation, cells were washed twice with PBS to remove unbound primary antibody. Then, fluorescently labeled secondary antibodies were added to both groups, and cells were incubated at room temperature in the dark for 30 minutes. After incubation, cells were washed twice with PBS, centrifuged, and the supernatant was discarded. Cells were resuspended in PBS, and cell fluorescence intensity was detected by flow cytometry. The overexpression level of AMHR2 on the surface of AMHR2-Luc 293 cells was analyzed, using the blank control group as a reference. The results showed that all four control antibodies could bind to AMHR2-Luc 293 cells. Figure 3 As shown, AMHR2-Luc 293 cells overexpressing AMHR2 can be used for subsequent bispecific antibody killing assays. Example 3

[0038] Screening for mouse antibodies against AMHR2: Ten female BALB / c mice aged 6-8 weeks were selected. After acclimatizing to the environment for one week, they were cross-immunized with human AMHR2 and mouse AMHR2 tagged with hFc. On day 0, 50 μg / mouse of antigen-CFA emulsion was injected into the footpads, and on days 14 and 28, 25 μg / mouse of antigen-IFA emulsion was injected into the footpads. On days 21 and 35, blood was collected from the tail vein, and serum antibody titers were detected by ELISA. Four mice with high serum titers were selected for antibody screening.

[0039] Titer detection: The specific procedure involved diluting the AMHR2-his antigen to a concentration of 1 μg / mL with coating buffer, adding 100 μL to each well of a 96-well ELISA plate, and incubating overnight at 4°C to complete the coating. The coating buffer was discarded, and the plate was washed three times with washing buffer. Blocking buffer was added to each well, and the plate was blocked at 37°C for 1 hour. After blocking, the plate was washed again, and AMHR2 immune serum was serially diluted, with 100 μL of diluted serum added to each well. Negative serum controls and blank controls were included, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was washed, HRP-labeled secondary antibody was added, and the plate was incubated at 37°C in the dark for 30 minutes. After washing, substrate solution was added, and the reaction was carried out for 10-15 minutes in the dark. The reaction was terminated by adding stop solution, and the absorbance at OD450 nm was immediately measured using an ELISA reader. The titer of the immune serum was determined using the negative serum control as a reference. The results showed that the serum titers of all four mice exceeded 20,000. Figure 4 As shown, it meets the requirements for antibody screening.

[0040] Hybridoma method: Splenic cell fusion: Three days prior to fusion, four mice were intraperitoneally injected with 50 μg / mouse of 1 mg / mL AMHR2 saline antigen solution. Splenic lymphocytes (half the number of lymphocytes from each mouse were retained for subsequent B cell sorting) were fused with Sp2 / 0 cells at a ratio of 10:1. After fusion, the cells were spaced at a density of 2.0 × 10⁻⁶ cells / mL. 5 Hybridoma cell clones were seeded at a density of cells / well in 96-well plates and cultured at 37°C with 5% CO2. After selective culture in HAT medium, the supernatant of hybridoma cell clones was screened by ELISA on day 7. Subcloning was performed on positive wells using a limiting dilution method three times, ultimately yielding 8 stable anti-AMHR2 positive hybridoma single-cell clones.

[0041] Sequence identification and analysis: Eight positive clones were sequenced, yielding five unique anti-AMHR2 antibody variable region sequences, including heavy chain and light chain variable region sequences. CDR region annotation was completed, and each sequence was novel with no duplication.

[0042] B-cell sorting method: Red blood cell lysis: Add red blood cell lysis buffer to the spleen lymphocyte suspension obtained by the above method, incubate at room temperature for 5 min, add 0.1% BSA-PBS to terminate lysis, centrifuge at 1000 r / min for 5 min, discard the supernatant, and resuspend the cells in 0.1% BSA-PBS; Dead cell removal: Add 7-AAD dye (final concentration 5 μg / mL), incubate at room temperature in the dark for 10 min to label dead cells for removal during subsequent flow cytometry sorting.

[0043] AMHR2-positive B cells were sorted by flow cytometry. 1. Cell staining Biotin-labeled AMHR2 protein (final concentration 2 μg / mL) was added to the spleen single-cell suspension and incubated at 4°C for 1 hour. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed and resuspended with 0.1% BSA-PBS. B cell surface marker antibodies (CD19-PE-Cy7, CD38-FITC, IgG-APC, and SA-PE, all diluted 1:100) were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice with 0.1% BSA-PBS, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended with 0.1% BSA-PBS, adjusting the cell concentration to 1 × 10⁶ cells / mL. 7 Cells / mL were filtered through a 300-mesh sieve and transferred into a flow cytometry sample tube. The sample was then stored in an ice bath in the dark for later use.

[0044] 2. Setting Flow Sorting Conditions Control tube settings: Set up blank control (unstained cells), isotype control (CD19 / CD38 / IgG isotype antibody staining), and single positive control (cells labeled with each fluorescent single antibody) for fluorescence compensation adjustment and gating settings; Gating strategy: Step 1: Use FSC / SSC to gate and exclude cell debris and clumps; Step 2: Exclude 7-AAD-positive dead cells and select live cell populations; Step 3: Select CD19 + IgG + CD38 + Mature B cell population; Step 4: Select cells that are positive for the biotin-labeled AMHR2 protein from the mature B cell population; these are AMHR2-specific B cells. Sorting parameters: Single cell sorting mode was used, and sorted single cells were directly collected into 96-well PCR plates containing 5 μL of cDNA synthesis buffer, with 50 cells per well; aseptic conditions were maintained during the sorting process.

[0045] Antibody variable region (VH / VK) gene amplification: cDNA synthesis: Follow the instructions of the single-cell cDNA synthesis kit to obtain a single-cell cDNA library; VH / VK gene-specific amplification: Using cellular cDNA as a template, PCR amplification was performed using universal primers for the antibody variable region (covering the FR1-FR4 regions of mouse IgG heavy chain VH and κ / λ light chain VK). PCR: Amplification of the full-length variable region fragment of the antibody, reaction system 25 μL, program: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ extension for 10 min; PCR product identification: Take 5 μL of PCR product and perform 1.5% agarose gel electrophoresis to identify the size of the amplified product (VH approximately 400 bp, VK approximately 350 bp). Select wells with positive amplification for sequencing.

[0046] Antibody variable region sequence analysis and expression vector construction: Sequencing and analysis: The PCR positive product was ligated into the pTT5 antibody display vector, transformed into E. coli DH5α, and plasmid was extracted; Fab sorting: The display library plasmid was diluted and transfected into 293 cells. Single-positive cells were sorted by flow cytometry using biotin-labeled AMHR2 protein. Cells were lysed, and heavy and light strands were amplified by PCR and sequenced.

[0047] Sequencing results analysis: Repetitive sequences were removed, and CDR regions were annotated using the IMGT database to obtain the unique anti-AMHR2 antibody VH / VK sequence, and the complementarity-determining region (CDR1 / CDR2 / CDR3) sequence was determined; Antibody expression vector construction: The VH / VK sequence of the murine anti-AMHR2 antibody was fused with the constant regions of murine IgG2a and human IgG1. The fusion gene was cloned into the eukaryotic expression vector pTT5 by restriction endonuclease digestion and T4 DNA ligase to construct the anti-AMHR2 chimeric antibody recombinant expression plasmid. Plasmid identification: The recombinant expression plasmid is identified by enzyme digestion and sequenced to ensure that there are no mutations, insertions or deletions in the sequence. The plasmid with the correct sequence is extracted for subsequent antibody expression.

[0048] A total of 11 unique antibody sequences were obtained by the two screening methods. The antibody VH and VK sequences are shown in Table 3, and the CDR sequence is shown in Table 4.

[0049] Table 3. Sequence listing of AMHR2 antibody VH and VK ; .

[0050] Table 4. AMHR2 antibody CDR region sequence listing ; . Example 4

[0051] Preparation of anti-AMHR2 antibody: Eleven variable region sequences of anti-AMHR2 antibodies obtained in Example 3 were fused with the constant regions of mouse IgG2a and human IgG1, respectively, to construct antibody expression plasmids. These plasmids were cloned into the pTT5 vector, and sequencing confirmed the absence of sequence mutations. The heavy and light chain plasmids of 11 mIgG2a antibodies obtained from hybridoma and B cells were mixed at a 1:2 ratio and transfected into 293 cells. After transfection, the cells were cultured at 37°C and 5% CO2 for 6 days, showing good cell growth and efficient antibody expression. The cell culture supernatant was collected, filtered through a 0.22 μm filter, and purified using a Protein G chromatography column. The purified antibodies were immediately neutralized with Tris-HCl buffer after elution. SDS-PAGE analysis showed that the purity of all 11 anti-AMHR2 antibodies was >95%, meeting the standard requirements for antibody application. Example 5

[0052] Assay of binding activity and species selectivity of anti-AMHR2 antibody: To confirm the species specificity of the candidate molecule and to select a suitable animal model, the anti-AMHR2 antibody prepared in Example 4 (hereinafter referred to as the candidate antibody molecule) was subjected to a species binding assay. The specific experimental steps are as follows: The binding activity of AMHR2 antibodies to AMHR2 protein in three species (human, mouse, and cynomolgus monkey) was measured using an ELISA method. The specific steps were as follows: 96-well microplates were coated with antigen molecules hAMHR2-His, mAHR2-His, and cynoAMHR2-His at 2 μg / mL (100 μL / well) and incubated overnight at 4°C. The next day, after washing and blocking, serially diluted candidate antibody molecules were added and incubated at room temperature for 1 hour. After washing, diluted anti-GAM-HRP was added and incubated at room temperature for 1 hour. After washing and development, the results were read using a microplate reader. See attached [link to results]. Figure 5a , Figure 5b , Figure 5cSee Table 5. The results showed that the EC50 (KD) of all 11 anti-AMHR2 antibodies was <0.3 nM with human AMHR2 recombinant protein, except for Ab13. The EC50 (KD) of the antibodies against mouse AMHR2 recombinant protein, except for Ab11, Ab13, Ab15, Ab24, and Ab25, was all less than 1 nM. The EC50 (KD) of the antibody against cynomolgus monkey AMHR2 recombinant protein was close to that of the antibody, around 0.3 nM, except for Ab13.

[0053] Table 5. Binding activity and species of anti-AMHR2 antibodies . Example 6

[0054] AMH protein blocks AMHR2 antibody binding to human AMHR2 protein: To confirm the blocking function of the candidate molecule on AMH / AMHR2 signal transduction, ELISA was used for verification. The specific experimental steps are as follows: Human AMHR2 protein was diluted to a concentration of 2 μg / mL with coating buffer, and 100 μL was added to each well of a 96-well microplate. The plate was incubated overnight at 4°C to complete the coating. The coating buffer was discarded, and the plate was washed three times with washing buffer. Blocking buffer was added to each well, and the plate was blocked at 37°C for 1 hour. After blocking, the plate was washed, and experimental, positive, and blank control groups were set up. The experimental group was incubated with serially diluted AMHR2 candidate molecule and control antibody, and simultaneously pre-incubated with a fixed concentration of 3 μg / mL AMH recombinant protein. The positive control group was incubated with an equal volume of AMHR2 antibody without AMH protein, and the blank control group was incubated with an equal volume of PBS. The plates were incubated at 37°C for 1 hour. After incubation, wash the sample, add HRP-labeled secondary antibody, and incubate at 37°C in the dark for 30 minutes. Wash again, add substrate solution, and react in the dark for 10-15 minutes. Stop the reaction by adding stop solution, and immediately measure the absorbance at OD450nm using a microplate reader. By comparing the absorbance of each group, the blocking effect of AMH protein on the binding of AMHR2 antibody to human AMHR2 protein can be analyzed. See the results below. Figure 6 The results showed that some control antibodies (AMBM6, AMBM7, and AMBM8) could be completely blocked by AMH protein, while Ab12, Ab21, and Ab37 could be partially blocked by recombinant AMH protein. Example 7

[0055] AMBM2 antibodies compete with other AMHR2 antibodies for binding to the human AMHR2 protein: To confirm whether the candidate molecule and the control antibody have the same binding epitope, verification was performed using an ELISA method. The specific experimental steps are as follows: Human AMHR2 protein was diluted to a concentration of 2 μg / mL with coating buffer, and 100 μL was added to each well of a 96-well microplate. The plate was incubated overnight at 4°C to complete the coating. The coating buffer was discarded, and the plate was washed three times with washing buffer. Blocking buffer was added to each well, and the plate was blocked at 37°C for 1 hour. After blocking and washing, an autoantibody competition group, a positive control group, and a blank control group were set up. The competition group was pre-incubated with a fixed concentration of 3 μg / mL AMBM2 antibody and serially diluted other AMHR2 antibodies. The positive control group was pre-incubated with an equal volume of other AMHR2 antibodies without AMBM2 antibody, and the blank control group was pre-incubated with an equal volume of PBS. The plates were incubated at 37°C for 1 hour. After incubation, wash the sample, add the corresponding HRP-labeled secondary antibody, and incubate at 37°C in the dark for 30 minutes. Wash again, add substrate solution, and react in the dark for 10-15 minutes. Stop the reaction by adding stop solution, and immediately measure the absorbance at OD450nm using a microplate reader. By comparing the absorbance differences among groups, the competitive effect of AMBM2 antibody on the binding of other AMHR2 antibodies to human AMHR2 protein can be analyzed. See the results below. Figure 7 The results showed that AMBM7 and AMBM8 could be competed for by AMBM2, while other antibodies were not, indicating that the candidate molecules have different epitopes from AMBM2. Example 8

[0056] Construction and preparation of TCE bispecific antibodies targeting AMHR2 and CD3: Construction of AMHR2×CD3 bispecific antibody: The AMHR2 antibody Ab26 selected above was combined with the CD3 antibody OKT3 to form a bispecific antibody with scFV at both ends (referred to as BAM26T3), the sequence of which is shown in the table below. A TCE bispecific antibody targeting AMHR2 and CD3 was designed using the scFv structure at both ends. Combined with KIH heterodimer technology and Fc mutation modification, the specific structure consists of two heavy chain fusion fragments forming a heterodimer: AMHR2 end: scFv-AMHR2-CH1-CH2-CH3 (containing KIH mutation T366W and Fc mutations L234A / L235A / P329G); CD3 end: scFv-CD3-CH1-CH2-CH3 (containing KIH mutations T366S / L368A / Y407V and Fc mutations L234A / L235A / P329G); no light chain fragments. In this design, scFv-AMHR2 is VH-AMHR2-(G4S)3-VK-AMHR2, and scFv-CD3 is VH-CD3-(G4S)3-VK-CD3. The flexible linker peptides ensure the spatial conformation of scFv and its antigen-binding activity. The KIH mutation ensures that the two heavy chains specifically form heterodimers, avoiding the formation of homodimers. The Fc mutation eliminates CDC and ADCC activities, reducing non-specific cell damage, while retaining the Protein A binding ability of the Fc fragment, facilitating subsequent purification. These constructed fragments are then ligated into the pTT5 expression vector for the subsequent production of bispecific antibodies.

[0057] In the bispecific antibody, the sequence of the AMHR2 heavy chain is shown in SEQ ID NO. 20, and the sequence of the AMHR2 terminal light chain is shown in SEQ ID NO. 21. The CD3 sequence is derived from the OKT3 sequence in patent US4361549A.

[0058] Table 6 AMHR2×CD3 Bispecific Antibodies .

[0059] Preparation of AMHR2×CD3 bispecific antibody: After confirming the sequence of the bispecific antibody by sequencing, the expression plasmid was transformed into DH5α cells and cultured for large-scale extraction. The plasmid was then extracted using an endotoxin-free large-scale extraction kit, following the manufacturer's instructions. The expression plasmid was co-transfected into 293 cells at a 1:1 ratio to express the bispecific antibody. After 7 days of culture, the culture supernatant was collected, purified by Protein G affinity chromatography, and dialyzed against 1×PBS. The antibody was then concentrated using ultrafiltration to achieve a concentration of 1 mg / mL. SDS-PAGE analysis showed that the purity of the purified bispecific antibody was >95%. Example 9

[0060] Assay for the activity of BAM26T3 antibody: PBMCs were used as effector cells. Antibodies induced T cell-mediated cell killing, leading to target cell lysis. The TDCC efficacy of the candidate antibody was assessed by detecting lucifrase release. Specific procedures included: resuscitating PBMCs; the following day, adjusting the density of 293-Luc-hAMHR2 cells that had reached the logarithmic growth phase to 1×10⁻⁶. 5 Add 50 μL of the solution to each well of a 96-well plate; the cell density is 1 × 10⁶ cells / mL. 6 PBMC cells (number / mL), 100 μL / well; add antibody (1 μg / ml, 3-fold serial dilution, 11 concentration points) 50 μL / well; incubate at 37°C and 5% CO2 for 24 hours.

[0061] Before the experiment, target cells overexpressing AMHR2 and stably expressing Luciferase, as well as human PBMCs, were resuscitated and routinely cultured to the logarithmic growth phase to ensure good cell viability (≥95% viability). Serially diluted AMHR2×CD3 bispecific TCE, luciferase detection substrate, and sterile 96-well plates were prepared, and all reagents and consumables were equilibrated at room temperature for 30 minutes. At the start of the experiment, target cell plating was performed. Logarithmically growing AMHR2-Luc293 cells were adjusted to a concentration of 1×10⁻⁶ cells using fresh culture medium. 5 cells / mL, then add 100 μL of cell suspension (1 × 10⁶ cells / mL) to each well of a 96-well white plate. 4 (1 target cell), and then effector cell seeding, taking cultured human PBMC cells and adjusting the concentration to 1×10⁶. 6 At an effector cell:target cell ratio of 10:1, add 100 μL of PBMC suspension to each well (each well containing 1 × 10⁶ cells / mL). 5After adding the target cells (100 effector cells), gently pipette 3-5 times to ensure thorough mixing and avoid excessively high or low cell concentrations in certain areas. Then, administer TCE. Start with 1 μg / mL of pre-prepared AMHR2×CD3TCE and perform 3-fold serial dilutions, creating 11 concentration gradients. Add the serially diluted TCE solution to the corresponding wells. Simultaneously, set up a target cell autofluorescence control group (target cells and culture medium only), an effector cell autofluorescence background control group (PBMCs and culture medium only), and a blank control group (culture medium only). The final volume of all wells should be uniformly 200 μL. After adding the samples, gently shake the culture plate to ensure thorough mixing. After adding the samples, place the culture plate in a 37°C, 5% CO2 incubator for 18-24 hours. After incubation, remove the culture plate and carefully discard the supernatant from each well, avoiding contact with adherent target cells. Then, add 50 μL of cell lysis buffer to each well, gently agitate the plate to ensure the lysis buffer evenly covers all cells at the bottom of the well, and incubate at room temperature for 10 minutes to allow for complete lysis of the target cells and release of Luciferase. After lysis, add 50 μL of luciferase substrate to each well, mix quickly and gently, and incubate in the dark for 10 minutes to ensure sufficient reaction between Luciferase and the substrate. Then, place the culture plate in a microplate reader and measure the fluorescence intensity (RLU) of each well, keeping it in the dark during the detection process to prevent fluorescence signal attenuation. After all wells have been tested, calculate the killing rate for each concentration group using the formula: Killing rate (%) = (Control group - Experimental group) ÷ Control group × 100%. After calculation, organize the data, fit a dose-killing effect curve, and analyze key efficacy indicators such as EC50 and maximum killing rate.

[0062] The results are as follows Figure 8 As shown in the figure. The results indicate that the BAM26T3 antibody of the present invention has significant killing activity against the target cells.

Claims

1. An antibody capable of cross-binding with human, mouse, and cynomolgus monkey AMHR2 proteins, characterized in that, The antibody includes one of the following anti-AMHR2 monoclonal antibodies: Ab11, Ab12, Ab13, Ab15, Ab16, Ab18, Ab24, Ab25, Ab26, Ab29, and Ab30.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody Ab11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.26-SEQ ID NO.28, respectively; the light chain variable region of Ab11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.29-SEQ ID NO.31, respectively. The heavy chain variable region of the monoclonal antibody Ab12 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.32-SEQ ID NO.34, respectively; the light chain variable region of Ab12 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.35-SEQ ID NO.37, respectively. The heavy chain variable region of the monoclonal antibody Ab13 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.38-SEQ ID NO.40, respectively; the light chain variable region of Ab13 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.41-SEQ ID NO.43, respectively. The heavy chain variable region of the monoclonal antibody Ab15 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.44-SEQ ID NO.46, respectively; the light chain variable region of Ab15 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.47-SEQ ID NO.49, respectively. The heavy chain variable region of the monoclonal antibody Ab16 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.50-SEQ ID NO.52, respectively; the light chain variable region of Ab16 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.53-SEQ ID NO.55, respectively. The heavy chain variable region of the monoclonal antibody Ab18 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.56-SEQ ID NO.58, respectively; the light chain variable region of Ab18 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.59-SEQ ID NO.61, respectively. The heavy chain variable region of the monoclonal antibody Ab24 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.62-SEQ ID NO.64, respectively; the light chain variable region of Ab24 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.65-SEQ ID NO.67, respectively. The heavy chain variable region of the monoclonal antibody Ab25 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.68-SEQ ID NO.70, respectively; the light chain variable region of Ab25 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.71-SEQ ID NO.73, respectively. The heavy chain variable region of the monoclonal antibody Ab26 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.74-SEQ ID NO.76, respectively; the light chain variable region of Ab26 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.77-SEQ ID NO.79, respectively. The heavy chain variable region of the monoclonal antibody Ab29 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.80-SEQ ID NO.82, respectively; the light chain variable region of Ab29 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.83-SEQ ID NO.85, respectively. The heavy chain variable region of the monoclonal antibody Ab30 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.86-SEQ ID NO.88, respectively; the light chain variable region of Ab30 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.89-SEQ ID NO.91, respectively.

3. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of monoclonal antibody Ab11 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab11 is shown in SEQ ID NO.5; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab12 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab12 is shown in SEQ ID NO.7; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab13 is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab13 is shown in SEQ ID NO.9; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab15 is shown in SEQ ID NO.10, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab15 is shown in SEQ ID NO.11; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab16 is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab16 is shown in SEQ ID NO.13; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab18 is shown in SEQ ID NO. As shown in NO.14, the amino acid sequence of the light chain variable region of monoclonal antibody Ab18 is shown in SEQ ID NO.15; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab24 is shown in SEQ ID NO.16, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab24 is shown in SEQ ID NO.17; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab25 is shown in SEQ ID NO.18, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab25 is shown in SEQ ID NO.19; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab26 is shown in SEQ ID NO.20, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab26 is shown in SEQ ID NO.21; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab29 is shown in SEQ ID NO.22, and the amino acid sequence of the light chain variable region of monoclonal antibody Ab29 is shown in SEQ ID NO.23; the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab30 is shown in SEQ ID NO. As shown in NO.24, the amino acid sequence of the light chain variable region of the monoclonal antibody Ab30 is shown in SEQ ID NO.

25.

4. A method for preparing an antibody capable of cross-binding with human, mouse, and cynomolgus monkey AMHR2 proteins as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Positive clones were obtained by cross-immunization with Balb / C mice, using hybridoma technology and B cell sorting technology. The antibody sequences were then identified by sequencing. The variable region sequence of the antibody was fused with the constant region of mouse IgG2a or human IgG1 to construct a recombinant expression vector, which was transfected into 293 cells and purified to obtain high-purity antibodies.

5. A bispecific T-cell connector targeting AMHR2×CD3, characterized in that, A combination of an antibody that can cross-bind human, mouse and cynomolgus monkey AMHR2 protein as described in any one of claims 1 to 3 and a CD3 antibody OKT3.

6. The application of an antibody as described in any one of claims 1 to 3, capable of cross-binding human, mouse, and cynomolgus monkey AMHR2 proteins, in the detection of human, mouse, and monkey AMHR2 proteins and in cross-species research.

7. The use of an antibody as described in any one of claims 1 to 3, capable of cross-binding human, mouse, and cynomolgus monkey AMHR2 proteins, in the preparation of diagnostic and therapeutic products for AMHR2-positive diseases.

8. The use of the AMHR2×CD3-targeting bispecific T-cell connector as described in claim 5 in the preparation of diagnostic and therapeutic products for AMHR2-positive diseases.

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