A b7h3 / pdl1 bispecific antibody and pharmaceutical composition and application thereof

Abstract

The present application relates to a kind of B7H3 / PDL1 bispecific antibody and its pharmaceutical composition and application.The bispecific antibody can be targeted to combine B7H3 and PDL1, it includes: monoclonal antibody unit, it is to PDL1 and includes 2 heavy chains and 2 light chains;Nanobody unit, it is to B7H3 and includes 2 identical nanobodies, wherein the N terminal of the 2 nanobodies is respectively connected with the C terminal of the Fc fragment of the 2 heavy chains of the monoclonal antibody unit by connecting peptide.The bispecific antibody of the present application can be combined with B7H3 and PDL1, can be released PDL1 to T cell while targeting tumor cell, show the antitumor activity of being superior to monoclonal antibody combined drug.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a B7H3 / PDL1 bispecific antibody, its pharmaceutical composition, and its applications. Background Technology

[0002] The B7-CD28 family, as co-stimulatory signals for T lymphocyte activation, plays a crucial role in T lymphocyte-mediated immune responses. Studies have shown that different B7 molecule types have positive or negative regulatory effects on immune cell responses. B7H3 (also known as CD276), a member of the B7 family, is primarily expressed on the surface of tumor cells. Chapoval AI et al. first discovered its effect on CD4+. + and CD8 + T cells exhibit co-stimulatory effects; B7H3 signaling induces cellular immunity and selectively enhances interferon-γ (IFN-γ) production under T cell receptor signaling. However, as research on B7H3 continues, its inhibitory functions have been gradually discovered; for example, it can inhibit CD4+. + T and CD8 + T cell proliferation. Furthermore, studies have shown that abnormal expression of B7H3 is associated with the occurrence, development, and metastasis of various cancers, and there is substantial evidence that its high expression is associated with poor prognosis in various malignant tumors.

[0003] B7H3 is highly expressed in all tested cancer types with limited heterogeneity and is rarely expressed in normal tissues. This suggests that B7H3 can be considered a tumor antigen (TA), offering potential for targeted therapy against B7H3-overexpressing tumor cells. Currently, B7H3-targeted therapy strategies mainly include blocking monoclonal antibodies, radioimmunotherapy, antibody-drug conjugates (ADCs), cytotoxic monoclonal antibodies, and bispecific antibodies (BsAbs). Enoblituzumab (MGA271), a humanized monoclonal antibody targeting B7H3 developed by MacroGenics, is based on antibody-dependent cell-mediated cytotoxicity (ADCC) and acts on various malignant tumors, such as melanoma, osteosarcoma, and Ewing's sarcoma. Enoblituzumab has demonstrated strong antitumor activity in various xenograft tumor models and showed no significant toxicity in primate safety evaluations. Enoblituzumab is currently in Phase II clinical trials and shows promising therapeutic potential.

[0004] Inhibitors targeting the immune checkpoints PD1 / PDL1 are undoubtedly a focal point of tumor immunotherapy. PDL1 is expressed on the surface of tumor cells, and theoretically, cytotoxic PDL1 inhibitory antibodies should have better anti-tumor effects. However, among the marketed PDL1 monoclonal antibodies, only Avelumab has been reported to mediate ADCC effects against tumors and has shown comparable safety profiles to other PDL1 antibodies. A key reason for this is that the effectiveness of ADCC largely depends on the abundance of antigen expression, and PDL1 cannot be considered a typical tumor antigen, and it exhibits significant heterogeneity within tumor cells.

[0005] PD1 / PDL1 pathway blocking antibodies are often used in combination with cytotoxic antibodies to enhance the efficacy of immunotherapy. The combination of enoblituzumab and pembrolizumab (a monoclonal antibody targeting PD1) can, on the one hand, block the immunosuppressive effect of the PD1 / PDL1 pathway, and on the other hand, enoblituzumab can exert a tumor-killing effect. This combination therapy has entered Phase II clinical trials (NCT04129320). Data show that the incidence of adverse reactions with combination therapy is similar to that of monotherapy, and the objective response rate (ORR) of combination therapy is superior to that of PD1 monoclonal antibody therapy. This demonstrates the feasibility of simultaneously blocking the B7H3 pathway and the PD1 / PDL1 pathway.

[0006] Bispecific antibodies (BsAbs) are genetically engineered antibodies that can simultaneously target two different antigenic epitopes. Bispecific antibodies against B7H3 have been developed, most of which rely on T cell redirection strategies, such as CD3 / B7H3 bispecific antibodies. However, theoretically, cytotoxic bispecific antibodies might be better suited for targeting tumor cells that highly express B7H3, as B7H3 serves as a tumor antigen with high abundance and low heterogeneity. Currently, only five bispecific antibody drugs have been approved for marketing, and the main reason limiting their application is issues with stability and activity. Therefore, obtaining bispecific antibodies with good stability and excellent biological activity remains a key focus for the industry. Summary of the Invention

[0007] One technical objective of this invention is to provide a method for preparing B7H3 / PDL1 bispecific antibody and its application.

[0008] On one hand, the present invention provides a B7H3 / PDL1 bispecific antibody that targets and binds to both B7H3 and PDL1, wherein the bispecific antibody comprises: A monoclonal antibody unit that targets PDL1 and comprises two heavy chains and two light chains; Nanobody unit targeting B7H3 and comprising two identical nanobodies (VHH). The N-terminus of each of the two nanobodies is connected to the C-terminus of the Fc fragment of each of the two heavy chains of the monoclonal antibody unit via a linker peptide.

[0009] In this invention, the linker peptide refers to a polypeptide containing glycine and serine and possessing certain elasticity and protease resistance.

[0010] In one embodiment, the amino acid sequence of the linker peptide is SEQ ID No.:11.

[0011] In one embodiment, the light chain variable region of the monoclonal antibody unit includes CDR1 with the amino acid sequence SEQ ID NO.: 1, CDR2 with the amino acid sequence SEQ ID NO.: 2, and CDR3 with the amino acid sequence SEQ ID NO.: 3; the heavy chain variable region of the monoclonal antibody unit includes CDR1 with the amino acid sequence SEQ ID NO.: 5, CDR2 with the amino acid sequence SEQ ID NO.: 6, and CDR3 with the amino acid sequence SEQ ID NO.: 7; and the nanobody includes CDR1 with the amino acid sequence SEQ ID NO.: 12, CDR2 with the amino acid sequence SEQ ID NO.: 13, and CDR3 with the amino acid sequence SEQ ID NO.: 14.

[0012] In one embodiment, the light chain variable region of the monoclonal antibody unit includes an amino acid sequence such as SEQ ID NO.: 4, and the heavy chain variable region of the monoclonal antibody unit includes an amino acid sequence such as SEQ ID NO.: 8.

[0013] In one embodiment, the light chain of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 9, and the heavy chain of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 10.

[0014] In one embodiment, the nanobody comprises an amino acid sequence such as SEQ ID NO.: 15. In one embodiment, the amino acid sequence of the light chain variable region of the monoclonal antibody unit is shown in SEQ ID NO.: 4, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody unit is shown in SEQ ID NO.: 8.

[0015] In one embodiment, the full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9, and the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10.

[0016] In one embodiment, the amino acid sequence of the nanobody is shown in SEQ ID NO.: 15. In one embodiment, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence is shown in SEQ ID NO.: 17.

[0017] On the other hand, the present invention also provides a polynucleotide encoding the B7H3 / PDL1 bispecific antibody.

[0018] In another aspect, the present invention also provides an expression vector comprising a polynucleotide encoding the B7H3 / PDL1 bispecific antibody.

[0019] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned B7H3 / PDL1 bispecific antibody and a pharmaceutically acceptable carrier.

[0020] On the other hand, the present invention provides the use of the B7H3 / PDL1 bispecific antibody in the preparation of a medicament for the prevention, diagnosis, treatment or adjuvant treatment of tumors.

[0021] In a specific embodiment, the drug inhibits tumors by binding to B7H3 and blocking the B7H3 signaling pathway, thereby mediating the ADCC effect.

[0022] In a specific embodiment, the drug inhibits tumors by binding to PD-L1, blocking the binding of PD-1 to PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes.

[0023] In a specific embodiment, the drug inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, binding to PD-L1, blocking the binding of PD-1 and PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes.

[0024] In a specific implementation, the tumor may be selected from one or more of the following: lung cancer, stomach cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma. Specifically, the tumor may be breast cancer, ovarian cancer, or melanoma.

[0025] Beneficial effects The bispecific antibody constructed in this application can bind to both B7H3 and PDL1 simultaneously, and can relieve the inhibition of T cells by PDL1 while targeting tumor cells, thus exhibiting superior antitumor activity compared to combination therapy with monoclonal antibodies.

[0026] In addition, compared with related monoclonal antibody combination therapy, the bispecific antibody of this application has the advantages of good compliance and controllable quality.

[0027] Furthermore, the stability characterization of the bispecific antibody in this application mainly focuses on the study of monomer purity and thermal stability. After a single affinity purification, the monomer content of the bispecific antibody can reach 95%, even superior to the purity achieved after multiple secondary purifications in the industry. Structural and activity analyses of the antibody after heat treatment demonstrate that the antibody can maintain a good molecular conformation and complete biological activity under harsh environments, which is beneficial for the industrial production and packaging storage of the antibody. Overall, this application constructs a B7H3 / PDL1 bispecific antibody in the form of IgG-VHH2; it exhibits good molecular stability and significantly superior in vitro activity (binding molecular and cellular levels) compared to Avelumab and MGA271. In vivo data demonstrate that, in B7H3… + In A375 tumor cells, the bispecific antibody exhibited superior antitumor activity compared to the combination therapy with the B7H3 monoclonal antibody. Therefore, the bispecific antibody presented in this application has broad application prospects due to its excellent developability and activity.

[0028] In summary, the method used in this invention represents a novel and cutting-edge approach to tumor treatment, and tumor immunotherapy holds the promise of revolutionizing the field of tumor treatment following surgery, chemotherapy, radiotherapy, and targeted therapy. The B7H3 / PDL1 bispecific antibody in this invention shows promise as a novel anti-tumor drug. Attached Figure Description

[0029] Figure 1 Affinity screening diagram of the anti-B7H3 VHH humanized antibody prepared in this application and B7H3.

[0030] Figure 2 : A schematic diagram of the structure of the B7H3 / PDL1 bispecific antibody prepared in this application. Figure 3 SDS-polyacrylamide gel electrophoresis image of the B7H3 / PDL1 bispecific antibody of this application.

[0031] Figure 4 : SEC-HPLC purity determination of the B7H3 / PDL1 bispecific antibody of this application.

[0032] Figure 5 The Tm value (DSF) of the B7H3 / PDL1 bispecific antibody of this application was determined.

[0033] Figure 6The B7H3 / PDL1 bispecific antibody of this application is bound to ELISA before and after heat treatment at 60°C, where a represents ELISA bound to PDL1 and b represents ELISA bound to B7H3.

[0034] Figure 7 The present application describes a B7H3 / PDL1 bispecific antibody binding ELISA, where a represents a binding ELISA with PDL1 and b represents a binding ELISA with B7H3.

[0035] Figure 8 The graphs show the affinity of the B7H3 / PDL1 bispecific antibody B7H3-his or PDL1-his at five different concentrations. a represents the affinity curve with PDL1, and b represents the affinity curve with B7H3.

[0036] Figure 9 The blocking curve of the B7H3 / PDL1 bispecific antibody PDL1 / CHO-PD1 in this application.

[0037] Figure 10 The B7H3 / PDL1 bispecific antibody of this application induces T cells to secrete IFN-γ.

[0038] Figure 11 The B7H3 / PDL1 bispecific antibody in this application promotes T cell proliferation.

[0039] Figure 12 The ADCC effect of the B7H3 / PDL1 bispecific antibody in this application on cancer cells, a represents the cytotoxic effect on human breast cancer cells MDA-MB-231, and b represents the cytotoxic effect on human ovarian clear cell carcinoma cells ES-2.

[0040] Figure 13 The in vivo tumor-suppressing effect of the B7H3 / PDL1 bispecific antibody in this application.

[0041] Figure 14 The effect of the B7H3 / PDL1 bispecific antibody of this application on mouse body weight. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the embodiments.

[0043] Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and can be obtained through commercial channels.

[0044] The present invention will be described below through specific embodiments to enable those skilled in the art to better understand the technical content of this application; however, these embodiments should not be construed as limiting the scope of the present invention.

[0045] Preparation Examples 1. Humanization of camel-derived anti-B7H3 Camel-derived anti-B7H3 nanobodies were humanized using the Germline gene of the human antibody gene as a template via the framework shuffling method. The corresponding VHH morphology libraries were generated through whole-body in vitro synthesis via overlap PCR. Then, clones of the VHH phage library were constructed, screened, and identified.

[0046] More precisely, a one-step strategy was used to humanize camel-derived anti-B7H3 nanobodies. Approximately 1000 clones were screened from this sub-library, and the selected positive clones were screened for phage-level thermostability using ELISA. 5 μg / ml huB7H3 antigen was coated onto highly absorbent 96-well ELISA plates, and the supernatant from overnight amplification of the screened phages was reacted. Clones exhibiting higher OD450 reads were selected.

[0047] The above-mentioned phage clones were sequenced to obtain the B7H3 VHH gene sequence. The N-terminus of this sequence was fused with the human Fc protein gene to construct and express B7H3-Fc. Using the biomembrane interference method, a Protein A probe was used to capture 100 nM of B7H3 VHH-Fc and bind it to a 2-fold diluted 200 nM concentration of B7H3 antigen. The KD value of the antibody-antigen binding was calculated. The results showed that 75-16 (amino acid sequence SEQ ID NO.: 15, CDR1 sequence SEQ ID NO.: 12, CDR2 sequence SEQ ID NO.: 13, CDR3 sequence SEQ ID NO.: 14) had the highest affinity for B7H3, with a KD reaching 3.24 × 10⁻⁶. -9 M (see) Figure 1 Therefore, it was chosen for the next step of constructing bispecific antibodies.

[0048] 2. Construction and expression of B7H3 / PDL1 bispecific antibody The amino acid sequences of the PDL1 monoclonal antibody light chain (amino acid sequence SEQ ID NO.: 9, variable region sequence SEQ ID NO.: 4, CDR1 sequence SEQ ID NO.: 1, CDR2 sequence SEQ ID NO.: 2, CDR3 sequence SEQ ID NO.: 3) and heavy chain (amino acid sequence SEQ ID NO.: 10, variable region sequence SEQ ID NO.: 8, CDR1 sequence SEQ ID NO.: 5, CDR2 sequence SEQ ID NO.: 6, CDR3 sequence SEQ ID NO.: 7) are derived from existing PDL1 hIgG1 humanized monoclonal antibodies, and the N-terminus of anti-B7H3 VHH (75-16 above) is linked to the C-terminus of the Fc fragment via a linker peptide (SEQ ID NO.: 11) (structure as shown). Figure 2 (As shown).

[0049] The DNA sequence was synthesized, subcloned into the pcDNA3.1 vector, and amplified in *E. coli*. The purified plasmid was transfected into HEK293 cells via PEI. The cells were then cultured in OPM-CD05 expression medium. After 6 days of culture, the cell culture supernatant was collected, and the antibody was purified using a protein A column. The purified IgG1 was dialyzed against phosphate-buffered saline (PBS), flash-frozen, and stored at -80°C.

[0050] The heavy chain amino acid sequence of the purified B7H3 / PDL1 bispecific antibody is SEQ ID NO.: 16, and the light chain amino acid sequence is SEQ ID NO.: 17.

[0051] Test section Unless otherwise specified, the term "bispecific antibody" in this application refers to the B7H3 / PDL1 bispecific antibody, which is also simply referred to as "B7H3 / PDL1 bispecific antibody" or "bispecific antibody".

[0052] Test method: Example 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 5 μg of bispecific antibody was mixed with protein reduction and non-reduction buffer, and the mixture was brought to a final volume of 10 μl with PBS. The mixture was heated at 100 °C for 10 minutes to fully denature the protein. 9 μl of this mixture was added to pre-made polyacrylamide gel wells (Bio-Rad). Separation was performed by first setting the voltage to 80 V for 30 minutes, then to 120 V for 60 minutes. The gel was stained with Coomassie Brilliant Blue for 30 minutes, and then destained with a destaining solution (acetic acid: ethanol: water = 1:3:6) for 15 minutes. This destaining process was repeated three times to remove the background. Images were then acquired using a gel imaging system. The results showed that the B7H3 / PDL1 bispecific antibody exhibited good monomer purity under non-reduction conditions. Under reduction conditions, two bands appeared (heavy and light chains) due to the opening of disulfide bonds between the light and heavy chains, with no impurities. Figure 3 ).

[0053] Example 2 Size Exclusion Chromatography (SEC-HPLC) SEC-HPLC analysis was used to assess the monomeric purity of the bispecific antibody. The B7H3 / PDL1 bispecific antibody was analyzed using a 1260 HPLC system (Agilent, Santa Clara, CA) on a Thermo MAbPac SEC-1, 5 µm, (7.8 × 300 mm) P / N 088460, and compared with PDL1 monoclonal antibody and B7H3 VHH-Fc. The mobile phase used was phosphate-buffered saline (PBS). The flow rate was set to 0.7 mL / min; the injection volume was 15 μl. SEC chromatograms were recorded by monitoring the absorbance at 280 nm using a UV detector at a constant temperature of 25 °C. Figure 4 As shown, all the antibodies tested had a very high proportion of monomer peaks, with the peak area ratio of monomer peaks reaching over 95%. This indicates that the bispecific antibodies have good monomer purity and a small number of aggregates under PBS buffer conditions.

[0054] Example 3: Differential scanning fluorescence (DSF) detection of antibody Tm value DSF was detected using a real-time PCR instrument (Biorad cfx96, USA). B7H3 / PDL1 bispecific antibody, B7H3 VHH-Fc, and PDL1 monoclonal antibody were diluted to 1 mg / mL in PBS. SYPRO Orange was diluted 1000-fold from a 5000-fold concentrated stock solution with ddH2O. 20 μl of sample was placed in a PCR tube, and SYPRO Orange working solution was added to the reaction to prevent bleaching. The mixture was briefly centrifuged, and the liquid was collected at the bottom of the PCR tube. The qPCR instrument was turned on, and the program was set to 25℃-95℃ with a temperature increase of 0.3℃ per second. Data were collected, and temperature and signal values ​​were plotted. The melting temperature (Tm) was calculated. The data showed that the Tm of the Fc and Fab of the B7H3 / PDL1 bispecific antibody were 69℃ and 90℃, respectively, demonstrating excellent heat resistance, and were similar to the Tm of the B7H3 VHH-Fc and PDL1 monoclonal antibody. Figure 5 ).

[0055] Example 4: Verification of antibody thermostability and binding activity using ELISA 96-well ELISA plates were coated overnight at 4°C with 2 µg / ml histagged PDL1 or B7H3 antigen protein. The next day, 100 μl of Casein blocking buffer was added to each well and the plates were blocked at 37°C for 1 hour. Three-fold serial dilutions of B7H3 / PDL1 bispecific antibody (for thermostability testing, the bispecific antibody was treated in a 60°C water bath for 1 hour) and monoclonal antibodies (for testing bispecific antibody binding to B7H3 using ELISA, the control monoclonal antibodies used were MGA271, Isotype hIgG1, and B7H3 VHH-Fc; for testing binding to PDL1, the control monoclonal antibodies were PDL1 monoclonal antibody, Avelumab, and Isotype hIgG1) were added to the wells. After incubation at 37°C for 1 hour, unbound antibodies were washed away with 0.1% PBST. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, USA). Color development was performed with 50 μl of 3,3',5,5'-tetramethylbenzidine substrate (TMB). After standing for 5 minutes, 50 μl of 2M sulfuric acid was added to stop the color development. The absorbance was then measured at OD450 nm using a SpectraMax M5e (Molecular Devices) microplate reader. A 4-parameter fitting method was used to plot the antibody concentration against the OD450 reading, and the EC50 was calculated. 50 . Figure 6Figures a and b show the binding curves of the bispecific antibody with PDL1 and B7H3 before and after treatment at 60°C for 1 hour. As can be seen from the figures, the binding curves of the bispecific antibody before and after heating largely overlap for both PDL1 and B7H3, indicating that the bispecific antibody can withstand the high temperature of 60°C and maintain strong binding activity against both PDL1 and B7H3 targets. On the other hand, the bispecific antibody, positive antibody, PDL1 monoclonal antibody, and B7H3 VHH-Fc showed similar binding abilities to both targets. The bispecific antibody, PDL1 monoclonal antibody, and control Avelumab bound to human PDL1 EC3... 50 The values ​​are 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively, which are in the same order of magnitude. Figure 7 a) Bispecific antibodies, B7H3 VHH-Fc, and MGA271 also exhibit similar binding activity to human B7H3, EC 50 The values ​​were 0.04105 nM, 0.02515 nM, and 0.05476 nM, respectively (Figure 7b).

[0056] Example 5: Determination of the affinity between bispecific antibodies and antigens using the BLI method. B7H3 / PDL1 bispecific antibody, PDL1 monoclonal antibody, and B7H3 VHH-Fc were diluted to 100 nM in sample buffer (0.02% Tween 20 and 0.1% BSA in PBS). The affinity of the bispecific antibody for specific human B7H3 and human PDL1 antigens was analyzed using OCTET 96. Protein A probes were used to immobilize the antibodies. B7H3-his and PDL1-his antigens were diluted to an initial concentration of 200 nM in sample buffer, and multiple antigen gradients were set up at 2-fold dilutions to bind with the antibodies, obtaining rate constants and affinity. Kon and Koff values ​​were calculated using software provided by the supplier, and the KD value of the antibody was obtained. As shown in the figure, the bispecific antibody has a high affinity for PDL1 and B7H3, with KD values ​​reaching 5.85 × 10⁻⁶. -10 M and 8.11×10 -9 M ( Figure 8 ).

[0057] Example 6: Flow cytometry detection of the ability of bispecific antibodies to block the PD1 / PDL1 pathway The ability of the B7H3 / PDL1 bispecific antibody to block the binding of human PDL1 and human PD1-CHO cells was evaluated by flow cytometry and compared with Avelumab and PDL1 monoclonal antibody. 2×10 5CHO-PD1 cells were evenly seeded in 96-well plates and incubated at room temperature for 30 minutes with a mixture of 400 nM starting, serially diluted antibodies (B7H3 / PDL1 bispecific antibody, Avelumab, PDL1 monoclonal antibody, IsotypehIgG1, B7H3 VHH-Fc) and biotinylated PDL1 antigen (50 nM). The mixture was then incubated with the cells at 4°C for 45 minutes. Unbound antigen was washed away with PBS. Cells were then stained with PE-streptavidin for fluorescence. The mean fluorescence intensity (MFI) of the PE channel in the flow cytometer was read, and the antibody concentration and MFI were plotted. The IC50 of the antibody was calculated using a four-parameter fitting method. 50 Flow cytometry analysis showed that the bispecific antibody could block the binding of PDL1 to CHO-PD1 cells, with an IC50 value of [missing information]. 50 The concentration was 106.4 nM and the blocking activity was similar to that of the PDL1 monoclonal antibody (IC50). 50 (94.20 nM), Avelumab (IC) 50 (115.0 nM) similar to ( Figure 9 ).

[0058] Example 7: Detection of the ability of bispecific antibodies to activate T cells using mixed lymphocyte reaction (MLR). Dendritic cells (DCs) were induced by culturing peripheral blood mononuclear cells (PBMCs) for 7 days in vitro using a mononuclear cell purification kit (Miltenyi Biotec, Germany) with 500 U / mL interleukin-4 (IL-4) and 250 U / mL GM-CSF. CD4 + T cells (1×10) 5 ) and allogeneic DCs (1.25×10 4 Co-culture was performed using RPMI 1640 complete medium containing 10% FBS under 5% CO2 and constant temperature of 37℃. Groups were established with no antibody and with different concentrations of B7H3 / PDL1 bispecific antibody, PDL1 monoclonal antibody, Avelumab, B7H3 VHH-Fc, MGA271, and Isotype hIgG1. After 5 days, the IFN-γ concentration in the culture supernatant was analyzed using an IFN-γ ELISA kit. MLR results indicated that the bispecific antibody could stimulate CD4+. +T cells secrete IFN-γ, and the T cell activation ability of bispecific antibodies is superior to that of PDL1 monoclonal antibodies and Avelumab at both low and high concentrations. In addition, when only B7H3 antibody is present, although it is not as obvious as PDL1 signal blocking antibody, it can still partially activate T cells because bispecific antibody blocks B7H3 inhibitory signal transduction (Figure 10).

[0059] Example 8: T cell proliferation experiment 1 μg / ml CD3 antibody (Clone HIT3a), 1 μg / ml CD28 antibody (Clone CD28.2), and 5 μg / ml human PDL1 were coated onto 96-well cell plates (Corning, USA) at 4°C for 1 hour. Control wells were coated with mouse IgG2a isotype control alone, or CD3 and CD28 antibodies were coated using the same method. CD4 was isolated using the Dynabeads™ CD4Positive Isolation Kit. + T cells. CD4 + T cells were cultured for 4 days at 37°C in RPMI 1640 medium containing 10% FBS (Gibco) in pre-coated 96-well plates with different concentrations of B7H3 / PDL1 bispecific antibody, Avelumab, and PDL1 monoclonal antibody. After 4 days, changes in T cell count were detected using a CCK8 assay kit. The data indicated that freshly isolated human CD4+ cells cultured in wells coated with anti-CD3 and anti-CD28 antibodies... + T cells exhibited increased proliferation, but their proliferative capacity significantly decreased when PDL1 was added to the well plate, confirming that PDL1 provided an inhibitory signal to T cells. Avelumab, PDL1 monoclonal antibody, and bispecific antibody significantly promoted T cell proliferation at concentrations of 100 nM and 500 nM (Figure 11).

[0060] Example 9: Antibody-dependent cytotoxicity (ADCC) The primary antitumor activity of MGA271 and Avelumab derives from the antibody's ADCC function, which is related to its IgG1 subtype; the bispecific antibody also possesses an IgG1 functional region. The antibody's ADCC was measured using an LDH cytotoxicity assay kit. Human PBMCs were purified from leukocyte packages using Ficoll gradient centrifugation, and NK cells were isolated from human PBMCs using negative-selective magnetic beads (Miltenyi Biotec, Auburn, CA). NK cells (3 × 10⁻⁶) 6 ) and MDA-MB-231, ES-2 cells (3×10 5At the start of the assay, different concentrations of bispecific antibody and Avelumab were added or not added, and the culture was co-cultured. After 18 hours, lactate dehydrogenase (LDH) secretion in the culture supernatant was analyzed by ELISA. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) + PDL1 + When MDA-MB-231 was used as the target cell, both the bispecific antibody and Avelumab exhibited ADCC activity, but the bispecific antibody showed stronger activity at low concentrations; when PDL1 was used... + Using ES-2 cells as target cells, the bispecific antibody was observed to have activity comparable to Avelumab at a dose of 100 nM, and also exhibited strong ADCC activity at low concentrations. Figure 12 ).

[0061] Example 10: In vivo antitumor activity study of bispecific antibodies Human PBMCs (6.67 × 10⁻⁶) 6 One day before inoculation with A375 tumor cells, 41 NPSG mice were injected via the tail vein, and the following day, 5 × 10⁵ cells were injected subcutaneously. 6 A375 tumor cells were inoculated. Subcutaneous tumor formation was observed five days after tumor inoculation. Ten animals were used in each group, divided into isotype control, monoclonal antibody combination therapy (Pembrolizumab + MGA271 and Avelumab + MGA271), monoclonal antibody, and bispecific antibody groups. Drug administration began on day 5 after successful model establishment and continued twice weekly until the end of the experiment. Tumor volume and animal weight were measured and recorded on days 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39, and 42. Efficacy and safety were evaluated based on tumor growth inhibition values ​​based on changes in relative tumor volume (TGIRTV) and animal body weight. The bispecific antibody maintained significantly stronger activity than the PD1 monoclonal antibody + MGA271 combination therapy throughout the experiment; with increasing experimental time, the bispecific antibody also gradually showed superior efficacy compared to the Avelumab + MGA271 group, with TGIs of 39.29% and 26.45% at the end of the experiment, respectively. Figure 13 Furthermore, no significant weight loss was observed in mice during the experiment, demonstrating the safety of this bispecific antibody treatment. Figure 14 ).

[0062] The test results from the above test examples show that the bispecific antibody constructed in this application can bind to both B7H3 and PDL1 simultaneously, and can relieve the inhibition of T cells by PDL1 while targeting tumor cells, thus exhibiting anti-tumor activity superior to that of monoclonal antibody combination therapy.

Claims

1. A B7H3 / PDL1 bispecific antibody that targets and binds to both B7H3 and PDL1, said bispecific antibody comprising: A monoclonal antibody unit that targets PDL1 and comprises two heavy chains and two light chains; Nanobody unit targeting B7H3 and comprising two identical nanobodies. The N-terminus of each of the two nanobodies is connected to the C-terminus of the Fc fragment of each of the two heavy chains of the monoclonal antibody unit via a linker peptide. The light chain variable region of the monoclonal antibody unit includes CDR1 with the amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence SEQ ID NO: 2, and CDR3 with the amino acid sequence SEQ ID NO: 3; the heavy chain variable region of the monoclonal antibody unit includes CDR1 with the amino acid sequence SEQ ID NO: 5, CDR2 with the amino acid sequence SEQ ID NO: 6, and CDR3 with the amino acid sequence SEQ ID NO: 7; and the nanobody includes CDR1 with the amino acid sequence SEQ ID NO: 12, CDR2 with the amino acid sequence SEQ ID NO: 13, and CDR3 with the amino acid sequence SEQ ID NO:

14.

2. The bispecific antibody according to claim 1, wherein, The linker peptide is a polypeptide containing glycine and serine and possessing elasticity and protease resistance.

3. The bispecific antibody according to claim 2, wherein, The amino acid sequence of the linker peptide is SEQ ID No:

11.

4. The bispecific antibody according to claim 1, wherein, The light chain variable region of the monoclonal antibody unit includes an amino acid sequence as shown in SEQ ID NO: 4; the heavy chain variable region of the monoclonal antibody unit includes an amino acid sequence as shown in SEQ ID NO: 8; and the nanobody includes an amino acid sequence as shown in SEQ ID NO:

15.

5. The bispecific antibody according to claim 1, wherein, The light chain of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO: 9, the heavy chain of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO: 10, and the nanobody comprises an amino acid sequence as shown in SEQ ID NO:

15.

6. The bispecific antibody according to claim 1, wherein, The full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO: 9; the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO: 10; and the amino acid sequence of the nanobody is shown in SEQ ID NO:

15.

7. The bispecific antibody according to claim 1, wherein, The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 16, and the light chain amino acid sequence is shown in SEQ ID NO:

17.

8. A polynucleotide encoding a B7H3 / PDL1 bispecific antibody as described in any one of claims 1 to 7.

9. An expression vector comprising the polynucleotide as described in claim 8.

10. A pharmaceutical composition comprising a therapeutically effective amount of the B7H3 / PDL1 bispecific antibody as described in any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

11. Use of the B7H3 / PDL1 bispecific antibody as described in any one of claims 1 to 7 in the preparation of a medicament for treating tumors, wherein, The tumor is selected from one or more of the following: lung cancer, stomach cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma.

12. The use according to claim 11, wherein, The tumor is breast cancer, ovarian cancer, or melanoma.

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