B7H3 / PDL1 bispecific antibodies and pharmaceutical compositions thereof and uses thereof

Abstract

The present invention relates to a B7H3 / PDL1 bispecific antibody, a pharmaceutical composition thereof, and uses thereof. The bispecific antibody can target and bind to both PDL1 and B7H3. The bispecific antibody comprises a monoclonal antibody unit that targets PDL1 and comprises two heavy chains and two light chains, and a nanobody unit that targets B7H3 and comprises two identical nanobodies (VHHs), the C-termini of the two nanobodies being linked via linker peptides to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit. The bispecific antibody of the present invention can bind to both B7H3 and PDL1, can target tumor cells while relieving PDL1-mediated T cell inhibition, and exhibits superior antitumor activity compared to a combination of monoclonal antibodies.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a B7H3 / PDL1 bispecific antibody and its pharmaceutical composition and uses. [Background technology]

[0002] The B7-CD28 family plays an important role in immune responses involving T lymphocytes as a costimulatory signal for T lymphocyte activation. Studies have shown that different types of B7 molecules positively or negatively regulate immune cell responses. B7H3 (also known as CD276), a member of the B7 family, is primarily expressed on the surface of tumor cells and has been shown by Chapoval AI et al. to bind to CD4 + and CD8 + B7H3 was first identified as having a costimulatory effect on T cells. B7H3 signaling induces cellular immunity and selectively enhances interferon-γ (IFN-γ) production in association with T cell receptor signaling. However, as research on B7H3 progressed, inhibitory functions of B7H3 were gradually discovered, e.g., CD4 + T cells and CD8 + It can inhibit 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 mounting evidence suggesting that high expression of B7H3 is correlated with poor prognosis in various malignant tumors.

[0003] B7H3 was highly expressed in all cancer types tested, with limited heterogeneity and little expression in normal tissues. This suggests that B7H3 may be a tumor antigen (TA), providing the potential for targeted therapy against tumor cells with high B7H3 expression. Currently, therapeutic strategies targeting B7H3 include blocking monoclonal antibodies, radioimmunotherapy, antibody-drug coupling (ADC), cytotoxic monoclonal antibodies, and bispecific antibodies (BsAbs). MacroGenics developed enoblituzumab (MGA271), a humanized monoclonal antibody targeting B7H3, which acts on various malignancies, including melanoma, osteosarcoma, and Ewing's sarcoma, via antibody-dependent cell-mediated cytotoxicity (ADCC). Enoblituzumab demonstrated potent antitumor activity in various xenograft tumor models and demonstrated no significant toxicity in primate safety studies. Enoblituzumab is currently in phase II clinical trials and shows promising therapeutic potential.

[0004] Inhibitors targeting the immune checkpoint PD1 / PDL1 are undoubtedly a focus of tumor immunotherapy. PDL1 is expressed on the surface of tumor cells, and cytotoxic PDL1-blocking antibodies theoretically demonstrate superior antitumor efficacy. However, among commercially available PDL1 monoclonal antibodies, only avelumab has been reported to mediate ADCC effects against tumors and exhibit a safety profile comparable to other PDL1 antibodies. The key reason for this is that ADCC effects are highly dependent on antigen expression, whereas PDL1 is not considered a typical tumor antigen and is highly heterogeneous within tumor cells.

[0005] PD1 / PDL1 pathway-blocking antibodies are often combined with cytotoxic antibodies to enhance the efficacy of immunotherapy. The combination of enoblituzumab and pembrolizumab, a monoclonal antibody targeting PD1, blocks the immunosuppression of the PD1 / PDL1 pathway on the one hand, while enoblituzumab exerts tumor-killing effects on the other. This combination therapy is currently undergoing a phase II clinical trial (NCT04129320). Results showed that the side effect rate of the combination therapy was comparable to that of monotherapy, and the objective response rate (ORR) of the combination therapy was 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 capable of simultaneously targeting two distinct antigen epitopes. While bispecific antibodies targeting B7H3 have been developed, most rely on T cell redirection strategies, such as CD3 / B7H3 bispecific antibodies. However, because B7H3 is characterized as a tumor antigen with high expression levels and low expression heterogeneity, cytotoxic bispecific antibodies may theoretically be more effective against tumor cells that highly express B7H3. Currently, only five bispecific antibody drugs have been approved for commercial sale, and their application is largely limited by stability and activity issues. Therefore, developing bispecific antibodies with good stability and excellent biological activity remains a key industry focus. Summary of the Invention

[0007] One technical object of the present invention is to provide methods and uses for producing B7H3 / PDL1 bispecific antibodies.

[0008] In one aspect, the present invention provides a B7H3 / PDL1 bispecific antibody that targets and binds B7H3 and PDL1, comprising: a monoclonal antibody unit that targets PDL1 and includes two heavy chains and two light chains; a nanobody unit that targets B7H3 and contains two identical nanobodies (VHHs); Including, the C-termini of the two Nanobodies are linked via linker peptides to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit, respectively; B7H3 / PDL1 bispecific antibodies are provided.

[0009] In the present invention, the linker peptide refers to a polypeptide segment that contains glycine and serine and has a certain degree of flexibility 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 comprises a CDR1 whose amino acid sequence is SEQ ID NO: 1, a CDR2 whose amino acid sequence is SEQ ID NO: 2, and a CDR3 whose amino acid sequence is SEQ ID NO: 3; the heavy chain variable region of the monoclonal antibody unit comprises a CDR1 whose amino acid sequence is SEQ ID NO: 5, a CDR2 whose amino acid sequence is SEQ ID NO: 6, and a CDR3 whose amino acid sequence is SEQ ID NO: 7; and the Nanobody comprises a CDR1 whose amino acid sequence is SEQ ID NO: 12, a CDR2 whose amino acid sequence is SEQ ID NO: 13, and a CDR3 whose amino acid sequence is SEQ ID NO: 14.

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

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

[0014] In one embodiment, the Nanobody comprises the amino acid sequence of SEQ ID NO: 15.

[0015] In one embodiment, the amino acid sequence of the light chain variable region of the monoclonal antibody unit is SEQ ID NO:4, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody unit is SEQ ID NO:8.

[0016] In one embodiment, the full-length amino acid sequence of the light chain of the monoclonal antibody unit is SEQ ID NO:9, and the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is SEQ ID NO:10.

[0017] In one embodiment, the amino acid sequence of said Nanobody is SEQ ID NO: 15.

[0018] In one embodiment, the bispecific antibody has a heavy chain amino acid sequence of SEQ ID NO: 16 and a light chain amino acid sequence of SEQ ID NO: 17.

[0019] In another aspect, the present invention further provides a polynucleotide encoding the B7H3 / PDL1 bispecific antibody.

[0020] In a further aspect, the present invention further provides an expression vector comprising a polynucleotide encoding said B7H3 / PDL1 bispecific antibody.

[0021] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the B7H3 / PDL1 bispecific antibody described above and a pharmaceutically acceptable carrier.

[0022] In another aspect, the present invention provides the use of said B7H3 / PDL1 bispecific antibody in the manufacture of a medicament for the prevention, diagnosis, treatment or adjuvant therapy of a tumor.

[0023] In a specific embodiment, the medicament inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, and mediating an ADCC effect.

[0024] In a specific embodiment, the medicament inhibits tumors by binding to PD-L1, inhibiting PD-1 binding to PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes.

[0025] In a specific embodiment, the medicament inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, and binding to PD-L1, inhibiting the binding of PD-1 to PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes.

[0026] In a specific embodiment, the tumor may be one or more selected from the group consisting of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, renal tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma. In particular, the tumor is breast cancer, ovarian cancer, or melanoma. [Effects of the Invention]

[0027] The bispecific antibody constructed in this study can bind to both B7H3 and PDL1, and can target tumor cells while reversing PDL1-mediated inhibition of T cells, demonstrating superior antitumor activity compared to the combined use of monoclonal antibodies.

[0028] Furthermore, the bispecific antibodies according to the present application have the advantage of better compliance and controllable quality compared to related monoclonal antibody combination therapies.

[0029] Furthermore, the stability characteristics of the bispecific antibody in this application are primarily reflected in studies of monomer purity and thermal stability. After a single affinity purification, the monomer content of the bispecific antibody can reach 95%, which is superior to the purity achieved by multiple double purifications in the industry. Analysis of the antibody structure and activity after heat treatment demonstrated that the antibody maintains good molecular conformation and intact biological activity even under harsh conditions, making it suitable for industrial antibody production, packaging, and storage. In summary, this application constructs a B7H3 / PDL1 bispecific antibody in the form of IgG-VHH2, which exhibits good molecular stability and significantly superior in vitro activity (combined molecular and cellular levels) to avelumab and MGA271. In vivo data also demonstrates that B7H3 + In A375 tumor cells, the antitumor activity of the bispecific antibody was superior to that of the B7H3 monoclonal antibody combination group. Therefore, the bispecific antibody of this application is expected to have a wide range of applications in the future due to its excellent usability and activity.

[0030] As described above, the method used in the present invention is a new, cutting-edge method for tumor treatment at present, and tumor immunotherapy is expected to be an innovation in the field of tumor treatment following surgery, chemotherapy, radiation therapy, and targeted therapy. The B7H3 / PDL1 bispecific antibody of the present invention is expected to be a novel antitumor drug. [Brief explanation of the drawings]

[0031] [Figure 1] 1 shows an affinity screening graph of the anti-B7H3 VHH humanized antibodies produced in the present application targeting B7H3. [Figure 2] FIG. 1 is a schematic structural diagram of the B7H3 / PDL1 bispecific antibody produced in the present application. [Figure 3] FIG. 1 shows an SDS-polyacrylamide gel electrophoresis pattern of the B7H3 / PDL1 bispecific antibody of the present application. [Figure 4] 1 shows the results of SEC-HPLC purity measurement of the B7H3 / PDL1 bispecific antibody of the present application. [Figure 5]1 shows the results of Tm value measurement (DSF) of the B7H3 / PDL1 bispecific antibody of the present application. [Figure 6] FIG. 1 shows binding ELISA of the B7H3 / PDL1 bispecific antibody of the present application before and after heat treatment at 60° C., where a shows binding ELISA with PDL1 and b shows binding ELISA with B7H3. [Figure 7] FIG. 1 shows binding ELISA of the B7H3 / PDL1 bispecific antibody according to the present application, where a shows binding ELISA with PDL1 and b shows binding ELISA with B7H3. [Figure 8] 1 shows BLI affinity assay plots of the B7H3 / PDL1 bispecific antibody according to the present application at five different concentrations of B7H3-his or PDL1-his, where a shows the affinity curve with PDL1 and b shows the affinity curve with B7H3. [Figure 9] 1 shows the PDL1 / CHO-PD1 blocking curve of the B7H3 / PDL1 bispecific antibody of the present application. [Figure 10] 1 is a graph showing the induction of IFN-γ secretion from T cells by the B7H3 / PDL1 bispecific antibody of the present application. [Figure 11] 1 is a graph showing the promotion of T cell proliferation by the B7H3 / PDL1 bispecific antibody of the present application. [Figure 12] FIG. 1 shows the ADCC effect of the B7H3 / PDL1 bispecific antibody of the present application against cancer cells, where (a) shows the cytotoxicity against MDA-MB-231 human breast cancer cells, and (b) shows the cytotoxicity against ES-2 human ovarian clear cell carcinoma cells. [Figure 13] 1 is a graph showing the in vivo tumor-suppressing effect of the B7H3 / PDL1 bispecific antibody of the present application. [Figure 14] 1 is a graph showing the effect of the B7H3 / PDL1 bispecific antibody according to the present application on mouse body weight. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be further described below in connection with specific embodiments, but the present invention is not limited to these embodiments.

[0033] Materials, reagents, instruments, and methods used in the following embodiments are conventional materials, reagents, instruments, and methods in the relevant technical fields and are commercially available, unless otherwise specified.

[0034] Hereinafter, the present invention will be described with reference to specific embodiments so that those skilled in the art can better understand the technical contents of the present application, but these embodiments should not be construed as limiting the scope of the present invention.

[0035] Manufacturing example 1. Humanization of camel-derived anti-B7H3 Using the germline gene of the human antibody gene as a template, a camel-derived anti-B7H3 nanobody was humanized using framework shuffling, and the corresponding VHH conformational rearrangement library was synthesized in vitro by overlap PCR. Subsequently, clones of the VHH phage library were constructed, screened, and characterized.

[0036] More precisely, a camel-derived anti-B7H3 nanobody was humanized using a one-step strategy. Approximately 1,000 clones were screened from this sublibrary, and the selected positive clones were screened for thermostability at the phage level by ELISA. In ELISA, 5 μg / ml of huB7H3 antigen was encapsulated in a high-adsorption 96-well ELISA plate, and the supernatant of the screened phage was reacted with overnight-amplified phage. Clones with higher OD450 values ​​were selected.

[0037] The sequence of the phage clone was determined, and the base sequence of the B7H3 VHH gene was obtained. The C-terminus was fused to a human Fc protein gene to construct and express B7H3-Fc. Using a biofilm interferometer, 100 nM of B7H3 VHH-Fc was probed using a protein A probe and allowed to bind to a starting concentration of 200 nM B7H3 antigen at a 2-fold dilution. The antibody KD value of the bound antigen was calculated. The KD value of the bound antigen was calculated, and 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) showed the highest affinity for B7H3, with a KD value of 3.24 x 10. -9 M was reached (see Figure 1), and it was therefore selected to apply to the next step in bispecific antibody construction.

[0038] 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 an existing humanized monoclonal antibody against PDL1 hIgG1, and the C-terminus of the 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 shown in Figure 2).

[0039] The DNA sequence was synthesized, subcloned into the pcDNA3.1 vector, and amplified in E. coli. The purified plasmid was transfected into HEK293 cells with PEI. The cells were then suspended and 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), snap-frozen, and stored at -80°C.

[0040] 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.

[0041] Measuring part Hereinafter, unless otherwise specified, the term "bispecific antibody" refers to a B7H3 / PDL1 bispecific antibody according to the present application, also abbreviated as "B7H3 / PDL1 biantibody" or "biantibody."

[0042] Measurement method: Example 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Five micrograms of bispecific antibody was mixed with protein reducing and non-reducing buffers and then added to PBS to make a 10-microliter solution. The mixture was heated at 100°C for 10 minutes to fully denature the protein, and 9 microliters was then added to a well of a precast polyacrylamide gel (Bio-Rad). The voltage was set to 80V for 30 minutes, followed by 120V for 60 minutes. The gel was then stained with Caumas Brilliant Blue stain for 30 minutes. The gel was destained with a destaining solution (acetic acid:ethanol:water = 1:3:6) for 15 minutes. The destaining process was repeated three times to reduce the background, and images were captured using a gel imager. The B7H3 / PDL1 bispecific antibody exhibited excellent monomer purity under non-reducing conditions. Under reducing conditions, the disulfide bonds between the light and heavy chains were opened, resulting in the appearance of two bands, one for the heavy chain and one for the light chain, and no impurity bands (Figure 3).

[0043] Example 2 Size Exclusion Chromatography (SEC-HPLC) SEC-HPLC analysis was used to evaluate the monomer purity of the bispecific antibodies. The B7H3 / PDL1 bispecific antibodies were analyzed using a Thermo MAbPac SEC-1, 5 μm, (7.8 × 300 mm) column (Product No. 088460) on a 1260 HPLC system (Agilent, Santa Clara, CA) and compared with the PDL1 monoclonal antibody, B7H3 VHH-Fc. Phosphate buffer solution (PBS) was used as the mobile phase. As shown in Figure 4, all of the detected antibodies had a very high proportion of monomer peaks, with the peak area percentage of the monomer peak reaching over 95%. This indicates that the bispecific antibodies had high monomer purity and low aggregate counts in PBS buffer.

[0044] Example 3: Detection of antibody Tm values ​​by differential scanning fluorescence (DSF) DSF was detected using a real-time PCR system (Biorad cfx96, USA). The B7H3 / PDL1 bispecific antibody, B7H3 VHH-Fc, and PDL1 monoclonal antibody were diluted to 1 mg / mL in PBS. A 5000x concentrated SYPRO Orange stock solution was diluted 1000x with ddH2O, and 20 μl of the sample was transferred to a PCR tube. To prevent bleaching, SYPRO Orange working solution was added to the reaction mixture. The PCR tube was centrifuged briefly to pool the liquid at the bottom. The qPCR system was then turned on and programmed to 25°C-95°C, with the temperature increasing by 0.3°C per second. Data were collected, and the temperature and signal values ​​were plotted to calculate the melting temperature (Tm). The data showed that the Tm of the Fc and Fab of the B7H3 / PDL1 bispecific antibody was 69°C and 90°C, respectively, demonstrating good high-temperature resistance and similar to the Tm of the B7H3 VHH-Fc and PDL1 monoclonal antibody (Figure 5).

[0045] Example 4: Validation of antibody thermostability and binding activity in combination with ELISA A 96-well plate was coated with 2 μg / ml of his-tagged PDL1 or B7H3 antigenic protein overnight at 4°C. The next day, 100 μl of casein blocking solution was added to each well for 1 hour at 37°C to block. A B7H3 / PDL1 bispecific antibody (for thermal stability testing, the bispecific antibody was incubated at 60°C in a water bath for 1 hour) or a monoclonal antibody (control monoclonal antibody: MGA271, isotype hIgG1, B7H3 VHH-Fc for B7H3 binding by ELISA; and avelumab, isotype hIgG1, PDL1 monoclonal antibody for PDL1 binding testing) was added to the wells at 3-fold dilutions. After 1 hour of incubation at 37°C, unbound antibody was washed away with 0.1% PBST. Bound antibody was detected with 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). The plate was then incubated for 5 minutes. The color development was stopped by adding 50 μl of 2 M sulfuric acid. Absorbance at OD450 nm was then measured using a SpectraMax M5e (Molecular Devices) plate reader. The antibody concentration was plotted against the OD450 reading using a four-parameter fit, and the EC 50 The EC values ​​were calculated. Figures 6a and 6b show the binding curves of the bispecific antibody targeting 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 are essentially overlapping for both PDL1 and B7H3. This indicates that the bispecific antibody can withstand the high temperature of 60°C and retains strong binding activity for its dual targets of PDL1 and B7H3. Meanwhile, the binding ability of the bispecific antibody is similar to that of the positive antibody, PDL1 monoclonal antibody, and B7H3 VHH-Fc, and the EC values ​​for binding to human PDL1 are similar to those of the bispecific antibody, PDL1 monoclonal antibody, and control avelumab. 50The EC values ​​were similar, i.e., 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively (Fig. 7a). For human B7H3, the bispecific antibodies B7H3 VHH-Fc and MGA271 also showed similar binding activities, with EC values ​​of 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively (Fig. 7b). 50 were 0.04105 nM, 0.02515 nM, and 0.05476 nM, respectively (Fig. 7b).

[0046] Example 5 Measurement of binding affinity of bispecific antibodies to antigens by BLI method The B7H3 / PDL1 bispecific antibody and PDL1 monoclonal antibody, B7H3 VHH-Fc, were diluted to 100 nM in sample buffer (PBS with 0.02% Tween 20 and 0.1% BSA). The affinity of the bispecific antibody for the specific human B7H3 and human PDL1 antigens was analyzed using an OCTET 96 column. Protein A was used as the probe to immobilize the antibody. The B7H3-his antigen and PDL1-his antigen were diluted in sample buffer to an initial concentration of 200 nM. Multiple antigen gradients were set up with two-fold dilutions to allow binding to the antibody. The rate constants and affinities were determined, and the K on and K off values ​​were calculated using software provided by the supplier to determine the antibody K values. As can be seen, the bispecific antibody exhibited high affinity for PDL1 and B7H3, with K values ​​of 5.85 x 10, respectively. -10 M and 8.11 x 10 -9 M was reached (Fig. 8).

[0047] Example 6: Detection of the ability of bispecific antibodies to block the PD1 / PDL1 pathway by flow cytometry The ability of the B7H3 / PDL1 bispecific antibody to inhibit binding of human PDL1 to human PD1-CHO cells was assessed by flow cytometry and compared with avelumab, a PDL1 monoclonal antibody. 5CHO-PD1 cells were uniformly spread on a 96-well culture plate and incubated with a mixture of 400 nM antibody (B7H3 / PDL1 bispecific antibody, avelumab, PDL1 monoclonal antibody, isotype hIgG1, B7H3 VHH-Fc) diluted 2-fold and biotinylated PDL1 antigen (50 nM) at room temperature for 30 minutes. This solution was then incubated with the cells for 45 minutes at 4°C, and unbound antigen was washed away with PBS. The cells were then fluorescently stained with PE-streptavidin. Finally, the mean fluorescence intensity (MFI) of the PE channel was read on a flow cytometer, and the antibody concentration vs. MFI was plotted. The IC of the antibody was calculated using a four-parameter fit. 50 Flow cytometry analysis revealed that the bispecific antibody had an IC of 106.4 nM. 50 PDL1 monoclonal antibody (IC 50 94.20 nM) and avelumab (IC 50 It was shown that it could block the binding of PDL1 to CHO-PD1 cells with a blocking activity similar to that of 115.0 nM (Figure 9).

[0048] Example 7 Detection of T cell activation by bispecific antibodies by mixed lymphocyte reaction (MLR) Dendritic cells (DCs) were induced by culturing monocytes isolated from peripheral blood mononuclear cells (PBMCs) in vitro for 7 days with 500 U / mL interleukin-4 (IL-4) and 250 U / mL GM-CSF using a monocyte purification kit (Miltenyi Biotec, Germany). + T cells (1×10 5 ) and the same type DC (1.25 x 10 4 ) were co-cultured in RPMI 1640 complete medium containing 10% FBS at 5% CO2 and 37°C. Groups were established with no antibody, or with different concentrations of B7H3 / PDL1 bispecific antibody, PDL1 monoclonal antibody, avelumab, B7H3 VHH-Fc, MGA271, or isotype hIgG1. Five days later, the IFN-γ concentration in the culture supernatant was analyzed using an IFN-γ ELISA kit. The MLR results indicated that the bispecific antibody significantly inhibited CD4 +The bispecific antibody was able to stimulate T cells to secrete IFN-γ, and its T cell activation ability was superior to that of the PDL1 monoclonal antibody and avelumab at both low and high concentrations. Furthermore, even in the presence of B7H3 antibody alone, the bispecific antibody partially activated T cells, although not as clearly as the PDL1 signal-blocking antibody, due to its ability to block B7H3 inhibitory signals (Figure 10).

[0049] Example 8 T cell proliferation experiment 1 μg / ml of CD3 antibody (Clone HIT3a), 1 μg / ml of CD28 antibody (Clone CD28.2), and 5 μg / ml of human PDL1 were coated onto a 96-well cell plate (Corning, USA) for 1 hour at 4°C. Control wells were coated separately with mouse IgG2a isotype control or CD3 or CD28 antibodies in the same manner. + T cells were isolated using Dynabeads TM CD4 was isolated using the CD4 Positive Isolation Kit. + T cells were cultured in pre-coated 96-well plates in RPMI1640 medium (Gibco) containing 10% FBS supplemented with different concentrations of B7H3 / PDL1 bispecific antibody, avelumab, and PDL1 monoclonal antibody at 37°C for 4 days. After 4 days, changes in T cell numbers were detected using the CCK8 kit. The data showed that freshly isolated human CD4 T cells cultured in anti-CD3 and anti-CD28 antibody-coated well plates showed a significant improvement in T cell numbers. + T cells showed increased proliferation, and the addition of PDL1 to the well plate significantly reduced their proliferation, confirming that PDL1 exerts an inhibitory signal on T cells. Avelumab, a PDL1 monoclonal antibody, and a bispecific antibody significantly promoted T cell proliferation at concentrations of 100 nM and 500 nM (Figure 11).

[0050] Example 9 Antibody-dependent cytotoxicity (ADCC) The primary antitumor effects of MGA271 and avelumab are due to the ADCC function associated with the IgG1 subtype of the antibody, and the bispecific antibody also contains an IgG1 functional domain. The ADCC of the antibody was measured using an LDH cytotoxicity detection kit. Human PBMCs were purified from leukocyte packages using Ficoll gradient centrifugation, and NK cells were isolated from the human PBMCs using negatively selective magnetic beads (Miltenyi Biotec, Auburn, CA). NK cells (3 × 10 6 ) and MDA-MB-231 and ES-2 cells (3 × 10 5 ) were co-cultured with or without the addition of different concentrations of the bispecific antibody avelumab at the start of the assay. After 18 hours, lactate dehydrogenase (LDH) secretion in the culture supernatant was analyzed by ELISA. + PDL1 + When MDA-MB-231 cells were used as target cells, both the bispecific antibody and avelumab demonstrated ADCC activity, but the activity of the bispecific antibody was stronger at lower concentrations. + When ES-2 cells were used as target cells, the activity of the bispecific antibody was comparable to that of avelumab at a dose of 100 nM, and it was observed that it exhibited strong ADCC activity even at low concentrations (Figure 12).

[0051] Example 10: In vivo antitumor activity test of bispecific antibodies Human PBMCs (6.67 × 10 6 ) was injected into 41 NPSG mice via the tail vein the day before A375 tumor cell inoculation, and 5 × 10 6A375 tumor cells were subcutaneously injected into the mice. Five days after tumor inoculation, subcutaneous tumor formation was observed. Each group consisted of 10 mice. The groups were divided into an isotype control group, a monoclonal antibody combination group (pembrolizumab + MGA271, avelumab + MGA271), and a bispecific antibody group. Drug treatment was administered on day 5 after successful model establishment and 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 after the start of the experiment. Efficacy and safety were evaluated based on tumor growth inhibition values, which were calculated based on changes in relative tumor volume (TGIRTV) and animal weight. The bispecific antibody maintained significantly stronger activity throughout the study than the PD1 monoclonal antibody + MGA271 combination group. As the study time increased, the bispecific antibody gradually demonstrated superiority over the avelumab + MGA271 combination group, with TGI at the end of the study of 39.29% and 26.45%, respectively (Figure 13). Furthermore, there was no significant weight loss during the study, demonstrating the safety of this bispecific antibody treatment (Figure 14).

[0052] The test results of the above test examples demonstrate that the bispecific antibody constructed in this application can bind to both B7H3 and PDL1, can target tumor cells while reversing the inhibition of T cells by PDL1, and exhibits superior antitumor activity compared to the combined use of monoclonal antibodies.

Claims

1. A B7H3 / PDL1 bispecific antibody that targets and binds to B7H3 and PDL1, a monoclonal antibody unit that targets PDL1 and includes two heavy chains and two light chains; a nanobody unit that targets B7H3 and contains two identical nanobodies; Including, the C-termini of the two Nanobodies are linked via linker peptides to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit, respectively; B7H3 / PDL1 bispecific antibody.

2. The linker peptide is a polypeptide segment containing glycine and serine and having a certain flexibility and protease resistance, and preferably, the amino acid sequence of the linker peptide is SEQ ID NO:

11. The bispecific antibody of claim 1.

3. the light chain variable region of the monoclonal antibody unit comprises CDR1 whose amino acid sequence is SEQ ID NO: 1, CDR2 whose amino acid sequence is SEQ ID NO: 2, and CDR3 whose amino acid sequence is SEQ ID NO: 3; the heavy chain variable region of the monoclonal antibody unit comprises CDR1 whose amino acid sequence is SEQ ID NO: 5, CDR2 whose amino acid sequence is SEQ ID NO: 6, and CDR3 whose amino acid sequence is SEQ ID NO: 7; and the Nanobody comprises CDR1 whose amino acid sequence is SEQ ID NO: 12, CDR2 whose amino acid sequence is SEQ ID NO: 13, and CDR3 whose amino acid sequence is SEQ ID NO: 14; Preferably, the light chain variable region of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 4, the heavy chain variable region of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 8, and said Nanobody comprises the amino acid sequence of SEQ ID NO: 15; More preferably, the light chain of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 9, the heavy chain of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 10, and said Nanobody comprises the amino acid sequence of SEQ ID NO: 15; More preferably, the full-length amino acid sequence of the light chain of the monoclonal antibody unit is SEQ ID NO: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is SEQ ID NO: 10, and the amino acid sequence of the Nanobody is SEQ ID NO:

15. The bispecific antibody of claim 1.

4. The bispecific antibody has a heavy chain amino acid sequence of SEQ ID NO: 16 and a light chain amino acid sequence of SEQ ID NO:

17. The bispecific antibody of claim 1.

5. A polynucleotide encoding the B7H3 / PDL1 bispecific antibody of any one of claims 1 to 4.

6. An expression vector comprising the polynucleotide of claim 5.

7. A pharmaceutical composition comprising a therapeutically effective amount of the B7H3 / PDL1 bispecific antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

8. 10. Use of a B7H3 / PDL1 bispecific antibody according to any one of claims 1 to 4 in the manufacture of a medicament for the prevention, diagnosis, treatment or adjuvant therapy of a tumor.

9. The medicament inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, and mediating ADCC effects, or The medicament inhibits tumors by binding to PD-L1, inhibiting the binding of PD-1 to PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes; Preferably, the medicament inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, and binding to PD-L1, inhibiting the binding of PD-1 to PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes.

9. The use according to claim 8.

10. The tumor is one or more selected from the group consisting of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, pancreatic cancer and Ewing's sarcoma, preferably, the tumor is breast cancer, ovarian cancer or melanoma; 9. The use according to claim 8.

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