B7H3 / PDL1 bispecific antibody, its pharmaceutical composition and use, and polynucleotide encoding the B7H3 / PDL1 bispecific antibody, polynucleotide-containing expression vector.

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] This invention belongs to the field of biopharmaceuticals, and more specifically, relates to a B7H3 / PDL1 bispecific antibody, its pharmaceutical composition, and its use. [Background technology]

[0002] The B7-CD28 family plays a crucial role in T lymphocyte-mediated immune responses as a co-stimulatory 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 Chapoval AI et al. have shown that it is associated with CD4 + and CD8 + It was first identified that B7H3 has a co-stimulatory 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 progresses, its inhibitory function has gradually been discovered, for example, CD4 + T cells and CD8 + It can inhibit the proliferation of T cells. Furthermore, studies have shown that abnormal expression of B7H3 is associated with the development, onset, and metastasis of various cancers, and there is much evidence suggesting that high expression of B7H3 is correlated with a poor prognosis in various malignancies.

[0003] B7H3 was highly expressed in all cancer types tested, with limited heterogeneity, and was hardly expressed in normal tissues. This suggests that B7H3 is a tumor antigen (TA), offering the potential for targeted therapy against tumor cells that highly express B7H3. Currently, therapeutic strategies targeting B7H3 include blocking monoclonal antibodies, radioimmunotherapy, antibody-drug coupling (ADCs), cytotoxicity-mediated monoclonal antibodies, and bispecific antibodies (BsAbs). Enobrituzumab (MGA271), a humanized monoclonal antibody targeting B7H3 developed by MacroGenics, acts on various malignancies such as melanoma, osteosarcoma, and Ewing's sarcoma based on antibody-dependent cell-mediated cytotoxicity (ADCC). Enobrituzumab has shown potent antitumor activity in various xenograft tumor models and has not shown significant toxicity in safety evaluations using primates. Enobrituzumab is currently in Phase II clinical trials and has shown promising potential as a treatment.

[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 inhibitory antibodies theoretically exhibit 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. A key reason for this is that the effect of ADCC is highly dependent on the expression level of the antigen, whereas PDL1 is not considered a typical tumor antigen and is highly heterogeneous 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 enobrituzumab and pembrolizumab, a monoclonal antibody targeting PD1, blocks immunosuppression in the PD1 / PDL1 pathway while simultaneously providing tumor-killing effects through enobrituzumab. This combination therapy is currently in a Phase II clinical trial (NCT04129320). The results showed that the incidence of side effects with the combination therapy was comparable to that of monotherapy, and the objective remission rate (ORR) was superior to that of PD1 monoclonal antibody therapy. This demonstrated the feasibility of simultaneously blocking both the B7H3 pathway and the PD1 / PDL1 pathway.

[0006] Bispecific antibodies (BsAbs) are genetically engineered antibodies that can simultaneously target two different antigenic 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 could theoretically act better on tumor cells that highly express B7H3. Currently, only five bispecific antibody drugs are approved for commercial sale, and the main reasons limiting their application are stability and activity issues. Therefore, obtaining bispecific antibodies that are both stable and biologically active remains a focus of industry efforts. [Overview of the project]

[0007] One technical objective of the present invention is to provide a method and use for producing a B7H3 / PDL1 bispecific antibody.

[0008] In one embodiment, the present invention relates to a B7H3 / PDL1 bispecific antibody that targets and binds to B7H3 and PDL1, A monoclonal antibody unit that targets PDL1 and contains two heavy chains and two light chains, Targeting B7H3, a nanobody unit containing two identical nanobodies (VHH), Includes, The C-terminuses of the two nanobodies are each linked to the C-terminuses of the two heavy chain Fc fragments of the monoclonal antibody unit via linker peptides. We provide a bispecific antibody for B7H3 / PDL1.

[0009] In the present invention, the linker peptide refers to a polypeptide segment containing glycine and serine, and having a certain degree of elasticity and protease resistance.

[0010] In one embodiment, the amino acid sequence of the linker peptide is sequence number 11.

[0011] In one embodiment, the light chain variable region of the monoclonal antibody unit includes 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 includes 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 includes 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.

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

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

[0014] In one embodiment, the nanobody includes 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 the 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 said 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 said B7H3 / PDL1 bispecific antibody and a pharmaceutically acceptable carrier.

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

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

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

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

[0026] In specific embodiments, the tumor may be one or more selected from the group consisting of lung cancer, stomach 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, hematological cancer, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma. In particular, the tumor may be breast cancer, ovarian cancer, or melanoma. [Effects of the Invention]

[0027] The bispecific antibody constructed in this invention can bind to both B7H3 and PDL1, target tumor cells while simultaneously resolving PDL1-mediated T cell inhibition, and exhibits superior antitumor activity compared to monoclonal antibody combination therapy.

[0028] Furthermore, the bispecific antibodies described in this invention have the advantages 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 mainly reflected in studies of monomer purity and thermal stability. A single affinity purification can achieve a monomer content of 95% for the bispecific antibody, which is superior to the purity achieved after multiple double purifications in the industry. Structural and activity analysis of the antibody after heat treatment demonstrates that it maintains good molecular conformation and intact biological activity even under harsh conditions, making it suitable for industrial production, packaging, and storage. In short, 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 (combination of molecular and cellular levels) compared to avelumab and MGA271. In vivo data also shows that B7H3 + In A375 tumor cells, the antitumor activity of the bispecific antibody was superior to that of the group using the B7H3 monoclonal antibody. Therefore, the bispecific antibody developed in this invention is expected to have a wide range of applications in the future due to its excellent availability and activity.

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

[0031] [Figure 1] This is an affinity screening graph for B7H3 targeting the anti-B7H3 VHH humanized antibody manufactured in this application. [Figure 2] This is a schematic structural diagram of the B7H3 / PDL1 bispecific antibody manufactured in this application. [Figure 3] This is an SDS-polyacrylamide gel electrophoresis map of the B7H3 / PDL1 bispecific antibody according to the present invention. [Figure 4] This is the result of SEC-HPLC purity measurement of the B7H3 / PDL1 bispecific antibody according to the present invention. [Figure 5]This is the Tm value measurement result (DSF) of the B7H3 / PDL1 bispecific antibody according to the present invention. [Figure 6] This figure shows the binding ELISA of the B7H3 / PDL1 bispecific antibody according to the present invention before and after heat treatment at 60°C, where a shows the binding ELISA with PDL1 and b shows the binding ELISA with B7H3. [Figure 7] This figure shows the conjugation ELISA of the B7H3 / PDL1 bispecific antibody according to the present invention, where a shows the conjugation ELISA with PDL1 and b shows the conjugation ELISA with B7H3. [Figure 8] These are BLI affinity assay plots of the B7H3 / PDL1 bispecific antibody according to the present invention 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] This is the PDL1 / CHO-PD1 blockade curve of the B7H3 / PDL1 bispecific antibody according to the present invention. [Figure 10] This graph shows the induction of IFN-γ secretion from T cells by the B7H3 / PDL1 bispecific antibody according to this invention. [Figure 11] This graph shows the promotion of T cell proliferation by the B7H3 / PDL1 bispecific antibody according to this invention. [Figure 12] This figure shows the ADCC effect of the B7H3 / PDL1 bispecific antibody according to the present invention on cancer cells. Figure a shows cytotoxicity against MDA-MB-231 in human breast cancer cells, and figure b shows cytotoxicity against ES-2 in human ovarian clear cell cancer cells. [Figure 13] This graph shows the in vivo tumor-suppressing effect of the B7H3 / PDL1 bispecific antibody described in this application. [Figure 14] This graph shows the effect of the B7H3 / PDL1 bispecific antibody developed in this application on the body weight of mice. [Modes for carrying out the invention]

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

[0033] The materials, reagents, apparatus, and methods used in the following embodiments are, unless otherwise specified, conventional materials, reagents, apparatus, and methods in the art and are commercially available.

[0034] The present invention will be described below with reference to specific embodiments so that those skilled in the art can better understand the technical content of this application. However, 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 human antibody gene Germline as a template, camel-derived anti-B7H3 nanobodies were humanized using the Framework shuffling method, and libraries of corresponding VHH conformational rearrangements were synthesized in vitro using overlap PCR. Subsequently, clones of the VHH phage libraries were constructed, and screening and characterization were performed.

[0036] More precisely, camel-derived anti-B7H3 nanobodies were humanized using a one-step strategy. Approximately 1000 clones were screened from this sublibrary, and the selected positive clones were screened for thermal stability at the phage level by ELISA. In the ELISA, 5 μg / ml of huB7H3 antigen was encapsulated in a highly adsorbent 96-well ELISA plate, and the supernatant of the screened phages was reacted with phages amplified overnight. Clones exhibiting higher OD450 measurements were selected.

[0037] The sequence of the above phage clone was determined, the base sequence of the B7H3 VHH gene was obtained, and its C-terminus was fused with the human Fc protein gene to construct and express B7H3-Fc. Using a biofilm interferometer, 100 nM B7H3 VHH-Fc was grasped with a protein A probe and conjugated to a 200 nM B7H3 antigen at a 2-fold dilution. The antibody KD value of the conjugated antigen was calculated. As a result of calculating the KD value of the conjugated antigen, 75-16 (amino acid sequence is SEQ ID NO: 15, CDR1 sequence is SEQ ID NO: 12, CDR2 sequence is SEQ ID NO: 13, CDR3 sequence is SEQ ID NO: 14) showed the highest affinity for B7H3, with a KD value of 3.24 × 10⁻¹⁶. -9 It reached M (see Figure 1). Therefore, it was selected to be applied to the next step in the construction of a bispecific antibody.

[0038] 2. Construction and expression of a B7H3 / PDL1 bispecific antibody The amino acid sequences of the PDL1 monoclonal antibody light chain (amino acid sequence is SEQ ID NO: 9, variable region sequence is SEQ ID NO: 4, CDR1 sequence is SEQ ID NO: 1, CDR2 sequence is SEQ ID NO: 2, CDR3 sequence is SEQ ID NO: 3) and heavy chain (amino acid sequence is SEQ ID NO: 10, variable region sequence is SEQ ID NO: 8, CDR1 sequence is SEQ ID NO: 5, CDR2 sequence is SEQ ID NO: 6, CDR3 sequence is SEQ ID NO: 7) are derived from an existing humanized monoclonal antibody against PDL1 hIgG1, with the C-terminus of anti-B7H3 VHH (75-16 above) being bound to the C-terminus of the Fc fragment via a linker peptide (SEQ ID NO: 11) (structure shown in Figure 2).

[0039] DNA sequences were synthesized, subcloned into a pcDNA3.1 vector, and amplified in E. coli. The purified plasmid was transfected into HEK293 cells using PEI. The cells were then suspended in OPM-CD05 expression medium and cultured. 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 with phosphate-buffered water (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 In the following, unless otherwise specified, the term "bispecific antibody" refers to the B7H3 / PDL1 bispecific antibody as defined in this application, and is also abbreviated as "B7H3 / PDL1 bispecific antibody" or "bispecific antibody."

[0042] Measurement method: Example 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 5 μg of bispecific antibody was mixed with protein reducing and non-reducing buffers, and a 10 μl system was prepared in PBS. After heating at 100°C for 10 minutes to sufficiently denature the protein, 9 μl was taken and added to the wells of a precast polyacrylamide gel (Bio-Rad). The voltage was first set to 80 V for 30 minutes, then to 120 V for 60 minutes for separation. The gel was then stained with Caumas Brilliant Blue for 30 minutes. The gel was destained with a destaining solution (acetic acid:ethanol:water = 1:3:6) for 15 minutes, and the destaining process was repeated three times to thin the background. Images were then acquired using a gel imager. As a result, the B7H3 / PDL1 bispecific antibody showed good monomer purity under non-reducing conditions, and under reducing conditions, two bands appeared, one for the heavy chain and one for the light chain, due to the opening of the disulfide bonds between the light and heavy chains, with no impurity bands observed (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 antibody was analyzed using a 1260 HPLC system (Agilent, Santa Clara, CA) with Thermo MAbPac SEC-1, 5 μm, (7.8 × 300 mm) P / N088460, and compared with the PDL1 monoclonal antibody and B7H3 VHH-Fc. Phosphate buffer (PBS) was used as the mobile phase. As shown in Figure 4, all detected antibodies had a very high proportion of monomer peaks, and the proportion of monomer peak area reached over 95%, indicating that the bispecific antibody had high monomer purity and a low number of aggregates under PBS buffer conditions.

[0044] Example 3 Detection of antibody Tm value by differential scanning fluorescence (DSF) 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. A 5000-fold concentrated stock solution of SYPRO Orange was diluted 1000-fold with ddH2O, and 20 μl of sample was collected in a PCR tube. To prevent whitening, a working solution of SYPRO Orange was added to the reaction mixture, and the liquid was pooled at the bottom of the PCR tube by instantaneous centrifugation. The qPCR instrument was powered on, the program was set to 25°C-95°C, and the temperature was increased by 0.3°C per second. Data was collected, the temperature and signal values ​​were graphed, and the melting temperature (Tm) was calculated. According to the data, the Tm of Fc and Fab of the B7H3 / PDL1 bispecific antibody was 69°C and 90°C, respectively, demonstrating good high-temperature tolerance, similar to the Tm of B7H3 VHH-Fc and PDL1 monoclonal antibody (Figure 5).

[0045] Example 4 Verification of the thermal stability and binding activity of antibodies combined with ELISA. 96-well plates were coated overnight at 4°C with 2 μg / ml his-tagged PDL1 or B7H3 antigenic protein. The following day, 100 μl of casein blocking solution was added to each well and blocked at 37°C for 1 hour. B7H3 / PDL1 bispecific antibody (if testing thermal stability, the bispecific antibody was treated in a water bath at 60°C for 1 hour) and monoclonal antibody (for the control monoclonal antibody used, MGA271, Isotype hIgG1, B7H3 VHH-Fc was used to examine B7H3 binding by the bispecific antibody via ELISA, and PDL1 monoclonal antibody avelumab, Isotype hIgG1 was used to examine PDL1 binding) were added to the wells at a 3-fold dilution. After incubation at 37°C for 1 hour, unbound antibodies were washed away with 0.1% PBST, and bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG(H+L) antibody (Jackson ImmunoResearch, USA). The antibodies were then colored with 50 μl of 3,3',5,5'-tetramethylbenzidine substrate (TMB), and the plate was allowed to stand for 5 minutes. 50 μl of 2 M sulfuric acid was added to stop the color development, and then the absorbance at OD450 nm was measured using a SpectraMax M5e (Molecular Devices) plate reader. The antibody concentration was plotted against the OD450 reading using a 4-parameter fit, and the EC (Emission Control Value) was measured. 50 The following was 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 figure, the binding curves of the bispecific antibody before and after heating basically overlap for both PDL1 and B7H3. This indicates that the bispecific antibody withstands high temperatures of 60°C and retains strong binding activity to the dual targets of PDL1 and B7H3. On the other hand, the binding ability of the bispecific antibody is similar to that of the positive antibody, the PDL1 monoclonal antibody, and B7H3 VHH-Fc, and the EC of the bispecific antibody, PDL1 monoclonal antibody, and the control avelumab for binding to human PDL1 50were at the same level respectively, that is, 0.3056 nM, 0.4407 nM and 0.1563 nM (Figure 7a); against human B7H3, the bispecific antibody, B7H3 VHH-Fc and MGA271 also showed similar binding activities, EC 50 were 0.04105 nM, 0.02515 nM and 0.05476 nM respectively (Figure 7b).

[0046] Example 5 Determination of binding affinity of bispecific antibody to antigen by BLI method The B7H3 / PDL1 bispecific antibody, PDL1 monoclonal antibody, and B7H3 VHH-Fc were diluted to 100 nM with sample buffer (0.02% Tween 20 and 0.1% BSA in PBS), and the affinity of the bispecific antibody for specific human B7H3 and human PDL1 antigens was analyzed using OCTET 96. Herein, the probe used is protein A, which is for immobilizing the antibody. The B7H3-his antigen and PDL1-his antigen were diluted with sample buffer to an initial concentration of 200 nM, multiple antigen gradients were set by 2-fold dilution to allow binding to the antibody, the rate constant and affinity were determined, the Kon value and Koff value were calculated using software provided by the supplier, and the KD value of the antibody was obtained. As can be seen from the figure, the bispecific antibody showed high affinity for PDL1 and B7H3, with KD values of 5.85×10 -10 M and 8.11×10 -9 M (Figure 8).

[0047] Example 6 Detection of the ability of bispecific antibody to block PD1 / PDL1 pathway by flow cytometry The ability of B7H3 / PDL1 bispecific antibody to inhibit the binding of human PDL1 to human PD1-CHO cells was evaluated by flow cytometry and compared with avelumab, a PDL1 monoclonal antibody. 2×10 5CHO-PD1 cells were uniformly spread in a 96-well culture plate and incubated at room temperature for 30 minutes with a mixture of 400 nM to 2-fold diluted antibody (B7H3 / PDL1 bispecific antibody, avelumab, PDL1 monoclonal antibody, Isotype hIgG1, B7H3 VHH-Fc) and biotinylated PDL1 antigen (50 nM). This solution was then incubated with the cells at 4°C for 45 minutes, and any unbound antigen was washed away with PBS. Subsequently, the cells were fluorescently stained with PE-streptavidin. Finally, the mean fluorescence intensity (MFI) of the PE channel was read using a flow cytometer, and the antibody concentration and MFI were plotted. The IC of the antibody was then measured using a 4-parameter fit. 50 The following was calculated. Flow cytometry analysis revealed that the bispecific antibody had an IC50 of 106.4 nM. 50 So, PDL1 monoclonal antibody (IC 50 (94.20 nM) and avelumab (IC) 50 It was shown that the binding of PDL1 to CHO-PD1 cells could be blocked with a blocking activity of approximately 115.0 nM (Figure 9).

[0048] Example 7 Detection of T cell activation by bispecific antibodies using mixed lymphocyte reaction (MLR) Dendritic cells (DCs) were induced by culturing monocytes isolated from peripheral blood mononuclear cells (PBMCs) in vitro for 7 days using a monocyte 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 the same type DC (1.25 × 10 4 The cells were co-cultured in RPMI 1640 complete medium containing 10% FBS under constant temperature conditions of 5% CO2 and 37°C. 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 added. After 5 days, the IFN-γ concentration in the culture supernatant was analyzed using an IFN-γ ELISA kit. From the MLR results, the bispecific antibody was found to be 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 PDL1 monoclonal antibody and avelumab at both low and high concentrations. Furthermore, even when only the B7H3 antibody was present, although not as clearly as with the PDL1 signal-blocking antibody, the bispecific antibody also blocked the B7H3 inhibitory signal, thereby partially activating T cells (Figure 10).

[0049] 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 in 96-well cell plates (Corning, USA) at 4°C for 1 hour. Control wells were individually coated in the same manner with mouse IgG2a isotype control or CD3 and CD28 antibodies. CD4 + T cells are Dynabeads TM CD4 was isolated using the CD4 Positive Isolation Kit. + T cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS, supplemented with different concentrations of B7H3 / PDL1 bispecific antibody, avelumab, and PDL1 monoclonal antibody, in pre-coated 96-well plates at 37°C for 4 days. After 4 days, changes in T cell count were detected using the CCK8 kit. The data showed that fresh isolated human CD4 cultured in well plates coated with anti-CD3 and anti-CD28 antibodies was also observed. + T cells showed increased proliferation, and a significant decrease in proliferative capacity was observed when PDL1 was added to the well plate, confirming that PDL1 provides 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).

[0050] Example 9 Antibody-dependent cytotoxicity (ADCC) The primary antitumor effects of MGA271 and avelumab are attributed to ADCC function related to the IgG1 subtype of the antibody, and the bispecific antibody also possesses an IgG1 functional region. The ADCC of the antibody was measured using an LDH cytotoxicity detection kit. Human PBMCs were purified from leukocyte packages using Ficol gradient centrifugation, and NK cells were isolated from human PBMCs using negative-selective magnetic beads (Miltenyi Biotec, Auburn, CA). NK cells (3 × 10⁶) 6 ) and MDA-MB-231, ES-2 cells (3 × 10 5 The cells were co-cultured with and without the addition of bispecific antibodies and avelumab at different concentrations at the start of measurement. After 18 hours, lactate dehydrogenase (LDH) secretion in the culture supernatant was analyzed by ELISA. B7H3 + PDL1 + When MDA-MB-231 was used as the target cell, both the bispecific antibody and avelumab showed ADCC activity, but the activity of the bispecific antibody was stronger at lower concentrations. PDL1 + 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 strong ADCC activity was observed even at low concentrations (Figure 12).

[0051] Example 10: In vivo antitumor activity test of bispecific antibodies Human PBMC (6.67 × 10 6 ) was injected into the tail vein of 41 NPSG mice the day before inoculation with A375 tumor cells, and the following day 5 × 10 6A375 tumor cells were injected subcutaneously. Subcutaneous tumor production was observed 5 days after tumor inoculation. Each group consisted of 10 animals, divided into an isotype control group, a monoclonal antibody combination group (pembrolizumab + MGA271, avelumab + MGA271), and a bispecific antibody group. The drugs were administered 5 days after successful model construction and twice weekly until the end of the experiment. Tumor volume and animal body 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 ​​based on changes in relative tumor volume (TGIRTV) and animal body weight. The bispecific antibodies maintained significantly stronger activity from start to finish than the PD1 monoclonal antibody + MGA271 combination group. The bispecific antibody gradually showed superiority over the avelumab + MGA271 combination group as the experiment duration increased, with TGI rates of 39.29% and 26.45%, respectively, at the end of the experiment (Figure 13). Furthermore, no severe weight loss occurred during the experiment, demonstrating the safety of this bispecific antibody treatment (Figure 14).

[0052] The test results from the above-mentioned test examples show that the bispecific antibody constructed in this invention can bind to both B7H3 and PDL1, can target tumor cells while reversing PDL1-mediated T cell inhibition, and exhibits superior antitumor activity compared to combination therapy with monoclonal antibodies.

Claims

1. A B7H3 / PDL1 bispecific antibody that targets and binds to B7H3 and PDL1, comprising: a monoclonal antibody unit targeting PDL1 and containing two heavy chains and two light chains; and a nanobody unit targeting B7H3 and containing two identical nanobodies, wherein the N-terminuses of the two nanobodies are linked via linker peptides to the C-terminuses of the Fc fragments of the two heavy chains of the monoclonal antibody unit. The B7H3 / PDL1 bispecific antibody is characterized in that the light chain variable region of the monoclonal antibody unit includes 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 includes 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 includes 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.

2. The bispecific antibody according to claim 1, wherein the amino acid sequence of the linker peptide is SEQ ID NO:

11.

3. The bispecific antibody according to claim 1, wherein the light chain variable region of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 4, the heavy chain variable region of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 8, and the nanobody comprises the amino acid sequence of SEQ ID NO:

15.

4. The bispecific antibody according to claim 3, wherein the light chain of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 9, the heavy chain of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 10, and the nanobody comprises the amino acid sequence of SEQ ID NO:

15.

5. The bispecific antibody according to claim 4, wherein 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.

6. The aforementioned 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 according to claim 1.

7. A polynucleotide encoding a B7H3 / PDL1 bispecific antibody according to any one of claims 1 to 6.

8. An expression vector comprising the polynucleotide described in claim 7.

9. A pharmaceutical composition comprising a therapeutically effective amount of a B7H3 / PDL1 bispecific antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

10. Use of the B7H3 / PDL1 bispecific antibody according to any one of claims 1 to 6 in the manufacture of a pharmaceutical product for the prevention, diagnosis, treatment or adjunctive therapy of tumors.

11. The use of the pharmacopoeia according to claim 10, wherein the pharmacopoeia inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, and mediating the ADCC effect, or the pharmacopoeia inhibits tumors by binding to PD-L1, inhibiting PD-1 from binding to PDL-1, activating T lymphocytes, and increasing the expression of IL-2 and IFN-γ in T lymphocytes.

12. The use of the pharmaceutical agent according to claim 10, wherein the pharmaceutical agent 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.

13. The use according to claim 10, wherein the tumor is one or more selected from the group consisting of lung cancer, stomach 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, hematological cancer, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma.

14. The use according to claim 10, wherein the tumor is breast cancer, ovarian cancer, or melanoma.

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