WO2024077547A1 - 一种b7h3/pdl1双特异性抗体及其药物组合物及应用 - Google Patents

一种b7h3/pdl1双特异性抗体及其药物组合物及应用 Download PDF

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WO2024077547A1
WO2024077547A1 PCT/CN2022/125089 CN2022125089W WO2024077547A1 WO 2024077547 A1 WO2024077547 A1 WO 2024077547A1 CN 2022125089 W CN2022125089 W CN 2022125089W WO 2024077547 A1 WO2024077547 A1 WO 2024077547A1
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seq
amino acid
acid sequence
pdl1
bispecific antibody
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French (fr)
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陈艺丽
王春河
李欢欢
刘国键
谭杰
唐磊
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达石药业(广东)有限公司
上海迈石生物技术有限公司
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Priority to PCT/CN2022/125089 priority Critical patent/WO2024077547A1/zh
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Definitions

  • the present invention belongs to the field of biopharmaceuticals, and specifically relates to a B7H3/PDL1 bispecific antibody and a pharmaceutical composition and application thereof.
  • B7H3 also known as CD276
  • B7H3 signaling induces cellular immunity and selectively enhances the production of interferon- ⁇ (IFN- ⁇ ) under T cell receptor signaling.
  • IFN- ⁇ interferon- ⁇
  • 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), providing the possibility of targeted therapy for tumor cells with high B7H3 expression.
  • B7H3 targeted therapy strategies mainly include blocking monoclonal antibodies, radioimmunotherapy, antibody-drug conjugates (ADCs), monoclonal antibodies that mediate cytotoxicity, and bispecific antibodies (BsAbs).
  • Enoblituzumab (MGA271), a humanized monoclonal antibody targeting B7H3 developed by MacroGenics, acts on a variety of malignancies such as melanoma, osteosarcoma, and Ewing's sarcoma based on antibody-dependent cell-mediated cytotoxicity (ADCC).
  • Enoblituzumab has been shown to have strong anti-tumor activity in a variety of xenograft tumor models and has no obvious toxicity in the safety evaluation of primates. Enoblituzumab is currently in Phase II clinical research and shows good therapeutic potential.
  • Inhibitors targeting the immune checkpoint PD1/PDL1 are undoubtedly the focus of tumor immunotherapy.
  • PDL1 is expressed on the surface of tumor cells, and cytotoxic PDL1 inhibitory antibodies theoretically have better anti-tumor effects.
  • Avelumab among the PDL1 monoclonal antibodies on the market can mediate ADCC effects against tumors and show comparable safety to other PDL1 antibodies.
  • One important reason is that the effect of ADCC mainly depends on the abundance of antigen expression, while PDL1 cannot be regarded as a typical tumor antigen, and it has great heterogeneity in tumor cells.
  • PD1/PDL1 pathway blocking antibodies are often used in combination with cytotoxic antibodies to enhance the effect of immunotherapy.
  • Enoblituzumab and Pembrolizumab a monoclonal antibody targeting PD1 are used in combination. On the one hand, they can block the immunosuppression of the PD1/PDL1 pathway, and on the other hand, they can exert a tumor-killing effect through Enoblituzumab.
  • This combination therapy has entered clinical phase II (NCT04129320). Data show that the incidence of adverse reactions in combination therapy is similar to that of monotherapy, and the objective response rate (ORR) of combination therapy is better than that of PD1 monoclonal antibody therapy. This proves the feasibility of simultaneously blocking the B7H3 pathway and the PD1/PDL1 pathway.
  • Bispecific antibodies are genetically engineered antibodies that can simultaneously target two different antigenic epitopes.
  • Bispecific antibodies targeting B7H3 have been developed, most of which rely on T cell redirection strategies, such as CD3/B7H3 bispecific antibodies.
  • cytotoxicity-based bispecific antibodies may better act on B7H3-high-expressing tumor cells because B7H3 can be used as a tumor antigen with high abundance and low heterogeneity.
  • bispecific antibody drugs have been approved for marketing, and the main reason limiting their application is stability and activity issues. Therefore, obtaining bispecific antibodies with good stability and excellent biological activity is still the direction of the industry's efforts.
  • a technical objective of the present invention is to provide a method for preparing a B7H3/PDL1 bispecific antibody and its application.
  • the present invention provides a B7H3/PDL1 bispecific antibody, which targets and binds to B7H3 and PDL1, and the bispecific antibody comprises:
  • a monoclonal antibody unit which is directed against PDL1 and includes 2 heavy chains and 2 light chains;
  • a Nanobody unit which is directed against B7H3 and comprises 2 identical Nanobodies (VHH),
  • the C-termini of the two nanobodies are respectively connected to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit through connecting peptides.
  • the connecting peptide refers to a polypeptide comprising glycine and serine and having certain elasticity and protease resistance.
  • amino acid sequence of the connecting peptide is SEQ ID No.:11.
  • the light chain variable region of the monoclonal antibody unit includes CDR1 with an amino acid sequence of SEQ ID NO.: 1, CDR2 with an amino acid sequence of SEQ ID NO.: 2, and CDR3 with an amino acid sequence of SEQ ID NO.: 3
  • the heavy chain variable region of the monoclonal antibody unit includes CDR1 with an amino acid sequence of SEQ ID NO.: 5, CDR2 with an amino acid sequence of SEQ ID NO.: 6, and CDR3 with an amino acid sequence of SEQ ID NO.: 7
  • the nanoantibody includes CDR1 with an amino acid sequence of SEQ ID NO.: 12, CDR2 with an amino acid sequence of SEQ ID NO.: 13, and CDR3 with an amino acid sequence of SEQ ID NO.: 14.
  • the light chain variable region of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 4, and the heavy chain variable region of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 8.
  • the light chain of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 9
  • the heavy chain of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 10.
  • the nanoantibody comprises an amino acid sequence such as SEQ ID NO.: 15.
  • amino acid sequence of the light chain variable region of the monoclonal antibody unit is as shown in SEQ ID NO.: 4, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody unit is as shown in SEQ ID NO.: 8.
  • the full-length amino acid sequence of the light chain of the monoclonal antibody unit is as shown in SEQ ID NO.: 9, and the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is as shown in SEQ ID NO.: 10.
  • amino acid sequence of the nanoantibody is as shown in SEQ ID NO.: 15.
  • the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO.: 16, and the light chain amino acid sequence is as shown in SEQ ID NO.: 17.
  • the present invention also provides a polynucleotide encoding the B7H3/PDL1 bispecific antibody.
  • the present invention also provides an expression vector comprising a polynucleotide encoding the B7H3/PDL1 bispecific antibody.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned B7H3/PDL1 bispecific antibody and a pharmaceutically acceptable carrier.
  • the present invention provides a use of the B7H3/PDL1 bispecific antibody in the preparation of a drug for preventing, diagnosing, treating or assisting in the treatment of tumors.
  • the drug inhibits tumors by binding to B7H3 and blocking the B7H3 signaling pathway, thereby mediating the ADCC effect.
  • 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- ⁇ in T lymphocytes.
  • the drug inhibits tumors by binding to B7H3, blocking the B7H3 signaling pathway, 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- ⁇ in T lymphocytes.
  • the tumor can be selected from one or more 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.
  • the tumor is breast cancer, ovarian cancer or melanoma.
  • the bispecific antibody constructed in the present application can bind to B7H3 and PDL1 simultaneously, and can relieve the inhibition of PDL1 on T cells while targeting tumor cells, and also shows anti-tumor activity that is superior to the combination of monoclonal antibodies.
  • the bispecific antibodies of the present application have the advantages of good compliance and controllable quality.
  • the stability characterization of the bispecific antibody in this application is mainly reflected in the study of monomer purity and thermal stability. After a single affinity purification, the monomer content of the bispecific antibody can reach 95%, which is even better than the purity after multiple secondary purifications in the industry.
  • the results of the structural and activity analysis of the antibody after heat treatment prove that the antibody can still maintain a good molecular conformation and complete biological activity under harsh conditions, which is conducive to the industrial production and packaging and storage of antibodies.
  • the present application constructs a B7H3/PDL1 bispecific antibody in the form of IgG-VHH 2 ; it shows good molecular stability and has significantly better in vitro activity (binding molecular level and cellular level) than Avelumab and MGA271.
  • the bispecific antibody of the present application has broad application prospects due to its excellent developability and activity.
  • the method used in the present invention is a new cutting-edge method for treating tumors.
  • Tumor immunotherapy is expected to become a revolution in the field of tumor treatment after surgery, chemotherapy, radiotherapy, and targeted therapy.
  • the B7H3/PDL1 bispecific antibody in the present invention is expected to become a new type of anti-tumor drug.
  • Figure 1 Affinity screening diagram of the anti-B7H3 VHH humanized antibody prepared in this application and B7H3.
  • FIG2 Schematic diagram of the structure of the B7H3/PDL1 bispecific antibody prepared in the present application.
  • FIG3 SDS-polyacrylamide gel electrophoresis diagram of the B7H3/PDL1 bispecific antibody of the present application.
  • FIG4 SEC-HPLC purity determination of the B7H3/PDL1 bispecific antibody of the present application.
  • FIG5 Tm value determination (DSF) of the B7H3/PDL1 bispecific antibody of the present application.
  • Figure 6 Binding ELISA of the B7H3/PDL1 bispecific antibody of the present application before and after heat treatment at 60°C, wherein a represents the binding ELISA with PDL1, and b represents the binding ELISA with B7H3.
  • FIG. 7 B7H3/PDL1 bispecific antibody binding ELISA of the present application, wherein a represents the binding ELISA with PDL1, and b represents the binding ELISA with B7H3.
  • FIG. 8 BLI affinity detection graph of the B7H3/PDL1 bispecific antibody B7H3-his or PDL1-his of the present application at 5 different concentrations, a represents the affinity curve with PDL1, and b represents the affinity curve with B7H3.
  • Figure 9 Blocking curve of the B7H3/PDL1 bispecific antibody PDL1/CHO-PD1 of the present application.
  • FIG. 10 The B7H3/PDL1 bispecific antibody of the present application induces T cells to secrete IFN- ⁇ .
  • FIG. 11 The B7H3/PDL1 bispecific antibody of the present application promotes T cell proliferation.
  • FIG. 12 ADCC effect of the B7H3/PDL1 bispecific antibody of the present 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.
  • FIG. 13 In vivo tumor inhibition effect of the B7H3/PDL1 bispecific antibody of the present application.
  • FIG. 14 Effects of the B7H3/PDL1 bispecific antibody of the present application on the body weight of mice.
  • the materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art and can be obtained through commercial channels.
  • the camel-derived anti-B7H3 nanoantibody was humanized using the framework shuffling method using the human antibody gene germline gene as a template.
  • the corresponding VHH framework shuffling library was generated by the whole in vitro synthesis of overlapping PCR. Then the VHH phage library was cloned, screened and identified.
  • a one-step strategy was used to humanize the camel-derived anti-B7H3 nanobody.
  • About 1,000 clones were screened from the sublibrary, and the selected positive clones were screened for phage-level thermal stability using the ELISA method.
  • 5 ⁇ g/ml huB7H3 antigen was coated in a highly adsorbable 96-well ELISA plate, and the supernatant after overnight amplification of the screened phage was reacted to select clones showing higher OD450 readings.
  • the above phage clone was sequenced to obtain the B7H3 VHH gene sequence, and its C-terminus was fused with the human Fc protein gene to construct and express B7H3-Fc.
  • the biomembrane interferometry method was applied to capture 100 nM of B7H3 VHH-Fc using Protein A probe and bind to 200 nM starting concentration and 2-fold diluted B7H3 antigen. The KD value of antibody binding to antigen was calculated.
  • amino acid sequences of the PDL1 monoclonal antibody light chain are from the existing PDL1hIgG1 humanized monoclonal antibody, and the C-terminus of the anti-B7H3 VHH (75-16 mentioned above) is connected to the C-terminus of the Fc fragment through a connecting peptide ((SEQ ID NO.: 11) (the structure is shown in Figure 2).
  • the DNA sequence was synthesized, subcloned into the pcDNA3.1 vector and amplified in E. coli.
  • the purified plasmid was transfected into HEK293 cells by PEI.
  • the cells were then suspended in OPM-CD05 expression medium for culture. After 6 days of culture, the cell culture supernatant was collected and the antibody was purified by protein A column.
  • the purified IgG1 was dialyzed with phosphate buffered saline (PBS), snap-frozen and stored at -80°C.
  • PBS phosphate buffered saline
  • 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.
  • bispecific antibody refers to the B7H3/PDL1 bispecific antibody in the present application, which is also referred to as “B7H3/PDL1 bispecific antibody” or "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 ⁇ m, (7.8 ⁇ 300 mm) P/N 088460 and compared with PDL1 mAb and B7H3 VHH-Fc.
  • the mobile phase used was phosphate buffered saline (PBS).
  • the flow rate was set to 0.7 mL/min; injection volume: 15 ⁇ l.
  • the SEC chromatogram was recorded by monitoring the absorbance at 280 nm using a UV detector at a constant temperature of 25 °C. As shown in Figure 4, all the antibodies tested had a very high proportion of monomer peaks, and the peak area ratio of the monomer peak reached more than 95%, indicating that the bispecific antibody had good monomer purity and a small number of aggregates under PBS buffer conditions.
  • DSF was detected by 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 times from a 5000-fold concentrated stock solution with ddH 2 O, 20 ⁇ l of sample was taken into a PCR tube, and the SYPRO Orange working solution was added to the reaction to prevent bleaching. The liquid was collected at the bottom of the PCR tube by instant centrifugation, and the qPCR instrument was turned on. The program was set to 25°C-95°C heating, with a temperature increase of 0.3°C per second.
  • Tm melting temperature
  • B7H3/PDL1 bispecific antibody when testing thermal stability, the bispecific antibody was treated in a 60°C water bath for 1 hour
  • monoclonal antibody when testing the binding of bispecific antibody to B7H3 by ELISA, the control monoclonal antibody used was MGA271, Isotype hIgG1, B7H3 VHH-Fc; when testing the binding to PDL1, the control monoclonal antibody was PDL1 monoclonal antibody, Avelumab, Isotype hIgG1) were added to the well plate in three-fold dilutions.
  • HRP horseradish peroxidase
  • H+L horseradish peroxidase
  • TMB 3,3',5,5'-tetramethylbenzidine substrate
  • Figures 6a and 6b show the binding curves of the bispecific antibody with PDL1 and B7H3 before and after treatment at 60°C for 1 hour. It can be seen from the figure that the binding curves of the bispecific antibody before and after heating basically overlap, regardless of whether it is for PDL1 or B7H3, which indicates that the bispecific antibody can withstand the high temperature of 60°C and maintain strong binding activity to the dual targets of PDL1 and B7H3. On the other hand, the bispecific antibody had similar binding abilities to the positive antibody, PDL1 monoclonal antibody, and B7H3 VHH-Fc for both targets.
  • the EC 50s of the bispecific antibody, PDL1 monoclonal antibody, and control Avelumab for binding to human PDL1 were 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively, which were of the same order of magnitude (Figure 7a).
  • the bispecific antibody, B7H3 VHH-Fc, and MGA271 also had similar binding activities to human B7H3, with EC 50s of 0.04105 nM, 0.02515 nM, and 0.05476 nM, respectively (Figure 7b).
  • 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), and the affinity of the bispecific antibody to specific human B7H3 and human PDL1 antigens was analyzed by OCTET 96, wherein the probe used was a protein A material to fix the antibody; B7H3-his and PDL1-his antigens were diluted to an initial concentration of 200 nM with sample buffer, and multiple antigen gradients were set up according to 2-fold dilution to bind to the antibody to obtain the rate constant and affinity, and the Kon and Koff values were calculated using the software provided by the supplier, and the KD value of the antibody was obtained. It can be seen from the figure that the bispecific antibody has a high affinity for PDL1 and B7H3, and the KD values reached 5.85 ⁇ 10-10 M and 8.11 ⁇ 10-9 M, respectively (Figure 8).
  • Example 6 Flow cytometry detection of the ability of bispecific antibodies to block the PD1/PDL1 pathway
  • 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 5 CHO-PD1 cells were evenly plated in a 96-well culture plate and incubated with a mixture of antibodies (B7H3/PDL1 bispecific antibody, Avelumab, PDL1 monoclonal antibody, Isotype hIgG1, B7H3 VHH-Fc) and biotinylated PDL1 antigen (50nM) starting at 400nM for 30 minutes at room temperature.
  • the mixed solution was then incubated with cells at 4°C for 45 minutes, and unbound antigens were washed away with PBS.
  • the cells were then fluorescently stained with PE-streptavidin.
  • MFI mean fluorescence intensity
  • the antibody concentration and MFI were plotted, and the IC 50 of the antibody was calculated using four-parameter fitting.
  • the results of flow cytometry analysis showed that the bispecific antibody was able to block the binding of PDL1 to CHO-PD1 cells with an IC 50 of 106.4 nM and a blocking activity similar to that of PDL1 monoclonal antibody (IC 50 of 94.20 nM) and Avelumab (IC 50 of 115.0 nM) ( FIG. 9 ).
  • PBMCs peripheral blood mononuclear cells
  • IL-4 interleukin-4
  • DCs dendritic cells
  • CD4 + T cells (1 ⁇ 10 5 ) and allogeneic DCs (1.25 ⁇ 10 4 ) were co-cultured in RPMI 1640 complete medium containing 10% FBS, with 5% CO 2 and a constant temperature of 37°C.
  • the cells were set up without antibody and with different concentrations of B7H3/PDL1 bispecific antibody, PDL1 monoclonal antibody, Avelumab, B7H3 VHH-Fc, MGA271, and Isotype hIgG1.
  • the IFN- ⁇ concentration in the culture supernatant was analyzed using an IFN- ⁇ ELISA detection kit.
  • the MLR results showed that the bispecific antibody could stimulate CD4 + T cells to secrete IFN- ⁇ , and the T cell activation ability of the bispecific antibody was superior to that of PDL1 monoclonal antibody and Avelumab at both low and high concentrations.
  • the bispecific antibody blocked B7H3 inhibitory signal transduction and partially activated T cells (Figure 10).
  • CD3 antibody (Clone HIT3a)
  • 1 ⁇ g/ml CD28 antibody (Clone CD28.2)
  • 5 ⁇ g/ml human PDL1 were coated on 96-well cell plates (Corning, USA) at 4°C for 1 hour, and control wells were coated with mouse IgG2a isotype control alone or CD3 and CD28 antibodies using the same method.
  • CD4 + T cells were isolated using Dynabeads TM CD4 Positive Isolation Kit.
  • CD4 + T cells were cultured in pre-coated 96-well plates with different concentrations of B7H3/PDL1 bispecific antibody, Avelumab and PDL1 monoclonal antibody at 37°C in RPMI1640 medium containing 10% FBS (Gibco) for 4 days. After 4 days, the CCK8 kit was used to detect changes in the number of T cells. The data showed that freshly isolated human CD4 + T cells cultured on well plates coated with anti-CD3 and anti-CD28 antibodies showed increased proliferation. When PDL1 was added to the well plates, the proliferation ability decreased significantly, which confirmed that PDL1 provided an inhibitory signal to T cells. Avelumab, PDL1 monoclonal antibody and bispecific antibody can significantly promote T cell proliferation at concentrations of 100nM and 500nM ( Figure 11).
  • the main antitumor effects of MGA271 and Avelumab are derived from the ADCC function of the antibodies, which is related to their IgG1 subtype.
  • the bispecific antibodies also have IgG1 functional regions.
  • the ADCC of the antibodies was measured using the LDH cytotoxicity detection kit.
  • Human PBMCs were purified from leukocytes using Ficoll gradient centrifugation and NK cells were isolated from human PBMCs using negative selection 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 different concentrations of bispecific antibodies and Avelumab at the beginning of the assay.
  • Human PBMC (6.67 ⁇ 10 6 ) were injected into 41 NPSG mice through the tail vein one day before inoculation of A375 tumor cells, and 5 ⁇ 10 6 A375 tumor cells were injected subcutaneously the next day. Five days after tumor inoculation, subcutaneous tumors were observed. There were 10 animals in each group, and the groups were set as isotype control, monoclonal antibody group combined with drug group (Pembrolizumab+MGA271 and Avelumab+MGA271), monoclonal and bispecific antibody groups. After the model was successfully established, the drug was administered on the 5th day, and the drug was administered twice a week until the end of the experiment.
  • the bispecific antibody constructed in the present application can bind to B7H3 and PDL1 at the same time, can relieve the inhibition of PDL1 on T cells while targeting tumor cells, and at the same time exhibits anti-tumor activity that is superior to the combination of monoclonal antibodies.

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Abstract

本发明涉及一种B7H3/PDL1双特异性抗体及其药物组合物及应用。所述双特异性抗体可靶向结合B7H3以及PDL1，其包括：单克隆抗体单元，其针对PDL1并且包括2条重链和2条轻链；纳米抗体单元，其针对B7H3并且包括2个相同的纳米抗体，其中，所述2个纳米抗体的C端分别通过连接肽与所述单克隆抗体单元的2条重链的Fc片段的C端连接。本发明的双特异性抗体可以同时结合B7H3和PDL1，可在靶向肿瘤细胞的同时解除PDL1对T细胞的抑制，表现出优于单克隆抗体联合用药的抗肿瘤活性。

Description

一种B7H3/PDL1双特异性抗体及其药物组合物及应用 技术领域
本发明属于生物制药领域，具体涉及一种B7H3/PDL1双特异性抗体及其药物组合物及应用。
背景技术
B7-CD28家族作为T淋巴细胞活化的共刺激信号，在T淋巴细胞参与的免疫反应中起着至关重要的作用。研究表明，不同的B7分子类型对免疫细胞反应有正向或负向调节。B7H3(也称为CD276)是B7家族的成员，主要表达在肿瘤细胞表面，Chapoval AI等人首次发现它对CD4 +和CD8 +T细胞具有共刺激作用，B7H3信号传导诱导细胞免疫并在T细胞受体信号传导下选择性增强干扰素-γ(IFN-γ)的产生。然而，随着对B7H3的研究不断深入，B7H3的抑制功能逐渐被发现，例如，它可以抑制CD4 +T和CD8 +T细胞的增殖。此外，研究表明B7H3的异常表达与多种癌症的发生、发展和转移有关，有大量证据表明，其高表达与各种恶性肿瘤的不良预后相关。
B7H3在所有测试的癌症类型中均高度表达，且异质性有限，在正常组织中很少表达。这表明B7H3可以被认为是一种肿瘤抗原(TA)，为针对B7H3高表达肿瘤细胞的靶向治疗提供了可能。目前，B7H3靶向治疗策略主要包括阻断性单克隆抗体、放射免疫疗法、抗体-药物偶联物(ADCs)、介导细胞毒性的单克隆抗体和双特异性抗体(BsAbs)等。由MacroGenics开发的靶向B7H3的人源化单克隆抗体Enoblituzumab(MGA271)基于抗体依赖性细胞介导的细胞毒性(ADCC)，作用于多种恶性肿瘤，如黑色素瘤、骨肉瘤和尤文氏肉瘤等。Enoblituzumab在多种异种移植肿瘤模型中被证明具有强的抗肿瘤活性，并且在灵长类动物的安全性评价中没有明显的毒性。Enoblituzumab目前处于II期临床研究阶段，并显示出良好的治疗潜力。
靶向免疫检查点PD1/PDL1的抑制剂无疑是肿瘤免疫治疗的焦点。PDL1表达于肿瘤细胞表面，具有细胞毒性的PDL1抑制性抗体理论上具有更好的抗肿瘤作用。然而据报道上市的PDL1单克隆抗体中只有Avelumab可介导针对肿瘤的ADCC效应，并显示出与其他PDL1抗体相当的安全 性。一个重要原因是ADCC的作用主要取决于抗原表达的丰度，而PDL1不能被视为典型的肿瘤抗原，而且它在肿瘤细胞中具有很大的异质性。
PD1/PDL1通路阻断抗体常与细胞毒抗体联合应用，以增强免疫治疗的效果。Enoblituzumab和Pembrolizumab(一种靶向PD1的单克隆抗体)联合使用，一方面，可以阻断PD1/PDL1通路的免疫抑制，另一方面，可通过Enoblituzumab发挥肿瘤杀伤作用。该联合疗法已进入临床II期(NCT04129320)。数据显示，联合治疗的不良反应发生率与单药治疗相似，且联合治疗的客观缓解率(ORR)优于PD1单克隆抗体治疗。这证明了同时阻断B7H3通路和PD1/PDL1通路的可行性。
双特异性抗体(BsAb)是可以同时靶向两个不同抗原表位的基因工程抗体。针对B7H3的双特异性抗体已经被开发出来，其中大部分依赖于T细胞重定向策略，例如CD3/B7H3双特异性抗体。然而，理论上基于细胞毒性的双特异性抗体可能更好地作用于B7H3高表达肿瘤细胞，因为B7H3可作为肿瘤抗原具有丰度高、异质性低的表达特点。目前，仅有5款双特异性抗体药物曾获批上市，而限制其应用的主要原因是稳定性和活性问题。因此，获得具有稳定性好以及优异生物活性的双特异性抗体仍是业界努力的方向。
发明内容
本发明的一个技术目的是提供一种B7H3/PDL1双特异性抗体制备方法及应用。
一方面，本发明提供一种B7H3/PDL1双特异性抗体，其靶向结合B7H3以及PDL1，所述双特异性抗体包括：
单克隆抗体单元，其针对PDL1并且包括2条重链和2条轻链；
纳米抗体单元，其针对B7H3并且包括2个相同的纳米抗体(VHH)，
其中，所述2个纳米抗体C端分别通过连接肽与所述单克隆抗体单元的2条重链的Fc片段的C端连接。
本发明中，所述连接肽是指一段包含甘氨酸和丝氨酸并且具有一定弹性及蛋白酶抗性的多肽。
在一个实施方式中，所述连接肽的氨基酸序列为SEQ ID No.:11。
在一个实施方式中，所述单克隆抗体单元的轻链可变区包括氨基酸序列为SEQ ID NO.：1的CDR1、氨基酸序列为SEQ ID NO.：2的CDR2以及氨基酸序列为SEQ ID NO.：3的CDR3，所述单克隆抗体单元的重链可变区包括氨基酸序列为SEQ ID NO.：5的CDR1、氨基酸序列为SEQ ID NO.：6的CDR2以及氨基酸序列为SEQ ID NO.：7的CDR3，以及所述纳米抗体包括氨基酸序列为SEQ ID NO.：12的CDR1、氨基酸序列为SEQ ID NO.：13的CDR2以及氨基酸序列为SEQ ID NO.：14的CDR3。
在一个实施方式中，所述单克隆抗体单元的轻链可变区包括如SEQ ID NO.：4的氨基酸序列，所述单克隆抗体单元的重链可变区包括如SEQ ID NO.：8的氨基酸序列。
在一个实施方式中，所述单克隆抗体单元的轻链包括如SEQ ID NO.：9的氨基酸序列，所述单克隆抗体单元的重链包括如SEQ ID NO.：10的氨基酸序列。
在一个实施方式中，所述纳米抗体包括如SEQ ID NO.：15的氨基酸序列。
在一个实施方式中，所述单克隆抗体单元的轻链可变区的氨基酸序列如SEQ ID NO.：4所示，所述单克隆抗体单元的重链可变区的氨基酸序列如SEQ ID NO.：8所示。
在一个实施方式中，所述单克隆抗体单元的轻链全长氨基酸序列如SEQ ID NO.：9所示，所述单克隆抗体单元的重链全长氨基酸序列如SEQ ID NO.：10所示。
在一个实施方式中，所述纳米抗体的氨基酸序列如SEQ ID NO.：15所示。
在一个实施方式中，所述双特异性抗体的重链氨基酸序列如SEQ ID NO.：16所示，轻链氨基酸序列如SEQ ID NO.：17所示。
另一方面，本发明还提供了一种编码所述B7H3/PDL1双特异性抗体的多核苷酸。
再一方面，本发明还提供了一种表达载体，其包含编码所述B7H3/PDL1双特异性抗体的多核苷酸。
另一方面，本发明提供一种药物组合物，其包含治疗有效量的上述B7H3/PDL1双特异性抗体，以及药学上可接受的载体。
另一方面，本发明提供一种所述B7H3/PDL1双特异性抗体在制备用于预防、诊断、治疗或辅助治疗肿瘤的药物中的应用。
在具体实施方式中，所述药物通过结合B7H3、阻断B7H3信号通路从而介导ADCC效应来抑制肿瘤。
在具体实施方式中，所述药物通过结合PD-L1、阻断PD-1与PDL-1结合、激活T淋巴细胞的药物、提高T淋巴细胞中IL-2、IFN-γ表达，从而抑制肿瘤。
在具体实施方式中，所述药物通过结合B7H3、阻断B7H3信号通路，以及通过结合PD-L1、阻断PD-1与PDL-1结合、激活T淋巴细胞的药物、提高T淋巴细胞中IL-2、IFN-γ表达，从而抑制肿瘤。
在具体实施方式，所述肿瘤可选自肺癌、胃癌、肝癌、结肠直肠癌、黑色素瘤、肾瘤、卵巢癌、前列腺癌、膀胱癌、乳腺癌、食管癌、大肠癌、鼻咽癌、脑肿瘤、宫颈癌、血癌、骨癌、淋巴癌、胰脏癌和尤文氏肉瘤中的一种或多种。特别地，所述肿瘤为乳腺癌、卵巢癌或黑色素瘤。
有益效果
本申请构建的双特异性抗体可以同时结合B7H3和PDL1，可在靶向肿瘤细胞的同时解除PDL1对T细胞的抑制，更表现出了优于单克隆抗体联合用药的抗肿瘤活性。
另外，与相关的单克隆抗体联合治疗相比，本申请的双特异性抗体具有依从性好、质量可控的优点。
进一步地，本申请中双特异性抗体的稳定性表征主要体现在为单体纯度、热稳定性的研究上。单次亲和纯化后，双特异性抗体单体含量可达95％，甚至优于业内多次二次纯化后的纯度。抗体经热处理后的结构和活性分析结果证明，该抗体在恶劣环境下仍能保持良好的分子构象和完整的生物活性，有利于抗体的工业化生产及包装贮存。总体来说，本申请构建了IgG-VHH 2形式的B7H3/PDL1双特异性抗体；它显示出良好的分子稳定性，并具有显著优于Avelumab和MGA271的体外活性(结合分子水平和细胞水平)。体内数据证明，在B7H3 +A375肿瘤细胞中，双特异性抗体的抗肿瘤活性优于B7H3单克隆抗体联合用药组。因此，本申请的双特异性抗体由于其优异的可开发性和活性，具有广阔的应用前景。
综上，本发明所用的方法是目前治疗肿瘤的一种新的前沿方法，肿瘤免疫治疗有望成为继手术、化疗、放疗、靶向治疗后肿瘤治疗领域的一场革 新。本发明中的B7H3/PDL1双特异性抗体有望成为一种新型的抗肿瘤药物。
附图说明
图1：本申请制备的抗B7H3 VHH人源化抗体与B7H3的亲和力筛选图。
图2：本申请制备的B7H3/PDL1双特异性抗体的结构示意图。
图3：本申请的B7H3/PDL1双特异性抗体的SDS-聚丙烯酰胺凝胶电泳图。
图4：本申请的B7H3/PDL1双特异性抗体的SEC-HPLC纯度测定。
图5：本申请的B7H3/PDL1双特异性抗体的Tm值测定(DSF)。
图6：本申请的B7H3/PDL1双特异性抗体60℃热处理前后结合ELISA，其中a表示与PDL1的结合ELSIA，b表示与B7H3的结合ELISA。
图7：本申请的B7H3/PDL1双特异性抗体结合ELISA，其中a表示与PDL1的结合ELISA，b表示与B7H3的结合ELISA。
图8：本申请的B7H3/PDL1双特异性抗体B7H3-his或PDL1-his 5种不同浓度下的BLI亲和力检测图，a表示与PDL1的亲和力曲线，b表示与B7H3的亲和力曲线。
图9：本申请的B7H3/PDL1双特异性抗体PDL1/CHO-PD1阻断曲线。
图10：本申请的B7H3/PDL1双特异性抗体诱导T细胞分泌IFN-γ。
图11：本申请的B7H3/PDL1双特异性抗体促进T细胞增殖。
图12：本申请的B7H3/PDL1双特异性抗体对癌细胞的ADCC作用，a表示对人乳腺癌细胞MDA-MB-231的细胞毒作用，b表示对人卵巢透明细胞癌细胞ES-2的细胞毒作用。
图13：本申请的B7H3/PDL1双特异性抗体的体内抑瘤效果。
图14：本申请的B7H3/PDL1双特异性抗体对小鼠体重的影响。
具体实施方式
下面结合具体实施例对本发明做进一步说明，但本发明不受实施例的限制。
以下实施例中所用材料、试剂、仪器和方法，未经特殊说明，均为本领域中的常规材料、试剂、仪器和方法，均可通过商业渠道获得。
具体实施方式
以下通过具体实施方式来描述本发明，以使本领域的技术人员更好的了解本申请的技术内容，然而这些实施方式不应理解为限制本发明的范围。
制备实施例
1.骆驼源anti-B7H3的人源化
用人抗体基因的Germline基因作为模板采用Framework shuffling方法将骆驼源anti-B7H3纳米抗体人源化，相应的VHH构架改组的文库都是通过重叠PCR的全体外合成生成的。然后构建VHH噬菌体文库的克隆，筛选和鉴定。
更准确地说，使用一步策略将骆驼源anti-B7H3纳米抗体人源化。从该亚文库中筛选出约1000个克隆，用ELISA方法对挑取的阳性克隆进行噬菌体水平热稳定性筛选，在高吸附力的96孔ELISA板中包被5μg/ml huB7H3抗原，将筛选的噬菌体过夜扩增后的上清进行反应，挑选显示出较高的OD450读值的克隆。
将上述噬菌体克隆进行测序，获取B7H3 VHH基因序列，并将其C末端与人Fc蛋白基因进行融合，构建B7H3-Fc并表达，应用生物膜干涉法，使用Protein A探针抓取100nM的B7H3 VHH-Fc，并与200nM起始浓度，2倍稀释的B7H3抗原结合，计算抗体结合抗原的KD值，结果表明，75-16(其氨基酸序列为SEQ ID NO.：15，CDR1序列为SEQ ID NO.：12，CDR2序列为SEQ ID NO.：13，CDR3序列为SEQ ID NO.：14)具有与B7H3最高的亲和力，KD至达到了3.24×10 -9M(见图1)，故挑选其应用于下一步双特异性抗体的构建。
2.B7H3/PDL1双特异性抗体的构建及表达
PDL1单抗轻链(氨基酸序列为SEQ ID NO.：9，可变区序列为SEQ ID NO.：4，CDR1序列为SEQ ID NO.：1，CDR2序列为SEQ ID NO.：2，CDR3序列为SEQ ID NO.：3)和重链(氨基酸序列为SEQ ID NO.：10，可变区序列为SEQ ID NO.：8，CDR1序列为SEQ ID NO.：5，CDR2序列为SEQ ID NO.：6，CDR3序列为SEQ ID NO.：7)的氨基酸序列来自已有 的PDL1hIgG1人源化单克隆抗体，并且抗B7H3 VHH(上述75-16)的C末端通过连接肽((SEQ ID NO.：11)连接到Fc片段的C末端(结构如图2所示)。
合成了DNA序列，并将其亚克隆到pcDNA3.1载体中并在大肠杆菌中扩增。通过PEI将纯化的质粒转染到HEK293细胞中。然后将细胞悬浮在OPM-CD05表达培养基中进行培养。培养6天后，收集细胞培养上清液，并通过蛋白A柱纯化抗体。纯化的IgG1用磷酸盐缓冲液(PBS)透析，速冻并保存在-80℃。
经过纯化得到的B7H3/PDL1双特异性抗体的重链氨基酸序列为SEQ ID NO.：16，轻链氨基酸序列为SEQ ID NO.：17。
测试部分
下文中，如未特别指出，术语“双特异性抗体”均指代本申请中的B7H3/PDL1双特异性抗体，其也简称为“B7H3/PDL1双抗”或“双抗”。
测试方法：
实施例1十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)
将5μg双特异性抗体与蛋白质还原和非还原缓冲液混合用PBS将体系补至10μl，经过100℃加热10分钟使蛋白充分变性，取9μl添加到预制聚丙烯酰胺凝胶孔(Bio-Rad)中。设置电压先80V 30分钟，后120V 60分钟进行分离后，凝胶用考马斯亮蓝染色液染色30分钟；用脱色溶液(乙酸：乙醇：水＝1:3:6)15分钟脱色，重复三次脱色使背景褪色，然后用凝胶成像仪获取图像。结果表明，非还原条件下B7H3/PDL1双特异性抗体具有良好的单体纯度，还原条件下，由于轻重链之间的二硫键被打开出现重链轻链两个条带，且无杂带(图3)。
实施例2尺寸排阻色谱(SEC-HPLC)
SEC-HPLC分析用于评估双特异性抗体的单体纯度。B7H3/PDL1双特异性抗体使用1260HPLC系统(Agilent，Santa Clara，CA)在Thermo MAbPac SEC-1,5μm,(7.8×300mm)P/N 088460上进行分析，同时与PDL1单抗、B7H3 VHH-Fc进行对比。使用的流动相是磷酸盐缓冲液(PBS)。流速设置为0.7mL/min；进样量：15μl。通过在恒温25℃下使用紫外检测器 监测280nm处的吸光度，记录SEC色谱图，如图4所示，所检测抗体均具有极高比例的单体峰，单体峰的峰面积比例达到95％以上，这表明双特异性抗体在PBS缓冲液条件下具有良好的单体纯度，聚集体数量少。
实施例3差示扫描荧光法(DSF)检测抗体的Tm值
DSF用实时PCR仪(Biorad cfx96，USA)检测。B7H3/PDL1双特异性抗体、B7H3 VHH-Fc、PDL1单抗在PBS中稀释至1mg/mL。将SYPRO Orange从5000倍浓缩原液用ddH 2O稀释1000倍，取20μl样品于PCR管，将SYPRO Orange的工作溶液添加到反应中以防止漂白，瞬时离心将液体汇集于PCR管底，打开qPCR仪器，设置程序为25℃-95℃升温，每秒升温0.3℃，收集数据，将温度和信号值作图，并计算熔解温度(Tm)，数据显示，B7H3/PDL1双特异性抗体的Fc、Fab的Tm分别为69℃，90℃，达到很好的耐高温能力，并与B7H3 VHH-Fc、PDL1单克隆抗体的Tm相近(图5)。
实施例4结合ELISA验证抗体的热稳定性及结合活性
96孔酶标板在4℃下用2μg/ml his-tagged PDL1或者B7H3抗原蛋白包被过夜，第二天每孔添加100μl Casein封闭液37℃封闭1小时。B7H3/PDL1双特异性抗体(测试热稳定性时，将双特异性抗体于60℃水浴处理1小时)、单克隆抗体(在用ELISA法测试双特异性抗体结合B7H3时，所使用的对照单克隆抗体为MGA271，Isotype hIgG1，B7H3 VHH-Fc；在测试与PDL1结合时，对照单克隆抗体为PDL1单抗、Avelumab、Isotype hIgG1)三倍倍比稀释液添加至孔板中。37℃孵育1小时后，用0.1％PBST洗去未结合的抗体，通过辣根过氧化物酶(HRP)偶联的山羊抗人IgG(H+L)抗体(Jackson ImmunoResearch,USA)检测结合的抗体，并用3,3',5,5'-四甲基联苯胺底物(TMB)50μl显色，静置5分钟后加入50μl 2M硫酸停止显色，然后用SpectraMax M5e(Molecular Devices)酶标仪在OD450nm处检测吸光度，并用4参数拟合的方法将抗体浓度与OD450读值作图，并计算EC 50。图6a、b表示的是双特异性抗体在经过60℃处理1小时前后与PDL1、B7H3的结合曲线，由图可知，不管是对于PDL1还是B7H3，双特异性抗体在加热前后的结合曲线基本重叠，这表明双特异性抗体能够耐受 60℃的高温且保持了对PDL1、B7H3双靶点的强结合活性。另一方面，双特异性抗体与阳性抗体以及PDL1单抗、B7H3 VHH-Fc对两靶点结合能力皆相似，双特异性抗体、PDL1单克隆抗体和对照Avelumab结合人PDL1的EC 50分别为0.3056nM、0.4407nM和0.1563nM，属同一数量级(图7a)；双特异性抗体、B7H3 VHH-Fc和MGA271与人B7H3也具有相近的结合活性，EC 50分别为0.04105nM、0.02515nM和0.05476nM(图7b)。
实施例5 BLI法测定双特异性抗体与抗原结合的亲和力
B7H3/PDL1双特异性抗体和PDL1单抗、B7H3 VHH-Fc在样品缓冲液(0.02％tween 20和0.1％BSA于PBS)中稀释至100nM，通过OCTET 96分析双特异性抗体与特定人B7H3和人PDL1抗原的亲和力，其中使用的探针为Protein A材质，用来固定抗体；B7H3-his、PDL1-his抗原用样品缓冲液稀释至初始浓度为200nM，并按照2倍稀释设置多个抗原梯度用以与抗体结合，以获得速率常数和亲和力，使用供应商提供的软件计算Kon以及Koff值，并得到抗体的KD值。由图可见双特异性抗体对PDL1和B7H3有很高的亲和力，KD值分别达到了5.85×10 -10M和8.11×10 -9M(图8)。
实施例6流式细胞术检测双特异性抗体阻断PD1/PDL1通路的能力
用流式细胞术评估B7H3/PDL1双特异性抗体阻断人PDL1和人PD1-CHO细胞结合的能力，并与Avelumab、PDL1单抗对比。将2×10 5个CHO-PD1细胞均匀铺在96孔培养板中，并与400nM起始，倍比稀释的抗体(B7H3/PDL1双特异性抗体、Avelumab、PDL1单抗、Isotype hIgG1、B7H3 VHH-Fc)和生物素化的PDL1抗原(50nM)的混合物一起室温孵育30分钟，混合溶液再与细胞于4℃孵育45分钟，用PBS洗掉未结合的抗原。然后用PE-链霉亲和素对细胞进行荧光染色。最后读取流式细胞仪中PE通道的平均荧光强度(MFI)，将抗体浓度和MFI作图，使用四参数拟合计算抗体的IC 50。流式细胞术分析结果表明，双特异性抗体能够阻断PDL1与CHO-PD1细胞的结合，其IC 50为106.4nM且阻断活性与PDL1单抗(IC 50为94.20nM)、Avelumab(IC 50为115.0nM)相近(图9)。
实施例7混合淋巴细胞反应(MLR)检测双特异性抗体激活T细胞的能力
通过使用单核细胞纯化试剂盒(Miltenyi Biotec,Germany)使用500U/mL白细胞介素-4(IL-4)和250U/mL GM-CSF在体外培养从外周血单个核细胞(PBMC)分离的单核细胞7天，诱导产生树突状细胞(DC)。CD4 +T细胞(1×10 5)和同种异体DCs(1.25×10 4)用含10％FBS的RPMI 1640完全培养基进行共培养，培养条件为5％CO 2，37℃恒温，并设置不加抗体以及添加不同浓度B7H3/PDL1双特异性抗体、PDL1单抗、Avelumab、B7H3 VHH-Fc、MGA271、Isotype hIgG1组别。5天后，利用IFN-γELISA检测试剂盒分析培养上清液中的IFN-γ浓度。MLR结果表明，双特异性抗体可以刺激CD4 +T细胞分泌IFN-γ，双特异性抗体的T细胞激活能力在低浓度或高浓度下皆优于PDL1单抗和Avelumab；除此之外，当仅存在B7H3抗体时，尽管没有PDL1信号阻断抗体明显，但由于双特异性抗体阻断了B7H3抑制性信号传导，也会部分激活T细胞(图10)。
实施例8 T细胞增殖实验
将1μg/ml CD3抗体(Clone HIT3a)、1μg/ml CD28抗体(Clone CD28.2)和5μg/ml human PDL1于4℃包被在96孔细胞板(Corning,USA)上1小时，对照孔单独包被小鼠IgG2a同种型对照或使用相同的方法包被CD3、CD28抗体。使用Dynabeads TMCD4Positive Isolation Kit分离CD4 +T细胞。CD4 +T细胞在预包被的96孔板中与不同浓度B7H3/PDL1双特异性抗体、Avelumab以及PDL1单抗一起在37℃下在含有10％FBS(Gibco)的RPMI1640培养基中培养4天。4天后，利用CCK8试剂盒检测T细胞数量变化，数据表明用抗CD3和抗CD28抗体包被的孔板培养的新鲜分离的人CD4 +T细胞表现出增殖增加，当孔板中添加PDL1时，增殖能力显著下降，这证实了PDL1向T细胞提供了抑制信号；Avelumab、PDL1单克隆抗体和双特异性抗体在浓度为100nM和500nM时可以显著促进T细胞增殖(图11)。
实施例9抗体依赖性细胞毒性(ADCC)
MGA271和Avelumab的主要抗肿瘤作用来源于抗体的ADCC功能，这与其IgG1亚型有关，双特异性抗体也具有IgG1功能区。使用LDH细胞毒性检测试剂盒测量抗体的ADCC，使用Ficoll梯度离心从白细胞包中纯化人PBMC并用负选择磁珠(Miltenyi Biotec，Auburn，CA)从人PBMC中分离 NK细胞。NK细胞(3×10 6)和MDA-MB-231、ES-2细胞(3×10 5)在测定开始时加入或不加不同浓度的双特异性抗体、Avelumab共培养。18小时后，通过ELISA分析培养上清液中的乳酸脱氢酶(LDH)分泌。当以B7H3 +PDL1 +MDA-MB-231用作靶细胞时，双特异性抗体和Avelumab均表现出了ADCC活性，但在低浓度下双特异性抗体的活性更强；使用PDL1 +ES-2细胞作为靶细胞，观察到双特异性抗体在100nM剂量下的活性与Avelumab相当，并且在低浓度下也能表现出很强的ADCC活性(图12)。
实施例10双特异性抗体的体内抗肿瘤活性研究
人PBMC(6.67×10 6)在接种A375肿瘤细胞前一天通过尾静脉注射到41只NPSG小鼠体内，次日皮下注射5×10 6个A375肿瘤细胞。肿瘤接种五天后，可观察到皮下肿瘤产生，每组10只动物，组别设置为同型对照、单克隆抗体组联合用药组(Pembrolizumab+MGA271和Avelumab+MGA271)单克和双特异性抗体组，模型成功后第5天给药，每周给药2次至实验结束。在实验的0、4、7、11、14、18、21、25、28、32、35、39和42天测量肿瘤体积和动物体重并记录。根据基于相对肿瘤体积(TGIRTV)和动物体重变化的肿瘤生长抑制值来评价疗效和安全性。双特异性抗体自始至终都比PD1单克隆抗体+MGA271联合应用组保持显著更强的活性；随着实验时间的增加，双特异性抗体也逐渐表现出优于Avelumab+MGA271组的效果，实验结束时TGI分别为39.29％和26.45％(图13)；另外实验期间没有小鼠体重的严重减轻，证明本双特异性抗体治疗的安全性(图14)。
从以上测试例的测试结果可以看出，本申请构建的双特异性抗体可以同时结合B7H3和PDL1，可在靶向肿瘤细胞的同时解除PDL1对T细胞的抑制，同时表现出了优于单克隆抗体联合用药的抗肿瘤活性。

Claims (10)

  1. 一种B7H3/PDL1双特异性抗体，其靶向结合B7H3以及PDL1，所述双特异性抗体包括：
    单克隆抗体单元，其针对PDL1并且包括2条重链和2条轻链；
    纳米抗体单元，其针对B7H3并且包括2个相同的纳米抗体，
    其中，所述2个纳米抗体的C端分别通过连接肽与所述单克隆抗体单元的2条重链的Fc片段的C端连接。
  2. 根据权利要求1所述的双特异性抗体，其中，所述连接肽为一段包含甘氨酸和丝氨酸并且具有一定弹性及蛋白酶抗性的多肽，优选地，所述连接肽的氨基酸序列为SEQ ID No.:11。
  3. 根据权利要求1所述的双特异性抗体，其中，
    所述单克隆抗体单元的轻链可变区包括氨基酸序列为SEQ ID NO.：1的CDR1、氨基酸序列为SEQ ID NO.：2的CDR2以及氨基酸序列为SEQ ID NO.：3的CDR3，所述单克隆抗体单元的重链可变区包括氨基酸序列为SEQ ID NO.：5的CDR1、氨基酸序列为SEQ ID NO.：6的CDR2以及氨基酸序列为SEQ ID NO.：7的CDR3，以及所述纳米抗体包括氨基酸序列为SEQ ID NO.：12的CDR1、氨基酸序列为SEQ ID NO.：13的CDR2以及氨基酸序列为SEQ ID NO.：14的CDR3；
    优选地，所述单克隆抗体单元的轻链可变区包括如SEQ ID NO.：4的氨基酸序列，所述单克隆抗体单元的重链可变区包括如SEQ ID NO.：8的氨基酸序列；以及所述纳米抗体包括如SEQ ID NO.：15的氨基酸序列；
    进一步，优选地，所述单克隆抗体单元的轻链包括如SEQ ID NO.：9的氨基酸序列，所述单克隆抗体单元的重链包括如SEQ ID NO.：10的氨基酸序列，以及所述纳米抗体包括如SEQ ID NO.：15的氨基酸序列；
    更优选地，所述单克隆抗体单元的轻链全长氨基酸序列如SEQ ID NO.：9所示，所述单克隆抗体单元的重链全长氨基酸序列如SEQ ID NO.：10所示；以及所述纳米抗体的氨基酸序列如SEQ ID NO.：15所示。
  4. 根据权利要求1所述的双特异性抗体，其中，所述双特异性抗体的重链氨基酸序列如SEQ ID NO.：16所示，轻链氨基酸序列如SEQ ID NO.：17所示。
  5. 一种编码如权利要求1至4任一项所述的B7H3/PDL1双特异性抗体的多核苷酸。
  6. 一种表达载体，其包含如权利要求5所述的多核苷酸。
  7. 一种药物组合物，其包含治疗有效量的如权利要求1至4任一项所述的B7H3/PDL1双特异性抗体，以及药学上可接受的载体。
  8. 一种如权利要求1至4任一项所述的B7H3/PDL1双特异性抗体在制备用于预防、诊断、治疗或辅助治疗肿瘤的药物中的用途。
  9. 根据权利要求8所述的用途，其中，所述药物通过结合B7H3、阻断B7H3信号通路从而介导ADCC效应来抑制肿瘤；或者
    所述药物通过结合PD-L1、阻断PD-1与PDL-1结合、激活T淋巴细胞的药物、提高T淋巴细胞中IL-2、IFN-γ表达，从而抑制肿瘤，
    优选地，所述药物通过结合B7H3、阻断B7H3信号通路，以及通过结合PD-L1、阻断PD-1与PDL-1结合、激活T淋巴细胞的药物、提高T淋巴细胞中IL-2、IFN-γ表达，从而抑制肿瘤。
  10. 根据权利要求8所述的用途，其中，所述肿瘤选自肺癌、胃癌、肝癌、结肠直肠癌、黑色素瘤、肾瘤、卵巢癌、前列腺癌、膀胱癌、乳腺癌、食管癌、大肠癌、鼻咽癌、脑肿瘤、宫颈癌、血癌、骨癌、淋巴癌、胰脏癌和尤文氏肉瘤中的一种或多种，优选地，所述肿瘤为乳腺癌、卵巢癌或黑色素瘤。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110627906A (zh) * 2019-10-10 2019-12-31 上海洛启生物医药技术有限公司 抗pd-l1/4-1bb双特异性抗体及其用途
CN110642948A (zh) * 2019-10-09 2020-01-03 达石药业(广东)有限公司 B7-h3纳米抗体、其制备方法及用途
CN113943371A (zh) * 2021-11-01 2022-01-18 达石药业(广东)有限公司 一种抗her2/抗pd-l1双功能抗体及其应用
WO2022105817A1 (en) * 2020-11-18 2022-05-27 Suzhou Transcenta Therapeutics Co., Ltd. Bi-functional molecules
CN114829403A (zh) * 2019-11-22 2022-07-29 爱必乐生物公司 抗pd-l1/抗b7-h3多特异性抗体及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110642948A (zh) * 2019-10-09 2020-01-03 达石药业(广东)有限公司 B7-h3纳米抗体、其制备方法及用途
CN110627906A (zh) * 2019-10-10 2019-12-31 上海洛启生物医药技术有限公司 抗pd-l1/4-1bb双特异性抗体及其用途
CN114829403A (zh) * 2019-11-22 2022-07-29 爱必乐生物公司 抗pd-l1/抗b7-h3多特异性抗体及其用途
WO2022105817A1 (en) * 2020-11-18 2022-05-27 Suzhou Transcenta Therapeutics Co., Ltd. Bi-functional molecules
CN113943371A (zh) * 2021-11-01 2022-01-18 达石药业(广东)有限公司 一种抗her2/抗pd-l1双功能抗体及其应用

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