WO2021244371A1 - Anti-pd-l1/vegf fusion protein - Google Patents

Anti-pd-l1/vegf fusion protein Download PDF

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WO2021244371A1
WO2021244371A1 PCT/CN2021/096084 CN2021096084W WO2021244371A1 WO 2021244371 A1 WO2021244371 A1 WO 2021244371A1 CN 2021096084 W CN2021096084 W CN 2021096084W WO 2021244371 A1 WO2021244371 A1 WO 2021244371A1
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cancer
fusion protein
seq
acid sequence
amino acid
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Chinese (zh)
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黄浩旻
邓岚
李理
朱祯平
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三生国健药业（上海）股份有限公司
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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Definitions

  • the present invention relates to the technical field of fusion proteins, and more specifically, to an anti-PD-L1/VEGF fusion protein.
  • PD-1 Human Programmed Cell Death Receptor-1
  • PD-1 is a type I membrane protein with 288 amino acids. It is one of the major known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy) , 54: 307-314). PD-1 is expressed on activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- 1). Ligand 2, programmed cell death-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in the body.
  • PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells.
  • B T lymphocytes
  • peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells.
  • PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and/or PD-L2), it negatively regulates antigen receptor signal transduction. At present, the structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al., Immunity 20:337-347 (2004); Lin et al., Proc. Natl. Acad. Sci.USA 105:3011-6 (2008)).
  • PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules.
  • the cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (Immunoreceptor Tyrosine Inhibition Motif) and ITSM (Immune Receptor Tyrosine Switch Motif).
  • PD-1 plays an important role in the immune evasion mechanism of tumors.
  • Tumor immunotherapy which uses the body’s own immune system to fight cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction. Therefore, how to break the tumor microenvironment has become an anti-tumor research Focus.
  • Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN- ⁇ in most PD-L1-negative tumor cell lines), It is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci.
  • PD-1 on tumor infiltrating lymphocytes
  • PD-L1 on tumor cells
  • Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors. It can be seen that blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-L1 has become a popular target for the development of tumor immunotherapy drugs. .
  • VEGFs vascular endothelial growth factor
  • VEGF-A165 VEGF-A165
  • VEGF vascular endothelial growth factor
  • VEGFR1-D2 competes for binding to VEGF and blocks the binding of VEGFR2 to VEGF, thereby blocking the signaling pathway, inhibiting endothelial cell proliferation and angiogenesis, thereby inhibiting the rapid proliferation and metastasis of tumors.
  • the purpose of the present invention is to provide a new anti-PD-L1/VEGF fusion protein that can simultaneously block PD-L1 and VEGF signal pathways.
  • the object of the present invention is also to provide a nucleic acid molecule encoding the fusion protein; to provide an expression vector containing the nucleic acid molecule; to provide a host cell containing the expression vector; to provide a method for preparing the fusion protein; to provide a method for preparing the fusion protein; Protein pharmaceutical composition; provide the application of the fusion protein or the pharmaceutical composition in the preparation of drugs for treating cancer; provide the method for the fusion protein or the pharmaceutical composition to treat cancer.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides an anti-PD-L1/VEGF fusion protein comprising an anti-PD-L1 antibody and the D2 domain of VEGFR1;
  • the heavy chain of the anti-PD-L1 antibody comprises a complementarity determining region HCDR1-3
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 11
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13
  • the light chain of the anti-PD-L1 antibody includes In the complementarity determining region LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16.
  • the N-terminus of the D2 domain of VEGFR1 is connected to the C-terminus of the heavy chain of the anti-PD-L1 antibody via a peptide linker L.
  • amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: shown in 18.
  • the anti-PD-L1 antibody is a monoclonal antibody.
  • the anti-PD-L1 antibody is a humanized antibody.
  • the anti-PD-L1 antibody is an IgG antibody.
  • amino acid sequence of the peptide linker L is shown in SEQ ID NO: 3.
  • amino acid sequence of the D2 domain of VEGFR1 is shown in SEQ ID NO: 1 or SEQ ID NO: 6.
  • the fusion protein is selected from M8-D2 and M8-D2-M2.
  • the D2 domain of VEGFR1 in M8-D2-M2 is truncated by 2 amino acids at the C-terminal relative to the D2 domain of VEGFR1 of the fusion protein M8-D2. These two amino acids are easy to fall off during the fermentation process, and the removal does not affect the efficacy .
  • the heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4 or SEQ ID NO: 7, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 5.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the fusion protein.
  • nucleic acid sequence encoding the heavy chain of the fusion protein is shown in SEQ ID NO: 8 or SEQ ID NO: 10
  • nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 9 .
  • nucleic acid molecule encoding the amino acid sequence of the above-mentioned fusion protein can be replaced, deleted, changed, inserted or added as appropriate to provide a homolog of the nucleic acid molecule.
  • the third aspect of the present invention provides an expression vector containing the above-mentioned nucleic acid molecule.
  • the fourth aspect of the present invention provides a host cell containing the above-mentioned expression vector.
  • the fifth aspect of the present invention provides a method for preparing a fusion protein, which includes the following steps:
  • the sixth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the above-mentioned fusion protein and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the seventh aspect of the present invention provides the use of the above-mentioned fusion protein and pharmaceutical composition in the preparation of drugs for the treatment of cancer.
  • the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial cancer, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer Cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer.
  • the cancer is colon cancer.
  • the anti-PD-L1/VEGF fusion protein of the present invention can be used alone or in combination with other anti-tumor drugs.
  • the cancer treatment drugs referred to in the present invention refer to drugs that inhibit and/or treat tumors, which may include delays in the development of tumor-related symptoms and/or reduction in the severity of these symptoms, and further include symptoms associated with existing tumors. Reduce and prevent the appearance of other symptoms, including reducing or preventing tumor metastasis.
  • the dosage of administration varies with the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. .
  • the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present invention.
  • the eighth aspect of the present invention provides a method of treating cancer, which comprises administering the above-mentioned fusion protein or pharmaceutical composition to a subject in need.
  • the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial cancer, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer Cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer.
  • the cancer is colon cancer.
  • the administered dose When the anti-PD-L1/VEGF fusion protein and the pharmaceutical composition thereof are administered to a subject, the administered dose must be a therapeutically effective amount.
  • the therapeutically effective amount refers to an amount effective in treating cancer.
  • the dosage will vary depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. With reference to the results of animal experiments and various situations, the total dose should not exceed a certain range.
  • the positive effect of the present invention is that the fusion protein of the present invention can bind to PD-L1 and VEGF with high affinity, and its affinity to PD-L1 is equivalent to that of the anti-PD-L1 monoclonal antibody positive control M8.
  • the determination of the affinity dissociation constant shows that it is The affinity of VEGF is higher than that of anti-VEGF monoclonal antibody positive control Bevacizumab.
  • the fusion protein of the present invention can effectively block the binding of PD-1 and PD-L1, and its blocking ability is equivalent to the anti-PD-L1 monoclonal antibody positive control M8, and it can effectively block the interaction between VEGF and its receptor KDR Its blocking ability is better than that of anti-VEGF monoclonal antibody positive control Bevacizumab.
  • the fusion protein of the present invention can significantly inhibit the growth of colon cancer transplantation tumors, and its anti-tumor effect takes effect quickly, and its anti-tumor effect is obviously better than that of the anti-PD-L1 monoclonal antibody positive control M8.
  • the fusion protein of the present invention has the potential to treat diseases related to PD-L1 and VEGF activity.
  • Figure 1 Schematic diagram of the structure of anti-PD-L1/VEGF bifunctional fusion protein
  • FIG. 1 Electrophoresis detection image of anti-PD-L1/VEGF bifunctional fusion protein
  • Figure 3A ELISA detection diagram of the affinity between anti-PD-L1/VEGF bifunctional fusion protein and PD-L1
  • Figure 3B ELISA detection diagram of the affinity of anti-PD-L1/VEGF bifunctional fusion protein and VEGF
  • Figure 4 FACS detection diagram of the binding affinity of anti-PD-L1/VEGF bifunctional fusion protein to the surface antigen of target cells
  • Figure 5 Cellular experiment detection diagram of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of PD-1 and PD-L1
  • Figure 6 Cellular experimental detection diagram of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of VEGF and receptor KDR
  • Figure 7 Anti-tumor effect of anti-PD-L1/VEGF bifunctional fusion protein on MC38-hPD-L1 xenograft tumor model
  • fusion protein refers to a new polypeptide sequence obtained by the fusion of two or more identical or different polypeptide sequences.
  • fusion refers to direct connection by peptide bonds or operative connection via one or more connecting peptides (peptide linkers).
  • connecting peptide peptide linker
  • connecting peptide linker refers to a short peptide that can connect two polypeptide sequences, generally a peptide of 2-30 amino acids in length.
  • the term "antibody (Ab)” refers to a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains.
  • Chain (H) composition Each heavy chain has a variable region (VH) at one end, followed by a constant region.
  • the heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain.
  • Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on.
  • the heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain.
  • the two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins.
  • humanized means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies.
  • framework residues can be changed to maintain binding affinity.
  • the terms "anti” and “binding” refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • SPR Surface Plasmon Resonance
  • an BIACORE instrument is used to measure the relative binding affinity of an antibody to the antigen.
  • the term "expression vector” refers to a conventional expression vector in the art that contains appropriate regulatory sequences, such as a promoter, a terminator, an enhancer, etc.
  • the expression vector may be a virus or a plasmid.
  • the expression vector preferably includes pDR1, pcDNA3.4(+), pDHFR or pTT5.
  • the term "host cell” refers to various conventional host cells in the art, as long as the vector can stably replicate itself and the nucleic acid molecule carried can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells are preferably selected from: COS, CHO, NS0, sf9, sf21, DH5 ⁇ , BL21(DE3), TG1, BL21(DE3), 293F Cell or 293E cell.
  • the term "effective amount” refers to the amount or dose that produces the expected effect in the treated individual after the pharmaceutical composition of the present invention is administered to the patient, and the expected effect includes the improvement of the individual's condition.
  • the anti-human PD-L1 antibody positive control M8 used in the following examples is derived from PCT/CN2020/090442, and its heavy chain and light chain amino acid sequences are SEQ ID NO: 2 and SEQ ID NO: 5 in the Sequence Listing Table 1, respectively. .
  • the heavy chain and light chain amino acid sequences of the anti-VEGF antibody positive control Bevacizumab used in the following examples are SEQ ID NO: 19 and SEQ ID NO: 20 in Table 1 of the Sequence Listing, respectively.
  • reagents and raw materials used in the following examples can be purchased from commercial sources.
  • an anti-PD-L1 monoclonal antibody and the D2 domain of VEGFR1 are connected in series to construct an anti-PD-L1/VEGF bifunctional fusion protein.
  • the schematic diagram of the structure is shown in FIG. 1.
  • the D2 domain of VEGFR1 in M8-D2-M2 is truncated by 2 amino acids at the C-terminal relative to the D2 domain of VEGFR1 of the fusion protein M8-D2. These two amino acids are easy to fall off during the fermentation process, and the removal does not affect the efficacy .
  • the nucleic acid sequence of the heavy chain of M8-D2 is SEQ ID NO: 8, and the nucleic acid sequence of the light chain is SEQ ID NO: 9.
  • the nucleic acid sequence of the heavy chain of M8-D2-M2 is SEQ ID NO: 10, and the nucleic acid sequence of the light chain is SEQ ID NO: 9.
  • the heavy chain and light chain DNA fragments of the anti-PD-L1/VEGF bifunctional fusion protein were subcloned into pcDNA3.4 vector (purchased from thermofisher, A14697), and the recombinant plasmid was extracted and transfected into CHO cells and/or 293F cells.
  • the protein was eluted in one step with 100 mM citric acid, pH 3.5 eluent, and the target sample was recovered and dialyzed Change the medium to PBS.
  • the purified protein was detected by HPLC, and the antibody molecule was in uniform state, and the monomer purity reached more than 97%.
  • the full-length protein molecule is greater than 180kD (theoretical molecular weight is 168kD), and the heavy chain is at At 70kD, the light chain is at 25-35kD.
  • Example 3 Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-PD-L1/VEGF bifunctional fusion protein to antigen
  • PD-L1-ECD-Fc protein (refer to WO2018/137576A1 for preparation method), plate with 100ng/well, overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the antibody to 100 nM with the diluent, dilute by 4 times to form 12 concentration gradients, and add them to the blocked microtiter plate successively, 100 ⁇ l/well, and place at 37°C for 1 hour.
  • Recombinant VEGF165 protein (purchased from acrobiosystems, Cat.#VE5-H4210) was coated with 100ng/well on the microplate, overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the antibody to 200 nM with the diluent, 4 times the dilution to form 12 concentration gradients, and add them to the blocked microtiter plate successively, 100 ⁇ l/well, and place at 37°C for 1 hour.
  • Example 4 Determination of binding affinity of anti-PD-L1/VEGF bifunctional fusion protein to target cell surface antigen by FACS method
  • the cells were washed twice with PBS containing 0.5% BSA to remove unbound antibodies, and then the cells were incubated with 100 ⁇ l of 1 ⁇ g/ml goat anti-human IgG-FITC (purchased from sigma, Cat. #F9512) at 4° C. for 30 minutes. Centrifuge at 300g for 5 minutes. Wash the cells twice with PBS containing 0.5% BSA to remove unbound secondary antibodies. Finally, resuspend the cells in 200 ⁇ l PBS. Use a Beckman Co ⁇ lter CytoFLEX flow cytometer to determine the antibody against PD-L1 on the surface of CHO cells. The binding affinity. The data obtained was fitted and analyzed by GraphPad Prism6 software.
  • the results of the experiment are shown in Figure 4.
  • the anti-PD-L1/VEGF bifunctional fusion protein and the positive control M8 monoclonal antibody can specifically bind to PD-L1 expressed on the cell surface with equivalent affinity.
  • the EC 50 of M8-D2 and M8 are respectively It is 1.25nM and 0.71nM.
  • the octet molecular interaction analyzer was used to determine the kinetic parameters of the binding and dissociation of the anti-PD-L1/VEGF bifunctional fusion protein and the antigen VEGF165 using the capture method.
  • the antibody at a concentration of 5 ⁇ g/ml was bound to AHC (purchased from PALL life sciences) , Cat.#18-5060) on the probe, dilute the antigen VEGF165 with 1 ⁇ HBS working solution (purchased from GE Healthcare, Cat.#14100669), set 6 concentration gradients with the highest concentration of 25nM to bind to the antibody, Dissociated in the working fluid.
  • the affinity dissociation constants of anti-PD-L1/VEGF bifunctional fusion protein M8-D2 and the positive control Bevacizumab are shown in Table 2 below. The results show that M8-D2 has a higher affinity for VEGF165 than Bevacizumab.
  • KD is the affinity constant
  • kon is the association rate constant
  • kdis is the dissociation rate constant
  • Example 6 Cellular experiment of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of PD-1 and PD-L1
  • PD-L1aAPC/CHO-K1 cells purchased from promega, Cat.#J1252 grown in the logarithmic phase, trypsinize them into single cells and transfer them to a white bottom permeable 96-well plate, 100 ⁇ l/well, 40,000 cells/well, Place at 37°C, 5% CO 2 , and incubate overnight.
  • the anti-PD-L1/VEGF bifunctional fusion protein, positive control M8, and isotype negative control antibody IgG1 were diluted three-fold into 2 ⁇ working solution, the highest concentration was 200 nM, and there were 10 concentration gradients.
  • the PD-1 effector cells (purchased from promega, Cat.#J1252) with a density of 1.4-2 ⁇ 10 6 /ml and a cell viability above 95% were trypsinized to 1.25 ⁇ 10 6 cells/ml.
  • Cell suspension Take the PD-L1aAPC/CHO-K1 cells plated the day before, discard the supernatant, add 40 ⁇ l of the antibody working solution of gradient dilution, and then add an equal volume of PD-1 effector cells. Place at 37°C, 5% CO 2 , and incubate for 6 hours. After the cells were incubated at 37°C for 6 hours, 80 ⁇ l of detection reagent Bio-Glo (purchased from promega, Cat.
  • Example 7 Cellular experiment of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of VEGF and receptor KDR
  • KDR cells purchased from promega, Cat.#GA1082 with a logarithmic growth density of about 80%-90% in adherent culture were taken, and the growth medium was discarded. After washing once with DPBS, use The solution (purchased from Sigma, Cat.#A6964) was digested, and the pancreatin was neutralized, and then centrifuged at 200g for 5 min. The cells were resuspended in DMEM medium (purchased from Gibco, Cat.#11995) containing 10% FBS. Count the blue cells, adjust the cell density to 40,000 cells/well, plate 50 ⁇ l/well, and incubate at 37°C with 5% CO 2.
  • VEGF Dilute VEGF to 30ng/ml with 10% FBS-containing DMEM medium, and dilute the antibody with VEGF-containing medium fold, 3 times dilution, 10 gradients.
  • Example 8 Anti-tumor effect of anti-PD-L1/VEGF bifunctional fusion protein on MC38-hPD-L1 xenograft tumor model
  • MC38-hPD-L1 cells are genetically modified mouse colon cancer MC38 cells by Beijing Biocytogenes Biotechnology Company to overexpress human PD-L1 and knock out mouse PD-L1 cells.
  • MC38-hPD-L1 cells resuspended in PBS were inoculated into B-hPD-L1 humanized mice at a concentration of 5 ⁇ 10 5 cells/0.1ml and a volume of 0.1ml/head (Biocytop (Beijing) Pharmaceutical Technology Co., Ltd. Company) subcutaneously on the right side.
  • the average tumor volume reached approximately 138 mm 3
  • the animals were randomly divided into groups, and the first administration was given on the day of grouping (day 0).
  • the dose of the test sample M8-D2-M2 was 23.2 mg/kg, the dose of the control M8 was set to 20 mg/kg, and the blank control group was given the same volume of normal saline.
  • the experimental results are shown in Figure 7. In this model, M8-D2-M2 and M8 can significantly inhibit tumor growth. At equimolar doses, the anti-tumor effect of M8-D2-M2 takes effect quickly, and the anti-tumor effect during administration is significantly better than that of M8 monoclonal antibody.
  • Example 9 Physical stability of anti-PDL1/VEGF bifunctional fusion protein.
  • DSC Different scanning calorimetry

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Abstract

Provided is an anti-PD-L1/VEGF fusion protein, containing an anti-PD-L1 antibody, a peptide linker L, and a D2 domain of VEGFR1, wherein the N-terminus of the D2 domain of VEGFR1 is connected to the C-terminus of a heavy chain of the anti-PD-L1 antibody by means of the peptide linker L. The fusion protein of the present invention has the potential to treat diseases related to PD-L1 and VEGF activity.

Description

一种抗PD-L1/VEGF融合蛋白An anti-PD-L1/VEGF fusion protein 技术领域Technical field
本发明涉及融合蛋白技术领域，更具体地，涉及一种抗PD-L1/VEGF融合蛋白。The present invention relates to the technical field of fusion proteins, and more specifically, to an anti-PD-L1/VEGF fusion protein.
背景技术Background technique
人程序性细胞死亡受体-1(PD-1)是一种有288个氨基酸的I型膜蛋白，是已知的主要免疫检查点(Immune Checkpoint)之一(Blank et al,2005,Cancer Immunotherapy,54:307-314)。PD-1表达在已经激活的T淋巴细胞，它与配体PD-L1(程序性细胞死亡受体-配体1，programmed cell death-Ligand 1)和PD-L2(程序性细胞死亡受体-配体2，programmed cell death-Ligand 2)结合可以抑制T淋巴细胞的活性及相关的体内细胞免疫反应。PD-L2主要表达在巨噬细胞和树突状细胞，而PD-L1则广泛表达于B、T淋巴细胞及外周细胞如微血管上皮细胞，肺、肝、心等组织细胞中。大量研究表明，PD-1和PD-L1的相互作用不但是维持体内免疫***平衡所必须，也是导致PD-L1表达阳性肿瘤细胞规避免疫监视的主要机制和原因。通过阻断癌细胞对PD-1/PD-L1信号通路的负调控，激活免疫***，能够促进T细胞相关的肿瘤特异性细胞免疫反应，从而打开了一扇新的肿瘤治疗方法的大门-肿瘤免疫疗法。Human Programmed Cell Death Receptor-1 (PD-1) is a type I membrane protein with 288 amino acids. It is one of the major known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy) , 54: 307-314). PD-1 is expressed on activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- 1). Ligand 2, programmed cell death-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in the body. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in the body, but also the main mechanism and reason that causes PD-L1 positive tumor cells to evade immune surveillance. By blocking the negative regulation of the PD-1/PD-L1 signaling pathway by cancer cells and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses, thereby opening the door to a new tumor treatment method-tumor Immunotherapy.
PD-1(由基因Pdcd1编码)为与CD28和CTLA-4有关的免疫球蛋白超家族成员。研究成果显示，当PD-1与其配体(PD-L1和/或PD-L2)结合时会负调节抗原受体信号转导。目前已弄清鼠PD-1结构以及小鼠PD-1与人PD-L1的共结晶结构(Zhang,X.等,Immunity 20:337-347(2004)；Lin等,Proc.Natl.Acad.Sci.USA 105:3011-6(2008))。PD-1及类似的家族成员为I型跨膜糖蛋白，其含有负责配体结合的Ig可变型(V-型)结构域和负责结合信号转导分子的胞质尾区。PD-1胞质尾区含有两个基于酪氨酸的信号转导模体ITIM(免疫受体酪氨酸抑制作用模体)和ITSM(免疫受体酪氨酸转换作用模体)。PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and/or PD-L2), it negatively regulates antigen receptor signal transduction. At present, the structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al., Immunity 20:337-347 (2004); Lin et al., Proc. Natl. Acad. Sci.USA 105:3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (Immunoreceptor Tyrosine Inhibition Motif) and ITSM (Immune Receptor Tyrosine Switch Motif).
PD-1在肿瘤的免疫逃避机制中起到了重要的作用。肿瘤免疫疗法，即利用人体自身的免疫***抵御癌症，是一种突破性的肿瘤治疗方法，但是肿瘤微环境可保护肿瘤细胞免受有效的免疫破坏，因此如何打破肿瘤微环境成为抗肿瘤研究的重点。现有研究成果已确定了PD-1在肿瘤微环境中的作用：PD-L1在许多小鼠和人肿瘤中表达(并在大多数PD-L1阴性肿瘤细胞系中可由IFN-γ诱导)，并被推定为介导肿瘤免疫逃避的重要靶点(Iwai Y.等,Proc.Natl.Acad.Sci.U.S.A.99:12293-12297(2002)；Strome S.E.等,Cancer Res.,63:6501-6505(2003))。通过免疫组织化学评估活组织检查，已经在人的很多原发性肿瘤中发现PD-1(在肿瘤浸润淋巴细胞上)和/或PD-L1在肿瘤细胞上的表达。这样的组织包括肺癌、肝癌、卵巢 癌、***、皮肤癌、结肠癌、神经胶质瘤、膀胱癌、乳腺癌、肾癌、食道癌、胃癌、口腔鳞状细胞癌、尿道上皮细胞癌和胰腺癌以及头颈肿瘤等。由此可见，阻断PD-1/PD-L1的相互作用可以提高肿瘤特异性T细胞的免疫活性，有助于免疫***清除肿瘤细胞，因此PD-L1成为开发肿瘤免疫治疗药物的热门靶点。PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, which uses the body’s own immune system to fight cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction. Therefore, how to break the tumor microenvironment has become an anti-tumor research Focus. Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN-γ in most PD-L1-negative tumor cell lines), It is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505) (2003)). Through immunohistochemical assessment of biopsies, the expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found in many primary human tumors. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors. It can be seen that blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-L1 has become a popular target for the development of tumor immunotherapy drugs. .
肿瘤的生长有两个阶段，从无血管的缓慢生长期到有血管的快速增殖期。如果肿瘤内部没有血管的生成，则原发肿瘤生长缓慢，转移无法实现。因此抑制肿瘤血管生成被认为是当前具有前途的肿瘤治疗方法之一。血管内皮生长因子(VEGFs)家族中，VEGF-A165(以下简称VEGF)是最丰富活跃的亚型。VEGF通过与II型受体VEGFR2结合，激活信号通路发生一系列级联反应，促进新生血管形成并维持其完整性。但I型受体VEGFR1与VEGF结合的能力远大于VEGFR2，发生作用部位主要是VEGFR1的胞外区D2结构域。VEGFR1-D2通过竞争结合VEGF，阻断VEGFR2与VEGF结合，从而阻断信号通路，抑制内皮细胞增殖与血管生成，从而抑制肿瘤的快速增殖与转移。There are two stages of tumor growth, from the avascular phase of slow growth to the vascularized phase of rapid proliferation. If there is no angiogenesis in the tumor, the primary tumor grows slowly and metastasis cannot be achieved. Therefore, the inhibition of tumor angiogenesis is considered as one of the promising tumor treatment methods. In the vascular endothelial growth factor (VEGFs) family, VEGF-A165 (hereinafter referred to as VEGF) is the most abundant and active subtype. VEGF binds to the type II receptor VEGFR2 to activate a series of cascade reactions in signal pathways, promote the formation of new blood vessels and maintain their integrity. However, the type I receptor VEGFR1 has a much greater ability to bind to VEGF than VEGFR2, and the main site of action is the D2 domain of the extracellular domain of VEGFR1. VEGFR1-D2 competes for binding to VEGF and blocks the binding of VEGFR2 to VEGF, thereby blocking the signaling pathway, inhibiting endothelial cell proliferation and angiogenesis, thereby inhibiting the rapid proliferation and metastasis of tumors.
发明内容Summary of the invention
本发明的目的在于提供一种新的抗PD-L1/VEGF融合蛋白，能同时阻断PD-L1和VEGF信号通路。本发明的目的还在于提供编码所述融合蛋白的核酸分子；提供包含所述核酸分子的表达载体；提供包含所述表达载体的宿主细胞；提供所述融合蛋白的制备方法；提供包含所述融合蛋白的药物组合物；提供所述融合蛋白或所述药物组合物在制备治疗癌症的药物中的应用；提供所述融合蛋白或所述药物组合物用于治疗癌症的方法。The purpose of the present invention is to provide a new anti-PD-L1/VEGF fusion protein that can simultaneously block PD-L1 and VEGF signal pathways. The object of the present invention is also to provide a nucleic acid molecule encoding the fusion protein; to provide an expression vector containing the nucleic acid molecule; to provide a host cell containing the expression vector; to provide a method for preparing the fusion protein; to provide a method for preparing the fusion protein; Protein pharmaceutical composition; provide the application of the fusion protein or the pharmaceutical composition in the preparation of drugs for treating cancer; provide the method for the fusion protein or the pharmaceutical composition to treat cancer.
为了达到上述目的，本发明提供了以下技术方案：In order to achieve the above objective, the present invention provides the following technical solutions:
本发明的第一个方面提供了一种抗PD-L1/VEGF融合蛋白，包含抗PD-L1抗体和VEGFR1的D2结构域；所述抗PD-L1抗体重链包含互补决定区HCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：11所示，HCDR2的氨基酸序列如SEQ ID NO：12所示，HCDR3的氨基酸序列如SEQ ID NO：13所示；所述抗PD-L1抗体轻链包含互补决定区LCDR1-3，其中LCDR1的氨基酸序列如SEQ ID NO：14所示，LCDR2的氨基酸序列如SEQ ID NO：15所示，LCDR3的氨基酸序列如SEQ ID NO：16所示。The first aspect of the present invention provides an anti-PD-L1/VEGF fusion protein comprising an anti-PD-L1 antibody and the D2 domain of VEGFR1; the heavy chain of the anti-PD-L1 antibody comprises a complementarity determining region HCDR1-3, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13; the light chain of the anti-PD-L1 antibody includes In the complementarity determining region LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16.
在一个优选的实施方案中，所述VEGFR1的D2结构域的N末端通过肽接头L连接至抗PD-L1抗体重链的C末端。In a preferred embodiment, the N-terminus of the D2 domain of VEGFR1 is connected to the C-terminus of the heavy chain of the anti-PD-L1 antibody via a peptide linker L.
在一个优选的实施方案中，所述抗PD-L1抗体重链可变区的氨基酸序列如SEQ ID NO：17所示，所述抗PD-L1抗体轻链可变区的氨基酸序列如SEQ ID NO：18所示。In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: shown in 18.
在一个优选的实施方案中，所述抗PD-L1抗体为单克隆抗体。In a preferred embodiment, the anti-PD-L1 antibody is a monoclonal antibody.
在一个优选的实施方案中，所述抗PD-L1抗体为人源化抗体。In a preferred embodiment, the anti-PD-L1 antibody is a humanized antibody.
在一个优选的实施方案中，所述抗PD-L1抗体为IgG类抗体。In a preferred embodiment, the anti-PD-L1 antibody is an IgG antibody.
在一个优选的实施方案中，所述肽接头L的氨基酸序列如SEQ ID NO：3所示。In a preferred embodiment, the amino acid sequence of the peptide linker L is shown in SEQ ID NO: 3.
在一个优选的实施方案中，所述VEGFR1的D2结构域的氨基酸序列如SEQ ID NO：1或SEQ ID NO：6所示。In a preferred embodiment, the amino acid sequence of the D2 domain of VEGFR1 is shown in SEQ ID NO: 1 or SEQ ID NO: 6.
在一个优选的实施方案中，所述融合蛋白选自M8-D2和M8-D2-M2。M8-D2-M2中的VEGFR1的D2结构域相对融合蛋白M8-D2的VEGFR1的D2结构域在C末端截断2个氨基酸，该两个氨基酸在发酵过程中容易脱落，且去除并不影响药效。In a preferred embodiment, the fusion protein is selected from M8-D2 and M8-D2-M2. The D2 domain of VEGFR1 in M8-D2-M2 is truncated by 2 amino acids at the C-terminal relative to the D2 domain of VEGFR1 of the fusion protein M8-D2. These two amino acids are easy to fall off during the fermentation process, and the removal does not affect the efficacy .
在一个优选的实施方案中，所述融合蛋白的重链氨基酸序列如SEQ ID NO：4或SEQ ID NO：7所示，所述融合蛋白的轻链氨基酸序列如SEQ ID NO：5所示。In a preferred embodiment, the heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4 or SEQ ID NO: 7, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 5.
本发明的第二个方面提供了一种核酸分子，所述核酸分子编码所述的融合蛋白。The second aspect of the present invention provides a nucleic acid molecule encoding the fusion protein.
在一个优选的实施方案中，所述核酸分子编码融合蛋白重链的核酸序列如SEQ ID NO：8或SEQ ID NO：10所示，编码其轻链的核酸序列如SEQ ID NO：9所示。In a preferred embodiment, the nucleic acid sequence encoding the heavy chain of the fusion protein is shown in SEQ ID NO: 8 or SEQ ID NO: 10, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 9 .
本领域技术人员知晓，编码上述融合蛋白的氨基酸序列的核酸分子可以适当引入替换、缺失、改变、***或增加来提供一个核酸分子的同系物。Those skilled in the art know that the nucleic acid molecule encoding the amino acid sequence of the above-mentioned fusion protein can be replaced, deleted, changed, inserted or added as appropriate to provide a homolog of the nucleic acid molecule.
本发明的第三个方面提供了一种表达载体，所述表达载体含有上述的核酸分子。The third aspect of the present invention provides an expression vector containing the above-mentioned nucleic acid molecule.
本发明的第四个方面提供了一种宿主细胞，所述宿主细胞含有上述的表达载体。The fourth aspect of the present invention provides a host cell containing the above-mentioned expression vector.
本发明的第五个方面提供了一种融合蛋白的制备方法，所述制备方法包括以下步骤：The fifth aspect of the present invention provides a method for preparing a fusion protein, which includes the following steps:
a)在表达条件下，培养如上所述宿主细胞，从而表达抗PD-L1/VEGF融合蛋白；a) Under expression conditions, culture the host cell as described above to express the anti-PD-L1/VEGF fusion protein;
b)分离并纯化步骤a)所述的融合蛋白。b) Isolation and purification of the fusion protein described in step a).
本发明的第六个方面提供了一种药物组合物，所述药物组合物包含有效量的上述的融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。The sixth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the above-mentioned fusion protein and one or more pharmaceutically acceptable carriers, diluents or excipients.
本发明的第七个方面提供了上述的融合蛋白、药物组合物在制备治疗癌症的药物中的用途。The seventh aspect of the present invention provides the use of the above-mentioned fusion protein and pharmaceutical composition in the preparation of drugs for the treatment of cancer.
根据本发明，所述癌症选自：黑色素瘤、胃癌、肾癌、尿路上皮癌、肺癌、肝癌、结直肠癌、膀胱癌、食道癌、***癌、胰腺癌、乳腺癌、卵巢癌、***、子宫癌、输卵管癌、原发性腹膜癌、甲状腺癌、胶质瘤、白血病、淋巴瘤、皮肤癌、头颈癌。According to the present invention, the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial cancer, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer Cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer.
在一个优选的实施方案中，所述癌症是结肠癌。In a preferred embodiment, the cancer is colon cancer.
本发明所述抗PD-L1/VEGF融合蛋白可以单独使用或与其它抗肿瘤药物联合使用。The anti-PD-L1/VEGF fusion protein of the present invention can be used alone or in combination with other anti-tumor drugs.
本发明所称的治疗癌症的药物，指具有抑制和/或***的药物，可以包括伴随肿瘤相关症状发展的延迟和/或这些症状严重程度的降低，进一步还包括已存在的肿瘤伴随症状的减轻并防止其他症状的出现，还包括减少或防止肿瘤的转移等。The cancer treatment drugs referred to in the present invention refer to drugs that inhibit and/or treat tumors, which may include delays in the development of tumor-related symptoms and/or reduction in the severity of these symptoms, and further include symptoms associated with existing tumors. Reduce and prevent the appearance of other symptoms, including reducing or preventing tumor metastasis.
本发明中抗PD-L1/VEGF融合蛋白及其药物组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。When the anti-PD-L1/VEGF fusion protein and its pharmaceutical composition of the present invention are administered to animals including humans, the dosage of administration varies with the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. , You can refer to the results of animal experiments and various situations, and the total dose cannot exceed a certain range. On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present invention.
本发明的第八个方面提供了一种治疗癌症的方法，包括向有需要的受试者施用上述融合蛋白或药物组合物。The eighth aspect of the present invention provides a method of treating cancer, which comprises administering the above-mentioned fusion protein or pharmaceutical composition to a subject in need.
根据本发明，所述癌症选自：黑色素瘤、胃癌、肾癌、尿路上皮癌、肺癌、肝癌、结直肠癌、膀胱癌、食道癌、***癌、胰腺癌、乳腺癌、卵巢癌、***、子宫癌、输卵管癌、原发性腹膜癌、甲状腺癌、胶质瘤、白血病、淋巴瘤、皮肤癌、头颈癌。According to the present invention, the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial cancer, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer Cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer.
在一个优选的实施方案中，所述癌症是结肠癌。In a preferred embodiment, the cancer is colon cancer.
所述抗PD-L1/VEGF融合蛋白及其药物组合物在向受试者施用时，给药剂量需为治疗有效量。所述治疗有效量是指在治疗癌症中有效果的量。具体的，所述抗PD-L1/VEGF融合蛋白及其药物组合物在向受试者施用时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。When the anti-PD-L1/VEGF fusion protein and the pharmaceutical composition thereof are administered to a subject, the administered dose must be a therapeutically effective amount. The therapeutically effective amount refers to an amount effective in treating cancer. Specifically, when the anti-PD-L1/VEGF fusion protein and its pharmaceutical composition are administered to a subject, the dosage will vary depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. With reference to the results of animal experiments and various situations, the total dose should not exceed a certain range.
本发明的积极效果在于：本发明的融合蛋白能与PD-L1和VEGF高亲和力结合，其与PD-L1的亲和力与抗PD-L1单抗阳性对照M8相当，亲和解离常数测定表明其与VEGF的亲和力高于抗VEGF单抗阳性对照Bevacizumab。本发明的融合蛋白能有效阻断PD-1与PD-L1的结合，其阻断能力与抗PD-L1单抗阳性对照M8相当，且其能够有效阻断VEGF与其受体KDR之间的相互作用，其阻断能力优于抗VEGF单抗阳性对照Bevacizumab。本发明的融合蛋白能显著抑制结肠癌移植瘤生长，其抑瘤作用起效快，抑瘤效果明显优于抗PD-L1单抗阳性对照M8。本发明的融合蛋白具有治疗PD-L1和VEGF活性相关的疾病的潜力。The positive effect of the present invention is that the fusion protein of the present invention can bind to PD-L1 and VEGF with high affinity, and its affinity to PD-L1 is equivalent to that of the anti-PD-L1 monoclonal antibody positive control M8. The determination of the affinity dissociation constant shows that it is The affinity of VEGF is higher than that of anti-VEGF monoclonal antibody positive control Bevacizumab. The fusion protein of the present invention can effectively block the binding of PD-1 and PD-L1, and its blocking ability is equivalent to the anti-PD-L1 monoclonal antibody positive control M8, and it can effectively block the interaction between VEGF and its receptor KDR Its blocking ability is better than that of anti-VEGF monoclonal antibody positive control Bevacizumab. The fusion protein of the present invention can significantly inhibit the growth of colon cancer transplantation tumors, and its anti-tumor effect takes effect quickly, and its anti-tumor effect is obviously better than that of the anti-PD-L1 monoclonal antibody positive control M8. The fusion protein of the present invention has the potential to treat diseases related to PD-L1 and VEGF activity.
附图说明Description of the drawings
图1：抗PD-L1/VEGF双功能融合蛋白结构示意图Figure 1: Schematic diagram of the structure of anti-PD-L1/VEGF bifunctional fusion protein
图2：抗PD-L1/VEGF双功能融合蛋白电泳检测图Figure 2: Electrophoresis detection image of anti-PD-L1/VEGF bifunctional fusion protein
图3A：抗PD-L1/VEGF双功能融合蛋白与PD-L1亲和力的ELISA检测图Figure 3A: ELISA detection diagram of the affinity between anti-PD-L1/VEGF bifunctional fusion protein and PD-L1
图3B：抗PD-L1/VEGF双功能融合蛋白与VEGF亲和力的ELISA检测图Figure 3B: ELISA detection diagram of the affinity of anti-PD-L1/VEGF bifunctional fusion protein and VEGF
图4：抗PD-L1/VEGF双功能融合蛋白对靶细胞表面抗原结合亲和力的FACS检测图Figure 4: FACS detection diagram of the binding affinity of anti-PD-L1/VEGF bifunctional fusion protein to the surface antigen of target cells
图5：抗PD-L1/VEGF双功能融合蛋白阻断PD-1与PD-L1结合的细胞实验检测图Figure 5: Cellular experiment detection diagram of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of PD-1 and PD-L1
图6：抗PD-L1/VEGF双功能融合蛋白阻断VEGF与受体KDR结合的细胞实验检测图Figure 6: Cellular experimental detection diagram of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of VEGF and receptor KDR
图7：抗PD-L1/VEGF双功能融合蛋白在MC38-hPD-L1移植瘤模型上的抗肿瘤作用图Figure 7: Anti-tumor effect of anti-PD-L1/VEGF bifunctional fusion protein on MC38-hPD-L1 xenograft tumor model
具体实施方式detailed description
以下实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法或本领域常规方法的详细描述，如核酸分子的制备方法，用于构建载体和质粒的方法，将编码蛋白的基因***到这样的载体和质粒的方法或将质粒引入宿主细胞的方法，宿主细胞的培养方法等。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual,2nd edition,Cold spring Harbor Laboratory Press。The following experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods or conventional methods in the art, such as methods for preparing nucleic acid molecules, methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or introducing plasmids Host cell method, host cell culture method, etc. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.
本发明中，术语“融合蛋白”是指由两个或多个相同或不同的多肽序列融合得到的新的多肽序列。术语“融合”是指由肽键直接连接或借助一个或多个连接肽(肽接头)有效连接。术语“连接肽(肽接头)”是指可以连接两个多肽序列的短肽，一般是长度为2-30个氨基酸的肽。In the present invention, the term "fusion protein" refers to a new polypeptide sequence obtained by the fusion of two or more identical or different polypeptide sequences. The term "fusion" refers to direct connection by peptide bonds or operative connection via one or more connecting peptides (peptide linkers). The term "connecting peptide (peptide linker)" refers to a short peptide that can connect two polypeptide sequences, generally a peptide of 2-30 amino acids in length.
本发明中，术语“抗体(Antibody，缩写Ab)”是指有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条重链的一端有可变区(VH)，其后是恒定区。重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。In the present invention, the term "antibody (Ab)" refers to a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains. Chain (H) composition. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain. Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on. The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda). The heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。In the present invention, the term "monoclonal antibody (monoclonal antibody)" refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins.
本发明中，术语“人源化”是指其CDR来源于非人物种(优选小鼠)抗体，抗体分子中残余的部分(包括框架区和恒定区)来源于人抗体。此外，框架区残基可被改变以维持结合亲和性。In the present invention, the term "humanized" means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies. In addition, framework residues can be changed to maintain binding affinity.
本发明中，术语“抗”和“结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M、10 -10M、10 -11M或更小的平衡解离常数(KD)结合该抗原。术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。 In the present invention, the terms "anti" and "binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. Generally, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less. The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, Surface Plasmon Resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or an ELISA is used to measure the relative binding affinity of an antibody to the antigen.
本发明中，术语“表达载体”是指包含适当的调控序列，例如启动子、终止子、增强子等的本领域的常规表达载体，所述表达载体可以是病毒或质粒。所述表达载体较佳地包括pDR1、pcDNA3.4(+)、pDHFR或pTT5。In the present invention, the term "expression vector" refers to a conventional expression vector in the art that contains appropriate regulatory sequences, such as a promoter, a terminator, an enhancer, etc. The expression vector may be a virus or a plasmid. The expression vector preferably includes pDR1, pcDNA3.4(+), pDHFR or pTT5.
本发明中，术语“宿主细胞”为本领域常规的各种宿主细胞，只要能使载体稳定地自行复制，且所携带的核酸分子可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地选自：COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)、TG1、BL21(DE3)、293F细胞或293E细胞。In the present invention, the term "host cell" refers to various conventional host cells in the art, as long as the vector can stably replicate itself and the nucleic acid molecule carried can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells are preferably selected from: COS, CHO, NS0, sf9, sf21, DH5α, BL21(DE3), TG1, BL21(DE3), 293F Cell or 293E cell.
本发明中，术语“有效量”是指本发明的药物组合物施用患者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善。In the present invention, the term "effective amount" refers to the amount or dose that produces the expected effect in the treated individual after the pharmaceutical composition of the present invention is administered to the patient, and the expected effect includes the improvement of the individual's condition.
以下实施例中涉及的序列信息总结在序列表表1中。The sequence information involved in the following examples is summarized in Table 1 of the Sequence Listing.
表1 序列表Table 1 Sequence Listing
Figure PCTCN2021096084-appb-000001
Figure PCTCN2021096084-appb-000001
Figure PCTCN2021096084-appb-000002
Figure PCTCN2021096084-appb-000002
Figure PCTCN2021096084-appb-000003
Figure PCTCN2021096084-appb-000003
Figure PCTCN2021096084-appb-000004
Figure PCTCN2021096084-appb-000004
Figure PCTCN2021096084-appb-000005
Figure PCTCN2021096084-appb-000005
以下实施例中使用的抗人PD-L1抗体阳性对照M8来源于PCT/CN2020/090442，其重链和轻链氨基酸序列分别为序列表表1中的SEQ ID NO：2和SEQ ID NO：5。The anti-human PD-L1 antibody positive control M8 used in the following examples is derived from PCT/CN2020/090442, and its heavy chain and light chain amino acid sequences are SEQ ID NO: 2 and SEQ ID NO: 5 in the Sequence Listing Table 1, respectively. .
以下实施例中使用的抗VEGF抗体阳性对照Bevacizumab的重链和轻链氨基酸序列分别为序列表表1中的SEQ ID NO：19和SEQ ID NO：20。The heavy chain and light chain amino acid sequences of the anti-VEGF antibody positive control Bevacizumab used in the following examples are SEQ ID NO: 19 and SEQ ID NO: 20 in Table 1 of the Sequence Listing, respectively.
以下实施例中所用试剂和原料若无特殊说明，均可从商业途径购得。Unless otherwise specified, the reagents and raw materials used in the following examples can be purchased from commercial sources.
实施例1.抗PD-L1/VEGF双功能融合蛋白构建Example 1. Construction of anti-PD-L1/VEGF bifunctional fusion protein
本发明采用了抗PD-L1单克隆抗体和VEGFR1的D2结构域串联的方式，构建了抗PD-L1/VEGF双功能融合蛋白，结构示意图如图1所示。In the present invention, an anti-PD-L1 monoclonal antibody and the D2 domain of VEGFR1 are connected in series to construct an anti-PD-L1/VEGF bifunctional fusion protein. The schematic diagram of the structure is shown in FIG. 1.
融合蛋白M8-D2Fusion protein M8-D2
将VEGFR1的D2结构域(SEQ ID NO：1)的N末端和抗PD-L1单克隆抗体M8的重链(SEQ ID NO：2)的C末端通过肽接头L(SEQ ID NO：3)连接起来，得到融合蛋白的重链(SEQ ID NO：4)，融合蛋白的轻链序列为SEQ ID NO：5。Connect the N-terminus of the D2 domain (SEQ ID NO: 1) of VEGFR1 and the C-terminus of the heavy chain (SEQ ID NO: 2) of the anti-PD-L1 monoclonal antibody M8 through a peptide linker L (SEQ ID NO: 3) Get the heavy chain of the fusion protein (SEQ ID NO: 4), and the light chain sequence of the fusion protein is SEQ ID NO: 5.
融合蛋白M8-D2-M2Fusion protein M8-D2-M2
将VEGFR1的D2结构域(SEQ ID NO：6)的N末端和抗PD-L1单克隆抗体M8的重链(SEQ ID NO：2)的C末端通过肽接头L(SEQ ID NO：3)连接起来，得到融合蛋白的重链(SEQ ID NO：7)，融合蛋白的轻链序列为SEQ ID NO：5。Connect the N-terminus of the D2 domain (SEQ ID NO: 6) of VEGFR1 and the C-terminus of the heavy chain (SEQ ID NO: 2) of the anti-PD-L1 monoclonal antibody M8 through a peptide linker L (SEQ ID NO: 3) Get the heavy chain of the fusion protein (SEQ ID NO: 7), and the light chain sequence of the fusion protein is SEQ ID NO: 5.
M8-D2-M2中的VEGFR1的D2结构域相对融合蛋白M8-D2的VEGFR1的D2结构域在C末端截断2个氨基酸，该两个氨基酸在发酵过程中容易脱落，且去除并不影响药效。The D2 domain of VEGFR1 in M8-D2-M2 is truncated by 2 amino acids at the C-terminal relative to the D2 domain of VEGFR1 of the fusion protein M8-D2. These two amino acids are easy to fall off during the fermentation process, and the removal does not affect the efficacy .
实施例2.抗PD-L1/VEGF双功能融合蛋白的表达与纯化Example 2. Expression and purification of anti-PD-L1/VEGF bifunctional fusion protein
M8-D2重链核酸序列为SEQ ID NO：8，轻链核酸序列为SEQ ID NO：9。M8-D2-M2重链核酸序列为SEQ ID NO：10，轻链核酸序列为SEQ ID NO：9。将抗PD-L1/VEGF双功能融合蛋白重链和轻链的DNA片段分别亚克隆到pcDNA3.4载体(购自thermofisher,A14697)中，提取重组质粒共转染CHO细胞和/或293F细胞。细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后上样至HiTrap MabSelect SuRe柱，用100mM柠檬酸，pH3.5的洗脱液一步洗脱蛋白，回收目标样品并透析换液至PBS。将纯化后的蛋白用HPLC检测，抗体分子状态均一，单体纯度达到97％以上。分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行检测，结果如图2所示，全长蛋白分子在大于180kD处(理论分子量168kD)，重链在70kD处，轻链在25-35kD处。The nucleic acid sequence of the heavy chain of M8-D2 is SEQ ID NO: 8, and the nucleic acid sequence of the light chain is SEQ ID NO: 9. The nucleic acid sequence of the heavy chain of M8-D2-M2 is SEQ ID NO: 10, and the nucleic acid sequence of the light chain is SEQ ID NO: 9. The heavy chain and light chain DNA fragments of the anti-PD-L1/VEGF bifunctional fusion protein were subcloned into pcDNA3.4 vector (purchased from thermofisher, A14697), and the recombinant plasmid was extracted and transfected into CHO cells and/or 293F cells. After the cells were cultured for 7 days, the culture solution was subjected to high-speed centrifugation, vacuum filtration with a microporous membrane, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with 100 mM citric acid, pH 3.5 eluent, and the target sample was recovered and dialyzed Change the medium to PBS. The purified protein was detected by HPLC, and the antibody molecule was in uniform state, and the monomer purity reached more than 97%. Add reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer, respectively, and test after boiling. The results are shown in Figure 2. The full-length protein molecule is greater than 180kD (theoretical molecular weight is 168kD), and the heavy chain is at At 70kD, the light chain is at 25-35kD.
实施例3.酶联免疫吸附法(ELISA)测定抗PD-L1/VEGF双功能融合蛋白对抗原的亲和力Example 3. Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-PD-L1/VEGF bifunctional fusion protein to antigen
3.1 ELISA检测抗PD-L1/VEGF双功能融合蛋白与PD-L1的亲和力3.1 ELISA to detect the affinity of anti-PD-L1/VEGF bifunctional fusion protein and PD-L1
自制重组PD-L1-ECD-Fc蛋白(制备方法参考WO2018/137576A1)，以100ng/孔包板，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗体至100nM，4倍比稀释形成12个浓度梯度，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，加入HRP标记的羊抗人Fab抗体(购自abcam，Cat.#ab87422)，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。 Homemade recombinant PD-L1-ECD-Fc protein (refer to WO2018/137576A1 for preparation method), plate with 100ng/well, overnight at 4°C. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the antibody to 100 nM with the diluent, dilute by 4 times to form 12 concentration gradients, and add them to the blocked microtiter plate successively, 100 μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat.#ab87422), and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and place at room temperature (20±5°C) in the dark for 5 minutes; add stop solution to each well to stop the substrate reaction, enzyme label instrument read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC 50.
实验结果如图3A所示，抗PD-L1/VEGF双功能融合蛋白和阳性对照M8单抗与PD-L1-ECD结合亲和力相当。M8、M8-D2、M8-D2-M2的EC 50分别为0.15nM、0.24nM、0.23nM。 The experimental results are shown in Figure 3A. The binding affinity of anti-PD-L1/VEGF bifunctional fusion protein and positive control M8 monoclonal antibody is equivalent to PD-L1-ECD. The EC 50 of M8, M8-D2, and M8-D2-M2 were 0.15nM, 0.24nM, 0.23nM, respectively.
3.2 ELISA检测抗PD-L1/VEGF双功能融合蛋白与VEGF的亲和力3.2 ELISA to detect the affinity of anti-PD-L1/VEGF bifunctional fusion protein and VEGF
将重组VEGF165蛋白(购自acrobiosystems，Cat.#VE5-H4210)以100ng/孔包被酶标板，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗体至200nM，4倍比稀释形成12个浓度梯度，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，加入HRP标记的羊抗人Fab抗体(购自abcam，Cat.#ab87422)，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。 Recombinant VEGF165 protein (purchased from acrobiosystems, Cat.#VE5-H4210) was coated with 100ng/well on the microplate, overnight at 4°C. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the antibody to 200 nM with the diluent, 4 times the dilution to form 12 concentration gradients, and add them to the blocked microtiter plate successively, 100 μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat.#ab87422), and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and place at room temperature (20±5°C) in the dark for 5 minutes; add stop solution to each well to stop the substrate reaction, enzyme label instrument read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC 50.
实验结果如图3B所示，M8-D2和阳性对照Bevacizumab单抗与VEGF结合的亲和力相当。M8-D2和Bevacizumab的EC 50分别为0.89nM和0.85nM。 The experimental results are shown in Figure 3B. The binding affinity of M8-D2 and the positive control Bevacizumab monoclonal antibody to VEGF is equivalent. The EC 50 of M8-D2 and Bevacizumab are 0.89nM and 0.85nM, respectively.
实施例4.FACS法测定抗PD-L1/VEGF双功能融合蛋白对靶细胞表面抗原的结合亲和力Example 4. Determination of binding affinity of anti-PD-L1/VEGF bifunctional fusion protein to target cell surface antigen by FACS method
本实验以细胞表面表达PD-L1的PD-L1aAPC/CHO-K1细胞(购自promega，Cat.#J1252)作为靶细胞，将靶细胞按照2×10 5/孔接种于96孔板，用含有0.5％BSA的PBS洗涤三次，每次300g离心5分钟，弃上清。将100μl按照3倍梯度从83.5nM连续稀释11个梯度的抗体作为一抗加入96孔板，将细胞悬浮后于4℃孵育1h。用含有0.5％BSA的PBS洗涤细胞两次以去除未结合的抗体，再将细胞与100μl的1μg/ml羊抗人IgG-FITC(购自sigma，Cat.#F9512)于4℃孵育30分钟。300g离心5分钟，用含有0.5％BSA的PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μlPBS中，通过Beckman Coμlter CytoFLEX流式细胞仪测定抗体对CHO细胞表面PD-L1的结合亲和力。所得数据通过GraphPad Prism6软件拟合分析。 In this experiment, cell surface PD-L1 expression in PD-L1aAPC / CHO-K1 cells (available from promega, Cat. # J1252) as target cells, the target cells according to 2 × 10 5 / well were seeded in 96-well plates, containing Wash three times with 0.5% BSA in PBS, centrifuge at 300g for 5 minutes each time, and discard the supernatant. 100 μl of antibodies, which were serially diluted with 11 gradients from 83.5 nM in a 3-fold gradient, were added as primary antibodies to a 96-well plate, and the cells were suspended and incubated for 1 h at 4°C. The cells were washed twice with PBS containing 0.5% BSA to remove unbound antibodies, and then the cells were incubated with 100 μl of 1 μg/ml goat anti-human IgG-FITC (purchased from sigma, Cat. #F9512) at 4° C. for 30 minutes. Centrifuge at 300g for 5 minutes. Wash the cells twice with PBS containing 0.5% BSA to remove unbound secondary antibodies. Finally, resuspend the cells in 200μl PBS. Use a Beckman Coμlter CytoFLEX flow cytometer to determine the antibody against PD-L1 on the surface of CHO cells. The binding affinity. The data obtained was fitted and analyzed by GraphPad Prism6 software.
实验结果如图4所示，抗PD-L1/VEGF双功能融合蛋白和阳性对照M8单抗可以特异性的结合细胞表面表达的PD-L1，并且亲和力相当，M8-D2和M8的EC 50分别为1.25nM和0.71nM。 The results of the experiment are shown in Figure 4. The anti-PD-L1/VEGF bifunctional fusion protein and the positive control M8 monoclonal antibody can specifically bind to PD-L1 expressed on the cell surface with equivalent affinity. The EC 50 of M8-D2 and M8 are respectively It is 1.25nM and 0.71nM.
实施例5.抗PD-L1/VEGF双功能融合蛋白对抗原VEGF的亲和解离常数KD的测定Example 5. Determination of the affinity dissociation constant KD of anti-PD-L1/VEGF bifunctional fusion protein to antigen VEGF
利用octet分子相互作用分析仪，使用捕获法测定抗PD-L1/VEGF双功能融合蛋白和抗原VEGF165结合解离的动力学参数，将浓度为5μg/ml的抗体结合在AHC(购自PALL life sciences，Cat.#18-5060)探针上，将抗原VEGF165用1×HBS工作液(购自GE Healthcare，Cat.#14100669)稀释，设最高浓度为25nM的6个浓度梯度与抗体结合，于HBS工作液中解离。抗PD-L1/VEGF双功能融合蛋白M8-D2与阳性对照Bevacizumab的亲和解离常数见下表表2。结果表明，M8-D2比Bevacizumab对VEGF165具有更高的亲和力。The octet molecular interaction analyzer was used to determine the kinetic parameters of the binding and dissociation of the anti-PD-L1/VEGF bifunctional fusion protein and the antigen VEGF165 using the capture method. The antibody at a concentration of 5 μg/ml was bound to AHC (purchased from PALL life sciences) , Cat.#18-5060) on the probe, dilute the antigen VEGF165 with 1×HBS working solution (purchased from GE Healthcare, Cat.#14100669), set 6 concentration gradients with the highest concentration of 25nM to bind to the antibody, Dissociated in the working fluid. The affinity dissociation constants of anti-PD-L1/VEGF bifunctional fusion protein M8-D2 and the positive control Bevacizumab are shown in Table 2 below. The results show that M8-D2 has a higher affinity for VEGF165 than Bevacizumab.
表2 亲和解离常数Table 2 Affinity dissociation constant
Figure PCTCN2021096084-appb-000006
Figure PCTCN2021096084-appb-000006
注：KD为亲和力常数；kon为结合速率常数；kdis为解离速率常数。Note: KD is the affinity constant; kon is the association rate constant; kdis is the dissociation rate constant.
实施例6.抗PD-L1/VEGF双功能融合蛋白阻断PD-1与PD-L1结合的细胞实验Example 6. Cellular experiment of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of PD-1 and PD-L1
取对数期生长的PD-L1aAPC/CHO-K1细胞(购自promega，Cat.#J1252)，胰酶消化成单个细胞后转移到白色底透96孔板，100μl/孔，40000细胞/孔，置于37℃，5％CO 2，孵育过夜。将抗PD-L1/VEGF双功能融合蛋白、阳性对照M8、同型阴性对照抗体IgG1按三倍梯度稀释成2×工作液，最高浓度200nM，共10个浓度梯度。同时，取密度在1.4-2×10 6/ml，细胞活率在95％以上的PD-1效应细胞(购自promega，Cat.#J1252)胰酶消化成1.25×10 6细胞/ml的单细胞悬液。取前一天铺好的PD-L1aAPC/CHO-K1细胞，弃掉上清，加入40μl梯度稀释的抗体工作液，再加入等体积的PD-1效应细胞。置于37℃，5％CO 2，孵育6小时。细胞于37℃孵育6小时后，每孔加入80μl检测试剂Bio-Glo(购自promega，Cat.#G7940)。室温孵育10分钟后，用SpectraMax i3x读取luminescence。所有数据均为双复孔，所得信号值取平均值后用4-parameter法拟合，用GraphPad Prism6进行数据分析。 Take the PD-L1aAPC/CHO-K1 cells (purchased from promega, Cat.#J1252) grown in the logarithmic phase, trypsinize them into single cells and transfer them to a white bottom permeable 96-well plate, 100μl/well, 40,000 cells/well, Place at 37°C, 5% CO 2 , and incubate overnight. The anti-PD-L1/VEGF bifunctional fusion protein, positive control M8, and isotype negative control antibody IgG1 were diluted three-fold into 2× working solution, the highest concentration was 200 nM, and there were 10 concentration gradients. At the same time, the PD-1 effector cells (purchased from promega, Cat.#J1252) with a density of 1.4-2×10 6 /ml and a cell viability above 95% were trypsinized to 1.25×10 6 cells/ml. Cell suspension. Take the PD-L1aAPC/CHO-K1 cells plated the day before, discard the supernatant, add 40 μl of the antibody working solution of gradient dilution, and then add an equal volume of PD-1 effector cells. Place at 37°C, 5% CO 2 , and incubate for 6 hours. After the cells were incubated at 37°C for 6 hours, 80 μl of detection reagent Bio-Glo (purchased from promega, Cat. #G7940) was added to each well. After 10 minutes of incubation at room temperature, read the luminescence with SpectraMax i3x. All data are double-replicated holes, and the obtained signal values are averaged and then fitted with the 4-parameter method, and the data is analyzed with GraphPad Prism6.
实验结果如图5所示，抗PD-L1/VEGF双功能融合蛋白与阳性对照M8均能够有效阻断PD-1与PD-L1之间的相互作用，并且阻断能力相当。M8、M8-D2和M8-D2-M2的IC 50分别为0.49nM、0.56nM和0.66nM。 The experimental results are shown in Figure 5. Both the anti-PD-L1/VEGF bifunctional fusion protein and the positive control M8 can effectively block the interaction between PD-1 and PD-L1, and the blocking ability is equivalent. The IC 50 of M8, M8-D2 and M8-D2-M2 are 0.49nM, 0.56nM and 0.66nM, respectively.
实施例7.抗PD-L1/VEGF双功能融合蛋白阻断VEGF与受体KDR结合的细胞实验Example 7. Cellular experiment of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of VEGF and receptor KDR
取贴壁培养的对数期生长的密度约在80％-90％的KDR细胞(购自promega， Cat.#GA1082)，弃掉生长培养基。用DPBS洗一次后，用
Figure PCTCN2021096084-appb-000007
solution(购自Sigma，Cat.#A6964)消化，中和胰酶后，200g离心5min，用含10％FBS的DMEM培养基(购自Gibco，Cat.#11995)将细胞重悬后，台盼蓝细胞计数，调整细胞密度以40000个/孔铺板，50μl/孔，置于37℃，5％CO 2孵育。用含10％FBS的DMEM培养基稀释VEGF至30ng/ml，用含VEGF的培养基倍比稀释抗体，3倍稀释，10个梯度。将稀释好的抗体加入细胞孔每孔25μl(最终VEGF浓度为10ng/ml，抗体起始浓度为50nM)，37℃孵育6h后，每孔加入75μl检测试剂Bio-Glo(购自promega，Cat.#G7940)。室温孵育10分钟后，用SpectraMax i3x读取luminescence。所有数据均为双复孔，所得信号值取平均值后用4-parameter法拟合，用GraphPad Prism6进行数据分析。 KDR cells (purchased from promega, Cat.#GA1082) with a logarithmic growth density of about 80%-90% in adherent culture were taken, and the growth medium was discarded. After washing once with DPBS, use
Figure PCTCN2021096084-appb-000007
The solution (purchased from Sigma, Cat.#A6964) was digested, and the pancreatin was neutralized, and then centrifuged at 200g for 5 min. The cells were resuspended in DMEM medium (purchased from Gibco, Cat.#11995) containing 10% FBS. Count the blue cells, adjust the cell density to 40,000 cells/well, plate 50μl/well, and incubate at 37°C with 5% CO 2. Dilute VEGF to 30ng/ml with 10% FBS-containing DMEM medium, and dilute the antibody with VEGF-containing medium fold, 3 times dilution, 10 gradients. Add the diluted antibody to each well of the cell in 25μl (final VEGF concentration is 10ng/ml, the initial antibody concentration is 50nM), after incubating at 37°C for 6h, add 75μl of detection reagent Bio-Glo (purchased from promega, Cat. #G7940). After 10 minutes of incubation at room temperature, read the luminescence with SpectraMax i3x. All data are double-replicated holes, and the obtained signal values are averaged and then fitted with the 4-parameter method, and the data is analyzed with GraphPad Prism6.
实验结果如图6所示，抗PD-L1/VEGF双功能融合蛋白与阳性对照Bevacizumab均能够有效阻断VEGF与其受体KDR之间的相互作用，并且抗PD-L1/VEGF双功能融合蛋白M8-D2、M8-D2-M2的阻断能力更优。M8-D2、M8-D2-M2和Bevacizumab的IC 50分别为0.46nM、0.39nM和1.22nM。 The experimental results are shown in Figure 6. Both anti-PD-L1/VEGF bifunctional fusion protein and positive control Bevacizumab can effectively block the interaction between VEGF and its receptor KDR, and anti-PD-L1/VEGF bifunctional fusion protein M8 -D2, M8-D2-M2 have better blocking ability. The IC 50 of M8-D2, M8-D2-M2 and Bevacizumab are 0.46nM, 0.39nM and 1.22nM, respectively.
实施例8.抗PD-L1/VEGF双功能融合蛋白在MC38-hPD-L1移植瘤模型上的抗肿瘤作用Example 8. Anti-tumor effect of anti-PD-L1/VEGF bifunctional fusion protein on MC38-hPD-L1 xenograft tumor model
MC38-hPD-L1细胞是由北京百奥赛图基因生物技术公司对小鼠结肠癌MC38细胞进行了基因改造，使其过量表达人源PD-L1，同时敲除鼠源PD-L1的细胞。将PBS重悬的MC38-hPD-L1细胞以5×10 5个/0.1ml浓度，0.1ml/只体积接种于B-hPD-L1人源化小鼠(百奥赛图(北京)医药科技股份有限公司)的右侧皮下。当平均肿瘤体积达到大约138mm 3后将动物随机分组，分组当天(第0天)进行首次给药。每两天给药1次，一共给药8次，第14天给药结束。受试样品M8-D2-M2的剂量为23.2mg/kg，对照M8的剂量设置为20mg/kg，空白对照组给以相同体积的生理盐水。整个实验过程中，每周2次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b 2。其中a、b分别表示长、宽。实验结果如图7所示，在此模型上M8-D2-M2和M8均能显著抑制肿瘤生长。在等摩尔剂量下，M8-D2-M2的抑瘤作用起效快，给药期间的抑瘤效果明显优于M8单抗。 MC38-hPD-L1 cells are genetically modified mouse colon cancer MC38 cells by Beijing Biocytogenes Biotechnology Company to overexpress human PD-L1 and knock out mouse PD-L1 cells. MC38-hPD-L1 cells resuspended in PBS were inoculated into B-hPD-L1 humanized mice at a concentration of 5×10 5 cells/0.1ml and a volume of 0.1ml/head (Biocytop (Beijing) Pharmaceutical Technology Co., Ltd. Company) subcutaneously on the right side. When the average tumor volume reached approximately 138 mm 3, the animals were randomly divided into groups, and the first administration was given on the day of grouping (day 0). It is administered once every two days for a total of 8 administrations, and the administration ends on the 14th day. The dose of the test sample M8-D2-M2 was 23.2 mg/kg, the dose of the control M8 was set to 20 mg/kg, and the blank control group was given the same volume of normal saline. Throughout the experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed at the same time. The calculation formula of tumor volume (TV) is: TV=1/2×a×b 2 . Among them, a and b represent length and width respectively. The experimental results are shown in Figure 7. In this model, M8-D2-M2 and M8 can significantly inhibit tumor growth. At equimolar doses, the anti-tumor effect of M8-D2-M2 takes effect quickly, and the anti-tumor effect during administration is significantly better than that of M8 monoclonal antibody.
实施例9.抗PDL1/VEGF双功能融合蛋白的物理稳定性。Example 9. Physical stability of anti-PDL1/VEGF bifunctional fusion protein.
利用DSC(Differential scanning calorimetry,差示扫描量热法)检测M8-D2-M2在PBS缓冲体系下的热稳定性。将样品置换到PBS缓冲液中，控制样品浓度在1mg/ml，利用MicroCal*Vp-Capillary DSC(Malvern)进行检测。检测前，将样品及空白缓冲液用0.22μm滤膜过滤。样品板每个孔加入400μl样品或空白缓冲液(设置6组空白缓冲对)，最后三对孔板加入ddH 2O，以备清洗用。样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到 100℃结束，扫描速率150℃/h。具体结果如下表3所示，M8-D2-M2蛋白表现出良好的热稳定性。 DSC (Differential scanning calorimetry) was used to detect the thermal stability of M8-D2-M2 in the PBS buffer system. Replace the sample with PBS buffer, control the sample concentration at 1 mg/ml, and use MicroCal*Vp-Capillary DSC (Malvern) for detection. Before testing, filter the sample and blank buffer with a 0.22μm filter membrane. Add 400μl sample or blank buffer to each well of the sample plate (set 6 groups of blank buffer pairs), and add ddH 2 O to the last three pairs of well plates for cleaning. After loading the sample plate, put on the plastic soft cover. The scanning temperature starts at 25°C and ends at 100°C, and the scanning rate is 150°C/h. The specific results are shown in Table 3 below. The M8-D2-M2 protein showed good thermal stability.
表3table 3
SampleSample TmOnset(℃)TmOnset(℃) Tm1(℃)Tm1(℃)
M8-D2-M2M8-D2-M2 66.2266.22 78.7678.76
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, the various modifications listed herein and the method changes in the invention are obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should all be included in the scope of the present invention.

Claims (16)

  1. 一种抗PD-L1/VEGF融合蛋白，其特征在于，其包含抗PD-L1抗体和VEGFR1的D2结构域；所述抗PD-L1抗体重链包含互补决定区HCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：11所示，HCDR2的氨基酸序列如SEQ ID NO：12所示，HCDR3的氨基酸序列如SEQ ID NO：13所示；所述抗PD-L1抗体轻链包含互补决定区LCDR1-3，其中LCDR1的氨基酸序列如SEQ ID NO：14所示，LCDR2的氨基酸序列如SEQ ID NO：15所示，LCDR3的氨基酸序列如SEQ ID NO：16所示。An anti-PD-L1/VEGF fusion protein, characterized in that it comprises an anti-PD-L1 antibody and the D2 domain of VEGFR1; the heavy chain of the anti-PD-L1 antibody comprises a complementarity determining region HCDR1-3, in which the amino acids of HCDR1 The sequence is shown in SEQ ID NO: 11, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13; the light chain of the anti-PD-L1 antibody includes the complementarity determining region LCDR1 -3, where the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16.
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述VEGFR1的D2结构域的N末端通过肽接头L连接至抗PD-L1抗体重链的C末端。The fusion protein of claim 1, wherein the N-terminus of the D2 domain of VEGFR1 is connected to the C-terminus of the heavy chain of the anti-PD-L1 antibody via a peptide linker L.
  3. 根据权利要求1所述的融合蛋白，其特征在于，所述抗PD-L1抗体重链可变区的氨基酸序列如SEQ ID NO：17所示，所述抗PD-L1抗体轻链可变区的氨基酸序列如SEQ ID NO：18所示。The fusion protein of claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: 17, and the light chain variable region of the anti-PD-L1 antibody The amino acid sequence of is shown in SEQ ID NO: 18.
  4. 根据权利要求2所述的融合蛋白，其特征在于，所述肽接头L的氨基酸序列如SEQ ID NO：3所示。The fusion protein of claim 2, wherein the amino acid sequence of the peptide linker L is shown in SEQ ID NO: 3.
  5. 根据权利要求1所述的融合蛋白，其特征在于，所述VEGFR1的D2结构域的氨基酸序列如SEQ ID NO：1或SEQ ID NO：6所示。The fusion protein according to claim 1, wherein the amino acid sequence of the D2 domain of VEGFR1 is as shown in SEQ ID NO: 1 or SEQ ID NO: 6.
  6. 根据权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的重链氨基酸序列如SEQ ID NO：4或SEQ ID NO：7所示，所述融合蛋白轻链氨基酸序列如SEQ ID NO：5所示。The fusion protein of claim 1, wherein the heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4 or SEQ ID NO: 7, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO. ：5 shows.
  7. 一种核酸分子，其特征在于，所述核酸分子编码根据权利要求1-6任一项所述的融合蛋白。A nucleic acid molecule, characterized in that it encodes the fusion protein according to any one of claims 1-6.
  8. 根据权利要求7所述的核酸分子，其特征在于，所述核酸分子编码融合蛋白重链的核酸序列如SEQ ID NO：8或SEQ ID NO：10所示，编码融合蛋白轻链的核酸序列如SEQ ID NO：9所示。The nucleic acid molecule according to claim 7, wherein the nucleic acid sequence encoding the heavy chain of the fusion protein is shown in SEQ ID NO: 8 or SEQ ID NO: 10, and the nucleic acid sequence encoding the light chain of the fusion protein is shown in SEQ ID NO: 9 is shown.
  9. 一种表达载体，其特征在于，所述表达载体含有根据权利要求7或8所述的核酸分子。An expression vector, characterized in that the expression vector contains the nucleic acid molecule according to claim 7 or 8.
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有根据权利要求9所述的表达载体。A host cell, characterized in that it contains the expression vector according to claim 9.
  11. 一种根据权利要求1-6任一项所述的融合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：A method for preparing a fusion protein according to any one of claims 1 to 6, characterized in that the preparation method comprises the following steps:
    a)在表达条件下，培养根据权利要求10所述的宿主细胞，从而表达抗PD-L1/VEGF融合蛋白；a) Under expression conditions, culture the host cell according to claim 10 to express the anti-PD-L1/VEGF fusion protein;
    b)分离并纯化步骤a)所述的融合蛋白。b) Isolation and purification of the fusion protein described in step a).
  12. 一种药物组合物，其特征在于，所述药物组合物包含有效量的根据权利要求1-6任一项所述的融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。A pharmaceutical composition, characterized in that it comprises an effective amount of the fusion protein according to any one of claims 1 to 6 and one or more pharmaceutically acceptable carriers, diluents or excipients. Shape agent.
  13. 根据权利要求1-6任一项所述的融合蛋白、或根据权利要求12所述的药物组合物在制备治疗癌症的药物中的用途。Use of the fusion protein according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 12 in the preparation of a medicine for the treatment of cancer.
  14. 根据权利要求13所述的用途，其特征在于，所述癌症选自：黑色素瘤、胃癌、肾癌、尿路上皮癌、肺癌、肝癌、结直肠癌、膀胱癌、食道癌、***癌、胰腺癌、乳腺癌、卵巢癌、***、子宫癌、输卵管癌、原发性腹膜癌、甲状腺癌、胶质瘤、白血病、淋巴瘤、皮肤癌、头颈癌。The use according to claim 13, wherein the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial cancer, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreas Cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer.
  15. 一种治疗癌症的方法，其特征在于，所述方法包括向有需要的受试者施用根据权利要求1-6任一项所述的融合蛋白、或根据权利要求12所述的药物组合物。A method for treating cancer, characterized in that the method comprises administering the fusion protein according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 12 to a subject in need.
  16. 根据权利要求15所述的方法，其特征在于，所述癌症选自：黑色素瘤、胃癌、肾癌、尿路上皮癌、肺癌、肝癌、结直肠癌、膀胱癌、食道癌、***癌、胰腺癌、乳腺癌、卵巢癌、***、子宫癌、输卵管癌、原发性腹膜癌、甲状腺癌、胶质瘤、白血病、淋巴瘤、皮肤癌、头颈癌。The method according to claim 15, wherein the cancer is selected from the group consisting of: melanoma, gastric cancer, kidney cancer, urothelial cancer, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreas Cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, head and neck cancer.
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