WO2021244371A1 - Protéine de fusion anti-pd-l1/vegf - Google Patents

Protéine de fusion anti-pd-l1/vegf 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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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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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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

Abstract

L'invention concerne une protéine de fusion anti-PD-L1/VEGF, contenant un anticorps anti-PD-L1, un lieur peptidique L et un domaine D2 du VEGFR1, l'extrémité N-terminale du domaine D2 du VEGFR1 étant reliée à l'extrémité C-terminale d'une chaîne lourde de l'anticorps anti-PD-L1 par l'intermédiaire du lieur peptidique L. La protéine de fusion de la présente invention a la capacité de traiter des maladies liées à l'activité de PD-L1 et du VEGF.
PCT/CN2021/096084 2020-06-02 2021-05-26 Protéine de fusion anti-pd-l1/vegf WO2021244371A1 (fr)

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CN109575140A (zh) * 2017-09-29 2019-04-05 北京比洋生物技术有限公司 靶向pd-1或pd-l1且靶向vegf家族的双靶向融合蛋白及其用途
WO2019168947A1 (fr) * 2018-02-28 2019-09-06 Ap Biosciences, Inc. Protéines bifonctionnelles combinant un blocage des points de contrôle pour une thérapie ciblée

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CN109575140A (zh) * 2017-09-29 2019-04-05 北京比洋生物技术有限公司 靶向pd-1或pd-l1且靶向vegf家族的双靶向融合蛋白及其用途
WO2019168947A1 (fr) * 2018-02-28 2019-09-06 Ap Biosciences, Inc. Protéines bifonctionnelles combinant un blocage des points de contrôle pour une thérapie ciblée

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