Anti-pd-l1 / vegf fusion protein

Abstract

Provided are anti-PD-L1 / VEGF fusion proteins comprising 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 the heavy chain of the anti-PD-L1 antibody through the peptide linker L. The fusion proteins of the present invention have the potential to treat diseases related to the activities of PD-L1 and VEGF.

Description

Technical Field

[0001] This invention relates to the field of fusion protein technology, and more specifically, to an anti-PD-L1 / VEGF fusion protein. Background Technology

[0002] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids and is one of the known major immune checkpoints (Blank et al., 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed on activated T lymphocytes. Its binding to ligands PD-L1 (programmed cell death-ligand 1) and PD-L2 (programmed cell death-ligand 2) can inhibit T lymphocyte activity and related in vivo cellular immune responses. PD-L2 is mainly expressed on macrophages and dendritic cells, while PD-L1 is widely expressed on B and T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, and heart tissue cells. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only essential for maintaining the balance of the immune system in the body, but also a major mechanism and cause for PD-L1-positive tumor cells to circumvent immune surveillance. By blocking the negative regulation of the PD-1 / PD-L1 signaling pathway by cancer cells, the immune system can be activated, promoting T cell-related tumor-specific cellular immune responses, thus opening the door to a new cancer treatment method—tumor immunotherapy.

[0003] PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily associated with CD28 and CTLA-4. Research shows that PD-1 negatively regulates antigen receptor signal transduction when it binds to its ligands (PD-L1 and / or PD-L2). The structure of mouse PD-1 and the co-crystallization structure of mouse PD-1 and human PD-L1 have been elucidated (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 containing a variable (V-type) Ig domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The PD-1 cytoplasmic tail region contains two tyrosine-based signal transduction motifs: ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor tyrosine switching motif).

[0004] PD-1 plays a crucial role in the immune evasion mechanisms of tumors. Tumor immunotherapy, which utilizes the body's own immune system to fight cancer, is a groundbreaking cancer treatment method. However, the tumor microenvironment can protect tumor cells from effective immune destruction; therefore, disrupting the tumor microenvironment has become a key focus of anti-tumor research. Existing research has identified 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), and is presumed to be an important target mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99:12293-12297 (2002); Strome SE et al., Cancer Res., 63:6501-6505 (2003)). Immunohistochemical evaluation of biopsies has revealed the expression of PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 on tumor cells in many primary human tumors. Such cancers 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, pancreatic cancer, and head and neck tumors. Therefore, blocking the interaction between PD-1 and PD-L1 can enhance the immune activity of tumor-specific T cells, helping the immune system to clear tumor cells. Consequently, PD-L1 has become a popular target for developing tumor immunotherapy drugs.

[0005] Tumor growth occurs in two phases: a slow, avascular growth phase followed by a rapid, vascularized proliferation phase. Without angiogenesis, the primary tumor grows slowly, and metastasis is impossible. Therefore, inhibiting tumor angiogenesis is considered one of the promising cancer treatment methods. Among the vascular endothelial growth factor (VEGF) family, VEGF-A165 (hereinafter referred to as VEGF) is the most abundant and active subtype. VEGF binds to the type II receptor VEGFR2, activating a series of cascade reactions in the signaling pathway, promoting angiogenesis and maintaining its integrity. However, the type I receptor VEGFR1 binds to VEGF much more readily than VEGFR2, primarily acting on the extracellular D2 domain of VEGFR1. VEGFR1-D2 competitively binds to VEGF, blocking the binding of VEGFR2 to VEGF, thereby blocking the signaling pathway, inhibiting endothelial cell proliferation and angiogenesis, and thus suppressing rapid tumor proliferation and metastasis. Summary of the Invention

[0006] The present invention aims to provide a novel anti-PD-L1 / VEGF fusion protein that can simultaneously block the PD-L1 and VEGF signaling pathways. Further objectives of the present invention include providing a nucleic acid molecule encoding the fusion protein; providing an expression vector comprising the nucleic acid molecule; providing a host cell comprising the expression vector; providing a method for preparing the fusion protein; providing a pharmaceutical composition comprising the fusion protein; providing the use of the fusion protein or the pharmaceutical composition in the preparation of a medicament for treating cancer; and providing a method for using the fusion protein or the pharmaceutical composition to treat cancer.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first aspect of the present invention provides an anti-PD-L1 / VEGF fusion protein comprising an anti-PD-L1 antibody and a D2 domain of VEGFR1; the heavy chain of the anti-PD-L1 antibody comprises complementarity-determining regions HCDR1-3, wherein 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 comprises complementarity-determining regions LCDR1-3, wherein 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.

[0009] In a preferred embodiment, the N-terminus of the D2 domain of VEGFR1 is linked to the C-terminus of the anti-PD-L1 antibody heavy chain via a peptide linker L.

[0010] In a preferred embodiment, the amino acid sequence of the variable region of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the variable region of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 18.

[0011] In a preferred embodiment, the anti-PD-L1 antibody is a monoclonal antibody.

[0012] In a preferred embodiment, the anti-PD-L1 antibody is a humanized antibody.

[0013] In a preferred embodiment, the anti-PD-L1 antibody is an IgG antibody.

[0014] In a preferred embodiment, the amino acid sequence of the peptide linker L is as shown in SEQ ID NO: 3.

[0015] 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.

[0016] 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 two amino acids at the C-terminus relative to the D2 domain of VEGFR1 in the fusion protein M8-D2. These two amino acids are easily detached during fermentation, and their removal does not affect the efficacy.

[0017] 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.

[0018] A second aspect of the invention provides a nucleic acid molecule that encodes the fusion protein.

[0019] In a preferred embodiment, the nucleic acid molecule encodes the heavy chain of the fusion protein as shown in SEQ ID NO: 8 or SEQ ID NO: 10, and the nucleic acid sequence encoding its light chain as shown in SEQ ID NO: 9.

[0020] Those skilled in the art will understand that nucleic acid molecules encoding the amino acid sequence of the aforementioned fusion protein can be appropriately substituted, deleted, altered, inserted, or added to provide a homologue of a nucleic acid molecule.

[0021] A third aspect of the present invention provides an expression vector containing the above-described nucleic acid molecules.

[0022] A fourth aspect of the present invention provides a host cell containing the above-described expression vector.

[0023] A fifth aspect of the present invention provides a method for preparing a fusion protein, the method comprising the following steps:

[0024] a) Under the expression conditions, host cells were cultured as described above to express the anti-PD-L1 / VEGF fusion protein;

[0025] b) Isolate and purify the fusion protein described in step a).

[0026] A sixth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the above-described fusion protein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0027] A seventh aspect of the present invention provides the use of the above-described fusion protein and pharmaceutical composition in the preparation of a medicament for treating cancer.

[0028] According to the present invention, the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial carcinoma, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, and head and neck cancer.

[0029] In a preferred embodiment, the cancer is colon cancer.

[0030] The anti-PD-L1 / VEGF fusion protein described in this invention can be used alone or in combination with other anti-tumor drugs.

[0031] The cancer treatment drug referred to in this invention refers to a drug that inhibits and / or treats tumors, and may include delaying the development of tumor-related symptoms and / or reducing the severity of these symptoms, further including alleviating existing tumor-related symptoms and preventing the occurrence of other symptoms, and also including reducing or preventing tumor metastasis, etc.

[0032] In this invention, when the anti-PD-L1 / VEGF fusion protein and its pharmaceutical composition are administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage can be determined by referring to animal experimental results and various other factors, but the total dosage should not exceed a certain range. Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this invention.

[0033] An eighth aspect of the present invention provides a method for treating cancer, comprising administering the above-described fusion protein or pharmaceutical composition to a subject in need.

[0034] According to the present invention, the cancer is selected from: melanoma, gastric cancer, kidney cancer, urothelial carcinoma, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, leukemia, lymphoma, skin cancer, and head and neck cancer.

[0035] In a preferred embodiment, the cancer is colon cancer.

[0036] When the anti-PD-L1 / VEGF fusion protein and its pharmaceutical composition are administered to subjects, the dosage must be a therapeutically effective amount. The therapeutically effective amount refers to an amount that is effective in treating cancer. Specifically, when the anti-PD-L1 / VEGF fusion protein and its pharmaceutical composition are administered to subjects, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. This may be based on results from animal experiments and various other factors, but the total dosage must not exceed a certain range.

[0037] The positive effects of this invention are as follows: The fusion protein of this invention can bind with high affinity to PD-L1 and VEGF. Its affinity for PD-L1 is comparable to that of the anti-PD-L1 monoclonal antibody positive control M8, and affinity dissociation constant measurements show that its affinity for VEGF is higher than that of the anti-VEGF monoclonal antibody positive control Bevacizumab. The fusion protein of this invention can effectively block the binding of PD-1 to PD-L1, with blocking ability comparable to that of the anti-PD-L1 monoclonal antibody positive control M8. Furthermore, it can effectively block the interaction between VEGF and its receptor KDR, with blocking ability superior to that of the anti-VEGF monoclonal antibody positive control Bevacizumab. The fusion protein of this invention can significantly inhibit the growth of colorectal cancer xenografts, with a rapid onset of tumor-suppressing effect and a significantly better tumor-suppressing effect than that of the anti-PD-L1 monoclonal antibody positive control M8. The fusion protein of this invention has the potential to treat diseases related to PD-L1 and VEGF activity. Attached Figure Description

[0038] Figure 1 Schematic diagram of the structure of the anti-PD-L1 / VEGF bifunctional fusion protein

[0039] Figure 2 Electrophoretic detection image of anti-PD-L1 / VEGF bifunctional fusion protein

[0040] Figure 3A ELISA assay diagram of the affinity of anti-PD-L1 / VEGF bifunctional fusion protein for PD-L1

[0041] Figure 3B ELISA assay of the affinity of the anti-PD-L1 / VEGF bifunctional fusion protein for VEGF

[0042] Figure 4 FACS assay of the binding affinity of the anti-PD-L1 / VEGF bifunctional fusion protein to target cell surface antigens.

[0043] Figure 5 : Cellular assay to detect the blocking of PD-1-PD-L1 binding by the anti-PD-L1 / VEGF bifunctional fusion protein

[0044] Figure 6: Cellular assay to detect the blocking effect of anti-PD-L1 / VEGF bifunctional fusion protein on VEGF binding to receptor KDR

[0045] Figure 7 Graph illustrating the anti-PD-L1 / VEGF bifunctional fusion protein's anti-tumor effect in the MC38-hPD-L1 xenograft model. Detailed Implementation

[0046] The following experimental examples are intended to further illustrate the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional or routine methods in the art, such as methods for preparing nucleic acid molecules, methods for constructing vectors and plasmids, methods for inserting protein-encoding genes into such vectors and plasmids, methods for introducing plasmids into host cells, methods for culturing host cells, etc. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press.

[0047] In this invention, the term "fusion protein" refers to a new polypeptide sequence obtained by fusing two or more identical or different polypeptide sequences. The term "fusion" refers to direct linkage by peptide bonds or effective linkage by one or more linking peptides (peptide adapters). The term "linking peptide (peptide adapter)" refers to a short peptide that can link two polypeptide sequences, generally a peptide with a length of 2-30 amino acids.

[0048] In this invention, the term "antibody (abbreviated Ab)" refers to a heterotetraglycoprotein of approximately 150,000 Daltons with identical structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other, with the light chain constant region including a domain CL. The light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. The constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are also covalently linked by interpeptide disulfide bonds formed between the hinge regions.

[0049] In this invention, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which are typically mixtures of different antibodies targeting different antigenic determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized through hybridoma culture without contamination by other immunoglobulins.

[0050] In this invention, the term "humanized" refers to a antibody whose CDR is derived from a non-human species (preferably mouse) antibody, and whose residual portions (including the frame region and constant region) are derived from human antibodies. Furthermore, the frame region residues can be modified to maintain binding affinity.

[0051] In this invention, the terms "antibody" and "binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11An antibody binds to an antigen with an equilibrium dissociation constant (KD) of M or less. The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of the antibody to the antigen.

[0052] In this invention, the term "expression vector" refers to a conventional expression vector in the art that contains appropriate regulatory sequences, such as promoters, terminators, enhancers, etc., and the expression vector may be a virus or a plasmid. The expression vector preferably includes pDR1, pcDNA3.4(+), pDHFR, or pTT5.

[0053] In this invention, the term "host cell" refers to any host cell conventional in the art, as long as it enables the vector to replicate stably and the carried nucleic acid molecules to be effectively expressed. The host cell includes prokaryotic expression cells and eukaryotic expression cells, and is preferably selected from: COS, CHO, NSO, sf9, sf21, DH5α, BL21(DE3), TG1, BL21(DE3), 293F cells, or 293E cells.

[0054] In this invention, the term "effective amount" refers to the amount or dose of the pharmaceutical composition of this invention that produces the desired effect in the treated individual after administration to a patient, the desired effect including improvement of the individual's condition.

[0055] The sequence information involved in the following embodiments is summarized in the sequence list Table 1.

[0056] Table 1 Sequence List

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] 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 1, respectively.

[0063] The heavy 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, respectively, as shown in Sequence Listing 1.

[0064] Unless otherwise specified, all reagents and raw materials used in the following examples are commercially available.

[0065] Example 1. Construction of anti-PD-L1 / VEGF bifunctional fusion protein

[0066] This invention utilizes a tandem approach of an anti-PD-L1 monoclonal antibody and the D2 domain of VEGFR1 to construct an anti-PD-L1 / VEGF bifunctional fusion protein, as shown in the schematic diagram below. Figure 1 As shown.

[0067] Fusion protein M8-D2

[0068] The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 1) and the C-terminus of the heavy chain of the anti-PD-L1 monoclonal antibody M8 (SEQ ID NO: 2) are linked together via peptide linker L (SEQ ID NO: 3) to obtain 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.

[0069] Fusion protein M8-D2-M2

[0070] The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 6) and the C-terminus of the heavy chain of the anti-PD-L1 monoclonal antibody M8 (SEQ ID NO: 2) are linked together via peptide linker L (SEQ ID NO: 3) to obtain 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.

[0071] The D2 domain of VEGFR1 in M8-D2-M2 is truncated by two amino acids at the C-terminus compared to the D2 domain of VEGFR1 in the fusion protein M8-D2. These two amino acids are easily detached during fermentation, and their removal does not affect the efficacy.

[0072] Example 2. Expression and purification of anti-PD-L1 / VEGF bifunctional fusion protein

[0073] The heavy chain nucleic acid sequence of M8-D2 is SEQ ID NO: 8, and the light chain nucleic acid sequence is SEQ ID NO: 9. The heavy chain nucleic acid sequence of M8-D2-M2 is SEQ ID NO: 10, and the light chain nucleic acid sequence is SEQ ID NO: 9. DNA fragments of the heavy and light chains of the anti-PD-L1 / VEGF bifunctional fusion protein were subcloned into the pcDNA3.4 vector (purchased from Thermofisher, A14697), and the recombinant plasmids were extracted and co-transfected into CHO cells and / or 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, filtered through a microporous membrane under vacuum, and then loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with 100mM citric acid, pH 3.5 elution buffer. The target sample was recovered and dialyzed to PBS. The purified protein was detected by HPLC. The antibody molecules were homogeneous, and the monomer purity reached over 97%. Electrophoresis loading buffers for reduced and non-reduced proteins were added, and the samples were boiled before detection. The results are as follows: Figure 2 As shown, the full-length protein molecule is located at more than 180 kD (theoretical molecular weight 168 kD), the heavy chain is located at 70 kD, and the light chain is located at 25-35 kD.

[0074] Example 3. Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-PD-L1 / VEGF bifunctional fusion protein for antigen.

[0075] 3.1 ELISA detection of the affinity between the anti-PD-L1 / VEGF bifunctional fusion protein and PD-L1

[0076] Recombinant PD-L1-ECD-Fc protein (preparation method according to WO2018 / 137576A1) was prepared at 100 ng / well and coated onto a plate, incubated overnight at 4°C. The plate was washed three times with PBST, and 200 μl / well of blocking buffer was added. After incubation at 37°C for 1 hour, the plate was washed once with PBST. The antibody was diluted to 100 nM with dilution buffer, and 12 concentration gradients were created by 4-fold serial dilution. 100 μl / well was added sequentially to the blocked ELISA plate, and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat.#ab87422) was added. The plate was incubated at 37°C for 30 minutes. After washing the plate three times with PBST, pat dry any remaining droplets on absorbent paper. Add 100 μl of TMB to each well and incubate at room temperature (20±5℃) in the dark for 5 minutes. Add stop solution to each well to terminate the substrate reaction. Read the OD value at 450 nm using a microplate reader. Analyze the data using GraphPad Prism6, plot the graphs, and calculate the EC50. 50 .

[0077] Experimental results are as follows Figure 3AAs shown, the anti-PD-L1 / VEGF bifunctional fusion protein and the positive control M8 monoclonal antibody exhibit comparable binding affinity to PD-L1-ECD. ECD binding affinity of M8, M8-D2, and M8-D2-M2 is also shown. 50 The values ​​are 0.15 nM, 0.24 nM, and 0.23 nM, respectively.

[0078] 3.2 ELISA detection of the affinity of the anti-PD-L1 / VEGF bifunctional fusion protein for VEGF

[0079] Recombinant VEGF165 protein (purchased from AcroBiosystems, Cat.#VE5-H4210) was coated onto microplates at 100 ng / well and incubated overnight at 4°C. The plates were washed three times with PBST, and 200 μl / well of blocking buffer was added. After incubation at 37°C for 1 hour, the plates were washed once with PBST. The antibody was diluted to 200 nM using dilution buffer, and 12 concentration gradients were created by 4-fold serial dilution. 100 μl / well of each gradient was added sequentially to the blocked microplates, and the plates were incubated at 37°C for 1 hour. After washing three times with PBST, HRP-labeled goat anti-human Fab antibody (purchased from Abcam, Cat.#ab87422) was added, and the plates were incubated at 37°C for 30 minutes. After washing the plate three times with PBST, pat dry any remaining droplets on absorbent paper. Add 100 μl of TMB to each well and incubate at room temperature (20±5℃) in the dark for 5 minutes. Add stop solution to each well to terminate the substrate reaction. Read the OD value at 450 nm using a microplate reader. Analyze the data using GraphPad Prism6, plot the graphs, and calculate the EC50. 50 .

[0080] Experimental results are as follows Figure 3B As shown, M8-D2 and the positive control Bevacizumab monoclonal antibody have comparable affinity for VEGF. The EC50 of M8-D2 and Bevacizumab... 50 The values ​​are 0.89 nM and 0.85 nM, respectively.

[0081] Example 4. FACS determination of the binding affinity of anti-PD-L1 / VEGF bifunctional fusion protein to target cell surface antigens.

[0082] This experiment used PD-L1aAPC / CHO-K1 cells (purchased from Promega, Cat.#J1252) expressing PD-L1 on their cell surface as target cells, and arranged the target cells at a ratio of 2×10⁻⁶. 5Cells were seeded in 96-well plates and washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 minutes each time, and the supernatant was discarded. 100 μl of antibody, serially diluted 11 times from 83.5 nM in a 3-fold gradient, was added to the 96-well plates as primary antibody. After resuspending the cells, they were incubated at 4°C for 1 h. Cells were washed twice with PBS containing 0.5% BSA to remove unbound antibody, and then incubated with 100 μl of 1 μg / ml goat anti-human IgG-FITC (purchased from Sigma, Cat.#F9512) at 4°C for 30 minutes. After centrifugation at 300g for 5 minutes, cells were washed twice with PBS containing 0.5% BSA to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μl of PBS, and the binding affinity of the antibody to PD-L1 on the surface of CHO cells was determined using a Beckman Coulter CytoFLEX flow cytometer. The obtained data were analyzed using GraphPad Prism6 software.

[0083] Experimental results are as follows Figure 4 As shown, 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, and their affinity is comparable. The EC50 of M8-D2 and M8... 50 The values ​​are 1.25 nM and 0.71 nM, respectively.

[0084] Example 5. Determination of the affinity dissociation constant KD of the anti-PD-L1 / VEGF bifunctional fusion protein to the antigen VEGF

[0085] The kinetic parameters of the binding and dissociation of the anti-PD-L1 / VEGF bifunctional fusion protein and the antigen VEGF165 were determined using the Octet molecular interaction analyzer via a capture method. Antibody at a concentration of 5 μg / ml was bound to an AHC probe (purchased from PALL Lifesciences, Cat. #18-5060). Antigen VEGF165 was diluted with 1×HBS working solution (purchased from GE Healthcare, Cat. #14100669), and bound to the antibody at six concentration gradients with a maximum concentration of 25 nM, followed by dissociation in HBS working solution. The affinity and dissociation constants of the anti-PD-L1 / VEGF bifunctional fusion protein M8-D2 and the positive control Bevacizumab are shown in Table 2. The results indicate that M8-D2 has a higher affinity for VEGF165 than Bevacizumab.

[0086] Table 2 Affinity dissociation constants

[0087]

[0088] Note: KD is the affinity constant; kon is the binding rate constant; kdis is the dissociation rate constant.

[0089] Example 6. Cellular experiment to block PD-1 binding to PD-L1 by anti-PD-L1 / VEGF bifunctional fusion protein

[0090] Log-phase PD-L1aAPC / CHO-K1 cells (purchased from Promega, Cat.#J1252) were trypsinized into single cells and transferred to white-bottomed 96-well plates (100 μl / well, 40,000 cells / well), incubated overnight at 37°C with 5% CO2. Anti-PD-L1 / VEGF bifunctional fusion protein, positive control M8, and isotype negative control antibody IgG1 were serially diluted three-fold to prepare 2× working solutions, with a maximum concentration of 200 nM, for a total of 10 concentration gradients. Simultaneously, cells with densities ranging from 1.4 to 2 × 10⁶ cells / well were... 6 / ml, PD-1 effector cells with a cell viability of over 95% (purchased from Promega, Cat.#J1252) were digested with trypsin to form 1.25×10⁶ cells / ml. 6 Single-cell suspensions were prepared at 1 / ml. PD-L1aAPC / CHO-K1 cells cultured the previous day were collected, the supernatant was discarded, and 40 μl of serially diluted antibody working solution was added, followed by an equal volume of PD-1 effector cells. The cells were incubated at 37°C with 5% CO2 for 6 hours. After 6 hours of incubation at 37°C, 80 μl of Bio-Glo assay reagent (purchased from Promega, Cat.#G7940) was added to each well. After incubation at room temperature for 10 minutes, luminescence was read using SpectraMax i3x. All data were in duplicate, and the average signal values ​​were fitted using the 4-parameter method. Data analysis was performed using GraphPad Prism6.

[0091] Experimental results are as follows Figure 5 As shown, both the anti-PD-L1 / VEGF bifunctional fusion protein and the positive control M8 effectively blocked the interaction between PD-1 and PD-L1, and their blocking abilities were comparable. The IC50 values ​​for M8, M8-D2, and M8-D2-M2 were... 50 The values ​​are 0.49 nM, 0.56 nM, and 0.66 nM, respectively.

[0092] Example 7. Cellular experiment to block VEGF binding to receptor KDR using anti-PD-L1 / VEGF bifunctional fusion protein.

[0093] Take KDR cells (purchased from Promega, Cat.#GA1082) that are in the logarithmic growth phase and have a density of approximately 80%-90% adherent, and discard the growth medium. Wash once with DPBS, then... Digested with solution (Sigma, Cat. #A6964), and after neutralizing trypsin, the cells were centrifuged at 200g for 5 min. Resuspended in DMEM medium (Gibco, Cat. #11995) containing 10% FBS, cells were counted with trypan blue, and the cell density was adjusted to 40,000 cells / well. 50 μl of the solution was added to each well, and the cells were incubated at 37°C with 5% CO2. VEGF was diluted to 30 ng / ml with DMEM medium containing 10% FBS. The antibody was serially diluted 3-fold in 10 gradients with VEGF-containing medium. 25 μl of the diluted antibody was added to each well (final VEGF concentration 10 ng / ml, initial antibody concentration 50 nM). After incubation at 37°C for 6 h, 75 μl of Bio-Glo assay reagent (Promega, Cat. #G7940) was added to each well. After incubation at room temperature for 10 min, luminescence was read using SpectraMax i3x. All data were obtained from two duplicate apertures. The average signal values ​​were then fitted using the 4-parameter method, and the data were analyzed using GraphPad Prism6.

[0094] Experimental results are as follows Figure 6 As shown, both the anti-PD-L1 / VEGF bifunctional fusion protein and the positive control Bevacizumab effectively blocked the interaction between VEGF and its receptor KDR, with the anti-PD-L1 / VEGF bifunctional fusion proteins M8-D2 and M8-D2-M2 exhibiting superior blocking ability. The IC50 values ​​of M8-D2, M8-D2-M2, and Bevacizumab were also compared. 50 The values ​​are 0.46 nM, 0.39 nM, and 1.22 nM, respectively.

[0095] Example 8. Antitumor effect of anti-PD-L1 / VEGF bifunctional fusion protein in MC38-hPD-L1 xenograft model

[0096] MC38-hPD-L1 cells were developed by Beijing Biocytogen Gene Biotechnology Co., Ltd. through genetic modification of mouse colon cancer MC38 cells to overexpress human PD-L1, while simultaneously knocking out mouse PD-L1. MC38-hPD-L1 cells resuspended in PBS were cultured at 5 × 10⁻⁶ cells / year. 5 At a concentration of 0.1 ml / mole, 0.1 ml / mole was injected subcutaneously into the right side of B-hPD-L1 humanized mice (Biocytok (Beijing) Pharmaceutical Technology Co., Ltd.). The tumor volume was approximately 138 mm². 3Animals were then randomly assigned to groups, and the first dose was administered on the day of grouping (day 0). Administered once every two days for a total of eight doses, ending on day 14. The dose of the test sample M8-D2-M2 was 23.2 mg / kg, the dose of the control M8 was 20 mg / kg, and the blank control group received the same volume of physiological saline. Throughout the experiment, the tumor diameter was measured twice a week, and the mice were weighed simultaneously. The tumor volume (TV) was calculated using the formula: TV = 1 / 2 × a × b 2 Where a and b represent length and width, respectively. The experimental results are as follows: Figure 7 As shown, both M8-D2-M2 and M8 significantly inhibited tumor growth in this model. At equimolar doses, M8-D2-M2 exhibited a faster onset of tumor inhibition, and its tumor-suppressing effect during administration was significantly superior to that of M8 monoclonal antibody.

[0097] Example 9. Physical stability of the anti-PDL1 / VEGF bifunctional fusion protein.

[0098] The thermal stability of M8-D2-M2 in a PBS buffer system was determined using DSC (Differential Scanning Calorimetry). The sample was displaced into PBS buffer, with a concentration controlled at 1 mg / ml, and detected using a MicroCal*Vp-Capillary DSC (Malvern). Before detection, the sample and blank buffer were filtered through a 0.22 μm filter. 400 μl of sample or blank buffer was added to each well of the sample plate (6 blank buffer pairs were set up), and ddH2O was added to the last three pairs of wells for washing. After sample loading, the plate was covered with a soft plastic cap. The scanning temperature started at 25℃ and ended at 100℃, with a scanning rate of 150℃ / h. The specific results are shown in Table 3 below; the M8-D2-M2 protein exhibited good thermal stability.

[0099] Table 3

[0100] Sample TmOnset(°C) Tm1 (°C) M8-D2-M2 66.22 78.76

[0101] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the 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 be included within the scope of this invention. sequence list <110> Zeda Biomedical Co., Ltd. <120> An anti-PD-L1 / VEGF fusion protein <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 100 <212> PRT <213> Artificial Sequence <400> 1 Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile 1 5 10 15 Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr 20 25 30 Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu 35 40 45 Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile 50 55 60 Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala 65 70 75 80 Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln 85 90 95 Thr Asn Thr Ile 100 <210> 2 <211> 447 <212> PRT <213> Artificial Sequence <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 4 <211> 557 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro Phe Val 450 455 460 Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg 465 470 475 480 Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr 485 490 495 Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile 500 505 510 Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys 515 520 525 Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr 530 535 540 Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile 545 550 555 <210> 5 <211> 214 <212> PRT <213> Artificial Sequence <400> 5 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 6 <211> 98 <212> PRT <213> Artificial Sequence <400> 6 Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile 1 5 10 15 Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr 20 25 30 Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu 35 40 45 Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile 50 55 60 Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala 65 70 75 80 Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln 85 90 95 Thr Asn <210> 7 <211> 555 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro Phe Val 450 455 460 Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg 465 470 475 480 Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr 485 490 495 Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile 500 505 510 Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys 515 520 525 Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr 530 535 540 Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn 545 550 555 <210> 8 <211> 1671 <212> DNA <213> Artificial Sequence <400> 8 caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg 60 acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcgggggaac agactataac 180 cccagcctga aatcccggct gaccatctct agatacca gtaagaatca agtgagcttt 240 aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga 300 ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcgagcacc 360 aagggacctt ccgtgtttcc cctcgccccc agctccaaaa gcaccagcgg cggaacagct 420 gctctcggct gtctcgtcaa ggattacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggagccctga caagcggcgt ccacaccttc cctgctgtcc tacagtcctc cggactgtac 540 agcctgagca gcgtggtgac agtccctagc agctccctgg gcacccagac atatatttgc 600 aacgtgaatc acaagcccag caacaccaag gtcgataaga aggtggagcc taagtcctgc 660 gacaagaccc acacatgtcc cccctgtccc gctcctgaac tgctgggagg cccttccgtg 720 ttcctgttcc cccctaagcc caaggacacc ctgatgattt ccaggacacc cgaggtgacc 780 tgtgtggtgg tggacgtcag ccacgaggac cccgaggtga aattcaactg gtacgtcgat 840 ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagtacaa ttccacctac 900 agggtggtgt ccgtgctgac cgtcctccat caggactggc tgaacggcaa agagtataag 960 tgcaaggtga gcaacaaggc cctccctgct cccatcgaga agaccatcag caaagccaag 1020 ggccagccca gggaacctca agtctatacc ctgcctccca gcagggagga gatgaccaag 1080 aaccaagtga gcctcacatg cctcgtcaag ggcttctatc cttccgatat tgccgtcgag 1140 tgggagtcca acggacagcc cgagaacaac tacaagacaa caccccccgt gctcgattcc 1200 gatggcagct tcttcctgta ctccaagctg accgtggaca agtccagatg gcaacaaggc 1260 aacgtcttca gttgcagcgt catgcatgag gccctccaca accactacac ccagaagagc 1320 ctctccctga gccctggaaa gggcggtggg ggaagtggag gcggtgggag cgacaccggc 1380 aggcccttcg tggagatgta cagcgaaatc cccgaaatca tccacatgac cgagggcagg 1440 gagctggtga tcccgtgcag ggtgaccagc cccaacatca ccgtgaccct gaagaagttc 1500 cccctggaca ccctgattcc cgacggcaag aggatcatct gggacagcag gaagggcttc 1560 atcatcagca acgccaccta caaggagatc ggcctgctga cctgcgaggc caccgtcaac 1620 ggccacctgt acaagaccaa ctacctgacc cacaggcaga ccaataccat c 1671 <210> 9 <211> 642 <212> DNA <213> Artificial Sequence <400> 9 gaaatcgtgc tgacacagag ccctgacttt ctgtccgtga cacccaagga gaaagtcact 60 atcacctgcc gggctagcca gtccatcgga accacaattc actggtacca gcagaagccc 120 gaccagagcc ctaagctgct gattaaatat gcctctcaga gtttctcagg cgtgccatcc 180 agatttagcg gctccgggtc tggaactgac ttcacactga ctatcaactc tgtcgaggca 240 gaagatgccg ctacctacta ttgtcagcag agtaattcat ggcccctgac ctttggcgcc 300 gggacaaagc tggaaattaa aagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc <210> 10 <211> 1665 <212> DNA <213> Artificial Sequence <400> 10 60. caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcggggac agactataac cccagcctga aatcccggct gaccatctct aggatacca gtaagaatca agtgagcttt aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcgagcacc 360 aagggacctt ccgtgtttcc cctcgcccc agctccaaaa gcaccagcgg cggaacagct 420 gctctcggct gtctcgtcaa ggattacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggagccctga caagcggcgt ccacaccttc cctgctgtcc tacagtcctc cggactgtac 540 agcctgagca gcgtggtgac agtccctagc agctccctgg gcacccagac atatatttgc 600 aacgtgaatc acaagcccag caacaccaag gtcgataaga aggtggagcc taagtcctgc 660 gacaagaccc acacatgtcc cccctgtccc gctcctgaac tgctgggagg cccttccgtg 720 ttcctgttcc cccctaagcc caaggacacc ctgatgattt ccaggacacc cgaggtgacc 780 tgtgtggtgg tggacgtcag ccacgaggac cccgaggtga aattcaactg gtacgtcgat 840 ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagtacaa ttccacctac 900 agggtggtgt ccgtgctgac cgtcctccat caggactggc tgaacggcaa agagtataag 960 tgcaaggtga gcaacaaggc cctccctgct cccatcgaga agaccatcag caaagccaag 1020 ggccagccca gggaacctca agtctatacc ctgcctccca gcagggagga gatgaccaag 1080 aaccaagtga gcctcacatg cctcgtcaag ggcttctatc cttccgatat tgccgtcgag 1140 tgggagtcca acggacagcc cgagaacaac tacaagacaa caccccccgt gctcgattcc 1200 gatggcagct tcttcctgta ctccaagctg accgtggaca agtccagatg gcaacaaggc 1260 aacgtcttca gttgcagcgt catgcatgag gccctccaca accactacac ccagaagagc 1320 ctctccctga gccctggaaa gggcggtggg ggaagtggag gcggtgggag cgacaccggc 1380 aggcccttcg tggagatgta cagcgaaatc cccgaaatca tccacatgac cgagggcagg 1440 gagctggtga tcccgtgcag ggtgaccagc cccaacatca ccgtgaccct gaagaagttc 1500 cccctggaca ccctgattcc cgacggcaag aggatcatct gggacagcag gaagggcttc 1560 atcatcagca acgccaccta caaggagatc ggcctgctga cctgcgaggc caccgtcaac 1620 ggccacctgt acaagaccaa ctacctgacc cacaggcaga ccaat 1665 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <400> 11 Gly Phe Ser Leu Thr Ser Tyr Gly Val His 1 5 10 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <400> 12 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Gln Leu Gly Leu Arg Ala Met Asp Tyr 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <400> 14 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <400> 15 Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 17 <211> 117 <212> PRT <213> Artificial Sequence <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 453 <212> PRT <213> Artificial Sequence <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 20 <211> 214 <212> PRT <213> Artificial Sequence <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An anti-PD-L1 / VEGF fusion protein, characterized in that, It contains an anti-PD-L1 antibody and a D2 domain of VEGFR1; the anti-PD-L1 antibody heavy chain contains complementarity-determining regions HCDR1-3, wherein 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 anti-PD-L1 antibody light chain contains complementarity-determining regions LCDR1-3, wherein 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 amino acid sequence of the D2 domain of the VEGFR1 is shown in SEQ ID NO: 1 or SEQ ID NO: 6; The N-terminus of the D2 domain of VEGFR1 is linked to the C-terminus of the anti-PD-L1 antibody heavy chain via a peptide linker L. The length of the peptide linker L is 2–30 amino acids.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the variable region of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO:

18.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the peptide linker L is shown in SEQ ID NO:

3.

4. The fusion protein according to claim 1, characterized in that, 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.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, characterized in that, 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.

7. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule according to claim 5 or 6.

8. A host cell, characterized in that, The host cell contains the expression vector according to claim 7.

9. A method for preparing a fusion protein according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: a) Under expression conditions, host cells according to claim 8 are cultured to express the anti-PD-L1 / VEGF fusion protein; b) Isolate and purify the fusion protein described in step a).

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of the fusion protein according to any one of claims 1-4 and one or more pharmaceutically acceptable carriers, diluents or excipients.

11. Use of the fusion protein according to any one of claims 1-4, or the pharmaceutical composition according to claim 10, in the preparation of a medicament for treating cancer, wherein the cancer is selected from: gastric cancer, kidney cancer, urothelial carcinoma, lung cancer, liver cancer, colorectal cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, fallopian tube cancer, primary peritoneal cancer, thyroid cancer, glioma, skin cancer, and head and neck cancer.

12. The use according to claim 11, wherein the skin cancer is melanoma.

Images