CN113754776A

CN113754776A - anti-PD-L1/VEGF fusion protein

Info

Publication number: CN113754776A
Application number: CN202010487517.3A
Authority: CN
Inventors: 黄浩旻; 邓岚; 李理; 朱祯平
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Zi Da Biological Medicine Co.,Ltd.
Priority date: 2020-06-02
Filing date: 2020-06-02
Publication date: 2021-12-07
Also published as: TW202146447A; CN115989243A; WO2021244371A1

Abstract

The invention relates to the technical field of fusion proteins, and particularly relates to an anti-PD-L1/VEGF fusion protein. The fusion protein comprises an anti-PD-L1 antibody, a peptide linker L, and the D2 domain of VEGFR 1. The N-terminus of the D2 domain of VEGFR1 was linked to the C-terminus of the anti-PD-L1 antibody heavy chain through a peptide linker L. The fusion protein has the potential of treating diseases related to PD-L1 and VEGF activity.

Description

anti-PD-L1/VEGF fusion protein

Technical Field

The invention relates to the technical field of fusion proteins, and particularly relates to an anti-PD-L1/VEGF fusion protein.

Background

Human programmed cell death receptor-1 (PD-1) is a 288 amino acid type I membrane protein and is one of the known major Immune checkpoints (Immune Checkpoint) (Blank et al,2005, Cancer Immunotherapy,54: 307-) -314. PD-1 is expressed in activated T lymphocytes, and binding of the ligands PD-L1 (programmed cell death receptor-Ligand 1) and PD-L2 (programmed cell death receptor-Ligand 2) can inhibit the activity of T lymphocytes and the related in vivo cellular immune response. 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. Numerous studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain immune system balance in vivo, but also a major mechanism and cause PD-L1 expressing positive tumor cells to circumvent immune surveillance. By blocking the negative regulation and control of cancer cells to PD-1/PD-L1 signal channels, the immune system is activated, and the tumor specific cellular immune response related to T cells can be promoted, thereby opening a new tumor treatment method, namely a tumor immunotherapy.

PD-1 (encoded by gene Pdcd 1) is an immunoglobulin superfamily member that is associated with CD28 and CTLA-4. The results of the study show that PD-1 negatively regulates antigen receptor signaling when bound to its ligand (PD-L1 and/or PD-L2). The murine PD-1 structure and the cocrystal structure of murine 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 that contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail responsible for binding to a signaling molecule. The PD-1 cytoplasmic tail contains two tyrosine-based signaling motifs, the ITIM (immunoreceptor tyrosine inhibition motif) and the ITSM (immunoreceptor tyrosine transduction motif).

PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, namely, cancer resistance by using the immune system of the human body, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction, so how to break the tumor microenvironment becomes the key point of anti-tumor research. The role of PD-1 in the tumor microenvironment has been determined by prior work: PD-L1 is expressed in a number of mouse and human tumors (and can be induced by IFN-. gamma. in most PD-L1 negative tumor cell lines) and is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al, Proc. Natl. Acad. Sci. U.S.A.99:12293-12297 (2002); Strome S.E. et al, Cancer Res.,63:6501-6505 (2003)). Biopsy evaluation by immunohistochemistry has revealed expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells in many primary tumors in humans. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, stomach cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer, as well as head and neck tumors, among others. Therefore, the blocking of the interaction of PD-1/PD-L1 can improve the immunocompetence of tumor specific T cells and is beneficial to the immune system to eliminate tumor cells, so that PD-L1 becomes a hot target for developing tumor immunotherapy drugs.

There are two phases of tumor growth, from the slow avascular growth phase to the rapid angiogenic proliferation phase. If no blood vessels are formed inside the tumor, the primary tumor grows slowly and metastasis cannot be achieved. Inhibition of tumor angiogenesis is therefore considered to be one of the currently promising approaches to tumor therapy. Among the Vascular Endothelial Growth Factor (VEGFs) family, VEGF-A165 (hereinafter referred to as VEGF) is the most abundant active subtype. VEGF, by binding to the type II receptor VEGFR2, activates a signaling pathway to undergo a cascade of reactions that promote neovascularization and maintain its integrity. However, the type I receptor VEGFR1 binds VEGF much more strongly than VEGFR2 and acts mainly at the extracellular domain D2 of VEGFR 1. VEGFR1-D2 blocks the binding of VEGFR2 and VEGF by competing with the binding of VEGF, thereby blocking the signal pathway, inhibiting the proliferation and angiogenesis of endothelial cells, and thus inhibiting the rapid proliferation and metastasis of tumors.

Disclosure of Invention

The invention aims to provide a novel anti-PD-L1/VEGF fusion protein which can block PD-L1 and VEGF signal channels simultaneously. It is also an object of the present invention to provide a nucleic acid molecule encoding said fusion protein; providing an expression vector comprising said 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; provides the application of the fusion protein or the pharmaceutical composition in preparing a medicament for treating cancer; methods of providing the fusion protein or the pharmaceutical composition for treating cancer are provided.

In order to achieve the purpose, the invention provides the following technical scheme:

the first aspect of the invention provides an anti-PD-L1/VEGF fusion protein comprising an anti-PD-L1 antibody and the D2 domain of VEGFR1, the N-terminus of the D2 domain of VEGFR1 being linked to the C-terminus of the heavy chain of the anti-PD-L1 antibody by a peptide linker L; the heavy chain of the anti-PD-L1 antibody comprises a complementarity determining region 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, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13 is shown in the figure; the light chain of the anti-PD-L1 antibody comprises a complementarity determining region 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, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 16.

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is as set forth 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 as SEQ ID NO: 18, respectively.

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

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

In a preferred embodiment, the anti-PD-L1 antibody is an IgG class antibody.

In a preferred embodiment, the amino acid sequence of the peptide linker L is as set forth in SEQ ID NO: 3, respectively.

In a preferred embodiment, the amino acid sequence of the D2 domain of VEGFR1 is as set forth in SEQ ID NO: 1 or SEQ ID NO: and 6.

In a preferred embodiment, the fusion protein is selected from the group consisting of M8-D2 and M8-D2-M2.

In a preferred embodiment, the heavy chain amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 4 or SEQ ID NO: 7, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO: 5, respectively.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding the fusion protein.

In a preferred embodiment, the nucleic acid molecule encodes the heavy chain of the fusion protein as set forth in SEQ ID NO: 8 or SEQ ID NO: 10, and the nucleic acid sequence encoding the light chain is shown as SEQ ID NO: shown at 9.

The skilled person will appreciate that the nucleic acid molecule encoding the amino acid sequence of the above fusion protein may suitably incorporate substitutions, deletions, alterations, insertions or additions to provide a nucleic acid molecule homologue.

In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described above.

In a fourth aspect, the present invention provides a host cell comprising the above-described expression vector.

The fifth aspect of the present invention provides a method for preparing a fusion protein, comprising the steps of:

a) culturing a host cell as described above under expression conditions such that an anti-PD-L1/VEGF fusion protein is expressed;

b) isolating and purifying the fusion protein of step a).

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

The seventh aspect of the invention provides the use of the fusion protein and the pharmaceutical composition in the preparation of a medicament for treating cancer.

According to the invention, the cancer is selected from: melanoma, gastric cancer, renal cancer, urothelial cancer, 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, head and neck cancer.

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

The invention has the positive effects that: the fusion protein can be combined with PD-L1 and VEGF with high affinity, the affinity of the fusion protein with PD-L1 is equivalent to that of anti-PD-L1 monoclonal antibody positive control M8, and affinity dissociation constant determination shows that the affinity of the fusion protein with VEGF is higher than that of anti-VEGF monoclonal antibody positive control Bevacizumab. The fusion protein can effectively block the combination of PD-1 and PD-L1, the blocking capability of the fusion protein is equivalent to that of anti-PD-L1 monoclonal antibody positive control M8, the fusion protein can effectively block the interaction between VEGF and a receptor KDR thereof, and the blocking capability of the fusion protein is superior to that of anti-VEGF monoclonal antibody positive control Bevacizumab. The fusion protein has the potential of treating diseases related to PD-L1 and VEGF activity.

Drawings

FIG. 1: structural schematic diagram of anti-PD-L1/VEGF bifunctional fusion protein

FIG. 2: electrophoresis detection picture of anti-PD-L1/VEGF double-function fusion protein

FIG. 3A: ELISA detection of affinity of anti-PD-L1/VEGF bifunctional fusion protein to PD-L1

FIG. 3B: ELISA detection of affinity of anti-PD-L1/VEGF bifunctional fusion protein and VEGF

FIG. 4: FACS detection of the binding affinity of the anti-PD-L1/VEGF bifunctional fusion protein to the target cell surface antigen

FIG. 5: cell experiment detection chart of anti-PD-L1/VEGF bifunctional fusion protein for blocking binding of PD-1 and PD-L1

FIG. 6: cell experiment detection chart for blocking combination of VEGF and receptor KDR by anti-PD-L1/VEGF bifunctional fusion protein

Detailed Description

The following experimental examples are further illustrative of the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods or methods conventional 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 methods for introducing plasmids into host cells, methods for culturing host cells, and the like. Such methods are well known to those having ordinary skill in the art and are described in a number of publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning, A Laboratory Manual,2nd edition, Cold spring Harbor Laboratory Press.

In the present invention, the term "fusion protein" refers to a novel polypeptide sequence obtained by fusing two or more identical or different polypeptide sequences. The term "fusion" refers to a linkage by peptide bonds either directly or by means of one or more connecting peptides (peptide linkers). The term "linker peptide" refers to a short peptide, typically a peptide of 2-30 amino acids in length, that can link two polypeptide sequences.

In the present invention, the term "Antibody (Ab), for short" refers to an isotetraglycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has at one end a variable region (VH) followed by a constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC) and the like. Heavy chain constant regions include IgG1, IgG2, IgG3, IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by interpoly disulfide bonds formed between the hinge regions.

In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with epitopes against different antigens), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.

In the present invention, the term "humanized" means that the CDRs are derived from an antibody of a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework and constant regions) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity.

In the present invention, the terms "anti-and" binding "refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. Typically, the antibody is present in an amount less than about 10^-7M, e.g. less than about 10^-8M、10^-9M、10^-10M、10^-11M or less binds the antigen with an equilibrium dissociation constant (KD). The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using Surface Plasmon Resonance (SPR for short) or the relative affinity of the binding of an antibody to an antigen is determined using ELISA.

In the present invention, the term "expression vector" refers to an expression vector conventional in the art, which may be a virus or a plasmid, comprising appropriate regulatory sequences, such as a promoter, a terminator, an enhancer, and the like. The expression vector preferably comprises pDR1, pcDNA3.4(+), pDFFR or pTT 5.

In the present invention, the term "host cell" is a variety of host cells that are conventional in the art, as long as the vector is stably self-replicating and the carried nucleic acid molecule can be efficiently expressed. Wherein the host cell comprises prokaryotic expression cells and eukaryotic expression cells, preferably comprising: COS, CHO, NS0, sf9, sf21, DH5 alpha, BL21(DE3), TG1, BL21(DE3) or 293E cells.

In the present invention, the term "effective amount" refers to an amount or dose that, upon administration of a pharmaceutical composition of the invention to a patient, produces a desired effect in the treated subject, including an improvement in the condition of the subject.

The sequence information referred to in the following examples is summarized in table 1 of the sequence listing.

TABLE 1 sequence listing

The anti-human PD-L1 antibody positive control M8 used in the following examples was derived from PCT/CN2020/090442, and its heavy and light chain amino acid sequences are SEQ ID NOs: 2 and SEQ ID NO: 5.

the heavy and light chain amino acid sequences of the anti-VEGF antibody positive control Bevacizumab used in the following examples are SEQ ID NOs: 19 and SEQ ID NO: 20.

the reagents and starting materials used in the following examples are commercially available unless otherwise specified.

Example 1 anti-PD-L1/VEGF bifunctional fusion protein construction

The invention adopts a mode of connecting an anti-PD-L1 monoclonal antibody and a D2 structural domain of VEGFR1 in series to construct an anti-PD-L1/VEGF bifunctional fusion protein, and the structural schematic diagram is shown in figure 1.

Fusion protein M8-D2

The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 1) and the C-terminus of the heavy chain of anti-PD-L1 monoclonal antibody M8 (SEQ ID NO: 2) were linked by a peptide linker L (SEQ ID NO: 3) to give a heavy chain of a fusion protein (SEQ ID NO: 4) whose light chain sequence is SEQ ID NO: 5.

fusion protein M8-D2-M2

The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 6) and the C-terminus of the heavy chain of anti-PD-L1 monoclonal antibody M8 (SEQ ID NO: 2) were linked by a peptide linker L (SEQ ID NO: 3) to give a heavy chain of a fusion protein (SEQ ID NO: 7) whose light chain sequence is SEQ ID NO: 5.

the D2 domain of VEGFR1 in M8-D2-M2 was truncated by 2 amino acids at the C-terminus relative to the D2 domain of VEGFR1 in fusion protein M8-D2, which were easily shed during fermentation and were removed without affecting drug efficacy.

Example 2 expression and purification of anti-PD-L1/VEGF bifunctional fusion proteins

The M8-D2 heavy chain nucleic acid sequence is SEQ ID NO: 8, the light chain nucleic acid sequence is SEQ ID NO: 9. the M8-D2-M2 heavy chain nucleic acid sequence is SEQ ID NO: 10, the light chain nucleic acid sequence is SEQ ID NO: 9. the DNA fragments of the heavy chain and the light chain of the anti-PD-L1/VEGF double-function fusion protein are respectively subcloned into a pcDNA3.4 vector (purchased from thermofisher, A14697), and recombinant plasmids are extracted to co-transfect CHO cells and/or 293F cells. After 7 days of cell culture, the culture fluid is subjected to high-speed centrifugation, vacuum filtration through a microfiltration membrane, then loaded on a HiTrap MabSelect SuRe column, protein is eluted by an eluent with 100mM citric acid and pH3.5 in one step, and a target sample is recovered and dialyzed to change the fluid to PBS. The purified protein is detected by HPLC, the molecular state of the antibody is uniform, and the purity of the monomer reaches more than 97 percent. Reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer are respectively added, and detection is carried out after boiling, and the result is shown in figure 2, wherein the full-length protein molecule is at a position (theoretical molecular weight 168kD) larger than 180kD, the heavy chain is at a position 70kD, and the light chain is at a position 25-35 kD.

Example 3 determination of affinity of anti-PD-L1/VEGF bifunctional fusion protein to antigen by enzyme-linked immunosorbent assay (ELISA) 3.1 detection of affinity of anti-PD-L1/VEGF bifunctional fusion protein to PD-L1 by ELISA

The recombinant PD-L1-ECD-Fc protein (the preparation method is referred to WO2018/137576A1) is prepared in a home-made way and is coated on a plate at 100 ng/hole overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The antibody was diluted to 100. mu.g/ml with a diluent, and diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the resultant mixture was sequentially added to the microplate after blocking, 100. mu.l/well, and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat. # ab87422) was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀。

The experimental results are shown in FIG. 3A, the anti-PD-L1/VEGF bifunctional fusion protein and the positive control M8 monoclonal antibody have equivalent binding affinity with PD-L1-ECD. EC of M8, M8-D2, M8-D2-M2₅₀0.15nM, 0.24nM, 0.23nM, respectively.

3.2ELISA detection of affinity of anti-PD-L1/VEGF bifunctional fusion protein and VEGF

Recombinant VEGF165 protein (purchased from acrobiosystems, Cat. # VE5-H4210) was coated onto microtiter plates at 100 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The antibody was diluted to 100. mu.g/ml with a diluent, and diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the resultant mixture was sequentially added to the microplate after blocking, 100. mu.l/well, and left at 37 ℃ for 1 hour. PBST plates were washed 3 times and HRP-labeled goat anti-human Fab antibody (from a) was addedbcam, Cat. # ab87422), 30 minutes at 37 ℃. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀。

The results are shown in FIG. 3B, where M8-D2 and the positive control Bevacizumab were comparable in affinity for VEGF binding. EC of M8-D2 and Bevacizumab₅₀0.89nM and 0.85nM, respectively.

Example 4 measurement of binding affinity of anti-PD-L1/VEGF bifunctional fusion protein to target cell surface antigen by FACS method

In this experiment, PD-L1aAPC/CHO-K1 cells (purchased from promega, Cat. # J1252) expressing PD-L1 on the cell surface were used as target cells, and the target cells were arranged in a 2X 10 order⁵Perwell was inoculated in a 96-well plate, washed three times with PBS containing 0.5% BSA, centrifuged at 300g each for 5 minutes, and the supernatant was discarded. Mu.l of 12-gradient antibody serially diluted from 3. mu.g/ml in a 3-fold gradient was added as a primary antibody to a 96-well plate, and the cells were suspended and incubated at 4 ℃ for 1 h. The cells were washed twice with PBS containing 0.5% BSA to remove unbound antibody and incubated with 100. mu.l of 1g/ml goat anti-human IgG-FITC (purchased from sigma, # Cat. # F9512) for 30 minutes at 4 ℃. The cells were centrifuged at 300g for 5min, washed twice with PBS containing 0.5% BSA to remove unbound secondary antibodies, and finally resuspended in 200 μ Ι PBS and the binding affinity of the antibody to CHO cell surface PD-L1 was determined by Beckman Co μ lter CytoFLEX flow cytometer. The data obtained were analyzed by GraphPad Prism6 software fitting.

The experimental results are shown in FIG. 4, the anti-PD-L1/VEGF bifunctional fusion protein and the positive control M8 monoclonal antibody can specifically bind to cell surface-expressed PD-L1 with equivalent affinity, EC of M8-D2 and M8₅₀1.25nM and 0.71nM, respectively.

Example 5 determination of affinity dissociation constant KD of anti-PD-L1/VEGF bifunctional fusion protein for antigen VEGF

The kinetic parameters of the binding and dissociation of the anti-PD-L1/VEGF bifunctional fusion protein and the antigen VEGF165 were determined by a capture method using an octet molecular interaction analyzer, and the antigen VEGF165 was dissociated in HBS working solution by binding the antibody at a concentration of 5. mu.g/ml to an AHC (available from PALLIFESCIES, Cat. #18-5060) probe, diluting the antigen VEGF165 with 1 XHBS working solution (available from GEHealthcare, Cat. #14100669), and setting 6 concentration gradients with a maximum concentration of 25nM to bind to the antibody. The affinity dissociation constant of the anti-PD-L1/VEGF bifunctional fusion protein M8-D2 and a positive control Bevacizumab is shown in Table 2 below. The results show that M8-D2 has higher affinity for VEGF165 than Bevacizumab.

TABLE 2 affinity dissociation constants

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

Example 6 cell experiment of anti-PD-L1/VEGF bifunctional fusion protein blocking the binding of PD-1 to PD-L1

Taking PD-L1aAPC/CHO-K1 cells (purchased from promega, Cat. # J1252) growing in logarithmic phase, trypsinizing into single cells, transferring to a white bottom-transparent 96-well plate, 100. mu.l/well, 40000 cells/well, placing at 37 ℃ and 5% CO₂And incubated overnight. The anti-PD-L1/VEGF double-function fusion protein, the positive control M8 and the isotype negative control antibody IgG1 are diluted into 2 Xworking solution according to a triple gradient, the highest concentration is 600nM, the lowest concentration is 0.09nM, and 9 concentration gradients are used in total. Simultaneously, the density is 1.4-2 multiplied by 10⁶PD-1 effector cells (from promega, Cat. # J1252) with a cell viability of 95% or more were trypsinized to 1.25X 10⁶Cells/ml single cell suspension. PD-L1aAPC/CHO-K1 cells plated on the previous day were taken, the supernatant was discarded, 40. mu.l of antibody working solution diluted in a gradient was added, and an equal volume of PD-1 effector cells was added. Standing at 37 deg.C for 5% CO₂And incubated for 6 hours. After incubation of the cells at 37 ℃ for 6 hours, 80. mu.l of detection reagent Bio-Glo (purchased from promega, Cat. # G7940) was added to each well. After incubation at room temperature for 10 minutes, luminescences were read with SpectraMax i3 x. All data were in duplicate wells, and the signal values averaged and fitted to the 4-parameter method, and the data were scored using GraphPad Prism6And (6) analyzing.

The experimental results are shown in FIG. 5, 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 abilities are equivalent. ICs for M8, M8-D2 and M8-D2-M2₅₀0.49nM, 0.56nM and 0.66nM, respectively.

Example 7 cell experiment of anti-PD-L1/VEGF bifunctional fusion protein blocking VEGF binding to receptor KDR

KDR cells (purchased from promega, Cat. # GA1082) grown in adherent culture at a density of approximately 80% -90% in log phase were removed from the growth medium. After washing once with DPBS, use

Digestion (purchased from Sigma, Cat. # A6964), neutralization of pancreatin, centrifugation at 200g for 5min, cell resuspension in 10% FBS-containing DMEM medium (purchased from GibcoCat. #11995), trypan blue cell counting, cell density adjustment at 40000/well plating, 50. mu.l/well, setting at 37 ℃, 5% CO₂. VEGF was diluted to 30ng/ml with DMEM medium containing 10% FBS, antibody was diluted two-fold with VEGF-containing medium, 3-fold dilution, 10 gradients. The diluted antibody was added to 25ul of wells per cell (final VEGF concentration 10ng/ml, starting antibody concentration 100nM), and after incubation for 6h at 37 ℃ 75 μ l of detection reagent Bio-Glo (purchased from promega, Cat. # G7940) was added to each well. After incubation at room temperature for 10 minutes, luminescences were read with SpectraMax i3 x. All data were in duplicate wells and the signal values averaged and fitted to the 4-parameter method and analyzed using GraphPad Prism 6.

The experimental results are shown in FIG. 6, the anti-PD-L1/VEGF bifunctional fusion protein and the positive control Bevacizumab can effectively block the interaction between VEGF and a receptor KDR thereof, and the blocking abilities of the anti-PD-L1/VEGF bifunctional fusion protein M8-D2 and M8-D2-M2 are better. ICs for M8-D2, M8-D2-M2 and Bevacizumab₅₀0.46nM, 0.39nM and 1.22nM, respectively.

Sequence listing

<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH

<120> an anti-PD-L1/VEGF fusion protein

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 100

<212> PRT

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 agagatacca 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 (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 600

ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 10

<211> 1665

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg 60

acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120

cctggaaagg gactggagtg gatcggcctg atttggtctg gcgggggaac agactataac 180

cccagcctga aatcccggct gaccatctct agagatacca 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 ccaat 1665

<210> 11

<211> 10

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 13

Gln Leu Gly Leu Arg Ala Met Asp Tyr

1 5

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 15

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln Gln Ser Asn Ser Trp Pro Leu Thr

1 5

<210> 17

<211> 117

<212> PRT

<213> Artificial Sequence (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 (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 (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 (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, comprising an anti-PD-L1 antibody and the D2 domain of VEGFR1, the N-terminus of the D2 domain of VEGFR1 being linked to the C-terminus of the heavy chain of the anti-PD-L1 antibody by a peptide linker L; the heavy chain of the anti-PD-L1 antibody comprises a complementarity determining region 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, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13 is shown in the figure; the light chain of the anti-PD-L1 antibody comprises a complementarity determining region 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, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 16.

2. The fusion protein of claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is as set forth 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 as SEQ ID NO: 18, respectively.

3. The fusion protein of claim 1, wherein the amino acid sequence of the peptide linker L is as set forth in SEQ ID NO: 3, respectively.

4. The fusion protein of claim 1, wherein the amino acid sequence of the D2 domain of VEGFR1 is as set forth in SEQ ID NO: 1 or SEQ ID NO: and 6.

5. The fusion protein of claim 1, wherein the heavy chain amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 4 or SEQ ID NO: 7, the amino acid sequence of the light chain of the fusion protein is shown as SEQ ID NO: 5, respectively.

6. A nucleic acid molecule encoding the fusion protein of any one of claims 1-5.

7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule encoding the heavy chain of the fusion protein has the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, and the nucleic acid sequence of the light chain of the encoding fusion protein is shown as SEQ ID NO: shown at 9.

8. An expression vector comprising the nucleic acid molecule of claim 6 or 7.

9. A host cell comprising the expression vector of claim 8.

10. A method for preparing the fusion protein according to any one of claims 1 to 5, comprising the steps of:

a) culturing the host cell of claim 9 under expression conditions, thereby expressing an anti-PD-L1/VEGF fusion protein;

b) isolating and purifying the fusion protein of step a).

11. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1-5 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

12. Use of the fusion protein of any one of claims 1-5, or the pharmaceutical composition of claim 11, in the manufacture of a medicament for the treatment of cancer.

13. The use of claim 12, wherein the cancer is selected from the group consisting of: melanoma, gastric cancer, renal cancer, urothelial cancer, 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, head and neck cancer.