Recombinant fusion protein targeting PD-L1 and VEGF, its preparation and use

Abstract

The present application provides a recombinant fusion protein comprising 1) an anti-PD-L1 antibody or an antigen-binding fragment thereof, and 2) a VEGF-binding peptide, wherein the N-terminus of the heavy chain or light chain of the anti-PD-L1 antibody or an antigen-binding fragment thereof is linked to the VEGF-binding peptide via a linker. Also provided are a nucleic acid molecule for encoding the recombinant fusion protein, an expression vector containing the nucleic acid molecule, a method for preparing the recombinant fusion protein, and a method for treating diseases related to PD-L1 and / or VEGF signaling using the recombinant fusion protein.

Description

Technical Field

[0001] This application claims priority to Chinese Patent Application No. CN202210639281.X, filed on May 31, 2022.

[0002] This application relates to recombinant fusion proteins targeting PD-L1, VEGF, and / or FcR, and their preparation and use, particularly their use in tumor treatment.

Background Art

[0003] Cancer cells have developed multiple mechanisms to evade the immune surveillance of host cells. For example, many tumors or cancer cells express high levels of PD-L1 and PD-L2 on their surfaces, both of which bind to PD-1 on the surface of T cells and induce T cell apoptosis.

[0004] In addition, cancer cell proliferation depends on a sufficient supply of nutrients. Cancer cells themselves can secrete factors that promote angiogenesis, such as vascular endothelial growth factor (VEGF). Inhibiting the activity of VEGF or its receptor stops the blood supply to solid tumors, thereby inhibiting their growth. PD-L1 and PD-1

[0005] PD-1 / PD-L1 is one of the most studied inhibitory immune checkpoints.

[0006] PD-L1, also known as programmed death ligand1 or CD274, is a transmembrane protein that plays an important role in the suppression of the immune system during some specific events such as tissue allotransplantation, autoimmune diseases, and cancer development. In the tumor microenvironment (TME), CD4 + Th1 helper cells and CD8 +T cells produce interferon-γ (IFN-γ), which on the one hand promotes macrophage-mediated tumor injury and on the other hand induces PD-L1 expression by macrophages and tumor cells. Tumor-specific CD8 + PD-1 on CD8 + T cells binds to PD-L1 expressed by various cells in the TME, leading to CD8

[0007] T cell anergy (Topalian, S. et al., (2016) Nat Rev Cancer 16(5): 275-287). PD-1 can also bind to PD-L2 and downregulate the immune system. VEGF and VEGFR

[0008] VEGF is a multifaceted growth factor central to tissue / wound repair programs and is classified into VEGF-A (also known as VEGF165), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PIGF. During the tissue healing process, VEGF, particularly VEGF-A, promotes neovascularization while downregulating immunity (Canic M, et al., (2009) J Surg Res.153:347-358; Leung DW, et al., (1989) Science.246:1306-1309; Voron T, et al., (2014) Front Oncol.4:70). Both of these properties of VEGF are central to the carcinogenesis process. This is because newly formed tumor blood vessels can supply blood and nutrients to tumor tissue, and inhibition of antitumor immunity can promote tumor cell proliferation (Hanahan D, Weinberg RA. (2011) Cell.144:646-674). In particular, VEGF is thought to exert its immunosuppressive effects through three main mechanisms: inhibition of dendritic cell (DC) maturation, reduction of T cell infiltration, and increase of inhibitory cells in the tumor microenvironment. Abnormal VEGF expression also contributes to the formation of abnormally structured new blood vessels that are prone to leakage, causing, for example, diabetic retinopathy, wet age-related macular degeneration, and the like. Bevacizumab (Avastin), an anti-VEGF monoclonal antibody drug approved by the FDA, functions to treat cancer (colon cancer, lung cancer), non-squamous non-small cell lung cancer, renal cell carcinoma, glioblastoma multiforme, ovarian cancer, and cervical cancer by inhibiting the biological activity of VEGF. Aflibercept, another protein drug targeting VEGF, has been approved in the United States and Europe for the treatment of wet macular degeneration (trade name Eylea) and metastatic colorectal cancer (Zaltrap).

[0009] VEGF receptors (VEGFRs) have three subtypes, VEGFR-1, VEGFR-2, and VEGFR-3, all of which have an extracellular portion consisting of seven immunoglobulin-like domains, a transmembrane region, and an intracellular portion. VEGFR-2 is thought to mediate almost all of the known cellular responses to VEGF, while VEGFR-1 sequesters VEGF from VEGFR-2 binding and regulates VEGFR-2 signaling. The most dangerous type of VEGF-A for human health binds to both VEGFR-1 and VEGFR-2. VEGFR antagonists are mainly used or are under investigation for treating cancer. Lenvima, which acts as a kinase inhibitor against VEGFR-1, VEGFR-2, and VEGFR-3 kinases, was approved in 2015 for the treatment of differentiated thyroid cancer and in 2016 for the treatment of advanced renal cell carcinoma in combination with everolimus. Fc and FcR

[0010] The fragment crystallizable region (Fc region) is the tail region of an antibody and is the domain that determines the effector function of the antibody, i.e., how it engages specific cell receptors or other defense proteins.

[0011] Fc receptors (FcRs) are proteins expressed on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. These cells contribute to the protective functions of the immune system.

[0012] The Fc region can interact with several proteins of the Fc receptor and complement system to activate the immune system and complement system. Therapeutic bispecific or multispecific fusion proteins / antibodies

[0013] Antibodies that target a single antigen can have limited efficacy. For example, for avelumab (BAVENCIO®), an approved anti-PD-L1 antibody, the overall response rate is only 33%.

[0014] Bispecific or trispecific fusion proteins have been developed in recent years, and these fusion proteins have shown good effects in preclinical and clinical trials.

[0015] Attaching additional binding moieties to conventional antibodies may seem conceptually straightforward, but such modifications can significantly alter the antibody structure and compromise their affinity and / or efficacy for each other (Wang S et al., (2021) EMBO Mol Med. 13(9):e14291). To optimize the in vivo efficacy and pharmacological properties, delicate design and manipulation are required for the selection of the primary and additional binding moieties (sequences), balanced affinity for the target, attachment sites (N- or C-terminus of the heavy or light chain), structural stability, linker length, and / or sequence (Shim H. (2020) Biomolecules. 10(3):360).

[0016] The citation or identification of any document in this application does not admit that such document is available as prior art against the present disclosure.

Summary of the Invention

[0017] This application provides a novel recombinant fusion protein comprising an anti-PD-L1 antibody and a VEGF-binding peptide. Compared with anti-PD-L1 antibodies and VEGF-binding proteins (including anti-VEGF antibodies), the recombinant fusion proteins of the present disclosure have equivalent activity against PD-L1 + , VEGF + and / or PD-L1 + VEGF + cells, and bind to PD-L1 + VEGF +It is possible to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) at an equivalent level with respect to cells, and / or to block PD-1-PD-L1 interaction and VEGF-VEGFR interaction with equivalent activity. In addition, the recombinant fusion protein of the present disclosure showed better antitumor effects in in vivo tests as compared to the combined use of a VEGF-binding protein and an anti-PD-L1 antibody.

[0018] Specifically, the present application discloses a recombinant fusion protein comprising: 1) an anti-PD-L1 antibody that specifically binds to PD-L1 or an antigen-binding fragment thereof, and 2) a VEGF-binding peptide that specifically binds to VEGF, wherein the VEGF-binding peptide and the anti-PD-L1 antibody or an antigen-binding fragment thereof are both linked. The anti-PD-L1 antibody or an antigen-binding fragment thereof may comprise a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region may comprise HV-CDR1, HV-CDR2, and HV-CDR3, wherein HV-CDR1, HV-CDR2, and HV-CDR3 may each comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the light-chain variable region may comprise LV-CDR1, LV-CDR2, and LV-CDR3, wherein LV-CDR1 and LV-CDR3 may each comprise the amino acid sequences of SEQ ID NOs: 4 and 5, respectively, and LV-CDR2 may comprise the amino acid sequence "YTS". The anti-PD-L1 antibody or an antigen-binding fragment thereof may comprise a heavy-chain constant region that may be linked to the C-terminus of the heavy-chain variable region. The heavy-chain constant region may comprise FcR-binding ability. In certain embodiments, the anti-PD-L1 antibody or an antigen-binding fragment thereof may comprise a heavy-chain variable region, a heavy-chain constant region, and a light-chain variable region, wherein the heavy-chain variable region may comprise HV-CDR1, HV-CDR2, and HV-CDR3, wherein HV-CDR1, HV-CDR2, and HV-CDR3 may each comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the light-chain variable region may comprise LV-CDR1, LV-CDR2, and LV-CDR3, wherein LV-CDR1 and LV-CDR3 may each comprise the amino acid sequences of SEQ ID NOs: 4 and 5, respectively, and LV-CDR2 may comprise the amino acid sequence "YTS", and wherein the heavy-chain constant region may comprise FcR-binding ability and is linked to the C-terminus of the heavy-chain variable region. The VEGF-binding peptide may be linked to the N-terminus of the heavy-chain variable region or to the light-chain variable region of the anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0019] The VEGF-binding peptide can be the extracellular Ig-like domain of vascular endothelial growth factor receptor 1 (VEGFR1). The VEGF-binding peptide can be the second extracellular Ig-like domain of VEGFR1 (VEGFR1D2). In certain embodiments, the VEGF-binding peptide can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11.

[0020] The heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment thereof can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In one embodiment, the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 7. The light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment thereof can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the light chain variable region can comprise the amino acid sequence of SEQ ID NO: 8. In one embodiment, the heavy chain variable region and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment thereof can each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 7 and 8, respectively. In certain embodiments, the heavy chain variable region and the light chain variable region of the anti-PD-L1 antibody or antigen-binding fragment thereof can each comprise the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0021] The heavy chain constant region can be a naturally occurring or engineered human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region or a functional fragment thereof. The heavy chain constant region having FcR binding ability can be a naturally occurring or engineered human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region or a functional fragment thereof. In certain embodiments, the heavy chain constant region having FcR binding ability can be a human IgG1 heavy chain constant region or a functional fragment thereof. In certain embodiments, the heavy chain constant region having FcR binding ability can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9.

[0022] The anti-PD-L1 antibody or antigen-binding fragment thereof can further comprise a light chain constant region, such as a human κ light chain constant region, or a functional fragment thereof, linked to the C-terminus of the light chain variable region. In certain embodiments, the light chain constant region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10.

[0023] The heavy chain of the anti-PD-L1 antibody or an antigen-binding fragment thereof may include a heavy chain variable region and a heavy chain constant region. The heavy chain may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In one embodiment, the heavy chain may include the amino acid sequence of SEQ ID NO: 15. The light chain of the anti-PD-L1 antibody or an antigen-binding fragment thereof may include a light chain variable region and a light chain constant region. The light chain may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In one embodiment, the light chain may include the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain and the light chain of the anti-PD-L1 antibody or an antigen-binding fragment thereof may each include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15 and 14, respectively. In certain embodiments, the heavy chain and the light chain of the anti-PD-L1 antibody or an antigen-binding fragment thereof may each include the amino acid sequences of SEQ ID NO: 15 and 14, respectively.

[0024] At least one paratope of the anti-PD-L1 antibody or an antigen-binding fragment thereof may be linked to a VEGF-binding peptide at the N-terminus of the heavy chain variable region or the light chain variable region that constitutes the paratope. In certain embodiments, each paratope of the anti-PD-L1 antibody or an antigen-binding fragment thereof may be linked to a VEGF-binding peptide at the N-terminus of the heavy chain variable region or the light chain variable region that constitutes the paratope. In certain embodiments, each paratope of the anti-PD-L1 antibody or an antigen-binding fragment thereof may be linked to a VEGF-binding peptide at the N-terminus of the heavy chain variable region that constitutes the paratope. In certain embodiments, each paratope of the anti-PD-L1 antibody or an antigen-binding fragment thereof may be linked to a VEGF-binding peptide at the N-terminus of the light chain variable region that constitutes the paratope.

[0025] The anti-PD-L1 antibody or antigen-binding fragment thereof of the present disclosure can be linked to a VEGF-binding peptide via a linker. The linker can be a peptide of 5 to 30, 10 to 30, 10 to 20, or 15 amino acid residues. The linker can be a GS linker, for example, -(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO: 12).

[0026] The recombinant fusion protein of the present disclosure can include a VEGF-binding peptide-linker-anti-PD-L1 heavy chain chain and an anti-PD-L1 light chain, wherein the VEGF-binding peptide-linker-anti-PD-L1 heavy chain chain can include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13, and the anti-PD-L1 light chain can include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the recombinant fusion protein of the present disclosure can include a VEGF-binding peptide-linker-anti-PD-L1 heavy chain chain and an anti-PD-L1 light chain, wherein the VEGF-binding peptide-linker-anti-PD-L1 heavy chain chain can include the amino acid sequence of SEQ ID NO: 13, and the anti-PD-L1 light chain can include the amino acid sequence of SEQ ID NO: 14. The amino acid sequences of SEQ ID NOs: 13 and 14 can be encoded by the nucleotide sequences of SEQ ID NOs: 23 and 24, respectively.

[0027] In one embodiment, the recombinant fusion protein of the present disclosure is: i) a first polypeptide chain comprising, from N-terminus to C-terminus, a VEGF-binding peptide, a linker, an anti-PD-L1 heavy chain variable region, and a heavy chain constant region; ii) a second polypeptide chain comprising, from N-terminus to C-terminus, an anti-PD-L1 light chain variable region and a light chain constant region; iii) a third polypeptide chain comprising, from N-terminus to C-terminus, a VEGF-binding peptide, a linker, an anti-PD-L1 heavy chain variable region, and a heavy chain constant region; and iv) A fourth polypeptide chain comprising an anti-PD-L1 light chain variable region and a light chain constant region from the N-terminus to the C-terminus comprising, wherein the anti-PD-L1 heavy chain variable region in the first polypeptide chain and the anti-PD-L1 light chain variable region in the second polypeptide chain can associate to specifically form an antigen-binding domain for PD-L1, and the anti-PD-L1 heavy chain variable region in the third polypeptide chain and the anti-PD-L1 light chain variable region in the fourth polypeptide chain can associate to specifically form an antigen-binding domain for PD-L1, and the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can associate with each other via, for example, a knob-into-hole approach, a covalent bond, and / or a disulfide bond.

[0028] The recombinant fusion protein of the present disclosure may comprise an anti-PD-L1 heavy chain and a chain of a VEGF-binding peptide-linker-anti-PD-L1 light chain, wherein the anti-PD-L1 heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, and the chain of the VEGF-binding peptide-linker-anti-PD-L1 light chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. In certain embodiments, the recombinant fusion protein of the present disclosure may comprise an anti-PD-L1 heavy chain and a chain of a VEGF-binding peptide-linker-anti-PD-L1 light chain, wherein the anti-PD-L1 heavy chain may comprise the amino acid sequence of SEQ ID NO: 15, and the chain of the VEGF-binding peptide-linker-anti-PD-L1 light chain may comprise the amino acid sequence of SEQ ID NO: 16. The amino acid sequences of SEQ ID NO: 15 and 16 may be encoded by the nucleotide sequences of SEQ ID NO: 25 and 26, respectively.

[0029] In one embodiment, the recombinant fusion protein of the present disclosure is: i) A first polypeptide chain comprising an anti-PD-L1 heavy chain variable region and a heavy chain constant region from the N-terminus to the C-terminus; ii) A second polypeptide chain comprising, from the N-terminus to the C-terminus, a VEGF-binding peptide, a linker, an anti-PD-L1 light chain variable region, and a light chain constant region; iii) A third polypeptide chain comprising, from the N-terminus to the C-terminus, an anti-PD-L1 heavy chain variable region and a heavy chain constant region; and iv) A fourth polypeptide chain comprising, from the N-terminus to the C-terminus, a VEGF-binding peptide, a linker, an anti-PD-L1 light chain variable region, and a light chain constant region, wherein the anti-PD-L1 heavy chain variable region in the first polypeptide chain and the anti-PD-L1 light chain variable region in the second polypeptide chain can associate to specifically form an antigen-binding domain for PD-L1, the anti-PD-L1 heavy chain variable region in the third polypeptide chain and the anti-PD-L1 light chain variable region in the fourth polypeptide chain can associate to specifically form an antigen-binding domain for PD-L1, and the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can associate with each other via, for example, a knob-into-hole approach, a covalent bond, and / or a disulfide bond.

[0030] This application also relates to the anti-PD-L1 antibody or antigen-binding fragment thereof of the present disclosure, which may include a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may include HV-CDR1, HV-CDR2, and HV-CDR3, where HV-CDR1, HV-CDR2, and HV-CDR3 may each include the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, where the light chain variable region may include LV-CDR1, LV-CDR2, and LV-CDR3, where LV-CDR1 and LV-CDR3 may each include the amino acid sequences of SEQ ID NOs: 4 and 5, respectively, and LV-CDR2 may include the amino acid sequence "YTS". The heavy chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. The light chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. The anti-PD-L1 antibody or antigen-binding fragment thereof may further include a heavy chain constant region and / or a light chain constant region. The heavy chain constant region may be a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof, linked to the C-terminus of the heavy chain variable region. In certain embodiments, the heavy chain constant region may have FcR binding ability or a functional fragment thereof. In certain embodiments, the heavy chain constant region may be a human IgG1 heavy chain constant region or a functional fragment thereof. In certain embodiments, the heavy chain constant region may include the amino acid sequence of SEQ ID NO: 9. The light chain constant region may be a human κ constant region or a functional fragment thereof linked to the C-terminus of the light chain variable region. In certain embodiments, the light chain constant region may include the amino acid sequence of SEQ ID NO: 10. The anti-PD-L1 antibody or antigen-binding fragment thereof of the present disclosure includes equivalent PD-L1 binding activity / affinity and equivalent PD-1-PD-L1 blocking activity as compared to prior art anti-PD-L1 antibodies such as atezolizumab. The anti-PD-L1 antibody or antigen-binding fragment thereof of the present disclosure may be capable of inducing ADCC and ADCP against PD-L1 + cells.

[0031] The present disclosure further provides a recombinant fusion protein of the present disclosure, a nucleic acid molecule encoding an anti-PD-L1 antibody or an antigen-binding fragment thereof, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or having the nucleic acid molecule integrated into its genome. A method for preparing the recombinant fusion protein, anti-PD-L1 antibody, or antigen-binding fragment thereof of the present disclosure using the host cell of the present disclosure is also provided, which comprises: (i) expressing the recombinant fusion protein, anti-PD-L1 antibody or antigen-binding fragment thereof in the host cell; and (ii) isolating the recombinant fusion protein, anti-PD-L1 antibody or antigen-binding fragment thereof from the host cell or its cell culture.

[0032] The present disclosure further provides a pharmaceutical composition that may comprise the recombinant fusion protein, anti-PD-L1 antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, or host cell of the present disclosure, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable adjuvant.

[0033] The pharmaceutical composition of the present disclosure can be used for treating a disease associated with VEGF and / or PD-L1 overexpression / signaling, or for preparing a medicament for treating a disease associated with VEGF and / or PD-L1 overexpression / signaling.

[0034] In one aspect, the present disclosure provides a method for treating or alleviating a disease associated with VEGF and / or PD-L1 signaling / overexpression in a subject in need thereof, which comprises administering to the subject a therapeutically acceptable amount of the pharmaceutical composition of the present disclosure.

[0035] The pharmaceutical composition of the present disclosure can be administered via an intraperitoneal, subcutaneous, or intravenous route. In one embodiment, the pharmaceutical composition of the present disclosure can be administered by intraperitoneal injection, subcutaneous injection, or intravenous injection.

[0036] Diseases associated with VEGF and / or PD-L1 signaling / overexpression can be cancers, including solid cancers and blood cancers. The cancers can be acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma, cervical cancer, or angiosarcoma.

[0037] Diseases associated with VEGF and / or PD-L1 signaling / overexpression can be age-related macular degeneration (AMD), diabetic retinopathy or liver fibrosis.

[0038] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be regarded as limiting. The contents of all references, GenBank entries, patents and patent application publications cited throughout this application are hereby expressly incorporated herein by reference.

Brief Description of the Drawings

[0039] The following detailed description, given by way of example and not intended to limit the application to only the specific embodiments described, can be best understood when read in conjunction with the accompanying drawings.

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Mode for Carrying Out the Invention

[0051] There are mainly three different approaches to targeting two or more pharmacologies for tumor growth. Most commonly, a patient can be given a cocktail of two or more different agents. This option allows for maximum flexibility with respect to possible combinations of agents and different dosages, but a) due to the increased tablet burden and different dosing schedules for each individual agent, patients are less likely to comply with the treatment; b) due to drug-drug interactions, there may be dosing contraindications; and c) there is a problem of increased risk of side effects of the agents. These problems can reduce the effectiveness of treatment and prevent the achievement of treatment goals, especially in the management of chronic diseases such as cancer.

[0052] The second approach relies on using a fixed-dose combination of agents in a single-dose form. This approach results in improved patient compliance as it reduces the tablet burden. The main drawback of fixed-dose combinations is mainly the limited choice of possible dose ratios between the active ingredients, which makes it more difficult to appropriately dose individual patients to achieve maximum efficacy with minimal side effects. In addition, the different pharmacokinetic properties of the components in the combination can lead to complex temporal inconsistencies in the pharmacodynamic action at the individual targets, thereby compromising the overall efficacy.

[0053] The third approach is the use of multifunctional agents that combine two or more pharmacologies in a single entity. The design and validation of such multifunctional entities are more complex and require extensive research on the optimal ratio of target activities, and the pharmacokinetics of the combined agents can result in the pharmacokinetics of the agents being coordinated at the molecular target level. Multifunctional molecules can also be used in fixed-dose combinations with other agents, thereby combining three or even four pharmacologies in a single tablet to further increase efficacy.

[0054] Through elaborate experiments, the inventors of the present disclosure have invented a novel recombinant multifunctional fusion protein that can adhere to tumors through three mechanisms of action. The first mechanism is to relieve the brake or inhibition on immune cells such as T cells by the PD-1 mediated inhibitory signal. The second mechanism is to prohibit angiogenesis in the tumor microenvironment, relieve immunosuppression, and thus limit target cell proliferation. The other mechanism is to promote the damage of target cells such as tumor cells by immune cells such as NK cells and macrophages. + This is to promote the damage of target cells such as tumor cells.

[0055] The recombinant fusion protein of the present disclosure contains an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein at least one paratope of the anti-PD-L1 antibody or an antigen-binding fragment thereof is linked to a VEGF-binding peptide at the N-terminus of the heavy chain or light chain constituting the paratope via a linker. The recombinant fusion protein can bind to VEGF, PD-L1, and FcR simultaneously. i) It blocks the interaction between PD-L1 on the surface of cancer / tumor cells and PD-L1 on the surface of immune cells (e.g., the surface of T cells), thereby relieving the brake of the PD-1 mediated inhibitory signal on T cells. ii) It binds to FcR on immune cells such as NK cells and macrophages, activating the damage of target cells such as tumor cells by NK cells or macrophages. And iii) It binds to VEGF in, for example, the immune microenvironment to reduce tumor angiogenesis, and thus reduce the supply of blood and nutrients to tumor cells. + This is to activate the damage of target cells such as tumor cells, and iii) It binds to VEGF in, for example, the immune microenvironment to reduce tumor angiogenesis, and thus reduce the supply of blood and nutrients to tumor cells.

[0056] In one embodiment, at least one paratope of an anti-PD-L1 antibody or an antigen-binding fragment thereof can be linked to a VEGF-binding peptide at the N-terminus of the heavy or light chain constituting the paratope via a linker. In another embodiment, each paratope of an anti-PD-L1 antibody or an antigen-binding fragment thereof can be linked to a VEGF-binding peptide at the N-terminus of the heavy or light chain constituting the paratope via a linker. In one embodiment, each paratope of an anti-PD-L1 antibody or an antigen-binding fragment thereof can be linked to a VEGF-binding peptide at the N-terminus of the heavy chain constituting the paratope via a linker. In one embodiment, each paratope of an anti-PD-L1 antibody or an antigen-binding fragment thereof can be linked to a VEGF-binding peptide at the N-terminus of the light chain constituting the paratope via a linker. The recombinant fusion protein of the present disclosure is small in size (15 - 180 kDa) and has a relatively long half-life of 5 - 10 days.

[0057] The three main components included in the recombinant fusion protein of the present disclosure are a VEGF-binding peptide, a linker, and an anti-PD-L1 antibody or an antigen-binding fragment thereof. Those skilled in the art will recognize that there are many design options for selecting the above three components. Since the immunogenicity of non-human animal proteins or peptides can cause allergies and other side effects, preferably, human-derived sequences are used in human cancer therapy. However, other animal proteins or peptides, which are humanized if necessary, can also be used in the present disclosure based on different application purposes.

[0058] Any extracellular Ig-like domain of any VEGFR (VEGFR1, VEGFR2, and VEGFR3) that can bind to VEGF, particularly VEGF-A, can be selected for the construction of the recombinant protein of the present disclosure. In one embodiment, the VEGFR in the recombinant fusion protein is VEGFR1, particularly the second extracellular Ig-like domain of VEGFR1 (VEGFR1D2).

[0059] VEGFR1D2 may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11, wherein VEGFR1D2 binds to VEGF molecules expressed by or around target cells, such as cancer / tumor cells, and thus may limit the growth of target cells. In one embodiment, VEGFR1D2 may include the amino acid sequence of SEQ ID NO: 11.

[0060] The linker mainly functions as a spacer between the VEGF binding peptide and the N-terminus of the heavy or light chain of the anti-PD-L1 antibody. The linker may be composed of amino acids linked together by peptide bonds, preferably 5-30, 10-30, 10-20, or 15 amino acids linked by peptide bonds, where the amino acids are selected from the 20 naturally occurring amino acids. As will be understood by those skilled in the art, one or more of these amino acids may be glycosylated or non-glycosylated. In one embodiment, 5-30, 10-30, 10-20, or 15 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine and lysine. In one embodiment, the linker is composed of a majority of sterically unhindered amino acids such as glycine and alanine. Exemplary linkers are polyglycine (especially Glys, poly(Gly-Ala)), and polyalanine. One exemplary suitable linker shown in the example below is -(Gly-Gly-Gly-Gly-Ser) 3 - (SEQ ID NO: 12) such as a GS linker.

[0061] The linker may also be a non-peptide linker. For example, an alkyl linker such as -NH-, -(CH 2 )s-C(O)- etc. may be used, where s = 2-20. These alkyl linkers may be further substituted by any non-sterically hindering group such as lower alkyl (e.g., C 1~4 lower acyl), halogen (e.g., Cl, Br), CN, NH2, phenyl, or the like.

[0062] In certain embodiments, the anti-PD-L1 antibody can be an isolated monoclonal antibody and includes two heavy chains and two light chains, each heavy chain including the amino acid sequence of SEQ ID NO: 15 and each light chain including the amino acid sequence of SEQ ID NO: 14. The Fab portion (or paratope) of the anti-PD-L1 antibody can bind to PD-L1 on the surface of target cells such as cancer / tumor cells, block the interaction between PD-L1 and PD-1 on the surface of immune cells such as T cells, and thus release the brake on PD-1-mediated inhibitory signals for immune cells. On the other hand, the Fc portion of the anti-PD-L1 antibody can bind to FcRs on immune cells such as NK cells and macrophages, and induce target cell damage by NK cells or macrophages. In certain embodiments, the heavy chain can include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, wherein the anti-PD-L1 antibody can bind to PD-L1, block the PD-1-PD-L1 interaction, and bind to FcRs on NK cells or macrophages and induce cancer cell damage by NK cells and / or macrophages. In certain embodiments, the light chain can include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14, wherein the anti-PD-L1 antibody can bind to PD-L1 and block the PD-1-PD-L1 interaction.

[0063] As used herein, the term "antibody" includes IgG, IgA, IgD, IgE, and IgM whole antibodies, and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (V H ) and a heavy chain constant region. The heavy chain constant region includes three domains, C H1 , C H2 , and C H3 . Each light chain includes a light chain variable region (V Land a light chain constant region. The light chain constant region contains one domain, C L including. V H and V L regions can be further subdivided into hypervariable regions called CDR regions, in which more conserved framework regions (FRs) are interspersed. Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The "functional fragment" of the heavy chain constant region refers to the portion of the heavy chain constant region that retains binding affinity for multiple immune system cells (e.g., effector cells) and the first component (C1q) of the complement system, such as the Fc region, the hinge Fc region, and the like.

[0064] The "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (e.g., the PD-L1 protein). It has been shown that the antigen-binding function of an antibody can be carried out by fragments of the full-length antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody are (i) a monovalent fragment consisting of V L , V H , C L and C H1 domains; (ii) an F(ab') 2 fragment, a bivalent fragment that may contain two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V H and C H1 domains; (iv) an Fv fragment consisting of V L and V H domains of a single arm of the antibody, (v) V HdAb fragments consisting of domains (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (viii) fragments containing heavy chain variable regions, light chain variable regions, and Fc regions or hinge-Fc regions. Further, Fv fragments, V L and V H two domains of are encoded by separate genes, but they can be linked by a synthetic linker using recombinant methods, and the synthetic linker can make them into a single protein chain, where V L and V H regions pair up to form a monovalent molecule (known as single-chain Fv (scFv)). Such single-chain antibodies are also intended to be included within the term. These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as full antibodies.

[0065] The heavy chain variable region CDRs and light chain variable region CDRs of the antibodies or antigen-binding fragments of the present disclosure are defined by the IMGT numbering system. As is well known in the art, the heavy and light chain variable region CDRs can also be determined, for example, by the Chothia, Kabat, AbM or Contact numbering systems / methods.

[0066] The term "antibody-dependent cell cytotoxicity", "antibody-dependent cell-mediated cytotoxicity", or "ADCC" refers to a cell-mediated immune defense in which immune effector cells actively lyse target cells such as cancer cells to which an antibody (e.g., an anti-PD-L1 antibody) or a VEGF-binding peptide is bound to a cell surface antigen.

[0067] The term "antibody-dependent cell phagocytosis" or "ADCP" refers to antibody-mediated phagocytosis of target cells by macrophages or neutrophils, where the antibody binds to an antigen epitope on the target cell via its Fab portion and binds to the FcR of macrophages or neutrophils via its Fc portion.

[0068] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, for example, non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, etc., with mammals such as non-human primates, sheep, dogs, cats, cows, and horses being preferred.

[0069] The term "EC 50 ", also known as the half-maximal effective concentration, refers to the concentration of a molecule (e.g., an antibody, a fusion protein, and the like) that produces 50% of the maximal effect.

[0070] The term "IC 50 ", also known as the half-maximal inhibitory concentration, refers to the concentration of an inhibitor (e.g., an antibody, a fusion protein, and the like) that inhibits a particular biological process by only half.

[0071] The term "therapeutically effective amount" means an amount of a molecule (e.g., an antibody, a fusion protein, or the like) sufficient to prevent or alleviate symptoms associated with a disease or medical condition (such as cancer). A therapeutically effective amount is understood to be in line with the context of the medical condition being treated, and the actual effective amount can be readily recognized by those skilled in the art.

[0072] As used herein, "sequence identity" refers to the percentage of nucleotide / amino acid residues in a subject sequence that are identical to the nucleotide / amino acid residues in a reference sequence after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity between the sequences. Alignment of a pair and multiple sequences for the purpose of determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to those skilled in the art, such as using publicly available computer software such as ClustalOmega, T-coffee, Kalign, and MAFFT.

[0073] In addition, the present disclosure provides a polynucleotide encoding a recombinant fusion protein, an anti-PD-L1 antibody, or an antigen-binding portion thereof, and an expression vector expressing the recombinant fusion protein. Examples of vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformation-competent artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and human artificial episomal chromosomes (HAECs).

[0074] The present disclosure provides a host cell containing the above expression vector. The host cell can be transformed or transfected using the expression vector. Suitable host cells include Escherichia coli (E. coli), yeast, and other eukaryotes. Preferably, E. coli, yeast, or a mammalian cell line (such as COS or CHO) is used.

[0075] In another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant fusion protein, an anti-PD-L1 antibody or an antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, or a host cell of the present disclosure, formulated with a pharmaceutically acceptable carrier. The pharmaceutical composition may contain excipients and / or adjuvants. Optionally, the composition may contain one or more additional pharmaceutically active ingredients, such as another antibody or another drug. The pharmaceutical composition of the present disclosure can also be administered, for example, in combination therapy with another immunostimulant, anticancer agent, antiviral agent, or vaccine.

[0076] The pharmaceutical composition can contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion aids or suspending aids, solubilizers, colorants, flavorants, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0077] The main vehicle or carrier in a pharmaceutical composition may be essentially aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally with other substances commonly used in injections added. For example, the vehicle or carrier can be neutral buffered saline, or saline mixed with serum albumin. Other exemplary pharmaceutical compositions can include Tris buffer, which may further contain sorbitol or a suitable substitution thereof, or acetate buffer. In one embodiment of the present disclosure, the composition can be prepared for storage in the form of a lyophilized cake or an aqueous solution by mixing a selected composition having a desired purity with an optional formulating agent (Remington's Pharmaceutical Sciences above). Further, the therapeutic composition can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0078] The pharmaceutical composition can be used for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active molecule can be coated in a substance that protects it from the action of acids or enzymes and other natural conditions that can inactivate it. As used herein, the phrase "parenteral administration" means a route of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, arterial, intracavitary, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions. Alternatively, the antibodies of the present disclosure can be administered via non-parenteral routes such as local, epidermal or mucosal routes of administration, e.g., nasal, oral, vaginal, rectal, sublingual or topical administration. In certain embodiments, the pharmaceutical composition can be administered via an intraperitoneal, subcutaneous, or intravenous route.

[0079] The pharmaceutical composition can be in the form of a sterile aqueous solution or dispersion. They can also be formulated as microemulsions, liposomes, or other regular structures suitable for high drug concentrations.

[0080] The amount of the active ingredient that can be combined with the carrier substance to produce a single-dose administration form varies depending on the subject to be treated and the specific administration method, and generally is the amount of the composition that produces a therapeutic effect. Generally, this amount can range from about 0.01% to about 99% of the active ingredient, out of 100 percent, of the active ingredient combined with a pharmaceutically acceptable carrier.

[0081] The dosing schedule is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, several divided doses can be administered over time, or the dose can be proportionally decreased or increased according to the dictates of the exigencies of the therapeutic situation. It is particularly advantageous to formulate the parenteral composition in unit dosage form for ease of administration and to uniformize the dosage. As used herein, the unit dosage form refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of the active molecule calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the recombinant protein can be administered as a sustained-release formulation, in which case the required dosing frequency is less.

[0082] For administration of the fusion protein, the dosage can range from about 0.0001 to 100 mg / kg of the recipient's body weight. An exemplary treatment schedule involves administration twice a week.

[0083] The "therapeutically effective dosage" of the fusion protein of the present disclosure preferably results in a decrease in the severity of the disease symptoms, an increase in the frequency and duration of periods free of disease symptoms, or a prevention of the disorders or impairments resulting from the suffering of the disease. For example, for the treatment of a subject having a tumor, the "therapeutically effective dosage" preferably inhibits tumor growth by at least about 40%, more preferably at least about 60%, more preferably at least about 80%, and more preferably at least about 99% compared to an untreated subject. The therapeutically effective amount of the fusion protein of the present disclosure can reduce tumor size or, alternatively, can alleviate symptoms in a subject who can typically be human or another mammal.

[0084] The pharmaceutical composition can be a sustained-release formulation, including implants, transdermal patches, and microencapsulation delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0085] The pharmaceutical composition can be administered via medical devices such as (1) needleless subcutaneous syringes (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinjection pumps (U.S. Patent No. 4,487,603); (3) transdermal devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patent Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0086] In certain embodiments, the fusion proteins of the present disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic fusion proteins of the present disclosure cross the blood-brain barrier, the fusion proteins can be formulated in liposomes, which can additionally include targeting moieties to enhance selective transport to specific cells or organs. See, for example, U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331.

[0087] In vivo gene therapy is also envisioned, in which a nucleic acid molecule encoding the fusion protein or a derivative thereof of the present disclosure is directly introduced into a subject. For example, a nucleic acid sequence encoding the recombinant fusion protein of the present disclosure is introduced into target cells via local injection of a nucleic acid construct, with or without using a suitable delivery vector such as an adeno-associated virus vector. Alternative viral vectors include, but are not limited to, retroviruses, adenoviruses, herpes simplex viruses, papillomavirus vectors. Physical transfer of viral vectors can be achieved in vivo by local injection of the desired nucleic acid construct, or other suitable delivery vectors containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or particle bombardment (gene gun).

[0088] The compositions of the present disclosure can be used alone or in combination with other therapeutic agents to enhance the therapeutic effect or to reduce potential side effects.

[0089] Another object of the present disclosure is to provide a method for preparing the recombinant fusion protein, anti-PD-L1 antibody, or antigen-binding fragment thereof described above. In one embodiment, the method comprises: (1) providing a nucleic acid molecule encoding a recombinant fusion protein, anti-PD-L1 antibody or antigen-binding fragment thereof; (2) constructing an expression vector containing the nucleic acid molecule of (1); (3) transfecting or transforming a suitable host cell with the expression vector of (2) and culturing the host cell to express the recombinant fusion protein, anti-PD-L1 antibody, or antigen-binding fragment thereof; and (4) purifying the recombinant fusion protein, anti-PD-L1 antibody, or antigen-binding fragment thereof. The preparation can be carried out by those skilled in the art using known techniques.

[0090] Another object of the present disclosure is to provide a method for treating cancer using the pharmaceutical composition of the present disclosure, which comprises administering an effective amount of the aforementioned pharmaceutical composition to a patient or subject in need thereof. In one embodiment, the pharmaceutical composition is used for treating cancers associated with VEGF and / or PD-L1 signaling / overexpression, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma, cervical cancer, or angiosarcoma.

[0091] The present disclosure also provides a method for treating other diseases associated with VEGF and / or PD-L1 signaling / overexpression, including age-related macular degeneration (AMD), diabetic retinopathy, and liver fibrosis, using the pharmaceutical composition of the present disclosure.

[0092] Herein, the present disclosure will be further described using the following non-limiting examples. Examples

[0093] The structures of exemplary fusion proteins of the present disclosure, IMM2518 and IMM2519, and the control protein are briefly described below.

[0094] IMM2515H is an IgG antibody that specifically binds to PD-L1 and contains two heavy chains and two light chains, wherein the amino acid sequences of the heavy chain variable region, heavy chain constant region, light chain variable region, and light chain constant region are set forth in SEQ ID NOs: 7, 9, 8, and 10, respectively. The amino acid sequences of the heavy chain and light chain are set forth in SEQ ID NOs: 15 and 14, respectively.

[0095] IMM2518 is an exemplary recombinant fusion protein of the present disclosure and contains two copies of VEGFR1D2 (SEQ ID NO: 11) linked to the N-terminus of the two heavy chains of IMM2515H via a linker (SEQ ID NO: 12) as shown in FIG. 1. In other words, IMM2518 contains two long chains of SEQ ID NO: 13 and two short chains of SEQ ID NO: 14.

[0096] IMM2519 is an exemplary recombinant fusion protein of the present disclosure and, as shown in FIG. 1, contains two copies of VEGFR1D2 (SEQ ID NO: 11) linked to the N-terminus of the two light chains of IMM2515H via a linker (SEQ ID NO: 12). In other words, IMM2519 contains two long chains of SEQ ID NO: 15 and two short chains of SEQ ID NO: 16.

[0097] IMM2510 is a recombinant fusion protein having a structure similar to IMM2518, differing only in the anti-PD-L1 antibody, and specifically contains two long chains of SEQ ID NO: 17 and two short chains of SEQ ID NO: 18.

[0098] VEGFR1D2-Fc is a fusion protein consisting of VEGFR1D2 and Fc and contains the amino acid sequence of SEQ ID NO: 19. Example 1. Construction of a vector expressing a recombinant fusion protein 1.1 IMM2518

[0099] The full-length sequence encoding IMM2518 was artificially designed. Specifically, for the long chain, 57 nucleotides encoding the signal peptide of mouse IgG1 heavy chain (SEQ ID NO: 21) were added to the 5'-end of the VEGF-binding peptide-linker-anti-PD-L1 heavy chain coding sequence (SEQ ID NO: 23), and a Kozak sequence (GCCGCCACC) was added to the 5'-end of the signal peptide sequence. Finally, HindIII and NheI restriction sites were added to the 5' and 3'-ends of the resulting sequence, respectively. For the short chain, the same signal sequence and Kozac sequence were added to the coding sequence of the anti-PD-L1 light chain (SEQ ID NO: 24), and HindIII and XbaI restriction sites were added to the 5' and 3'-ends of the resulting sequence, respectively. The two resulting sequences were artificially synthesized and subcloned into pMac-H and pMac-L vectors, respectively. 1.2 IMM2519

[0100] A full-length sequence encoding IMM2519 was artificially designed. Specifically, for the long chain, 57 nucleotides encoding the signal peptide of mouse IgG1 heavy chain (SEQ ID NO: 21) were added to the 5' end of the anti-PD-L1 heavy chain coding sequence (SEQ ID NO: 25), and a Kozak sequence (GCCGCCACC) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction sites were added to the 5' and 3' ends of the resulting sequence, respectively. For the short chain, the same signal sequence and Kozac sequence were added to the coding sequence of VEGF binding peptide-linker-anti-PD-L1 light chain (SEQ ID NO: 26), and HindIII and XbaI restriction sites were added to the 5' and 3' ends of the resulting sequence, respectively. The two resulting sequences were artificially synthesized and subcloned into pMac-H and pMac-L vectors, respectively. Example 2. Preparation and quality analysis of recombinant fusion protein

[0101] The expression vectors constructed were used to transiently express proteins in CHO-S cells. Generally, 1) CHO-S cells at a density of 1×10 6 / ml were seeded in transient transfection medium (TransFx-CTMCHO Transient Transfection Medium, Hyclone) containing 6 mM glutamine one day before transient transfection; 2) The heavy / long chain expression vector and the light / short chain expression vector, having a total of 1 μg / ml of DNA and a mass ratio of 1:1, were added to OPTI-MEM culture medium (Gibco) in a volume that accounted for 1 / 20 of the total transient transfection system; 3) PEI (MW40,000 polyetherimide hydrochloride, polysciences) prepared at 1 mg / ml was added to OPTI-MEM culture medium (Gibco) in a volume that accounted for 1 / 20 of the transient transfection system at a PEI:DNA ratio of 4:1; 4) The PEI dilution was gradually added to the DNA dilution, mixed, and incubated at room temperature for 20 minutes; 5) The PEI / DNA mixture was added to the cell culture, and the cells were incubated at 37°C and 5% CO2 cultured in an incubator while being shaken at 110 rpm; 6) a transfection enhancer (1 mM sodium butyrate, 0.25% V / V DMSO) was added 2 days later, and the temperature was decreased to 33 °C for culturing; 7) when the cell viability decreased to 50% or less, centrifugation was performed at 3000 rpm for 5 minutes, the supernatant was collected, and subjected to affinity purification with Protein A.

[0102] As specified in Table 1, the SEC-HPLC diagram showed that IMM2518 had a relatively high purity, a relatively low amount of aggregation, and slight degradation. Table 1 SEC-HPLC results of the recombinant fusion protein of the present disclosure

Table 1

[0103] For the VEGF binding assay, recombinant human VEGF-165 protein (Cat#11066-HNAH, Sino Biologicals) was prepared in carbonate-bicarbonate coating buffer (Cat#C3041, Sigma-Aldrich), transferred to ELISA plates at 50 ng / well, and the plates were placed in a refrigerator at 4 °C overnight. The plates were blocked with 150 μl / well of 3% skim milk at room temperature for 2 hours. Next, 100 μl of serially diluted recombinant fusion protein was added to the plates, incubated at 37 °C for 1 hour, and washed 5 times using PBS-T with 0.05% Tween® 20. The plates were then added with HRP rabbit anti-human IgG Fc (Cat#: 309-036-008, Jackson ImmunoResearch Lab) and incubated at 37 °C for 1 hour. The plates were washed 5 times using PBS-T, 100 μL of TMB was added, and subsequently, 500 μL of 1N H 2 SO 4was added and the absorbance was measured using a microplate reader.

[0104] As shown in Figure 2, the two recombinant fusion proteins of the present disclosure had similar VEGF-165 binding activities. Example 4. Recombinant fusion protein bound to PD-L1

[0105] For the PD-L1 binding assay, 100 μl of 1×10 6 / ml CHO-PD-L1 cells (Cat#YMAK-C006, ImmuneOnco) were incubated with 100 μl of serial diluted recombinant fusion proteins and control proteins IMM2515H and hIgG1-Fc (SEQ ID NO: 20) at 4°C for 40 minutes, respectively. After washing with cold PBS, the cells were incubated with an anti-human IgG-Fc antibody conjugated with FITC (Cat#F9512, Sigma) for 40 minutes. The cells were then washed twice and subjected to FACS analysis.

[0106] The results are shown in Figure 3. The binding activities of IMM2518 and IMM2519 to PD-L1 + cells were 2.6-fold and 4.2-fold lower than those of the single-specific anti-PD-L1 antibody IMM2515H.

[0107] The recombinant fusion proteins were also tested for their binding activities to CHO-PD-L1 cells together with control proteins IMM2515H, IMM2510, atezolizumab, and hIgG1-Fc according to the above protocol.

[0108] As shown in Figure 4, the binding activity of IMM2518 to PD-L1 + cells was similar to that of IMM2510. Example 5. Binding affinity of the recombinant fusion protein for VEGF and PD-L1

[0109] The binding affinity of the recombinant fusion protein for the antigen was tested in the Kinetics Assay mode using a label-free biomolecular analyzer (Access Medical Systems, Gator bio) with Q buffer (10 mM, pH 7.4, PBS + 0.02% Tween® + 0.2% BSA) as the assay buffer and a human Fc probe as the assay probe. Here, IMM2518 was serially diluted 2-fold starting from 10 μg / ml (a total of 5 concentrations), and the antigens used were 10 μg / ml VEGF165 (Cat#11066-HNAH, sino Biological) and 10 μg / ml PD-L1-his (Cat#90251-C08H, sino Biological). The probe was regenerated using a regeneration buffer at pH 2.0 (10 mM Gly + 150 mM NaCl, pH 2.0), and the analyzer was set at 25°C. Table 2 Affinity of the recombinant fusion protein for VEGF and PD-L1 measured by BLI

Table 2

[0110] The test results showed that when IMM2518 bound to VEGF165, the dissociation rate was very low, resulting in no specific dissociation steady state. The PD-L1 affinity of IMM2518 was 0.441 nM. Example 6. Blocking activity of the recombinant fusion protein against PD-1-PD-L1

[0111] For the analysis of PD-1-PD-L1 blocking activity, 50 μl of 5 × 10 5 / ml CHO-PD-L1 cells (Cat#YMAK-C006, ImmuneOnco) were each incubated with 50 μl of serial diluted recombinant fusion proteins and control proteins (IMM2515H, IMM2510, atezolizumab and hIgG1-Fc) at 4°C for 45 minutes, 50 μl of PD1-mFc (self-made by ImmuneOnco, SEQ ID NO: 22) was added, and incubated at 4°C for 45 minutes. After washing with cold PBS, the cells were incubated with a PE-conjugated anti-mouse IgG-Fc antibody (Biolegend, Cat#405307). The cells were then washed twice and subjected to FACS analysis.

[0112] As shown in Figure 5, PD-L1 + The ability of IMM2518 to block the binding of PD-1 protein to PD-L1 cells was 2.2-fold and 3-fold lower than that of IMM2515H and atezolizumab, respectively, and was close to that of IMM2510. Example 7. Blocking activity of recombinant fusion proteins against VEGF activity

[0113] The blocking ability against VEGF activity was measured using Jurkat-CVR cells constructed by ImmuneOnco. The CVR (chimeric VEGFR1D2 receptor) consisting of the second extracellular domain of VEGFR1, the CD8α hinge domain, the transmembrane region and intracellular domain of CD28, and the CD3z signaling domain contains the amino acid sequence of SEQ ID NO: 6. 50 μl of 5×10 5 / ml Jurkat-CVR cells were incubated with 50 μl of serial diluted recombinant fusion proteins and control proteins (VEGFR1D2-Fc (SEQ ID NO: 19), IMM2510, bevacizumab, and hIgG1-Fc) at 37°C for 45 minutes, respectively, 50 μl of VEGF-Fc (self-made by ImmuneOnco, SEQ ID NO: 27) was added, and incubated overnight at 37°C. After washing with cold PBS, the cells were incubated with an anti-human CD69 antibody conjugated with PE (Biolegend, Cat# 310906). The cells were then washed twice and subjected to FACS analysis.

[0114] As shown in Figure 6, the blocking ability of IMM2518 against VEGF activity was slightly lower than that of VEGFR1D2-Fc and bevacizumab, and was close to that of IMM2510. Example 8. PD-L1 Binding Specificity of Recombinant Fusion Proteins

[0115] Human PD-L1 (Cat#10084-H08H, sino Biological), mouse PD-L1 (Cat#50010-M08H, sino Biological), monkey PD-L1 (Cat#90251-C08H, sino Biological), rat PD-L1 (Cat#80450-R08H, sino Biological), and human PD-L2 (Cat#10292-H08H, sino Biological) in carbonate-bicarbonate coating buffer (Cat#C3041, Sigma) were coated onto the plate at 50 ng / well, and the plate was placed in the refrigerator at 4°C overnight. The plate was blocked with 150 μl / well of 3% skim milk for 2 hours at room temperature. The plate was added with 100 μl of serially diluted recombinant fusion protein, incubated at 37°C for 1 hour, and washed 5 times using PBS-T with 0.05% Tween® 20. The plate was added with HRP rabbit anti-human IgG Fc (Cat#:309-036-008, Jackson ImmunoResearchLab) and incubated at room temperature for 1 hour. The plate was washed 5 times using PBS-T, added with 100 μL of TMB, and subsequently added with 500 μL of 1N H 2 SO 4 to stop the color development, and the absorbance was measured in a microplate reader.

[0116] As shown in Figure 7, IMM2518 bound to human PD-L1 and cross-reacted with monkey PD-L1, but did not cross-react with rat PD-L1, mouse PD-L1, or human PD-L2. Example 9. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by recombinant fusion protein

[0117] 50 μl of 6×10 5 / ml Raji-PD-L1 cells (Cat#21888-25 mg, Sigma) labeled with CFSE dye, and 100 μl of 6×10 5NK92MI cells (Cat#CRL-2408, ATCC) at / ml were mixed at a ratio of 1:2, and the mixed cells were cultured at 37°C for 4 hours with 50 μl of serial diluted IMM2518 or IMM2515H. The cell culture was then added with propidium iodide (PI, Cat#P4170, Sigma) at a final concentration of 5 μg / ml and subjected to FACS to measure the PI signal from CFSE-labeled target cells. The ADCC-mediated cell lysis rate was calculated as % lysis (or % ADCC) = (% PI-positive cells treated with IMM2518 or positive control - % PI-positive cells treated with negative control protein) / (100 - % PI-positive cells treated with negative control protein) × 100.

[0118] The results are shown in Figure 8A. The ADCC induced by IMM2518 against Raji-PD-L1 cells was 6-fold lower than that induced by IMM2515H.

[0119] 50 μl of 5×10 treated with trypsin 5 / ml RKO cells (Cat#TCHu116, Cell Collection of Chinese Academy of Sciences) or HCC827 cells (Cat#TCHu153, Cell Collection of Chinese Academy of Sciences), and 50 μl of 5×10 of NK92MI cells (Cat#CRL-2408, ATCC) stably expressing FcγRIIIa (158V) as effector cells were mixed at a ratio of 1:1, and the mixed cells were cultured at 37°C, 5% CO 5 / ml with 50 μl of serial diluted IMM2518, IMM2515H, IMM2510, atezolizumab, or hIgG1-Fc 2It was cultured overnight. Next, 20 μl of CCK-8 (Beyotime, Cat# C0039) was added to the cell culture medium, incubated in an incubator for 3 hours, and the absorbance at 450 nm was measured. The ADCC-mediated cell lysis rate was calculated as % lysis (or % ADCC) = (OD450 of effector cells and target cells in the negative control group - OD450 of IMM2518 or effector cells and target cells treated with the positive control) / (OD450 of effector cells and target cells in the negative control group - OD450 of effector cells in the negative control group) × 100%.

[0120] As shown in FIGS. 8B and 8C, the ADCC induced by IMM2518 against RKO cells and HCC827 cells was 6-fold and 33-fold lower than that induced by IMM2515H, respectively. Example 10. Antibody-dependent cell phagocytosis (ADCP) induced by the recombinant fusion protein

[0121] 2.5×10 5 / ml of THP-1 cells (Cat# TCHu 57, Cell Collection of Chinese Academy of Sciences) in 100 μl of complete culture medium were seeded on a 96-well flat-bottom plate containing 200 ng / ml PMA (Sigma, Cat# 379346-1MG) and cultured for 48 hours. RKO cells (Cat# TCHu116, Cell Collection of Chinese Academy of Sciences) as target cells were treated with trypsin and labeled with CFSE (Cat# 21888-25mg, Sigma). 100 μl of 5.0×10 5 / ml CFSE-labeled RKO cells were mixed with THP-1 cells on a 96-well plate at an effector:target ratio of 1:2, and the mixed cells were incubated with 50 μl of serial diluted IMM2518, IMM2515H, IMM2510, atezolizumab, or hIgG1-Fc at 37 °C, 5% CO 2It was cultured for 2 hours. After incubation, the supernatant was discarded, and the plates were washed 5 times with 250 μl / well of PBS to remove unbound target cells. THP-1 cells were suspended by repeated pipetting and subjected to FACS to measure the CFSE signal from THP-1 macrophages.

[0122] As shown in Figure 9, the phagocytic activities induced by IMM2518, IMM2515H, and IMM2510 against RKO tumor cells were comparable. Example 11. The recombinant fusion protein had good antitumor effects.

[0123] The in vivo antitumor efficacy of the recombinant fusion protein of the present disclosure was tested using humanized C57BL / 6hPD-1 mice (Gempharmtech). For cell passage, MC38-hPD-L1 cells (mouse colon tumor cell line, AppTec) were cultured in monolayer in vitro and digested twice a week using trypsin-EDTA. When the cell saturation density reached 80% - 90% and the total count met the requirements, the cells were harvested and counted.

[0124] In the right axilla of the mice, 3×10 in 100 μl of PBS 5 MC38-hPD-L1 cells were subcutaneously seeded. On day 0, 36 animals with an average tumor size of 60 - 90 mm 3 were selected, and 6 mice in each of the 6 groups were randomly assigned. From this day on, the mice were intraperitoneally administered with combinations of DPBS, IMM2510 (6.0 mg / kg), IMM2518 (6.0 mg / kg), IMM2515H (5.0 mg / kg), VEGFR1D2-Fc (2.5 mg / kg), and IMM2515H (5.0 mg / kg) + VEGFR1D2-Fc (2.5 mg / kg) twice a week for 4 weeks. Tumor size and animal body weight were measured twice a week.

[0125] The tumor volume (V) was (length × width 2) / 2. Tumor growth inhibition (TGI) was calculated by the formula: tumor growth inhibition rate = (1 - change in tumor volume in the treatment group / change in tumor volume in the control group) × 100%.

[0126] Data were presented as mean ± standard error. Dunnett's multiple comparison test was used to compare between two groups, and a p-value less than 0.05 was considered statistically significant.

[0127] As shown in Figure 10, in mice treated with a single-specificity targeting agent or a combination of two single-specificity targeting agents, tumor size increased slowly compared to the DPBS group, and an obvious slow increase in tumor size was observed in mice treated with IMM2510 or IMM2518. The antitumor efficacy of IMM2518 was higher than that of IMM2510. Here, the TGI of IMM2510 treatment was 74.07%, and the TGI of IMM2518 treatment was 97.47%. Example 12. Antitumor effects of recombinant fusion proteins administered in different ways

[0128] Following the test steps of Example 11, the recombinant fusion proteins were tested at lower doses for their in vivo antitumor effects with different administration routes.

[0129] 3×10 in 100 μl of PBS was subcutaneously inoculated into the right axilla of humanized C57BL / 6 hPD-1 mice 5 MC38-hPD-L1 cells. On day 0, animals with an average tumor size of 60 - 90 mm 3 were selected, and 6 mice in each of the 4 groups were randomly assigned. From this day on, DPBS via intraperitoneal (IP) injection, IMM2518 (4.0 mg / kg) via intraperitoneal (IP) injection, IMM2518 (4.0 mg / kg) via subcutaneous (SC) injection, and IMM2518 (4.0 mg / kg) via tail vein injection were administered to the mice twice a week for 4 weeks, respectively. Tumor size and animal body weight were measured twice a week.

[0130] The mouse tumor sizes of each group during the experiment are shown in Figure 11. It can be seen that when IMM2518 was administered at a relatively low dose, it still significantly inhibited tumor growth compared to the DPBS group, and the effect was the same among the groups of intraperitoneal injection, subcutaneous injection, and intravenous injection.

[0131] The sequences of this application are summarized below.

Table 3

[0132] This application has been described above in connection with one or more embodiments, but this application is not limited to those embodiments, and the description is intended to include all alternatives and equivalents that may be included within the spirit and scope of the appended claims. All references cited herein are hereby incorporated by reference in their entirety.

[0133] [Item 1] A recombinant fusion protein comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof and a VEGF-binding peptide, The anti-PD-L1 antibody or its antigen-binding fragment thereof includes a heavy-chain variable region, a heavy-chain constant region, and a light-chain variable region. The heavy-chain variable region includes HV-CDR1, HV-CDR2, and HV-CDR3. The light-chain variable region includes LV-CDR1, LV-CDR2, and LV-CDR3. The HV-CDR1, the HV-CDR2, the HV-CDR3, the LV-CDR1, and the LV-CDR3 each include the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, and 5, respectively. The LV-CDR2 includes the amino acid sequence "YTS". The heavy-chain constant region is linked to the C-terminus of the heavy-chain variable region and includes FcR-binding ability. The VEGF-binding peptide includes the second extracellular Ig-like domain (VEGFR1D2) of vascular endothelial growth factor receptor 1 (VEGFR1). The VEGF-binding peptide is linked to the N-terminus of the heavy-chain variable region or the light-chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment thereof. Recombinant fusion protein. [Item 2] The recombinant fusion protein according to Item 1, wherein the VEGF-binding peptide includes the amino acid sequence of SEQ ID NO: 11. [Item 3] The recombinant fusion protein according to Item 1, wherein the heavy-chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the light-chain variable region includes the amino acid sequence of SEQ ID NO: 8. [Item 4] The recombinant fusion protein according to Item 1, wherein the VEGF-binding peptide is linked to the N-terminus of the heavy-chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment thereof. [Item 5] The recombinant fusion protein according to Item 1, wherein the heavy-chain constant region includes the amino acid sequence of SEQ ID NO: 9. [Item 6] The recombinant fusion protein according to Item 1, wherein the VEGF-binding peptide is linked to the anti-PD-L1 antibody or its antigen-binding fragment thereof via a linker. [Item 7] The linker is -(Gly-Gly-Gly-Gly-Ser) 3The recombinant fusion protein according to item 6, which is (Accession number: 12). [Item 8] The recombinant fusion protein according to item 1, further comprising a light chain constant region, wherein the light chain constant region contains the amino acid sequence of Accession number: 10 and is linked to the C-terminus of the light chain variable region. [Item 9] i) A chain of a VEGF-binding peptide-linker-anti-PD-L1 antibody heavy chain variable region-heavy chain constant region containing the amino acid sequence of Accession number: 13, and a chain of an anti-PD-L1 antibody light chain variable region-light chain constant region containing the amino acid sequence of Accession number: 14; or ii) A chain of an anti-PD-L1 antibody heavy chain variable region-heavy chain constant region containing the amino acid sequence of Accession number: 15, and a chain of a VEGF-binding peptide-linker-anti-PD-L1 antibody light chain variable region-light chain constant region containing the amino acid sequence of Accession number: 16 The recombinant fusion protein according to item 1, comprising. [Item 10] A nucleic acid molecule encoding the recombinant fusion protein according to any one of items 1 to 9. [Item 11] An expression vector comprising the nucleic acid molecule according to item 10. [Item 12] A host cell comprising the expression vector according to item 11, or comprising the nucleic acid molecule according to item 10 integrated into its genome. [Item 13] A pharmaceutical composition comprising the recombinant fusion protein according to any one of items 1 to 9, the nucleic acid molecule according to item 10, the expression vector according to item 11, or the host cell according to item 12, and at least one pharmaceutically acceptable carrier. [Item 14] Use of the pharmaceutical composition according to item 13 in the manufacture of a medicament for treating a disease related to PD-L1 and / or VEGF signaling, wherein the disease is cancer, age-related macular degeneration (AMD), diabetic retinopathy, or liver fibrosis. [Item 15] The cancer is the use according to item 14, selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma multiforme, cervical cancer, and angiosarcoma.

Claims

1. A recombinant fusion protein comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof and a VEGF-binding peptide, wherein the anti-PD-L1 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, a heavy chain constant region, and a light chain variable region, the heavy chain variable region comprises HV-CDR1, HV-CDR2, and HV-CDR3, the light chain variable region comprises LV-CDR1, LV-CDR2, and LV-CDR3, the HV-CDR1, the HV-CDR2, the HV-CDR3, the LV-CDR1, and the LV-CDR3 each comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, and 5, the LV-CDR2 comprises the amino acid sequence "YTS", the heavy chain constant region is linked to the C-terminus of the heavy chain variable region and comprises FcR-binding ability, the VEGF-binding peptide comprises the second extracellular Ig-like domain of vascular endothelial growth factor receptor 1 (VEGFR1) (VEGFR1D2), the VEGF-binding peptide is linked to the N-terminus of the heavy chain variable region or the light chain variable region of the anti-PD-L1 antibody or the antigen-binding fragment thereof, a recombinant fusion protein.

2. The recombinant fusion protein according to claim 1, wherein the VEGF-binding peptide comprises the amino acid sequence of SEQ ID NO:

11.

3. The recombinant fusion protein according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

4. The recombinant fusion protein according to claim 1, wherein the VEGF-binding peptide is linked to the N-terminus of the heavy chain variable region of the anti-PD-L1 antibody or the antigen-binding fragment thereof.

5. The recombinant fusion protein according to claim 1, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

9.

6. The recombinant fusion protein according to claim 1, wherein the VEGF-binding peptide is linked to the anti-PD-L1 antibody or the antigen-binding fragment thereof via a linker.

7. The linker is -(Gly-Gly-Gly-Gly-Ser) ３ - (SEQ ID NO: 12), the recombinant fusion protein according to claim 6.

8. The recombinant fusion protein according to claim 1, further comprising a light chain constant region, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 10 and is linked to the C-terminus of the light chain variable region.

9. i) a chain of a VEGF-binding peptide-linker-anti-PD-L1 antibody heavy chain variable region-heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 13, and a chain of an anti-PD-L1 antibody light chain variable region-light chain constant region comprising the amino acid sequence of SEQ ID NO: 14; or ii) a chain of an anti-PD-L1 antibody heavy chain variable region-heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 15, and a chain of a VEGF-binding peptide-linker-anti-PD-L1 antibody light chain variable region-light chain constant region comprising the amino acid sequence of SEQ ID NO: 16 The recombinant fusion protein according to claim 1, comprising the same.

10. A nucleic acid molecule encoding the recombinant fusion protein according to any one of claims 1 to 9.

11. An expression vector comprising the nucleic acid molecule according to claim 10.

12. A host cell comprising the expression vector according to claim 11, or comprising the nucleic acid molecule according to claim 10 integrated into its genome.

13. A pharmaceutical composition comprising the recombinant fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the expression vector according to claim 11, or the host cell according to claim 12, and at least one pharmaceutically acceptable carrier.

14. Use of the pharmaceutical composition according to claim 13 in the manufacture of a medicament for treating a disease associated with PD-L1 and / or VEGF signaling, wherein the disease is cancer, age-related macular degeneration (AMD), diabetic retinopathy, or liver fibrosis.

15. The use according to claim 14, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma multiforme, cervical cancer, and angiosarcoma.

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