Application of multi-domain fusion protein

Abstract

The invention provides application of a multi-structural-domain fusion protein in preparation of a medicine for treating or preventing tumors. The fusion protein comprises an anti-PD-L1 single-domain antibody fragment, an antagonistic VEGF fragment and a TGF-beta binding fragment. The provided multi-structural domain fusion protein with anticancer activity can organically combine the functions of blocking PD-L1 / PD-1 interaction by the anti-PD-L1 monoclonal antibody, reducing microvascular growth and inhibiting metastatic diseases by the anti-VEGF monoclonal antibody, relieving tolerance of cancer cells to TGF-beta signals by a TGF-beta receptor and enhancing immune response together in one antibody fusion protein molecule, so that the anti-cancer activity of the anti-cancer fusion protein is improved, and the anti-cancer activity of the anti-cancer fusion protein is improved. Therefore, the compound can be used for treating or preventing tumors.

Description

Uses of multi-domain fusion proteins Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to use of a multi-domain fusion protein with anti-cancer activity. Background Art

[0002] In the era of the widespread rise of cancer therapeutics, protein engineering technology has continued to advance. Research into the mechanisms of antibody therapy is also deepening, and multi-targeted drugs (such as polyclonal antibodies or multi-domain fusion proteins) are gradually emerging. These drugs are expected to enhance the specificity and effectiveness of tumor immunotherapy by increasing the number of targets. In theory, in specific treatment settings, multi-targeted drugs can achieve more significant efficacy than single-agent combination therapy. Compared with bispecific antibodies, multi-target drugs such as multi-antibodies or multi-domain fusion proteins have broader clinical application prospects. While binding to different tumor surface antigens, they can also bridge and activate immune cells. In recent years, this treatment method has been widely recognized in theory. However, to date, the research and development of most multi-specific antibody drugs is still in its infancy, and the fastest-progressing ones are in clinical phase 1 or 2, such as Sanofi's SAR-443579 (targeting CD16 / NKp46 / CD123), CStone Pharmaceuticals' NM21-1480 (targeting PD-L1 / 4-1BB / HSA), and AbbVie and HARPOON's HPN-217 (targeting BCMA / CD3 / HSA). Their safety and efficacy still require more data verification and support in the future.

[0003] In addition, although anti-PD-1 / PD-L1 monoclonal antibodies as immune checkpoint inhibitors have been widely used in the treatment of various tumors, the proportion of patients who benefit is still not high, especially for "cold tumors", the efficacy of immune checkpoint inhibitors is not obvious. For example, M7824, which is a fusion of the anti-PD-L1 monoclonal antibody Avelumab and TGF-β receptor II (TGF-βTrap), has been terminated in clinical trials due to reasons such as insufficient efficacy. This shows that immune checkpoint inhibitors can also be combined with other targets to form multi-target drugs to further improve the immune status of the tumor microenvironment and enhance efficacy. Therefore, there is an urgent need for a better and safer dual antibody or multi-antibody (or multi-domain fusion protein) to meet the unmet clinical needs of cancer patients.

[0004] Summary of the Invention

[0005] The present invention aims to provide a multi-domain fusion protein with anti-cancer activity, which can be used as a therapeutic agent for treating and / or preventing tumors, such as lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, sarcoma, head and neck cancer, nasopharyngeal cancer, cervical cancer, bile duct cancer and hematological malignancies, in particular lung cancer, breast cancer, liver cancer and cervical cancer.

[0006] To achieve the above objectives and other related objectives, the present invention provides, on the one hand, the use of a fusion protein in the preparation of a medicament for treating or preventing tumors, wherein the fusion protein comprises an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment.

[0007] Another aspect of the present invention provides a method for treating or preventing tumors, comprising administering a therapeutically effective amount of a fusion protein to a patient in need thereof, wherein the fusion protein comprises an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment.

[0008] On the other hand, the present invention provides a multi-domain fusion protein for treating or preventing tumors, wherein the fusion protein comprises an anti-PD-L1 single-domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment.

[0009] Another aspect of the present invention provides use of an isolated polynucleotide or a construct containing the polynucleotide in preparing a drug for treating or preventing tumors, wherein the isolated polynucleotide encodes the above-mentioned fusion protein.

[0010] Another aspect of the present invention provides a use of an expression system in preparing a drug for treating or preventing tumors, wherein the expression system contains the above-mentioned construct or the above-mentioned polynucleotide is integrated into the genome.

[0011] Another aspect of the present invention provides a use of a pharmaceutical composition in preparing a drug for treating or preventing tumors, wherein the pharmaceutical composition comprises the above-mentioned fusion protein or the culture of the above-mentioned expression system. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 TAF-6 and The area under the plasma concentration-time curve;

[0013] Figure 2: Antitumor efficacy of multi-domain fusion proteins in the MDA-MB-231 breast cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0014] Figure 3. Antitumor efficacy of multi-domain fusion proteins in the MDA-MB-231 breast cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0015] Figure 4 Multi-domain fusion protein TAF-6, M7824 analogs, Anti-tumor efficacy in the MDA-MB-231 breast cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0016] Figure 5: Antitumor efficacy of multi-domain fusion proteins in a Calu-6 lung cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0017] Figure 6 Multi-domain fusion protein TAF-6, M7824 analogs, Anti-tumor efficacy in a Calu-6 lung cancer subcutaneous xenograft mouse model with a humanized PBMC immune system;

[0018] Figure 7: Antitumor efficacy of multi-domain fusion proteins in a HCT116 colon cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0019] Figure 8: Antitumor efficacy of multi-domain fusion proteins in a Huh-7 liver cancer subcutaneous transplant mouse model with humanized PBMC immune system;

[0020] Figure 9 Multi-domain fusion protein TAF-6, M7824 analogs, Anti-tumor efficacy in a Huh-7 liver cancer subcutaneous xenograft mouse model with a humanized PBMC immune system;

[0021] Figure 10 Antitumor efficacy of multi-domain fusion proteins in the HT1080 sarcoma subcutaneous transplant mouse model with humanized PBMC immune system.

[0022] Figure 11: Antitumor efficacy of multi-domain fusion proteins in a HeLa cervical cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0023] FIG12 shows the anti-tumor efficacy of multi-domain fusion proteins in the CRC-034 colorectal cancer PDX subcutaneous transplant mouse model with humanized PBMC immune system;

[0024] Figure 13 shows the anti-tumor efficacy of the multi-domain fusion protein in the HuCCT1 cholangiocarcinoma subcutaneous xenograft mouse model with humanized PBMC immune system.

[0025] FIG14 shows the anti-tumor efficacy of multi-domain fusion proteins in a SJSA osteosarcoma subcutaneous xenograft mouse model with humanized PBMC immune system;

[0026] Figure 15: Antitumor efficacy of multi-domain fusion proteins in a NUGC-4 / hCLDN18.2 gastric cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

[0027] Figure 16 shows the anti-tumor efficacy of the multi-domain fusion protein in the OV90 ovarian cancer subcutaneous transplant mouse model with humanized PBMC immune system. DETAILED DESCRIPTION

[0028] After extensive exploration and research, the inventors of the present invention unexpectedly discovered a fusion protein molecule that can combine the functions of anti-PD-L1 monoclonal antibodies to block the PD-L1 / PD-1 interaction, antagonistic VEGF monoclonal antibodies to reduce microvascular growth and inhibit metastatic disease, and TGF-β receptors to improve TGF-β-induced T cell dysfunction and enhance immune response in the tumor microenvironment, thereby exhibiting excellent tumor inhibitory effects. The present invention was completed on this basis.

[0029] The first aspect of the present invention provides a fusion protein for the preparation of a drug for treating or preventing tumors, wherein the fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment. In the above-mentioned fusion protein, the anti-PD-L1 single domain antibody fragment can generally be used to block the PD-L1 / PD-1 interaction, increase the expression of IFN-γ and / or IL-2 in T lymphocytes, and thus inhibit tumor growth. The antagonist VEGF fragment can generally include an Fc portion that can bind to the FcRn receptor, thereby extending the half-life in vivo, and can also bind to effector cells expressing Fc receptors to play a role in killing cancer cells. The TGF-β binding fragment can enhance the killing function of tumor-infiltrating T cells against tumor cells by clearing TGF-β overexpressed in the tumor microenvironment.

[0030] A second aspect of the present invention provides a method for treating or preventing tumors, comprising administering a therapeutically effective amount of a fusion protein to a patient in need thereof, wherein the fusion protein comprises an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment.

[0031] The third aspect of the present invention provides a fusion protein for treating or preventing tumors, wherein the fusion protein comprises an anti-PD-L1 single-domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment.

[0032] In some embodiments, the above-mentioned anti-PD-L1 single-domain antibody fragment can generally be a polypeptide or protein fragment that can specifically bind to PD-L1. In the anti-PD-L1 single-domain antibody fragment, the corresponding antibody light chain is usually missing, and only the fragment corresponding to the heavy chain variable region is present. The binding properties of the anti-PD-L1 single-domain antibody fragment can generally be determined by the three complementarity determining regions (CDRs) it includes. The CDR region can be arranged in order with the framework region (FR), and the FR region does not directly participate in the binding reaction. These CDRs can form a loop structure, and the β-folds formed by the FRs therebetween are close to each other in the spatial structure, forming the antigen binding site of the antibody. For example, the complementarity determining region (CDR) of the above-mentioned anti-PD-L1 single-domain antibody fragment can include an amino acid sequence such as CDR1 shown in one of SEQ ID NOs. 1 to 5, CDR2 shown in one of SEQ ID NOs. 6 to 9, and CDR3 shown in one of SEQ ID NOs. 10 to 15.

[0033] In a specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include: CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.6, and CDR3 with an amino acid sequence as shown in SEQ ID NO.10.

[0034] In another specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include: CDR1 with an amino acid sequence as shown in SEQ ID NO.2, CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and CDR3 with an amino acid sequence as shown in SEQ ID NO.11.

[0035] In another specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include: CDR1 with an amino acid sequence as shown in SEQ ID NO.3, CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and CDR3 with an amino acid sequence as shown in SEQ ID NO.12.

[0036] In another specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include: CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.8, and CDR3 with an amino acid sequence as shown in SEQ ID NO.13.

[0037] In another specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include: CDR1 with an amino acid sequence as shown in SEQ ID NO.2, CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and CDR3 with an amino acid sequence as shown in SEQ ID NO.14.

[0038] In another specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include: CDR1 with an amino acid sequence as shown in SEQ ID NO.5, CDR2 with an amino acid sequence as shown in SEQ ID NO.9, and CDR3 with an amino acid sequence as shown in SEQ ID NO.15.

[0039] The above-mentioned anti-PD-L1 single-domain antibody fragment may further include a framework region (FR). As described above, the CDR region may be arranged in order with the FR region. For example, the anti-PD-L1 single-domain antibody fragment may include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The framework region FR includes FR1 whose amino acid sequence may be as shown in SEQ ID No. 49, FR2 whose amino acid sequence is as shown in one of SEQ ID Nos. 50 to 52, FR3 whose amino acid sequence is as shown in one of SEQ ID Nos. 53 to 55, and FR4 whose amino acid sequence is as shown in SEQ ID No. 56.

[0040] In a specific embodiment of the present invention, the frame region FR includes:

[0041] The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.50, FR3 shown in SEQ ID NO.53; FR4 shown in SEQ ID NO.56, or

[0042] The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.51, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or

[0043] The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or

[0044] The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.55; and FR4 shown in SEQ ID NO.56.

[0045] In another specific embodiment of the present invention, the anti-PD-L1 single-domain antibody fragment may include: a) a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID Nos. 16 to 21; or, b) a polypeptide fragment having an amino acid sequence having a sequence identity of more than 80% with one of SEQ ID Nos. 16 to 21 and having the function of the polypeptide fragment specified in a). Specifically, the polypeptide fragment in the above b) specifically refers to: an amino acid sequence as shown in one of SEQ ID Nos. 16 to 21, obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the functions of a polypeptide fragment as shown in one of SEQ ID Nos. 16 to 21, for example, the ability to specifically bind to PD-L1, the ability to block the PD-L1 / PD-1 interaction, thereby blocking the PD-L1 / PD1 pathway, the ability to increase the expression of IFN-γ and / or IL-2 in T lymphocytes, or the ability to inhibit tumor growth. The amino acid sequence of the anti-PD-L1 single-domain antibody fragment in b) above may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or greater identity to one of SEQ ID Nos. 16 to 21. The anti-PD-L1 single-domain antibody fragment may typically be derived from alpaca (Vicugna pacos), for example, its CDR region may be derived from alpaca. The anti-PD-L1 single-domain antibody fragment may typically be humanized, for example, its framework region may be derived from human.

[0046] Herein, sequence identity refers to the percentage of identical residues in the compared sequences. The sequence identity of two or more sequences can be calculated using software well known in the art, such as those available from NCBI.

[0047] In some embodiments, the antagonist VEGF fragment can generally be a polypeptide or protein fragment capable of antagonizing VEGF. For example, the antagonist VEGF fragment can be a monoclonal antibody, etc. For another example, the antagonist VEGF fragment can be bevacizumab, etc.

[0048] In a specific embodiment of the present invention, the antagonist VEGF fragment may include:

[0049] c) a polypeptide fragment whose heavy chain and light chain amino acid sequences are shown in SEQ ID Nos. 22 and 23, respectively;

[0050] d) A polypeptide fragment whose heavy chain and light chain amino acid sequences have more than 80% sequence identity with SEQ ID No. 22 and 23, respectively, and has the function of the polypeptide fragment defined in c). Specifically, the amino acid sequence in the above d) specifically refers to: an amino acid sequence as set forth in one of SEQ ID Nos. 22 to 23 obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and having the functions of a polypeptide fragment as set forth in one of SEQ ID Nos. 22 to 23, for example, the function of antagonizing VEGF, or the function of an Fc portion that binds to an FcRn receptor, thereby prolonging the in vivo half-life, and also binding to effector cells expressing Fc receptors to kill cancer cells. The amino acid sequence in d) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or greater identity to one of SEQ ID Nos. 22-23. The antagonist VEGF fragment may typically be derived from mouse (Mus musculus), for example, its CDR region may be derived from mouse. The antagonist VEGF fragment may typically be humanized, for example, its framework region may be derived from human.

[0051] In some embodiments, the aforementioned TGF-β binding fragments can typically specifically bind to various TGF-β isoforms (e.g., TGF-β1, TGF-β2, and TGF-β3). TGF-β isoforms are typically highly expressed in various malignant tumors and are likely one of the key factors contributing to poor clinical treatment outcomes. For example, the TGF-β binding fragment can be a fragment of the extracellular domain of TGF-βRⅡ (TGF-β receptor II).

[0052] In a specific embodiment of the present invention, the TGF-β binding fragment may include:

[0053] e) a polypeptide fragment whose amino acid sequence is shown in SEQ ID No. 24;

[0054] f) A polypeptide fragment having an amino acid sequence with more than 80% sequence identity with SEQ ID No. 24 and having the function of the polypeptide fragment defined in e). Specifically, the amino acid sequence in f) above specifically refers to: an amino acid sequence as shown in SEQ ID No. 24 obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and having the functions of the polypeptide fragment as shown in SEQ ID No. 24, for example, it can bind to various TGF-β isoforms (e.g., TGF-β1, TGF-β2 and TGF-β3, etc.), thereby eliminating overexpressed TGF-β in the tumor microenvironment, and can also enhance the cytotoxicity of tumor-infiltrating T cells against tumor cells. The amino acid sequence in f) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or greater identity with SEQ ID No. 24. The TGF-β binding fragment may generally be derived from human (homo sapiens).

[0055] In some embodiments, the fusion protein may further include a connecting peptide fragment. The fusion protein may generally include multiple connecting peptide fragments, and connecting peptide fragments may be provided between at least some of the domains or each domain. For example, a connecting peptide may be provided between an anti-PD-L1 single domain antibody fragment and an antagonist VEGF fragment. For another example, a connecting peptide may be provided between an antagonist VEGF fragment and a TGF-β binding fragment. The connecting peptide fragment may generally be a flexible polypeptide rich in G, S and / or A (mainly composed of glycine (G), serine (S) and / or alanine (A)) of appropriate length, so that adjacent protein domains can move freely relative to each other. For example, the amino acid sequence of the connecting peptide fragment may include sequences such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG)nA, etc., wherein n is selected from an integer between 1 and 10. In a specific embodiment of the present invention, the length of the amino acid sequence of the connecting peptide fragment can be 3-30, 3-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-22, 22-24, 24-26, 26-28, or 28-30. In a more preferred embodiment of the present invention, the connecting peptide fragment can include a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NOs. 34-36.

[0056] In some embodiments, the fusion protein can be linear. For example, the fusion protein can include an anti-PD-L1 single-domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment in order from the N-terminus to the C-terminus. The fusion protein can also have a structure similar to that of a monoclonal antibody. For example, the anti-PD-L1 single-domain antibody fragment can be located at the N-terminus of the heavy chain of the antagonist VEGF fragment. For another example, the anti-PD-L1 single-domain antibody fragment can be located at the N-terminus of the light chain of the antagonist VEGF fragment. For another example, the TGF-β binding fragment can be located at the C-terminus of the heavy chain of the antagonist VEGF fragment. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein may include the sequence shown in SEQ ID NO.23, SEQ ID NO.25-33, for example, the amino acid sequence of the fusion protein may include the heavy chain shown in SEQ ID NO.25 and the light chain sequence shown in SEQ ID NO.26, the heavy chain shown in SEQ ID NO.25 and the light chain sequence shown in SEQ ID NO.27, the heavy chain shown in SEQ ID NO.25 and the light chain sequence shown in SEQ ID NO.28, the heavy chain shown in SEQ ID NO.25 and the light chain sequence shown in SEQ ID NO.29, the heavy chain shown in SEQ ID NO.30 and the light chain sequence shown in SEQ ID NO.27, the heavy chain shown in SEQ ID NO.30 and the light chain sequence shown in SEQ ID NO.29, the heavy chain shown in SEQ ID NO.31 and the light chain sequence shown in SEQ ID NO.23, the heavy chain shown in SEQ ID NO.32 and the light chain sequence shown in SEQ ID NO.23, and the heavy chain shown in SEQ ID NO.33 and the light chain sequence shown in SEQ ID NO.23.

[0057] A fourth aspect of the present invention provides the use of an isolated polynucleotide or a construct containing such a polynucleotide in the preparation of a medicament for treating or preventing tumors, wherein the isolated polynucleotide encodes the aforementioned fusion protein. The polynucleotide may be RNA, DNA, or cDNA, among others. Methods for providing such isolated polynucleotides should be known to those skilled in the art. For example, they can be prepared by automated DNA synthesis and / or recombinant DNA techniques, or they can be isolated from suitable natural sources. Suitable methods for constructing such constructs should be known to those skilled in the art. For example, such constructs can be constructed by in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. More specifically, they can be constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vectors herein generally refer to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. Generally, suitable vectors can contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers. For example, these promoters may include, but are not limited to, the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, and may include, for example, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence. Insertion of an enhancer sequence into the vector enhances transcription. Enhancers are cis-acting DNA elements, typically about 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0058] A fifth aspect of the present invention provides an expression system for use in preparing a drug for treating or preventing tumors, wherein the expression system comprises the above-mentioned construct or an exogenous isolated polynucleotide integrated into its genome, thereby expressing the above-mentioned fusion protein. The above-mentioned expression system can be a host cell. Any cell suitable for expression by an expression vector can serve as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells. Methods for introducing the construct into host cells should be known to those skilled in the art, and can include, for example, microinjection, gene gun techniques, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.

[0059] A sixth aspect of the present invention provides the use of a pharmaceutical composition for the preparation of a medicament for treating or preventing tumors, the pharmaceutical composition comprising the fusion protein or culture of the expression system described herein. The fusion protein or culture in such pharmaceutical compositions is generally present in a therapeutically effective amount. As used herein, a "therapeutically effective amount" generally refers to an amount that, after an appropriate administration period, reduces the severity of disease symptoms, increases the frequency and duration of symptom-free periods, or prevents impairment or disability caused by the pain of the disease. The ability to inhibit tumor growth can be evaluated in animal model systems that are predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be measured in vitro using assays known to those skilled in the art. A therapeutically effective amount of a fusion protein or pharmaceutical composition generally reduces tumor size or otherwise alleviates symptoms in a subject. A skilled artisan can select an appropriate therapeutically effective amount based on the specific circumstances, for example, the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. The prescription of treatment (e.g., dosage determination, etc.) can be determined by a physician, typically considering factors including, but not limited to, the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors.

[0060] The pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier may include various excipients and diluents. These carriers themselves are not essential active ingredients and are not excessively toxic after administration. Suitable carriers should be well known to those skilled in the art. For example, a comprehensive discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0061] The tumor, for example, can be a solid tumor or a blood tumor, specifically lung cancer, melanoma, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, sarcoma (which can be fibrosarcoma or osteosarcoma, etc.), head and neck cancer, nasopharyngeal cancer, cervical cancer and bile duct cancer. Hematological malignancies include classical Hodgkin's lymphoma, etc. These cancers can be in the early, middle or late stages, such as metastatic cancer, etc.

[0062] As used herein, the term "treatment" encompasses preventative, curative, or palliative procedures that result in a desired pharmaceutical and / or physiological effect. A preferred therapeutic effect is one that can medically alleviate one or more symptoms of a disease, completely eliminate the disease, or inhibit or delay the onset of the disease and / or reduce the risk of disease progression or worsening.

[0063] In the present invention, "individual" generally includes humans, non-human primates, or other mammals (such as dogs, cats, horses, sheep, pigs, cows, etc.), which can benefit from treatment using the preparation, kit or combination preparation.

[0064] In some embodiments, the multi-domain fusion protein with anti-cancer activity can organically combine the functions of anti-PD-L1 monoclonal antibody to block PD-L1 / PD-1 interaction, anti-VEGF monoclonal antibody to reduce microvascular growth and inhibit metastatic disease, and TGF-β receptor to relieve cancer cells' tolerance to TGF-β signals and enhance immune response in a single antibody fusion protein molecule, thereby being used to treat tumors and having good industrial prospects.

[0065] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0066] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0067] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the prior art grasped by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0068] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0069] In the following examples, the control drugs used include Atezolizumab (trade name ), Bevacizumab (trade name ).

[0070] Example 1 Construction and recombinant expression of fusion protein

[0071] The amino acid sequences of the multi-domain fusion protein in Table 1, PD-L1-Fc fusion protein (SEQ ID NO. 47), and Fc-TGFβRII fusion protein (SEQ ID NO. 48) were converted into base sequences according to the codon preference of CHO cells. A HindIII restriction site and a Kozak sequence (GCCACC) were introduced at the 5' end of the heavy chain and light chain coding sequences, respectively, and a stop codon and an EcoRI restriction site were introduced at the 3' end. Full-length DNA was obtained by gene synthesis (Universal Biosystems (Anhui) Co., Ltd.). The synthesized heavy and light chain encoding genes were double-digested with HindIII-HF (purchased from NEB, R3104V) and EcoRI-HF (purchased from NEB, R3101V), respectively, and recovered using an agarose gel DNA / PCR product mini-recovery kit (purchased from Biomiga). The resulting DNA was ligated with the pCDNA3.1(+) vector, which had also been double-digested with HindIII and EcoRI, using T4 ligase (purchased from NEB, M0202V). The resulting DNA was then transformed into Top10 competent cells and plated on LB ampicillin-resistant plates. Clones were identified and sequenced to construct the heavy and light chain expression plasmids based on pCDNA3.1(+). The heavy and light chain expression plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Biomiga, BW-PD3511-02) and mixed at a 1:1 ratio. Dilute 1.0 mg of the mixed plasmid to 25 mL using Wayne293 Expression Medium (purchased from Zhongshan Kangsheng, A21501). Add 3.0 mg of PEI (linear, 25KD, Polysciences, Inc.) to 25 mL using Wayne293 Expression Medium, mix thoroughly, and incubate at room temperature for 30 minutes. Count Hek293F cells (viability >95%) growing in logarithmic phase, centrifuge at 1100 rpm for 10 minutes, discard the supernatant, and resuspend the cells in 450 mL of Wayne293 Expression Medium. Add the plasmid-PEI mixture to the cell suspension and culture in a shaking incubator at 37°C, 5% CO2 for 7 days. Centrifuge and remove the supernatant for subsequent protein purification.

[0072] Table 1 Amino acid sequence and coding sequence of multi-domain fusion protein

[0073] Example 2 Purification of multi-domain fusion protein

[0074] 2.1 Anti-PD-L1 single domain antibody is located at the N-terminus of the heavy chain of anti-VEGF monoclonal antibody

[0075] The cell fermentation supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borglon Biotechnology Co., Ltd.). The column was equilibrated with 20 mM PB, 0.15 M NaCl (pH = 7.0) and eluted with 100% 0.1 M Gly-HCl (pH = 3.0). The eluate was pre-spiked with 10% 1 M Tris-HCl (pH = 8.5). The 100% eluate was diluted to a conductivity < 3 ms / cm. The supernatant was adjusted to pH 7.0 and loaded onto a DSP column (Borglon Biotechnology Co., Ltd.). Elution was performed at 15% and 100% (20 mM PB, 0.5 M NaCl, pH 7.0). The 15% elution fraction was the target protein. Protein concentration was determined using UV280.

[0076] 2.2 Anti-PD-L1 single domain antibody is located at the N-terminus of the light chain of anti-VEGF monoclonal antibody

[0077] The cell fermentation supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borgron Biotechnology Co., Ltd.). The column was equilibrated with 20 mM PB, 0.15 M NaCl (pH 7.0) and eluted with 100% 0.1 M Gly-HCl (pH 3.0). The eluate was pre-spiked with 10% 1 M Tris-HCl (pH 8.5). The 100% eluate was diluted to a conductivity of 4 ms / cm and loaded onto a Super Q (TOSOH) column. The column was equilibrated with 20 mM Tris, pH 8, and eluted with 500 mM NaCl + 20 mM Tris (pH 8.0). Elution was performed at 35% and 100%, respectively. Excess light chains were removed by flow-through, yielding the 35% elution fraction, which was the target protein. Protein concentration was determined using UV280.

[0078] Purity was determined using SEC-HPLC-UV analysis. Detector: Agilent 1100LC; detection wavelength: 214 nm; mobile phase: 150 mM pH 7.0 PB + 5% isopropanol; column: Superdex 200 Increase 5 / 150GL; run time: 15 minutes; column temperature: 25°C. The results showed purities greater than 95%.

[0079] Purification of PD-L1-Fc fusion protein (SEQ ID NO. 47) and Fc-TGFβRII fusion protein (SEQ ID NO. 48): The cell fermentation supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borglon Biotechnology Co., Ltd.). The column was equilibrated with 20 mM PB, 0.15 M NaCl (pH = 7.0) and eluted with 100% 0.1 M Gly-HCl (pH = 3.0). The eluate was pre-neutralized with 10% 1 M Tris-HCl (pH = 8.5).

[0080] Example 3 Identification of the function of multi-domain fusion proteins in vitro

[0081] 3.1 In vitro activity testing of anti-PD-L1 single domain antibody fragments:

[0082] The CD5L-OKT3scFv-CD14 gene sequence (GenBank: ADN42857.1) was synthesized and digested with HindIII-EcoRI (Takara) and inserted into the vector pCDNA3.1 to construct pCDNA3.1-antiCD3TM. Using the human PD-L1 gene (GenBank: NM_014143.2) as a template, the PD-L1 fragment was amplified with high fidelity and recombinantly ligated into pCDNA3.1-antiCD3TM to construct pCDNA3.1-antiCD3TM-PDL1. CHO cells (Thermo) were transfected, followed by G418 selection for 10-14 days to generate the stable cell line CHO-antiCD3TM-PDL1.

[0083] The resulting fragment was amplified using the human PD1 gene (GenBank: NP_005009.2) as a template and recombined with the PB513B1-dual-puro vector (Ubao Biotech) digested with HindIII-BamHI (Takara) to construct the plasmid pB-PD1. High-fidelity amplification was performed using pGL4.30 (Ubao Biotech) as a template. The resulting fragment was recovered and recombined with the pB-PD1 vector digested with SfiI-XbaI (Takara) to construct the pB-NFAT-Luc2p-PD1 plasmid. After successful plasmid construction, the plasmid was extracted using an endotoxin-free plasmid extraction kit (Biomiga) and used for transfection into Jurkat cells (Stem Cell Bank, Chinese Academy of Sciences). Referring to the method described in patent CN 107022571A, Jurkat cells were treated with 0.1 mg / ml poly-D-lysine to achieve a relatively adherent state. They were then transfected according to the instructions in the lipofectamine transfection kit (Lipofectamine 3000; Invitrogen). On the third day, pressure selection was performed using RPMI1640 medium (Thermo) supplemented with 10% FBS and 2.5 μg / ml puromycin. Thereafter, the medium was replenished at regular intervals, and the puromycin content was gradually increased to 4 μg / ml after cell viability recovered. This ultimately resulted in a monoclonal Jurkat-NFAT-Luc2p-PD1 cell line.

[0084] Take CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells and count them, and adjust the cell density to 4×10 6 / ml, add 25 μl to each cell per well of a 96-well plate; serially dilute the fusion protein sample prepared in Example 2 with 1% BSA and add 50 μl to the cells; incubate at 37°C, 5% CO2 for 6 hours, then add 10 μl of luciferase substrate (Promega, E2620) to each well, shake on a shaker for 2 minutes, and read the results. The procedure was as described in the kit instructions.

[0085] 3.2 In vitro activity detection of antagonistic VEGF fragments:

[0086] HEK293 cells were plated in 6-well cell culture plates, with 1.0 × 10 cells per well. 6 The cells were cultured overnight in a 37°C, 5% CO2 incubator. The transfection system was prepared according to the transfection reagent instructions, containing 1.0 μg of pcDNA-KDR plasmid and 4 μg of pGL4.30 plasmid. 48 hours after transfection, cells were expanded to 10 cm cell culture dishes and supplemented with 200 μg / ml G418 and 100 μg / ml Hygromycin. Fresh pressurized culture medium was replaced every 3 days until obvious clones emerged. The cells were digested and plated on 96-well cell culture plates. After a single clone emerged, chemiluminescence was detected after 6 hours of stimulation with 0.1 μg / ml VEGF. Clones with a clear signal response were selected and further expanded. Finally, a single HEK293-NFAT-KDR clone was obtained. HEK293-NFAT-KDR cells were plated at a density of 40,000 per well and digested with Accutase. The digested cells were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in assay medium (DMEM + 5% FBS). The cells were counted and the cell density was adjusted to 1.6×10 6 / ml; plate a 96-well cell culture plate, 25ul per well; use analytical culture medium to prepare a VEGF solution at a concentration of 60ng / ml; add 25ul to the cell culture plate, 25ul per well; use analytical culture medium to prepare the fusion protein prepared in Example 2, add 25ul to the cell culture plate, incubate at 37°C, 5% CO2 for 6h; add 10ul Bright-Glo luciferase assay reagent (Promega, E2620) to each well, shake for 2 minutes, transfer 80ul of the lysate to an enzyme-labeled white plate, and read the results with a microplate reader.

[0087] 3.3 In vitro cell activity assay of TGF-β binding fragments:

[0088] Mouse breast cancer 4T1 cells were cultured until they were about 90% confluent (10 cm dish), digested with trypsin, and plated at 4×10 5 The cells were plated into 6-well plates and cultured overnight. The pGL4.48[luc2P SBE Hygro] plasmid samples were extracted and used 4T1 cells were transfected. 24 hours after transfection, the resulting TGFβ-4T1 cells were trypsinized and transferred to a 10 cm culture dish. The cells were pressure-selected using RPMI 1640 medium supplemented with 10% FBS and 150 μg / ml hygromycin (InvivoGen, Cat no.: ant-hg-1). After 10-15 days of pressure selection, TGFβ-4T1 cells were plated at 2 / well for monoclonal selection. Monoclonal cells were stimulated with TGFβ1 (Novoprotein, 10 μg, Cat no.: CA59) to verify the transfection efficacy of the monoclonal cells, and finally, TGFβ-4T1 monoclonal cells were obtained.

[0089] TGFβ-4T1 cells were grown to about 90% confluence, and about 2.5 ml of 0.25% trypsin was added for digestion at room temperature for 2 minutes. Cells adhered to the culture dish were pipetted off and dispersed by pipetting in the trypsin solution. The entire digestion process took 5 minutes. Complete medium (RPMI 1640 + 10% FBS) was added to terminate the digestion, and the cells were continued to be pipetted until the cells were evenly dispersed. The cells were transferred to a 50 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 2 ml of complete medium was added to resuspend the cells. The cell density was measured using a cell counter. The cells were diluted with complete medium (RPMI 1640 + 10% FBS) to a cell density of 2×10 5 / ml; the diluted cells (density 2×10 5 Cells were plated (96-well plate) with 100 μl per well and a cell density of 2 × 10 4 The 96-well plate was placed in a 37°C incubator and cultured overnight; the fusion protein sample prepared in Example 2 was diluted to the specified concentration using RPMI1640 + 0.2% FBS medium (containing 2 ng / ml TGFβ1) (each protein sample was placed at room temperature for 1 hour after dilution); the supernatant of TGFβ-4T1 cells cultured overnight in a 37°C incubator was discarded, 50 μl RPMI 1640 + 0.2% FBS medium was added, and then 50 μl of protein solutions of different concentrations were added; the 96-well plate was placed in a 37°C incubator and incubated for a further 3 hours, after which 10 μl Bright-Glo luciferase detection reagent (Promega, E2620) was added and shaken for 3 minutes; and the readings were read using a microplate reader.

[0090] The measurement results of the anti-PD-L1 activity, anti-VEGF activity, and anti-TGFβ activity of each multi-domain fusion protein are shown in Table 2. As shown in Table 2, each multi-domain fusion protein has good in vitro cell activity.

[0091] Table 2 In vitro cell activity of multi-domain fusion proteins

[0092] Example 4 Pharmacokinetics of multi-domain fusion protein in C57BL / 6 mice

[0093] C57BL / 6 mice were divided into 4 groups: Mice were divided into two groups: a low-dose (1 mg / kg) and a high-dose (10 mg / kg) group (Roche), and a low-dose (1 mg / kg) and high-dose (10 mg / kg) group (8 mice per group, half male and half female). Drugs were administered via a single tail vein injection, and pharmacokinetic samples were collected at different time points. Samples were collected before and 1 hour, 6 hours, 24 hours, 48 ​​hours, 78 hours, 120 hours, 144 hours, 168 hours, 192 hours, and 216 hours after drug administration. Serum drug concentrations in pharmacokinetic samples were quantified by ELISA. VEGF-coated plates were used, and the secondary antibody, Goat anti-Human IgG Fc, was HRP-conjugated to the drug. Detection was performed using the TMB assay. Concentration values ​​were converted to values ​​based on the signal-concentration relationship of the standard curve. Key pharmacokinetic parameters were calculated using a non-compartmental model using PK Solver software.

[0094] The results are shown in Figure 1. C57BL / 6 mice were given The pharmacokinetic parameters were similar to those of the high-dose and low-dose groups of TAF-6. The dosage ratio of the low-dose group to the high-dose group was 1:10. max The ratio is 1:9.3, AUC last The ratio is 1:8.9, T max All were 1h, exposure (C max and AUC last The increase of TAF-6 in the low-dose and high-dose groups was 1:10. max The ratio is 1:8.6, AUC last The ratio is 1:8.5, Tmax is 1h, and the exposure (C max and AUC last ) increases in a dose-proportional manner. and between the high and low dose groups of TAF-6, AUC and C max All showed a dose-linear correlation. The difference in AUC between PD-L1 and TAF-6 was inferred to be the PD-L1 target-mediated clearance (TMDD) effect.

[0095] Example 5 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Breast Cancer MDA-MB-231 Mice

[0096] The in vivo efficacy of the multi-domain fusion protein of the present invention was determined by modeling MDA-MB-231 (human breast cancer) cells in mice with humanized human PBMC immune systems (M-NSG mice). Female M-NSG mice aged 6-8 weeks were selected and inoculated with MDA-MB-231 cells (10*10E6 + 25% Matrigel). On the 7th day, PBMC (5*10E6 / 0.2ml) was injected into the tail vein. Tumor volume and body weight were then observed. Mice with tumor volumes between 140-260 mm were selected. 3 Mice between 1 and 2 years old were randomly divided into 6 groups based on their tumor volume and body weight, with 7 mice in each group. Drug administration began on the day of grouping. Tumor-bearing mice with tumors that were too large or too small were eliminated. Twice a week, intraperitoneal injections of: PBS, isotype control IgG1, positive control (Roche), TAF-6, TAF-7, and combination therapy (see Table 3 for detailed regimens) were administered for approximately three weeks. The PD-L1-Fc fusion protein (SEQ ID NO. 47) is a fusion protein of an anti-PD-L1 single-domain antibody and human IgG Fc (with the anti-PD-L1 single-domain antibody located at the N-terminus of the Fc, aPD-L1-Fc). The Fc-TGFβRII fusion protein (SEQ ID NO. 48) is a fusion protein of human IgG Fc and TGFβRII (with TGFβRII located at the C-terminus of the Fc).

[0097] Before grouping and before the end of the experiment, blood was collected from the mouse orbital vein. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized. During the experiment, the body weight of animals in Groups 3 and 4 (measured twice a week) was generally stable, and no experimental animals died. The tumor volume of Groups 3 and 4 (measured twice a week) was smaller than that of Group 6 combined administration, and significantly smaller than that of the corresponding single drug in Group 2. The results are shown in Figure 2.

[0098] Table 3

[0099] Example 6 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Breast Cancer MDA-MB-231 Mice

[0100] Human breast cancer MDA-MB-231 tumors were inoculated subcutaneously in the right anterior flank of female NCG mice. PBMC cells were inoculated into the mice one day after tumor inoculation. When the tumors grew to 53 mm 3The mice were divided into 6 groups with 10 mice in each group, namely: Vehicle group, TAF-6 low dose (2 mg / kg, ip, tiw×9) group, TAF-6 medium dose (6 mg / kg, ip, tiw×9) group, TAF-6 high dose (18 mg / kg, ip, tiw×9) group, (4 mg / kg, ip, tiw×9) group, (4 mg / kg, ip, tiw × 9) group. Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume and dosing time was recorded. Where tiw indicates dosing three times a week, ip indicates intraperitoneal injection, and tiw × 9 indicates dosing three times a week for a total of nine doses.

[0101] Blood was collected from the orbital venous plexus 2 days before grouping and at the end of the experiment. FACS analysis showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the proportion of CD45+ cells increased over time. At the end of the experiment, tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed, and serum and tumors were collected and fixed. Tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed: TAF-6 low-dose group, TAF-6 medium-dose group, TAF-6 high-dose group, Group, The tumor growth inhibition rates of the groups were 41%, 34%, 60%, 23%, and 32%, respectively. The tumor volumes of all groups were significantly smaller than those of the Vehicle group (all p<0.05), and the tumor volume of the TAF-6 high-dose group was significantly smaller than that of the group (p<0.01), There was no significant difference in tumor volume between the groups (p>0.05). The results are shown in Figure 3.

[0102] In summary, the test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized breast cancer MDA-MB-231 subcutaneous transplant tumor model and effectively inhibits tumor growth. Its anti-tumor effect is significantly better than The tumor inhibitory effect increases with increasing dose.

[0103] In another independent experiment with the same tumor model, the antitumor activity of the test drug and the positive drug M7824 analog (prepared by our laboratory according to M7824 patent US9676863B2) was compared. Four groups of 6 mice were set up, namely: Vehicle group, TAF-6 (4.2 mg / kg, ip, tiw×8 times; later the dose was adjusted to 8.4 mg / kg, ip, tiw×4 times) group, M7824 analog (3.6 mg / kg, ip, tiw×8 times; later the dose was adjusted to 7.2 mg / kg, ip, tiw×4 times) group, The results are shown in Figure 4. At equimolar concentrations, TAF-6 showed significantly better antitumor effects than the M7824 analog.

[0104] Example 7 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Calu-6 Lung Cancer Mice

[0105] Calu-6 human lung cancer cells were inoculated subcutaneously in the right anterior flank of male NCG mice. PBMC cells were inoculated into the mice 4 days before tumor cell inoculation. When the tumor grew to 50 mm 3 The mice were divided into 7 groups with 8 mice in each group, namely: Vehicle group, TAF-6 low-dose (2 mg / kg, ip, tiw×10) group, TAF-6 medium-dose (7 mg / kg, ip, tiw×10) group, TAF-6 high-dose (25 mg / kg, ip, tiw×10) group, (5mg / kg, ip, tiw×10) group, (5mg / kg, ip, tiw×10) group, (5+5mg / kg, ip, tiw×10) group. Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume of tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed, and serum and tumors were collected. Tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0106] Three days before grouping and at the end of the experiment (PG-D23), blood was collected from the orbital vein of the mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0107] During the treatment period, the mice in each group ate and drank normally, their body weights were generally stable, and no experimental animals died.

[0108] At the end of the experiment (PG-D23), TAF-6 low-dose group, TAF-6 medium-dose group, TAF-6 high-dose group, Group, Group, The tumor growth inhibition rates of the groups were 62%, 62%, 75%, 13%, 34%, and 32%, respectively. The tumor volumes of all groups except the control group were significantly smaller than those of the Vehicle group (all p<0.01), and the tumor volumes of the TAF-6 low-, medium-, and high-dose groups were significantly smaller than those of the control group. Group, Group and The results are shown in Table 4 and Figure 5.

[0109] In summary, the test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized lung cancer Calu-6 subcutaneous transplant tumor model, effectively inhibiting tumor growth, and the tumor inhibition effect is significantly better than and and The tumor inhibitory effect was enhanced with increasing dose.

[0110] Table 4 Antitumor effect of the test substances on human lung cancer Calu-6 (tumor volume)

[0111] Note: a . Mean ± standard error.

[0112] In another independent experiment with the same tumor model, the anti-tumor activity of the test drug was compared with that of the positive drug M7824 analog. Four groups were set up, with 6 mice in each group, namely: Vehicle group, TAF-6 (7 mg / kg, ip, TIW×8 times) group, M7824 analog (6 mg / kg, ip, TIW×8 times) group, (5 mg / kg, ip, TIW×8 times) group. The results are shown in Figure 6. At equimolar concentrations, TAF-6 had a significantly better anti-tumor effect than M7824 analogs and

[0113] Example 8 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Colon Cancer HCT116 Mice

[0114] The in vivo efficacy of the multi-domain fusion protein of the present invention was determined by modeling HCT-116 (human colon cancer) cells in mice with humanized human PBMC immune systems (NOG mice). Female NOG mice aged 7-9 weeks were selected and inoculated with HCT-116 cells (3*10E6 + Matrigel). PBMC (5*10E6 / 0.2ml) was injected into the tail vein on the third day after tumor cell inoculation. Tumor volume and body weight were then observed. 3 The mice between 1 and 2 weeks old were randomly divided into 6 groups according to their tumor volume and body weight, with 8 mice in each group. Drugs were administered on the day of grouping: Vehicle group, TAF-6 low-dose (2 mg / kg, ip, tiw×3) group, TAF-6 medium-dose (7 mg / kg, ip, tiw×3) group, TAF-6 high-dose (25 mg / kg, ip, tiw×3) group, (5mg / kg, ip, tiw×3) group, (5+5mg / kg, ip, tiw×3) group. Tumor volume and body weight were measured twice a week, and the relationship between changes in body weight and tumor volume of tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed, and serum and tumors were collected. Tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0115] Before grouping and the end of the experiment, blood was collected from the orbital vein of the mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0116] The tumor inhibition activity is shown in Figure 7. The test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized colon cancer HCT-116 subcutaneous transplant tumor model, effectively inhibiting tumor growth, and the high-dose and medium-dose groups have significantly better tumor inhibition effects than the At the same molar concentration, the TAF-6 group (7 mg / kg, ip, tiw×3) (5+5mg / kg,ip,tiw×3) group, TAF-6 was still superior to The efficacy of the group.

[0117] Example 9 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Hepatocellular Carcinoma Huh-7 Mice

[0118] Human liver cancer Huh-7 cells were inoculated subcutaneously in the right anterior flank of male NCG mice. PBMC cells were inoculated into the mice 45 days before tumor cell inoculation. When the tumor grew to 50 mm 3The mice were divided into 5 groups with 10 mice in each group, namely: Isotype group, TAF-6 (2 mg / kg, ip, tiw×8) group, TAF-6 (7 mg / kg, ip, tiw×8) group, (5mg / kg, ip, tiw×8) group, (5 mg / kg, ip, tiw × 8) group. Tumor volume and body weight were measured twice a week, and the relationship between changes in body weight and tumor volume of tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed, and serum and tumors were collected. Tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0119] Before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0120] During the administration period, the mice in each group ate and drank normally, and their body weight remained generally stable.

[0121] Before the end of the experiment (PG-D18), TAF-6 (2 mg / kg, ip, tiw×8) group, TAF-6 (7 mg / kg, ip, tiw×8) group, Group, The tumor growth inhibition rates of the groups were 57%, 74%, 18%, and 67%, respectively. The tumor volumes of all groups were significantly smaller than those of the Isotype group (all p<0.01), and the tumor volumes of the TAF-6 (7 mg / kg, ip, tiw×8) group were significantly smaller than those of the group (all p<0.01), The tumor volume of the group was significantly smaller than The results are shown in Figure 8.

[0122] In summary, the test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized liver cancer Huh-7 subcutaneous transplant tumor model, effectively inhibiting tumor growth, and its anti-tumor effect is significantly better than The tumor inhibitory effect increases with increasing dose.

[0123] In another independent experiment with the same tumor model, the anti-tumor activity of the test drug was compared with that of the positive drug M7824 analog. Four groups were set up, with 6 mice in each group, namely: Vehicle group, TAF-6 (7 mg / kg, ip, tiw x 9; later the dose was adjusted to 14 mg / kg, ip, tiw x 2) group, M7824 analog (6 mg / kg, ip, tiw x 9; later the dose was adjusted to 12 mg / kg, ip, tiw x 2) group, (5 mg / kg, ip, tiw x 9; later the dose was adjusted to 10 mg / kg, ip, tiw x 2) group. As shown in Figure 9, the anti-tumor effect of TAF-6 was significantly superior to that of the M7824 analog.

[0124] Example 10 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Sarcoma HT1080 Mice

[0125] Human sarcoma HT1080 cells were inoculated subcutaneously in the right anterior flank of male NCG mice. PBMC cells were inoculated into the mice 7 days before tumor cell inoculation. When the tumor grew to 56 mm 3 The mice were divided into 5 groups with 8 mice in each group, namely: Vehicle group, TAF-6 low dose (2 mg / kg, ip, tiw×9) group, TAF-6 medium dose (7 mg / kg, ip, tiw×9) group, TAF-6 high dose (25 mg / kg, ip, tiw×9) group, The tumor volume and body weight of the mice were measured weekly, and the relationship between the changes in body weight and tumor volume and the administration time was recorded.

[0126] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0127] Before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0128] Before the end of the experiment (PG-D19), TAF-6 low-dose group, TAF-6 medium-dose group, TAF-6 high-dose group, The tumor growth inhibition rates of the TAF-6 medium-dose group, TAF-6 high-dose group and The tumor volume of the TAF-6 group was significantly smaller than that of the Vehicle group (all p < 0.05), and the anti-tumor effects of the TAF-6 medium-dose group and the TAF-6 high-dose group were better than those of the The results are shown in Figure 10.

[0129] Example 11 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Hela Mice

[0130] Human cervical cancer Hela cells were inoculated subcutaneously in the right anterior flank of female NCG mice. One day after tumor cell inoculation, human PBMC cells were inoculated into the mice. When the tumor grew to 52 mm 3 The drugs were divided into 7 groups with 8 rats in each group, namely: Vehicle group, TAF-6 low dose (2 mg / kg, ip, tiw×10) group, TAF-6 medium dose (7 mg / kg, ip, tiw×10) group, TAF-6 high dose (25 mg / kg, ip, tiw×10) group, (5mg / kg, ip, tiw×10) group, (5mg / kg, ip, tiw×10) group, The dosage was calculated based on equimolar concentrations (5 + 5 mg / kg, ip, tiw × 10). Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume of tumor-bearing mice and the time of administration was recorded.

[0131] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0132] When grouping and at the end of the experiment, blood was collected from the orbital venous plexus of the mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0133] At the end of the experiment (PG-D21), TAF-6 low-dose group, TAF-6 medium-dose group, TAF-6 high-dose group, Group, Group, The tumor growth inhibition rates of the TAF-6 groups were 51%, 70%, 78%, 39%, 60%, and 65%, respectively. The tumor volumes of each group were significantly smaller than those of the Vehicle group (all p < 0.01). The tumor volumes of the TAF-6 medium and high dose groups were significantly smaller than those of the Vehicle group (all p < 0.01). group (p<0.05; p<0.01), and the results are shown in Figure 11.

[0134] In summary, the test drug TAF-6 (7 mg / kg, 25 mg / kg) has a significant anti-tumor effect on the PBMC humanized human cervical cancer Hela subcutaneous transplant tumor model, and the anti-tumor effect is significantly better than The tumor inhibitory effect increases with increasing dose.

[0135] Example 12 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Colorectal Cancer CRC-034PDX Model Mice

[0136] The tumor mass of the human colorectal cancer xenograft model CRC-034 (PDX, Beijing Yikang Pharmaceutical Technology Co., Ltd.) was inoculated subcutaneously in the right anterior flank of male NCG mice. PBMC cells were inoculated into the mice 10 days after tumor cell inoculation. When the tumor grew to 53 mm 3 Around 8:00 p.m., mice were divided into three groups, each with 10 mice: the isotype group, the TAF-6 (7 mg / kg, ip, tiw) group, and the M7824 analog (6 mg / kg, ip, tiw) group. Dosage was calculated based on equimolar concentrations. Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume and dosing time was recorded.

[0137] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0138] Three days before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0139] Before the end of the experiment (PG-D22), the tumor growth inhibition rates of the TAF-6 group and the M7824 analog group were 56% and 4%, respectively. The tumor volume in the TAF-6 group was significantly smaller than that in the Isotype group and the M7824 analog group (p < 0.05). There was no significant difference between the M7824 analog group and the Isotype group (p > 0.05). The results are shown in Figure 12.

[0140] In summary, the test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized CRC-034 colorectal cancer PDX subcutaneous transplanted tumor mouse model, and its anti-tumor effect is significantly better than that of the M7824 analog.

[0141] Example 13 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Cholangiocarcinoma HuCCT1 Mice

[0142] Human hepatobiliary carcinoma HuCCT1 cells were inoculated subcutaneously in the right anterior flank of male NCG mice. PBMC cells were inoculated into the mice one day after tumor cell inoculation. When the tumor grew to 53 mm 3 The mice were divided into four groups (8 mice each) for drug administration at approximately 12 hours (approximately 24 hours). The groups included: Vehicle, TAF-6 (2 mg / kg, ip, tiw), TAF-6 (10 mg / kg, ip, tiw), and M7824 analog (1.7 mg / kg, ip, tiw). Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume and drug administration time was recorded.

[0143] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0144] One day before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0145] At the end of the experiment (PG-D32), the tumor volumes of the TAF-6 (2 mg / kg, 10 mg / kg) and M7824 analog groups were significantly smaller than those of the Vehicle group (p < 0.01). The results are shown in FIG13 .

[0146] In summary, the test drug TAF-6 (2 mg / kg, 10 mg / kg) has a significant anti-tumor effect on the PBMC humanized human hepatobiliary carcinoma HuCCT1 subcutaneous transplant tumor model.

[0147] Example 14 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Osteosarcoma SJSA Mice

[0148] Human osteosarcoma SJSA-1 cells were inoculated subcutaneously in the right anterior flank of male NCG mice. PBMC cells were inoculated into the mice 2 days after tumor cell inoculation. When the tumor grew to 42 mm 3 The mice were divided into four groups (8 mice each) for drug administration at approximately 12 hours (approximately 24 hours). These groups included the vehicle group, the TAF-6 (2 mg / kg, ip, tiw) group, the TAF-6 (7 mg / kg, ip, tiw) group, and the M7824 analog (6 mg / kg, ip, tiw) group. Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume and drug administration time was recorded.

[0149] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0150] One day before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0151] At the end of the experiment (PG-D17), the tumor growth inhibition rates of the TAF-6 (2 mg / kg, ip, tiw) group, the TAF-6 (7 mg / kg, ip, tiw) group, and the M7824 analog group were 61%, 74%, and 45%, respectively. The tumor volumes of each group were significantly smaller than those of the Vehicle group (p<0.001, p<0.001, p<0.001). The tumor volumes of the two TAF-6 dose groups were significantly smaller than those of the M7824 analog group (p<0.01, p<0.01). The results are shown in Figure 14.

[0152] In conclusion, TAF-6 at doses of 2 mg / kg and 7 mg / kg had significant antitumor effects on the PBMC humanized human osteosarcoma SJSA-1 subcutaneous transplant tumor model, and TAF-6 at 7 mg / kg had a similar effect to the M7824 analog (TGI) at equimolar doses. TV was 45%, p<0.01) and had superior anti-tumor effect.

[0153] Example 15 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Gastric Cancer NUGC-4 / hCLDN18.2 Mice

[0154] Human gastric cancer NUGC-4 / hCLDN18.2 cells and PBMC cells were mixed and inoculated subcutaneously in the right anterior flank of male NCG mice. 3 The mice were divided into four groups (8 mice each) for drug administration at approximately 12 hours. The groups included: Vehicle, a low-dose TAF-6 (2 mg / kg, ip, tiw) group, a medium-dose TAF-6 (6 mg / kg, ip, tiw) group, and a high-dose TAF-6 (20 mg / kg, ip, tiw) group. Tumor volume and body weight were measured twice weekly, and the relationship between changes in body weight and tumor volume and drug administration time was recorded.

[0155] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0156] At the end of the experiment, peripheral blood and tumors of mice were collected. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0157] At the end of the experiment (PG-D23), the tumor growth inhibition rates of the TAF-6 low-dose group, TAF-6 medium-dose group, and TAF-6 high-dose group were 91%, 100%, and 102%, respectively. The tumor volumes of each group were significantly smaller than those of the Vehicle group (p<0.001, p<0.001, and p<0.001). The tumor volumes of the TAF-6 medium-dose group and TAF-6 high-dose group were significantly smaller than those of the TAF-6 low-dose group (p<0.01, p<0.001). The results are shown in Figure 15.

[0158] In summary, the test drug TAF-6 at doses of 2 mg / kg, 6 mg / kg, and 20 mg / kg had significant anti-tumor effects on the PBMC co-inoculated human gastric cancer NUGC-4 / hCLDN18.2 subcutaneous transplant tumor model, and the tumor inhibition effect showed a dose-dependent response trend.

[0159] Example 16 Tumor Suppressive Activity of Multi-Domain Fusion Protein in PBMC Humanized Ovarian Cancer OV90 Mice

[0160] PBMC cells were inoculated subcutaneously in the right anterior flank of female B-NDG B2m KO plus mice. The next day, human ovarian cancer OV90 cells were inoculated into the mice. When the tumors grew to 54 mm 3 The mice were divided into three groups, each with 8 mice: the vehicle group, the TAF-6 (2 mg / kg, ip, every 2 days) group, and the TAF-6 (6 mg / kg, ip, every 2 days) group. Tumor volume and body weight were measured weekly, and the relationship between changes in body weight and tumor volume and dosing schedule was recorded. Q2D means dosing every other day.

[0161] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated. TV (%) and statistical analysis was performed.

[0162] One day before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of mice. FACS test results showed the presence of human CD45-positive cells in the peripheral blood of mice in each group, and the CD45+ ratio increased over time, indicating that the mouse immune system was successfully humanized.

[0163] At the end of the experiment (PG-D24), the tumor growth inhibition rates of the TAF-6 (2 mg / kg, ip, Q2D) group and the TAF-6 (6 mg / kg, ip, Q2D) group were 68% and 79%, respectively. The tumor volumes of the two groups were significantly smaller than those of the Vehicle group (p<0.01, p<0.01). The results are shown in Figure 16.

[0164] In summary, the test drug TAF-6 at doses of 2 mg / kg and 6 mg / kg had significant anti-tumor effects on the PBMC humanized human ovarian cancer OV90 subcutaneous transplant tumor model.

[0165] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Use of a multi-domain fusion protein in the preparation of a drug for treating or preventing tumors, wherein the fusion protein comprises an anti-PD-L1 single-domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment.

2. The use according to claim 1, characterized in that The complementarity determining regions of the anti-PD-L1 single domain antibody fragment include: The amino acid sequence is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, and CDR3 shown in SEQ ID NO.10; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 11; or The amino acid sequence is CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.7, and CDR3 shown in SEQ ID NO.12; or The amino acid sequence is CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.13; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 14; or The amino acid sequence is CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.

15.

3. The use according to claim 1, characterized in that The anti-PD-L1 single domain antibody fragment further includes a framework region, wherein the framework region FR includes FR1 to FR4 with the amino acid sequences shown below: The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.50, FR3 shown in SEQ ID NO.53; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.51, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.55; and FR4 shown in SEQ ID NO.

56.

4. The use according to claim 1, characterized in that The anti-PD-L1 single domain antibody fragment comprises: a) a polypeptide fragment having an amino acid sequence as shown in any one of SEQ ID NOs. 16 to 21; or b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to one of SEQ ID NOs. 16 to 21 and having the function of the polypeptide fragment defined in a); and / or, the anti-PD-L1 single domain antibody fragment is derived from alpaca; And / or, the anti-PD-L1 single domain antibody fragment is humanized.

5. The use according to claim 1, characterized in that The antagonist VEGF fragment is bevacizumab. Preferably, the antagonist VEGF fragment comprises: c) a polypeptide fragment whose amino acid sequence is shown in one of SEQ ID NOs. 22 to 23; or d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity with one of SEQ ID NOs. 22 to 23 and having the function of the polypeptide fragment defined in c); and / or, the antagonistic VEGF fragment is of mouse origin; And / or, the antagonist VEGF fragment is humanized.

6. The use according to claim 1, characterized in that The TGF-β binding fragment is a TGF-βRⅡ extracellular domain fragment. Preferably, the TGF-β binding fragment includes: e) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 24; or f) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No. 24 and having the function of the polypeptide fragment defined in e); And / or, the TGF-β binding fragment is of human origin.

7. The use according to claim 1, characterized in that The fusion protein further includes a connecting peptide fragment. Preferably, the connecting peptide fragment is rich in G, S and / or A. More preferably, the connecting peptide is selected from a flexible polypeptide chain consisting of G glycine and / or S serine and / or A alanine, and the length of the connecting peptide is 3 to 30 amino acids.

8. The use according to claim 7, characterized in that The connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NOs. 34-36; And / or, a connecting peptide is provided between the anti-PD-L1 single domain antibody fragment and the antagonist VEGF fragment; And / or, a connecting peptide is provided between the antagonistic VEGF fragment and the TGF-β binding fragment.

9. The use according to claim 1, characterized in that The fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment from the N-terminus to the C-terminus; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the heavy chain of the antagonist VEGF fragment; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain of the antagonist VEGF fragment; And / or, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the antagonist VEGF fragment.

10. The use according to claim 1, characterized in that The amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.23 or SEQ ID NO.25-33; Or, the amino acid sequence of the fusion protein includes the sequences shown in SEQ ID NO.25 and SEQ ID NO.26, SEQ The sequence shown in SEQ ID NO.25 and SEQ ID NO.27, the sequence shown in SEQ ID NO.25 and SEQ ID NO.28, the sequence shown in SEQ ID NO.25 and SEQ ID NO.29, the sequence shown in SEQ ID NO.30 and SEQ ID NO.27, the sequence shown in SEQ ID NO.30 and SEQ ID NO.29, the sequence shown in SEQ ID NO.31 and SEQ ID NO.23, the sequence shown in SEQ ID NO.32 and SEQ ID NO.23, and the sequence shown in SEQ ID NO.33 and SEQ ID NO.

23.

11. The use according to any one of claims 1 to 10, characterized in that: The tumor is selected from the group consisting of lung cancer, melanoma, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, sarcoma, head and neck cancer, nasopharyngeal cancer, cervical cancer, bile duct cancer, and hematological malignancies.

12. A method for treating or preventing tumors, comprising administering to a patient a therapeutically effective amount of a multi-domain fusion protein, wherein the fusion protein comprises an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment.

13. The method according to claim 12, characterized in that The complementarity determining regions of the anti-PD-L1 single domain antibody fragment include: The amino acid sequence is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, and CDR3 shown in SEQ ID NO.10; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 11; or The amino acid sequence is CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.7, and CDR3 shown in SEQ ID NO.12; or The amino acid sequence is CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.13; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 14; or The amino acid sequence is CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.

15.

14. The method according to claim 12, characterized in that The anti-PD-L1 single domain antibody fragment further includes a framework region, wherein the framework region FR includes FR1 to FR4 with the amino acid sequences shown below: The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.50, FR3 shown in SEQ ID NO.53; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.51, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.55; and FR4 shown in SEQ ID NO.

56.

15. The method according to claim 12, characterized in that The anti-PD-L1 single domain antibody fragment comprises: a) a polypeptide fragment having an amino acid sequence as shown in any one of SEQ ID NOs. 16 to 21; or b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to one of SEQ ID NOs. 16 to 21 and having the function of the polypeptide fragment defined in a); and / or, the anti-PD-L1 single domain antibody fragment is derived from alpaca; And / or, the anti-PD-L1 single domain antibody fragment is humanized.

16. The method according to claim 12, characterized in that The antagonist VEGF fragment is bevacizumab. Preferably, the antagonist VEGF fragment comprises: c) a polypeptide fragment whose amino acid sequence is shown in one of SEQ ID NOs. 22 to 23; or d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity with one of SEQ ID NOs. 22 to 23 and having the function of the polypeptide fragment defined in c); and / or, the antagonistic VEGF fragment is of mouse origin; And / or, the antagonist VEGF fragment is humanized.

17. The method according to claim 12, characterized in that The TGF-β binding fragment is a TGF-βRⅡ extracellular domain fragment. Preferably, the TGF-β binding fragment includes: e) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 24; or f) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No. 24 and having the function of the polypeptide fragment defined in e); And / or, the TGF-β binding fragment is of human origin.

18. The method according to claim 12, characterized in that The fusion protein further includes a connecting peptide fragment. Preferably, the connecting peptide fragment is rich in G, S and / or A. More preferably, the connecting peptide is selected from a flexible polypeptide chain consisting of G glycine and / or S serine and / or A alanine, and the length of the connecting peptide is 3 to 30 amino acids.

19. The method according to claim 18, characterized in that The connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NOs. 34-36; And / or, a connecting peptide is provided between the anti-PD-L1 single domain antibody fragment and the antagonist VEGF fragment; And / or, a connecting peptide is provided between the antagonistic VEGF fragment and the TGF-β binding fragment.

20. The method according to claim 12, wherein The fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment from the N-terminus to the C-terminus; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the heavy chain of the antagonist VEGF fragment; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain of the antagonist VEGF fragment; And / or, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the antagonist VEGF fragment.

21. The method according to claim 12, wherein The amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.23 or SEQ ID NO.25-33; Or, the amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.25 and SEQ ID NO.26, the sequence shown in SEQ ID NO.25 and SEQ ID NO.27, the sequence shown in SEQ ID NO.25 and SEQ ID NO.28, the sequence shown in SEQ ID NO.25 and SEQ ID NO.29, the sequence shown in SEQ ID NO.30 and SEQ ID NO.27, the sequence shown in SEQ ID NO.30 and SEQ ID NO.29, the sequence shown in SEQ ID NO.31 and SEQ ID NO.23, the sequence shown in SEQ ID NO.32 and SEQ ID NO.23, and the sequence shown in SEQ ID NO.33 and SEQ ID NO.

23.

22. The method according to any one of claims 12 to 21, characterized in that The tumor is selected from the group consisting of lung cancer, melanoma, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, sarcoma, head and neck cancer, nasopharyngeal cancer, cervical cancer, bile duct cancer, and hematological malignancies.

23. A multi-domain fusion protein for treating or preventing tumors, wherein the fusion protein comprises an anti-PD-L1 single-domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment.

24. The fusion protein for use according to claim 23, characterized in that The complementarity determining regions of the anti-PD-L1 single domain antibody fragment include: The amino acid sequence is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, and CDR3 shown in SEQ ID NO.10; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 11; or The amino acid sequence is CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.7, and CDR3 shown in SEQ ID NO.12; or The amino acid sequence is CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.13; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 14; or The amino acid sequence is shown in CDR1 of SEQ ID NO.5, CDR2 of SEQ ID NO.9, CDR3 shown in NO.

15.

25. The fusion protein for use according to claim 23, characterized in that The anti-PD-L1 single domain antibody fragment further includes a framework region, wherein the framework region FR includes FR1 to FR4 with the amino acid sequences shown below: The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.50, FR3 shown in SEQ ID NO.53; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.51, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.54; FR4 shown in SEQ ID NO.56, or The amino acid sequence is FR1 shown in SEQ ID NO.49, FR2 shown in SEQ ID NO.52, FR3 shown in SEQ ID NO.55; and FR4 shown in SEQ ID NO.

56.

26. The fusion protein for use according to claim 23, characterized in that The anti-PD-L1 single domain antibody fragment comprises: a) a polypeptide fragment having an amino acid sequence as shown in any one of SEQ ID NOs. 16 to 21; or b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to one of SEQ ID NOs. 16 to 21 and having the function of the polypeptide fragment defined in a); and / or, the anti-PD-L1 single domain antibody fragment is derived from alpaca; And / or, the anti-PD-L1 single domain antibody fragment is humanized.

27. The fusion protein for use according to claim 23, characterized in that The antagonist VEGF fragment is bevacizumab. Preferably, the antagonist VEGF fragment comprises: c) a polypeptide fragment whose amino acid sequence is shown in one of SEQ ID NOs. 22 to 23; or d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity with one of SEQ ID NOs. 22 to 23 and having the function of the polypeptide fragment defined in c); and / or, the antagonistic VEGF fragment is of mouse origin; And / or, the antagonist VEGF fragment is humanized.

28. The fusion protein for use according to claim 23, characterized in that The TGF-β binding fragment is a TGF-βRⅡ extracellular domain fragment. Preferably, the TGF-β binding fragment includes: e) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 24; or f) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No. 24 and having the function of the polypeptide fragment defined in e); And / or, the TGF-β binding fragment is of human origin.

29. The fusion protein for use according to claim 23, characterized in that The fusion protein further includes a connecting peptide fragment. Preferably, the connecting peptide fragment is rich in G, S and / or A. More preferably, the connecting peptide is selected from a flexible polypeptide chain consisting of G glycine and / or S serine and / or A alanine, and the length of the connecting peptide is 3 to 30 amino acids.

30. The fusion protein for use according to claim 29, characterized in that The connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NOs. 34-36; And / or, a connecting peptide is provided between the anti-PD-L1 single domain antibody fragment and the antagonist VEGF fragment; And / or, a connecting peptide is provided between the antagonistic VEGF fragment and the TGF-β binding fragment.

31. The fusion protein for use according to claim 23, characterized in that The fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonist VEGF fragment, and a TGF-β binding fragment from the N-terminus to the C-terminus; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the heavy chain of the antagonist VEGF fragment; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain of the antagonist VEGF fragment; And / or, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the antagonist VEGF fragment.

32. The fusion protein for use according to claim 23, characterized in that The amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.23 or SEQ ID NO.25-33; Or, the amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.25 and SEQ ID NO.26, the sequence shown in SEQ ID NO.25 and SEQ ID NO.27, the sequence shown in SEQ ID NO.25 and SEQ ID NO.28, the sequence shown in SEQ ID NO.25 and SEQ ID NO.29, the sequence shown in SEQ ID NO.30 and SEQ ID NO.27, the sequence shown in SEQ ID NO.30 and SEQ ID NO.29, the sequence shown in SEQ ID NO.31 and SEQ ID NO.23, the sequence shown in SEQ ID NO.32 and SEQ ID NO.23, and the sequence shown in SEQ ID NO.33 and SEQ ID NO.

23.

33. Use of an isolated polynucleotide or a construct containing the polynucleotide in the preparation of a medicament for treating or preventing tumors, wherein the isolated polynucleotide encodes the fusion protein according to any one of claims 23 to 32.

34. Use of an expression system in preparing a drug for treating or preventing tumors, wherein the expression system comprises the construct according to claim 33 or an exogenous isolated polynucleotide according to claim 33 integrated into its genome.

35. Use of a pharmaceutical composition in preparing a drug for treating or preventing tumors, characterized in that: The pharmaceutical composition comprises the fusion protein according to any one of claims 23 to 32, or the culture of the expression system according to claim 34.

36. The fusion protein for use according to any one of claims 23 to 32, characterized in that The tumor is selected from the group consisting of lung cancer, melanoma, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, sarcoma, head and neck cancer, nasopharyngeal cancer, cervical cancer, bile duct cancer, and hematological malignancies.

Images