WO2024041477A1

WO2024041477A1 - Use of multi-domain fusion protein

Info

Publication number: WO2024041477A1
Application number: PCT/CN2023/113995
Authority: WO
Inventors: 黄岩山; 黄才
Original assignee: 浙江道尔生物科技有限公司
Priority date: 2022-08-22
Filing date: 2023-08-21
Publication date: 2024-02-29

Abstract

Provided is use of a multi-domain fusion protein in the preparation of a medicament for treating or preventing tumors. The fusion protein comprises an anti-PD-L1 single-domain antibody fragment, a VEGF-antagonizing fragment and a TGF-β binding fragment. The provided multi-domain fusion protein having anti-cancer activity can, in one antibody fusion protein molecule, organically combine the function of an anti-PD-L1 monoclonal antibody in blocking the interaction of PD-L1/PD-1, the function of an anti-VEGF monoclonal antibody in reducing the microvascular growth and inhibiting metastatic diseases, and the function of a TGF-β receptor in relieving the tolerance of cancer cells to TGF-β signals and enhancing the immune response, thus being useful for treating or preventing tumors.

Description

Uses of multi-domain fusion proteins

Technical field

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

Background technique

In an era when tumor treatment drugs are on the rise, protein engineering technology has been continuously developed. The research on the mechanism of antibody treatment is also in-depth, and multi-target drugs (such as polyantibodies or multi-domain fusion proteins) are gradually emerging, which are expected to enhance the specificity and effectiveness of tumor immunotherapy on the basis of increasing targets. Theoretically, in specific treatment settings, multi-target drugs can achieve more obvious effects than combination therapy with single drugs. Compared with dual antibodies, multi-target drugs such as polyantibodies 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 The method has been widely recognized in theory. However, so far, the development of most multi-specific antibody drugs is still in its infancy, and the fastest-growing ones are in clinical phase 1 or 2, such as Sanofi SAR-443579 (targeting CD16 /NKp46/CD123), CStone Pharmaceuticals’ NM21-1480 (targeting PD-L1/4-1BB/HSA), AbbVie and HARPOON’s HPN-217 (targeting BCMA/CD3/HSA), their safety and efficacy More data validation and support in the future is still needed.

In addition, although anti-PD-1/PD-L1 monoclonal antibodies as immune checkpoint inhibitors have been widely used in various tumor treatments, the proportion of patients who benefit is still not high, especially for "cold tumor" tumor types. , the efficacy of immune checkpoint inhibitors is not obvious. For example, the clinical trial of M7824, a fusion of the anti-PD-L1 monoclonal antibody Avelumab and TGF-β receptor II (TGF-βTrap), has been terminated due to insufficient efficacy and other reasons. 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 dual or multiple antibody (or multi-domain fusion protein) with better efficacy and safety to meet the unmet clinical needs of tumor patients.

Contents of the invention

The object of the present invention is to provide a multi-domain fusion protein with anti-cancer activity, which can be used as a therapeutic agent for the treatment and/or prevention of tumors, such as lung cancer, melanoma, gastric cancer, ovarian cancer, and colon cancer. , liver cancer, kidney cancer, bladder cancer, breast cancer, sarcoma, head and neck cancer, nasopharyngeal cancer, cervical cancer, cholangiocarcinoma and hematological malignancies, especially lung cancer, breast cancer, liver cancer and cervical cancer.

In order to achieve the above objects and other related objects, one aspect of the present invention provides the use of fusion proteins in the preparation of drugs for treating or preventing tumors. The fusion proteins include anti-PD-L1 single domain antibody fragments, antagonistic VEGF fragments, TGF- beta junction Combine the fragments.

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, the fusion protein comprising an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and TGF-β Combine fragments.

Another aspect of the present invention provides a multi-domain fusion protein for treating or preventing tumors. The fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment.

Another aspect of the present invention provides the use of an isolated polynucleotide encoding the above-mentioned fusion protein or a construct containing the polynucleotide in the preparation of a medicament for treating or preventing tumors.

Another aspect of the present invention provides the use of an expression system containing the above-mentioned construct or the exogenous above-mentioned polynucleotide integrated into the genome in the preparation of drugs for treating or preventing tumors.

Another aspect of the present invention provides the use of a pharmaceutical composition in the preparation of a drug for treating or preventing tumors. The pharmaceutical composition includes the above-mentioned fusion protein or the culture of the above-mentioned expression system.

Description of drawings

Figure 1 TAF-6 and The area under the blood drug concentration-time curve plot;

Figure 2 The tumor inhibitory effect of multi-domain fusion protein in the MDA-MB-231 breast cancer subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 3 The tumor inhibitory effect of multi-domain fusion protein in the MDA-MB-231 breast cancer subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 4 Multi-domain fusion protein TAF-6, M7824 analog, Tumor inhibitory efficacy in the subcutaneous transplantation mouse model of MDA-MB-231 breast cancer with humanized PBMC immune system;

Figure 5 The tumor inhibitory effect of multi-domain fusion protein in the Calu-6 lung cancer subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 6 Multi-domain fusion protein TAF-6, M7824 analog, Anti-tumor efficacy in the Calu-6 lung cancer subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 7 The tumor inhibitory effect of multi-domain fusion protein in the HCT116 colon cancer subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 8 The tumor inhibitory effect of multi-domain fusion protein in the mouse model of subcutaneous transplantation of Huh-7 liver cancer with humanized PBMC immune system;

Figure 9 Multi-domain fusion protein TAF-6, M7824 analog, Antitumor efficacy in the mouse model of subcutaneous transplantation of Huh-7 liver cancer with humanized PBMC immune system;

Figure 10 Multi-domain fusion protein in PBMC immune system humanized HT1080 sarcoma subcutaneously transplanted tumor mice Tumor inhibitory efficacy in the model.

Figure 11 The tumor inhibitory effect of multi-domain fusion protein in the Hela cervical cancer subcutaneous xenograft mouse model with humanized PBMC immune system;

Figure 12 The tumor inhibitory effect of multi-domain fusion protein in the CRC-034 colorectal cancer PDX subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 13 The tumor inhibitory effect of multi-domain fusion protein in the mouse model of subcutaneous transplantation of HuCCT1 cholangiocarcinoma with humanized PBMC immune system.

Figure 14 The anti-tumor effect of multi-domain fusion protein in the SJSA osteosarcoma subcutaneous xenograft mouse model with humanized PBMC immune system;

Figure 15 The tumor inhibitory effect of multi-domain fusion protein in the NUGC-4/hCLDN18.2 gastric cancer subcutaneous transplant mouse model with humanized PBMC immune system;

Figure 16 Anti-tumor efficacy of multi-domain fusion protein in the OV90 ovarian cancer subcutaneous transplant mouse model with humanized PBMC immune system.

Detailed ways

After extensive exploration and research, the inventor unexpectedly discovered a fusion protein molecule that can block PD-L1/PD-1 interaction with anti-PD-L1 monoclonal antibodies and reduce microvessel growth by antagonizing VEGF monoclonal antibodies. Combined with the function of inhibiting metastatic disease, and the TGF-β receptor improving T cell dysfunction caused by TGF-β in the tumor microenvironment and enhancing the immune response, it has excellent tumor suppressive effects. On this basis, this study was completed invention.

A first aspect of the present invention provides the use of a fusion protein in the preparation of drugs for treating or preventing tumors. The fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment. Among the above-mentioned fusion proteins, anti-PD-L1 single domain antibody fragments can usually be used to block PD-L1/PD-1 interaction and increase the expression of IFN-γ and/or IL-2 in T lymphocytes, thereby inhibiting tumor growth. Antagonistic VEGF fragments usually include an Fc part that can bind to the FcRn receptor, thereby extending the half-life in the body, and can also bind to effector cells expressing Fc receptors to kill cancer cells. TGF-β binding fragments can improve the killing function of tumor-infiltrating T cells against tumor cells by clearing overexpressed TGF-β in the tumor microenvironment.

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

A third aspect of the present invention provides a fusion protein for treating or preventing tumors, the fusion protein comprising anti-PD-L1 Single domain antibody fragments, antagonistic VEGF fragments, and TGF-β binding fragments.

In some embodiments, the above-mentioned anti-PD-L1 single domain antibody fragment can generally be a polypeptide or protein fragment capable of specifically binding to PD-L1. Anti-PD-L1 single domain antibody fragments usually lack the corresponding antibody light chain, and only have the fragment corresponding to the heavy chain variable region. The binding properties of anti-PD-L1 single domain antibody fragments can usually be determined by the three complementarity determining regions (CDRs) they include. The CDR regions can be ordered with the framework region (FR, framework region). The FR region Not directly involved in binding reactions. These CDRs can form a cyclic structure, and the β-sheets formed by the FRs between them are close to each other in 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 CDR1 whose amino acid sequence is shown in one of SEQ ID NO. 1 to 5, and CDR1 shown in one of SEQ ID NO. 6 to 9. CDR2, CDR3 shown in one of SEQ ID NO.10~15.

In a specific embodiment of the present invention, the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 whose amino acid sequence is shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, SEQ ID NO. CDR3 shown in 10.

In another specific embodiment of the present invention, the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 whose amino acid sequence is shown in SEQ ID NO.2, CDR2 shown in SEQ ID NO.7, and SEQ ID NO. CDR3 shown in .11.

In another specific embodiment of the present invention, the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 whose amino acid sequence is shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.7, and SEQ ID NO. CDR3 shown in .12.

In another specific embodiment of the present invention, the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 whose amino acid sequence is shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.8, and SEQ ID NO. CDR3 shown in .13.

In another specific embodiment of the present invention, the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 whose amino acid sequence is shown in SEQ ID NO.2, CDR2 shown in SEQ ID NO.7, and SEQ ID NO. CDR3 shown in .14.

In another specific embodiment of the present invention, the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 whose amino acid sequence is shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.9, and SEQ ID NO. CDR3 shown in .15.

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

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

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; FR4 shown in SEQ ID NO.56.

In another specific embodiment of the present invention, the anti-PD-L1 single domain antibody fragment can include: a) a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID No. 16-21; or, b) an amino acid sequence with SEQ One of the polypeptide fragments of ID No. 16 to 21 has more than 80% sequence identity and has the function of the polypeptide fragment defined in a). Specifically, the polypeptide fragment in b) above specifically refers to: the amino acid sequence shown in one of SEQ ID No. 16~21, which has been substituted, deleted or added one or more (specifically, it can be 1-50, 1 -30, 1-20, 1-10, 1-5, or 1-3) amino acids, or one or more (specifically, can be 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and has an amino acid sequence such as SEQ ID No. 16 to 21. The functional polypeptide fragment of a polypeptide fragment, for example, can be the ability to specifically bind to PD-L1, or can be a blocker of PD-L1/PD-1 interaction, thereby blocking PD-L1 /PD1 pathway can also have the function of increasing the expression of IFN-γ and/or IL-2 in T lymphocytes, or it can also have the function of inhibiting tumor growth. The amino acid sequence of the anti-PD-L1 single domain antibody fragment in b) above can be 80%, 85%, 90%, 93%, 95%, 97%, or 99% identical to one of SEQ ID No. 16-21 Consistency of the above. The above-mentioned anti-PD-L1 single domain antibody fragment can usually be derived from alpaca (Vicugna pacos), for example, its CDR region can be derived from alpaca. The above-mentioned anti-PD-L1 single domain antibody fragment can usually be humanized, for example, its framework region can be derived from human.

In this article, sequence identity refers to the percentage of identical residues in the compared sequences. The sequence identity of the sequences of two or more entries can be calculated using computational software well known in the art and available from, for example, NCBI.

In some embodiments, the above-mentioned VEGF-antagonizing fragments may generally be polypeptides or protein fragments capable of antagonizing VEGF. For example, the above-mentioned antagonistic VEGF fragment may be a monoclonal antibody or the like. For another example, the above-mentioned antagonistic VEGF fragment may be bevacizumab or the like.

In a specific embodiment of the present invention, the antagonistic VEGF fragment may include:

c) Polypeptide fragments with heavy chain and light chain amino acid sequences as shown in SEQ ID No. 22 and 23 respectively;

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 have the function of the polypeptide fragment defined in c). Specifically, the amino acid sequence in d) above specifically refers to: the amino acid sequence shown in one of SEQ ID No. 22~23 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30 , 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or add one at the N-terminus and/or C-terminus or more (specifically, it can be 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids. and has the function of a polypeptide fragment whose amino acids are represented by one of SEQ ID No. 22-23, for example, it can be a function of antagonizing VEGF, or it can be a function of the Fc part that binds to FcRn receptors, This can extend the half-life in the body, and can also combine with effector cells expressing Fc receptors to kill cancer cells. The amino acid sequence in d) may be 80%, 85%, 90%, 93%, 95%, 97%, or more than 99% identical to one of SEQ ID Nos. 22 to 23. The above-mentioned antagonistic VEGF fragment can usually be derived from mice (Mus musculus), for example, its CDR region can be derived from mice. The above-mentioned antagonistic VEGF fragment can usually be humanized, for example, its framework region can be derived from human.

In some embodiments, the above-mentioned TGF-β binding fragment can generally specifically bind to each TGF-β isoform (e.g., TGF-β1, TGF-β2, TGF-β3, etc.), and the TGF-β isoform is usually in High expression in a variety of malignant tumors is likely to be one of the important factors leading to poor clinical treatment results. For example, the TGF-β binding fragment may be a structural fragment of the extracellular domain of TGF-βRII (TGF-β receptor II).

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

e) A polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 24;

f) A polypeptide fragment whose amino acid sequence has more than 80% sequence identity with SEQ ID No. 24 and has the function of the polypeptide fragment defined in e). Specifically, the amino acid sequence in f) above specifically refers to: the amino acid sequence shown in SEQ ID No. 24 after substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1-20 , 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more (specifically It can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has amino acids The functional polypeptide fragment of the polypeptide fragment shown in SEQ ID No. 24, for example, can bind to each TGF-β isoform (for example, TGF-β1, TGF-β2 and TGF-β3, etc.), thereby clearing tumor microorganisms. Overexpression of TGF-β in the environment can also enhance the killing function of tumor-infiltrating T cells against tumor cells. The amino acid sequence in f) may have 80%, 85%, 90%, 93%, 95%, 97%, or more than 99% identity with SEQ ID No. 24. The above-mentioned TGF-β binding fragments can usually be derived from humans (homo sapiens).

In some embodiments, the fusion protein may also include a linker peptide fragment. The fusion protein may generally include multiple connecting peptide fragments, and at least part of the structural domains or between each structural domain may be provided with connecting peptide fragments. For example, against A connecting peptide can be provided between the PD-L1 single domain antibody fragment and the antagonistic VEGF fragment. For another example, a connecting peptide can be provided between the antagonistic VEGF fragment and the TGF-β binding fragment. The above-mentioned connecting peptide fragment can usually be a flexible polypeptide rich in G, S and/or A (mainly composed of glycine (G), serine (S) and/or alanine (A)) of suitable length, thereby making the phase Neighboring protein domains are free to move relative to each other. For example, the amino acid sequence of the connecting peptide fragment may include (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG )nA and other sequences, where 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 may include a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 34-36.

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

A fourth aspect of the present invention provides the use of an isolated polynucleotide or a construct containing the polynucleotide, wherein the isolated polynucleotide encodes the above-mentioned fusion protein, in the preparation of a medicament for treating or preventing tumors. The above-mentioned polynucleotide may be RNA, DNA, cDNA, etc. Methods of providing such isolated polynucleotides will be known to those skilled in the art. For example, it can be prepared by methods such as automated DNA synthesis and/or recombinant DNA technology, or it can be isolated from suitable natural sources. Suitable methods for constructing the above constructs will be known to those skilled in the art. For example, the construct can be constructed through in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and other methods. More specifically, it can be constructed by inserting the above-mentioned isolated polynucleotide into the multiple cloning site of the expression vector. . Expression vectors in the present invention 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 adenovirus, retrovirus or other vectors. Generally speaking, a suitable vector may 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 be lac or trp promoters including but not limited to E. coli; lambda phage PL promoter; eukaryotic promoters including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters , Pichia pastoris methanol oxidase promoter and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes may be used to provide phenotypic traits for selection of transformed host cells, and may include, but are not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or for tetracycline or ampicillin resistance in E. coli, etc. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence. If the enhancer sequence is inserted into the vector, transcription will be enhanced. The enhancer is a cis-acting factor of DNA, usually about It has 10 to 300 base pairs and acts on promoters to enhance gene transcription.

The fifth aspect of the present invention provides the use of an expression system in the preparation of drugs for the treatment or prevention of tumors. The expression system contains the above-mentioned construct or the exogenous above-mentioned isolated polynucleotide integrated into its genome, Thus, the above-mentioned fusion protein can be expressed. The above-mentioned expression system can be a host cell, and any cell suitable for expression of the expression vector can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; filamentous cell. Fungal cells, or higher eukaryotic cells, such as mammalian cells. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes Melanoma cells, animal cells, etc. The method of introducing the construct into the host cell should be known to those skilled in the art. For example, microinjection, gene bombardment, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation can be used. method etc.

A sixth aspect of the present invention provides the use of a pharmaceutical composition comprising a culture of the fusion protein or expression system of the present invention in the preparation of a medicament for treating or preventing tumors. In the above pharmaceutical composition, the content of the fusion protein or culture is usually a therapeutically effective amount. In the present invention, "therapeutically effective dose" generally refers to an amount that, after an appropriate period of administration, can reduce the severity of disease symptoms, increase the frequency and duration of disease-free periods, or prevent symptoms caused by disease pain. Impairment or disability. The ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of the fusion protein or pharmaceutical composition can usually reduce the size of the tumor or otherwise relieve the subject's symptoms. Those skilled in the art can select an appropriate therapeutically effective amount based on the actual situation, 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., determination of dosage, etc.) may be determined by a physician, typically taking into account factors including, but not limited to, The disease being treated, the individual patient's condition, delivery site, method of administration, and other factors.

The above-mentioned pharmaceutical composition may also include a pharmaceutically acceptable carrier. The above-mentioned carriers may include various excipients and diluents which are not necessarily active ingredients themselves and which do not cause excessive toxicity after administration. Suitable carriers will be well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable carriers can be found, for example, in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).

The tumor, for example, can be a solid tumor or a hematological 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). tumors, etc.), head and neck cancer, nasopharyngeal cancer, cervical cancer and cholangiocarcinoma, hematological malignant tumors including classic Hodgkin lymphoma, etc. These cancers can be early, intermediate or late, such as metastatic cancer.

In the present invention, the term "treatment" includes preventive, curative or palliative procedures that result in a desired pharmaceutical and/or physiological effect. A better treatment effect means that the medical treatment can reduce one or more symptoms of the disease or completely eliminate the disease, or block or delay the occurrence of the disease and/or reduce the risk of the disease development or worsening.

In the present invention, "individuals" generally include humans, non-human primates, or other mammals (such as dogs, cats, horses, sheep, pigs, cattle, etc.), which can be treated by using the preparation, kit or combination benefit from treatment.

In some embodiments, the multi-domain fusion protein with anti-cancer activity can combine anti-PD-L1 monoclonal antibody to block PD-L1/PD-1 interaction and anti-VEGF in one antibody fusion protein molecule. The functions of monoclonal antibodies in reducing microvessel growth and inhibiting metastatic disease, as well as the functions of TGF-β receptors that relieve cancer cells' tolerance to TGF-β signals and enhance immune responses are organically combined, so they can be used to treat tumors and have good efficacy. Industrialization prospects.

The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, It is not intended to limit the scope of the present invention.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art and the description of the present invention by those skilled in the art, methods, equipment, and materials similar to those described in the embodiments of the present invention can also be used. or any equivalent prior art methods, equipment and materials to implement the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology. These techniques have been well described in the existing literature. For details, see 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, 1987and 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 (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

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

Example 1 Construction and recombinant expression preparation of fusion protein

According to the codon preference of CHO cells, the amino acid sequence of the multi-domain fusion protein, PD-L1-Fc fusion protein (SEQ ID NO.47) and Fc-TGFβRII fusion protein (SEQ ID NO.48) in Table 1 were respectively converted into Base sequence, and introduce HindIII enzyme cleavage site and Kozak sequence (GCCACC) at the 5' end of the heavy chain and light chain coding sequence respectively, introduce stop codon and EcoRI enzyme cleavage site at the 3' end through gene synthesis (general Biological Systems (Anhui) Co., Ltd.) to obtain full-length DNA. The synthesized heavy chain and light chain encoding genes were double digested with HindIII-HF (purchased from NEB, R3104V) and EcoRI-HF (purchased from NEB, R3101V), and agarose gel DNA/PCR product mini-recovery reagent was used cassette (purchased from Biomiga) was used for gel recovery, and the pCDNA3.1(+) vector, which was also double digested with HindIII and EcoRI, was ligated with T4 ligase (purchased from NEB, M0202V), transformed into Top10 competent cells, and coated on Culture on LB ampicillin-resistant plates. Clones were picked for identification and confirmed by sequencing, and heavy chain and light chain expression plasmids based on pCDNA3.1(+) were constructed. Use the endotoxin-free plasmid extraction kit (purchased from Biomiga, BW-PD3511-02) to extract the heavy chain and light chain expression plasmids respectively, and mix the two at a ratio of 1:1. Take 1.0 mg of the mixed plasmid and dilute it to 25 mL using Wayne293 expression medium (purchased from Zhongshan Kangsheng, A21501); take 3.0 mg PEI (linear, 25KD, Polysciences, Inc.), dilute it to 25 mL using Wayne293 expression medium and add it to plasmid solution, mix well, and incubate at room temperature for 30 minutes. Take the Hek293F cells growing in the logarithmic phase (viability rate >95%) and count them; centrifuge at 1100 rpm for 10 minutes, discard the supernatant, and use 450 mL of Wayne293 Resuspend cells in expression medium. Add the above plasmid-PEI mixture to the cell suspension, culture it in a 37°C, 5% CO ₂ shaking incubator for 7 days, and then centrifuge to take the supernatant for subsequent protein purification.

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

Example 2 Purification of multi-domain fusion proteins

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

Adjust the cell fermentation supernatant to pH 7.0 and load it onto a Protein A affinity chromatography column (Borgron Biotechnology Co., Ltd.). The balance solution is 20mM PB, 0.15M NaCl (pH=7.0), 100% 0.1M Gly-HCl (pH=3.0) is eluted; 10% 1M Tris-HCl (pH=8.5) is added to the eluent in advance. 100% eluate was diluted to conductivity <3ms/cm, and the supernatant was adjusted to pH 7.0 and loaded onto a DSP chromatography column (Borgron Biotechnology Co., Ltd.), with 15% and 100% elution (20mM PB, 0.5) respectively. M NaCl, pH7.0). 15% of the elution fraction is obtained, which is the target protein. The protein concentration was determined using UV280 method.

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

Adjust the cell fermentation supernatant to pH 7.0 and load it onto a Protein A affinity chromatography column (Borgron Biotechnology Co., Ltd.). The balance solution is 20mM PB, 0.15M NaCl (pH=7.0), 100% 0.1M Gly-HCl (pH=3.0) is eluted; 10% 1M Tris-HCl (pH=8.5) is added to the eluent in advance. 100% eluate was diluted to conductivity 4ms/cm, and loaded onto the Super Q (TOSOH) chromatography column. The equilibrium solution was 20mM Tris, pH 8, and the eluent was 500mM NaCl+20mM Tris (pH=8.0), 35% respectively. and 100% elution, remove excess light chains through flow-through, and obtain 35% elution fraction, which is the target protein. The protein concentration was determined using UV280 method.

Purity testing uses SEC-HPLC-UV analysis. Detector: Agilent 1100LC; detection wavelength: 214nm; mobile phase: 150mM pH7.0 PB+5% isopropanol; column: Superdex 200 Increase 5/150GL; running time: 15 minutes; column temperature 25°C. Test results show that the purity is greater than 95%.

Purification of PD-L1-Fc fusion protein (SEQ ID NO.47) and Fc-TGFβRII fusion protein (SEQ ID NO.48): Adjust the cell fermentation supernatant to pH 7.0 and load it into Protein A affinity chromatography Column (Borgron Biotechnology Co., Ltd.), the equilibrium solution is 20mM PB, 0.15M NaCl (pH=7.0), 100% 0.1M Gly-HCl (pH=3.0) elution; the eluent is pre-added with 10% 1M Tris -HCl (pH=8.5) was used for neutralization.

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

3.1 In vitro activity detection of anti-PD-L1 single domain antibody fragments:

The CD5L-OKT3scFv-CD14 gene sequence (GenBank: ADN42857.1) was synthesized, 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 obtained by high-fidelity amplification was recombinantly ligated and inserted into pCDNA3.1-antiCD3TM to construct pCDNA3.1-antiCD3TM-PDL1. CHO cells (Thermo) were transfected and then selected with G418 for 10-14 days to generate the stable cell line CHO-antiCD3TM-PDL1.

The amplified fragment was amplified using the human PD1 gene (GenBank: NP_005009.2) as a template, and recombinantly ligated with the PB513B1-dual-puro vector (Ubao Biotech) digested with HindIII-BamHI (Takara) to construct plasmid pB-PD1. High-fidelity amplification was performed using pGL4.30 (Ubao Biotechnology) as a template, and the obtained fragment was recovered and recombinantly ligated with the pB-PD1 vector digested by SfiI-XbaI (Takara) to construct the pB-NFAT-Luc2p-PD1 plasmid. After the plasmid was successfully constructed, the endotoxin-free plasmid extraction kit (Biomiga) was used to extract the plasmid and used to transfect Jurkat cells (Stem Cell Bank of the Chinese Academy of Sciences). Referring to the method in patent CN 107022571A, Jurkat cells are treated into a relatively adherent state by using 0.1mg/ml poly-D-lysine, and then according to the lipofectamine transfection kit (Lipofectamine 3000; invitrogen) Transfection instructions for Jurkat cells were carried out; on the third day, RPMI1640 medium (Thermo) containing 10% FBS and 2.5 μg/ml puromycin was used for pressurized selection; thereafter, the medium was added at regular intervals, and the After the cell viability was restored, the puromycin content was gradually increased to 4 μg/ml. Finally, the monoclonal Jurkat-NFAT-Luc2p-PD1 cell line was obtained.

Take CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells and count them. Adjust the cell density to 4×10 ⁶ /ml. Add 25 μl to each cell in each well of the 96-well plate; perform gradient dilution with 1% BSA respectively. Add 50 μl of the fusion protein sample prepared in Example 2 to the cells; after co-culture for 6 hours at 37°C and 5% _CO2 , add 10 μl of luciferase substrate (Promega, E2620) to each well, shake on a oscillator for 2 minutes, and read. . The operation is as shown in the kit instructions.

3.2 In vitro activity detection of antagonistic VEGF fragments:

HEK293 cells were spread on a 6-well cell culture plate, with 1.0×10 ⁶ cells per well, and cultured overnight in a 37°C, 5% CO ₂ incubator. according to Transfection reagent instructions are used to prepare the transfection system, in which pcDNA-KDR Plasmid 1.0μg, pGL4.30 plasmid 4μg. 48 hours after transfection, the cells were expanded to a 10cm cell culture dish, and G418 200μg/ml and Hygromycin 100μg/ml were added. Replace fresh pressurized medium every 3 days until obvious colonies grow. Digest the cells and spread them on a 96-well cell culture plate. After the single clones grow out, stimulate with 0.1 μg/ml VEGF for 6 hours and then detect the chemiluminescence. Select clones with obvious signal response to continue amplification and culture. Finally, monoclonal HEK293-NFAT-KDR was obtained. HEK293-NFAT-KDR cells were plated at a density of 40,000 cells/well and digested with Accutase; the digested cells were collected and centrifuged at 1000 rpm for 5 minutes; the supernatant was discarded and analysis culture medium (DMEM+5% FBS) was added to resuspend the cells. ; Count cells and adjust cell density to 1.6×10 ⁶ /ml; spread 96-well cell culture plate, 25ul per well; use analysis culture medium to prepare VEGF solution, concentration is 60ng/ml; add to cell culture plate, 25ul per well ; Use the analysis culture medium to prepare the fusion protein prepared in Example 2, and add it to the cell culture plate, 25ul per well; incubate and culture at 37°C, 5% CO ₂ for 6 hours; add 10ul Bright-Glo luciferase detection reagent to each well (Promega, E2620), shake for 2 minutes, transfer 80ul of lysate to an enzyme-labeled white plate, and read with a microplate reader.

3.3 In vitro cell activity detection of TGF-β binding fragments:

Mouse breast cancer 4T1 cells were cultured to about 90% confluence (10cm round dish), digested with trypsin, plated into a 6-well plate at 4×10 ⁵ cells/well, and cultured overnight. The extracted pGL4.48[luc2P SBE Hygro] plasmid sample was Transfection of 4T1 cells. 24 hours after transfection, the obtained TGFβ-4T1 cells were trypsinized and transferred to a 10cm culture dish, and cultured in RPMI 1640 with 10% FBS and 150ug/ml hygromycin (InvivoGen, Cat no.:ant-hg-1). Cells are screened under pressure. TGFβ-4T1 cells after pressure screening for 10 to 15 days were plated at 2 cells/well for monoclonal screening, and TGFβ1 (Novoprotein, 10ug, Cat no.: CA59) was used to stimulate the monoclonal cells to verify the transfection of the monoclonal effect, and finally obtained TGFβ-4T1 monoclonal cells.

When the TGFβ-4T1 cells are about 90% full, add about 2.5 ml of 0.25% trypsin for digestion and digest at room temperature for 2 minutes; pipe down the cells attached to the culture dish and pipet in the trypsin solution to disperse them. , the entire digestion process takes ~5 minutes, add complete culture medium (RPMI 1640+10% FBS) to terminate the digestion, and continue to pipet the cells until the cells are evenly dispersed; transfer the cells to a 50m centrifuge tube, centrifuge at 1000 rpm for 5 minutes; discard the supernatant, and add Resuspend the cells in 2 ml of complete culture medium, and measure the cell density with a cell counter; use complete culture medium (RPMI 1640+10% FBS) to dilute the cells to a cell density of 2×10 ⁵ cells/ml; place the diluted cells ( The cell density is 2×10 ⁵ /ml) and plated (96-well plate), 100ul per well, the cell density is 2×10 ⁴ /well, the 96-well plate is placed in a 37°C incubator for overnight culture; in Example 2 The prepared fusion protein samples were diluted to the specified concentration using RPMI1640+0.2% FBS medium (containing 2ng/ml TGFβ1) (each protein sample was diluted and left at room temperature for 1 hour); the supernatant was discarded from TGFβ-4T1 cells cultured overnight in a 37°C incubator. , add 50ul RPMI 1640+0.2% FBS culture medium, then add 50 μl of protein solutions of different concentrations; place the 96-well plate in a 37°C incubator and continue to incubate for 3 hours, then add 10 μl of Bright-Glo luciferase detection reagent (Promega, E2620), and shake for 3 minutes; Microplate reader reading.

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 can be seen from Table 2, each multi-domain fusion protein has good in vitro cell activity.

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

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

C57BL/6 mice were divided into 4 groups, namely (Roche) low dose (1mg/kg) and high dose (10mg/kg), TAF-6 low dose (1mg/kg) and high dose groups (10mg/kg), 8 animals in each group, half male and half female. The mice were given a single dose of drug through tail vein injection, and drug samples were collected at different time points. The collection time is 1h, 6h, 24h, 48h, 78h, 120h, 144h, 168h, 192h, 216h before and after the drug. The serum drug concentration in the pharmaceutical samples was quantitatively detected by ELISA method. VEGF coated plate, secondary antibody Goat anti-Human IgG Fc, HRP conjugated drug, detected by TMB method, regressed and converted into concentration value according to the relationship between signal and concentration value of the standard curve, and calculated using the non-compartmental model of PK Solver software Main pharmacokinetic parameters.

The results are shown in Figure 1. C57BL/6 mice were administered The pharmacokinetic parameters were similar to those of the TAF-6 high- and low-dose groups. The dose value ratio of the low and high dose groups was 1:10, the ratio of C _max was 1:9.3, the ratio of AUC _last was 1:8.9, T _max was both 1h, and the exposure (C _max and AUC _last ) increased Increased dose proportionally. The dose value ratio of TAF-6 low and high dose groups is 1:10, the ratio of C _max is 1:8.6, the ratio of AUC _last is 1:8.5, Tmax is 1h, and the exposure (C _max and AUC _last ) The increase was dose proportional. therefore, And between the high and low dose groups of TAF-6, AUC and C _max were dose linearly related. The difference in AUC with TAF-6 is presumed to be the PD-L1 target-mediated clearance effect (TMDD).

Example 5 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized breast cancer MDA-MB-231 mice

MDA-MB-231 (human breast cancer) cells were used to model the human PBMC immune system in humanized mice (M-NSG mice) in vivo to determine the in vivo efficacy of the multi-domain fusion protein of the present invention. Screen 6-8 week old female M-NSG mice, inoculate the mice with MDA-MB-231 cells (10*10E6+Matrigel 25%), and inject PBMC (5*10E6/0.2ml) into the tail vein on the 7th day. The tumor volume and body weight were observed, and mice with tumor volumes between 140-260 mm ³ were selected and randomly divided into 6 groups based on their tumor volume and body weight, with 7 mice in each group. Administration began on the day of grouping. Tumor-bearing mice with tumors that are too large/too small will be eliminated. Intraperitoneal injection twice a week: PBS, isotype control IgG1, positive control (Roche), TAF-6, TAF-7 and combined administration (see Table 3 for details of the protocol), for a total of about 3 weeks. PD-L1-Fc fusion protein (SEQ ID NO. 47) is a fusion protein of anti-PD-L1 single domain antibody and human IgG Fc (anti-PD-L1 single domain antibody is located at the N-terminus of Fc, aPD-L1-Fc), Fc-TGFβRII fusion protein (SEQ ID NO. 48) is a fusion protein of human IgG Fc and TGFβRII (TGFβRII is located at the C-terminus of Fc).

Before grouping and before the end of the experiment, blood was collected from the orbital veins of the mice. FACS test results showed that there were human CD45-positive cells in the peripheral blood of mice in each group, and the proportion of CD45+ increased over time, indicating that the mouse immune system was successfully humanized. During the experiment, the body weights of the animals in Groups 3 and 4 (measured twice a week) were generally stable, and no experimental animals died. The tumor volumes of Groups 3 and 4 (measured twice a week) were smaller than those in Group 6 after combined administration. And it was significantly less than the corresponding single drug in group 2 The results are shown in Figure 2.

table 3

Example 6 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized breast cancer MDA-MB-231 mice

The human breast cancer MDA-MB-231 tumor mass was inoculated subcutaneously into the right front flank of female NCG mice. One day after the tumor mass was inoculated, PBMC cells were inoculated into the mice. When the tumor grew to about 53 mm ³ Administration was divided into groups, with a total of 6 groups, 10 animals in each group, namely: Vehicle group, TAF-6 low-dose (2mg/kg, ip, tiw×9) group, TAF-6 medium-dose (6mg/kg, ip, tiw ×9) group, TAF-6 high dose (18mg/kg, ip, tiw×9) group, (4mg/kg,ip,tiw×9) group, (4mg/kg,ip,tiw×9) group. Tumor volume and body weight were measured every week, and the body weight of tumor-bearing mice was recorded. Changes in tumor weight and volume as a function of administration time. Among them, tiw means administration three times a week, ip means intraperitoneal injection; tiw×9 means administration three times a week, for a total of 9 administrations.

Blood was taken from the orbital venous plexus 2 days before grouping and at the end of the experiment. FACS testing showed that there were human CD45-positive cells in the peripheral blood of mice in each group, and the proportion of CD45+ increased with time. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were stripped, weighed, and photographed, and serum was collected and tumors fixed. Calculate the tumor growth inhibition rate TGI _TV (%) and conduct statistical analysis: 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, except Outside the group, the tumor volume of each group was significantly smaller than that of the Vehicle group (p<0.05), and the tumor volume of the TAF-6 high-dose group was significantly smaller than that of the Vehicle group. group (p<0.01), There was no significant difference in tumor volume between groups (p>0.05). The results are shown in Figure 3.

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, effectively inhibiting tumor growth, and its anti-tumor effect is significantly better than And the tumor inhibitory effect increases with increasing dose.

In another independent experiment with the same tumor model, the anti-tumor activity of the test drug and the positive drug M7824 analog (prepared by our laboratory according to the M7824 patent US9676863B2) was compared. Set up 4 groups with 6 animals in each group, namely: Vehicle group, TAF-6 (4.2 mg/kg, ip, tiw × 8 times; the final dose is adjusted to 8.4 mg/kg, ip, tiw × 4 times) group, and M7824 Analog (3.6 mg/kg, ip, tiw × 8 times; later dose adjustment to 7.2 mg/kg, ip, tiw × 4 times) group, (3mg/kg, ip, tiw×8 times; later dose adjustment to 6mg/kg, ip, tiw×4 times) group. The results are shown in Figure 4. At equimolar concentration doses, the anti-tumor effect of TAF-6 is significantly better than that of M7824 analogues.

Example 7 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized lung cancer Calu-6 mice

Human lung cancer Calu-6 cells were inoculated subcutaneously into the right front flank of male NCG mice. PBMC cells were inoculated into the mice 4 days before tumor cell inoculation. When the tumors grew to ^about 50 mm, they were administered in groups. A total of 7 groups, 8 animals in each group, respectively: Vehicle group, TAF-6 low-dose (2mg/kg, ip, tiw×10) group, TAF-6 medium-dose (7mg/kg, ip, tiw×10) group, TAF-6 high dose (25mg/kg, ip, tiw×10) group, (5mg/kg,ip,tiw×10) group, (5mg/kg,ip,tiw×10) group, (5+5mg/kg,ip,tiw×10) group. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were peeled off, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate TGI _TV (%) was calculated and statistically analyzed.

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 that there were human CD45-positive cells in the peripheral blood of mice in each group, and the proportion of CD45+ increased over time, indicating that the mice were immune. System humanization was successful.

During the treatment period, the mice in each group ate and drank normally, their body weight was generally stable, and no experimental animals died.

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, except Outside the group, the tumor volume of each group was significantly smaller than that of the Vehicle group (all p<0.01), and the tumor volume of the TAF-6 low-, medium-, and high-dose groups was significantly smaller than that of the Vehicle group (all p<0.01). Group, Group and group (p<0.01). The results are shown in Table 4 and Figure 5.

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 anti-tumor effect is significantly better than and and combination, and the tumor inhibitory effect is enhanced with increasing dose.

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

Note: ^a _. Mean ± standard error.

In another independent experiment with the same tumor model, the tumor inhibitory activity of the test drug was compared with the positive drug M7824 analogue. Set up 4 groups, with 6 animals in each group, namely: Vehicle group, TAF-6 (7mg/kg, ip, TIW×8 times) group, M7824 analogue (6mg/kg, ip, TIW×8 times) group, (5mg/kg, ip, TIW×8 times) group. The results are shown in Figure 6. At equimolar concentration doses, the anti-tumor effect of TAF-6 is significantly better than that of M7824 analogues and

Example 8 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized colon cancer HCT116 mice

HCT-116 (human colon cancer) cells were used to model the human PBMC immune system in humanized mice (NOG mice) in vivo to determine the in vivo efficacy of the multi-domain fusion protein of the present invention. Female NOG mice aged 7-9 weeks were screened. The mice were inoculated with HCT-116 cells (3*10E6+Matrigel). On the third day after tumor cells were inoculated, PBMC (5*10E6/0.2ml) were injected into the tail vein. The tumor volume was then observed. and body weight, select mice whose tumor volume is between ^60-100mm3 , and randomly divide them into 6 groups according to their tumor volume and body weight, with 8 mice in each group. Administration will begin on the day of grouping: Vehicle group, TAF-6 low dose ( 2mg/kg, ip, tiw×3) group, TAF-6 medium dose (7mg/kg, ip, tiw×3) group, TAF-6 high dose (25mg/kg, ip, tiw×3) group, (5mg/kg,ip,tiw×3) group, (5+5mg/kg,ip,tiw×3) group. The tumor volume and body weight were measured twice a week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were peeled off, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate TGI _TV (%) was calculated and statistically analyzed.

Before grouping and before the end of the experiment, blood was collected from the orbital veins of the mice. FACS test results showed that there were human CD45-positive cells in the peripheral blood of mice in each group, and the proportion of CD45+ increased over time, indicating that the mouse immune system was successfully humanized.

The antitumor 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 anti-tumor effect in the high-dose and medium-dose groups was significantly better than Comparison under the same molar concentration dose, that is, TAF-6 (7mg/kg, ip, tiw×3) group (5+5mg/kg,ip,tiw×3) group, TAF-6 can still be observed to be better than group efficacy.

Example 9 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized liver cancer Huh-7 mice

Human liver cancer Huh-7 cells were inoculated subcutaneously into the right front flank of male NCG mice. PBMC cells were inoculated into the mice 45 days before tumor cell inoculation, and were administered in groups when the tumors grew to ^about 50 mm. 5 groups, 10 animals in each group, respectively: Isotype group, TAF-6 (2mg/kg, ip, tiw×8) group, TAF-6 (7mg/kg, ip, tiw×8) group, (5mg/kg,ip,tiw×8) group, (5mg/kg,ip,tiw×8) group. The tumor volume and body weight were measured twice a week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were peeled off, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate TGI _TV (%) was calculated and statistically analyzed.

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

During the administration period, the mice in each group ate and drank normally, and their body weight was generally stable.

Before the end of the experiment (PG-D18), TAF-6 (2mg/kg, ip, tiw×8) group, TAF-6 (7mg/kg, ip, tiw×8) Group, Group, The tumor growth inhibition rates of the groups were 57%, 74%, 18%, and 67% respectively, except Outside the group, the tumor volume of each group was significantly smaller than that of the Isotype group (p<0.01), and the tumor volume of the TAF-6 (7mg/kg, ip, tiw×8) group was significantly smaller than that of the Isotype group (p<0.01). group (all p<0.01), group tumor volume was significantly smaller than group (p<0.05). The results are shown in Figure 8.

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 the anti-tumor effect is significantly better than And the tumor inhibitory effect increases with increasing dose.

In another independent experiment with the same tumor model, the anti-tumor activity of the test drug was compared with the positive drug M7824 analogue. The groups were divided into 4 groups, with 6 animals in each group, namely: Vehicle group, TAF-6 (7mg/kg, ip, tiw x 9; the final dose was adjusted to 14mg/kg, ip, tiw x 2) group, M7824 analogue ( 6 mg/kg, ip, tiw x 9; later dose adjustment to 12 mg/kg, ip, tiw x 2) group, (5mg/kg, ip, tiw x 9; later dose adjustment to 10mg/kg, ip, tiw x 2) group. The results are shown in Figure 9. The anti-tumor effect of TAF-6 is significantly better than that of M7824 analogues.

Example 10 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized sarcoma HT1080 mice

Human sarcoma HT1080 cells were inoculated subcutaneously into the right front flank of male NCG mice. PBMC cells were inoculated into the mice 7 days before tumor cell inoculation. When the tumors grew to ^about 56 mm, they were administered in groups, for a total of 5 groups. , 8 animals in each group, respectively: Vehicle group, TAF-6 low-dose (2mg/kg, ip, tiw×9) group, TAF-6 medium-dose (7mg/kg, ip, tiw×9) group, TAF- 6 high dose (25mg/kg, ip, tiw×9) group, (5mg/kg,ip,tiw×9) group. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were peeled off, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate TGI _TV (%) was calculated and statistically analyzed.

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

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 middle-dose group, TAF-6 high-dose group and The tumor volume of the TAF-6 medium-dose group and the TAF-6 high-dose group were significantly smaller than those 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 The results are shown in Figure 10.

Example 11 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized HeLa mice

Human cervical cancer HeLa cells were inoculated subcutaneously into the right front flank of female NCG mice. One day after the tumor cells were inoculated, human PBMC cells were inoculated into the mice. When the tumors grew to ^about 52 mm, they were administered in groups. A total of 7 sets of 8 Only, respectively: Vehicle group, TAF-6 low-dose (2mg/kg, ip, tiw×10) group, TAF-6 medium-dose (7mg/kg, ip, tiw×10) group, TAF-6 high-dose ( 25mg/kg,ip,tiw×10) group, (5mg/kg,ip,tiw×10) group, (5mg/kg,ip,tiw×10) group, (5+5mg/kg,ip,tiw×10). Doses are calculated based on equimolar concentrations. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

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

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 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 TAF-6 medium and high-dose groups Tumor volumes were significantly smaller than group (p<0.05;p<0.01), the results are shown in Figure 11.

In summary, the test drug TAF-6 (7mg/kg, 25mg/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 And the tumor inhibitory effect increases with increasing dose.

Example 12 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized colorectal cancer CRC-034PDX model mice

The human colorectal cancer patient tissue xenograft model CRC-034 (PDX, Beijing Yikang Pharmaceutical Technology Co., Ltd.) tumor mass was inoculated subcutaneously in the right anterior flank of male NCG mice. 10 days after tumor cell inoculation, PBMC cells were Inoculated into mice, when the tumors grew to ^about 53mm, they were divided into groups and administered. There were 3 groups, 10 mice in each group, namely: Isotype group, TAF-6 (7mg/kg, ip, tiw) group, M7824 analog (6mg/kg,ip,tiw) group. Doses are calculated based on equimolar concentrations. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

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

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 56%, respectively. 4%. The tumor volume of the TAF-6 group was significantly smaller than that of the Isotype group and the M7824 analog group (both 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.

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

Example 13 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized cholangiocarcinoma HuCCT1 mice

Human hepatobiliary cancer HuCCT1 cells were inoculated subcutaneously into the right front flank of male NCG mice. PBMC cells were inoculated into the mice one day after the tumor cells were inoculated. When the tumors grew to ^about 53 mm, they were administered in groups. A total of 4 groups, 8 animals in each group, respectively: Vehicle group, TAF-6 (2mg/kg, ip, tiw) group, TAF-6 (10mg/kg, ip, tiw) group, M7824 analogue (1.7mg/kg ,ip,tiw) group. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

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

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 (both p<0.01). The results are shown in Figure 13.

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.

Example 14 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized osteosarcoma SJSA mice

Human osteosarcoma SJSA-1 cells were inoculated subcutaneously into the right front flank of male NCG mice. PBMC cells were inoculated into the mice 2 days after the tumor cells were inoculated. When the tumors grew to ^about 42 mm, they were administered in groups. There are 4 groups in total, 8 animals in each group, namely: Vehicle group, TAF-6 (2mg/kg, ip, tiw) group, TAF-6 (7mg/kg, ip, tiw) group, M7824 analogue (6mg/kg ,ip,tiw) group. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

One day before the grouping and at the end of the experiment, blood was collected from the orbital venous plexus of the mice. The FACS test results showed that there were human CD45-positive cells in the peripheral blood of the mice in each group, and the proportion of CD45+ increased over time, indicating that the mouse immune system was humanized. achievement.

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

In summary, TAF-6 at doses of 2 mg/kg and 7 mg/kg has significant anti-tumor effects on the PBMC humanized human osteosarcoma SJSA-1 subcutaneous xenograft tumor model, and TAF-6 at 7 mg/kg has an equimolar dose with Compared with the M7824 analogue (TGI _TV of 45%, p<0.01), the anti-tumor effect was superior.

Example 15 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized gastric cancer NUGC-4/hCLDN18.2 mice

Human gastric cancer NUGC-4/hCLDN18.2 cells and PBMC cells were mixed evenly and then inoculated subcutaneously into the right front flank of male NCG mice. When the tumors grew to ^about 54 mm, they were administered in groups, with a total of 4 groups. 8 animals, namely: Vehicle group, TAF-6 (2mg/kg, ip, tiw) low-dose group, TAF-6 (6mg/kg, ip, tiw) medium-dose group, TAF-6 (20mg/kg, ip , tiw) high-dose group. The tumor volume and body weight were measured twice a week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

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

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, and the tumor volumes in each group were Significantly smaller than the Vehicle group (p<0.001, p<0.001, p<0.001), the tumor volume of the TAF-6 medium-dose group and TAF-6 high-dose group was significantly smaller than the TAF-6 low-dose group (p<0.01, p<0.001 ), the results are shown in Figure 15.

In summary, 2 mg/kg, 6 mg/kg, and 20 mg/kg of the test drug TAF-6 all have significant anti-tumor effects on the human gastric cancer NUGC-4/hCLDN18.2 subcutaneous xenograft tumor model co-inoculated with PBMC, and the tumor The inhibitory effect showed a dose-dependent response trend.

Example 16 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized ovarian cancer OV90 mice

PBMC cells were inoculated subcutaneously into the right front flank of female B-NDG B2m KO plus mice. The next day, human ovarian cancer OV90 cells were inoculated into the mice and administered in groups when the tumors grew to ^about 54 mm. 3 sets of 8 Only, respectively: Vehicle group, TAF-6 (2mg/kg, ip, Q2D) group, TAF-6 (6mg/kg, ip, Q2D) group. The tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. Q2D means dosing once every two days.

One day before grouping and at the end of the experiment, blood was collected from the orbital venous plexus of the mice. The FACS test results showed that there were human CD45-positive cells in the peripheral blood of mice in each group, and the proportion of CD45+ increased over time, indicating that the mouse immune system was humanized. success.

At the end of the experiment (PG-D24), the tumor growth inhibition rates of the TAF-6 (2 mg/kg, i.p., Q2D) group and TAF-6 (6 mg/kg, i.p., 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.

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

The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims

The use of multi-domain fusion proteins in the preparation of drugs for treating or preventing tumors, wherein the fusion proteins include anti-PD-L1 single domain antibody fragments, antagonistic VEGF fragments, and TGF-β binding fragments.
The use according to claim 1, wherein the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes:

The amino acid sequence is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, 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, 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, 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, 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, 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.
The use according to claim 1, wherein the anti-PD-L1 single domain antibody fragment also includes a framework region, and the framework region FR includes FR1 to FR4 whose amino acid sequence is as follows:

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; FR4 shown in SEQ ID NO.56.
The use according to claim 1, wherein the anti-PD-L1 single domain antibody fragment includes:

a) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 16~21; or,

b) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID NO. 16 to 21 and has 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.
The use according to claim 1, wherein the antagonistic VEGF fragment is bevacizumab. Preferably, the antagonistic VEGF fragment includes:

c) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 22~23; or,

d) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID NO. 22 to 23 and has the function of the polypeptide fragment defined in c);

And/or, the antagonistic VEGF fragment is derived from mice;

And/or, the antagonistic VEGF fragment is humanized.
The use according to claim 1, wherein 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 whose amino acid sequence has more than 90% sequence identity with SEQ ID No. 24 and has the function of the polypeptide fragment defined by e);

And/or, the TGF-β binding fragment is derived from human.
The use according to claim 1, characterized in that the fusion protein also includes a connecting peptide fragment. Preferably, the connecting peptide fragment is rich in G, S and/or A, and more preferably, the connecting peptide is selected from A flexible polypeptide chain composed of free G glycine and/or S serine and/or A alanine, and the length of the connecting peptide is 3 to 30 amino acids.
The use according to claim 7, wherein the connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 34-36;

And/or, there is a connecting peptide between the anti-PD-L1 single domain antibody fragment and the antagonistic VEGF fragment;

And/or, there is a connecting peptide between the antagonistic VEGF fragment and the TGF-β binding fragment.
The use according to claim 1, wherein the fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment in order 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 that antagonizes the VEGF fragment;

And/or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain that antagonizes the VEGF fragment;

And/or, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the antagonistic VEGF fragment.
The use according to claim 1, characterized in that the amino acid sequence of the fusion protein includes the sequence shown in one of SEQ ID NO. 23 and 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 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, SEQ ID NO. .30 and the sequence shown in 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, SEQ ID NO.32 and the sequence shown in SEQ ID NO.23, SEQ ID NO.33 and the sequence shown in SEQ ID NO.23.
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 carcinoma, nasopharyngeal cancer, cervical cancer, cholangiocarcinoma and hematological malignancies.
A method for treating or preventing tumors, comprising administering to a patient a therapeutically effective amount of a multi-domain fusion protein, the fusion protein including an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment.
The method according to claim 12, wherein the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes:

The amino acid sequence is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, 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, 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, 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, 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, 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.
The method according to claim 12, wherein the anti-PD-L1 single domain antibody fragment further includes a framework region, and the framework region FR includes FR1 to FR4 with the following amino acid sequence:

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, SEQ ID FR3 shown in 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; FR4 shown in SEQ ID NO.56.
The method of claim 12, wherein the anti-PD-L1 single domain antibody fragment includes:

a) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 16~21; or,

b) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID NO. 16 to 21 and has the function of a) defined polypeptide fragment;

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.
The method according to claim 12, wherein the antagonistic VEGF fragment is bevacizumab. Preferably, the antagonistic VEGF fragment includes:

c) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 22~23; or,

d) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID NO. 22 to 23 and has the function of the polypeptide fragment defined in c);

And/or, the antagonistic VEGF fragment is derived from mice;

And/or, the antagonistic VEGF fragment is humanized.
The method according to claim 12, wherein 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 whose amino acid sequence has more than 90% sequence identity with SEQ ID No. 24 and has the function of the polypeptide fragment defined by e);

And/or, the TGF-β binding fragment is derived from human.
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, and more preferably, the connecting peptide is selected from A flexible polypeptide chain composed of free G glycine and/or S serine and/or A alanine, and the length of the connecting peptide is 3 to 30 amino acids.
The method according to claim 18, wherein the connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 34-36;

And/or, there is a connecting peptide between the anti-PD-L1 single domain antibody fragment and the antagonistic VEGF fragment;

And/or, there is a connecting peptide between the antagonistic VEGF fragment and the TGF-β binding fragment.
The method according to claim 12, wherein the fusion protein sequentially includes anti- PD-L1 single domain antibody fragment, antagonistic VEGF fragment, TGF-β binding fragment;

And/or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the heavy chain that antagonizes the VEGF fragment;

And/or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain that antagonizes the VEGF fragment;

And/or, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the antagonistic VEGF fragment.
The method according to claim 12, characterized in that the amino acid sequence of the fusion protein includes the sequence shown in one of SEQ ID NO. 23 and 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, SEQ ID NO.25 and SEQ ID The sequence shown in 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, SEQ ID NO.30 and SEQ ID NO. The sequence shown in 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, the sequence shown in SEQ ID NO.33 and SEQ ID NO.23 sequence shown.
The method according to any one of claims 12-21, wherein 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, cholangiocarcinoma and hematological malignancies.
A multi-domain fusion protein for treating or preventing tumors, wherein the fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment.
The fusion protein for use according to claim 23, wherein the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes:

The amino acid sequence is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, 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, 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, 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, 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, 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, SEQ ID CDR3 shown in NO.15.
The fusion protein for use according to claim 23, wherein the anti-PD-L1 single domain antibody fragment further includes a framework region, and the framework region FR includes FR1 to FR4 whose amino acid sequence is as follows:

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; FR4 shown in SEQ ID NO.56.
The fusion protein for use according to claim 23, wherein the anti-PD-L1 single domain antibody fragment includes:

a) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 16~21; or,

b) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID NO. 16 to 21 and has the function of a) defined polypeptide fragment;

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.
The fusion protein according to claim 23, wherein the antagonistic VEGF fragment is bevacizumab. Preferably, the antagonistic VEGF fragment includes:

c) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 22~23; or,

d) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID NO. 22 to 23 and has the function of the polypeptide fragment defined in c);

And/or, the antagonistic VEGF fragment is derived from mice;

And/or, the antagonistic VEGF fragment is humanized.
The fusion protein according to claim 23, wherein 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 whose amino acid sequence has more than 90% sequence identity with SEQ ID No. 24 and has the function of the polypeptide fragment defined in e);

And/or, the TGF-β binding fragment is derived from human.
The fusion protein for use according to claim 23, wherein the fusion protein further includes a connecting peptide fragment, preferably, the connecting peptide fragment is rich in G, S and/or A, and more preferably, the connecting peptide fragment is rich in G, S and/or A. The connecting peptide is selected from a flexible polypeptide chain composed of Gglycine and/or Sserine and/or Aalanine, and the length of the connecting peptide is 3 to 30 amino acids.
The fusion protein for use according to claim 29, wherein the connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NO. 34-36;

And/or, there is a connecting peptide between the anti-PD-L1 single domain antibody fragment and the antagonistic VEGF fragment;

And/or, there is a connecting peptide between the antagonistic VEGF fragment and the TGF-β binding fragment.
The fusion protein for use according to claim 23, wherein the fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF-β binding fragment in order 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 that antagonizes the VEGF fragment;

And/or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain that antagonizes the VEGF fragment;

And/or, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the antagonistic VEGF fragment.
The fusion protein for use according to claim 23, wherein the amino acid sequence of the fusion protein includes the sequence shown in one of SEQ ID NO. 23 and 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, SEQ ID NO.25 and SEQ ID The sequence shown in 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, SEQ ID NO.30 and SEQ ID NO. The sequence shown in 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, the sequence shown in SEQ ID NO.33 and SEQ ID NO.23 sequence shown.
Use of an isolated polynucleotide or a construct containing the polynucleotide encoding the fusion protein of any one of claims 23-32 in the preparation of a medicament for treating or preventing tumors .
The use of an expression system containing the construct of claim 33 or the exogenous isolated polynucleoside of claim 33 integrated into its genome in the preparation of a drug for treating or preventing tumors. acid.
The use of a pharmaceutical composition in the preparation of drugs for the treatment or prevention of tumors, characterized in that the pharmaceutical composition includes the fusion protein of any one of claims 23-32, or the expression of claim 34 Systematic cultures.
The fusion protein for use according to any one of claims 23 to 32, wherein the tumor is selected from the group consisting of lung cancer, melanoma, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, and breast cancer. , sarcoma, head and neck cancer, nasopharyngeal cancer, cervical cancer, cholangiocarcinoma and hematological malignancies.