Multi-domain fusion protein and its application

Abstract

The present invention provides a fusion protein, which comprises an anti-PD-L1 single domain antibody fragment, a VEGF antagonist fragment, and a TGF-β binding fragment. The multi-domain fusion protein with anti-cancer activity provided by the present invention can organically combine the functions of an anti-PD-L1 monoclonal antibody that blocks the interaction of PD-L1 / PD-1, an anti-VEGF monoclonal antibody that reduces the growth of capillaries and inhibits metastatic disease, and a TGF-β receptor that releases the resistance of cancer cells to TGF-β signals and enhances immune responses in a single antibody fusion protein molecule, and can be used to treat tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a multi-domain fusion protein having anti-cancer activity, a method for producing the same, and its use.

Background Art

[0002] One of the differences between tumor cells and normal cells lies in their high metabolic requirements during growth. Tumor cells are supplied with nutrients and oxygen from blood vessels, process metabolic products, and promote the formation of new blood vessels on existing blood vessels. Among the angiogenesis-promoting factors secreted from tumors, human vascular endothelial growth factor (VEGF), especially VEGF-A, is an important factor that causes tumor angiogenesis (Non-Patent Document 1). Therefore, by inhibiting the VEGF signaling pathway, the progression of many tumors can be restricted. For example, Bevacizumab (trade name Avastin (registered trademark)) is a humanized anti-VEGF monoclonal antibody that can block the interaction between VEGF and VEGF receptors (Flt-1 and KDR) on the surface of endothelial cells by binding to VEGF. Currently, the application of the Bevacizumab monoclonal antibody to the treatment of metastatic colorectal cancer, advanced, metastatic or recurrent non-small cell lung cancer, recurrent glioblastoma, etc. has been approved by the FDA.

[0003] Transforming growth factor TGF-β is a type of cytokine that can maintain tissue homeostasis by regulating cell growth, differentiation, proliferation, and survival. In the early stage of tumors, the TGF-β pathway can control tumors by promoting cell cycle arrest and apoptosis. However, at the late stage of tumors, TGF-β can promote the growth, invasion, and metastasis of cancer cells by inhibiting cytotoxic T cells, and ultimately escape from tumors. Such a functional conversion is called the "TGF-β paradox". The TGFβ signaling pathway is T HInducing the differentiation from cells to Tregs, attenuating the activation of CD8+ effector T cells, restricting the development of central memory T cells, and fundamentally affecting the functions of tumor-infiltrating T cells. In mammals, TGF-β mainly exists in three subtypes: TGF-β1, TGF-β2, and TGF-β3. When TGF-β is highly expressed, tumors can escape from immune surveillance. Consistently, the progression and poor prognosis of tumors are associated with non-small cell lung cancer (NSCLC), colorectal cancer (CRC), gastric cancer, and prostate cancer with high expression of TGF-β (Non-Patent Document 2). This indicates that antagonism against TGF-β is a new potential direction in tumor treatment.

[0004] Currently, the anti-PD-1 / PD-L1 monoclonal antibody, an immune checkpoint inhibitor, is widely applied in tumor treatment. In addition, M7824, which is a fusion of the anti-PD-L1 monoclonal antibody avelumab and TGF-β receptor II (TGF-β Trap), has advanced to the clinical stage. However, there is no report on the combination of anti-PD-1 / PD-L1 monoclonal antibody, TGF-β antagonist, and anti-VEGF monoclonal antibody so far.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a multi-domain fusion protein having anti-cancer activity, a method for producing the same, and its use in order to solve the problems of the prior art.

Means for Solving the Problems

[0007] In order to achieve the above object and other related objects, the present invention provides, in one aspect, a fusion protein. The fusion protein includes an anti-PD-L1 single-domain antibody fragment, a VEGF antagonist fragment, and a TGF-β binding fragment.

[0008] In another aspect, the present invention provides a separated polynucleotide encoding the above fusion protein.

[0009] In another aspect, the present invention provides a construct. The construct includes the above separated polynucleotide.

[0010] In another aspect, the present invention provides an expression system. The expression system includes the above construct, or the above polynucleotide with a foreign gene integrated into the genome.

[0011] In another aspect, the present invention provides a method for producing the above fusion protein. The method includes culturing the above expression system under appropriate conditions, expressing the fusion protein in the expression system, and separating and purifying the fusion protein to provide the fusion protein.

[0012] In another aspect, the present invention provides the use of the above fusion protein or a culture of the above expression system in the manufacture of a drug.

[0013] In another aspect, the present invention provides a drug composition including the above fusion protein or a culture of the above expression system.

Brief Description of the Drawings

[0014]

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Modes for Carrying Out the Invention

[0015] Through extensive exploration and research, the inventor of the present invention unexpectedly discovered a kind of fusion protein molecule. The said fusion protein molecule can combine the function of an anti-PD-L1 monoclonal antibody that blocks the interaction of PD-L1 / PD-1, the function of a VEGF antagonist monoclonal antibody that reduces the growth of capillaries and suppresses metastatic diseases, and the function of a TGF-β receptor that improves the functional abnormality of T cells caused by TGF-β in the tumor microenvironment and enhances the immune response, and has an excellent tumor suppression effect. Based on this, the present invention was completed.

[0016] In a first aspect, the present invention provides a fusion protein. The said fusion protein contains an anti-PD-L1 single-domain antibody fragment, a VEGF antagonist fragment, and a TGF-β binding fragment. In the above-mentioned fusion protein, the anti-PD-L1 single-domain antibody fragment can usually be used to suppress tumor growth by blocking the interaction of PD-L1 / PD-1 and improving the expression of IFN-γ and / or IL-2 in T cells. Also, the VEGF antagonist fragment may usually contain an Fc portion capable of binding to the FcRn receptor. Thereby, it can extend the in vivo half-life and also exhibit the effect of killing cancer cells by binding to effector cells expressing the Fc receptor. Further, the TGF-β binding fragment can improve the tumor cell killing function of tumor-infiltrating T cells by removing TGF-β overexpressed in the tumor microenvironment.

[0017] The fusion protein provided by the present invention may contain an anti-PD-L1 single-domain antibody fragment. Usually, the above-mentioned anti-PD-L1 single-domain antibody fragment can be a polypeptide or protein fragment that can specifically bind to PD-L1. Usually, the anti-PD-L1 single-domain antibody fragment lacks the corresponding antibody light chain and has only a fragment corresponding to the heavy chain variable region. The binding characteristics of the anti-PD-L1 single-domain antibody fragment can usually be determined by three complementarity determining regions (CDRs) contained in the fragment. The CDR regions can be arranged in an orderly manner with the framework regions (FRs). The FR regions are not directly involved in the binding reaction. These CDRs can form a cyclic structure. The CDRs are close to each other within the spatial structure through the β-sheets formed by the FRs between them and constitute the antigen-binding site of the antibody. For example, the complementarity determining regions (CDRs) of the above-mentioned anti-PD-L1 single-domain antibody fragment may include CDR1 whose amino acid sequence is shown by any one of SEQ ID NOs. 1 to 5, CDR2 whose amino acid sequence is shown by any one of SEQ ID NOs. 6 to 9, and CDR3 whose amino acid sequence is shown by any one of SEQ ID NOs. 10 to 15.

[0018] In a specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include CDR1 whose amino acid sequence is shown by SEQ ID NO. 1, CDR2 whose amino acid sequence is shown by SEQ ID NO. 6, and CDR3 whose amino acid sequence is shown by SEQ ID NO. 10.

[0019] In another specific embodiment of the present invention, the complementarity determining regions of the anti-PD-L1 single-domain antibody fragment include CDR1 whose amino acid sequence is shown by SEQ ID NO. 2, CDR2

[0020] shown by SEQ ID NO. 7, and CDR3 shown by SEQ ID NO. 11. The complementarity-determining regions include CDR1 with the amino acid sequence shown in SEQ ID NO.3, SEQ CDR2 with the amino acid sequence shown in SEQ ID NO.7, and CDR3 with the amino acid sequence shown in SEQ ID NO.12.

[0021] In another specific embodiment of the present invention, the complementarity-determining regions of the anti-PD-L1 single-domain antibody fragment include CDR1 with the amino acid sequence shown in SEQ ID NO.4, SEQ CDR2 with the amino acid sequence shown in SEQ ID NO.8, and CDR3 with the amino acid sequence shown in SEQ ID NO.13.

[0022] In another specific embodiment of the present invention, the complementarity-determining regions of the anti-PD-L1 single-domain antibody fragment include CDR1 with the amino acid sequence shown in SEQ ID NO.2, SEQ CDR2 with the amino acid sequence shown in SEQ ID NO.7, and CDR3 with the amino acid sequence shown in SEQ ID NO.14.

[0023] In another specific embodiment of the present invention, the complementarity-determining regions of the anti-PD-L1 single-domain antibody fragment include CDR1 with the amino acid sequence shown in SEQ ID NO.5, SEQ CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.15.

[0024] The above anti-PD-L1 single-domain antibody fragment may further include a framework region (FR). As described above, the CDR regions can be arranged in an orderly manner with the FR regions. For example, the anti-PD-L1 single-domain antibody fragment may sequentially include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The framework region FR includes FR1 with the amino acid sequence shown in SEQ ID NO.49, FR2 with the amino acid sequence shown in any one of SEQ ID NOs.50 - 52, FR3 with the amino acid sequence shown in any one of SEQ ID NOs.53 - 55, and FR4 with the amino acid sequence shown in SEQ ID NO.56.

[0025] In a specific embodiment of the present invention, the framework region FR includes FR1 with an amino acid sequence shown by SEQ ID NO.49, FR2 with an amino acid sequence shown by SEQ ID NO.50, FR3 with an amino acid sequence shown by SEQ ID NO.53, FR4 with an amino acid sequence shown by SEQ ID NO.56, or FR1 with an amino acid sequence shown by SEQ ID NO.49, FR2 with an amino acid sequence shown by SEQ ID NO.51, FR3 with an amino acid sequence shown by SEQ ID NO.54, FR4 with an amino acid sequence shown by SEQ ID NO.56, or FR1 with an amino acid sequence shown by SEQ ID NO.49, FR2 with an amino acid sequence shown by SEQ ID NO.52, FR3 with an amino acid sequence shown by SEQ ID NO.54, FR4 with an amino acid sequence shown by SEQ ID NO.56, or FR1 with an amino acid sequence shown by SEQ ID NO.49, FR2 with an amino acid sequence shown by SEQ ID NO.52, FR3 with an amino acid sequence shown by SEQ ID NO.55, FR4 with an amino acid sequence shown by SEQ ID NO.56.

[0026] In another specific embodiment of the present invention, the anti-PD-L1 single domain antibody fragment may include: a) a polypeptide fragment with an amino acid sequence shown by any one of SEQ ID NOs. 16 to 21, or b) a polypeptide fragment having a sequence identity of 80% or more with any one of the amino acid sequences shown by SEQ ID NOs. 16 to 21 and having the function of the polypeptide fragment defined in a). Specifically, the polypeptide fragment of b) may be obtained by substitution, deletion or addition of one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5 or 1 to 3) amino acids in the amino acid sequence shown by any one of SEQ ID NOs. 16 to 21, or may have one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5 or 1 to 3) amino acids added or deleted at the N-terminus and / or C-terminus It is obtained by adding the amino acids of ( ). Moreover, a polypeptide fragment having the function of a polypeptide fragment whose amino acid sequence is represented by any one of SEQ ID NOs. 16 to 21 may, for example, have the ability to specifically bind to PD-L1, may be able to block the PD-L1 / PD-1 pathway by blocking the interaction between PD-L1 and PD-1, may have the function of improving the expression of IFN-γ and / or IL-2 in T cells, or may have the function of suppressing tumor growth. The amino acid sequence of the anti-PD-L1 single-domain antibody fragment in b) above may have 80%, 85%, 90%, 93%, 95%, 97% or 99% or more identity with any one of SEQ ID NOs. 16 to 21. Usually, the above anti-PD-L1 single-domain antibody fragment is available from alpaca (Vicugna pacos), and for example, the CDR region may be obtained from alpaca. Also, usually, the above anti-PD-L1 single-domain antibody fragment may be humanized, and for example, the framework region may be obtained from humans.

[0027] In this text, sequence identity means the percentage of identical residues in the sequences to be compared. Using computational software well-known in the art, sequence identity can be calculated for two or more target sequences. These software can be obtained, for example, from NCBI.

[0028] The fusion protein provided by the present invention may contain a VEGF antagonist fragment. Usually, the above VEGF antagonist fragment can be a polypeptide or protein fragment capable of antagonizing VEGF. For example, the above VEGF antagonist fragment may be a monoclonal antibody or the like. Also, for example, the above VEGF antagonist fragment may be bevacizumab or the like.

[0029] In a specific embodiment of the present invention, the VEGF antagonist fragment may include: c) a polypeptide fragment having an amino acid sequence shown in any of SEQ ID NOs. 22 to 23; d) a polypeptide fragment having an amino acid sequence having 80% or more sequence identity with any of SEQ ID NOs. 22 to 23 and having the function of the polypeptide fragment defined in c). Specifically, the amino acid sequence in d) above means that the amino acid sequence shown in any of SEQ ID NOs. 22 to 23 is obtained through substitution, deletion or addition of one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3 amino acids), or is obtained by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3 amino acids) amino acids to the N-terminus and / or C-terminus. And the polypeptide fragment having the function of the polypeptide fragment having an amino acid sequence shown in any of SEQ ID NOs. 22 to 23 may, for example, have a function of antagonizing VEGF or may have a function of an Fc portion capable of binding to the FcRn receptor. Thereby, it is possible to extend the in vivo half-life and also to exhibit an action of killing cancer cells by binding to effector cells expressing the Fc receptor. The amino acid sequence in d) above may have 80%, 85%, 90%, 93%, 95%, 97% or 99% or more identity with any of SEQ ID NOs. 22 to 23. Usually, the above VEGF antagonist fragment is available from Mus musculus. For example, the CDR region may be obtained from a mouse. Also, usually, the above VEGF antagonist fragment may be humanized. For example, the framework region may be obtained from a human.

[0030] The fusion protein provided by the present invention may include a TGF-β binding fragment. Usually, the above TGF-β binding fragment can specifically bind to each isomer of TGF-β (for example, TGF-β1, TGF-β2, TGF-β3, etc.). Usually, the isomers of TGF-β It is highly likely to be highly expressed in various types of malignant tumors and become one of the important factors that deteriorate the clinical treatment effect. For example, the TGF-β binding fragment may be a fragment of the extracellular region structure of TGF-βRII (transforming growth factor-β receptor II).

[0031] In a specific embodiment of the present invention, the TGF-β binding fragment may include: e) a polypeptide fragment having an amino acid sequence shown in SEQ ID NO.24; f) a polypeptide fragment having an amino acid sequence with 80% or more sequence identity to SEQ ID NO.24 and having the function of the polypeptide fragment defined in e). Specifically, the amino acid sequence in f) means that the amino acid sequence shown in SEQ ID NO.24 is obtained through substitution, deletion or addition of one or more (specifically, it can be 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) amino acids, or is obtained by adding one or more (specifically, it can be 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) amino acids to the N-terminus and / or C-terminus. And the polypeptide fragment having the function of the polypeptide fragment with the amino acid sequence shown in SEQ ID NO.24 can bind to each isomer of TGF-β (such as TGF-β1, TGF-β2 and TGF-β3, etc.), for example. Thereby, it becomes possible to remove the TGF-β overexpressed in the tumor microenvironment and also possible to improve the killing function of tumor cells by tumor-infiltrating T cells. The amino acid sequence in f) may have 80%, 85%, 90%, 93%, 95%, 97% or 99% or more identity to SEQ ID NO.24. Usually, the above-mentioned TGF-β binding fragment is available from Homo sapiens.

[0032] The fusion protein provided by the present invention may further contain a linker peptide fragment. Usually, the fusion protein can contain a plurality of linker peptide fragments, and linker peptide fragments may be provided between at least some domains or between each domain. For example, a linker peptide may be provided between an anti-PD-L1 single domain antibody fragment and a VEGF antagonist fragment. Also, for example, a linker peptide may be provided between a VEGF antagonist fragment and a TGF-β binding fragment. Usually, the above linker peptide fragment can be a flexible polypeptide of an appropriate length rich in G, S and / or A (mainly composed of glycine (G), serine (S) and / or alanine (A)). Thereby, the domains of adjacent proteins can be made relatively freely movable with respect to each other. For example, the amino acid sequence of the linker peptide fragment may include sequences such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG)nA, etc. Here, n is selected from integers of 1 to 10 of In a specific embodiment of the present invention, the length of the amino acid sequence of the linker peptide fragment may be 3 to 30, 3 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 12, 12 to 14, 14 to 16, 16 to 18, 18 to 20, 20 to 22, 22 to 24, 24 to 26, 26 to 28 or 28 to 30. In a more preferred specific embodiment of the present invention, the linker peptide fragment may contain a polypeptide fragment whose amino acid sequence is represented by any one of SEQ ID NOs. 34 to 36.

[0033] In the fusion protein provided by the present invention, the fusion protein may be linear. For example, the fusion protein may sequentially contain an anti-PD-L1 single domain antibody fragment, a VEGF antagonist fragment, and a TGF-β binding fragment from the N-terminus to the C-terminus. Further, the fusion protein may have a structure similar to that of a monoclonal antibody. For example, the anti-PD-L1 single domain antibody fragment may be located at the N-terminus of the heavy chain of the VEGF antagonist fragment. Also, for example, the anti-PD-L1 single domain antibody fragment may be located at the N-terminus of the light chain of the VEGF antagonist fragment. Also, for example, the TGF-β binding fragment may be located at the C-terminus of the heavy chain of the VEGF antagonist fragment and may also be good. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein may include a sequence represented by any one of SEQ ID NO.23, SEQ ID NO.25 to SEQ ID NO.33. For example, the amino acid sequence of the fusion protein may include the sequences represented by SEQ ID NO.25 and SEQ ID NO.26, the sequences represented by SEQ ID NO.25 and SEQ ID NO.27, the sequences represented by SEQ ID NO.25 and SEQ ID NO.28, the sequences represented by SEQ ID NO.25 and SEQ ID NO.29, the sequences represented by SEQ ID NO.30 and SEQ ID NO.27, the sequences represented by SEQ ID NO.30 and SEQ ID NO.29, the sequences represented by SEQ ID NO.31 and SEQ ID NO.23, the sequences represented by SEQ ID NO.32 and SEQ ID NO.23, and the sequences represented by SEQ ID NO.33 and SEQ ID NO.23.

[0034] In a second aspect, the present invention provides a separated polynucleotide encoding the fusion protein provided in the first aspect of the present invention. The above polynucleotide can be RNA, DNA, cDNA, etc. The method for providing the separated polynucleotide should be known to those skilled in the art. For example, it may be prepared and obtained by methods such as automated DNA synthesis and / or recombinant DNA technology, or may be separated from an appropriate natural source.

[0035] In a third aspect, the present invention provides a structure. The above structure contains the isolated polynucleotide provided in the second aspect of the present invention. The method for appropriately producing the above structure should be known to those skilled in the art. For example, the structure can be obtained by producing it by methods such as in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. More specifically, it can be produced by inserting the above isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector in the present invention usually refers to various commercially available expression vectors well-known in the art, such as, for example, bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses (e.g., adenoviruses, retroviruses), or other vectors. Usually, an appropriate vector may contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers. For example, these promoters include the lac or trp promoter of Escherichia coli, the PL promoter of phage λ, eukaryotic promoters (including the CMV major immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris), and other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses, but are not limited thereto. The marker gene can be used to confer phenotypes and traits for selecting transformed host cells. For example, it may include, but is not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance used for Escherichia coli. When expressing the above polynucleotide, the expression vector may contain an enhancer sequence. Inserting an enhancer sequence into the vector can enhance transcription. An enhancer is a cis element of DNA and usually has about 10 to 300 base pairs. The enhancer acts on the promoter to enhance the transcription of the gene.

[0036] In a fourth aspect, the present invention provides an expression system. To be able to express the above fusion protein, the expression system includes the structure provided in the third aspect of the present invention, or the polynucleotide provided in the second aspect of the present invention in which a foreign gene is integrated into the genome. The above expression system may also be a host cell. Any cell applicable to the expression of an expression vector can be 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 or a filamentous fungal cell, or a higher eukaryotic cell such as a mammalian cell. Representative examples include bacterial cells of Escherichia coli, Streptomyces genus, Salmonella typhimurium, for example, fungal cells such as yeast, filamentous fungi, plant cells such as insect cells of Drosophila S2 or Sf9, animal cells such as CHO, COS, 293 cells, or Bowes malignant melanoma cells. The method for introducing the structure into the host cell should be known to those skilled in the art, and for example, methods such as microinjection method, particle gun method, electroporation method, virus-mediated transformation method, electron shock method, calcium phosphate coprecipitation method, etc. can be used.

[0037] In a fifth aspect, the present invention provides a method for producing the fusion protein provided in the first aspect of the present invention. Those skilled in the art can select an appropriate method to produce the above fusion protein. For example, the production method may include the following. That is, the expression system provided in the fourth aspect of the present invention is cultured under appropriate conditions, the fusion protein is expressed in the expression system, and a culture containing the fusion protein is collected. Then, the fusion protein is provided by separation and purification.

[0038] In a sixth aspect, the present invention provides the use of the fusion protein provided in the first aspect of the present invention and the culture of the expression system provided in the fourth aspect of the present invention in the manufacture of a drug. The above drug can be a drug used for the treatment of tumors. The tumor can be, for example, cancer or a solid tumor, specifically, lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, hematological malignancy, sarcoma, head and neck cancer, nasopharyngeal cancer, etc. Further, these cancers can be in the early, middle or late stage, and can be, for example, metastatic cancers.

[0039] In a seventh aspect, the present invention provides a drug composition comprising the fusion protein provided in the first aspect of the present invention or the culture of the expression system provided in the fourth aspect of the present invention. Usually, the content of the fusion protein or the culture in the above drug composition is a therapeutically effective amount. In the present invention, usually, the "therapeutically effective amount" refers to an amount that can significantly reduce the symptoms of a disease, increase the frequency and duration of the disease-free period, or prevent damage or loss of function caused by the pain of the disease after an appropriate administration period. The ability to suppress tumor growth can be evaluated in an animal model system for predicting the therapeutic effect on human tumors. Alternatively, it may be evaluated by examining the ability to suppress cell growth. Such suppression can be measured in vitro through experiments known to those skilled in the art. With a therapeutically effective amount of the fusion protein and the drug composition, usually, it is possible to reduce the size of the tumor. Alternatively, the symptoms of the subject can be alleviated in other ways. Those skilled in the art may select an appropriate therapeutically effective amount according to the actual situation, such as the size of the subject, the severity of the subject's symptoms, and the specific composition or administration route selected. The treatment prescription (for example, determination of the dosage, etc.) may be determined by a doctor. Also, the factors usually considered include, but are not limited to, the disease to be treated, the individual situation of the patient, the delivery site, the application method, and other factors.

[0040] The pharmaceutical composition provided by the present invention may further contain a pharmaceutically acceptable vector. The above-mentioned vector may contain various excipients and diluents, but these vectors themselves are not essential active ingredients and do not have excessive toxicity after administration. Appropriate vectors should be well-known to those skilled in the art. For example, sufficient discussion on pharmaceutically acceptable vectors can be obtained from Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).

[0041] In an eighth aspect, the present invention provides a treatment method comprising administering to an individual a therapeutically effective amount of the fusion protein provided in the first aspect of the present invention, a culture of the expression system provided in the fourth aspect of the present invention, or the pharmaceutical composition provided in the seventh aspect of the present invention.

[0042] In the present invention, "treatment" includes preventive, curative or palliative operations that can bring about desirable pharmaceutical and / or physiological effects. The therapeutic effect preferably means that one or more symptoms of the disease can be medically reduced, or the disease can be completely removed, or the occurrence of the disease can be arrested or delayed, and / or the risk of progression or exacerbation of the disease can be reduced.

[0043] In the present invention, "individual" usually includes humans, non-human primates or other mammals (for example, dogs, cats, horses, sheep, pigs, cows, etc.). An "individual" can benefit from treatment using the above-mentioned preparation, reagent kit or formulation.

[0044] In the present invention, the above-mentioned fusion protein, culture of the expression system or pharmaceutical composition may be used as a single active ingredient, or may be combined with other drugs for administration in combination therapy. For example, the above-mentioned multi-domain fusion protein having anti-cancer activity, culture of the expression system or pharmaceutical composition may be combined with at least one other anti-tumor drug. Further, for example, the above-mentioned multi-domain fusion protein having anti-cancer activity, culture of the expression system or pharmaceutical composition may be used in combination with an antibody targeting other tumor-specific antigens.

[0045] The multi-domain fusion protein having anti-cancer activity provided by the present invention can organically combine the function of an anti-PD-L1 monoclonal antibody that blocks the interaction of PD-L1 / PD-1, the function of an anti-VEGF monoclonal antibody that reduces the growth of capillaries and suppresses metastatic diseases, and the function of a TGF-β receptor that relieves the resistance of cancer cells to the TGF-β signal and enhances the immune response in one antibody fusion protein molecule. Therefore, it can be used for the treatment of tumors and has good prospects for industrialization.

[0046] Hereinafter, embodiments of the present invention will be described through specific 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. Furthermore, the present invention can also be implemented or applied by other different specific embodiments. In addition, each detail of this specification can be supplemented or modified in various ways on the premise of not departing from the spirit of the present invention based on different viewpoints and applications.

[0047] 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 specific specific implementation plans described below. Further, it should be further understood that the terms used in the examples of the present invention are for describing specific specific implementation plans and do not limit the protection scope of the present invention.

[0048] When numerical ranges are shown in the examples, unless otherwise specified in the present invention, it should be construed that any of the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Also, unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. Furthermore, in addition to the specific methods, devices, and materials used in the examples, based on the understanding of the prior art by those skilled in the art and the description of the present invention, the present invention may be realized using any methods, devices, and materials in the prior art that are similar or equivalent to the methods, devices, and materials described in the examples of the present invention.

[0049] Unless otherwise explained, all the experimental methods, detection methods, and production methods disclosed in the present invention use common techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are fully described in the prior literature. Specifically, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001, Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates, the series METHODS IN ENZYMOLOGY, Academic Press, San Diego, Wolffe, CHROMATIN For reference, see documents such as 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.

Example

[0050] Example 1 Construction, Recombination, Expression, and Production of Fusion Protein Based on the codon bias of CHO cells, the amino acid sequences of the multi-domain fusion proteins in Table 1, the PD-L1-Fc fusion protein (SEQ ID NO.47), and the Fc-TGFβRII fusion protein (SEQ ID NO.48) were each converted into nucleotide sequences. Then, a HindIII enzyme cleavage site and a Kozak sequence (GCCACC) were introduced at the 5' end of the coding sequences of the heavy and light chains, respectively, and a stop codon and an EcoRI enzyme cleavage site were introduced at the 3' end to obtain the full-length DNA by gene synthesis (General Biosystems (Anhui) Co., Ltd.). Next, the synthesized heavy and light chain coding genes were each subjected to double digestion with HindIII-HF (purchased from NEB. R3104V) and EcoRI-HF (purchased from NEB. R3101V), and were cut and recovered from the gel using an agarose gel DNA / PCR product small amount recovery reagent kit (purchased from Biomiga). Then, using T4 ligase (purchased from NEB. M0202V), it was ligated to the pCDNA3.1(+) vector that had been similarly subjected to double digestion with HindIII and EcoRI, transformed into the Top10 competent state, and then spread on an LB ampicillin-resistant plate and cultured. Subsequently, clones were selected and identified, the sequences were confirmed, and expression plasmids for the heavy and light chains based on pCDNA3.1(+) were constructed respectively. Next, using an endotoxin-free plasmid maxi kit (purchased from Biomiga. BW-PD3511-02), the heavy and light chain expression plasmids were each extracted and mixed in a 1:1 ratio. Then, 1.0 mg of the mixed plasmid was taken and diluted to 25 mL using Wayne293 expression medium (purchased from Zhongshan Kangsheng. A21501). Subsequently, 3.0 mg of PEI (linear, 25KD, Polyscience) was taken, diluted to 25 ml using Wayne293 expression medium, added to the plasmid solution, mixed uniformly, and then cultured at room temperature for 30 minutes. Also, Hek293F cells in the logarithmic growth phase (viability > 95%) were taken and counted. This was centrifuged at 1100 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended using 450 mL of Wayne293 expression medium.Then, the above plasmid-PEI mixture was added to the cell suspension, cultured in a shaking incubator at 37°C and 5% CO2 for 7 days, then centrifuged to collect the supernatant, which was used for subsequent protein purification.

[0051]

Table 1

Example

[0052] Example 2 Purification of the multi-domain fusion protein 2.1 When the anti-PD-L1 single-domain antibody is located at the N-terminus of the heavy chain of the anti-VEGF monoclonal antibody After adjusting the cell fermentation supernatant to pH 7.0, the sample was loaded onto a Protein A affinity chromatography column (Bogelong Biotechnology Co., Ltd.), and the equilibration solution was 20 mM PB, 0.15 M NaCl (pH = 7.0), and eluted with 100% 0.1 M Gly-HCl (pH = 3.0). 10% 1 M Tris-HCl (pH = 8.5) was added to the eluate in advance. Next, the 100% eluate was diluted to a conductivity < 3 ms / cm, the supernatant was adjusted to pH 7.0, and then the sample was loaded onto a DSP chromatography column (Bogelong Biotechnology Co., Ltd.) and eluted with 15% and 100% respectively (20 mM PB, 0.5 M NaCl, pH 7.0). As a result, the 15% elution component, which is the target protein, was obtained. Also, the protein concentration was measured by the UV280 method.

[0053] 2.2 When the anti-PD-L1 single-domain antibody is located at the N-terminus of the light chain of the anti-VEGF monoclonal antibody After adjusting the cell fermentation supernatant to pH 7.0, the sample was loaded onto a Protein A affinity chromatography column (Bogelong Biotechnology Co., Ltd.), and the equilibration solution was 20 mM Using PB, 0.15 M NaCl (pH = 7.0), it was eluted with 100% 0.1 M Gly-HCl (pH = 3.0). 10% 1 M Tris-HCl (pH = 8.5) was added to the eluate in advance. Next, the 100% eluate was diluted to a conductivity of 4 ms / cm, and the sample was loaded onto a Super Q (Tosoh Corporation) chromatography column. The equilibration solution was 20 mM Tris, pH 8, and the elution solution was 500 mM NaCl + 20 mM Tris (pH = 8.0), and elution was performed at 35% and 100% respectively. Then, the extra light chain was removed by flow-through, and the 35% elution component, which is the target protein, was obtained. Also, the protein concentration was measured by the UV280 method.

[0054] SEC-HPLC-UV analysis was used for purity check. The detector was an Agilent 1100 LC, the detection wavelength was 214 nm, the mobile phase was 150 mM pH 7.0 PB + 5% isopropanol, the chromatography column was a Superdex 200 Increase 5 / 150 GL, the running time was 15 minutes, and the column temperature was 25°C. As a result of the detection, it was shown that the purity exceeded 95% in all cases.

[0055] Purification of PD-L1-Fc fusion protein (SEQ ID NO.47) and Fc-TGFβRII fusion protein (SEQ ID NO.48): After adjusting the cell fermentation supernatant to pH 7.0, the sample was loaded onto a Protein A affinity chromatography column (Bogolong Biotechnology Co., Ltd.), and the equilibration solution was 20 mM PB, 0.15 M NaCl (pH = 7.0), and it was eluted with 100% 0.1 M Gly-HCl (pH = 3.0). 10% 1 M Tris-HCl (pH = 8.5) was added to the eluate in advance for neutralization.

Example

[0056] In vitro functional identification of the multi-domain fusion protein in Example 3 3.1 Detection of in vitro activity of anti-PD-L1 single-domain antibody fragment: The gene sequence of CD5L-OKT3scFv-CD14 (GenBank: ADN42857.1) was synthesized, enzymatically cleaved using HindIII-EcoRI (Takara), and then inserted into the vector pCDNA3.1 to generate pCDNA3.1-antiCD3TM. Next, using the human PD-L1 gene (GenBank: NM_014143.2) as a template, a PDL1 fragment was obtained by high-fidelity amplification and inserted into pCDNA3.1-antiCD3TM by recombinant ligation to generate pCDNA3.1-antiCD3TM-PDL1. Subsequently, CHO cells (Thermo Fisher Scientific) were transfected and then selected using G418 for 10 - 14 days to produce the stable cell line CHO-antiCD3TM-PDL1.

[0057] A fragment obtained by amplification using the human PD1 gene (GenBank: NP_005009.2) as a template was recombinantly ligated with the PB513B1-dual-puro vector (Youbao Biotechnology Co., Ltd.) digested with HindIII-BamHI (Takara) to produce plasmid pB-PD1. Next, pGL4.30 (Youbao Biotechnology Co., Ltd.) was amplified with high fidelity as a template, and the recovered fragment was recombinantly ligated with the pB-PD1 vector digested with SfiI-XbaI (Takara) to produce the pB-NFAT-Luc2p-PD1 plasmid. After successful production of the plasmid, the plasmid was extracted using an endotoxin-free plasmid maxi kit (Biomiga) and used for transfection into Jurkat cells (Chinese Academy of Sciences Stem Cell Bank). Referring to the method of Chinese Patent Application Publication No. 107022571A, after treating Jurkat cells with 0.1 mg / ml poly-D-lysine to make them in a monolayer culture state, transfection was performed on Jurkat cells based on the transfection instructions of the liposome transfection reagent kit (Lipofectamine 3000, Invitrogen). Then, on the third day, pressure screening was performed using RPMI 1640 medium (Thermo Fisher Scientific) containing 10% FBS and 2.5 μg / ml puromycin. Thereafter, the medium was replenished at regular intervals, and after the cell viability recovered, the puromycin content was gradually increased to 4 μg / ml. As a result, finally, a monoclonal Jurkat-NFAT-Luc2p-PD1 cell line was obtained.

[0058] CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells were collected and counted, and the cell density was adjusted to 4×10 6After adjusting the concentration to 1 / ml, 25 μl of each cell was added per well to a 96-well microplate. Next, the fusion protein samples prepared in Example 2 were serially diluted using 1% BSA, and 50 μl was added to the cells. After co-culture for 6 hours at 37°C and 5% CO2, 10 μl of luciferase substrate (Promega, E2620) was added per well, and the value was read after shaking for 2 minutes on a shaker. The procedure was as described in the reagent kit.

[0059] 3.2 In vitro activity detection of VEGF antagonistic fragments: HEK293 cells were plated in a 6-well cell culture plate at 1.0 × 10 cells per well. 6 The cells were plated and cultured overnight in a 37°C, 5% CO2 incubator. The transfection reagent Lipofectamine® 3000 was used according to its manufacturer's instructions. A transfection system was prepared with 1.0 μg of pcDNA-KDR plasmid and 4 μg of pGL4.30 plasmid. After 48 hours of transfection, the cells were grown to fill a 10 cm cell culture dish. G418 at 200 μg / ml and hygromycin at 100 μg / ml were added to the medium. The medium was replaced with fresh pressurized medium every 3 days until a clear clone group grew. The cells were then digested and plated in a 96-well cell culture plate. After the monoclonal growth, the cells were stimulated with 0.1 μg / ml VEGF for 6 hours to detect the chemiluminescence status. The clones with clear signal response were selected and cultured for further growth. Thus, a monoclonal HEK293-NFAT-KDR was finally obtained. The HEK293-NFAT-KDR cells were plated at a density of 40,000 cells / well and digested with Accutase. The digested cells were then collected and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in assay medium (DMEM + 5% FBS). The cells were counted and found to have a cell density of 1.6 × 10 6After adjusting to [[ / ml]], it was plated in a 96-well cell culture plate at 25 μl per well. Next, a VEGF solution was prepared using the analysis culture medium, and the concentration was set to 60 ng / ml, and 25 μl per well was added to the cell culture plate. Then, the fusion protein prepared and obtained in Example 2 was prepared with the analysis culture medium, added to the cell culture plate at 25 μl per well, and incubated and cultured at 37 °C and 5% CO2 for 6 h. Also, 10 μl of Bright-Glo luciferase assay reagent (Promega, E2620) was added to each well, shaken for 2 minutes, then 80 μl of lysis solution was transferred to an enzyme-labeled white plate, and the numerical value of the enzyme-linked immunosorbent assay device was read.

[0060] 3.3 In vitro cell activity detection of TGF-β binding fragment: Mouse breast cancer cells 4T1 were cultured until the cells reached about 90% confluence (10 cm petri dish) and digested with pancreatin. Then, the cells were plated in a 6-well microplate at 4×10 5 cells / well and cultured overnight. Next, the extracted pGL4.48[luc2P SBE Hygro] plasmid sample was transfected into 4T1 cells with Lipofectamine® 3000. Then, 24 h after transfection, the obtained TGFβ-4T1 cells were digested with pancreatin and transferred to a 10 cm petri dish, and the cells were subjected to pressure screening using RPMI 1640 medium containing 10% FBS and 150 μg / ml hygromycin (InvivoGen, Cat no.: ant-hg-1). Subsequently, TGFβ-4T1 cells 10 - 15 d after pressure screening were plated at 2 cells / well for monoclonal screening. Then, the monoclonal cells were stimulated with TGFβ1 (Novoprotein, 10 μg, Cat no.: CA59) to verify the transfection effect of the monoclonal, and finally TGFβ-4T1 monoclonal cells were obtained.

[0061] After the TGFβ-4T1 cells grew to reach about 90%, about 2.5 ml of 0.25% pancreatin was added for digestion. The digestion was carried out at room temperature for 2 min. Also, the cells attached to the wall of the petri dish were pipetted and dispersed by pipetting into the pancreatin solution. The total time required for the entire digestion process was ~5 min. Subsequently, complete medium (RPMI 1640 + 10% FBS) was added to stop the digestion, and pipetting was continued until the cells were uniformly dispersed. Next, the cells were transferred to a 50 m centrifuge tube and centrifuged at 1000 rpm for 5 min. Then, the supernatant was discarded, 2 ml of complete medium was added to resuspend the cells, and the cell density was measured with a cell counter. Subsequently, the cells were diluted with complete medium (RPMI 1640 + 10% FBS), and the cell density was adjusted to 2×10 5 cells / ml. The diluted cells (density 2×10 5 cells / ml) were plated at 100 μl per well at a cell density of 2×10 4 cells / well (96-well microplate). The 96-well microplate was placed in an incubator at 37°C and cultured overnight. Also, the fusion protein samples prepared in Example 2 were diluted to a predetermined concentration with RPMI 1640 + 0.2% FBS medium (containing 2 ng / ml of TGFβ1) (each protein sample was left at room temperature for 1 h after dilution). Subsequently, the supernatant was discarded from the TGFβ-4T1 cells cultured overnight in an incubator at 37°C, 50 μl of RPMI 1640 + 0.2% FBS medium was added, and then 50 μl of protein solutions with different concentrations were added. Then, the 96-well microplate was placed in an incubator at 37°C and continuously cultured for 3 h, after which 10 μl of Bright-Glo luciferase assay reagent (Promega, E2620) was added, shaken for 3 min, and the numerical value of the enzyme-linked immunosorbent assay device was read.

[0062] The measurement results of the anti-PD-L1 activity, anti-VEGF activity, and anti-TGFβ activity of each multi-domain fusion protein were as shown in Table 2. As is clear from Table 2, each multi-domain fusion protein had good in vitro cell activity.

[0063] [Table 2] [Examples]

[0064] Pharmacokinetics of Multidomain Fusion Protein in C57BL / 6 Mice C57BL / 6 mice were divided into four groups: a low-dose group (1 mg / kg) and a high-dose group (10 mg / kg) of avastin (Roche), and a low-dose group (1 mg / kg) and a high-dose group (10 mg / kg) of TAF-6. Each group consisted of 8 mice, with an equal number of males and females. The drugs were administered to the mice via a single injection into the tail vein, and drug metabolism samples were collected cross-sectionally at different time points. The sampling times were before administration and 1 h, 6 h, 24 h, 48 h, 78 h, 120 h, 144 h, 168 h, 192 h, and 216 h after administration. Then, the serum drug concentrations in the drug metabolism samples were quantitatively detected by ELISA. VEGF was coated on the plate, and the secondary antibody Goat anti-Human IgG Fc and HRP were conjugated to the drug and detected by the TMB method. Based on the relationship between the signal and concentration value of the standard curve, the concentration value was converted by regression, and the main pharmacokinetic parameters were calculated using the non-compartmental model of PK Solver software.

[0065] As a result, as shown in Figure 1, the high- and low-dose groups administered with avastin and TAF-6 in C57BL / 6 mice had similar drug metabolism parameters. Also, the ratio of the dose values of the low- and high-dose avastin groups was 1:10, C max ratio was 1:9.3, AUC last ratio was 1:8.9, and T max was 1 h for all. And the increase in exposure (C max and AUC last ) showed a proportional increase with the dose. Also, the ratio of the dose values of the low- and high-dose TAF-6 groups was 1:10, C max ratio was 1:8.6, AUC last ratio was 1:8.5, and T maxThey were all 1 h. And the increase in exposure (C max and AUC last ) showed a proportional increase with the dose. Therefore, between the high- and low-dose groups of abastin and TAF-6, both AUC and C max showed a linear correlation with the dose. The difference in AUC between abastin and TAF-6 is presumed to be the PD-L1 target-mediated disappearance effect (TMDD).

Example

[0066] Example 5 Antitumor Activity of the Multidomain Fusion Protein in PBMC-Humanized Breast Cancer MDA-MB-231 Mice Using MDA-MB-231 (human breast cancer) cells, a model was constructed in the body of mice (M-NSG mice) humanized with the human PBMC immune system to measure the in vivo pharmacological effects of the multidomain fusion protein in the present invention. Female M-NSG mice aged 6 - 8 weeks were selected, and MDA-MB-231 cells (10×10E6 + Matrigel 25%) were transplanted into the mice. On the 7th day, after injecting PBMC (5×10E6 / 0.2 ml) into the tail vein, tumor volume and body weight were observed. Then, mice with a tumor volume between 140 - 260 mm 3 were selected and randomly divided into 6 groups based on tumor volume and body weight. Each group consisted of 7 mice, and administration was started on the day of grouping. Also, among the tumor-bearing mice, those with a tumor volume that was too large or too small were excluded. Then, twice a week, PBS, IgG1 as an isotype control, Tecentriq (Roche) as a positive control, TAF-6, TAF-7, and combination drugs were intraperitoneally injected (see Table 3 for the protocol). Administration was carried out for about 3 weeks. The PD-L1-Fc fusion protein (SEQ ID NO.47) is a fusion protein of an anti-PD-L1 single-domain antibody and human IgG Fc (the anti-PD-L1 single-domain antibody is located at the N-terminus of Fc), and the 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).

[0067] Blood was collected from the orbital veins of mice before grouping and before the end of the experiment. As a result of the FACS test, it was shown that human CD45-positive cells were present in the peripheral blood of mice in each group. Moreover, the ratio of CD45 increased with time, indicating that humanization of the mouse immune system was successful. During the experiment, the body weights of the animals in Group 3 and Group 4 (measured twice a week) were almost stable, and no deaths of the experimental animals occurred. In addition, the tumor volumes of Group 3 and Group 4 (measured twice a week) were smaller than those of Group 6 with combined administration, and significantly smaller than those of Group 2 corresponding to tecentriq alone. The results were as shown in Figure 2.

[0068]

Table 3

Example

[0069] Example 6 Anti-tumor Activity of Multi-domain Fusion Protein in PBMC-humanized Breast Cancer MDA-MB-231 Mice Tumor masses of human breast cancer MDA-MB-231 were transplanted subcutaneously into the right anterior axillary abdomen of female NCG mice. Then, one day after transplantation of the tumor mass, PBMC cells were transplanted into the mice, and when the tumor grew to about 53 mm 3 in size, they were divided into a total of 6 groups for administration. Each group consisted of 10 animals, namely, the Vehicle group, the TAF-6 low (2 mg / kg, i.p., ti w×9) group, the TAF-6 medium (6 mg / kg, i.p., tiw×9) group, the TAF-6 high (18 mg / kg, i.p., tiw×9) group, the tecentriq (4 mg / kg, i.p., tiw×9) group, and the avastin (4 mg / kg, i.p., tiw×9) group. Then, the tumor volume and body weight were measured weekly, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

[0070] Blood samples were collected from the orbital venous plexus 2 days before subgrouping and at the end of the experiment. As a result of FACS tests, it was shown that human CD45-positive cells were present in the peripheral blood of mice in each group. Moreover, the ratio of CD45 increased with time. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were dissected and weighed and photographed, and serum was collected and the tumors were fixed. Then, the tumor growth inhibition rate TGI TV (%) was calculated and statistically analyzed. As a result, the tumor growth inhibition rates of the low TAF-6 group, medium TAF-6 group, high TAF-6 group, Tencentriq group, and Avastin group were 41%, 34%, 60%, 23%, and 32% respectively. In addition, except for the Tencentriq group, the tumor volumes of each group were significantly smaller than those of the Vehicle group (all p < 0.05). The tumor volume of the high TAF-6 group was significantly smaller than that of the Tencentriq group (p < 0.01), and there was no significant difference in the tumor volumes between the Tencentriq group and the Avastin group (p > 0.05). The results were as shown in Figure 3.

[0071] As described above, the test drug TAF-6 had a significant antitumor effect on the PBMC-humanized breast cancer MDA-MB-231 subcutaneous transplantation tumor model and effectively inhibited tumor growth. Moreover, its antitumor effect was significantly superior to that of Tencentriq. And the tumor suppression effect was enhanced with the increase of the dosage.

[0072] In an independent experiment in another same tumor model, the antitumor activities of the test drug and a similar body of the positive drug M7824 (prepared in our laboratory based on US Patent No. 9,676,863 B2 of M7824) were compared. Each group had 6 mice, and 4 groups were set up: the Vehicle group, the TAF-6 (4.2 mg / kg, i.p., tiw × 8 times. The later dosage was adjusted to 8.4 mg / kg, i.p., tiw × 4 times) group, the M7824 similar body (3.6 mg / kg, i.p., tiw × 8 times. The later dosage was adjusted to 7.2 mg / kg, i.p., tiw × 4 times) group, and the Avastin (3 mg / kg, i.p., tiw × 8 times. The later dosage was adjusted to 6 mg / kg, i.p., tiw × 4 times) group. As a result, as shown in Figure 4, when the molar concentrations and dosages were the same, the antitumor effect of TAF-6 was significantly superior to that of the M7824 similar body.

Example

[0073] Example 7 Anti-tumor activity of the multi-domain fusion protein in PBMC humanized lung cancer Calu-6 mice Human lung cancer Calu-6 cells were transplanted subcutaneously into the right anterior flank of male NCG mice. Also, PBMC cells were transplanted into the mice 4 days before transplanting the tumor cells. And when the tumor grew to about 50 mm 3 in size, they were divided into a total of 7 groups for administration. Each group consisted of 8 mice, namely, the Vehicle group, the low-dose TAF-6 (2 mg / kg, i.p., tiw×10) group, the medium-dose TAF-6 (7 mg / kg, i.p., tiw×10) group, the high-dose TAF-6 (25 mg / kg, i.p., tiw×10) group, the Tecentriq (5 mg / kg, i.p., tiw×10) group, the Avastin (5 mg / kg, i.p., tiw×10) group, and the Tecentriq + Avastin (5 + 5 mg / kg, i.p., tiw×10) group. The tumor volume and body weight were measured weekly, and the relationship between the changes in the 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 dissected, weighed and photographed, and serum and tumors were collected. Then, the tumor growth inhibition rate TGI TV (%) was calculated and statistically analyzed.

[0074] Blood was collected from the orbital veins of the mice 3 days before grouping and at the end of the experiment (PG-D23). As a result of the FACS test, it was shown that human CD45-positive cells were present in the peripheral blood of the mice in each group. Moreover, the ratio of CD45 increased with time, indicating that the humanization of the immune system of the mice was successful.

[0075] During the treatment period, the mice in each group ate and drank normally, and their body weights were almost stable. Also, no deaths of the experimental animals occurred.

[0076] At the end of the experiment (PG-D23), the tumor growth inhibition rates of the low TAF-6 group, medium TAF-6 group, high TAF-6 group, tesentriq group, abatacept group, and tesentriq + abatacept group were 62%, 62%, 75%, 13%, 34%, and 32%, respectively. Also, except for the tesentriq group, the tumor volumes of each group were significantly smaller than those of the Vehicle group (all p < 0.01). The tumor volumes of the low, medium, and high TAF-6 groups were all significantly smaller than those of the tesentriq group, abatacept group, and tesentriq + abatacept group, respectively (all p < 0.01). The results were as shown in Table 4 and Figure 5.

[0077] As described above, the test drug TAF-6 had a significant antitumor effect on the PBMC humanized lung cancer Calu-6 subcutaneous xenograft tumor model and effectively inhibited tumor growth. Also, the tumor inhibitory effect was significantly superior to that of tesentriq, abatacept, and the combination of tesentriq and abatacept. Moreover, the tumor inhibitory effect was enhanced with the increase in the dosage.

[0078]

Table 4

[0079] In an independent experiment in another identical tumor model, the antitumor activity of the test drug was considered in comparison with an analog of the positive drug M7824. Six animals were in each group, and four groups were set up: the Vehicle group, the TAF-6 (7 mg / kg, i.p., TIW × 8 times) group, the M7824 analog (6 mg / kg, i.p., TIW × 8 times) group, and the abatacept (5 mg / kg, i.p., TIW × 8 times) group. As a result, as shown in Figure 6, at the same molar concentration and dosage, the antitumor effect of TAF-6 was significantly superior to that of the M7824 analog and abatacept.

Example

[0080] Example 8 Antitumor Activity of a Multidomain Fusion Protein in PBMC Humanized Colorectal Cancer HCT116 Mice Using HCT-116 (human colon cancer) cells, a model was constructed in the body of humanized mice (NOG mice) with the human PBMC immune system to measure the in vivo pharmacological effects of the multi-domain fusion protein in the present invention. Female NOG mice aged 7 - 9 weeks were selected and transplanted with HCT-116 cells (3*10E6 + Matrigel). Then, on the 3rd day after transplantation of tumor cells, PBMC (5*10E6 / 0.2 ml) was injected into the tail vein, and then tumor volume and body weight were observed. Mice with a tumor volume between 60 and 100 mm 3 were selected. Subsequently, based on tumor volume and body weight, with 8 mice in each group, they were randomly divided into 6 groups: Vehicle group, low-dose TAF-6 (2 mg / kg, i.p., tiw×3) group, medium-dose TAF-6 (7 mg / kg, i.p., tiw×3) group, high-dose TAF-6 (25 mg / kg, i.p., tiw×3) group, Tencentriq (5 mg / kg, i.p., tiw×3) group, and Tencentriq + Avastin (5 + 5 mg / kg, i.p., tiw×3) group. Administration was started on the day of grouping. Then, tumor volume and body weight were measured twice a week, and the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were dissected, weighed and photographed, and serum and tumors were collected. Then, the tumor growth inhibition rate TGI TV (%) was calculated and statistically analyzed.

[0081] When blood was collected from the orbital veins of mice before grouping and before the end of the experiment, the results of the FACS test showed that human CD45-positive cells were present in the peripheral blood of mice in each group. Moreover, the ratio of CD45 increased with time, indicating that the humanization of the mouse immune system was successful.

[0082] The inhibition of tumor activity was as shown in Figure 7. The test drug TAF-6 had a significant antitumor effect on the PBMC humanized colon cancer HCT116 subcutaneous xenograft tumor model, effectively suppressing tumor growth. Moreover, the tumor suppression effects of the high-dose group and the medium-dose group were significantly superior to those of cetuximab. Also, when comparing the TAF-6 group (7 mg / kg, i.p., tiw×3) with the cetuximab + bevacizumab group (5 + 5 mg / kg, i.p., tiw×3) at the same molar concentration and dosage, it was observed that TAF-6 had a therapeutic effect not inferior to that of the cetuximab + bevacizumab group and even superior to it.

Example

[0083] Example 9 Antitumor Activity of Multidomain Fusion Protein in PBMC Humanized Liver Cancer Huh-7 Mice Human liver cancer Huh-7 cells were transplanted subcutaneously into the right anterior flank of male NCG mice. Also, PBMC cells were transplanted into the mice 45 days before transplanting the tumor cells. Then, when the tumor grew to about 50 mm 3 in size, they were divided into a total of 5 groups for administration. Each group consisted of 10 mice, namely the Isotype group, the TAF-6 low (2 mg / kg, i.p., tiw×8) group, the TAF-6 high (7 mg / kg, i.p., tiw×8) group, the cetuximab (5 mg / kg, i.p., tiw×8) group, and the bevacizumab (5 mg / kg, i.p., tiw×8) group. Then, the tumor volume and body weight were measured twice a week, and the relationship between the changes in the 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 dissected for weighing and photography, and serum and tumors were collected. Then, the tumor growth inhibition rate TGI TV (%) was calculated and statistically analyzed.

[0084] Blood was collected from the orbital venous plexus of the mice before grouping and at the end of the experiment. As a result of the FACS test, it was shown that human CD45-positive cells were present in the peripheral blood of the mice in each group. Moreover, the ratio of CD45 increased with time, indicating that the humanization of the mouse immune system was successful.

[0085] During the administration period, the mice in each group ate and drank normally, and their body weights were almost stable.

[0086] Before the end of the experiment (PG-D18), the tumor growth inhibition rates of the low TAF-6 group, high TAF-6 group, Tencentrik group, and abastin group were 57%, 74%, 18%, and 67%, respectively. Also, except for the Tencentrik group, the tumor volumes of each group were significantly smaller than those of the Isotype group (all p < 0.01). The tumor volumes of the high TAF-6 group were all significantly smaller than those of the Tencentrik group (all p < 0.01), and the tumor volumes of the abastin group were significantly smaller than those of the Tencentrik group (p < 0.05). The results were as shown in Figure 8.

[0087] As described above, the test drug TAF-6 had a significant antitumor effect on the PBMC-humanized liver cancer Huh-7 subcutaneous transplantation tumor model and effectively inhibited tumor growth. Also, the antitumor effect was significantly superior to that of Tencentrik. Moreover, the tumor suppression effect was enhanced with the increase in the dosage.

[0088] In an independent experiment in another identical tumor model, the antitumor activity of the test drug was considered in comparison with an analog of the positive drug M7824. Each group consisted of 6 mice, and they were divided into 4 groups: the Vehicle group, the TAF-6 (7 mg / kg, i.p., tiw×9. The later dosage was adjusted to 14 mg / kg, i.p., tiw×2) group, the M7824 analog (6 mg / kg, i.p., tiw×9. The later dosage was adjusted to 12 mg / kg, i.p., tiw×2) group, and the abastin (5 mg / kg, i.p., tiw×9. The later dosage was adjusted to 10 mg / kg, i.p., tiw×2) group. As a result, as shown in Figure 9, the antitumor effect of TAF-6 was significantly superior to that of the M7824 analog.

Example

[0089] Example 10 Antitumor Activity of the Multidomain Fusion Protein in PBMC-Humanized Sarcoma HT1080 Mice Human sarcoma HT1080 cells were transplanted subcutaneously into the right anterior flank of male NCG mice. Also, PBMC cells were transplanted into the mice 7 days before transplanting the tumor cells. And when the tumor grew to about 56 mm 3 in size, they were divided into a total of 5 groups and administered. Each group consisted of 8 animals, namely the Vehicle group, the low TAF-6 (2 mg / kg, i.p., tiw×9) group, the medium TAF-6 (7 mg / kg, i.p., tiw×9) group, the high TAF-6 (25 mg / kg, i.p., tiw×9) group, and the Tencentriq (5 mg / kg, i.p., tiw×9) group. And the tumor volume and body weight were measured weekly, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.

[0090] At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were dissected and weighed and photographed, and serum and tumors were collected. And the tumor growth inhibition rate TGI TV (%) was calculated and statistically analyzed.

[0091] When blood was collected from the orbital venous plexus of the mice before grouping and at the end of the experiment, as a result of the FACS test, it was shown that human CD45-positive cells were present in the peripheral blood of the mice in each group. And the ratio of CD45 increased with time, indicating that humanization of the mouse immune system was successful.

[0092] Before the end of the experiment (PG-D19), the tumor growth inhibition rates of the low TAF-6 group, the medium TAF-6 group, the high TAF-6 group, and the Tencentriq group were 19%, 33%, 41%, and 28%, respectively. Also, the tumor volumes of the medium TAF-6 group and the Tencentriq group were significantly smaller than those of the Vehicle group (both p<0.05). And there was no significant difference in the tumor volume among the groups (p>0.05). The results were as shown in Figure 10.

[0093] As described above, the present invention eliminates various drawbacks in the prior art and has a high industrial utility value.

[0094] The above embodiments merely exemplarily illustrate the principles and effects of the present invention and do not limit the present invention. Those skilled in the art can supplement or modify the above embodiments on the premise of not departing from the spirit and scope of the present invention. Therefore, all equivalent supplements or modifications completed by those skilled in the art without departing from the spirit and technical idea disclosed in the present invention shall also be included in the scope of the claims of the present invention.

Claims

1. A fusion protein comprising an anti-PD-L1 single domain antibody fragment, a VEGF antagonist fragment, and a TGF-β binding fragment, wherein the complementarity determining regions of the anti-PD-L1 single domain antibody fragment are CDR1 having an amino acid sequence shown by SEQ ID NO. 1, CDR2 having an amino acid sequence shown by SEQ ID NO. 6, and CDR3 having an amino acid sequence shown by SEQ ID NO. 10, or CDR1 having an amino acid sequence shown by SEQ ID NO. 4, CDR2 having an amino acid sequence shown by SEQ ID NO. 8, and CDR3 having an amino acid sequence shown by SEQ ID NO. 13, or CDR1 having an amino acid sequence shown by SEQ ID NO. 2, CDR2 having an amino acid sequence shown by SEQ ID NO. 7, and CDR3 having an amino acid sequence shown by SEQ ID NO. 14, or CDR1 having an amino acid sequence shown by SEQ ID NO. 5, CDR2 having an amino acid sequence shown by SEQ ID NO. 9, and CDR3 having an amino acid sequence shown by SEQ ID NO. 15, and the fusion protein comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 single domain antibody fragment, a VEGF antagonist fragment, and a TGF-β binding fragment, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the heavy chain of the VEGF antagonist fragment, or the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain of the VEGF antagonist fragment, the TGF-β binding fragment is located at the C-terminus of the heavy chain of the VEGF antagonist fragment, the VEGF antagonist fragment comprises the antigen-binding site of the bevacizumab monoclonal antibody, the amino acid sequence of the heavy chain CDR1 of the VEGF antagonist fragment is GYTFTNYG, the amino acid sequence of the heavy chain CDR2 of the VEGF antagonist fragment is INTYTGEP, the amino acid sequence of the heavy chain CDR3 of the VEGF antagonist fragment is AKYPHYYGSSHWYFDV, and the amino acid sequence of the light chain CDR1 of the VEGF antagonist fragment is QDISNY, the amino acid sequence of the light chain CDR2 of the VEGF antagonist fragment is FTS, and the amino acid sequence of the light chain CDR3 of the VEGF antagonist fragment is QQYS TVPWT, The TGF-β binding fragment is a fragment of the extracellular region structure of TGF-βRII, a fusion protein. **Claim 2** The anti-PD-L1 single domain antibody fragment further includes a framework region FR, wherein the framework region FR includes FR1 with an amino acid sequence shown in SEQ ID NO. 49, FR2 with an amino acid sequence shown in any one of SEQ ID NOs. 50 to 52, FR3 with an amino acid sequence shown in any one of SEQ ID NOs. 53 to 55, and FR4 with an amino acid sequence shown in SEQ ID NO.

56. The fusion protein according to claim 1, characterized by including these. **Claim 3** The framework region FR includes FR1 with an amino acid sequence shown in SEQ ID NO. 49, FR2 with an amino acid sequence shown in SEQ ID NO. 50, FR3 with an amino acid sequence shown in SEQ ID NO. 53, and FR4 with an amino acid sequence shown in SEQ ID NO. 56, or includes FR1 with an amino acid sequence shown in SEQ ID NO. 49, FR2 with an amino acid sequence shown in SEQ ID NO. 51, FR3 with an amino acid sequence shown in SEQ ID NO. 54, and FR4 with an amino acid sequence shown in SEQ ID NO. 56, or includes FR1 with an amino acid sequence shown in SEQ ID NO. 49, FR2 with an amino acid sequence shown in SEQ ID NO. 52, FR3 with an amino acid sequence shown in SEQ ID NO. 54, and FR4 with an amino acid sequence shown in SEQ ID NO. 56, or includes FR1 with an amino acid sequence shown in SEQ ID NO. 49, FR2 with an amino acid sequence shown in SEQ ID NO. 52, FR3 with an amino acid sequence shown in SEQ ID NO. 55, and FR4 with an amino acid sequence shown in SEQ ID NO.

56. The fusion protein according to claim 2, characterized by including these. **Claim 4** The anti-PD-L1 single domain antibody fragment a) includes a polypeptide fragment with an amino acid sequence shown in any one of SEQ ID NOs. 16 to 21, and optionally, the anti-PD-L1 single domain antibody fragment is derived from alpaca, and optionally, the anti-PD-L1 single domain antibody fragment is humanized. The fusion protein according to claim 1, characterized by these. **Claim 5** The TGF-β binding fragment is e) a polypeptide fragment having an amino acid sequence shown by SEQ ID NO. 24, or f) a polypeptide fragment having an amino acid sequence with 90% or more sequence identity to SEQ ID NO. 24 and having the function of the polypeptide fragment specified in e) above, and Optionally, the TGF-β binding fragment is of human origin, and the fusion protein according to claim 1 is characterized thereby.

6. The fusion protein according to claim 1, further comprising a linking peptide fragment.

7. The linking peptide fragment has an amino acid sequence comprising at least one selected from the group consisting of the sequences of (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGG A)nA, or (GGGGG)nA, and n is selected from integers of 1 to 10, and the fusion protein according to claim 6 is characterized thereby.

8. The linking peptide fragment is selected from flexible polypeptide chains composed of G glycine and / or S serine and / or A alanine, and the length of the linking peptide fragment is a length in which 3 to 30 amino acids are connected, and the fusion protein according to claim 6 is characterized thereby.

9. The linking peptide fragment includes a polypeptide fragment having an amino acid sequence shown by any one of SEQ ID NOs. 34 to 36, Optionally, a linking peptide fragment is provided between the anti-PD-L1 single domain antibody fragment and the VEGF antagonist fragment, Optionally, a linking peptide fragment is provided between the VEGF antagonist fragment and the TGF-β binding fragment, and the fusion protein according to any one of claims 6 to 8 is characterized thereby.

10. The amino acid sequence of the fusion protein includes a sequence shown by any one of SEQ ID NO. 23, SEQ ID NOs. 25 to 33, Alternatively, the amino acid sequence of the fusion protein is the sequence shown in SEQ ID NO. 25 and SEQ ID NO. 26, the sequence shown in SEQ ID NO. 25 and SEQ ID NO. 27, the sequence shown in SEQ ID NO. 25 and SEQ ID NO. 28, the sequence shown in SEQ ID NO. 25 and SEQ ID NO. 29, the sequence shown in SEQ ID NO. 30 and SEQ ID NO. 27, the sequence shown in SEQ ID NO. 30 and SEQ ID NO. 29, the sequence shown in SEQ ID NO. 31 and SEQ ID NO. 23, the sequence shown in SEQ ID NO. 32 and SEQ ID NO. 23, or the sequence shown in SEQ ID NO. 33 and SEQ ID NO.

23. The fusion protein according to claim 1, characterized by comprising the sequence.

11. A separated polynucleotide encoding the fusion protein according to any one of claims 1 to 10.

12. A construct comprising the separated polynucleotide according to claim 11.

13. An expression system comprising the construct according to claim 12, or the polynucleotide according to claim 11 in which a foreign gene is integrated into the genome.

14. A method for producing the fusion protein according to any one of claims 1 to 10, comprising culturing the expression system according to claim 13 under appropriate conditions, expressing the fusion protein in the expression system, and separating and purifying the fusion protein to provide the fusion protein.

15. Use of the fusion protein according to any one of claims 1 to 10, or a culture of the expression system according to claim 13, in the manufacture of a drug.

16. The use according to claim 15, characterized in that the drug is selected from drugs used for the treatment of tumors.

17. The tumor is selected from lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, hematological malignancies, sarcoma, head and neck cancer or nasopharyngeal cancer. The use according to claim 16, characterized by being selected.

18. A drug composition comprising the fusion protein according to any one of claims 1 to 10, or a culture of the expression system according to claim 13.

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