WO2022174781A1 - 多结构域融合蛋白及其应用 - Google Patents

多结构域融合蛋白及其应用 Download PDF

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WO2022174781A1
WO2022174781A1 PCT/CN2022/076610 CN2022076610W WO2022174781A1 WO 2022174781 A1 WO2022174781 A1 WO 2022174781A1 CN 2022076610 W CN2022076610 W CN 2022076610W WO 2022174781 A1 WO2022174781 A1 WO 2022174781A1
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seq
fragment
fusion protein
amino acid
acid sequence
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PCT/CN2022/076610
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French (fr)
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黄岩山
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浙江道尔生物科技有限公司
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Priority to CN202280007712.9A priority Critical patent/CN116761814A/zh
Priority to JP2023550320A priority patent/JP2024507856A/ja
Priority to US18/277,996 priority patent/US20240124587A1/en
Priority to KR1020237031894A priority patent/KR20230147675A/ko
Priority to CA3211427A priority patent/CA3211427A1/en
Priority to AU2022221660A priority patent/AU2022221660A1/en
Priority to EP22755554.7A priority patent/EP4282884A1/en
Publication of WO2022174781A1 publication Critical patent/WO2022174781A1/zh

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    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of biotechnology, in particular to a multi-domain fusion protein with anticancer activity and a preparation method and use thereof.
  • VEGF vascular endothelial growth factor
  • Bevacizumab trade name
  • Bevacizumab a humanized anti-VEGF monoclonal antibody that binds to VEGF and prevents VEGF from interacting with endothelial cell surface VEGF receptors (Flt-1 and KDR).
  • Bevacizumab is currently FDA-approved for the treatment of metastatic colorectal cancer, advanced, metastatic or recurrent non-small cell lung cancer, and recurrent glioblastoma.
  • Transforming growth factor TGF- ⁇ is a cytokine that maintains tissue homeostasis by regulating cell growth, differentiation, proliferation and survival.
  • TGF- ⁇ pathway can control tumors by promoting cell cycle arrest and apoptosis in the early stages of tumors; but in the late stages of tumors, TGF- ⁇ can also inhibit cytotoxic T cells and promote cancer cell proliferation and invasion. and metastasis ultimately allows tumor escape, a functional switch known as the "TGF- ⁇ paradox".
  • TGF ⁇ signaling induces TH1 cell differentiation into Tregs, attenuates CD8 + effector T cell activation, and restricts central memory T cell development, fundamentally affecting tumor-infiltrating T cell function.
  • TGF- ⁇ In mammals, TGF- ⁇ mainly has three subtypes: TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3.
  • TGF- ⁇ By expressing TGF- ⁇ at high levels, tumors are protected from immune surveillance. Consistent with this, non-small cell lung cancer (NSCLC), renal cell carcinoma (CRC), gastric and prostate cancers with high TGF- ⁇ expression are associated with tumor progression and poor prognosis (Marin-Acevedo et al., Journal of Hematology). & Oncology, 11:39, 2018). This shows that TGF- ⁇ antagonism will be a potential new direction for tumor therapy.
  • NSCLC non-small cell lung cancer
  • CRC renal cell carcinoma
  • gastric and prostate cancers with high TGF- ⁇ expression are associated with tumor progression and poor prognosis (Marin-Acevedo et al., Journal of Hematology). & Oncology, 11:39, 2018). This shows that TGF- ⁇ antagonism will be
  • anti-PD-1/PD-L1 monoclonal antibodies as immune checkpoint inhibitors have been widely used in tumor therapy.
  • the anti-PD-L1 monoclonal antibody Avelumab fused to TGF- ⁇ receptor II (TGF- ⁇ Trap) has entered the clinical stage of M7824.
  • TGF- ⁇ Trap TGF- ⁇ receptor II
  • the combination of anti-PD-1/PD-L1 mAb, TGF- ⁇ antagonist and anti-VEGF mAb has not yet been reported.
  • the purpose of the present invention is to provide a multi-domain fusion protein with anti-cancer activity and a preparation method and use thereof, so as to solve the problems in the prior art.
  • one aspect of the present invention provides a fusion protein, which includes an anti-PD-L1 single-domain antibody fragment, an antagonistic VEGF fragment, and a TGF- ⁇ binding fragment.
  • Another aspect of the present invention provides an isolated polynucleotide encoding the above-mentioned fusion protein.
  • Another aspect of the present invention provides a construct comprising the isolated polynucleotide described above.
  • Another aspect of the present invention provides an expression system comprising the above-mentioned construct or the above-mentioned exogenous polynucleotide integrated into the genome.
  • Another aspect of the present invention provides a method for preparing the above-mentioned fusion protein, comprising: culturing the above-mentioned expression system under suitable conditions to express the fusion protein, and isolating and purifying to provide the fusion protein.
  • Another aspect of the present invention provides the use of the above-mentioned fusion protein or the culture of the above-mentioned expression system in the preparation of medicine.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein, or a culture of the above-mentioned expression system.
  • Figure 10 Antitumor efficacy of multi-domain fusion protein in PBMC immune system humanized HT1080 sarcoma subcutaneous xenograft tumor model.
  • the inventor of the present invention unexpectedly found a fusion protein molecule, which can block PD-L1/PD-1 interaction by anti-PD-L1 monoclonal antibody, and antagonize VEGF monoclonal antibody to reduce microvascular growth. Combined with the inhibition of metastatic disease, and TGF- ⁇ receptors to improve T cell dysfunction caused by TGF- ⁇ in the tumor microenvironment and enhance the immune response, it has excellent tumor suppressive effect. invention.
  • the first aspect of the present invention provides a fusion protein, the fusion protein includes an anti-PD-L1 single domain antibody fragment, an antagonistic VEGF fragment, and a TGF- ⁇ binding fragment.
  • anti-PD-L1 single-domain antibody fragments can usually be used to block the PD-L1/PD-1 interaction and increase the expression of IFN- ⁇ and/or IL-2 in T lymphocytes, thereby inhibiting tumor growth.
  • Antagonistic VEGF fragments can generally include an Fc portion that can bind to FcRn receptors, thereby prolonging half-life in vivo, and can also bind to effector cells expressing Fc receptors to kill cancer cells.
  • TGF- ⁇ -binding fragments can improve the killing function of tumor-infiltrating T cells on tumor cells by removing over-expressed TGF- ⁇ in the tumor microenvironment.
  • an anti-PD-L1 single domain antibody fragment can be included.
  • the above-mentioned anti-PD-L1 single-domain antibody fragment can generally be a polypeptide or protein fragment that can specifically bind to PD-L1.
  • the corresponding antibody light chain is usually deleted, and only the fragment corresponding to the variable region of the heavy chain is present.
  • the binding properties of anti-PD-L1 single-domain antibody fragments can usually be determined by the three complementarity determining regions (CDRs, complementarity determining regions) included.
  • the CDR regions can be arranged in order with the framework regions (FR, framework regions), and the FR regions Not directly involved in the binding reaction.
  • 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. 1-5, and one of SEQ ID NO. 6-9 CDR2, CDR3 shown in one of SEQ ID NO.10 ⁇ 15.
  • the complementarity determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 with amino acid sequence shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.6, SEQ ID NO. CDR3 shown in 10.
  • the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 with amino acid sequence shown in SEQ ID NO.2, CDR2 shown in SEQ ID NO.7, SEQ ID NO. .11 shown CDR3.
  • 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.
  • the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 with amino acid sequence shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.8, SEQ ID NO. CDR3 shown in .13.
  • the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 with amino acid sequence shown in SEQ ID NO.2, CDR2 shown in SEQ ID NO.7, SEQ ID NO. .14 CDR3 shown.
  • the complementarity determining region of the anti-PD-L1 single domain antibody fragment includes: CDR1 with amino acid sequence shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.9, SEQ ID NO. CDR3 shown in .15.
  • the above-mentioned anti-PD-L1 single-domain antibody fragment may also include a framework region (FR, framework region).
  • the CDR regions can be arranged in order with the FR regions, for example, the anti-PD-L1 single domain antibody fragment can include FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 in order from N-terminus to C-terminus.
  • the framework region FR includes FR1 whose amino acid sequence can be shown in SEQ ID No. 49, FR2 whose amino acid sequence is shown in one of SEQ ID No. 50-52, and FR2 whose amino acid sequence is shown in one of SEQ ID No. 53-55. FR3 is shown, and FR4 whose amino acid sequence is shown in SEQ ID No. 56.
  • 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.
  • the anti-PD-L1 single domain antibody fragment may include: a) a polypeptide fragment whose amino acid sequence is as shown in one of SEQ ID Nos. 16 to 21; One of ID Nos. 16 to 21 has more than 80% sequence identity and has the function of the polypeptide fragment as defined in a).
  • the polypeptide fragment in the above b) specifically refers to: the amino acid sequence shown in one of SEQ ID No.
  • 16 to 21 has undergone substitution, deletion or addition of one or more (specifically, 1-50, 1 -30, 1-20, 1-10, 1-5, or 1-3) amino acids, or by adding one or more (specifically can be N-terminal and/or C-terminal) amino acids 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and have an amino acid sequence such as one of SEQ ID No.16-21
  • the function of the polypeptide fragment shown in a polypeptide fragment for example, can be the ability to specifically bind to PD-L1, or it can be the blocking of PD-L1/PD-1 interaction, which can block PD-L1
  • the /PD1 pathway can also be a function of increasing the expression of IFN- ⁇ and/or IL-2 in T lymphocytes, and it can also be a function of inhibiting tumor growth.
  • the amino acid sequence of the anti-PD-L1 single domain antibody fragment in the above b) can be 80%, 85%, 90%, 93%, 95%, 97%, or 99% with one of SEQ ID No. 16 to 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 fragments may generally be humanized, for example, the framework regions thereof may be derived from humans.
  • sequence identity refers to the percentage of identical residues in the sequences participating in the alignment. Sequence identity of two or more entry sequences can be calculated using computational software well known in the art, such software available from NCBI, for example.
  • an antagonistic VEGF fragment can be included.
  • the above-mentioned VEGF-antagonizing fragment can generally be a polypeptide or protein fragment capable of antagonizing VEGF.
  • the above-mentioned VEGF antagonistic fragment may be a monoclonal antibody or the like.
  • the above-mentioned VEGF antagonistic fragment may be bevacizumab and the like.
  • the antagonistic VEGF fragment may include:
  • the amino acid sequence in the above d) specifically refers to: the amino acid sequence shown in one of SEQ ID No.
  • the amino acid sequence in d) can be more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% identical to one of SEQ ID Nos. 22 to 23.
  • the above-mentioned VEGF antagonistic fragment can usually be derived from a mouse (Mus musculus), for example, the CDR region thereof can be derived from a mouse.
  • the VEGF-antagonizing fragments described above can generally be humanized, eg, their framework regions can be derived from humans.
  • a TGF- ⁇ binding fragment can be included.
  • the above-mentioned TGF- ⁇ -binding fragments can usually specifically bind to each TGF- ⁇ isoform (for example, TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3, etc.), and TGF- ⁇ isoforms are usually highly expressed in various malignant tumors. , is likely to be one of the important factors leading to poor clinical treatment effect.
  • the TGF-beta binding fragment can be a TGF-beta RII (TGF-beta receptor II) extracellular domain structural fragment.
  • the TGF- ⁇ binding fragment may comprise:
  • 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).
  • amino acid sequence in the above f) specifically refers to: the amino acid sequence shown in SEQ ID No. 24 is substituted, deleted or added one or more (specifically, 1-50, 1-30, 1-20 1, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or adding one or more (specifically, N-terminal and/or C-terminal) amino acids 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 function of the polypeptide fragment shown in SEQ ID No.
  • the polypeptide fragment for example, can bind to each TGF- ⁇ isoform (eg, TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3, etc.), thereby eliminating tumor microbes.
  • TGF- ⁇ in the environment can also enhance the killing function of tumor-infiltrating T cells against tumor cells.
  • Said amino acid sequence in f) can be more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% identical to SEQ ID No. 24.
  • the above-mentioned TGF-beta binding fragments can generally be derived from humans (homo sapiens).
  • a linker peptide fragment can also be included.
  • a plurality of linker peptide fragments may be generally included, and at least some of the domains or between each of the domains may be provided with linker peptide fragments.
  • a linker peptide may be provided between the anti-PD-L1 single domain antibody fragment and the VEGF antagonistic fragment.
  • a linker peptide may be provided between the antagonistic VEGF fragment and the TGF- ⁇ binding fragment.
  • the above-mentioned linker 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, so that the Neighboring protein domains are free to move relative to each other.
  • the amino acid sequence of the linker peptide fragment may include, for example, (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG) )nA etc. sequence, wherein n is selected from integers between 1-10.
  • 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.
  • the connecting peptide fragment may comprise a polypeptide fragment whose amino acid sequence is shown in one of SEQ ID NO. 34-36.
  • the fusion protein can be linear, for example, the fusion protein can sequentially include anti-PD-L1 single domain antibody fragment, antagonistic VEGF fragment, and TGF- ⁇ binding fragment from the N-terminus to the C-terminus.
  • the fusion protein can also have a structure similar to that of a monoclonal antibody.
  • an anti-PD-L1 single-domain antibody fragment can be located at the N-terminus of the heavy chain of the VEGF fragment, and for another example, an anti-PD-L1 single-domain antibody fragment can be located at the antagonism VEGF.
  • the N-terminus of the light chain of the fragment as another example, the TGF-beta binding fragment can be positioned C-terminus of the heavy chain of the antagonizing VEGF fragment.
  • the amino acid sequence of the fusion protein may include the sequence shown in one of SEQ ID NO. 23 and SEQ ID NO.
  • the amino acid sequence of the fusion protein may include SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.25 and SEQ ID NO.27, SEQ ID NO.25 and SEQ ID NO.28, SEQ ID NO.28
  • SEQ ID NO.28 The sequences shown in NO.25 and SEQ ID NO.29, the sequences shown in SEQ ID NO.30 and SEQ ID NO.27, the sequences shown in SEQ ID NO.30 and SEQ ID NO.29, the sequences shown in SEQ ID NO. 31 and the sequence shown in 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.
  • the second aspect of the present invention provides an isolated polynucleotide encoding the fusion protein provided by the first aspect of the present invention.
  • the above-mentioned polynucleotide may be RNA, DNA, cDNA, or the like.
  • Methods of providing such isolated polynucleotides should be known to those skilled in the art. For example, they can be prepared by methods such as automated DNA synthesis and/or recombinant DNA technology, or they can be isolated from suitable natural sources.
  • a third aspect of the present invention provides a construct comprising the isolated polynucleotide provided by the second aspect of the present invention.
  • Appropriate methods of constructing such constructs should be known to those skilled in the art.
  • the construct can be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc., and more specifically, it can be constructed by inserting the above-mentioned isolated polynucleotide into the multiple cloning site of the expression vector. .
  • the expression vector in the present invention generally refers 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.
  • 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.
  • these promoters may be lac or trp promoters including but not limited to E.
  • Marker genes can be used to provide a phenotypic trait for selection of transformed host cells, for example, can include, but are not limited to, dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), Or for tetracycline or ampicillin resistance in E. coli etc.
  • the expression vector may also include an enhancer sequence. If an enhancer sequence is inserted into the vector, transcription will be enhanced.
  • An enhancer is a cis-acting factor of DNA, usually about There are 10 to 300 base pairs and act on the promoter to enhance transcription of the gene.
  • the fourth aspect of the present invention provides an expression system comprising the construct provided by the third aspect of the present invention or the exogenous polynucleotide provided by the second aspect of the present invention integrated into the genome, so as to express the above-mentioned fusion protein.
  • the above-mentioned expression system can be a host cell, and any cell suitable for 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; Fungal cells, or higher eukaryotic cells such as mammalian cells.
  • 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 Animal cells of melanoma cells, etc.
  • Methods for introducing constructs into host cells should be known to those skilled in the art, for example, microinjection, biolistic, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation can be used method, etc.
  • the fifth aspect of the present invention provides a method for preparing the fusion protein provided in the first aspect of the present invention.
  • a suitable method to prepare the fusion protein may include: under suitable conditions
  • the expression system provided by the fourth aspect of the present invention is cultured to express the fusion protein, the culture containing the fusion protein is collected, and then separated and purified to provide the fusion protein.
  • the sixth aspect of the present invention provides the use of the fusion protein provided by the first aspect of the present invention and the culture of the expression system provided by the fourth aspect of the present invention in preparing medicine.
  • the above-mentioned drug can be a drug used for the treatment of tumors, for example, can be cancer or solid tumor, specifically can be lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classic Hodge Golden lymphoma, hematological malignancies, sarcomas, head and neck cancer, and nasopharyngeal cancer, etc., these cancers can be early, intermediate or advanced stage, such as metastatic cancer, etc.
  • the seventh aspect of the present invention provides a pharmaceutical composition comprising the culture of the fusion protein provided by the first aspect of the present invention or the expression system provided by the fourth aspect of the present invention.
  • the content of fusion protein or culture is usually a therapeutically effective amount.
  • a "therapeutically effective amount” generally refers to an amount that, after an appropriate period of administration, can result in a reduction in the severity of symptoms of a disease, an increase in the frequency and duration of asymptomatic periods of the disease, or in the prevention of symptoms caused by the suffering of the disease. injury or disability.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors.
  • a therapeutically effective amount of the fusion protein, pharmaceutical composition is generally capable of reducing tumor size, or otherwise alleviating symptoms in a subject.
  • Those skilled in the art can select an appropriate therapeutically effective amount according to the actual situation, for example, it can be the size of the subject, the severity of the subject's symptoms and the selected specific composition or route of administration.
  • the prescription of treatment eg, determination of dosage, etc.
  • a pharmaceutically acceptable carrier may also be included.
  • the above-mentioned carriers may include various excipients and diluents which are not themselves essential to the active ingredient and which are not unduly toxic after administration. Suitable carriers will be well known to those skilled in the art, for example, a thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).
  • the eighth aspect of the present invention provides a method of treatment, comprising: administering to an individual a therapeutically effective amount of the fusion protein provided by the first aspect of the present invention, the culture of the expression system provided by the fourth aspect of the present invention, or the seventh aspect of the present invention
  • the pharmaceutical composition provided by the aspect comprising: administering to an individual a therapeutically effective amount of the fusion protein provided by the first aspect of the present invention, the culture of the expression system provided by the fourth aspect of the present invention, or the seventh aspect of the present invention
  • the pharmaceutical composition provided by the aspect comprising: administering to an individual a therapeutically effective amount of the fusion protein provided by the first aspect of the present invention, the culture of the expression system provided by the fourth aspect of the present invention, or the seventh aspect of the present invention
  • the pharmaceutical composition provided by the aspect comprising: administering to an individual a therapeutically effective amount of the fusion protein provided by the first aspect of the present invention, the culture of the expression system provided by the fourth aspect of the present invention, or the seventh aspect of the present invention
  • treatment includes prophylactic, curative or palliative manipulations that result in desired pharmaceutical and/or physiological effects.
  • a better therapeutic effect is one that medically reduces one or more symptoms of the disease or completely eliminates the disease, or blocks, delays the onset of the disease and/or reduces the risk of developing or worsening the disease.
  • “individual” generally includes humans, non-human primates, or other mammals (such as dogs, cats, horses, sheep, pigs, cattle, etc.), which can be obtained by utilizing the formulation, kit or combination benefit from treatment.
  • the fusion protein, the culture of the expression system, or the pharmaceutical composition can be used as a single active ingredient, or can be used in combination with other agents, so as to be administered in combination therapy.
  • the above-mentioned multi-domain fusion protein with anticancer activity, the culture of the expression system, or the pharmaceutical composition can be combined with at least one other antitumor drug.
  • the above-described multi-domain fusion proteins with anticancer activity, cultures of expression systems, or pharmaceutical compositions can be used in combination with antibodies targeting other tumor-specific antigens.
  • the multi-domain fusion protein with anti-cancer activity provided by the present invention can block the PD-L1/PD-1 interaction with anti-PD-L1 monoclonal antibody, and the anti-VEGF monoclonal antibody can reduce microvessel in one antibody fusion protein molecule.
  • Growth and inhibition of metastatic disease, and the function of TGF- ⁇ receptors to relieve cancer cells' tolerance to TGF- ⁇ signals and enhance immune response are organically combined, so that they can be used for tumor treatment and have good industrialization prospects.
  • the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques have been well described in the existing literature. For details, please refer to 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; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M. Wassarman and A.P. Wolfe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, et al.
  • the amino acid sequences of the multi-domain fusion protein, PD-L1-Fc fusion protein (SEQ ID NO. 47) and Fc-TGF ⁇ RII fusion protein (SEQ ID NO. 48) in Table 1 were respectively converted into Base sequence, and introduced HindIII restriction site and Kozak sequence (GCCACC) at the 5' end of the heavy chain and light chain coding sequences respectively, and introduced a stop codon and EcoRI restriction site at the 3' end by gene synthesis (general Biosystems (Anhui) Co., Ltd.) obtained full-length DNA.
  • GCCACC HindIII restriction site and Kozak sequence
  • the synthetic heavy chain and light chain coding genes were double digested by HindIII-HF (purchased from NEB, R3104V) and EcoRI-HF (purchased from NEB, R3101V), and the reagents were recovered in small amounts with agarose gel DNA/PCR products
  • the cassette purchased from Biomiga
  • the clones were picked for identification and confirmed by sequencing, and the heavy chain and light chain expression plasmids based on pCDNA3.1(+) were constructed respectively.
  • the heavy chain and light chain expression plasmids were extracted separately with endotoxin-removing plasmid large-scale kit (purchased from Biomiga, BW-PD3511-02), and the two were mixed 1:1.
  • the anti-PD-L1 single domain antibody is located at the N-terminus of the heavy chain of the anti-VEGF mAb
  • the 100% eluate was diluted to conductance ⁇ 3ms/cm, and the supernatant was adjusted to pH 7.0 and loaded onto a DSP column (Borgron Biotechnology Co., Ltd.), with 15% and 100% elution (20mM PB, 0.5 M NaCl, pH 7.0). 15% of the elution fraction was obtained, which was the target protein.
  • the protein concentration was determined by UV280 method.
  • the anti-PD-L1 single domain antibody is located at the N-terminus of the light chain of the anti-VEGF monoclonal antibody
  • the 100% eluate was diluted to a conductivity of 4ms/cm, and the sample was loaded on a Super Q (TOSOH) column.
  • the protein concentration was determined by UV280 method.
  • Detector Agilent 1100LC; Detection wavelength: 214nm; Mobile phase: 150mM pH7.0PB+5% isopropanol; Chromatographic column: Superdex 200Increase 5/150GL; Running time: 15 minutes; Column temperature 25°C. Test results show that the purity is greater than 95%.
  • 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.
  • the human PD-L1 gene (GenBank: NM_014143.2) was used as a template, and the PD-L1 fragment was obtained by high-fidelity amplification, and the PD-L1 fragment was recombined and inserted into pCDNA3.1-antiCD3TM to construct pCDNA3.1-antiCD3TM-PDL1.
  • CHO cells (Thermo) were transfected and then selected with G418 for 10-14d to generate the stable cell line CHO-antiCD3TM-PDL1.
  • the obtained fragment was amplified with the human PD1 gene (GenBank: NP_005009.2) as the template, and then recombined with the PB513B1-dual-puro vector (Youbao Bio) digested by HindIII-BamHI (Takara) to construct the plasmid pB-PD1.
  • High-fidelity amplification was carried out with pGL4.30 (Youbao Bio) as the template, and the obtained fragment was recovered and recombined with the pB-PD1 vector digested by SfiI-XbaI (Takara) to construct the pB-NFAT-Luc2p-PD1 plasmid.
  • the plasmid was extracted with endotoxin-removing plasmid extraction kit (Biomiga) and used to transfect Jurkat cells (Stem Cell Bank of Chinese Academy of Sciences).
  • endotoxin-removing plasmid extraction kit Biomiga
  • Jurkat cells were treated to a relatively adherent state by using 0.1 mg/ml of poly-D-lysine, and then according to the lipofection kit (Lipofectamine 3000; invitrogen)
  • the transfection instructions for transfection of Jurkat cells were carried out; on the third day, pressurized selection was carried out with RPMI1640 medium (Thermo) containing 10% FBS and 2.5 ⁇ g/ml puromycin; After the recovery of cell viability, the content of puromycin was gradually increased to 4 ⁇ g/ml.
  • the monoclonal Jurkat-NFAT-Luc2p-PD1 cell line was obtained.
  • CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells Take CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells and count them, adjust the cell density to 4 ⁇ 10 6 /ml, and add 25 ⁇ l to each well of each cell in a 96-well plate; use 1% BSA for gradient dilution respectively.
  • the fusion protein sample prepared in Example 2 was added to the cells in 50 ⁇ l; after co-cultivation at 37°C and 5% CO 2 for 6 h, 10 ⁇ l of luciferase substrate (Promega, E2620) was added to each well, shaken on a shaker for 2 min, and read . The operation is as described in the kit.
  • HEK293 cells were plated in 6-well cell culture plates, 1.0 x 10 6 cells per well, and cultured overnight in a 37°C, 5% CO 2 incubator. according to The transfection reagent instructions were used to prepare a transfection system, including 1.0 ⁇ g of pcDNA-KDR plasmid and 4 ⁇ g of pGL4.30 plasmid. 48 hours after transfection, cells were expanded into 10 cm cell culture dishes, and G418 200 ⁇ g/ml and Hygromycin 100 ⁇ g/ml were added. The pressurized medium was replaced with fresh pressurized medium every 3 days until a clear colony of colonies emerged. The cells were digested and plated in a 96-well cell culture plate.
  • the chemiluminescence was detected after stimulation with 0.1 ⁇ g/ml VEGF for 6 hours, and the clones with obvious signal response were selected to continue to expand and cultivate.
  • the monoclonal HEK293-NFAT-KDR was finally 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 the cells were resuspended by adding assay medium (DMEM+5% FBS) ; cell count, adjust the cell density to 1.6 ⁇ 10 6 /ml; lay 96-well cell culture plate, 25ul per well; use the assay medium to prepare VEGF solution, the concentration is 60ng/ml; add to the cell culture plate, 25ul per well ; Use the analytical medium to prepare the fusion protein prepared in Example 2, add it to the cell culture plate, 25ul per well; incubate at 37°C, 5% CO 2 for 6h; 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
  • Mouse breast cancer cell 4T1 cells were cultured to about 90% confluence (10 cm round dish), digested with trypsin, and plated at 4 ⁇ 10 5 cells/well in a 6-well plate, and cultured overnight.
  • the extracted pGL4.48[luc2P SBE Hygro] plasmid sample was 4T1 cells were transfected. 24h after transfection, the resulting TGF ⁇ -4T1 cells were trypsinized and transferred to a 10cm dish, cultured with 10% FBS and 150ug/ml hygromycin (InvivoGen, Cat no.: ant-hg-1) in RPMI 1640 Pressurized screening of cells.
  • TGF ⁇ -4T1 cells after pressure screening for 10-15 days were plated at 2 cells/well for monoclonal screening, and TGF ⁇ 1 (Novoprotein, 10ug, Cat no.: CA59) was used to stimulate monoclonal cells to verify monoclonal transfection Finally, TGF ⁇ -4T1 monoclonal cells were obtained.
  • TGF ⁇ -4T1 cells were confluent to about 90%, add about 2.5ml of 0.25% trypsin for digestion, and digest at room temperature for 2 minutes; the cells adhered to the culture dish were pipetted down and dispersed in the trypsin solution by pipetting , the whole digestion process takes ⁇ 5min, add complete 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 and centrifuge at 1000rpm for 5min; discard the supernatant and add 2ml complete medium to resuspend the cells, and use a cell counter to measure the cell density; dilute the cells with complete medium (RPMI 1640+10% FBS) to make the cell density 2 ⁇ 10 5 cells/ml; put the diluted cells ( The density is 2 ⁇ 10 5 cells/ml) plating (96-well plate), each well is 100ul, the cell density is 2 ⁇ 10 4 cells/well, and the 96-well plate is
  • mice C57BL/6 mice were divided into 4 groups, Avastin (Roche) low-dose (1mg/kg) and high-dose (10mg/kg), TAF-6 low-dose (1mg/kg) and high-dose groups (10mg/kg) ), 8 in each group, half male and half female.
  • the mice were given a single dose of the drug by tail vein injection, and the pharmacokinetic samples were collected at different time points. The collection time was before the drug, 1h, 6h, 24h, 48h, 78h, 120h, 144h, 168h, 192h, 216h after the drug.
  • the serum drug concentration in the pharmacokinetic 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 to concentration value according to the relationship between signal and concentration value of standard curve, and calculated using non-compartmental model of PK Solver software Main pharmacokinetic parameters.
  • Avastin low-dose and high-dose groups have a dose-to-value ratio of 1:10, Cmax ratio of 1:9.3, AUClast ratio of 1:8.9, Tmax for both 1h, and exposure ( Cmax and AUClast ) of The increase was dose proportional.
  • TAF-6 low-dose and high-dose groups have a dose-to-value ratio of 1:10, Cmax ratio of 1:8.6, AUC last ratio of 1:8.5, Tmax for both 1h, exposure ( Cmax and AUClast ) The increase was dose proportional.
  • AUC and C max were dose-linearly correlated.
  • the in vivo efficacy of the multi-domain fusion proteins of the present invention was determined by in vivo modeling of MDA-MB-231 (human breast cancer) cells in human PBMC immune system humanized mice (M-NSG mice). Screening 6-8 week old female M-NSG mice, mice were inoculated with MDA-MB-231 cells (10*10E6 + Matrigel 25%), and PBMC (5*10E6/0.2ml) was injected into the tail vein on the 7th day. The tumor volume and body weight were observed. Mice with tumor volume between 140-260 mm 3 were selected and randomly divided into 6 groups according to their tumor volume and body weight, with 7 mice in each group. Tumor-bearing mice were eliminated if the tumor volume was too large or too small.
  • 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), Fc-TGF ⁇ RII fusion protein ( SEQ ID NO. 48) is a fusion protein of human IgG Fc and TGF ⁇ RII (TGF ⁇ RII is located at the C-terminus of Fc).
  • Example 6 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized breast cancer MDA-MB-231 mice
  • the tumor mass of human breast cancer MDA-MB-231 was subcutaneously inoculated into the right anterior flank of female NCG mice, and PBMC cells were inoculated into the mice 1 day after tumor mass inoculation.
  • a total of 6 groups, 10 animals in each group respectively: Vehicle group, TAF-6 low (2mg/kg, ip, tiw ⁇ 9) group, TAF-6 medium (6 mg/kg, ip, tiw ⁇ 9) ) group, TAF-6 high (18mg/kg, ip, tiw ⁇ 9) group, Tecentriq (4mg/kg, ip, tiw ⁇ 9) group, Avastin (4mg/kg, ip, tiw ⁇ 9) group.
  • the tumor volume and body weight were measured every week, and the relationship between the changes of body weight and tumor volume of tumor-bearing mice and the administration time was recorded.
  • test drug TAF-6 has a significant anti-tumor effect on PBMC humanized breast cancer MDA-MB-231 subcutaneous xenograft model, effectively inhibits tumor growth, and its anti-tumor effect is significantly better than Tecentriq, And the tumor inhibitory effect was enhanced with the increase of dose.
  • 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 in the right anterior flank of male NCG mice, PBMC cells were inoculated into mice 4 days before tumor cell inoculation, and administered in groups when the tumor grew to about 50 mm 3 .
  • mice in each group ate and drank normally, and the body weight was stable, and there was no death of the experimental animals.
  • the tumor growth inhibition rates of TAF-6 low group, TAF-6 medium group, TAF-6 high group, Tecentriq group, Avastin group, and Tecentriq+Avastin group were 62%, 62%, and 62%, respectively. 75%, 13%, 34%, and 32%.
  • the tumor volume in each group was significantly smaller than that in the Vehicle group (all p ⁇ 0.01), and the tumor volumes in the TAF-6 low, medium and high groups were significantly smaller than those in the Tecentriq group, respectively , Avastin group and Tecentriq+Avastin group (all p ⁇ 0.01).
  • Table 4 and Figure 5 The results are shown in Table 4 and Figure 5.
  • test drug TAF-6 has a significant anti-tumor effect on PBMC humanized lung cancer Calu-6 subcutaneous xenograft model, effectively inhibits tumor growth, and its tumor-inhibitory effect is significantly better than that of Tecentriq, Avastin, Tecentriq and Tecentriq. Avastin combined, and the tumor suppressive effect increased with increasing dose.
  • the tumor inhibitory activity of the test drug was compared with that of the positive drug M7824 analog.
  • Set up 4 groups of 6 animals namely: Vehicle group, TAF-6 (7mg/kg, i.p., TIW x 8 times) group, M7824 analog (6mg/kg, i.p., TIW x 8 times) group, Avastin (5mg/kg, i.p., TIW x 8 times) group.
  • TAF-6 7mg/kg, i.p., TIW x 8 times
  • M7824 analog (6mg/kg, i.p., TIW x 8 times) group
  • Avastin 5mg/kg, i.p., TIW x 8 times
  • 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 in vivo human PBMC immune system humanized mice (NOG mice) to determine the in vivo efficacy of the multi-domain fusion proteins of the present invention. 7-9 weeks old female NOG mice were screened, the mice were inoculated with HCT-116 cells (3*10E6+Matrigel), and PBMC (5*10E6/0.2ml) was injected into the tail vein on the third day after inoculation with tumor cells, and the tumor volume was observed after and body weight, mice with tumor volume between 60-100 mm 3 were selected and randomly divided into 6 groups according to their tumor volume and body weight, with 8 mice in each group.
  • TGI TV (%) was calculated and analyzed statistically.
  • the tumor suppressor activity is shown in Figure 7.
  • the test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized colon cancer HCT116 subcutaneous xenograft model, effectively inhibiting tumor growth, and the high-dose and medium-dose groups have significantly better tumor-inhibiting effects than Tecentriq.
  • TAF-6 7mg/kg, i.p., tiw ⁇ 3) group compared with Tecentriq+Avastin (5+5mg/kg, i.p., tiw ⁇ 3) group
  • TAF-6 can still observe no difference. Inferior to, or even better than, the efficacy of the Tecentriq + Avastin group.
  • Example 9 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized liver cancer Huh-7 mice
  • Human hepatoma Huh-7 cells were inoculated subcutaneously in the right anterior flank of male NCG mice, PBMC cells were inoculated into mice 45 days before tumor cell inoculation, and administered in groups when the tumor grew to about 50 mm 3 .
  • 5 groups, 10 in each group respectively: Isotype group, TAF-6 low (2mg/kg, ip, tiw ⁇ 8) group, TAF-6 high (7mg/kg, ip, tiw ⁇ 8) group, Tecentriq ( 5mg/kg, ip, tiw ⁇ 8) group, Avastin (5mg/kg, ip, tiw ⁇ 8) group.
  • the tumor volume and body weight were measured twice a week, and the relationship between the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.
  • the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected.
  • the tumor growth inhibition rate TGI TV (%) was calculated and analyzed statistically.
  • mice in each group ate and drank normally, and their body weight was stable.
  • the tumor growth inhibition rates of the TAF-6 low group, TAF-6 high group, Tecentriq group, and Avastin group were 57%, 74%, 18%, and 67%, respectively.
  • the tumor volume in each group was significantly smaller than that in the Isotype group (all p ⁇ 0.01)
  • the tumor volume in the TAF-6 high group was significantly smaller than that in the Tecentriq group (all p ⁇ 0.01)
  • the tumor volume in the Avastin group was significantly smaller than that in the Tecentriq group (p ⁇ 0.05).
  • the results are shown in Figure 8.
  • test drug TAF-6 has a significant anti-tumor effect on the PBMC humanized liver cancer Huh-7 subcutaneously transplanted tumor model, effectively inhibiting the tumor growth, the anti-tumor effect is significantly better than that of Tecentriq, and the tumor inhibitory effect increased with increasing dose.
  • the tumor inhibitory activity of the test drug was compared with that of the positive drug M7824 analog.
  • Vehicle group TAF-6 (7mg/kg, i.p., tiw x 9; later dose adjusted to 14mg/kg, i.p., tiw x 2) group
  • M7824 analog 6mg/kg, i.p., tiw x 9; post dose adjusted to 12mg/kg, i.p., tiw x 2) group
  • Avastin 5mg/kg, i.p., tiw x 9; post dose adjusted to 10mg/kg, i.p., tiw x 2) 2 teams.
  • the results are shown in Figure 9, the anti-tumor effect of TAF-6 was significantly better than that of the M7824 analog.
  • Example 10 Tumor inhibitory activity of multi-domain fusion protein in PBMC humanized sarcoma HT1080 mice
  • Human-derived sarcoma HT1080 cells were inoculated subcutaneously into the right anterior flank of male NCG mice, and PBMC cells were inoculated into mice 7 days before tumor cell inoculation.
  • 8 in each group respectively: Vehicle group, TAF-6 low (2mg/kg, ip, tiw ⁇ 9) group, TAF-6 medium (7mg/kg, ip, tiw ⁇ 9) group, TAF-6 high (25mg/kg, ip, tiw ⁇ 9) group, Tecentriq (5mg/kg, ip, tiw ⁇ 9) group.
  • the tumor volume and body weight were measured every week, and the relationship between the changes of body weight and tumor volume of tumor-bearing mice and the administration time was recorded.
  • the tumor-bearing mice were euthanized, the tumors were removed, weighed, photographed, and serum and tumors were collected.
  • the tumor growth inhibition rate TGI TV (%) was calculated and analyzed statistically.
  • the tumor growth inhibition rates of TAF-6 low group, TAF-6 medium group, TAF-6 high group, and Tecentriq group were 19%, 33%, 41%, and 28%, respectively.
  • the tumor volume in the middle group and the Tecentriq group was significantly smaller than that in the Vehicle group (both p ⁇ 0.05), and there was no significant difference in tumor volume between the groups (p>0.05).
  • the results are shown in Figure 10.
  • the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

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Abstract

本发明提供一种融合蛋白，所述融合蛋白包括抗PD-L1单域抗体片段、拮抗VEGF片段、TGF-β结合片段。本发明所提供的具有抗癌活性的多结构域融合蛋白，能够在一个抗体融合蛋白分子中，将抗PD-L1单抗阻断PD-L1/PD-1相互作用、抗VEGF单抗减少微血管生长和抑制转移性疾病，以及TGF-β受体解除癌细胞对TGF-β信号的耐受、增强免疫应答的功能有机地结合在一起，从而可以用于治疗肿瘤。

Description

多结构域融合蛋白及其应用 技术领域
本发明涉及生物技术领域，特别是涉及一种具有抗癌活性的多结构域融合蛋白及其制备方法和用途。
背景技术
肿瘤细胞和正常细胞不同的地方之一，在其生长的高代谢需求。肿瘤细胞依赖血管提供营养物质和氧气，处理代谢产物，并在现有血管上，促进新生血管的生成。在肿瘤分泌的促血管生成因子中，人血管内皮生长因子(VEGF)，特别是VEGF-A是导致肿瘤血管生成的一个重要因素(Josep Garcia等人,2020，Cancer Treatment Reviews 86：1-2)。因此,对VEGF信号通路的抑制可限制多种肿瘤的进展。比如贝伐珠单抗(Bevacizumab,商品名
Figure PCTCN2022076610-appb-000001
)，一种人源化抗-VEGF单克隆抗体，可与VEGF结合，阻止VEGF与内皮细胞表面VEGF受体(Flt-1和KDR)相互作用。目前贝伐珠单抗被FDA批准用于转移性结直肠癌，晚期、转移性或复发性非小细胞肺癌和复发性胶质母细胞瘤等的治疗。
转化生长因子TGF-β是一种细胞因子，可通过调节细胞的生长、分化、增殖和生存来维持组织稳态。尽管TGF-β通路在肿瘤的早期阶段，可通过促进细胞周期停滞和凋亡来控制肿瘤；但在肿瘤的晚期阶段，TGF-β也能通过抑制细胞毒性T细胞并促进癌细胞的增殖、侵袭和转移最终使得肿瘤逃逸，这种功能转换被称为“TGF-β悖论”。TGFβ信号通路诱导T H1细胞向Tregs分化，削弱CD8+效应T细胞的激活，并限制中央记忆T细胞的发育，从根本上影响肿瘤浸润T细胞功能。哺乳动物中，TGF-β主要有TGF-β1、TGF-β2、TGF-β3三种亚型。通过高水平表达TGF-β，肿瘤得以免受免疫监视。与此相一致的是，高表达TGF-β的非小细胞肺癌(NSCLC)、肾细胞癌(CRC)，胃癌和前列腺癌均与肿瘤进展和不良预后相关(Marin-Acevedo等人，Journal of Hematology & Oncology，11:39，2018)。这说明，TGF-β拮抗将是肿瘤治疗的一个潜在新方向。
目前，作为免疫检查点抑制剂的抗PD-1/PD-L1单抗已广泛应用于肿瘤治疗。抗PD-L1单抗Avelumab与TGF-β受体II(TGF-βTrap)融合而成的M7824已经进入临床阶段。而抗PD-1/PD-L1单抗、TGF-β拮抗剂和抗VEGF单抗的组合却仍未见报道。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种具有抗癌活性的多结构域融合蛋白及其制备方法和用途，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明一方面提供一种融合蛋白，所述融合蛋白包括抗PD-L1单域抗体片段、拮抗VEGF片段、TGF-β结合片段。
本发明另一方面提供一种分离的多核苷酸，编码上述的融合蛋白。
本发明另一方面提供一种构建体，所述构建体含有上述的分离的多核苷酸。
本发明另一方面提供一种表达系统，所述表达系统含有上述的构建体或基因组中整合有外源的上述的多核苷酸。
本发明另一方面提供上述的融合蛋白的制备方法，包括：在合适的条件下培养如上述的表达系统，使之表达所述融合蛋白，分离、纯化以提供所述融合蛋白。
本发明另一方面提供上述的融合蛋白、或上述的表达系统的培养物在制备药物中的用途。
本发明另一方面提供一种药物组合物，包括上述的融合蛋白、或上述的表达系统的培养物。
附图说明
图1TAF-6与Avastin的血药浓度-时间曲线下面积图；
图2多结构域融合蛋白在PBMC免疫系统人源化MDA-MB-231乳腺癌皮下移植瘤模型中的抑瘤药效；
图3多结构域融合蛋白在PBMC免疫系统人源化MDA-MB-231乳腺癌皮下移植瘤模型中的抑瘤药效；
图4多结构域融合蛋白TAF-6、M7824类似物、Avastin在PBMC免疫系统人源化MDA-MB-231乳腺癌皮下移植瘤模型中的抑瘤药效；
图5多结构域融合蛋白在PBMC免疫系统人源化Calu-6肺癌皮下移植瘤模型中的抑瘤药效；
图6多结构域融合蛋白TAF-6、M7824类似物、Avastin在PBMC免疫系统人源化Calu-6肺癌皮下移植瘤模型中的抑瘤药效；
图7多结构域融合蛋白在PBMC免疫系统人源化HCT116结肠癌皮下移植瘤模型中的抑瘤药效；
图8多结构域融合蛋白在PBMC免疫系统人源化Huh-7肝癌皮下移植瘤模型中的的抑瘤药效；
图9多结构域融合蛋白TAF-6、M7824类似物、Avastin在PBMC免疫系统人源化Huh-7肝癌皮下移植瘤模型中的抑瘤药效；
图10多结构域融合蛋白在PBMC免疫系统人源化HT1080肉瘤皮下移植瘤模型中的抑瘤药效。
具体实施方式
本发明发明人经过大量探索研究，意外地发现了一种融合蛋白分子，该融合蛋白分子可以将抗PD-L1单抗阻断PD-L1/PD-1相互作用、拮抗VEGF单抗减少微血管生长和抑制转移性疾病、以及TGF-β受体改善肿瘤微环境中TGF-β引起的T细胞功能异常和增强免疫应答的功能结合在一起，具有优良的肿瘤抑制效果，在此基础上完成了本发明。
本发明第一方面提供一种融合蛋白，所述融合蛋白包括抗PD-L1单域抗体片段、拮抗VEGF片段、TGF-β结合片段。上述融合蛋白中，抗PD-L1单域抗体片段通常可以用于阻断PD-L1/PD-1相互作用，提高T淋巴细胞中IFN-γ和/或IL-2表达，从而抑制肿瘤生长。拮抗VEGF片段通常可以包括可以与FcRn受体结合的Fc部分，从而可以延长体内半衰期，同时也可与表达Fc受体的效应细胞结合起到杀伤癌细胞的作用。TGF-β结合片段可以通过清除肿瘤微环境中过量表达的TGF-β，提高肿瘤浸润T细胞对肿瘤细胞的杀伤功能。
本发明所提供的融合蛋白中，可以包括抗PD-L1单域抗体片段。上述抗PD-L1单域抗体片段通常可以是能够特异性与PD-L1结合的多肽或蛋白片段。抗PD-L1单域抗体片段中，通常缺失对应的抗体轻链，而只有重链可变区对应的片段。抗PD-L1单域抗体片段的结合特性通常可以由其包括的3个互补决定区(CDR，complementarity determining region)来决定，CDR区可以与框架区(FR，framework region)有序排列，FR区不直接参与结合反应。这些CDR可以形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，构成了抗体的抗原结合位点。例如，上述抗PD-L1单域抗体片段的互补决定区(CDR)可以包括氨基酸序列如SEQ ID NO.1～5其中之一所示的CDR1，SEQ ID NO.6～9其中之一所示的CDR2，SEQ ID NO.10～15其中之一所示的CDR3。
在本发明一具体实施例中，抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.1所示的CDR1，SEQ ID NO.6所示的CDR2，SEQ ID NO.10所示的CDR3。
在本发明另一具体实施例中，抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.2所示的CDR1，SEQ ID NO.7所示的CDR2，SEQ ID NO.11所示的CDR3。
在本发明另一具体实施例中，抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.3所示的CDR1，SEQ ID NO.7所示的CDR2，SEQ ID NO.12所示的CDR3。
在本发明另一具体实施例中，抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.4所示的CDR1，SEQ ID NO.8所示的CDR2，SEQ ID NO.13所示的CDR3。
在本发明另一具体实施例中，抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.2所示的CDR1，SEQ ID NO.7所示的CDR2，SEQ ID NO.14所示的CDR3。
在本发明另一具体实施例中，抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.5所示的CDR1，SEQ ID NO.9所示的CDR2，SEQ ID NO.15所示的CDR3。
上述抗PD-L1单域抗体片段中，还可以包括框架区(FR，framework region)。如上所述，CDR区可以与FR区有序排列，例如，抗PD-L1单域抗体片段自N端至C端可以依次包括FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。框架区FR包括氨基酸序列可以如SEQ ID No.49所示的FR1、氨基酸序列如SEQ ID No.50～52其中之一所示的FR2、氨基酸序列如SEQ ID No.53～55其中之一所示的FR3、和氨基酸序列如SEQ ID No.56所示的FR4。
在本发明一具体实施例中，所述框架区FR包括：
氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.50所示的FR2，SEQ ID NO.53所示的FR3；SEQ ID NO.56所示的FR4，或
氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.51所示的FR2，SEQ ID NO.54所示的FR3；SEQ ID NO.56所示的FR4，或
氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.52所示的FR2，SEQ ID NO.54所示的FR3；SEQ ID NO.56所示的FR4，或
氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.52所示的FR2，SEQ ID NO.55所示的FR3；SEQ ID NO.56所示的FR4。
在本发明另一具体实施例中，抗PD-L1单域抗体片段可以包括：a)氨基酸序列如SEQ ID No.16～21其中之一所示的多肽片段；或，b)氨基酸序列与SEQ ID No.16～21其中之一具有80％以上序列一致性且具有a)限定的多肽片段的功能的多肽片段。具体的，上述b)中的多肽片段具体指：氨基酸序列如SEQ ID No.16～21其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，且具有氨基酸序列如SEQ ID No.16～21其中之一所示的多肽片段的功能的多肽片段，例如，可以是与PD-L1特异性结合的能力，也可以是对PD-L1/PD-1相互作用的阻断，从而可以阻断PD-L1/PD1通路，也可以是提高 T淋巴细胞中IFN-γ和/或IL-2表达的功能，还可以是抑制肿瘤生长的功能。上述b)中的抗PD-L1单域抗体片段的氨基酸序列可以与SEQ ID No.16～21其中之一具有80％、85％、90％、93％、95％、97％、或99％以上的一致性。上述抗PD-L1单域抗体片段通常可以来源于羊驼(Vicugna pacos)，例如，其CDR区可以来源于羊驼。上述抗PD-L1单域抗体片段通常可以是人源化的，例如，其框架区可以来源于人。
本文中，序列一致性(sequence identity)指参与对比的序列中相同残基的百分比。可采用本领域周知的计算软件计算两条或多条目的序列的序列一致性，这些软件可获自如NCBI。
本发明所提供的融合蛋白中，可以包括拮抗VEGF片段。上述拮抗VEGF片段通常可以是能够拮抗VEGF的多肽或蛋白片段。例如，上述拮抗VEGF片段可以是单克隆抗体等。再例如，上述拮抗VEGF片段可以是贝伐珠单抗等。
在本发明一具体实施例中，拮抗VEGF片段可以包括：
c)氨基酸序列如SEQ ID No.22～23其中之一所示的多肽片段；
d)氨基酸序列与SEQ ID No.22～23其中之一具有80％以上序列一致性且具有c)限定的多肽片段的功能的多肽片段。具体的，上述d)中的氨基酸序列具体指：如SEQ ID No.22～23其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID No.22～23其中之一所示的多肽片段的功能的多肽片段，例如，可以是拮抗VEGF的功能，还可以是与FcRn受体结合的Fc部分的功能，从而可以延长体内半衰期，同时也可与表达Fc受体的效应细胞结合起到杀伤癌细胞的作用。所述d)中的氨基酸序列可与SEQ ID No.22～23其中之一具有80％、85％、90％、93％、95％、97％、或99％以上的一致性。上述拮抗VEGF片段通常可以来源于小鼠(Mus musculus)，例如，其CDR区可以来源于小鼠。上述拮抗VEGF片段通常可以是人源化的，例如，其框架区可以来源于人。
本发明所提供的融合蛋白中，可以包括TGF-β结合片段。上述TGF-β结合片段通常可以特异性地结合各TGF-β异构体(例如，TGF-β1、TGF-β2和TGF-β3等)，TGF-β异构体通常在多种恶性肿瘤中高表达，很可能是导致临床治疗效果不佳的重要因素之一。例如，TGF-β结合片段可以是TGF-βRⅡ(TGF-β受体II)胞外区结构片段。
在本发明一具体实施例中，TGF-β结合片段可以包括：
e)氨基酸序列如SEQ ID No.24所示的多肽片段；
f)氨基酸序列与SEQ ID No.24具有80％以上序列一致性且具有e)限定的多肽片段的功能的多肽片段。具体的，上述f)中的氨基酸序列具体指：如SEQ ID No.24所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID No.24所示的多肽片段的功能的多肽片段，例如，可以结合各TGF-β异构体(例如，TGF-β1、TGF-β2和TGF-β3等)，从而可以清除肿瘤微环境中过量表达的TGF-β，还可以提高肿瘤浸润T细胞对肿瘤细胞的杀伤功能。所述f)中的氨基酸序列可与SEQ ID No.24具有80％、85％、90％、93％、95％、97％、或99％以上的一致性。上述TGF-β结合片段通常可以来源于人(homo sapiens)。
本发明所提供的融合蛋白中，还可以包括连接肽片段。所述融合蛋白中，通常可以包括多个连接肽片段，至少部分的结构域或各结构域之间均可以设有连接肽片段。例如，抗PD-L1单域抗体片段、拮抗VEGF片段之间可以设有连接肽。再例如，拮抗VEGF片段、TGF-β结合片段之间可以设有连接肽。上述连接肽片段通常可以为一段长度合适的富含G、S和/或A(主要由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成)的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动。例如，所述连接肽片段的氨基酸序列可以包括如(GS)n、(GGS)n、(GGSG)n、(GGGS)nA、(GGGGS)nA、(GGGGS)nG、(GGGGA)nA、(GGGGG)nA等序列，其中，n选自1-10之间的整数。在本发明一具体实施例中，所述连接肽片段的氨基酸序列的长度可以为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、或28-30。在本发明一更优选的具体实施例中，所述连接肽片段可以包括氨基酸序列如SEQ ID NO.34-36其中之一所示的多肽片段。
本发明所提供的融合蛋白中，融合蛋白可以是线性的，例如，融合蛋白可以自N端至C端可以依次包括抗PD-L1单域抗体片段、拮抗VEGF片段、TGF-β结合片段。融合蛋白也可以具有类似于单克隆抗体的结构，例如，抗PD-L1单域抗体片段可以位于拮抗VEGF片段的重链的N端，再例如，抗PD-L1单域抗体片段可以位于拮抗VEGF片段的轻链的N端，再例如，TGF-β结合片段可以位于拮抗VEGF片段的重链的C端。在本发明一具体实施例中，所述融合蛋白的氨基酸序列可以包括SEQ ID NO.23、SEQ ID NO.25-33其中之一所示的序列，例如，所述融合蛋白的氨基酸序列可以包括SEQ ID NO.25和SEQ ID NO.26所示的序列、SEQ ID NO.25和SEQ ID NO.27所示的序列、SEQ ID NO.25和SEQ ID NO.28所示的序列、SEQ ID NO.25和SEQ ID NO.29所示的序列、SEQ ID NO.30和SEQ ID NO.27所示 的序列、SEQ ID NO.30和SEQ ID NO.29所示的序列、SEQ ID NO.31和SEQ ID NO.23所示的序列、SEQ ID NO.32和SEQ ID NO.23所示的序列、SEQ ID NO.33和SEQ ID NO.23所示的序列。
本发明第二方面提供一种分离的多核苷酸，编码本发明第一方面所提供的融合蛋白。上述多核苷酸可以是RNA、DNA或cDNA等。提供所述分离的多核苷酸的方法对于本领域技术人员来说应该是已知的。例如，可以通过自动DNA合成和/或重组DNA技术等方法制备获得，也可以从适合的天然来源加以分离。
本发明第三方面提供一种构建体，上述构建体含有本发明第二方面所提供的分离的多核苷酸。合适的构建上述构建体的方法对于本领域技术人员来说应该是已知的。例如，所述构建体可以通过体外重组DNA技术、DNA合成技术、体内重组技术等方法构建获得，更具体的，可以由上述的分离的多核苷酸插入到表达载体的多克隆位点构建而成。本发明中的表达载体通常指本领域熟知的各种市售表达载体等，例如可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。通常来说，合适的载体可以包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如，这些启动子可以是包括但不限于大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、毕赤酵母的甲醇氧化酶启动子和其它一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。标记基因可用于提供用于选择转化的宿主细胞的表型性状，例如，可以是包括但不限于真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性等。当所述的多核苷酸被表达时，表达载体中还可以包括增强子序列，如果在载体中插入增强子序列，则将会使转录得到增强，增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本发明第四方面提供一种表达系统，所述表达系统含有本发明第三方面所提供的构建体或基因组中整合有外源的本发明第二方面所提供的多核苷酸，从而可表达上述的融合蛋白。上述表达系统可以是宿主细胞，任何适用于表达载体进行表达的细胞都可以作为宿主细胞，例如，宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；丝状真菌细胞、或是高等真核细胞，如哺乳动物细胞。代表性的例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、丝状真菌、植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。将构建体导入宿主细胞 的方法对于本领域技术人员来说应该是已知的，例如，可以显微注射法、基因枪法、电穿孔法、病毒介导的转化法、电子轰击法、磷酸钙沉淀法等方法。
本发明第五方面提供本发明第一方面所提供的融合蛋白的制备方法，本领域技术人员可选择合适的方法以制备所述融合蛋白，例如，所述制备方法可以包括：在合适的条件下培养本发明第四方面所提供的表达系统，使之表达所述融合蛋白，收集含有所述融合蛋白的培养物，而后分离及纯化以提供所述融合蛋白。
本发明第六方面提供本发明第一方面所提供的融合蛋白、本发明第四方面所提供的表达系统的培养物在制备药物中的用途。上述药物可以是用于治疗肿瘤的药物，例如，可以是癌症或实体肿瘤，具体可以是肺癌、黑色素瘤、胃癌、卵巢癌、结肠癌、肝癌、肾癌、膀胱癌、乳腺癌、经典霍奇金淋巴瘤、血液恶性肿瘤、肉瘤、头颈癌和鼻咽癌等，这些癌症可以为早期、中期或晚期，例如转移癌等。
本发明第七方面提供一种药物组合物，包括本发明第一方面所提供的融合蛋白或本发明第四方面所提供的表达系统的培养物。上述药物组合物中，融合蛋白或培养物的含量通常为治疗有效量的。本发明中，“治疗有效量”通常指一用量在经过适当的给药期间后，能够导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。治疗有效量的融合蛋白、药物组合物通常能够减小肿瘤大小，或者以其他方式缓解对象的症状。本领域技术人员可以根据实际情况选择合适的治疗有效量，例如，可以是对象的大小、对象症状的严重性和选择的特定组合物或给药途径。治疗的处方(例如，对剂量的决定等)可以是由医生确定的，通常考虑的因素包括但不限于所治疗的疾病、患者个体的情况、递送部位、施用方法以及其它因素等。
本发明所提供的药物组合物中，还可以包括药学上可接受的载体。上述载体可以包括各种赋形剂和稀释剂，这些载体本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体对于本领域技术人员来说应该是熟知的，例如，在Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.，1991)中可找到关于药学上可接受的载体的充分讨论。
本发明第八方面提供一种治疗方法，包括：向个体施用治疗有效量的本发明第一方面所提供的融合蛋白、本发明第四方面所提供的表达系统的培养物、或本发明第七方面所提供的药物组合物。
本发明中，“治疗”一词包括可导致理想的药学和/或生理效果的预防性、治愈性或缓和性 操作。治疗效果较佳是指医疗上可减少疾病的一种或多种症状或者完全消除疾病，或阻滞、延迟疾病的发生和/或降低疾病发展或恶化的风险。
本发明中，“个体”通常包括人类、非人类的灵长类，或其他哺乳动物(如狗、猫、马、羊、猪、牛等)，其可因利用所述制剂、试剂盒或联合制剂进行治疗而获益。
本发明中，上述融合蛋白、表达系统的培养物、或药物组合物可以作为是单一有效成分，也可以与其他药剂联用，从而在联合治疗中施用。例如，可以将上述具有抗癌活性的多结构域融合蛋白、表达系统的培养物、或药物组合物联合其他至少一种抗肿瘤药物。再例如，可以将上述具有抗癌活性的多结构域融合蛋白、表达系统的培养物、或药物组合物与靶向其它肿瘤特异性抗原的抗体联合使用。
本发明所提供的具有抗癌活性的多结构域融合蛋白，能够在一个抗体融合蛋白分子中，将抗PD-L1单抗阻断PD-L1/PD-1相互作用、抗VEGF单抗减少微血管生长和抑制转移性疾病，以及TGF-β受体解除癌细胞对TGF-β信号的耐受、增强免疫应答的功能有机地结合在一起，从而可以用于治疗肿瘤，具有良好的产业化前景。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN  MOLECULAR BIOLOGY，John Wiley & Sons，New York，1987 and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1 融合蛋白的构建和重组表达制备
根据CHO细胞密码子偏好性分别将表1中多结构域融合蛋白氨基酸序列、PD-L1-Fc融合蛋白(SEQ ID NO.47)及Fc-TGFβRII融合蛋白(SEQ ID NO.48)各自转换成碱基序列，并分别在重链和轻链编码序列的5’端引入HindIII酶切位点和Kozak序列(GCCACC)，在3’端引入终止密码子及EcoRI酶切位点通过基因合成(通用生物系统(安徽)有限公司)获得全长DNA。将合成的重链和轻链编码基因分别进行HindIII-HF(购自NEB，R3104V)和EcoRI-HF(购自NEB，R3101V)双酶切，并用琼脂糖凝胶DNA/PCR产物小量回收试剂盒(购自Biomiga)进行切胶回收，与同样进行HindIII和EcoRI双酶切的pCDNA3.1(+)载体用T4连接酶(购自NEB，M0202V)进行连接，转化Top10感受态，涂布在LB氨苄青霉素抗性平板上培养。挑取克隆进行鉴定，并测序确认，分别构建了基于pCDNA3.1(+)的重链和轻链表达质粒。用去内毒素质粒大抽试剂盒(购自Biomiga，BW-PD3511-02)分别提取重链和轻链表达质粒，并将两者1:1混合。取1.0mg混合质粒，使用Wayne293表达培养基(购自中山康晟，A21501)稀释至25mL；取3.0mg PEI(线性，25KD，Polysciences，Inc.)，使用Wayne293表达培养基稀释至25mL后加入到质粒溶液中，混匀，室温孵育30分钟。取对数期生长的Hek293F细胞(活率>95％)，计数；1100rpm，离心10分钟，弃上清，用450mLWayne293表达培养基重悬细胞。将上述质粒-PEI混合物，加入到细胞悬液中，37℃，5％CO 2摇床培养箱中培养7天后，离心取上清，用于后续蛋白纯化。
表1 多结构域融合蛋白氨基酸序列和编码序列
Figure PCTCN2022076610-appb-000002
Figure PCTCN2022076610-appb-000003
实施例2 多结构域融合蛋白的纯化
2.1抗PD-L1单域抗体位于抗VEGF单抗的重链N端
将细胞发酵上清调至pH7.0后上样到Protein A亲和层析柱(博格隆生物技术有限公司)，平衡液为20mM PB,0.15M NaCl(pH＝7.0)，100％0.1M Gly-HCl(pH＝3.0)洗脱；洗脱液预先加入10％1M Tris-HCl(pH＝8.5)。100％洗脱液稀释至电导<3ms/cm，上清液调pH7.0上样至DSP层析柱(博格隆生物技术有限公司)，分别15％和100％洗脱(20mM PB，0.5M NaCl，pH7.0)。得到15％洗脱组分，即为目的蛋白。采用UV280法测定蛋白浓度。
2.2抗PD-L1单域抗体位于抗VEGF单抗的轻链N端
将细胞发酵上清调至pH7.0后上样到Protein A亲和层析柱(博格隆生物技术有限公司)，平衡液为20mM PB,0.15M NaCl(pH＝7.0)，100％0.1M Gly-HCl(pH＝3.0)洗脱；洗脱液预先加入10％1M Tris-HCl(pH＝8.5)。100％洗脱液稀释至电导4ms/cm，上样于Super Q(TOSOH)层析柱，平衡液为20mM Tris,pH8，洗脱液为500mM NaCl+20mM Tris(pH＝8.0)，分别35％和100％洗脱，通过流穿去除过量轻链，得到35％洗脱组分，即为目的蛋白。采用UV280法测定蛋白浓度。
纯度检测使用SEC-HPLC-UV分析。检测器：Agilent 1100LC；检测波长：214nm；流动相：150mM pH7.0PB+5％异丙醇；色谱柱：Superdex 200Increase 5/150GL；运行时间：15分钟；柱温25℃。检测结果显示纯度都大于95％。
PD-L1-Fc融合蛋白(SEQ ID NO.47)及Fc-TGFβRII融合蛋白(SEQ ID NO.48)的纯化：将细胞发酵上清调至pH7.0后上样到Protein A亲和层析柱(博格隆生物技术有限公司)，平衡液为20mM PB,0.15M NaCl(pH＝7.0)，100％0.1M Gly-HCl(pH＝3.0)洗脱；洗脱液预先加入10％1M Tris-HCl(pH＝8.5)用于中和。
实施例3 鉴定多结构域融合蛋白在体外的功能
3.1抗PD-L1单域抗体片段的体外活性检测：
合成CD5L-OKT3scFv-CD14基因序列(GenBank:ADN42857.1)，并用 HindIII-EcoRI(Takara)酶切，插入载体pCDNA3.1，构建pCDNA3.1-antiCD3TM。以人PD-L1基因(GenBank:NM_014143.2)为模板，高保真扩增得到PD-L1片段重组连接插入pCDNA3.1-antiCD3TM，构建pCDNA3.1-antiCD3TM-PDL1。转染CHO细胞(Thermo)，然后用G418选择10-14d来产生稳定细胞系CHO-antiCD3TM-PDL1。
以人PD1基因(GenBank:NP_005009.2)为模板扩增所得片段，与经HindIII-BamHI(Takara)酶切的PB513B1-dual-puro载体(优宝生物)重组连接，构建质粒pB-PD1。以pGL4.30(优宝生物)为模板进行高保真扩增，回收所得片段，与经SfiI-XbaI(Takara)酶切的pB-PD1载体重组连接，构建pB-NFAT-Luc2p-PD1质粒。质粒成功构建后用去内毒素质粒大抽试剂盒(Biomiga)提取质粒用于转染Jurkat细胞(中国科学院干细胞库)。参考专利CN 107022571A中的方法，通过使用0.1mg/ml的多聚-D-赖氨酸将Jurkat细胞处理成相对贴壁的状态，然后根据脂质体转染试剂盒(Lipofectamine 3000；invitrogen)中的转染说明对Jurkat细胞进行转染；第三天用含有10％FBS和2.5μg/ml嘌呤霉素的RPMI1640培养基(Thermo)进行加压筛选；此后每隔一段时间补加培养基，待细胞活率恢复后逐渐增加嘌呤霉素的含量至4μg/ml。最终获得单克隆Jurkat-NFAT-Luc2p-PD1细胞株。
取CHO-antiCD3TM-PDL1、Jurkat-NFAT-Luc2p-PD1细胞并计数，调整细胞密度为4×10 6/ml，96孔板中每孔每个细胞各加入25μl；分别用1％BSA梯度稀释实施例2中制备的融合蛋白样品，并加入50μl至细胞中；37℃、5％CO 2共培养6h后，每孔加入10μl荧光素酶底物(Promega,E2620)，振荡器上震荡2min，读数。操作如试剂盒说明。
3.2拮抗VEGF片段的体外活性检测：
HEK293细胞铺6孔细胞培养板，每孔1.0×10 6细胞，37℃，5％CO 2培养箱中过夜培养。按照
Figure PCTCN2022076610-appb-000004
转染试剂说明书配制转染体系，其中pcDNA-KDR质粒1.0μg、pGL4.30质粒4μg。转染48小时后，细胞放大到10cm细胞培养皿，添加G418 200μg/ml、Hygromycin 100μg/ml。每3天更换一次新鲜加压培养基，至有明显的克隆团长出。消化细胞，铺96孔细胞培养板，待单克隆长出后，使用0.1μg/ml VEGF刺激6小时后检测化学发光情况，挑选信号响应明显的克隆继续放大培养。最终获得单克隆HEK293-NFAT-KDR。HEK293-NFAT-KDR细胞按照4万个/每孔密度铺板，使用Accutase进行消化；收集消化后的细胞，1000rpm离心5分钟；弃上清，加入分析培养液(DMEM+5％FBS)重悬细胞；细胞计数，调整细胞密度至1.6×10 6/ml；铺96孔细胞培养板， 每孔25ul；使用分析培养液配制VEGF溶液，浓度为60ng/ml；加入到细胞培养板中，每孔25ul；使用分析培养液配制实施例2中制备获得的融合蛋白，加入到细胞培养板中，每孔25ul；37℃，5％CO 2孵育培养6h；每孔加入10ul Bright-Glo荧光素酶检测试剂(Promega，E2620)，震荡2分钟转移80ul裂解液至酶标白板中，酶标仪读数。
3.3 TGF-β结合片段的体外细胞活性检测：
小鼠乳腺癌细胞4T1细胞培养长满至90％左右(10cm圆皿)，胰酶消化，按照4×10 5个/孔将细胞铺板至6孔板中，培养过夜。抽提得到的pGL4.48[luc2P SBE Hygro]质粒样品采用
Figure PCTCN2022076610-appb-000005
对4T1细胞转染。转染24h后，得到的TGFβ-4T1细胞采用胰酶消化并转移至10cm培养皿，采用10％FBS和150ug/ml潮霉素(InvivoGen，Cat no.:ant-hg-1)的RPMI 1640培养基对细胞进行加压筛选。加压筛选10～15d后的TGFβ-4T1细胞按照2个/孔进行铺板以进行单克隆筛选，采用TGFβ1(Novoprotein，10ug，Cat no.:CA59)对单克隆细胞进行刺激验证单克隆的转染效果，最终获得TGFβ-4T1单克隆细胞。
TGFβ-4T1细胞长满至90％左右，加入2.5ml左右0.25％胰酶进行消化，室温消化2min；将贴壁在培养皿上的细胞吹打下来，并在胰酶溶液中吹打使其分散开来，整个消化过程使用时间～5min，加入完全培养基(RPMI 1640+10％FBS)终止消化，并继续吹打细胞至细胞分散均匀；细胞转移至50m离心管中，1000rpm离心5min；弃上清，加入2ml完全培养基使细胞重悬，采用细胞计数仪测量细胞密度；采用完全培养基(RPMI 1640+10％FBS)稀释细胞，使细胞密度为2×10 5个/ml；将稀释好的细胞(密度为2×10 5个/ml)铺板(96孔板)，每孔100ul，细胞密度为2×10 4个/孔，96孔板置于37℃培养箱中进行培养过夜；实施例2中制备的融合蛋白样品采用RPMI 1640+0.2％FBS培养基(含2ng/ml TGFβ1)稀释至指定浓度(各蛋白样品稀释后室温放置1h)；37℃培养箱过夜培养的TGFβ-4T1细胞弃去上清，加入50ul RPMI 1640+0.2％FBS培养基，然后加入50μl不同浓度的蛋白溶液；96孔板置于37℃培养箱中继续孵育3h后加入10ul Bright-Glo荧光素酶检测试剂(Promega，E2620)，震荡3min；酶标仪读数。
各多结构域融合蛋白的抗PD-L1活性、抗VEGF活性、抗TGFβ活性的测量结果如表2所示。由表2可知，各多结构域融合蛋白均具有良好的体外细胞活性。
表2 多结构域融合蛋白的体外细胞活性
Figure PCTCN2022076610-appb-000006
Figure PCTCN2022076610-appb-000007
实施例4 多结构域融合蛋白在C57BL/6小鼠体内的药代动力学
C57BL/6小鼠分为4组，分别为Avastin(Roche)低剂量(1mg/kg)和高剂量(10mg/kg)、TAF-6低剂量(1mg/kg)和高剂量组(10mg/kg)，每组8只，雌雄各半。小鼠尾静脉注射单次给予药物，分时间点交叉采集药代样品。采集时间为药前、药后1h、6h、24h、48h、78h、120h、144h、168h、192h、216h。通过ELISA方法定量检测药代样品中血清药物浓度。VEGF包板，二抗Goat anti-Human IgG Fc，HRP结合药物，采用TMB法进行检测，根据标准曲线的信号和浓度值关系进行回归转化为浓度值，并使用PK Solver软件的非房室模型计算主要药代动力学参数。
结果如图1所示，C57BL/6小鼠给药Avastin和TAF-6高低剂量组，药代参数相近。Avastin低、高剂量组给药剂量值比为1：10，C max之比为1：9.3，AUC last之比为1：8.9，T max均为1h，暴露量(C max和AUC last)的增加呈剂量比例性增加。TAF-6低、高剂量组给药剂量值比为1：10，C max之比为1：8.6，AUC last之比为1：8.5，Tmax均为1h，暴露量(C max和AUC last)的增加呈剂量比例性增加。因此，Avastin及TAF-6组别高低剂量组之间，AUC及C max均呈剂量线性相关。Avastin和TAF-6的AUC差异，推定为PD-L1靶点介导的清除效应(TMDD)。
实施例5 多结构域融合蛋白在PBMC人源化乳腺癌MDA-MB-231小鼠体内的肿瘤抑制活性
利用MDA-MB-231(人乳腺癌)细胞在人PBMC免疫系统人源化小鼠(M-NSG小鼠)体内建模来测定本发明的多结构域融合蛋白体内药效。筛选6-8周龄雌性M-NSG小鼠，小鼠接种MDA-MB-231细胞(10*10E6+基质胶25％)，于第7天尾静脉注射PBMC(5*10E6/0.2ml)，后观察瘤体积及体重，选择肿瘤体积均在140-260mm 3之间的小鼠，依据其肿瘤体积和体重随机分为6组，每组7只，分组当天开始给药。荷瘤小鼠肿瘤体积过大/过小淘汰。每周2次腹腔注射：PBS、同型对照IgG1、阳性对照Tecentriq(Roche)、TAF-6、TAF-7及联合给药(方案详见表3)，共给药3周左右。PD-L1-Fc融合蛋白(SEQ ID NO.47)为抗PD-L1单域抗体与人IgG Fc的融合蛋白(抗 PD-L1单域抗体位于Fc的N端)、Fc-TGFβRII融合蛋白(SEQ ID NO.48)为人IgG Fc与TGFβRII的融合蛋白(TGFβRII位于Fc的C端)。
在分组前以及实验结束前，从小鼠眼眶静脉采血，FACS检测结果显示各组小鼠外周血中存在人CD45阳性细胞，并且CD45比例随着时间而增高，表明小鼠免疫系统人源化成功。实验过程中组3和组4动物体重(每周两次测量)大体稳定，并未出现实验动物死亡情况，组3和组4的肿瘤体积(每周两次测量)小于组6联合给药，并且显著小于组2相应单药Tecentriq，结果如图2所示。
表3
Figure PCTCN2022076610-appb-000008
实施例6 多结构域融合蛋白在PBMC人源化乳腺癌MDA-MB-231小鼠体内的肿瘤抑制活性
将人源乳腺癌MDA-MB-231瘤块接种于雌性NCG小鼠右侧前胁肋部皮下，肿瘤瘤块接种后1天将PBMC细胞接种于小鼠体内，在肿瘤生长至53mm 3左右时分组给药，共6组，每组10只，分别为：Vehicle组、TAF-6低(2mg/kg,i.p.,tiw×9)组、TAF-6中(6mg/kg,i.p.,tiw×9)组、TAF-6高(18mg/kg,i.p.,tiw×9)组、Tecentriq(4mg/kg,i.p.,tiw×9)组、Avastin(4mg/kg,i.p.,tiw×9)组。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。
分组前2天和实验结束时眼眶静脉丛取血，FACS检测显示各组小鼠外周血中存在人CD45阳性细胞，并且CD45比例随着时间增高。实验结束时，将荷瘤鼠安乐死，剥离肿瘤称重、拍照，采集血清和肿瘤固定。计算肿瘤生长抑制率TGI TV(％)并进行统计学分析：TAF-6低组、TAF-6中组、TAF-6高组、Tecentriq组、Avastin组的肿瘤生长抑制率分别为41％、34％、60％、23％、32％，除Tecentriq组外，各组肿瘤体积均显著小于Vehicle组(p均<0.05)，TAF-6高组肿瘤体积显著小于Tecentriq组(p<0.01)，Tecentriq、Avastin组间肿瘤体积无显著性差异(p>0.05)。结果如图3所示。
综上所述，受试药物TAF-6对PBMC人源化乳腺癌MDA-MB-231皮下移植瘤模型具有显著的抗肿瘤作用，有效地抑制了肿瘤生长，其抗肿瘤作用显著优于Tecentriq，且肿瘤抑制作用随剂量增加而增强。
在另一个相同肿瘤模型的独立实验中，考察受试药物和阳性药物M7824类似物(本实验室根据M7824专利US9676863B2制备)的抑瘤活性对比。设置4组，每组6只，分别为：Vehicle组、TAF-6(4.2mg/kg,i.p.,tiw x 8次；后剂量调整为8.4mg/kg,i.p.,tiw x 4次)组、M7824类似物(3.6mg/kg,i.p.,tiw x 8次；后剂量调整为7.2mg/kg,i.p.,tiw x 4次)组、Avastin(3mg/kg,i.p.,tiw x 8次；后剂量调整为6mg/kg,i.p.,tiw x 4次)组。结果如图4所示，在等摩尔浓度剂量下，TAF-6抗肿瘤作用显著优于M7824类似物。
实施例7 多结构域融合蛋白在PBMC人源化肺癌Calu-6小鼠体内的肿瘤抑制活性
将人源肺癌Calu-6细胞接种于雄性NCG小鼠右侧前胁肋部皮下，肿瘤细胞接种前4天将PBMC细胞接种于小鼠体内，在肿瘤生长至50mm 3左右时分组给药，共7组，每组8只，分别为：Vehicle组、TAF-6低(2mg/kg,i.p.,tiw×10)组、TAF-6中(7mg/kg,i.p.,tiw×10)组、TAF-6高(25mg/kg,i.p.,tiw×10)组、Tecentriq(5mg/kg,i.p.,tiw×10)组、Avastin(5mg/kg,i.p.,tiw×10)组、Tecentriq+Avastin(5+5mg/kg,i.p.,tiw×10)组。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。实验结束时，将荷瘤鼠安乐死，剥离肿瘤称重、拍照，采集血清和肿瘤。计算肿瘤生长抑制率TGI TV(％)并进行统计学分析。
分组前3天和实验结束时(PG-D23)，从小鼠眼眶静脉采血，FACS检测结果显示各组小鼠外周血中存在人CD45阳性细胞，并且CD45比例随着时间而增高，表明小鼠免疫系统人源化成功。
治疗期间，各组小鼠正常摄食饮水，体重大体稳定，未出现实验动物死亡情况。
实验结束时(PG-D23)，TAF-6低组、TAF-6中组、TAF-6高组、Tecentriq组、Avastin组、Tecentriq+Avastin组的肿瘤生长抑制率分别为62％、62％、75％、13％、34％、32％，除Tecentriq组外，各组肿瘤体积均显著小于Vehicle组(p均<0.01)，TAF-6低、中、高组肿瘤体积均分别显著小于Tecentriq组、Avastin组及Tecentriq+Avastin组(p均<0.01)。结果如表4和图5所示。
综上所述，受试药物TAF-6对PBMC人源化肺癌Calu-6皮下移植瘤模型具有显著的抗肿瘤作用，有效地抑制了肿瘤生长，抑瘤作用显著优于Tecentriq、Avastin及Tecentriq与Avastin联合，且肿瘤抑制作用随剂量增加而增强。
表4 受试物对人肺癌Calu-6的抑瘤作用(肿瘤体积)
Figure PCTCN2022076610-appb-000009
注： a.均数±标准误。
在另一个相同肿瘤模型的独立实验中，考察受试药物的抑瘤活性和阳性药物M7824类似物对比。设置4组，每组6只，分别为：Vehicle组、TAF-6(7mg/kg,i.p.,TIW x 8次)组、M7824类似物(6mg/kg,i.p.,TIW x 8次)组、Avastin(5mg/kg,i.p.,TIW x 8次)组。结果如图6所示，在等摩尔浓度剂量下，TAF-6抗肿瘤作用显著优于M7824类似物及Avastin。
实施例8 多结构域融合蛋白在PBMC人源化结肠癌HCT116小鼠体内的肿瘤抑制活性
利用HCT-116(人结肠癌)细胞在人PBMC免疫系统人源化小鼠(NOG小鼠)体内建模来测定本发明的多结构域融合蛋白体内药效。筛选7-9周龄雌性NOG小鼠，小鼠接种HCT-116细胞(3*10E6+基质胶)，于接种肿瘤细胞第三天尾静脉注射PBMC(5*10E6/0.2ml)，后观察瘤体积及体重，选择肿瘤体积均在60-100mm 3之间的小鼠，依据其肿瘤体积和体重随机分为6组，每组8只，分组当天开始给药：Vehicle组、TAF-6低(2mg/kg,i.p.,tiw×3)组、TAF-6中(7mg/kg,i.p.,tiw×3)组、TAF-6高(25mg/kg,i.p.,tiw×3)组、Tecentriq(5mg/kg,i.p.,tiw×3)组、Tecentriq+Avastin(5+5mg/kg,i.p.,tiw×3)组。每周两次测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。实验结束时，将荷瘤鼠安乐死，剥离肿瘤称重、拍照，采集血清和肿瘤。计算肿瘤生长抑制率TGI TV(％)并进行统计学分析。
在分组前以及实验结束前，从小鼠眼眶静脉采血，FACS检测结果显示各组小鼠 外周血中存在人CD45阳性细胞，并且CD45比例随着时间而增高，表明小鼠免疫系统人源化成功。
抑瘤活性如图7所示。受试药物TAF-6对PBMC人源化结肠癌HCT116皮下移植瘤模型具有显著的抗肿瘤作用，有效地抑制了肿瘤生长，且高剂量和中剂量组抑瘤作用显著优于Tecentriq。同摩尔浓度剂量下，即TAF-6中(7mg/kg,i.p.,tiw×3)组对比Tecentriq+Avastin(5+5mg/kg,i.p.,tiw×3)组，TAF-6仍能观察到不劣于，甚至优于Tecentriq+Avastin组的疗效。
实施例9 多结构域融合蛋白在PBMC人源化肝癌Huh-7小鼠体内的肿瘤抑制活性
将人源肝癌Huh-7细胞接种于雄性NCG小鼠右侧前胁肋部皮下，肿瘤细胞接种前45天将PBMC细胞接种于小鼠体内，在肿瘤生长至50mm 3左右时分组给药，共5组，每组10只，分别为：Isotype组、TAF-6低(2mg/kg,i.p.,tiw×8)组、TAF-6高(7mg/kg,i.p.,tiw×8)组、Tecentriq(5mg/kg,i.p.,tiw×8)组、Avastin(5mg/kg,i.p.,tiw×8)组。每周两次测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。实验结束时，将荷瘤鼠安乐死，剥离肿瘤称重、拍照，采集血清和肿瘤。计算肿瘤生长抑制率TGI TV(％)并进行统计学分析。
分组前和实验结束时，从小鼠眼眶静脉丛共采血，FACS检测结果显示各组小鼠外周血中存在人CD45阳性细胞，并且CD45比例随着时间而增高，表明小鼠免疫系统人源化成功。
给药期间，各组小鼠正常摄食饮水，体重大体稳定。
实验结束前(PG-D18)，TAF-6低组、TAF-6高组、Tecentriq组、Avastin组的肿瘤生长抑制率分别为57％、74％、18％、67％，除Tecentriq组外，各组肿瘤体积均显著小于Isotype组(p均<0.01)，TAF-6高组肿瘤体积均显著小于Tecentriq组(p均<0.01)，Avastin组肿瘤体积显著小于Tecentriq组(p<0.05)。结果如图8所示。
综上所述，受试药物TAF-6对PBMC人源化肝癌Huh-7皮下移植瘤模型具有显著的抗肿瘤作用，有效地抑制了肿瘤生长，抗肿瘤作用显著优于Tecentriq，且肿瘤抑制作用随剂量增加而增强。
在另一个相同肿瘤模型的独立实验中，考察受试药物的抑瘤活性和阳性药物M7824类似物对比。分组设置4组，每组6只，分别为：Vehicle组、TAF-6(7mg/kg,i.p.,tiw x 9；后剂量调整为14mg/kg,i.p.,tiw x 2)组、M7824类似物(6mg/kg,i.p.,tiw x 9；后剂量调整为12mg/kg,i.p.,tiw x 2)组、Avastin(5mg/kg,i.p.,tiw x 9；后剂量调整为10mg/kg,i.p.,tiw x 2)组。结果如图9所示，TAF-6抗肿瘤作用显著优于M7824类似物。
实施例10 多结构域融合蛋白在PBMC人源化肉瘤HT1080小鼠体内的肿瘤抑制活性
将人源肉瘤HT1080细胞接种于雄性NCG小鼠右侧前胁肋部皮下，肿瘤细胞接种前7天将PBMC细胞接种于小鼠体内，在肿瘤生长至56mm 3左右时分组给药，共5组，每组8只，分别为：Vehicle组、TAF-6低(2mg/kg,i.p.,tiw×9)组、TAF-6中(7mg/kg,i.p.,tiw×9)组、TAF-6高(25mg/kg,i.p.,tiw×9)组、Tecentriq(5mg/kg,i.p.,tiw×9)组。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。
实验结束时，将荷瘤鼠安乐死，剥离肿瘤称重、拍照，采集血清和肿瘤。计算肿瘤生长抑制率TGI TV(％)并进行统计学分析。
分组前和实验结束时，从小鼠眼眶静脉丛采血，FACS检测结果显示各组小鼠外周血中存在人CD45阳性细胞，并且CD45比例随着时间而增高，表明小鼠免疫系统人源化成功。
实验结束前(PG-D19)，TAF-6低组、TAF-6中组、TAF-6高组、Tecentriq组肿瘤生长抑制率分别为19％、33％、41％、28％，TAF-6中组和Tecentriq组肿瘤体积显著小于Vehicle组(p均<0.05)，各组间肿瘤体积无显著性差异(p>0.05)，结果如图10所示。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (19)

  1. 一种融合蛋白，所述融合蛋白包括抗PD-L1单域抗体片段、拮抗VEGF片段、TGF-β结合片段。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述抗PD-L1单域抗体片段的互补决定区包括：氨基酸序列如SEQ ID NO.1～5其中之一所示的CDR1，SEQ ID NO.6～9其中之一所示的CDR2，SEQ ID NO.10～15其中之一所示的CDR3。
  3. 如权利要求2所述的融合蛋白，其特征在于，所述抗PD-L1单域抗体片段的互补决定区包括：
    氨基酸序列如SEQ ID NO.1所示的CDR1，SEQ ID NO.6所示的CDR2，SEQ ID NO.10所示的CDR3；或
    氨基酸序列如SEQ ID NO.2所示的CDR1，SEQ ID NO.7所示的CDR2，SEQ ID NO.11所示的CDR3；或
    氨基酸序列如SEQ ID NO.3所示的CDR1，SEQ ID NO.7所示的CDR2，SEQ ID NO.12所示的CDR3；或
    氨基酸序列如SEQ ID NO.4所示的CDR1，SEQ ID NO.8所示的CDR2，SEQ ID NO.13所示的CDR3；或
    氨基酸序列如SEQ ID NO.2所示的CDR1，SEQ ID NO.7所示的CDR2，SEQ ID NO.14所示的CDR3；或
    氨基酸序列如SEQ ID NO.5所示的CDR1，SEQ ID NO.9所示的CDR2，SEQ ID NO.15所示的CDR3。
  4. 如权利要求2所述的融合蛋白，其特征在于，所述抗PD-L1单域抗体片段还包括框架区，所述框架区FR包括氨基酸序列如SEQ ID No.49所示的FR1、氨基酸序列如SEQ ID No.50～52其中之一所示的FR2、氨基酸序列如SEQ ID No.53～55其中之一所示的FR3、和氨基酸序列如SEQ ID No.56所示的FR4。
  5. 如权利要求4所述的融合蛋白，其特征在于，所述框架区FR包括氨基酸序列如下所示的FR1～FR4：
    氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.50所示的FR2，SEQ ID NO.53所示的FR3；SEQ ID NO.56所示的FR4，或
    氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.51所示的FR2，SEQ ID NO.54所示的FR3；SEQ ID NO.56所示的FR4，或
    氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.52所示的FR2，SEQ ID NO. 54所示的FR3；SEQ ID NO.56所示的FR4，或
    氨基酸序列如SEQ ID NO.49所示的FR1，SEQ ID NO.52所示的FR2，SEQ ID NO.55所示的FR3；SEQ ID NO.56所示的FR4。
  6. 如权利要求2所述的融合蛋白，其特征在于，所述抗PD-L1单域抗体片段包括：
    a)氨基酸序列如SEQ ID No.16～21其中之一所示的多肽片段；
    或，b)氨基酸序列与SEQ ID No.16～21其中之一具有90％以上序列一致性且具有a)限定的多肽片段的功能的多肽片段；
    和/或，所述抗PD-L1单域抗体片段来源于羊驼；
    和/或，所述抗PD-L1单域抗体片段是人源化的。
  7. 如权利要求1所述的融合蛋白，其特征在于，所述拮抗VEGF片段为贝伐珠单抗，优选的，所述拮抗VEGF片段包括：
    c)氨基酸序列如SEQ ID No.22～23其中之一所示的多肽片段；或，
    d)氨基酸序列与SEQ ID No.22～23其中之一具有90％以上序列一致性且具有c)限定的多肽片段的功能的多肽片段；
    和/或，所述拮抗VEGF片段来源于鼠；
    和/或，所述拮抗VEGF片段是人源化的。
  8. 如权利要求1所述的融合蛋白，其特征在于，所述TGF-β结合片段为TGF-βRⅡ胞外区结构域片段，优选的，所述TGF-β结合片段包括：
    e)氨基酸序列如SEQ ID No.24所示的多肽片段；或，
    f)氨基酸序列与SEQ ID No.24具有90％以上序列一致性且具有e)限定的多肽片段的功能的多肽片段；
    和/或，所述TGF-β结合片段来源于人。
  9. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白还包括连接肽片段，优选的，所述连接肽片段富含G、S和/或A，更优选的，所述连接肽选自由G甘氨酸和/或S丝氨酸和/或A丙氨酸组成的柔性多肽链，所述连接肽的长度为3～30个氨基酸。
  10. 如权利要求9所述的融合蛋白，其特征在于，所述连接肽片段包括氨基酸序列如SEQ ID NO.34-36其中之一所示的多肽片段；
    和/或，所述抗PD-L1单域抗体片段、拮抗VEGF片段之间设有连接肽；
    和/或，所述拮抗VEGF片段、TGF-β结合片段之间设有连接肽。
  11. 如权利要求1～10任一权利要求所述的融合蛋白，其特征在于，所述融合蛋白自N端 至C端依次包括抗PD-L1单域抗体片段、拮抗VEGF片段、TGF-β结合片段；
    和/或，所述抗PD-L1单域抗体片段位于拮抗VEGF片段的重链的N端；
    和/或，所述抗PD-L1单域抗体片段位于拮抗VEGF片段的轻链的N端；
    和/或，所述TGF-β结合片段位于拮抗VEGF片段的重链的C端。
  12. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列包括SEQ ID NO.23、SEQ ID NO.25-33其中之一所示的序列；
    或，所述融合蛋白的氨基酸序列包括SEQ ID NO.25和SEQ ID NO.26所示的序列、SEQ ID NO.25和SEQ ID NO.27所示的序列、SEQ ID NO.25和SEQ ID NO.28所示的序列、SEQ ID NO.25和SEQ ID NO.29所示的序列、SEQ ID NO.30和SEQ ID NO.27所示的序列、SEQ ID NO.30和SEQ ID NO.29所示的序列、SEQ ID NO.31和SEQ ID NO.23所示的序列、SEQ ID NO.32和SEQ ID NO.23所示的序列、SEQ ID NO.33和SEQ ID NO.23所示的序列。
  13. 一种分离的多核苷酸，编码如权利要求1～12任一权利要求所述的融合蛋白。
  14. 一种构建体，所述构建体含有如权利要求13所述的分离的多核苷酸。
  15. 一种表达系统，所述表达系统含有如权利要求14所述的构建体或基因组中整合有外源的如权利要求13所述的多核苷酸。
  16. 如权利要求1-12任一权利要求所述的融合蛋白的制备方法，包括：在合适的条件下培养如权利要求15所述的表达系统，使之表达所述融合蛋白，分离、纯化以提供所述融合蛋白。
  17. 如权利要求1-12任一权利要求所述的融合蛋白、或如权利要求15所述的表达系统的培养物在制备药物中的用途。
  18. 如权利要求17所述的用途，其特征在于，所述药物选自用于治疗肿瘤的药物；优选地，所述肿瘤选自肺癌、黑色素瘤、胃癌、卵巢癌、结肠癌、肝癌、肾癌、膀胱癌、乳腺癌、经典霍奇金淋巴瘤、血液恶性肿瘤、肉瘤、头颈癌和鼻咽癌。
  19. 一种药物组合物，包括如权利要求1-12之任一权利要求所述的融合蛋白、或如权利要求15所述的表达系统的培养物。
PCT/CN2022/076610 2021-02-22 2022-02-17 多结构域融合蛋白及其应用 WO2022174781A1 (zh)

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