WO2022174452A1 - Protéine de fusion bifonctionnelle ayant une activité anticancéreuse - Google Patents

Protéine de fusion bifonctionnelle ayant une activité anticancéreuse Download PDF

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WO2022174452A1
WO2022174452A1 PCT/CN2021/077284 CN2021077284W WO2022174452A1 WO 2022174452 A1 WO2022174452 A1 WO 2022174452A1 CN 2021077284 W CN2021077284 W CN 2021077284W WO 2022174452 A1 WO2022174452 A1 WO 2022174452A1
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seq
fusion protein
amino acid
acid sequence
fragment
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PCT/CN2021/077284
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Chinese (zh)
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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 PCT/CN2021/077284 priority Critical patent/WO2022174452A1/fr
Priority to CN202180003798.3A priority patent/CN117295769A/zh
Publication of WO2022174452A1 publication Critical patent/WO2022174452A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of biotechnology, in particular to a bifunctional fusion protein with anticancer activity and a preparation method and application 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.
  • Immunotherapy focuses on activating the immune system and truly changes the immune status of the tumor microenvironment.
  • the first immune checkpoint inhibitor targeting CTLA-4 was approved by the FDA in 2011.
  • five antibody drugs targeting PD-1/PD-L1 targets were launched in three years, pushing the research on immune checkpoints to a hot spot.
  • anti-PD-1/PD-L1 monoclonal antibodies have high efficacy and broad spectrum in tumor treatment, they also have obvious shortcomings, such as limited efficacy ( ⁇ 30%), drug resistance, and toxic and side effects.
  • ⁇ drugs can often achieve effects that cannot be achieved by single drugs.
  • Roche uses anti-PD-L1 monoclonal antibody (Atezolizumab) and Bevacizumab to treat hepatocellular carcinoma (HCC), which achieved dual endpoints (significantly prolonged OS and PFS) in Phase III clinical trials (IMbrave 150), and has been approved by the FDA in 2020;
  • the triple therapy Atezolizumab + Bevacizumab + chemotherapy significantly prolongs the progression-free survival (PFS) of non-small cell lung cancer (NSCLC) phase III clinical (IMpower 150), and was approved by the FDA in 2018.
  • the purpose of the present invention is to provide a bifunctional fusion protein with anti-cancer activity and its preparation method and use, which are used to solve the problems in the prior art.
  • one aspect of the present invention provides a fusion protein, the fusion protein includes an anti-PD-L1 single domain antibody fragment and an antagonistic VEGF 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.
  • FIG. 1 is a schematic diagram showing the tumor-inhibiting effect of the bifunctional fusion protein in Example 4 of the present invention in M-NSG mice.
  • the inventor of the present invention unexpectedly found a fusion protein molecule, which can reduce tumor angiogenesis and reduce tumor angiogenesis by blocking PD-L1/PD-1 interaction and blocking VEGF signaling pathway.
  • the immunosuppression is relieved, and the invention has excellent tumor suppressing effect, and the present invention is completed on this basis.
  • the first aspect of the present invention provides a fusion protein, the fusion protein includes an anti-PD-L1 single domain antibody fragment and an antagonistic VEGF 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.
  • the variable region portion of the antagonistic VEGF fragment can bind to VEGF, thereby blocking tumor angiogenesis and "starving" cancer cells.
  • the antagonistic VEGF fragment can also usually include an Fc part, which can bind to the FcRn receptor, thereby prolonging the half-life in vivo, and can also bind to the effector cells expressing the Fc receptor to kill cancer cells.
  • 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 Usually 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 can include FR1 whose amino acid sequence is shown in SEQ ID No. 38, FR2 whose amino acid sequence is shown in one of SEQ ID No. 39-41, and FR2 whose amino acid sequence is shown in SEQ ID No. 42-44.
  • the frame region FR includes:
  • the amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.39, FR3 shown in SEQ ID NO.42; FR4 shown in SEQ ID NO.45, or
  • the amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.40, FR3 shown in SEQ ID NO.43; FR4 shown in SEQ ID NO.45, or
  • the amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.41, FR3 shown in SEQ ID NO.43; FR4 shown in SEQ ID NO.45, or
  • amino acid sequences are FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.41, FR3 shown in SEQ ID NO.44; FR4 shown in SEQ ID NO.45.
  • 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. 22-23.
  • 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 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.
  • 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 fusion protein can be linear, for example, the fusion protein can include anti-PD-L1 single domain antibody fragment and antagonizing VEGF fragment in sequence from the N-terminus to the C-terminus.
  • the fusion protein can also have a structure similar to that of a monoclonal antibody, for example, an anti-PD-L1 single-domain antibody fragment can be located at the N-terminus of the heavy chain of the antagonizing VEGF fragment, or an anti-PD-L1 single-domain antibody fragment can be located in the antagonizing VEGF fragment. N-terminus of the light chain.
  • the amino acid sequence of the fusion protein may include the sequence shown in one of SEQ ID NO.
  • the amino acid sequence of the fusion protein may include SEQ ID NO. 22 and SEQ ID NO.24, SEQ ID NO.22 and SEQ ID NO.25, SEQ ID NO.22 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID
  • 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 techniques, 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 Gold lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, etc.
  • These cancers can be early, intermediate or advanced stage, such as metastatic cancer.
  • 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
  • the term "treatment” includes prophylactic, curative or palliative treatment that results in the desired pharmaceutical and/or physiological effect.
  • the effect refers to medically reducing one or more symptoms of the disease or completely eliminating the disease, or retarding, delaying the onset of the disease and/or reducing 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 bifunctional 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-mentioned bifunctional 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 bifunctional fusion protein with anti-cancer activity provided by the present invention can block the interaction of PD-L1/PD-1 with anti-PD-L1 monoclonal antibody, and the anti-VEGF monoclonal antibody can reduce the growth of microvessels in one antibody fusion protein molecule. It is organically combined with the function of inhibiting metastatic disease, so that it can be used to treat tumors and has a good industrialization prospect.
  • 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. Wolfffe, 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 bifunctional fusion proteins in Table 1 were converted into base sequences, and HindIII restriction sites and Kozak sequences (GCCACC) were introduced at the 5' ends of the heavy chain and light chain coding sequences, respectively, A stop codon and an EcoRI restriction site were introduced at the 3' end to obtain full-length DNA by gene synthesis (General Biosystems (Anhui) Co., Ltd.).
  • 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% eluent was diluted to a conductivity of 4ms/cm, and the sample was loaded on a Super Q (TOSOH) chromatography column.
  • the protein concentration was determined by UV280 method.
  • Detector Agilent 1100 LC; detection wavelength: 214nm; mobile phase: 150mM pH7.0 PB+5% isopropanol; chromatographic column: Superdex 200 Increase 5/150 GL; running time: 15 minutes; column temperature 25°C. Test results show that the purity is greater than 95%.
  • CD5L-OKT3scFv-CD14 (GenBank: ADN42857.1) was synthesized, digested with HindIII-EcoRI (Takara), and inserted into the vector pCDNA3.1 to construct pCDNA3.1-antiCD3TM.
  • human PD-L1 (GenBank: NM_014143.2) as the template, the PD-L1 fragment was obtained by high-fidelity amplification, and the CMV promoter sequence was introduced at the 5' end of the sequence by overlapping PCR, and the recombinant ligation was 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 human PD1 (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 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.
  • 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 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% CO2 for 6
  • MDA-MB-231 human breast cancer
  • M-NSG mice huPBMC immune system humanized mice
  • Intraperitoneal injection twice a week PBS, isotype control IgG1, positive control Avelumab (Merck), DAF-5a, DAF-6a and combined administration (see Table 3 for details), for about 3 weeks. Blood was collected from the orbit before grouping and before the end of the experiment. Animal body weight (measured twice a week), tumor volume (measured twice a week) during the experiment. The results are shown in Figure 1.
  • the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

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Abstract

La présente invention relève du domaine technique de la biologie et concerne en particulier, une protéine de fusion bifonctionnelle ayant une activité anticancéreuse, son procédé de préparation et son utilisation. La présente invention concerne une protéine de fusion. La protéine de fusion comprend un fragment d'anticorps à domaine unique anti-PD-L1 et un fragment de VEGF antagoniste. La protéine de fusion bifonctionnelle ayant une activité anticancéreuse décrite dans la présente invention a les fonctions d'un anticorps monoclonal anti-PD-L1 qui bloque l'interaction PD-L1/PD-1, et d'un anticorps monoclonal anti-VEGF qui inhibe la croissance microvasculaire et les maladies métastatiques qui ont été combinées organiquement dans une molécule de protéine de fusion d'anticorps. De cette manière, la présente invention peut être utilisée pour le traitement de tumeurs, et présente de bonnes perspectives d'industrialisation.
PCT/CN2021/077284 2021-02-22 2021-02-22 Protéine de fusion bifonctionnelle ayant une activité anticancéreuse WO2022174452A1 (fr)

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CN202180003798.3A CN117295769A (zh) 2021-02-22 2021-02-22 一种具有抗癌活性的双功能融合蛋白

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107814845A (zh) * 2016-09-14 2018-03-20 浙江特瑞思药业股份有限公司 新的抗pd‑1纳米抗体及其应用
CN109096396A (zh) * 2017-06-20 2018-12-28 华兰生物工程股份有限公司 一种抗pd-l1人源化纳米抗体及其应用
CN109942712A (zh) * 2019-04-01 2019-06-28 华博生物医药技术（上海）有限公司 抗pd-l1/vegf双功能抗体及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107814845A (zh) * 2016-09-14 2018-03-20 浙江特瑞思药业股份有限公司 新的抗pd‑1纳米抗体及其应用
CN109096396A (zh) * 2017-06-20 2018-12-28 华兰生物工程股份有限公司 一种抗pd-l1人源化纳米抗体及其应用
CN109942712A (zh) * 2019-04-01 2019-06-28 华博生物医药技术（上海）有限公司 抗pd-l1/vegf双功能抗体及其用途

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