WO2008145013A1 - Protéine hybride comprenant un peptide de ciblage de cd13 et lidamycine - Google Patents

Protéine hybride comprenant un peptide de ciblage de cd13 et lidamycine Download PDF

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WO2008145013A1
WO2008145013A1 PCT/CN2008/001014 CN2008001014W WO2008145013A1 WO 2008145013 A1 WO2008145013 A1 WO 2008145013A1 CN 2008001014 W CN2008001014 W CN 2008001014W WO 2008145013 A1 WO2008145013 A1 WO 2008145013A1
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protein
ldp
fusion protein
ngr
chromophore
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PCT/CN2008/001014
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Chinese (zh)
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WO2008145013A8 (fr
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Yanbo Zheng
Yongsu Zhen
Boyang Shang
Xiujun Liu
Shuying Wu
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Institute Of Medicinal Biotechnology Chinese Academy Of Medical Sciences
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to anti-tumor targeted drugs. More specifically, the present invention relates to a fortified fusion protein consisting of a CD13 targeting peptide and lidamycin, comprising an activated enediyne chromophore AE bound to LDP, a process for the preparation thereof and an antitumor targeted drug Applications.
  • Targeted drugs are generally composed of a targeting molecule that binds to a specific receptor at the tumor site and a "warhead" drug that kills the cell. Miniaturization and high efficiency have become the development trend of targeted drugs.
  • Tumor targeting peptides are a class of oligopeptides that bind to specific receptors at the tumor site. Compared with the antibody and its fragments, such targeted oligopeptides have a smaller molecular weight and a weaker immunogenicity, and are less likely to cause a response to human antibodies.
  • Lidamycin (LDM, also known as C-1027) is an actinomycete isolated from the soil of Qianjiang County, Hubei Province, China (Streptomyces globi sporus C-1027, strain accession number: CGMCC No 0704) Large molecule anti-tumor antibiotic produced.
  • the invention relates to a protein comprising: (1) a fusion protein comprising a CD13 targeting peptide and a lidamycin prosthetic protein LDP, and (2) an activated enediyne chromophore bound to LDP AE.
  • the NGR (Asn-Gly-Arg) peptide is a tumor targeting peptide obtained by an in vivo phage library selection technique such as Arap, which specifically binds to the CD13 isoform expressed by tumor neovascular endothelial cells.
  • CD13, aminopept idase N (APN; EC 3. 4. 11. 2 )
  • Arap in vivo phage library selection technique
  • CD13, aminopept idase N is a transmembrane glycoprotein with a molecular weight of approximately 150 kDa (the degree of glycosylation is different, the molecular weight is also different, and the difference is formed.
  • Isomer having metalloproteinase activity, catalyzing the hydrolysis of the amino terminal amino acid of the polypeptide, especially the neutral amino acid.
  • CD13 is expressed on the cell surface of various tissues, and CD13 expressed by tumor neovascular endothelial cells is an isomer of normal tissue CD13.
  • the high expression of CD13 in tumor vascular endothelial cells can promote the formation of new blood vessels.
  • the mechanism may be related to the degradation of extracellular matrix by CD13 aminopeptidase activity, promoting endothelial cell invasion and regulating the activity of certain growth factors and cytokines.
  • Certain tumor cells such as breast cancer cells and human melanoma cells, are also highly expressed, and these highly expressed cells may also be targeted sites for NGR peptides. Although the NGR peptide is anisotropic, it has no cytotoxic effect.
  • the NGR peptide is coupled with a highly effective "warhead" drug to truly exert its targeted killing effect.
  • Arap et al. coupled the NGR peptide with doxorubicin, which significantly improved the antitumor effect of doxorubicin and reduced toxicity.
  • the CD13 targeting peptide preferably comprises NGR, more preferably comprises CNGRC;
  • the CD13 targeting peptide is the amino acid sequence NGR, and in another preferred embodiment, the CD13 targeting peptide is the amino acid sequence CNGRC.
  • the CD13 targeting peptide is sometimes referred to as the CD13 targeting peptide NGR.
  • Lidamycin (L idamycin, LDM, also known as C-1027) is the Institute from the lake of China A macromolecular antitumor antibiotic produced by an actinomycete (Streptomyces globi sporus C-1027, strain accession number: CGMCC No. 0704) isolated from the soil of Qianjiang County, Northern province.
  • Lidamycin consists of two parts: one is an active chromophore (ACT enediyne, AE, relative molecular mass 843Da) containing a 9-membered enediyne structure, which has strong cytotoxicity but is unstable; It is a 110 amino acid acidic prosthetic protein (l idaprote in, LDP, relative molecular mass 10 506 Da), which protects the chromophore. The chromophore and the prosthetic protein are combined by non-covalent bonds, which can be resolved and reconstructed. Lidamycin has a strong killing effect on tumor cells.
  • active chromophore ACT enediyne, AE, relative molecular mass 843Da
  • LDP relative molecular mass 10 506 Da
  • the molecular weight of LDM is known to be 11349. 1120 Da.
  • the molecular weight of the basal protein LDP is 10505. 7830 Da, and the molecular weight of the chromophore is 843. 3295 Da.
  • Lidamycin has a strong killing effect on tumor cells. In vivo experiments in animals have shown that lidamycin has significant effects on transplanted mouse colon cancer26 and human liver cancer Bel-7402 and cecal cancer Hce-8693 transplanted into rats. Efficacy (Chinese Journal of Antibiotics, 1994, 19: 164-8). Molecular pharmacology studies have shown that its main mechanism of action is to induce specific DNA breaks and induce apoptosis. It is known that the chromophore of LDM. and the prosthetic protein are bound by non-covalent bonds, and the combination of the two is specific and robust. Moreover, LDM can be resolved and molecularly enhanced.
  • the present invention refers to the "Lida zero-prostaglandin LDP" package.
  • a wild-type protein such as a protein having the sequence of amino acids 7 to 116 of SEQ ID NO: 2, also includes variants that retain at least substantially its function, such as, but not limited to, the lidamycin prosthetic protein LDP wild-type protein.
  • Homologous protein may be the amino acid sequence of a polypeptide having a LD wild-type protein of the lidamycin prosthetic protein, but one or more of them (eg, 1-25, 1-20, 1-15,
  • amino acid residues have been conservatively altered, in particular by conservative substitutions of different amino acid residues, and the resulting polypeptides are useful in the practice of the invention.
  • Amino acid substitutions are known in the art. The rules that result in such substitutions include the substitution rules described by Dayhof, M. D. (1978, National Biomedical Research Fund, Washington, D. C., Vol. 5, Supplement 3). More specifically, conservative amino acid substitutions occur within a family of amino acids that are associated with their acidity, polarity, or side chain size.
  • substitutions in any particular group such as substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, replacement of threonine with serine, or any other amino acid residue
  • Amino acid residues associated with a base structure such as amino acid residues having similar acidity, polarity, side chain size, or similarity in some combinations thereof, generally do not have a significant effect on the function of the polypeptide.
  • Substantially homologous proteins and polypeptides are characterized by having one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5) amino acid substitutions, deletions or additions. These changes preferably affect smaller transformations, ie conservative amino acid substitutions and other substitutions that do not significantly affect the folding and activity of the protein or polypeptide; small deletions, usually small deletions of from 1 to about 30 amino acids; and small amino or Carboxyl end Extension, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension (affinity tag) that facilitates purification, such as a polyhistidine bundle, protein A (Ni ls Son et al, EMBO J. 4: 1075, 1985; Ni l sson et al., Methods in Enzymology, 198: 3, 1991). DNA encoding the affinity tag is available from the product supplier.
  • DNA encoding the affinity tag is available from the
  • the fusion protein may or may not contain a linker between the CD13 targeting peptide and the lidamycin prosthetic protein LDP.
  • the connector can be 1 - 20 amino acids long, preferably 1 - 10, for example 1 - 5 or 1 - 3.
  • the linker can be any linker known in the art and can be suitably selected by those skilled in the art and should generally not significantly impede or be able to increase the target peptide NGR targeting and/or the strength of the drug.
  • the fusion protein may also contain other amino acid sequences, which can be suitably selected by those skilled in the art, and generally should not significantly hinder or increase the targeting peptide NGR. Targeted and / or lidamycin toxicity.
  • the fusion protein comprises a purification tag such as a histidine hexamer tag.
  • the fusion protein comprises a signal peptide, e.g., a signal peptide for directing secretion of the fusion protein outside of its expression host cell.
  • the signal peptide can be any signal peptide known in the art and can be suitably selected by those skilled in the art.
  • the signal peptide can be excised after secretion.
  • the protein shield of the present invention consists of a fusion protein NGR-LDP formed by a CD13 targeting peptide and a lignin-based proprotein LDP and an activated enediyne chromophore AE bound to LDP.
  • the fusion protein comprises the amino acid sequence set forth in positions 1 to 116 of SEQ ID NO: 2, or the amino acid sequence shown in positions 1 to 124 of SEQ ID NO: 2, or SEQ ID NO: 2 - of the amino acid sequence of positions 22 to I 24 bits.
  • the amino acid number is shown in SEQ ID NO: 2 of the Sequence Listing.
  • the protein of the present invention is a fortified fusion protein NGR-LDP-AE constructed from the following examples, which is a fusion protein NGR-formed by a CD13 targeting peptide and a Lidasin prostaglandin LDP. LDP (molecular weight 12.
  • the NGR-LDP gene is 441 bp in length and encodes 146 amino acids.
  • the amino acid sequence thereof is shown in SEQ ID NO: 2 (amino acid sequence shown in positions 22 to 124), and the nucleic acid sequence is shown in SEQ ID NO: 1.
  • the NGR gene is 15 bp long and encodes 5 amino acids.
  • the signal peptide gene before NGR is 66 bp long and encodes 22 amino acids. It is excised when the protein is secreted into the periplasmic cavity.
  • the LDP gene is 330 bp long and encodes 110 amino acids.
  • the LDP gene is linked by a 3 bp sequence encoding one amino acid; the carboxy-terminal histidine hexamer gene is 18 bp long and encodes 6 amino acids; the Xho I cleavage site between the histidine hexamer tag and the LDP gene is long. 6 bp, encoding 2 amino acids; stop codon 3 bp.
  • the protein of the present invention is capable of carrying a lidadamycin-targeted tumor, i.e., targeting tumor blood vessel endothelial cells, such that lidamycin exerts a tumor cytotoxic effect.
  • the invention features an isolated nucleic acid molecule comprising: a nucleotide sequence encoding a fusion protein of the invention.
  • the invention also relates to expression vectors, such as plasmid vectors or viral vectors, comprising a nucleic acid molecule of the invention.
  • the invention also relates to host cells comprising the expression vector, such as prokaryotic host cells, E. coli cells or eukaryotic cells such as animal cells.
  • the invention also relates to a process for the preparation of a protein of the invention, said method comprising:
  • step (3) may comprise: purifying the purified fusion protein NGR-LDP with, for example, a lidamycin-active chromophore AE prepared by methanol extraction, by molecular ratio 1: 1-1: 50 For example, 1:5, at a temperature of 4 degrees Celsius-room temperature, sufficient to assemble the chromophore, for example 6 - 48 hours, for example 10 - 24 hours, for example 12 hours, the excess chromophore is removed by, for example, PD-10 column chromatography.
  • the invention further relates to the use of the above-described protein or nucleic acid molecule or expression vector of the invention for the preparation of an anti-tumor targeted drug.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising (a therapeutically effective amount) a protein or nucleic acid molecule of the invention described above or an expression vector and a pharmaceutically acceptable carrier.
  • the invention also relates to a protein or nucleic acid molecule or expression vector of the invention for use in the treatment of a tumor.
  • the invention further relates to a method of treating a tumor comprising administering to a patient, including a human and an animal, a therapeutically effective amount of a protein or nucleic acid molecule or expression vector or pharmaceutical composition of the invention described above.
  • the treatment includes improvement or reduction of symptoms, slowing of tumor development, shrinkage or disappearance of tumor volume, and the like.
  • the tumor usually refers to a malignant tumor (cancer), especially a cancer with high CD13 expression, and may be a solid cancer such as, but not limited to, fibrosarcoma, liver cancer, breast cancer, lung cancer, bladder cancer, gastric cancer, melanoma, nasal Pharyngeal cancer, esophageal cancer, and intestinal cancer.
  • the cancer can be a metastatic cancer, such as a metastatic breast cancer.
  • Cancer can be thin blood Cellular cancer, such as lymphoma or leukemia.
  • the dosage and manner of administration can be selected by the clinician according to known criteria. Suitable dosages of the compounds and compositions of the invention may depend on the type of cancer to be treated, the severity and duration of the disease, the size of the tumor, the degree of metastasis, whether the purpose of administering the drug is prophylactic or therapeutic, prior treatment, patient's Clinical history and response to the drug, as well as the judgment of the attending physician.
  • the compounds and compositions of the invention are suitable for administration to a patient, either in a single treatment or by a series of treatments.
  • the compound/composition can be administered by intravenous or subcutaneous injection.
  • the course of the treatment can be monitored by conventional methods and analytical tests based on criteria known to the physician or other skilled artisan.
  • NGR-LDP-AE is prepared by DNA recombination technology, which is a fusion protein of NGR and LDP, NGR-LDP, and then assembled by chromophore AE. Specific steps can include:
  • the protein of the invention can be prepared by a process comprising the following steps:
  • step A can be carried out as follows:
  • the sngr ldp gene is amplified by PCR using a plasmid containing the LDP gene as a template, the primer sequence contains a signal peptide and an NGR peptide coding sequence, and Nde I is introduced on both sides of the sngr ldp.
  • the Xho I restriction site the amplified sngr ldp gene was cloned into the T vector to obtain pGMTsngr ldp.
  • the sngr ldp fragment obtained by digesting pGMTsngr ldp with Nde I and Xho I can be cloned into the same double-digested pET-30a (+) vector to construct the recombinant expression plasmid pET30sngr ldp.
  • pET30sngr ldp may be transferred into Escherichia coli BL21 Star TM (DE3), to obtain secretory expression of fusion protein was induced by IPTG NGR-LDP.
  • the fusion protein NGR-LDP can be isolated and purified by ammonium sulfate precipitation and affinity chromatography, followed by dialysis and concentration to prepare a high-purity fusion protein.
  • the purified fusion protein NGR-LDP and the lidamycin-active chromophore AE prepared by methanol extraction can be assembled at a molecular ratio of 1: 5 for 12 hours at room temperature to assemble a chromophore, PD.
  • the -10 column chromatography was used to remove excess chromophore, and the enhanced fusion protein NGR-LDP-AE was obtained.
  • the invention also provides the biological activity test of the enhanced fusion protein as follows: fusion protein NGR-LDP binding activity analysis;
  • Enhancement of the cytotoxic effect of the fusion protein NGR-LDP-AE on tumor cells enhancement of the animal-based therapeutic regimen of the fusion protein NGR-LDP-AE.
  • the advantage and positive effect of the present invention is that the enhanced fusion protein NGR-LDP-AE is an anti-tumor targeted drug, and its targeting sequence is composed of only 5 amino acids CNGRC, the molecular weight is small, the immunogenicity is weak, and the receptor is a tumor.
  • the CD13 isomer specifically expressed by vascular endothelial cells has good targeting and the drug is easy to reach the tumor site; While lidamycin has a strong cytotoxic effect, it is a highly effective "warhead" drug. Therefore, the enhanced fusion protein NGR-LDP-AE of the present invention is expected to be a novel miniaturized and highly effective targeted anti-tumor drug with good clinical application prospects.
  • Figure 1 PCR amplification of the sngrldp gene.
  • 1-DNA molecular weight standard (DL 2 000 ); 2-gr amplification of the sngr ldp fragment.
  • FIG. 1 Recombinant T vector digestion and PCR identification. 1-pGMTsngr ldp ;
  • Figure 3 Recombinant plasmid pET30sngrldp Restriction enzyme analysis.
  • 1-DNA molecular weight standard (DL 2 000 ); 2-pET30sngr ldp / Nde I + Xho I;
  • Figure 4 SDS-PAGE analysis of NGR-LDP induced expression in E. coli.
  • 1- low molecular weight protein standard 2-the supernatant of the fermentation broth after the induction of the transformant; 3-the supernatant of the fermentation broth before the induction of the transformant; the supernatant of the fermentation broth after the induction of the bacterium; The liquid supernatant part.
  • Figure 5 SDS-PAGE analysis of expression product localization. 1-low molecular weight protein standard; 2-fermentation supernatant supernatant protein; 3-periplasmic cavity protein; 4-cytoplasmic soluble fraction; 5-cytoplasmic insoluble fraction.
  • Figure 6 Wes tern-blot analysis of expression product identification and localization. 1-fermentation supernatant supernatant protein; 2-periplasmic cavity protein; 3-cytoplasmic soluble fraction; 4-cytoplasmic insoluble fraction.
  • Figure 7 SDS-PAGE analysis of the fusion protein after purification by ammonium sulfate precipitation and metal chelate chromatography.
  • 1-low molecular weight protein standard 2-Affinity chromatographically purified sample; 3-fermented supernatant supernatant protein; 4-ammonium sulfate precipitated total protein.
  • Figure 8 ELISA for detection of NGR-LDP on human fibrosarcoma HT-1080 cells and humans Binding activity of breast cancer MCF-7 cells
  • Figure 9 ELISA assay for the binding activity of NGR-LDP and rLDP to human fibrosarcoma HT-1080 cells
  • Figure 10 Competitive ELISA detection of the effect of NGR peptide on the binding of NGR-LDP to human fibrosarcoma HT-1080 cells
  • Figure 11 MTT assay Cytotoxicity of NGR-LDP-AE, LDM and NGR-LDP on human fibrosarcoma HT-1080 cells
  • Figure 12 MTT assay Cytotoxicity of NGR-LDP-AE, LDM and NGR-LDP on human breast cancer MCF-7 cells
  • Figure 13 Inhibitory effect of enhanced fusion protein NGR-LDP-AE on the growth of subcutaneously transplanted liver cancer H22 in mice.
  • Cont ro l represents a control group; NGR-LDP (2.0) represents 2. Omg/kg NGR-LDP group; LDM (0.05) represents 0.05 mg/kg LDM group; NGR-LDP-AE (0. 2) 2mg/kg NGR-LDP-AE group; NGR-LDP-AE (0.4) represents 0. 4mg/kg NGR-LDP-AE group; NGR-LDP-AE (0.8) represents 0. 8 mg/kg NGR-LDP-AE group. See Example 7. Preservation of biological materials
  • Upstream primer a 5 ' -GG AATTCCATATG AA AT ACCTGCTGCCG ACCGCTGCTGCTGG Nde I
  • Downstream primer b 5' -GTTACTCGAGGCCGAAGGTCAGAGCCACGTG -3
  • the plasmid pEFL containing LDP gene (patent application number: CN03150240. 7, publication number: CN1475506) was used as a template and amplified according to conventional PCR conditions.
  • a sngr ldp fragment containing a signal peptide, an NGR peptide, and an LDP coding sequence of about 0.4 kb (Fig. 1), a signal peptide and an NGR peptide coding sequence in the primer sequence, and Nde I /Xho introduced on both sides of the sngr ldp I cleavage site.
  • This fragment was ligated into the T vector (TGM-T vector of Tiangen Biochemical Technology Co., Ltd.) to obtain pGMTsngr ldp, transformed into E. coll DH5 ⁇ , screened for blue and white spots, positive clones were verified by PCR and double enzyme digestion (Fig. 2), The T7 promoter was primer sequencing (sequence of Beijing Huada Gene Research Center).
  • pGMTsngr ldp was digested with Nde I /Xho I, and the restriction fragment was ligated into the same digested pET-30a ( + ) (Novagen) vector, and the ligated product was transformed into CO/ / DH5 a, 37 ⁇ after culture.
  • the monoclonal antibody was picked, and the plasmid was extracted after a small amount of culture, and the Nde I / Xho I double restriction enzyme digestion (Fig. 3) was carried out, and the T7 promoter was used as the primer sequencing (Beijing Huada Gene Research Center was sequenced, and the obtained fusion protein encoded nucleic acid sequence was obtained. See Sequence Listing SEQ ID N0: 1).
  • the recombinant plasmid containing the sngr ldp gene was named pET30sngr ldp.
  • the pET-30a (+) used in the present invention has a sequence encoding a histidine tail of the histidine at the 3' end of the multiple cloning site. After translation, Hi s6-Tag facilitates the expression and identification of the fusion protein. And isolated and purified.
  • the constructed pET30sngr ldp plasmid was transformed into E. coli BL21 StarTM (DE3) (product of Invi trogen), monoclonal was selected, and 0D 6 was cultured in small amounts. .
  • E. coli BL21 StarTM DE3 (product of Invi trogen)
  • IPTG isopropyl- ⁇ -D-thiogalactoside 37 ⁇ induced for 8 hours.
  • the supernatant was precipitated with trichloroacetic acid, and the expression of the target protein was detected by 15% SDS-PAGE (Fig. 4).
  • Samples were prepared from medium supernatant, perivascular shield, cytosolic soluble and insoluble fractions, analyzed by SDS-PAGE (Fig.
  • the bacterial fermentation broth was centrifuged at 10 000 g for 10 minutes, and the supernatant was collected.
  • Ammonium sulfate was added at a concentration of 390 g/L at 4 t with gentle agitation. 4 Place at rest for half an hour, centrifuge at 4, 10 000g for 20 minutes, and collect the pellet.
  • the precipitate obtained per 100 ml of the fermentation broth was dissolved with 2 ml of 1 Ni-NTA Bind Buffer (300 mM NaCl, 50 mM NaH 2 P0 4 , 10 mM imidazole, pH 8.0) and dialyzed against the same solution.
  • Ni-NTA Hi s -Bind resin ( Novagen ) was loaded into the column and equilibrated with about 5 volumes of 1 ⁇ Ni-NTA Bind Buffer until A 2S . Return to the baseline and stabilize, the flow rate does not exceed 2 ml / min.
  • the sample was dialyzed against 0. 45 ⁇ ⁇ was slowly added to the membrane after filtration chromatography column, 1 X Ni-NTA Bind Buffer column was washed with 5-10 bed volumes of up to A 28. Stable, collected effluent, flow rate 0. 5-1 ml / min.
  • the column was washed with 5-10 bed volumes of lx Ni-NTA Wash Buffer (300 mM NaCl, 50 mM NaH 2 P0 4 , 20 mM imidazole, pH 8.0) until A 2 S . Stable, collect the effluent.
  • the target protein bound to the column was eluted with 5 column volumes of lx Ni-NTA Elution Buffer (300 mM NaCl, 50 mM NaH 2 P0 4 , 250 mM imidazole, pH 8.0), according to A 28 .
  • the eluted fractions were collected and analyzed by SDS-PAGE (Fig. 7).
  • the eluate containing the protein of interest was dialyzed against PBS ( ⁇ 7.4), and concentrated by ultrafiltration. Concentration of the protein of interest. Protein quantification showed that about 10 mg of the target protein with a purity of more than 95% was obtained per liter of fermentation broth.
  • Detastatin lyophilized product prepared from Lidamycin producing bacteria (CGMCC NO.0704, published in Chinese Patent Application No. 00121527.2) according to methods known in the art) 10 mg, shaken for 5 minutes by adding 5 ml of cold methanol , -20 was placed for 1 hour, oscillated once in the middle; centrifuged at 0, 12 000 rpm for 20 minutes, the supernatant contained a chromophore, and the precipitate was a prosthetic protein, which was repeatedly extracted twice.
  • the methanol solution is naturally evaporated and the above operation is carried out at a low temperature and protected from light.
  • a certain volume and concentration of the fusion protein was dissolved in 0.01 mol/L phosphate (pH 7.0) buffer, and the chromophore was added.
  • the molecular ratio of the fusion protein to the chromophore was 1:5, and the volume ratio was 50:1.
  • the shaker was shaken slowly for 12 hours for molecular assembly.
  • the reaction solution was separated by PD-10 (Sephadex G-25 column, Pharmacia product), and the enhanced fusion protein NGR-LDP-AE was collected after UV detection at 2 80 nm. Excess unbound chromophore.
  • Tumor cells in logarithmic growth phase were inoculated into 96 well plates at a density of 2 ⁇ 10 4 cells/well, and cultured for 24 hours at 37°C, plus 4 pre-cooled 0.05% glutaraldehyde 50 ⁇ 1 / well, at 41C The cells were fixed for 15 minutes. After blocking 1% of BSA, the fusion protein was added to each well. After incubation for 1-2 hours at 37 C, His-tag antibody (product of Novagen) was used as the primary antibody, HRP-labeled sheep. Anti-mouse IgG (product of Santa Cruz) was incubated with secondary antibody, 100 ⁇ l of reaction substrate was added to each well, and the absorbance at 492 nm was measured by a microplate reader.
  • His-tag antibody product of Novagen
  • NGR-LDP is stronger than human fibrosarcoma HT-10 80 cells and human breast cancer MCF-7 cells expressing CD13.
  • the combination ( Figure 8), and the recombinant NAD peptide-free Lidamycin prosthetic protein LDP) was essentially unbound ( Figure 9).
  • the free NGR peptide significantly inhibited the binding of NGR-LDP to HT-1080 cells. effect, The inhibitory rate of the NGR peptide was about 40.6%, and the inhibition rate of the 1 mM NGR peptide was about 73.7% (Fig. 10).
  • the LDP sequence in the fusion protein did not affect the binding of the NGR sequence to the target site.
  • the mode of administration and dosage of the animal test treatment are designed.
  • Sixty Kunming mice weighing 18g-22g were randomly divided into 6 groups, 10 in each group.
  • the mouse liver cancer H22 ascites was taken, and the number of cells diluted with physiological saline was 7. 5 107 ml, and was inoculated subcutaneously into the armpits of Kunming mice according to 0.2 ml/mouse.
  • NGR-LDP-AE was administered to saline, lidamycin, NGR-LDP, and three dose groups at 4 and 10 days, respectively, with a tail vein injection of 0.2 ml/mouse.
  • NGR-LDP-AE The tumor inhibition rate of NGR-LDP-AE was 94.8%, which was significantly higher than 0.05 mg/kg (tolerance dose) of free lidomycin (66.9%) (Table 1).
  • Table 1 Inhibitory effect of enhanced fusion protein on growth of mouse liver cancer H22 (experimental day 16)
  • mice body weight, tumor volume
  • CGMCC China General Microbiological Culture Collection Center

Abstract

La présente invention concerne une protéine hybride NGR-LDP-AE qui comprend un peptide de ciblage du CD13, une apoprotéine lidamycine (LDP) et un chromophore énédiyne (AE) sous forme active associé au LDP. Cette protéine hybride peut servir pour certains traitements de cancer précis.
PCT/CN2008/001014 2007-05-25 2008-05-26 Protéine hybride comprenant un peptide de ciblage de cd13 et lidamycine WO2008145013A1 (fr)

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CN101070350B (zh) * 2007-05-25 2011-03-23 中国医学科学院医药生物技术研究所 针对cd13的靶向肽与力达霉素构成的强化融合蛋白ngr-ldp-ae
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