WO2010022639A1 - Protéine de fusion à double mutant à spécificité de cible - Google Patents

Protéine de fusion à double mutant à spécificité de cible Download PDF

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WO2010022639A1
WO2010022639A1 PCT/CN2009/073441 CN2009073441W WO2010022639A1 WO 2010022639 A1 WO2010022639 A1 WO 2010022639A1 CN 2009073441 W CN2009073441 W CN 2009073441W WO 2010022639 A1 WO2010022639 A1 WO 2010022639A1
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fusion protein
mgnrh
sequence
pe38m4a
target
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PCT/CN2009/073441
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Chinese (zh)
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张俊英
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北京博翱泰生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention belongs to the field of biotechnology and relates to a fusion protein having target cell-specific cytotoxicity. Specifically, the present invention relates to a human gonadotropin releasing hormone mutant (mGnRH) and a recombinant Pseudomonas aeruginosa exotoxin A mutant (PE38m4a).
  • mGnRH human gonadotropin releasing hormone mutant
  • PE38m4a recombinant Pseudomonas aeruginosa exotoxin A mutant
  • cytokines or receptor proteins on their surface, such as epidermal growth factor receptor (EGF-R) and transforming growth factor alpha receptor (TGF ci-R).
  • EGF-R epidermal growth factor receptor
  • TGF ci-R transforming growth factor alpha receptor
  • certain cytotoxic agents such as Pseudomonas aeruginosa exotoxin (PE), diphtheria toxin, cholera toxin, staphylococcal endotoxin, and ricin, etc.
  • PE Pseudomonas aeruginosa exotoxin
  • TGF- ⁇ or bFGF molecules as targeting agents.
  • hybrid molecules direct the entire molecule to the target cell by its ability to direct the target tumor cells and partially kill the target cell through its toxin.
  • PEA Pseudomonas aeruginosa exotoxin A
  • the region I includes the la region (amino acid 1-252) which mediates cell binding and the lb region (amino acid 365-399) which has not yet been clarified.
  • the PE molecule can be modified using biochemical or recombinant DNA techniques to prepare various modified PE fragments having one or more amino acid deletions or substitutions in the PE molecule.
  • the LA region of the PE molecule is generally deleted, and only the enzyme is contained.
  • the PE-A protein having a molecular weight of about 40 kDa is called PE40.
  • TGF ⁇ - ⁇ 40 hybrid fusion protein
  • the clinical utility of TGF ci-PE40 is dependent on its ability to bind and kill cells with epidermal growth factor receptors.
  • U.S. Patent No. 5,428,143 discloses the construction of a hybrid protein for selectively killing HIV-infected cells and a chimeric gene encoding the hybrid protein.
  • the hybrid protein described therein consists of human CD4 containing an HIV binding site and a cytotoxic protein (PE40) which kills HI V infection.
  • W097/15325 describes an immunogenic carrier system comprising GnRH chemically bound to Pseudomonas aeruginosa exotoxin, which can be used as a vaccine to induce high concentrations of anti-GnRH antibodies in animals and thus can be used for control Pregnancy, reduced reproductive hormone-driven behavior, and treatment of steroid-responsive tumors.
  • the object of the present invention is to provide a target-specific double mutant fusion protein which is a fusion toxin obtained by fusing a mutant gonadotropin releasing hormone mGnRH and a recombinant Pseudomonas aeruginosa exotoxin A mutant, characterized in that the recombinant green
  • the exotoxin of Pseudomonas is the amino acid 365-380 which removes the la and lb regions, and the C-terminal amino acids Glu610, Leu612 and Lys613 are artificially mutated to the PEA molecules of Lys610, Glu612 and Leu613, respectively, ie, PE38m4a.
  • the mutant gonadotropin releasing hormone mGnRH whose gene is synthesized by a gene encoding the Escherichia coli bias codon, and the amino acid sequence is: Met-Gly-G i u-Hi s-Trp-Ser- Tyr-Trp-Leu-Arg-Pro-Gly-His (SEQ ID NO: 4).
  • the target-specific double mutant fusion protein has the gene sequence as shown in the sequence listing SEQ ID NO: 1.
  • the target-specific double mutant fusion protein of the present invention is prepared by the following method;
  • step (3) transforming E. coli with the expression vector of step (2);
  • the fusion protein is recovered and purified from the cell culture.
  • Step (l) The mGnRH gene sequence was artificially designed to synthesize the Ncol endonuclease recognition sequence, the mGnRH nucleotide sequence consisting of the E. coli bias codon, and the 6th Gly mutation was the Trp-Ndel endonuclease recognition sequence.
  • the gene described in the step (2) is shown in the sequence listing SEQ ID NO: 1.
  • the vector is pET27.
  • the Escherichia coli described in the step (3) is Escherichia coli BL21 ( ⁇ DE3).
  • a further object of the invention is the use of a target-specific double mutant fusion protein for the treatment of a tumor drug associated with a gonadotropin releasing hormone receptor
  • GnRH was isolated and purified from animals by Schally in 1971, and its structure was synthesized and then synthesized, and the Nobel Prize of 1976 was obtained.
  • GnRH is a decapeptide containing no free amino acid and carboxyl group, and its molecular structure is: P-Glu-Hi s-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 (SEQ ID NO: 3)
  • amino acids 4-6 form a ⁇ -transition, which is hairpin-shaped, which is suitable for binding to receptors.
  • the second and third positions are important for biological activity, and the sixth position plays an important role in maintaining hairpin conformation.
  • the 4th to 10th amino acids are involved in receptor binding, and replacement of the above amino acid residues may result in loss of vigor or geometrical enhancement.
  • the requirement for the synthesis of long-acting GnRH agonists is to stabilize the molecular structure, making it less susceptible to enzymatic hydrolysis, increasing binding to circulating proteins and membranes, and increasing affinity for the GnRH receptor.
  • the 6-position is an analog of D-amino acid and a substituted GlylO amide group.
  • This GnRH agonist is not only resistant to protease hydrolysis but also has a high affinity for receptors.
  • the introduction of a bulky hydrophobic group at position 6 further increases the affinity for the receptor. Such substitutions stabilize the "active" configuration of the release hormone analog, increasing binding to circulating proteins, thereby extending half-life.
  • GnRH antagonists The theoretical basis for early development of GnRH antagonists is similar to that of GnRH agonists, which improves binding to receptors but produces unacceptable histamine release side effects. Therefore, the focus of developing the next generation of GnRH antagonists has focused on both improving efficacy and reducing histamine release.
  • hormones and cytokine receptors have abnormally high expression on tumor cells, such as EGFR, GnRHR and so on.
  • hormones and cytokines have relatively small molecules, simple structure, and convenient gene manipulation. Therefore, as a carrier of immunotoxins, it is highly feasible.
  • many cytokines and hormones are used as carriers for immunotoxins, such as IL-2, IL-4, EGF, GnRH, etc., and the expressed recombinant immunotoxin proteins have specific cytotoxic effects.
  • the GnRH receptor was first discovered in the pituitary.
  • GnRH and its receptors have been found on the pituitary and hindbrain cells of vertebrate bullfrogs.
  • Type I receptors and I Type GnRH binds
  • type II receptor binds to type II GnRH
  • the cross-reaction between the two is extremely low.
  • a large number of experiments have been carried out: (1) The distribution of GnRH receptors on the cell membrane of human placenta (including endometrium, myometrium, ovary and testis), placenta and brain tissue. (2) It is not excluded that there is affinity for other normal tissue membranes and the presence of low GnRH receptors, such as the liver. (3) GnRH receptors are mainly distributed on liver cancer, gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, endometrial cancer, uterine fibroids, breast cancer and prostate cancer cell membranes.
  • GnRH refers to an analog or derivative of native GnRH capable of binding to a cell having a steroid hormone receptor on its surface and which elicits anti-GnRH antibody production when administered to a mammalian host at a high dose.
  • Natural GnRH also known as luteinizing hormone releasing hormone (LHRH or LRH), is a decapeptide molecule having the amino acid sequence shown below:
  • amino acid symbols used in the formula are three-letter abbreviations commonly used in the art, and wherein pGlu represents pyroglutamic acid. Since pyroglutamic acid cannot be synthesized in genetic engineering expression, it is generally mutated to glutamine.
  • An mGnRH analog or derivative that may be suitable for use in the present invention is Trp 6 -mGnRH. The results showed that amino acids 3 to 6 in the molecule are essential for maintaining GnRH activity, while Ser 4 and Tyr 5 play a key role in receptor binding. There is evidence that ovarian cells, particularly granulosa cells, have a GnRH receptor on their surface.
  • the guide portion designed in the fusion toxin of the present invention is a mutant mGnRH amino acid sequence: Met-Gly-GI-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-His (SEQ ID NO : 4) ;
  • the amino acid symbols used in the formula are three-letter abbreviations commonly used in the art, in which a codon for Met-Gly is added to the N-terminus for gene synthesis, and the eighth amino acid of the new sequence is at the gene level.
  • the mutation is Trp to enhance the ability of the expression product to bind to the corresponding receptor; and the E. coli bias codon is introduced therein to make the expression product suitable for expression in E. coli.
  • the biological activity analysis of the prepared mGnRH-PE38m4a fusion protein has clearly demonstrated that mGnRH_PE38m4a has cytotoxic activity against tumor cells derived from various sexual organs including A549 cells, but not for normal cells of various tissue sources. Killing activity (see Example 2).
  • IC 5 refers to kill the target cells reached a concentration of 50% in the control group chimeric toxin required.
  • the "IC 5 " value is detected using a standard MTT method.
  • PE is a single-stranded toxin protein of 613 amino acids with a molecular weight of 66 kDa, a precursor of 638 amino acids, and a height of 25 amino acids cut in the secretory process.
  • Hydrophobic guide peptide It is known from the crystal structure of PE that it is spatially divided into three domain (Down, D) regions, namely, I region, I I region, and I I I region (see Figure 1). The I region is at the amino terminus (N-terminus) of PE, which is 1/3 of the molecule, and has an antiparallel ⁇ structure.
  • the la region consists of the first to 252 amino acids
  • the lb region consists of the 365th to 399th amino acids.
  • the I I region consists of 253 to 364 amino acids and is a central region composed of 6-7 a_helices.
  • the I I I region consists of the 400th to 613th amino acids and is the carboxy terminal region, which accounts for 1/3 of the molecule.
  • the toxin has eight cysteines in its molecular structure, forming four disulfide bonds, which are the determining factors for the formation of the tertiary structure.
  • PEA The structural function of PEA is characterized by its three structural functional regions, which are capable of cytotoxicity, such as cell binding, translocation and ADP-ribosylation, which are required for cytotoxicity in a single peptide chain, to ascertain the functions of each region and to analyze the structure of each region.
  • cytotoxicity such as cell binding, translocation and ADP-ribosylation
  • the functional relationship is mainly based on the study of PEA gene mutations.
  • the la region functions to recognize and bind to target cell surface receptors.
  • the deletion of the la region produces a PE40 with a molecular weight of 40,000, which retains the intact ADP-ribosylation function, but the cytotoxicity is reduced to 1% due to the loss of PE40.
  • the result of cell binding capacity Site-directed mutagenesis studies on the la region revealed that the cytotoxicity of PE mutated when the 57th lysine (Lys57) was induced to glutamate (Glu) was reduced to 1% due to the mutation of the toxin with the target cell surface. The reason for the loss of binding ability.
  • Lys57 is located on the surface of PE molecules, which is consistent with its receptor binding ability.
  • Lys57 is a key amino acid site in the la region.
  • three basic amino acids, His246, Arg247 and His249, located in the la region, are also important sites for maintaining the biological activity of PE.
  • the mutant PE Glu246, 247, 249 and PE Glu57, 246, 247, 249 the above basic amino acid has been replaced by Glu, and the hydrogen bond linking the la region to the II region is blocked, and the mutant molecule is more than the natural PE. Stretching increases the sensitivity to proteases and shortens the half-life of circulation in mice, and therefore, they show low cytotoxicity to animals.
  • the lb region is located between the la region and the I I I region in a three-dimensional structure, and most of the deletion in this region does not affect the biological activity of PE.
  • the amino acids 365 to 381 of the lb region were deleted to form PE38 molecules.
  • the PE38 molecule has a molecular weight of 2000 Da less than PE40, and 3 to 4 key epitopes are deleted, so the antigenicity is significantly reduced.
  • Zone II plays a leading role in the transmembrane translocation of toxins.
  • region II When the region II is deleted, although its cell-binding ability and ADP-ribosylation activity persist, the cytotoxicity is lost, indicating that the region II is necessary for the toxin translocation function. Insertion of membrane or transmembrane into the membrane of the strong hydrophobic region of zone II is an important part of the translocation process.
  • the I I I region has three functions, one of which is to catalyze the ADP-ribosylation of EF-2, and the second to direct the enzymatically active fragment of the toxin into the endoplasmic reticulum.
  • the three activate Caspases-3 enzymes involved in apoptosis, which initiate apoptosis of the cells and cause apoptosis.
  • Al lured suggested that a fracture zone in the I I I region is the active center of the enzyme.
  • Glu553 localized in the fracture region is a key active site, Glu553 is involved in the process of binding to NAD, and point mutation of Glu553 to Asp leads to at least 1% reduction of ADP-ribosylation activity, and complete loss of activity after deletion. .
  • Residues Arg458, Arg467 and Trp466 in several other fracture regions are also involved in the interaction of I I I with NAD.
  • Tyr481 When Tyr481 is substituted with Phe, it causes ADP-ribosyltransferase activity to decrease but does not decrease NAD glycoside hydrolase activity, and is presumably involved in interaction with EF-2; Trp558 is not directly involved in NAD-binding process but ADP-ribosylation Required.
  • region III is located in the a-helix consisting of amino acid residues 421 to 432, which is located at the distal end of the catalytic center of the enzyme, and plays a key role in the molecular construction of the catalytic site of the III region. effect.
  • the surface of the I I I region near the Arg490 is another protease target region. Loss or mutation of Arg490 can result in the production of protease-resistant molecules, and deletion or mutation of Arg492 causes a decrease in ADP-ribosylation activity.
  • the function of the C-terminal specific amino acid sequence of the I I I region is to mediate the translocation of PE from the endocytic vesicle to the endoplasmic reticulum.
  • This specific sequence is a fragment of five amino acid residues of Arg609-Glu610-Asp61 1- Leu612_Lys613 (S ⁇ REDLK). Its deletion results in loss of cytotoxicity of PE, but has no effect on its ADP-ribosylation activity.
  • the PE molecule used to construct the fusion protein of the present invention is based on the deletion of the PE region of the la region and most of the lb region, retaining 609 Arg and 611 Asp, and mutating Glu610, Leu612, Lys613 to Lys610, Glu612 and Leu613, and formed a new amino acid combination RKDEL, namely PE38m4a.
  • the original sequence gene of the gonadotropin-releasing hormone of the targeting agent or the recognition molecule of the anti-tumor chimeric toxin of the present invention is not suitable for genetic engineering expression, and the PE38m4a multi-site mutation as a cytotoxic component, for the convenience of gene manipulation, artificial
  • the gene sequence of the following components was designed and synthesized: Ncol endonuclease recognition sequence - mGnRH nucleotide sequence consisting of E. coli bias codon, wherein the 6th Gly mutation is Trp-Ndel endonuclease
  • the sequence _PE38m4a nucleotide sequence-EcoRI endonuclease recognition sequence was identified.
  • the 5' and 3' ends of the synthetic gene sequence introduce Nco l and EcoRI endonuclease cleavage sites, respectively, and result in sticky ends suitable for ligation.
  • the gene into the expression vector pET27 (Merck Co.) can be cloned according to recombinant techniques known to those skilled in the art (see, for example, Sambrook et al., Mol ecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, 1989). Construct pET27-mGnRH-PE38m4a.
  • the recombinant vector system is then transformed into prokaryotic cells such as E. coli cells to produce the mGnRH-PE38m4a fusion protein.
  • restriction enzyme digestion was used to identify the correctness of the sequence joining direction and possible mutations, and finally the sequence was determined by Sanger dideoxy chain termination method to further confirm.
  • the recombinant protein gene will be operably linked to appropriate expression control sequences, such as connecting to suitable for use in E. coli T 7, trp promoter or ⁇ promoter, ribosome binding site and the transcription termination signal.
  • the recombinant plasmid of the present invention can be transformed into a host cell of choice using known transformation methods, such as calcium chloride treatment for prokaryotic cells.
  • the transformed positive cells can be selected based on the antibiotic resistance conferred by the antibiotic resistance gene contained on the plasmid.
  • the fusion protein can be isolated and purified according to methods known in the art.
  • the bacterial cells can be collected by centrifugation from the fermentation culture and lysed with lysozyme and ultrasonic waves, then ultracentrifuged and subjected to stepwise precipitation by adding saturated ammonium sulfate to a low concentration phosphate (about 20 mM) solution.
  • the desired mGnRH-PE38m4a recombinant protein was purified by ion exchange chromatography (IEC) and size exclusion chromatography (SEC) in that order.
  • IEC ion exchange chromatography
  • SEC size exclusion chromatography
  • it may be purified by methods such as salting out, affinity chromatography and preparative gel electrophoresis (see R. Scopes, Prote in Purif icafion, Springer-Verlag, N, Y., 1982).
  • the elution fractions of each column were analyzed by SDS-PAGE electrophoresis (Laemml i, Nature 227: 680-689, 1970) using a polyacrylamide gel, and polyclonal anti-PE antiserum was used and immunoblotted. The law detects it.
  • a tumor cell inhibition assay was performed on the recombinant fusion protein purified product to detect the cytotoxicity of the fusion protein (IC 5 ).
  • the 125 1-labeled native GnRH and the fusion protein were competitively substituted on the surface of A549 cells to determine the affinity of mGnRH in the fusion protein to the native GnRH for cell surface receptors.
  • the mGnRH-PE38m4a fusion protein of the present invention can be used as a basic active ingredient, and one or more pharmaceutically acceptable carriers or excipients can be added to prepare a pharmaceutical composition suitable for clinical use.
  • the carrier or excipient includes, but is not limited to, phosphate buffered saline, physiological saline, isotonic glucose solution, dextran, dextran, and the like.
  • one or more other natural, synthetic or recombinant active compounds which are auxiliary or synergistic with the fusion proteins of the invention may be added to the pharmaceutical compositions of the invention.
  • a human serum albumin, a low molecular weight peptide, a glycine or a lysine, and a metal cation may be added to the pharmaceutical composition of the present invention.
  • a protein protectant and selected from polyethylene glycol, carboxymethyl Stabilizer for cellulose, polyglycine, glutathione.
  • the pharmaceutical composition of the present invention can be administered by a conventional administration route, particularly a parenteral route, for example, by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous or intramucosal routes.
  • the effective dosage of the pharmaceutical composition of the present invention may range from a few nanograms to several tens of milligrams per kilogram of body weight per day, but the specific dosage for each particular patient will depend on the nature and severity of the disease or pathological condition to be treated, the patient's Age, weight, ability to respond to drugs, and mode of administration.
  • the mechanism of action is not clear, our laboratory has demonstrated that the mGnRH-PE38m4a protein pair of the present invention includes colon cancer HT-29 cells, ovarian cancer 0VCAR3 cells, and cervical adenocarcinoma HeLa cells.
  • tumor cell lines such as HepG-2 cells have obvious specific binding activity and cytotoxicity; normal cells have almost no lethal effect; fusion protein is 9-10 times higher than natural PEA activity; and the cost is low, the method is simple, and more Suitable for industrial production.
  • Figure 1 shows the construction of a recombinant plasmid for expression of mGnRH-PE38m4a.
  • Figure 2 shows the saturation curves of 125 I-mGnRH-PE38m4a (A) and 125 I_GnRH (country) binding to A549 cell membrane surface receptors.
  • Figure 3 shows a curve in which 125 1 -labeled mGnRH-PE38m4a and GnRH compete with A549 cell membrane surface receptors for competition with unlabeled different concentrations of GnRH or mGnRH-PE38m4a, respectively.
  • ⁇ Representing the different concentrations of GnRH to compete for the curve of 125 I-mGnRH-PE38m4a, the country represents a different concentration of mGnRH_PE38m4a to compete for the curve of 125 I_GnRH.
  • T 4 ligase Promega was cloned in a direct PGEM-T vector and transformed into E. coli JM105, obtaining engineered strains containing PGEM-T / mGnRH-PE38m4a plasmid.
  • SEQ ID NO: 1 shows the nucleotide sequence of the measured mGnRH-PE38m4a recombinant gene.
  • SEQ ID NO: 2 shows the amino acid sequence deduced from the nucleotide sequence of the measured mGnRH-PE38m4a recombinant gene.
  • Figure 1 shows the construction of the recombinant plasmid pET27-mGnRH-PE38m4a.
  • E. coli BL21 ( ⁇ DE3) carrying the recombinant plasmid pET27-mGnRH-PE38m4a (containing the T 7 RNA polymerase promoter gene) (Studier, FW and Mof f att, BA, J. Mol, Biol, 189: 113- 130, 1986) Culture in LB medium containing kanamycin (50 ⁇ g/ml) at 37 °C, when 0D 6 . . When it was about 0. 4 ⁇ 0. 6, ImM isopropylthio- ⁇ _D_galactoside (IPTG) (final concentration ImM) was added, and incubation was continued at 37 °C for 3-4 hours to induce expression of the target product.
  • IPTG isopropylthio- ⁇ _D_galactoside
  • the bacteria are collected by centrifugation, and the bacteria are crushed by an appropriate method, and then the precipitate and the supernatant are collected by centrifugation, and the expression form and expression amount of the target protein are determined by SDS-PAGE electrophoresis.
  • SDS-PAGE electrophoresis showed that the target protein was expressed in soluble secretion, and the expression amount was about 20% of total bacterial protein.
  • the buffer containing the target protein supernatant was added to a buffer concentration of 50 mM Tri s-HCl, pH 8.0, ImM EDTA, 4 °C hollow fiber ultrafilter (Mi l ipore) for 30 minutes, and centrifuged at 4 ° C. (20, 000 g, 30 minutes), the supernatant (soluble fraction) is the crude extract of mGnRH-PE38m4a.
  • the crude extract of mGnRH-PE38m4a was buffer-balanced on a DEAE-Sepharose Fast Flow column (Pharmacia), washed in a continuous gradient with TE buffer (20 mM Tri s-HCl, pH 8.0, ImM EDTA) containing 0 to 0.5 M NaCl. Remove and collect the peak fraction of each component of the protein. The peak of the target component was subjected to a small hollow fiber ultrafilter (Mi l ipore) for 30 minutes and concentrated by ultrafiltration. The concentrate was buffered by using 20 mM Tri s-HCl, pH 8.0, ImM EDTA, 0.15 M NaCl.
  • the solution was equilibrated with a 1.6 X 100 cm Sephacryl-100 column (Pharmacia) and eluted with TE buffer (20 mM Tris. HC1, pH 8.0, ImM EDTA) containing 0.15 M NaCl.
  • TE buffer 20 mM Tris. HC1, pH 8.0, ImM EDTA
  • the active peak fraction was collected and again passed through a high pressure liquid chromatography column (Shimadzu, Japan), and the protein peak (A 28 ) fraction was collected and thoroughly dialyzed in 30 mM PBS, and dialyzed and stored at -20 ° C until use.
  • the purity of the protein thus purified is >95%.
  • the purified mGnRH-PE38m4a protein was identified by binding assay of tumor cell A549 with 125 1 labeled natural GnRH and mGnRH_PE38m4a and polyclonal anti-PE antiserum.
  • Example 2 Target specificity and biological activity analysis of 125 1 labeled native GnRH and mGnRH-PE38m4a fusion proteins
  • 125 1 label of GnRH and mGnRH_PE38m4a Weigh 1 mg Lodogen dissolved in 0.5 ml of chloroform, take 50 ⁇ l (100 ⁇ ⁇ to the bottom of the tube, blow dry with nitrogen, add the polypeptide without protective agent Or protein semi-finished product 0. 4ml, added Na 125 I 5mCi reaction at room temperature for 12min, the reaction process is continuously shaken, so that the reaction is sufficient.
  • the 125 1 labeled mixture was separated and purified using a Sepharyl S-200 HR gel column (1 X 50 cm). Tube, take the tube with the highest radioactivity for testing.
  • RPMI-1640 complete medium Monolayer adherent cultures were counted and counted in RPMI-1640 complete medium at 5% CO 2 , 37 °C. Add 125 1 labeled GnRH, mGnRH-PE38m4a (5 ⁇ Ci/well, diluted in BSA-PBS) of the same CPM to each well, and perform serial dilutions of the series. After 1 hour, RPMI-1640 complete medium wash 5 The cells were collected by trypsinization, pipetting, and counted by Y (see Figure 2 for results).
  • both 125 1-labeled GnRH and mGnRH_PE38m4a bind to the surface of tumor cell A549 in a dose-dependent manner until saturation is reached, and the binding ability of 125 I-mGnRH-PE38m4a is shown to be > 125 I-GnRH, The ability to bind to normal cells was extremely low, thus confirming that the purified mGnRH-PE38m4a fusion protein in the present invention can bind to receptor-positive cells in a dose-dependent manner.
  • GnRH, mGnRH_PE38m4a and 125 1 labeled GnRH, mGnRH_PE38m4a competition binding assay The peptide or protein binding to A549 cells was performed essentially as described by Qayum, A. et al. (Br. J. Cancer 62: 96-99, 1990). Specific competition and substitution studies.
  • the cultured A549 monolayer cells were counted and added to 125 I_mGnRH of the radioactive marker Y counting unit at the inflection point of the test saturation curve and incubated at 37 ° C for 1 hour. Wash RPMI-1640 complete medium 5 times, add unlabeled mGnRH-PE38m4a (0.
  • Figure 3 shows a curve in which China's different concentrations of mGnRH-PE38m4a compete for 125 I_GnRH. ⁇ Representing a different concentration of GnRH to compete for the curve of 125 I-mGnRH-PE38m4a.
  • the label can be substituted by adding unlabeled mGnRH-PE38m4a or GnRH at an increasing concentration, and it can be seen that the concentration of GnRH substituted 125 I-mGnRH-PE38m4a is mGnRH- PE38m4a replaced about 7-8 times the concentration of 125 I_GnRH, indicating that the mGnRH_PE38m4a fusion protein was subjected to mutation treatment, and the mGnRH component was more than 7-fold more binding to the target cell than the unmutated GnRH.
  • Example 3 Cytotoxicity test
  • Quantitative samples were filtered and sterilized. Different amounts of samples were added to each cell well by equal dilution method, and the total volume was 100 ⁇ l, 5% C0 2 , and cultured at 37 °C for 12 h. Add 100 ⁇ l ⁇ staining reagent, 5% C0 2 , 37 °C for 4 h, measure the absorbance at 490 nm, and calculate the concentration of recombinant toxin on 50% cell death of various tumors and normal cells ( IC 5 .), while using Pseudomonas aeruginosa exotoxin A (PEA, Sigma) as a control, compares the changes in activity of the fusion toxin and natural PEA. The results are shown in Table 1. Table 1: ICs of purified mGnRH-PE38m4a and native PEA for tumor and normal cells:
  • Table 1 shows the cytotoxicity of purified mGnRH-PE38m4a and native PEA on certain tumor cells and certain normal cell cultures.
  • IC 5 The value refers to the concentration of the fusion protein required to kill the target cells to 50% of the control group.
  • A549 is a human lung adenocarcinoma cell line
  • L0V0 is a human intestinal cancer cell line
  • MKN45 is a human gastric adenocarcinoma cell line
  • Bcap37 is a human breast cancer cell line
  • QGY is a human liver cancer cell line
  • PC-3M is a human prostate cancer cell line
  • A375 is a human melanoma cell line.
  • KB is a human oral cancer cell line
  • normal cells are human embryo kidney and human embryo liver culture strain.
  • the purified mGnRH-PE38m4a of the present invention can kill human lung adenocarcinoma, human intestinal cancer, human gastric adenocarcinoma, human breast cancer, human liver cancer, human prostate cancer, human melanoma, Human oral cancer cell line, and IC 5 .
  • the value is lower, and has no killing effect on normal cell human embryonic kidney and human embryo liver culture strain; natural PEA has IC 5 on the same kind of tumor cells. Both are higher than the fusion protein IC 5 .
  • the value, the difference between the two is about 10 times, which shows that the fusion protein is 9-10 times more effective than the natural PEA activity.
  • the test also showed that PEA has a lethal effect on almost all cells, while mGnRH-PE38m4a has no lethal effect on normal cells.

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  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur une protéine de fusion à double mutant à spécificité de cible qui est construite à partir d'une protéine de fusion d'un mutant d'hormone de libération de la gonadotropine humaine (mGnRH) et d'un mutant de l'exotoxine A de Pseudomonas aeruginosa recombinant (PE38m4a). La protéine mGnRH-PE38m4a de la présente invention a une activité de liaison spécifique significative et une cytoxicité pour des lignées de cellule tumorale comprenant les cellules HT-29 du cancer du côlon, les cellules OVCAR3 du cancer des ovaires, les cellules HeLa de l'adénocarcinome du col de l'utérus et les cellules HepG-2 du cancer du foie et similaire; et n'a essentiellement aucun effet létal sur des cellules normales, et a une activité 9 à 10 fois supérieure à PEA naturel, et est rentable, facile à produire et appropriée pour une production industrielle.
PCT/CN2009/073441 2008-08-25 2009-08-24 Protéine de fusion à double mutant à spécificité de cible WO2010022639A1 (fr)

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CN2008100511124A CN101343328B (zh) 2008-08-25 2008-08-25 靶特异性双突变体融合蛋白质

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WO2015143581A1 (fr) * 2014-03-24 2015-10-01 北京博翱泰生物技术有限公司 Protéine de fusion à double mutant à spécificité de cible et son procédé de fabrication

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CN101343328B (zh) * 2008-08-25 2011-06-01 北京博翱泰生物技术有限公司 靶特异性双突变体融合蛋白质
CN102161998B (zh) * 2011-01-14 2013-01-09 中国人民解放军军事医学科学院附属医院 基于b7-1-pe40kdel外毒素融合基因的dna疫苗及其用途
CN102603897B (zh) * 2011-12-08 2014-10-08 山东省科学院生物研究所 含引导肽和GnRH-PE39KDEL的融合蛋白和核酸及其用途
CN110357968B (zh) * 2018-04-08 2023-08-25 吉林省汇融生物科技有限公司 抗肿瘤融合蛋白及其制法和应用

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WO1999049059A2 (fr) * 1998-03-24 1999-09-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procedes servant a diagnostiquer le cancer au moyen d'une toxine chimere
CN1840546A (zh) * 2005-04-01 2006-10-04 北京诺思兰德生物技术有限责任公司 具有导向杀灭肿瘤细胞的重组融合蛋白
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WO2015143581A1 (fr) * 2014-03-24 2015-10-01 北京博翱泰生物技术有限公司 Protéine de fusion à double mutant à spécificité de cible et son procédé de fabrication

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