WO2017147820A1 - Vecteur de médicament à double ciblage - Google Patents

Vecteur de médicament à double ciblage Download PDF

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WO2017147820A1
WO2017147820A1 PCT/CN2016/075329 CN2016075329W WO2017147820A1 WO 2017147820 A1 WO2017147820 A1 WO 2017147820A1 CN 2016075329 W CN2016075329 W CN 2016075329W WO 2017147820 A1 WO2017147820 A1 WO 2017147820A1
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target
vegf
rgd
egf
dual
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PCT/CN2016/075329
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Chinese (zh)
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张正
蓝耿立
王信二
张顺福
李佳哲
詹佩嘉
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法玛科技顾问股份有限公司
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Priority to CN201680083086.6A priority Critical patent/CN108699164B/zh
Priority to PCT/CN2016/075329 priority patent/WO2017147820A1/fr
Publication of WO2017147820A1 publication Critical patent/WO2017147820A1/fr

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    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

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  • the present invention relates to a novel target drug carrier, and in particular to a dual target drug carrier having two target molecules.
  • Cancer is the leading cause of death in the United States, and cancer mortality continues to increase. Cancer is the inability of cells to divide, grow and differentiate normally. The initial clinical manifestations of cancer are extremely uneven, with almost 70 cancers occurring in human organs and tissues, and some of these similar cancer types belong to many different molecular diseases. Unfortunately, some cancers may have no actual symptoms until late in the course of the disease, making treatment and prognosis extremely difficult.
  • Cancer treatment usually includes surgery, chemotherapy, and/or radiation therapy. All current therapies have serious side effects and reduce the quality of life. Most chemotherapeutic drugs act on both normal and cancerous tissues. Therefore, one of the challenges in treating cancerous tumors is to give cancer cells maximum killing while minimizing damage to healthy tissue. Depending on the drug (eg, intravenous) and nature of the route of administration (eg, its physical and pharmacokinetic properties), typically only a small fraction of the administered dose reaches the target cell, with the remainder acting on other tissues or rapidly disappearing.
  • drug eg, intravenous
  • nature of the route of administration eg, its physical and pharmacokinetic properties
  • Tumor neovascular endothelial cells express a large number of specific membrane proteins, including ⁇ v ⁇ 3, ⁇ v ⁇ 5, integrin, and vascular-related growth factors.
  • RGD short-chain peptides
  • tumor cells are highly unstable and variability, and are resistant to drugs during treatment. At present, various drugs and diagnostic agents are still slow and ineffective in improving the survival of cancer patients.
  • the present invention provides a novel vector that can be used to diagnose and treat cancer.
  • the dual target drug carrier of the invention is a fusion protein platform, which can effectively reduce the threshold and cost of the pharmaceutical technology.
  • the invention provides a dual target drug carrier comprising a first target molecule and a second target molecule.
  • the first or second target molecule specifically binds to vascular endothelial cells in tumor cells or/and tumor microenvironment
  • the first and second target molecules include, but are not limited to, arginine-glycine-aspartate (RGD), asparagine-glycine-arginine (NGR) ), cyclic NGR (cyclic asparagine-glycine-arginine), internalization of RGD (iRGD, internalization-arginine-glycine-aspartate), cystine-glycine-aspartate Acid-lysine-arginine-threonine-arginine-glycine-alanine (CGNKRTRGA), stomach Gastrin, bombesin, octreotide or a derivative thereof.
  • RGD arginine-glycine-aspartate
  • NGR asparagine-glycine-arginine
  • cyclic NGR cyclic asparagine-glycine-arginine
  • internalization of RGD iRGD, internalization-arginine-glycine-aspartate
  • Macromolecular peptides include, but are not limited to, epidermal growth factor (EGF), anti-EGFR (epidermal growth factor receptor) antibody, vascular endothelial growth factor (VEGF), anti-VEGFR ( Vascular Endothelial Growth Factor Receptor, anti-HER2 (human epidermal growth factor receptor 2) antibody, hepatocyte growth factor receptor (HGFR), anti-HIV - HGFR antibody, tumor necrosis factor (TNF) or anti-TNF antibody.
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • anti-HER2 human epidermal growth factor receptor 2
  • HGFR hepatocyte growth factor receptor
  • TNF tumor necrosis factor
  • the first target molecule is linked to the second target molecule by a linker.
  • the connector is a peptide having 5 to 20 amino acids.
  • the linker comprises, but is not limited to, GG, PGGGG or GGGGSGGGGS, wherein G represents glycine, P represents proline, and S represents serine.
  • a radioisotope is further included.
  • the invention further provides a pharmaceutical composition comprising the above dual target pharmaceutical carrier and a pharmaceutically acceptable carrier.
  • a liposome is further included.
  • nanoparticles are further included.
  • Figure 1A shows purified VEGF, RGD-VEGF, RGD4C-VEGF, EGF, SDS-PAGE electropherogram of RGD-EGF and RGD4C-EGF protein.
  • Figure 1B is a SDS-PAGE electrophoresis map of marker, VEGF, RGD-VEGF, and RGD4C-VEGF, wherein M is marker, 1 is VEGF (15.3 Da), 2 is RGD-VEGF (15.6 Da), and 3 is RGD4C-VEGF ( 16.3Da).
  • FIGS. 2A-2F are binding curves/bar graphs of RGD-VEGF and RGD4C-VEGF and ⁇ v ⁇ 3, VEGFR1, VEGFR2.
  • RGD-EGF and RGD4C-EGF bind to ⁇ v ⁇ 3 and EGFR, respectively.
  • Figure 2A is a graph showing the binding of RGD-VEGF and RGD4C-VEGF to ⁇ v ⁇ 3.
  • Figure 2B is a bar graph of competition of RGDfV polypeptide with RGD-VEGF and RGD4C-VEGF in combination with ⁇ v ⁇ 3.
  • Figure 2C is a graph showing the binding of RGD-VEGF and RGD4C-VEGF to VEGFR1.
  • Figure 2D is a graph showing the binding of RGD-VEGF and RGD4C-VEGF to VEGFR2.
  • Figure 2E is a bar graph of competition of VEGFR2 antibodies by RGD-VEGF and RGD4C-VEGF in combination with VEGFR2.
  • Figure 2F is a graph showing the binding of RGD-EGF and RGD4C-EGF to ⁇ v ⁇ 3.
  • Figure 3A is a graph showing the binding of U87MG (expressing ⁇ v ⁇ 3 and EGFR) cells to RGD-VEGF and RGD4C-VEGF.
  • RGD-VEGF and RGD4C-VEGF can bind to ⁇ v ⁇ 3 and EGFR, respectively.
  • Figure 3B is a bar graph of binding of U87MG (expressing ⁇ v ⁇ 3 and EGFR) cells to RGD-VEGF and RGD4C-VEGF.
  • Figure 3C is a graph showing the binding of HUVEC (expressing VEGFR1, VEGFR2 and ⁇ v ⁇ 3) cells to RGD-VEGF, RGD4C-VEGF and NGR-VEGF.
  • RGD-VEGF, RGD4C-VEGF and NGR-VEGF can bind to express VEGFR1 Cells of VEGFR2 and ⁇ v ⁇ 3.
  • Figure 3D is a bar graph of binding of HUVEC (expressing VEGFR1, VEGFR2 and ⁇ v ⁇ 3) cells to RGD-VEGF, RGD4C-VEGF and NGR-VEGF.
  • Figures 4A-4D are cell adhesion assays. After coating RGD-VEGF and RGD4C-VEGF on the cell plate, the number of U87MG cells and HUVEC cells attached to the culture plate can be increased. If a large amount of RGD peptide chain is added to compete, the adsorption of cells can be reduced. 4A shows that RGD-VEGF and RGD4C-VEGF are applied to the cell plate to increase the number of U87MG cells attached to the culture plate, and FIG. 4B shows the amount of cells attached to the culture plate after the addition of the RGDfV polypeptide.
  • Figure 4C-4D shows that RGD-VEGF and RGD4C-VEGF bind to HUVEC cells, and then compete with RGDfV polypeptide or VEGFR2 recombinant protein, respectively, in terms of absorbance and percentage.
  • Figures 5A-5B show that RGD-EGF and RGD4C-EGF or RGD-VEGF and RGD4C-VEGF activate a signaling pathway downstream of EGFR or VEGFR, respectively.
  • the EGF or VEGF of the dual target drug carrier of the invention retains its original biological properties.
  • M of Figure 5A is Marker
  • C is the control group (no protein added)
  • E is EGF (25 nM)
  • RE is RGD-EGF (25 nM)
  • R4C-E is RGD4C-EGF (25 nM)
  • C of Figure 5B For the control group (no protein added), RV is RGD-VEGF, R4-V RGD4C-VEGF.
  • Figure 6 is a graph showing the stability of a radiolabeled fusion protein in a buffer solution (HEPES, 4 ° C) and serum (37 ° C).
  • the radiolabeled fusion protein was stored in Hepes buffer (4 ° C) and fetal bovine serum (37 ° C), and the radiochemical purity was still higher than 90% after 24 hours, indicating the radioactive In-111-labeled fusion protein.
  • the serum stability is excellent.
  • Fig. 7 is a fluorescent photograph.
  • the RGD-VEGF and RGD4C-VEGF marker recombinant proteins can bind to cells.
  • Figure 8 is a single photon and computed tomography (SPECT/CT) angiogram of radiolabeled recombinant protein.
  • U87MG subcutaneous tumor mice were injected with 111 In-DTPA (Diethylene triamine pentacetate aci)-EGF, 111 In-DTPA-RGD-EGF and 111 In-DTPA-RGD4C-EGF.
  • Small animal single photon and computed tomography (SPECT/CT) angiography was performed at 4, 8 and 24 hours.
  • SPECT/CT computed tomography
  • the image shows that after the In-111 marker recombinant protein is applied, the specific accumulation of the tumor in the tumor (expressed as the tumor/muscle accumulation ratio) increases with time, reaching a peak at 8 hours after the drug injection, at this time 111 In
  • the tumor/muscle accumulation ratio of -DTPA-RGD4C-EGF was 4.4, slightly higher than 3.6 of 111 In-DTPA-RGD-EGF, and much higher than 1.7 of 111 In-DTPA-EGF.
  • the invention provides a dual target drug carrier comprising a first target molecule and a second target molecule.
  • the target molecule of the present invention is a peptide, antibody or protein.
  • the target molecules of the invention can be chemically modified to increase stability.
  • Chemically modified peptides or peptide analogs include any chemically functional equivalent that increases stability and/or potency either in vivo or in vitro.
  • a peptide analog refers to an amino acid derivative of any peptide.
  • Peptide analogs include, but are not limited to, modified branches upon synthesis, combined with unsaturated amino acids and/or derivatives thereof, cross-linking and other methods that increase the peptide identity.
  • a branched modification includes a modification of an amine group.
  • the target molecule can bind to the lesion tissue in vivo and in vitro, especially for vascular endothelial cells of tumor, cancer tissue/cell and tumor microenvironment. Therefore, when the target peptide is conjugated to a reporter molecule (bioimaging fluorescent molecule or radiation), the tumor target peptide can directly display the tumor location and contribute to the diagnosis of cancer.
  • Conjugation as used in the present invention means that two molecules that contribute to the treatment/diagnosis of a tumor, such as a tumor target peptide and a reporter molecule, are bound by sufficient affinity. Conjugation can be accomplished by covalent, non-covalent bonds or other forms of bonding, for example, coating.
  • the target molecule of the present invention may be a small molecule peptide and/or a macropeptide.
  • Small molecule peptides include, but are not limited to, lysine-glycine-aspartate (RGD), asparagine-glycine-arginine (NGR), cyclic NGR, internalization RGD (iRGD), cystine - glycine-aspartate-lysine-arginine-threonine-arginine-glycine-alanine (CGNKRTRGA), gastrin, bombesin, octreotide Octreotide) or a derivative thereof.
  • RGD lysine-glycine-aspartate
  • NGR asparagine-glycine-arginine
  • iRGD internalization RGD
  • gastrin bombe
  • Macromolecular peptides include, but are not limited to, epidermal growth factor (EGF), anti-EGFR antibodies, vascular endothelial growth factor (VEGF), anti-VEGFR antibodies, anti-HER2 antibodies, hepatocyte growth factor receptor (HGFR), Anti-HGFR antibody, tumor necrosis factor (TNF) or anti-TNF antibody.
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • HGFR hepatocyte growth factor receptor
  • TNF tumor necrosis factor
  • the target molecule can form a conjugate with an imaging substance or a radiotherapeutic substance (radiopharmaceutical or isotope).
  • Radioactive imaging materials include, but are not limited to, 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm , 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb and 67 Ga.
  • Radiopharmaceuticals include, but are not limited to, 111 In hydroxyquinoline, 131 I sodium iodide, 99m Tc phenyl bromide, 99m Tc red blood cells, 123 I sodium iodide, 99m Tc Exametazime, 99m Tc large particle polymeric albumin, 99m Tc-methylene phosphonate, 99m Tc Mertiatide, 99m Tc hydroxymethylene diphosphonate, 99m Tc triamine pentaacetic acid, 99m Tc Pertechnetate, 99m TcSestamibi, 99m Tc sulphur colloid, 99m Tc Tetrofosmin, 201 Tl and 133 Xe .
  • Radiation isotopes include, but are not limited to, 52 Fe, 52 mMn, 55 Co, 64 Cu, 67 Ga, 68 Ga, 99 mTc, 111 In, 123 I, 125 I, 131 I, 32 P, 47 Sc, 67 Cu, 90 Y, 109 Pd, 111 Ag, 149 Pm, 186 Re, 188 Re, 211 At, 212 Bi, 213 Bi, 105 Rh, 153 Sm, 177 Lu and 198 Au.
  • Non-gastrointestinal as used in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, articular, arterial, synovial, focal or intracranial injections.
  • the "linker” as used in the present invention refers to a sequence of 2 to 50 synthetic amino acids, preferably 5 to 20 synthetic amino acids, which are linked to two peptide sequences, for example, to link two peptide structures.
  • the connector of the present invention can link two amino acid sequences through a peptide bond.
  • the linker peptide of the present invention is a linear sequence linking the first portion to the second portion.
  • the replaceable connector may be (GGGGS)n, (G)n, (EAAAK)n, (XP)n or (PAPAP)n, eg, GG, GGSG, GGGS, GGGGS, GGSGG, GGGSGG, GGGSGGG, GGGSGGGS, GGGSGGGGS, ASGG, GGGSASGG, SGCGS, GGGGSGGGG, GGSHG, SGCGGGS, and AACAA, wherein said G represents glycine; said S represents serine; said E represents glutamic acid; said A represents alanine; K represents lysine; P represents valine; said H represents histidine; and C represents cystine.
  • the dual target drug carrier of the present invention can carry a tumor therapeutic drug or It is a substance (such as a therapeutic radionuclide) that becomes a tumor target drug carrier.
  • a drug capable of molecular imaging can also be carried as a molecular imaging probe for tumor diagnosis.
  • the double target developer carrier of the invention has the effect of tumor diagnosis.
  • EGF, RGD-EGF, RGD4C-EGF, VEGF, RGD-VEGF and RGD4C-VEGF genes were constructed in the expression vector of pET28a(+) with Nco I and Xho I restriction enzyme cleavage sites.
  • the C-terminus (ie, the carbon terminus) of RGD and RGD4C is ligated to the N-terminus (ie, the nitrogen terminus) of EGF or VEGF, and the C-terminus of EGF or VEGF has a His 6 flag.
  • the His 6 marker facilitates subsequent purification and analysis, wherein the gene sequences of RGD-EGF, RGD4C-EGF, RGD-VEGF, and RGD4C-VEGF comprise the linker GG.
  • the constructed pET28a(+) EGF, RGD-EGF, RGD4C-EGF, VEGF, RGD-VEGF and RGD4C-VEGF expression vectors were transfected into E. coli.DH5 ⁇ to produce and preserve vector DNA. The sequence was confirmed by DNA sequencing.
  • Example 1 The vector of Example 1 was sent to E. coli BL21 (DE3) by heat shock, and screened and confirmed by kanamycin (50 ⁇ g/mL).
  • the E. coli BL21 (DE3) strain carrying the protein expression vector was induced with 1 mM IPTG for 16 hours at 37 °C.
  • the bacterial solution was sterilized by a French press at a pressure of 30 PSI, centrifuged at 13,000 g for 30 minutes, and samples of the supernatant and the pellet were collected for SDS-PAGE electrophoresis analysis, and observed by SDS-PAGE. Performance to the target fusion protein. If it is in the form of an insoluble body, the bacteria are present in the form of an insoluble body.
  • Subsequent protein production receives a pellet after the disruption for protein purification.
  • the lysed target fusion protein was filtered through a 0.45 ⁇ m filter, followed by a 1:50 ratio of refolding buffer (20 mM Tris, 0.1 Mm GSSG, 1 mM GSH, 1 mM EDTA pH 8.0). Dilution refolding.
  • the target protein sample was slowly added to the refolding buffer, and the concentration of the target fusion protein after dilution was not more than 0.2 mg/ml, and the renaturation was carried out at 4 ° C for 12 hours, and then filtered through a 0.45 ⁇ m filter to remove the precipitate.
  • Target protein was carried out at 4 ° C for 12 hours, and then filtered through a 0.45 ⁇ m filter to remove the precipitate.
  • the protein refolding solution was passed through a nickel column at a flow rate of 5 mL/min.
  • the column was first washed with buffer A (20 mM Tris (pH 8.0), 500 mM NaCl, 20 mM imidazole, 0.5 mM PMSF), followed by washing, using buffer A and buffer B (20 mM Tris (pH 8.0), The ratio of the imidazole of the solution was adjusted in proportion to 500 mM NaCl, 500 mM imidazole, 0.5 mM PMSF) to wash out the target fusion protein. Since the collected target fusion protein still has other impurity proteins, the fusion protein was isolated using a gel column (HiPrap 26/60S-100).
  • EGF EGF
  • RGD-EGF NGR-EGF
  • RGD4C-EGF VEGF
  • RGD-VEGF RGD-VEGF
  • NGR-VEGF NGR-VEGF
  • RGD4C-VEGF target fusion proteins target fusion proteins to cells was analyzed by ELISA cell binding assay.
  • 25 ⁇ g/well of integrin ( ⁇ v ⁇ 3) receptor dissolved in PBS (calcium and magnesium ions) was placed in a 96well ELISA microplate, and cultured at 4 ° C for 12 hours, and then reacted with 3% BSA at 25 ° C for 1 hour.
  • the serially diluted target fusion protein was added to culture at 25 ° C for 2 hours, and after washing with PBS buffer (containing calcium and magnesium ions), the mouse anti-His-HRP antibody was used to detect the binding of the protein to the receptor, and the wavelength was measured. The absorbance at 450 nm is used to quantify the amount of protein bound to the receptor.
  • the obtained data using the prism software for nonlinear curve fitting can obtain the dissociation constant K d value of protein binding, which can be used as a comparison of the binding ability of the protein to different receptors.
  • the mouse anti-His-HRP antibody was used to detect binding of the protein to the receptor, and the absorbance at a wavelength of 450 nm was measured to quantify the amount of protein bound to the receptor.
  • the obtained data using the prism software for nonlinear curve fitting can obtain the dissociation constant Kd value of protein binding, which can be used as a comparison of the binding ability of the protein to different receptors.
  • the dissociation constant K d values of EGF, RGD-EGF, RGD4C-EGF and EGFR were 2.38, 20.35 and 14.33 nM, respectively.
  • the dissociation constant K d values of VEGF, RGD-VEGF, RGD4C-VEGF and Integrin were 0, 5.0 and 1.0, respectively.
  • VEGF, RGD-VEGF, VEGFR1 solution RGD4C-VEGF with a dissociation constant K d values of 15.3,11.3 and 11.4.
  • the dissociation constant K d values of VEGF, RGD-VEGF, RGD4C-VEGF and VEGFR2 were 22.2, 12.1 and 13.9, respectively.
  • various dual-target fusion proteins can bind to more than two receptors, with the ability to dual target.
  • RGD-EGF and RGD4C-EGF can bind to Integrin ( ⁇ v ⁇ 3) and EGFR, respectively, and RGD-VEGF and RGD4C-VEGF can bind to Integrin ( ⁇ v ⁇ 3), VEGFR1 and VEGFR2, respectively.
  • This example uses MDA-MB468 (highly expressed EGFR), MDA-MB231 (moderately expressed EGFR), MCF-7 (low performance), HT1080 (expressed APN), U87MG (expressed ⁇ v ⁇ 3) and HUVEC (expressed VEGFR and ⁇ v ⁇ 3) Cell lines to analyze the specific binding of the dual-target fusion protein to different receptors.
  • MDA-MB468, MDA-MB231, MCF-7, HT1080, U87MG and HUVEC cells were seeded in a 96-well microplate at a dose of 20,000 cells/well, and cultured at 37 ° C for 12 hours. After the cells were attached, they were fixed with 4% paraformaldehyde hyde precooled at 4 ° C, and then incubated at room temperature for 15 minutes. After the fixed cells were incubated with 3% FBS at 25 ° C for 1 hour, serially diluted target fusion proteins were added and incubated at 25 ° C for 1 hour.
  • the mouse anti-His-HRP antibody was used to detect the binding of the protein to the cells, and then the TMP of the HRP was used for color measurement.
  • the absorbance at a wavelength of 450 nm was used for quantify the amount of protein bound to the cells.
  • VEGF, RGD-VEGF, RGD4C- VEGF, NGR-VEGF and VEGFR intergrin solution on HUVEC cells with a dissociation constant K d values of 12.7,11.9,6.4 and 9.4nM.
  • RGD-EGF, RGD4C-EGF, RGD-VEGF, and RGD4C-VEGF can bind to cells expressing the corresponding receptor.
  • This example uses an ECM cell adhesion analysis system to analyze the adhesion characteristics of cells.
  • Different concentrations of the target fusion protein were coated per well in a 96-well microplate and incubated at 16 ° C for 12 hours. After the protein was attached, 3% BSA was added and incubated at 25 ° C for 1 hour.
  • Tumor cells U87MG, 5 ⁇ 10 5 cells/well
  • Tumor cells that had been starved for one hour were added to a 96-well microplate, and after incubation at 37 ° C for 2 hours, they were washed with D-PBS to remove cells not bound to the protein.
  • MTT was added and incubated at 37 ° C for 1 hour.
  • the surviving cells metabolized the MTT agent to formazan blue-violet crystals.
  • the wax was dissolved in DMSO to detect the absorbance at a wavelength of 570 nm.
  • the cell attachment characteristics were calculated by the following formula.
  • FIGS 4A-D RGD-VEGF and RGD4C-VEGF were applied to the cell disk After that, the number of U87MG cells and HUVEC cells attached to the culture plate can be increased, and if a large amount of RGD peptide chain is added to compete, the adsorption of cells can be reduced.
  • Figure 4A shows that RGD-VEGF and RGD4C-VEGF are coated on the cell plate to increase the amount of U87MG cells attached to the culture plate
  • Figure 4B shows the amount of cells attached to the plate after the addition of the RGDfV polypeptide.
  • Figure 4C-4D shows that RGD-VEGF and RGD4C-VEGF bind to HUVEC cells, and then compete with RGDfV polypeptide or VEGFR2 recombinant protein, respectively, in terms of absorbance and percentage.
  • the fusion protein activates the cellular receptor and its downstream signaling molecule phosphorylation.
  • Various human tumor cells were cultured with 25nM target fusion protein at 37 ° C for 60 minutes to collect intracellular protein extracts, and the phosphorylation reaction of cell receptors and their downstream signal molecules was analyzed by Western blotting method.
  • RGD-EGF and RGD4C-EGF were co-cultured with MDA-MB468 cells expressing EGFR; or RGD-VEGF and RGD4C-VEGF were co-cultured with HUVEC vascular endothelial cells expressing VEGFR1 and VEGFR2.
  • the signaling molecules downstream of EGFR (Fig. 5A) or VEGFR (Fig. 5B) were analyzed.
  • RGD-EGF and RGD4C-EGF or RGD-VEGF and RGD4C-VEGF can activate the signaling pathway downstream of EGFR or VEGFR, respectively.
  • the results show that the double target molecule of the present invention not only has the ability to bind to a relatively biomarker molecule, but a large peptide molecule such as EGF or VEGF retains its original biological properties.
  • p-SCN-Bn-DTPA 2-(4-Iso thiocyanatobenzyl-diethylenetriaminepentaacetic acid, p-SCN-Bn-DTPA) is dissolved in HEPES buffer solution, and RGD-EGF is added.
  • RGD4C-EGF was reacted at room temperature for 1 hour.
  • the unreacted small molecule reactant was removed by colloidal chromatography (Sephadax G25) to obtain a radioactive marker precursor protein such as DTPA-RGD-EGF and DTPA-RGD4C-EGF.
  • HEPES buffer solution An appropriate amount of HEPES buffer solution and radioactive ( 111 InCl 3 , 67 GaCl 3 , 90 YCl 3 and 177 LuCl 3 , etc.) were added to the above-mentioned fusion protein solution bound to DTPA, and reacted at room temperature for 1 hour. Excess EDTA was added to chelate the radioactive metal ions not bound to the protein, and purified by membrane filtration centrifugation and colloidal chromatography. The activity was measured and the radiochemical purity of the product was analyzed by radioactive thin layer analysis. The stability of the fusion protein in buffer solution (HEPES, 4 ° C) and serum (37 ° C).
  • radioactive 111 InCl 3 , 67 GaCl 3 , 90 YCl 3 and 177 LuCl 3 , etc.
  • Tumor cells or vascular cells were cultured in a 12-well cell culture dish at 37 ° C for one day in a CO 2 incubator.
  • the medium in the culture plate was removed, and the cells were washed with 1 mL of PBS, and then VEGF, RGD-VEGF and RGD4C-VEGF were separately added, and the cells were allowed to stand at 37 ° C for 2 hours in a CO 2 incubator.
  • Tumor mice (tumor size about 50-100 mm 3 ) were injected with 500 ⁇ Ci of radiolabeled recombinant protein in the tail vein, respectively.
  • Small animal SPECT/CT angiography was performed at 1, 2, 4, 8, 24, 48 and 72 hours after the injection. After the end of the angiography, the image was processed by software, and the tumor to muscle ratio was calculated by ROI (region of interest) analysis to evaluate the dynamic accumulation of the drug in the living body of the animal.
  • ROI region of interest
  • Computed tomography angiography.
  • the image shows that after the In-111 marker recombinant protein is applied, the specific accumulation of the tumor in the tumor (expressed as the tumor/muscle accumulation ratio) increases with time, reaching a peak at 8 hours after the drug injection, at this time 111 In
  • the tumor/muscle accumulation ratio of -DTPA-RGD4C-EGF was 4.4, slightly higher than 3.6 of 111 In-DTPA-RGD-EGF, and much higher than 1.7 of 111 In-DTPA-EGF.
  • RGD4C-EGF and RGD-EGF fusion proteins have dual targeting ability, and the accumulation of tumors is significantly higher than that of EGF recombinant protein with only single target ability;
  • the sequence of RGD4C-EGF fusion protein has better binding ability to integrin ⁇ v ⁇ 3, which makes it accumulate in tumors slightly better than RGD-EGF fusion protein with linear RGD sequence.

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Abstract

L'invention concerne un vecteur de médicament à double ciblage comprenant une première molécule de ciblage et une seconde molécule de ciblage. La molécule de ciblage comprend un peptide, un anticorps ou une protéine. La molécule de ciblage est capable de se lier à un récepteur spécifique, ou à une protéine ou une glycoprotéine spécifique. La molécule cible peut également être conjuguée à un matériau de radiothérapie pour former un conjugué.
PCT/CN2016/075329 2016-03-02 2016-03-02 Vecteur de médicament à double ciblage WO2017147820A1 (fr)

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CN201680083086.6A CN108699164B (zh) 2016-03-02 2016-03-02 双重标靶药物载体
PCT/CN2016/075329 WO2017147820A1 (fr) 2016-03-02 2016-03-02 Vecteur de médicament à double ciblage

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PCT/CN2016/075329 WO2017147820A1 (fr) 2016-03-02 2016-03-02 Vecteur de médicament à double ciblage

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108187064A (zh) * 2017-11-28 2018-06-22 南京大学 一种类弹性蛋白-抗EGFR纳米抗体-iRGD的双靶向融合蛋白阿霉素偶联体的制法和用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113307857B (zh) * 2021-01-14 2022-11-01 艾时斌 从表皮生长因子、凝集素和Tat蛋白衍生的支架蛋白

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1766115A (zh) * 2005-09-06 2006-05-03 中国人民解放军第四军医大学 肿瘤血管导向肽与人干扰素α－2b的融合蛋白的制备方法
CN101124243A (zh) * 2004-12-23 2008-02-13 莫尔梅德股份有限公司 缀合产物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372172B1 (en) * 1997-12-19 2002-04-16 Kimberly-Clark Worldwide, Inc. Nonwoven webs having improved softness and barrier properties

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101124243A (zh) * 2004-12-23 2008-02-13 莫尔梅德股份有限公司 缀合产物
CN1766115A (zh) * 2005-09-06 2006-05-03 中国人民解放军第四军医大学 肿瘤血管导向肽与人干扰素α－2b的融合蛋白的制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108187064A (zh) * 2017-11-28 2018-06-22 南京大学 一种类弹性蛋白-抗EGFR纳米抗体-iRGD的双靶向融合蛋白阿霉素偶联体的制法和用途
CN108187064B (zh) * 2017-11-28 2021-05-28 南京大学 一种类弹性蛋白-抗EGFR纳米抗体-iRGD的双靶向融合蛋白阿霉素偶联体的制法和用途

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