WO2021115258A1 - 一种靶向fgfr的拮抗短肽 - Google Patents

一种靶向fgfr的拮抗短肽 Download PDF

Info

Publication number
WO2021115258A1
WO2021115258A1 PCT/CN2020/134550 CN2020134550W WO2021115258A1 WO 2021115258 A1 WO2021115258 A1 WO 2021115258A1 CN 2020134550 W CN2020134550 W CN 2020134550W WO 2021115258 A1 WO2021115258 A1 WO 2021115258A1
Authority
WO
WIPO (PCT)
Prior art keywords
short peptide
fgfr
fgfr1
antagonistic
targeting
Prior art date
Application number
PCT/CN2020/134550
Other languages
English (en)
French (fr)
Inventor
吴建章
李物兰
陈玲姿
范蕾
蔡跃飘
李校堃
Original Assignee
温州医科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 温州医科大学 filed Critical 温州医科大学
Priority to US17/768,296 priority Critical patent/US20240101600A1/en
Publication of WO2021115258A1 publication Critical patent/WO2021115258A1/zh

Links

Images

Classifications

    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the technical field of short peptide pharmacology, in particular to an antagonistic short peptide targeting FGFR.
  • Fibroblast growth factor and its receptor drive important developmental signal transduction pathways that affect cell proliferation, migration and survival.
  • Abnormal FGF signaling plays a role in many cancers. Turner, N. and Grose, R. (2010) Nat. Rev. Cancer 10: 116-29.
  • the FGFR family consists of FGFR1, FGFR2, FGFR3 and FGFR4.
  • FGFR is a tyrosine kinase activated by gene amplification, mutation or chromosomal translocation or rearrangement in some tumors.
  • FGFR1 amplification was found in squamous cell lung cancer and estrogen receptor-positive breast cancer
  • FGFR2 amplification was found in gastric cancer and breast cancer
  • FGFR2 mutation was observed in endometrial cancer
  • FGFR3 was observed in bladder cancer. mutation.
  • the FGFR fusion gene has also been reported in a variety of blood and solid tumor cancers.
  • FGFR1-ERLIN2 in breast cancer
  • FGFR2-KIAA1967 in squamous cell lung cancer and FGFR3 translocation in multiple myeloma (4,14).
  • some fusions occur in different cancers.
  • the FGFR3-TACC3 fusion occurs in glioblastoma, bladder cancer, and squamous cell carcinoma.
  • no FGFR inhibitor has been approved for marketing. Finding an effective FGFR targeted inhibitor has important research value.
  • Targeted inhibitors are mainly divided into small molecule inhibitors, antibody drugs, peptide inhibitors and their derivatives. Compared with small molecule inhibitors, peptide inhibitors have high affinity and specificity, and their adverse reactions are relatively low. Compared with antibody drugs, peptide inhibitors have low molecular weight and stronger tissue penetration activity. In view of the above advantages of peptide inhibitors, more and more scholars are favored. As of 2015, more than 60 peptide inhibitors have been approved for marketing worldwide, and more than 140 peptide inhibitors are in clinical research. However, research on FGFR antagonistic peptides has still stopped in the preclinical research stage.
  • the lag in the study of FGFR antagonistic peptides is mainly related to its easy degradation and short half-life.
  • the purpose of the present invention is to provide an antagonistic short peptide targeting FGFR.
  • the short peptide compound P48 can effectively target and inhibit FGFR1, FGFR2, FGFR3, and exhibits good anti-tumor activity in vivo and in vitro.
  • an antagonistic short peptide targeting FGFR including short peptide compound P48, which can target and bind to the extramembrane immunoglobulin domain of FGFR, short peptide
  • the amino acid sequence of compound P48 is Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH2, wherein FGFR is at least one of FGFR1, FGFR2 and FGFR3.
  • the short peptide compound P48 can target the extramembrane immunoglobulin domain of FGFR.
  • the medicament treats diseases related to abnormal FGFR disorders by antagonizing the signal pathway of at least one of FGFR1, FGFR2 and FGFR3 in the antibody.
  • the abnormal FGFR disorder includes the expression level of at least one of FGFR1, FGFR2, and FGFR3 exceeding a predetermined threshold level and a mutation of at least one of FGFR1, FGFR2, and FGFR3.
  • the drug is used to treat tumors caused by abnormalities in at least one of FGFR1, FGFR2 and FGFR3.
  • the short peptide compound P48 in the FGFR antagonistic short peptide inhibits the growth and invasion of tumor cells by inhibiting at least one of the FGFR1 pathway, the FGFR2 pathway and the FGFR3 pathway.
  • a medicine for treating diseases related to FGFR abnormalities including the above-mentioned FGFR-targeting antagonistic short peptide or a pharmaceutically acceptable salt or ester thereof.
  • the pharmaceutical preparations are injections, tablets, capsules, aerosols, suppositories, films, controlled release, sustained release or nano preparations.
  • a nucleic acid molecule whose sequence includes the nucleic acid sequence encoding the FGFR-targeting antagonistic short peptide and the short peptide before the amino group modification, and the FGFR-targeting antagonistic short peptide and the short peptide sequence before the amino modification is Ser -Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser.
  • a vector comprising the nucleic acid molecule as described above.
  • a recombinant cell comprising the vector as described above.
  • a method for preparing an antagonistic short peptide targeting FGFR as described above comprising culturing a recombinant cell as described above and purifying to obtain an antagonistic short peptide targeting FGFR and a short peptide before amino group modification, said antagonistic short peptide targeting FGFR
  • the short peptide sequence before the amino modification is Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser, and then the antagonistic short peptide targeting FGFR is modified by the amino group at the end of the short peptide before the amino modification
  • the antagonistic short peptide targeting FGFR as described in claim 1 is obtained.
  • a method for diagnosing diseases related to FGFR abnormalities and disorders including FGFR1, FGFR2, and FGFR3 expression levels exceeding a preset threshold level and mutations of at least one of FGFR1, FGFR2, and FGFR3
  • the method includes the use of an antagonistic short peptide targeting FGFR as described above.
  • a method for preventing and/or treating diseases related to abnormal FGFR disorders including the expression level of at least one of FGFR1, FGFR2 and FGFR3 exceeding a preset threshold level and at least one of FGFR1, FGFR2 and FGFR3
  • the method includes administering a drug as described above.
  • the advantage of the present invention is that compared with the prior art, the inventors obtained the short peptide compound P48 through unremitting efforts, and found that it has a stable secondary structure and a relatively long half-life.
  • the short peptide compound P48 can effectively target and inhibit FGFR, exhibits good anti-tumor activity in vivo and in vitro, and is expected to become a candidate peptide inhibitor drug for cancer treatment.
  • Figure 1 is a schematic diagram of the structure, stability and binding ability to FGFR1 of the short peptide compound P48 in an embodiment of the present invention
  • Figure 2 shows the binding ability of short peptide compound P48 with FGFR1C (Figure 2A), FGFR2B ( Figure 2B) and FGFR3B ( Figure 2C) according to the embodiment of the present invention
  • Figure 3 is a schematic diagram showing the detection of the inhibitory activity of the short peptide compound P48 on the FGFR1 signaling pathway in the highly transformed human embryonic kidney cells HEK-293 and fibroblasts MEF-WT and Balb/c 3T3 according to the embodiment of the present invention;
  • Figure 4 is a schematic diagram of the inhibitory activity of the short peptide compound P48 on the FGFR1 signaling pathway in various tumor cell lines according to the embodiment of the present invention
  • Figure 5 is a schematic diagram of the in vitro anti-tumor activity of short peptide compound P48 according to an embodiment of the present invention.
  • Figure 6 is a schematic diagram of the in vivo anti-tumor activity of the short peptide compound P48 of the embodiment of the present invention.
  • An antagonistic short peptide targeting FGFR disclosed in the present invention includes the short peptide compound P48, which can target the extramembrane immunoglobulin domain of FGFR, and the amino acid sequence of the FGFR antagonistic short peptide is:
  • the short peptide compound P48 can target the extramembrane immunoglobulin domain of FGFR1.
  • the binding ability of the short peptide compound P48 and FGFR1 is significantly better than that of FGFR2 and FGFR3.
  • the short peptide compound P48 has the best binding ability to FGFR1C.
  • the medicament treats diseases related to abnormal FGFR disorders by antagonizing the signal pathway of at least one of FGFR1, FGFR2 and FGFR3 in the antibody.
  • the abnormal FGFR disorder includes the expression level of at least one of FGFR1, FGFR2, and FGFR3 exceeding a predetermined threshold level and a mutation of at least one of FGFR1, FGFR2, and FGFR3.
  • the expression level of at least one of FGFR1, FGFR2, and FGFR3 exceeds a preset threshold level, which means that at least one of FGFR1, FGFR2, and FGFR3 has a high expression level.
  • the drug is used to treat tumors caused by abnormalities in at least one of FGFR1, FGFR2 and FGFR3.
  • the short peptide compound P48 in the FGFR antagonistic short peptide inhibits the growth and invasion of tumor cells by inhibiting at least one of the FGFR1 pathway, the FGFR2 pathway and the FGFR3 pathway.
  • the short peptide compound P48 has a stable secondary structure and relatively prolonged half-life, and has obvious specificity for target selection. Therefore, it has excellent medical and market prospects as a targeted drug.
  • a drug for treating diseases related to abnormal FGFR disorders including the above-mentioned antagonistic short peptide targeting at least one of FGFR1, FGFR2 and FGFR3 or a pharmaceutically acceptable salt or ester thereof.
  • the pharmaceutical preparations are injections, tablets, capsules, aerosols, suppositories, films, controlled release, sustained release or nano preparations.
  • the FGFR-targeting antagonistic short peptide provided by the present invention can be artificially synthesized by well-known techniques for synthesizing polypeptides or cloned and encoded using standard recombinant DNA procedures to obtain the above-mentioned FGFR-targeting antagonistic short peptide before its amino group modification.
  • the nucleic acid sequence of the short peptide, the short peptide sequence before the amino modification of the FGFR-targeted antagonistic short peptide is Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser.
  • a nucleic acid molecule whose sequence includes the nucleic acid sequence encoding the FGFR-targeting antagonistic short peptide and the nucleic acid sequence of the short peptide before the amino group modification, the FGFR-targeting antagonistic short peptide and the short peptide before the amino group modification
  • the sequence is Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser.
  • the nucleic acid includes single-stranded or double-stranded DNA or RNA, and the nucleic acid can be operably linked to an expression control sequence, that is, to a sequence necessary for realizing the expression of the encoding nucleic acid sequence.
  • expression control sequence may include a promoter, an enhancer, Ribosome binding site and/or transcription termination sequence.
  • a vector comprising the nucleic acid as described above.
  • the vector such as a plasmid, phagemid, phage, viral vector or retroviral vector, the nucleic acid sequence encoding the antagonistic short peptide targeting FGFR and the short peptide before amino modification is inserted into the vector, the vector may additionally include an origin of replication And/or selection marker genes.
  • the nucleic acid molecule can be linked to a vector containing a selection marker for propagation in the host.
  • a selection marker for propagation in the host.
  • the plasmid vector is introduced into the precipitate, such as calcium phosphate precipitation or rubidium chloride precipitation, or complexes with charged lipids, or into carbon-based atomic clusters (such as fullerenes). If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before being applied to the host cell.
  • the vector of the present invention is an expression vector in which the nucleic acid molecule is operably linked to one or more control sequences, thereby allowing transcription and optional expression in prokaryotic and/or eukaryotic host cells.
  • the expression of the nucleic acid molecule includes the transcription of the nucleic acid molecule, preferably into translatable mRNA.
  • Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually contain regulatory sequences that ensure transcription initiation and optional polyadenylation signals that ensure transcription termination and transcription stability. Other regulatory elements may include transcription enhancers and translation enhancers.
  • Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the lac, trp or tac promoter in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are AOXI or GAL-1 in yeast Promoter or CMV (cytomegalovirus)-, SV40 (simian virus 40)-, RSV-promoter (Rous sarcoma virus) in mammalian and other animal cells, CMV-enhancer, SV40-enhancer or ball Protein introns.
  • regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as SV40-polyadenylation sites or tk-polyadenylation sites.
  • suitable expression vectors are known in the art.
  • the vector is an expression vector and/or gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retrovirus, vaccinia virus, adeno-associated virus, herpes virus, or bovine papilloma virus can be used to deliver the polynucleotide or vector of the present invention to a target cell population.
  • the recombinant viral vector can be constructed using methods well known to those skilled in the art.
  • the nucleic acid molecules of the present invention can be reconstituted into liposomes for delivery to target cells.
  • the present invention relates to a host comprising the nucleic acid molecule or vector as described above.
  • the nucleic acid molecule or vector can be introduced into the host by transformation, transfection or transduction according to any method known in the art.
  • a recombinant cell is a host cell carrying the nucleic acid molecule or vector.
  • the host can be any prokaryotic or eukaryotic cell, such as bacteria, insects, fungi, plants, animals, mammals or preferably human cells.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, especially those of the species S. cerevisiae.
  • the term "prokaryotic” is meant to include all bacteria that can be transformed or transfected with polynucleotides to express the variant polypeptides of the invention.
  • Prokaryotic hosts may include Gram-negative and Gram-positive bacteria, such as Escherichia coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis.
  • the polynucleotide encoding the mutant form of the variant polypeptide of the present invention can be used to transform or transfect a host using any technique generally known to those of ordinary skill in the art. Methods of preparing fused operably linked genes and expressing them in bacteria or animal cells are well known in the art.
  • the genetic constructs and methods described herein can be used to express the variant antibodies, antibody fragments, or derivatives thereof of the present invention in, for example, a prokaryotic host.
  • an expression vector containing a promoter sequence that promotes efficient transcription of the inserted nucleic acid molecule is used in combination with the host.
  • An expression vector usually contains an origin of replication, a promoter and a terminator, and specific genes that can provide phenotypic selection for transformed cells.
  • the transformed prokaryotic host can be grown in a fermentor and cultured according to techniques known in the art to achieve optimal cell growth.
  • the antibody, antibody fragment or derivative thereof of the present invention can then be isolated from the growth medium, cell lysate or cell membrane fraction.
  • the isolation and purification of antibodies, antibody fragments or derivatives thereof expressed by microorganisms or other methods of the present invention can be carried out by any conventional means, such as preparative chromatographic separation and immunological separation, such as those methods involving the use of monoclonal or polyclonal antibodies.
  • the method for preparing the FGFR-targeting antagonistic short peptide as described above includes culturing the above-mentioned recombinant cell and purifying to obtain the FGFR-targeting antagonistic short peptide.
  • the use of the diagnostic reagent specifically includes the step of detecting the expression level of at least one of FGFR1, FGFR2, and FGFR3 through the above-mentioned FGFR-targeting antagonistic short peptide.
  • a method for diagnosing diseases related to abnormal FGFR disorders, FGFR abnormal disorders including expression levels of at least one of FGFR1, FGFR2 and FGFR3 exceeding a preset threshold level and at least one of FGFR1, FGFR2 and FGFR3 The method includes using the above-mentioned antagonistic short peptide targeting FGFR.
  • the method includes contacting the antibody of the present invention with a cell or tissue suspected of carrying one or more of FGFR1, FGFR2, and FGFR3 on its surface.
  • a suitable method for detecting FGFR4 expression in a sample may be enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry (IHC).
  • a method for preventing and/or treating diseases related to abnormal FGFR disorders including the expression level of at least one of FGFR1, FGFR2 and FGFR3 exceeding a preset threshold level and at least one of FGFR1, FGFR2 and FGFR3
  • the method includes administering the above-mentioned drug.
  • the present invention obtains the FGFR1 antagonistic short peptide, whose amino acid sequence is as follows (Example 1):
  • the short peptide compound P48 can inhibit the FGFR1 signaling pathway including cervical cancer, melanoma, lung cancer, and gastric cancer in a concentration-dependent manner (Example 3) .
  • the short peptide compound P48 has the same inhibitory effect on FGFR1 signaling as SSR, and its activity is weaker than that of AZD4547. This may be related to its different binding types, that is, the short peptide compound P48 may be similar to SSR and is extra-membrane Inhibitor, and AZD4547 is an intra-membrane inhibitor.
  • the short peptide compound P48 can significantly inhibit the viability of cancer cells.
  • the short peptide compound P48 can inhibit cell migration in a concentration-dependent manner (Example 4). Through flow cytometry experiments, it was found that the short peptide compound P48 can effectively block the cell cycle in the G0/G1 phase (Example 4).
  • the short peptide compound P48 can significantly inhibit the growth of gastric cancer cells SGC-7901, and has a good inhibitory effect on the FGFR1 signaling pathway (including phosphorylated FLG, FRS2 ⁇ , ERK1/2 protein) (implementation) Example 5).
  • the short peptide compound P48 can inhibit the expression of Ki67 in a concentration-dependent manner (Example 5).
  • the above results indicate that the short peptide compound P48 can also inhibit cell proliferation by inhibiting the FGFR1 pathway in vivo to play a good anti-tumor activity (Experimental Example 5).
  • the present invention provides the application of an efficient and stable FGFR1 antagonist peptide P48 in the preparation of anti-tumor drugs.
  • the anti-tumor drugs can selectively inhibit the FGFR1 signaling pathway to inhibit cell proliferation and migration and play a role in treating tumors.
  • the present invention also provides a pharmaceutical composition for treating tumors, which contains a therapeutically effective amount of active ingredients and pharmaceutical excipients, the active ingredients being the above-mentioned FGFR1 antagonistic short peptide or its pharmaceutically acceptable salt and pharmaceutical excipients .
  • pharmaceutical excipients refer to conventional pharmaceutical carriers in the pharmaceutical field, for example: diluents such as starch, sucrose, dextrin, lactose, pregelatinized starch, microcrystalline cellulose, calcium phosphate, etc.; wetting agents such as distilled water, Ethanol; binders such as starch slurry, cellulose derivatives, povidone, gelatin, polyethylene glycol, sodium alginate solution, etc.; disintegrants such as dry starch, sodium carboxymethyl starch, low-substituted qiongpropyl fiber Vegetarian, effervescent disintegrant, etc.; lubricants such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycols, sodium lauryl sulfate, etc.; coloring agents such as titanium dioxide, sunset yellow, methylene Blue, medicinal iron oxide red, etc.; in addition, other adjuvants such as flavors and sweeten
  • compositions of the present invention can be prepared according to conventional production methods in the pharmaceutical field.
  • the active ingredient is mixed with one or more carriers and then prepared into the desired dosage form.
  • the pharmaceutical preparation forms include granules, injections, tablets, capsules, aerosols, suppositories, films, dripping pills, ointments, controlled release or sustained release formulations or nano formulations.
  • the present invention can be administered to patients in need of such treatment in the form of a composition by oral, nasal inhalation, rectal or parenteral administration.
  • b The initial conformation of P9 and P48 and the superimposed image of the conformation at 4ns, 8ns, 12ns, 16ns and 20ns.
  • C The half-life of P9 and P48 in human plasma.
  • b Analysis of key residues to maintain the stability of the bFGF-FGFR1 complex.
  • Figure 2 (A) SPR detects the interaction between FGFR1C and P48; (B) SPR detects the interaction between FGFR2B and P48; (C) SPR detects the interaction between FGFR3B and P48.
  • Figure 3 Detection of the inhibitory activity of P48 on the FGFR1 signaling pathway in highly transformed human embryonic kidney cells HEK-293 and fibroblasts MEF-WT, Balb/c 3T3.
  • A The short peptide compound P48 is pretreated for 10 minutes or AZD4547/PD173074 is pretreated for 2 hours, and then bFGF is incubated for 15 minutes to detect the inhibition of FGFR1 and its downstream proteins in HEK-293, MEF-WT, Balb/c3T3 cells .
  • B After P48 pretreatment for 10 minutes or AZD4547/SSR treatment for 2 hours, bFGF was stimulated for 15 minutes, and the inhibition of PLC ⁇ by the drug was detected.
  • C Flow cytometry analysis of the ability of P48 to bind to FGFR1 in MEF-WT and FGFR1-FGFR2-FRS2 ⁇ knockout MEF cells. *, p ⁇ 0.05; ***, p ⁇ 0.001, vs Control.
  • Figure 4 Inhibitory activity of short peptide compound P48 on FGFR1 signaling pathway in various tumor cell lines.
  • bFGF was incubated for 15 minutes to detect the inhibition of FGFR1 and its downstream proteins by the drug.
  • B After P48 pretreatment for 10 minutes or AZD4547/SSR pretreatment for 2 hours, bFGF was incubated for 15 minutes to detect the inhibitory activity of the drug on phosphorylated FGFR1 and its downstream proteins in SGC-7901 cells.
  • C bFGF/aFGF stimulation for 15 minutes or KGF stimulation for 20 minutes, and the inhibitory activity of the short peptide compound P48 on phosphorylated FGFR1 and its downstream proteins in SGC-7901 cells was detected.
  • EGF stimulates for 10 minutes, and detects the inhibitory activity of the short peptide compound P48 on phosphorylated EGFR and ERK1/2 in PC-9 cells.
  • E Flow cytometry to detect the binding ability of short peptide compound P48 with FGFR1 in SGC-7901 cells.
  • Figure 5 In vitro anti-tumor activity of short peptide compound P48.
  • Figure 6 Anti-tumor activity of short peptide compound P48 in vivo.
  • A Tumor volume measurement of the control group and the administration group.
  • B Tumor weighing.
  • C The inhibitory effect of short peptide compound P48 on the phosphorylation level of FLG and its downstream proteins in nude mice.
  • D Immunohistochemical detection of the inhibitory activity of the short peptide compound P48 on the pro-proliferation protein Ki67.
  • the Amber11 program was used to analyze the amino acid sequence of the degraded short peptide P48 of P9, and the results are shown in Figure 1A.
  • Use the Tleap module of the Amber11 program to add hydrogen atoms to the peptide chain Add a truncated tetrahedral TIP3P solvent box at the distance, and add counter ions to the two systems to keep the system electrically neutral.
  • the system performs 20ns NPT system simulation to obtain the optimal spatial structure, and the result is shown in Figure 1B.
  • Figure 1C shows the half-life of P9 and P48 in plasma.
  • the experimental procedure is as follows: the plasma is centrifuged to remove the supernatant, and 60 ⁇ l peptide is added to incubate for different times (P9 incubate for 5, 10, 15, 20, 25, 30, 45 minutes, P48 incubate for 1, 2, 3, 4, 5, 10, 12 hours ), stop with 200 ⁇ l 5% glacial acetic acid, solid phase extraction: first activate with 1.5ml methanol, then wash with 1.5ml H 2 O, take 400 ⁇ l sample and load, wash with 1ml H 2 O, wash with 2ml 30% acetonitrile, collect the sample, freeze Dry and analyze by liquid chromatography.
  • Figure 1D shows the molecular dynamics simulation of the FGF2-FGFR1 complex system to determine the key amino acid residues that maintain the binding of FGF2-FGFR1.
  • the experimental steps are as follows: select the FGF2-FGFR1 protein crystal structure (PDB ID:1CVS) to construct the initial structure of the FGF2-FGFR1 complex system simulated by molecular dynamics.
  • the system uses the Amber99SB force field, and the Sander module is used to minimize the energy of the system.
  • MM-GBSA molecular mechanics generalized Bonn/surface area
  • FGFR1 coordinates are selected from FGFR1 in the A chain of FGF2-FGFR1 protein crystal structure (PDB ID: 1CVS).
  • PDB ID: 1CVS Use the Tleap module of the Amber11 program to add hydrogen atoms to the system. Add a truncated tetrahedral TIP3P solvent box at the distance, and add counter ions to the two systems to keep the system electrically neutral.
  • the system is simulated in 20ns NPT system, and the experimental results are shown in Figure 1E.
  • Figure 1F shows the interaction between P48 and FGFR1.
  • the experimental procedure is as follows: wash the sensor chip with 30 ⁇ l/min PBS, and activate it with an activation buffer of 40 mM EDAC and 10 mM sNHS. After bFGF and FGFR1 flow into the sensor chip, P48 (50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 300 ⁇ M, 400 ⁇ M) is injected into the sensor surface, and the data obtained is analyzed by BIAevaluation software.
  • FIG. 3A shows the inhibition of FGFR1 and its downstream proteins (FRS2 ⁇ , ERK1/2, AKT) in HEK-293, MEF-WT, Balb/c3T3 cells by P48/AZD4547/PD173074.
  • Figure 3B shows the inhibition of P48/AZD4547/SSR on the downstream protein PLC ⁇ of FGFR1 in MEF-WT cells.
  • Figure 3C shows the ability of P48 to bind to FGFR1 in MEF-WT and FGFR1-FGFR2-FRS2 ⁇ knockout MEF cells.
  • the experimental procedure is as follows: MEF-WT and MEF KO (FGFR1-FGFR2-FRS2 ⁇ ) cells (1 ⁇ 10 6 cells/ml) were seeded in a 35mm culture dish. After the cells adhered overnight, the cells were washed with PBS, and FITC-conjugated P48 (1, 10, 100, 1000 nM) was added and placed in an incubator for 30 minutes. The cells were fixed with 1% paraformaldehyde in the dark and washed with PBS. The expression level of FITC was measured at fluorescence channel 1 (FL-1) (530nm), and the excitation wavelength was 488nm. CellQuest software (Becton Dickinson) was used for data analysis.
  • Cervical cancer cell Hela229, melanoma B16-F10, lung cancer cell NCI-H460, gastric cancer cell SGC-7901, MGC-803 were respectively inoculated into 6-well plates. After the cells adhered overnight, they were pretreated with P48 for 10 minutes or AZD4547/SSR for 2 hours, and incubated with bFGF/aFGF for 15 minutes or KGF for 20 minutes.
  • Figure 4A shows the inhibition of FGFR1 and its downstream proteins (FRS2 ⁇ , ERK1/2, AKT) in Hela229, B16-F10, NCI-H460, MGC-803, and SGC-7901 cells stimulated by bFGF by P48/AZD4547.
  • Figure 4B shows the inhibition of FGFR1 and its downstream proteins (FRS2 ⁇ , ERK1/2) in SGC-7901 cells by P48/AZD4547/SSR under bFGF stimulation.
  • Figure 4C shows the inhibition of FGFR1 and its downstream proteins (FRS2 ⁇ , ERK1/2) in SGC-7901 cells by P48 under the stimulation of bFGF/aFGF/KGF.
  • Figure 4D shows the inhibition of EGFR and ERK1/2 in PC-9 cells by P48 under EGF stimulation.
  • Figure 4E shows the binding of P48 to FGFR1 in SGC-7901 cells, and the experimental procedure is the same as Figure 2C of the embodiment.
  • Hela229 (1500 cells/100 ⁇ l), B16-F10 (1000 cells/100 ⁇ l), NCI-H460 (4000 cells/100 ⁇ l) and SGC7901 (4000 cells/100 ⁇ l) cells were seeded in 96-well plates. After overnight adherence, replace the original medium with 0.1% FBS-containing DMEM or RPMI-1640 medium and continue culturing for 24 hours. Add the mixture of P48 and bFGF and bFGF alone into the corresponding wells. After incubating for 48 hours, add the MTT solution (5 mg/mL) prepared by phosphate buffered solution (PBS) to the cells in each well at 37°C. Treat for another 4 hours.
  • PBS phosphate buffered solution
  • the experimental procedure is as follows: add 10% FBS-containing medium (500 ⁇ l) to the lower surface of the transwell insertion chamber, inoculate the cells together with the drug into the upper chamber, and culture with serum-free medium (200 ⁇ l). After culturing in the incubator for 24 hours, the cells in the lower chamber were fixed with pre-cooled methanol and stained with crystal violet, while the cells in the upper chamber were removed with a cotton swab. Finally, capture the migrating cells under an optical microscope. The results in Fig. 5C show that P48 can block gastric cancer cells SGC-7901 in G0/G1 phase in a concentration-dependent manner.
  • the experimental procedure is as follows: SGC-7901 cells (6 ⁇ 10 5 cells/well) were seeded in a petri dish with a diameter of 60 mm. The cells were treated with a mixture of P48 and bFGF, bFGF and carrier (PBS) for 24 hours, washed with PBS, and fixed in 75% ice-cold ethanol overnight. The cells were then stained with 500 ⁇ L of propidium iodide (PI) containing ribonuclease (550825, BD Biosciences 35 Clontech, San Jose, CA, USA) at 4° C. away from light for 10 minutes, and filtered with 200 mesh gauze. Cell cycle analysis was performed in a FACS Calibur flow cytometer (BD Biosciences, CA).
  • PI propidium iodide
  • SGC-7901 cells were injected subcutaneously into the back of BALB/c nude mice. After 10 days, the mice were randomly divided into 4 groups, each with 12 mice, and the mice were administered intraperitoneally.
  • the administration group consisted of model group (normal saline group), AZD4547 group (20mg/kg.d), P48 group (32mg/kg.d, 64mg/kg.d) group. Use a vernier caliper to measure the length and width of the tumor.
  • the body weight of the mice was recorded every 3 days, and the tumor size was recorded on the day of the death of the mice. After 28 days, the nude mice were sacrificed for dissection, and tissue samples were taken out for further study.
  • A represents the weight change of the mouse.
  • B represents the volume and size of the tumor.
  • C indicates the expression of phosphorylated FLG, FRS2 ⁇ , and ERK1/2 in vivo after P48 treatment.
  • D represents the expression of Ki67 in vivo after P48 treatment.
  • the inventors obtained the short peptide compound P48, and found that it has a stable secondary structure and a relatively long half-life.
  • the short peptide compound P48 can effectively target and inhibit FGFR1, exhibit good anti-tumor activity in vivo and in vitro, and is expected to become a candidate peptide inhibitor drug for cancer treatment.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明公开了一种FGFR拮抗短肽，即短肽化合物P48，其氨基酸序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH 2，短肽化合物P48通过抑制FGFR通路，抑制细胞增殖和迁移。

Description

一种靶向FGFR的拮抗短肽 技术领域
本发明涉及短肽药物学技术领域，具体涉及一种靶向FGFR的拮抗短肽。
背景技术
成纤维细胞生长因子及其受体(FGFR)驱动影响细胞增殖、迁移和存活的重要发育信号传导途径。异常的FGF信号传导在许多癌症中起作用。Turner,N.和Grose,R.(2010)Nat.Rev.Cancer 10:116-29。FGFR家族由FGFR1、FGFR2、FGFR3和FGFR4组成。FGFR是在一部分肿瘤中通过基因扩增、突变或染色体易位或重排激活的酪氨酸激酶。例如在鳞状细胞肺癌和雌激素受体阳性乳腺癌中发现FGFR1的扩增，在胃癌和乳腺癌中发现FGFR2扩增，在子宫内膜癌中观察到FGFR2突变，在膀胱癌中观察到FGFR3突变。FGFR融合基因也在多种血液和实体瘤型癌症中被报道。例如，乳腺癌中的FGFR1-ERLIN2，鳞状细胞肺癌中的FGFR2-KIAA1967，多发性骨髓瘤中的FGFR3易位t(4,14)。注意到，一些融合发生在不同的癌症中。例如，FGFR3-TACC3融合发生在胶质母细胞瘤、膀胱癌和鳞状细胞癌中。目前尚无FGFR抑制剂批准上市，寻找有效的FGFR靶向抑制剂具有重要的研究价值。
靶向抑制剂主要分为小分子抑制剂、抗体药物、肽类抑制剂及其衍生物。相对于小分子抑制剂而言，肽类抑制剂具有高亲和力和特异性，其不良反应较低。与抗体药物相比，肽类抑制剂分子量低，对组织的渗透活性更强。鉴于肽类抑制剂的上述优势，受到越来越多学者的青睐。截止至2015年，全球已有超60种肽抑制剂批准上市，140余种肽抑制剂处于临床研究。然而，有关FGFR拮抗肽的研究仍止步于临床前研究阶段。
造成FGFR拮抗肽研究滞后主要与其易降解、半衰期短相关。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种靶向FGFR的拮抗短肽。短肽化合物P48能有效靶向抑制FGFR1、FGFR2、FGFR3，在体内外表现出良好的抗肿瘤活性。
为实现上述目的，本发明提供了如下技术方案：一种靶向FGFR的拮抗短肽，包括短肽化合物P48，所述短肽化合物P48可靶向结合FGFR的膜外免疫球蛋白域，短肽化合物P48的氨基酸序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH2，其中FGFR为FGFR1、FGFR2和FGFR3中的至少一种。
所述短肽化合物P48可靶向结合FGFR的膜外免疫球蛋白域。
如上述的一种靶向FGFR的拮抗短肽作为活性成分在药物中的应用。
所述药物为通过拮抗体内FGFR1、FGFR2和FGFR3中的至少一种的信号通路治疗与FGFR异常失调相关的疾病。
FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变。
所述药物为治疗与FGFR1、FGFR2和FGFR3中的至少一种异常失调导致的肿瘤。
FGFR拮抗短肽中的短肽化合物P48通过抑制FGFR1通路、FGFR2通路和FGFR3通路中的至少一种，抑制肿瘤细胞的生长和侵袭。
一种治疗与FGFR异常失调相关疾病的药物，包括如上述的一种靶向FGFR的拮抗短肽或其药学上可接受的盐、酯。
还包括药学上可接受的辅料。
所述药物制剂为注射剂、片剂、胶囊剂、气雾剂、栓剂、膜剂、控释、缓释剂或纳米制剂。
一种核酸分子，其序列中包括编码如上述的靶向FGFR的拮抗短肽其氨基修饰前的短肽的核酸序列，所述靶向FGFR的拮抗短肽其氨基修饰前的短肽序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser。
一种载体，其包含如上述的核酸分子。
一种重组细胞，其包括如上述的载体。
制备如上述的靶向FGFR的拮抗短肽方法，所述方法包括培养如上述的重组细胞和纯化得到靶向FGFR的拮抗短肽其氨基修饰前的短肽，所述靶向FGFR的拮抗短肽其氨基修饰前的短肽序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser，然后将靶向FGFR的拮抗短肽其氨基修饰前的短肽端部通过氨基修饰得到如权利要求1所述的靶向FGFR的拮抗短肽。
如上述的靶向FGFR的拮抗短肽作为诊断试剂的应用。
一种诊断与FGFR异常失调相关的疾病的方法，FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变，所述方法包括使用如上述的靶向FGFR的拮抗短肽。
一种预防和/或治疗FGFR异常失调相关的疾病的方法，FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变，所述方法包括给予如上述的药物。
本发明的优点是：与现有技术相比，本发明人通过不懈努力，获得了短肽化合物P48，并发现其具有稳定的二级结构及相对较长的半衰期。此外，短肽化合物P48能有效靶向抑制FGFR，在体内外表现出良好的抗肿瘤活性，有望成为癌症治疗的候选肽类抑制剂药物。
下面结合说明书附图和具体实施例对本发明作进一步说明。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，根据这些附图获得其他的附图仍属于本发明的范畴。
图1为本发明实施例短肽化合物P48的结构、稳定性和对FGFR1的结合能力分析示意图；
图2为本发明实施例短肽化合P48与FGFR1C(图2A)、FGFR2B(图2B)、FGFR3B(图2C)的结合能力；
图3为本发明实施例在高度转化的人胚肾细胞HEK-293及成纤维细胞MEF-WT、Balb/c 3T3中检测短肽化合物P48对FGFR1信号通路的抑制活性示意图；
图4为本发明实施例短肽化合物P48在多种肿瘤细胞株中对FGFR1信号通路的抑制活性示意图；
图5为本发明实施例短肽化合物P48的体外抗肿瘤活性示意图；
图6为本发明实施例短肽化合物P48的体内抗肿瘤活性示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
本发明在以下的实施例中进一步说明。这些实施例只是为了说明的目的，而不是用来限制本发明的范围。
本发明公开的一种靶向FGFR的拮抗短肽，包括短肽化合物P48，所述短肽化合物P48可靶向结合FGFR的膜外免疫球蛋白域，FGFR拮抗短肽的氨基酸序列为：
Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH 2，其中FGFR为FGFR1、FGFR2和FGFR3中的至少一种。
所述短肽化合物P48可靶向结合FGFR1的膜外免疫球蛋白域。短肽化合物P48与FGFR1的结合能力相比FGFR2和FGFR3明显较优。进一步地，短肽化合物P48与FGFR1C的结合能力最优。
如上述的靶向FGFR拮抗短肽作为活性成分在药物中的应用。
所述药物为通过拮抗体内FGFR1、FGFR2和FGFR3中的至少一种的信号通路治疗与FGFR异常失调相关的疾病。
FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变。FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平即指FGFR1、FGFR2和FGFR3中的至少一种为高表达水平。
所述药物为治疗与FGFR1、FGFR2和FGFR3中的至少一种异常失调导致的肿瘤。
FGFR拮抗短肽中的短肽化合物P48通过抑制FGFR1通路、FGFR2通路和FGFR3通路中的至少一种，抑制肿瘤细胞的生长和侵袭。
作为优选的，短肽化合物P48具有稳定的二级结构及相对延长的半衰期，对靶点选择具有明显的特异性，因此其具有极佳的作为靶向药的医学前景和市 场前景。
一种治疗与FGFR异常失调相关疾病的药物，包括如上述的一种靶向FGFR1、FGFR2和FGFR3中的至少一种的拮抗短肽或其药学上可接受的盐、酯。
进一步地，还包括药学上可接受的辅料。
进一步地，所述药物制剂为注射剂、片剂、胶囊剂、气雾剂、栓剂、膜剂、控释、缓释剂或纳米制剂。
本发明所提供的靶向FGFR的拮抗短肽可以通过众所周知的用于合成多肽的技术人工合成或使用标准重组DNA程序克隆编码并表达来获得上述的靶向FGFR的拮抗短肽其氨基修饰前的短肽的核酸序列，所述靶向FGFR的拮抗短肽其氨基修饰前的短肽序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser。
一种核酸分子，其序列中包括编码如上述的靶向FGFR的拮抗短肽的核酸序列其氨基修饰前的短肽的核酸序列，所述靶向FGFR的拮抗短肽其氨基修饰前的短肽序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser。所述核酸包括单链或双链DNA或RNA，该核酸可以操作连接于表达控制序列，即连接于实现编码核酸序列的表达所必需的序列，这样的表达控制序列可以包括启动子、增强子，核糖体结合位点和/或转录终止序列。
一种载体，其包含如上述的核酸。所述载体例如质粒、噬粒、噬菌体、病毒载体或逆转录病毒载体，编码靶向FGFR的拮抗短肽其氨基修饰前的短肽的核酸序列插入所述载体中，该载体可以另外包含复制起点和/或选择标志基因。
核酸分子可以与包含用于在宿主中繁殖的选择标志物的载体连接。通常，将质粒载体引入沉淀物中，例如磷酸钙沉淀或氯化铷沉淀中，或具有带电荷脂质的复合物中，或引入基于碳的原子簇(例如富勒烯)中。如果载体是病毒，则可以在应用到宿主细胞之前使用适当的包装细胞系对其进行体外包装。
本发明的载体是表达载体，其中核酸分子可操作地连接至一或多个控制序列，从而允许在原核和/或真核宿主细胞中转录和任选地表达。所述核酸分子的表达包括核酸分子的转录，优选转录成可翻译的mRNA。确保在真核细胞，优选哺乳动物细胞中表达的调节元件是本领域技术人员众所周知的。它们通常包 含确保转录起始的调节序列和确保转录终止和转录稳定的任选的多聚腺苷酸信号。其他调节元件可包括转录增强子和翻译增强子。允许在原核宿主细胞中表达的可能的调控元件包括，例如大肠杆菌中的lac，trp或tac启动子，以及允许在真核宿主细胞中表达的调控元件的例子是酵母中的AOXI或GAL-1启动子或在哺乳动物和其他动物细胞中的CMV(巨细胞病毒)-，SV40(猿猴病毒40)-，RSV-启动子(劳斯肉瘤病毒)，CMV-增强子，SV40-增强子或球蛋白内含子。除了负责转录起始的元件外，此类调节元件还可在多核苷酸下游包含转录终止信号，例如SV40-多聚腺苷酸位点或tk-多聚腺苷酸位点。在这种情况下，合适的表达载体是本领域已知的。优选地，所述载体是表达载体和/或基因转移或靶向载体。衍生自病毒例如逆转录病毒、牛痘病毒、腺相关病毒、疱疹病毒或牛乳头瘤病毒的表达载体可用于将本发明的多核苷酸或载体递送至靶细胞群。可以使用本领域技术人员众所周知的方法来构建重组病毒载体。替代性地，可以将本发明的核酸分子重构入脂质体，以递送至靶细胞。此外，本发明涉及包含如上所述的核酸分子或载体的宿主。可以根据本领域已知的任何方法通过转化，转染或转导将核酸分子或载体引入宿主。
一种重组细胞，即为携带所述的核酸分子或载体的宿主细胞，宿主可以是任何原核或真核细胞，例如细菌，昆虫，真菌，植物，动物，哺乳动物或优选人细胞。优选的真菌细胞是，例如，酵母属的那些细胞，特别是酿酒酵母(S.cerevisiae)种的那些细胞。术语“原核的”是指包括可以用多核苷酸转化或转染以表达本发明的变体多肽的所有细菌。原核宿主可以包括革兰氏阴性和革兰氏阳性细菌，例如大肠杆菌，鼠伤寒沙门氏菌，粘质沙雷氏菌和枯草芽孢杆菌。可以使用本领域普通技术人员通常已知的任何技术，将编码本发明变体多肽的突变形式的多核苷酸用于转化或转染宿主。制备融合的可操作连接的基因并在细菌或动物细胞中表达它们的方法是本领域众所周知的。本文所述的遗传构建体和方法可用于在例如原核宿主中表达本发明的变体抗体、抗体片段或其衍生物。通常，将含有促进启动子序列的表达载体与宿主结合使用，所述启动子序列促进插入的核酸分子的有效转录。表达载体通常包含复制起点，启动子和终 止子以及能够提供转化细胞表型选择的特定基因。转化的原核宿主可以在发酵罐中生长并根据本领域已知的技术培养以实现最佳的细胞生长。然后可以从生长培养基，细胞裂解物或细胞膜流分(fraction)中分离出本发明的抗体，抗体片段或其衍生物。本发明的微生物或其他方式表达的抗体、抗体片段或其衍生物的分离和纯化可以通过任何常规手段进行，例如制备色谱分离和免疫学分离，例如涉及使用单克隆或多克隆抗体的那些方法。
制备如上述的靶向FGFR的拮抗短肽方法，所述方法包括培养如上述的重组细胞和纯化得到靶向FGFR的拮抗短肽。
如上述的靶向FGFR的拮抗短肽作为诊断试剂的应用。使用该诊断试剂具体包括通过上述的靶向FGFR的拮抗短肽检测FGFR1、FGFR2和FGFR3中的至少一种的表达水平的步骤。
一种用于诊断与FGFR异常失调相关的疾病的方法，FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变，所述方法包括使用上述的靶向FGFR的拮抗短肽。该方法包括使本发明的抗体与怀疑在其表面上携带FGFR1、FGFR2和FGFR3中的一种或多种受体的细胞或组织接触。用于检测样品中FGFR4表达的合适方法可以是酶联免疫吸附测定(ELISA)或免疫组织化学(IHC)。
一种预防和/或治疗FGFR异常失调相关的疾病的方法，FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变，所述方法包括给予上述的药物。
具体而言，本发明获得所述FGFR1拮抗短肽，其氨基酸序列如下(实施例1)：
Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH 2
实验结果表明，与前体肽P9相比，本发明发现的短肽化合物P48具有稳定的二级结构及相对延长的半衰期。此外，经分子对接实验发现短肽化合物P48 与FGFR1间能形成稳定的氢键。经SPR实验发现P48能与bFGF竞争性结合FGFR1，浓度越高，结合力越强(实施例1)。
同时，Western blot实验发现，在经高度转化的人胚肾细胞HEK-293及成纤维细胞MEF-WT，Balb/c 3T3中，短肽化合物P48能浓度依赖性的抑制由bFGF诱导的FGFR1激活(实施例2)。此外，短肽化合物P48对FGFR1信号的抑制作用与SSR相仿，其对下游PLCγ无明显抑制作用(实施例2)。选用野生型及FGFR1-FGFR2-FRS2α敲除的MEF细胞，通过流式细胞仪检测发现，短肽化合物P48仅对野生型MEF细胞有结合作用(实施例2)。
接着，选用FGFR1异常失调的多种肿瘤细胞株，经Western blot实验发现，短肽化合物P48能浓度依赖性的抑制包括宫颈癌、黑色素瘤、肺癌、胃癌在内的FGFR1信号通路(实施例3)。此外，在胃癌细胞株中，短肽化合物P48对FGFR1信号的抑制作用也与SSR相当，其活性弱于AZD4547，这可能与其不同结合类型相关，即短肽化合物P48可能与SSR相似，为膜外抑制剂，而AZD4547为膜内抑制剂。经流式细胞术实验进一步发现，短肽化合物P48对细胞膜表面的FGFR1位点表现出良好的结合作用(实施例3)。进一步地，通过Western blot实验发现，短肽化合物P48仅能抑制由bFGF诱导的FGFR1激活，对aFGF、KGF、EGF刺激无效(实施例3)。
此外，在Hela229、B16-F10、NCI-H460和SGC-7901细胞中，短肽化合物P48能明显抑制癌细胞的活力。另外，在胃癌细胞SGC-7901中，与对照组相比，短肽化合物P48能浓度依赖性的抑制细胞迁移(实施例4)。通过流式周期实验发现，短肽化合物P48还能有效的将细胞周期阻滞在G0/G1期(实施例4)。
最后，在移植瘤小鼠模型中，短肽化合物P48能明显抑制胃癌细胞SGC-7901的生长，对FGFR1信号通路(包括磷酸化FLG、FRS2α、ERK1/2蛋白)表现出良好的抑制作用(实施例5)。另外，通过免疫组化实验发现，与对照组相比，短肽化合物P48能浓度依赖性抑制Ki67的表达(实施例5)。以上结果表明短肽化合物P48在体内也能通过抑制FGFR1通路，抑制细胞增殖起到良好的抗肿瘤活性(实验例5)。
如上所述，我们的研究结果表明，短肽化合物P48具有良好的稳定性，且能通过抑制FGFR1通路发挥抗肿瘤作用，具有开发为抗肿瘤药物的前景。
因此，本发明提供了一种高效稳定的FGFR1拮抗肽P48在制备抗肿瘤药物中的应用，抗肿瘤药物通过选择性抑制FGFR1信号通路抑制细胞增殖和迁移发挥治疗肿瘤的作用。
本发明还提供了一种用于治疗肿瘤的药物组合物，其含有治疗有效量的活性成分和药用辅料，所述活性成分为上述FGFR1拮抗短肽或其可药用盐及其药用辅料。本文中所用“药用辅料”指药学领域常规的药物载体，例如：稀释剂如淀粉、蔗糖、糊精、乳糖、预胶化淀粉、微晶纤维素、磷酸钙等；润湿剂如蒸馏水、乙醇；粘合剂如淀粉浆、纤维素衍生物、聚维酮、明胶、聚乙二醇、海藻酸钠溶液等；崩解剂如干淀粉、羧甲基淀粉钠、低取代羌丙基纤维素、泡腾崩解剂等；润滑剂如硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇类、十二烷基硫酸钠等；着色剂如二氧化钛、日落黄、亚甲蓝、药用氧化铁红等；另外，还可以在组合物中加入其它辅剂如香味剂、甜味剂等。
本发明药物组合物的各种剂型可以按照药学领域的常规生产方法制备。例如使活性成分与一种或多种载体混合，然后将其制成所需的剂型。所述药物的制剂形式包括颗粒剂、注射剂、片剂、胶囊剂、气雾剂、栓剂、膜剂、滴丸剂、软膏剂、控释或缓释剂或纳米制剂。本发明可以组合物的形式通过口服，鼻吸入、直肠或者肠胃外给药的方式施用于需要这种治疗的患者。用于口服时，可将其制成常规的固体制剂如片剂、粉剂、粒剂、胶囊等，制成液体制剂如水或油悬浮剂或其它液体制剂如糖浆、酏剂等；用于肠胃外给药时，可将其制成注射用的溶液、水或油性悬浮剂等。
参见图1：短肽化合物P48的结构、稳定性和对FGFR1的结合能力分析
(A)前体肽P9和降解短肽P48的序列。(B)a：P9和P48主链中C原子的RMSD随分子动力学模拟时间而变化。b：P9和P48的初始构象及在4ns、8ns、12ns、16ns和20ns的构象叠合图。(C)P9和P48在人血浆中的半衰期。(D)a：bFGF-FGFR1复合物的分子动力学模拟和自由能计算。b：维持 bFGF-FGFR1复合物稳定性的关键残基分析。(E)FGFR1B和P48的分子对接。(F)SPR检测FGFR1B和P48的相互作用。
图2：(A)SPR检测FGFR1C和P48的相互作用；(B)SPR检测FGFR2B和P48的相互作用；(C)SPR检测FGFR3B和P48的相互作用。
图3：在高度转化的人胚肾细胞HEK-293及成纤维细胞MEF-WT、Balb/c 3T3中检测P48对FGFR1信号通路的抑制活性。
(A)短肽化合物P48预处理10分钟或AZD4547/PD173074预处理2小时后，bFGF孵育15分钟，检测药物对HEK-293、MEF-WT、Balb/c3T3细胞中FGFR1及其下游蛋白的抑制情况。(B)P48预处理10分钟或AZD4547/SSR处理2小时后，bFGF刺激15分钟，检测药物对PLCγ的抑制情况。(C)流式细胞术分析P48在MEF-WT和FGFR1-FGFR2-FRS2α敲除的MEF细胞中与FGFR1的结合能力。*，p<0.05；***，p<0.001，vs Control。
图4：短肽化合物P48在多种肿瘤细胞株中对FGFR1信号通路的抑制活性。
(A)短肽化合物P48预处理10分钟后，bFGF孵育15分钟，检测药物对FGFR1及其下游蛋白的抑制情况。(B)P48预处理10分钟或AZD4547/SSR预处理2小时后，bFGF孵育15分钟，检测药物对SGC-7901细胞中磷酸化FGFR1及其下游蛋白的抑制活性。(C)bFGF/aFGF刺激15分钟或KGF刺激20分钟，检测短肽化合物P48对SGC-7901细胞中磷酸化FGFR1及其下游蛋白的抑制活性。(D)EGF刺激10分钟，检测短肽化合物P48对PC-9细胞中磷酸化EGFR和ERK1/2的抑制活性。(E)流式细胞术检测短肽化合物P48在SGC-7901细胞中与FGFR1的结合能力。
图5：短肽化合物P48的体外抗肿瘤活性。
(A)用MTT法检测短肽化合物P48对癌细胞的生长抑制作用。(B)通过细胞侵袭实验检测短肽化合物P48对SGC-7901细胞迁移的抑制活性。(C)流式细胞术分析短肽化合物P48对细胞周期的阻滞作用。***，p<0.001，vs bFGF。
图6：短肽化合物P48的体内抗肿瘤活性。
(A)对照组和给药组的肿瘤体积测量。(B)肿瘤重量称量。(C)短肽化 合物P48对裸鼠体内FLG及其下游蛋白的磷酸化水平的抑制作用。(D)免疫组化检测短肽化合物P48对促增殖蛋白Ki67的抑制活性。
实施例1
短肽化合物P48的空间结构、稳定性及对FGFR1结合活性分析：
应用Amber11程序对P9的降解短肽P48进行氨基酸序列分析，结果见图1A。用Amber11程序Tleap模块中的sequence命令构建P9和P48肽链初始结构。用Amber11程序Tleap模块添加氢原子，在肽链
Figure PCTCN2020134550-appb-000001
距离处添加截断的四面体TIP3P溶剂盒，在两个体系中分别添加抗衡离子以保持体系呈电中性。体系进行20ns的NPT系宗模拟，得到最优空间结构，结果见图1B。图1C表示P9和P48在血浆中的半衰期。实验步骤如下：血浆离心去上清，加60μl肽孵育不同时间(P9孵育5、10、15、20、25、30、45分钟，P48孵育1、2、3、4、5、10、12小时)，用200μl 5％冰醋酸终止，固相萃取：先用1.5ml甲醇活化，再用1.5mlH 2O洗，取400μl样品上样，1mlH 2O洗，2ml 30％乙腈洗脱收集样品，冷冻干燥，进行液相色谱分析。图1D表示FGF2-FGFR1复合物体系的分子动力学模拟，确定维持FGF2-FGFR1结合的关键性氨基酸残基。实验步骤如下：选择FGF2-FGFR1蛋白质晶体结构(PDB ID:1CVS)构建分子动力学模拟的FGF2-FGFR1复合物体系初始结构。用Amber11程序Tleap模块给体系添加氢原子，在复合物
Figure PCTCN2020134550-appb-000002
距离处添加截断的四面体TIP3P溶剂盒，体系中添加抗衡离子以保持体系呈电中性。体系采用Amber99SB力场，用Sander模块对体系进行能量最小化。计算结合自由能时，选取体系分子动力学模拟所得轨迹中的最后1ns的轨迹计算，删除其中溶剂水分子和抗衡离子，每10ps取一次构象，共得到101个结构。用分子力学广义波恩/表面积(MM-GBSA)方法计算FGF2和FGFR1之间的结合自由能。结合自由能和氨基酸残基能量分解的计算用均采用Amber11提供的MMPBSA.py脚本进行。上述实验步骤可确定FGFR1结合位点，接着，在Discovery Studio Visualizer 3.5将P48置于FGFR1结合位点处，并保存相应复合物体系文件P48-FGFR1.pdb，作为分子动力学模拟初始结构文件。其中，FGFR1坐标选自FGF2-FGFR1蛋白质晶体结构(PDB ID:1CVS)A链中的 FGFR1。用Amber11程序Tleap模块给体系添加氢原子，在复合物
Figure PCTCN2020134550-appb-000003
距离处添加截断的四面体TIP3P溶剂盒，两个体系中分别添加抗衡离子以保持体系呈电中性。体系进行20ns的NPT系宗模拟，实验结果见图1E。图1F(图1F中的为FGFR1B蛋白质)表示P48与FGFR1的相互作用。实验步骤如下：用30μl/min PBS洗涤传感器芯片，用40mM EDAC和10mM sNHS的激活型缓冲液进行活化。bFGF、FGFR1流入传感器芯片后，将P48(50μM、100μM、200μM、300μM、400μM)注入到传感器表面，使用BIAevaluation软件分析所得数据。
进一步地，研究P48与FGFR1C、FGFR2B、FGFR3B的相互作用，实验步骤同上，实验结果如图2所示，可以看出P48与FGFR1C(图2A)、FGFR2B(图2B)、FGFR3B(图2C)一定程度均可结合，结合图1F分析，可以看出P48与FGFR1结合度相比FGFR2、FGFR3更强，其中，FGFR1C结合度最强。
实施例2
短肽化合物P48对高度转化的人胚肾细胞及成纤维细胞FGFR1信号通路的抑制活性：
将人胚肾细胞HEK-293及成纤维细胞MEF-WT、Balb/c 3T3分别接种于6孔板。细胞过夜贴壁后，用P48预处理10分钟或AZD4547/PD173074/SSR预处理2小时，bFGF孵育15分钟。图3A表示P48/AZD4547/PD173074对HEK-293、MEF-WT、Balb/c3T3细胞中FGFR1及其下游蛋白(FRS2α、ERK1/2、AKT)的抑制情况。图3B表示P48/AZD4547/SSR对MEF-WT细胞中FGFR1下游蛋白PLCγ的抑制情况。图3C表示P48在MEF-WT和FGFR1-FGFR2-FRS2α敲除的MEF细胞中与FGFR1的结合能力。实验步骤如下：将MEF-WT和MEF KO(FGFR1-FGFR2-FRS2α)细胞(1×10 6个细胞/ml)接种于35mm培养皿中。细胞过夜贴壁后，PBS洗涤细胞，加入与FITC-共轭的P48(1、10、100、1000nM)置于培养箱中孵育30分钟。于暗处用1％多聚甲醛固定细胞并用PBS洗涤。在荧光通道1(FL-1)(530nm)处测定FITC的表达水平，激发波长为488nm。应用CellQuest软件(Becton Dickinson)进行数据分析。
实施例3
短肽化合物P48对多种肿瘤细胞FGFR1信号通路的抑制活性：
将宫颈癌细胞Hela229、黑色素瘤B16-F10、肺癌细胞NCI-H460及胃癌细胞SGC-7901、MGC-803分别接种于6孔板。细胞过夜贴壁后，用P48预处理10分钟或AZD4547/SSR预处理2小时，bFGF/aFGF孵育15分钟或KGF孵育20分钟。图4A表示在bFGF刺激下，P48/AZD4547对Hela229、B16-F10、NCI-H460、MGC-803、SGC-7901细胞中FGFR1及其下游蛋白(FRS2α、ERK1/2、AKT)的抑制情况。图4B表示在bFGF刺激下，P48/AZD4547/SSR对SGC-7901细胞中FGFR1及其下游蛋白(FRS2α、ERK1/2)的抑制情况。图4C表示在bFGF/aFGF/KGF刺激下，P48对SGC-7901细胞中FGFR1及其下游蛋白(FRS2α、ERK1/2)的抑制情况。图4D表示在EGF刺激下，P48对PC-9细胞中EGFR及ERK1/2的抑制情况。图4E表示P48在SGC-7901细胞中与FGFR1的结合，实验步骤同实施例图2C。
实施例4
短肽化合物P48的体外抗肿瘤活性：
将Hela229(1500个细胞/100μl)、B16-F10(1000个细胞/100μl)、NCI-H460(4000个细胞/100μl)及SGC7901(4000个细胞/100μl)细胞接种于96孔板。过夜贴壁后，用含0.1％FBS的DMEM或RPMI-1640培养基替代原培养基继续培养24小时。将P48与bFGF的混合物及单独的bFGF分别加入相对应孔内，孵育48小时后，将磷酸盐缓冲溶液(PBS)制备的MTT溶液(5mg/mL)在37℃下加入各孔中的细胞，再处理4小时。然后吸出MTT，每孔加入150μL DMSO，震动10分钟，最后在490nm纳米波长下用酶标仪定量(SpectraMax M2/M2e，Molecular Devices，Sunnyvale，USA)测定OD值。通过与bFGF组的比较来计算药物生长抑制率的百分比，结果见图5A。图5B结果表示P48能浓度依赖性的抑制胃癌细胞SGC-7901的迁移。实验步骤如下：将含有10％FBS的培养基 (500μl)加入到transwell插入室的下表面，将细胞连同药物一起接种到上室，用无血清培养基(200μl)进行培养。培养箱内培养24小时后，将下室细胞用预冷的甲醇固定并用结晶紫染色，同时用棉签除去上室细胞。最后，在光学显微镜下捕获迁移细胞。图5C结果表示P48能浓度依赖性的使胃癌细胞SGC-7901阻滞在G0/G1期。实验步骤如下：将SGC-7901细胞(6×10 5个细胞/孔)接种在直径为60mm的培养皿中。用P48和bFGF的混合物、bFGF和载体(PBS)处理细胞24小时，用PBS洗涤，固定在75％冰冷的乙醇中过夜。然后将细胞用含有核糖核酸酶(550825，BD Biosciences 35Clontech，San Jose，CA，USA)的500μL碘化丙啶(PI)在4℃远离光线染色10分钟，并用200目纱布过滤。在FACS Calibur流式细胞仪(BD Biosciences，CA)中进行细胞周期分析。
实施例5
短肽化合物P48的体外抗肿瘤活性：
将SGC-7901细胞皮下注射到BALB/c裸鼠背部。10天后，将小鼠随机分为4组，每组12只，进行腹腔给药。给药组设模型组(生理盐水组)、AZD4547组(20mg/kg.d)、P48组(32mg/kg.d,64mg/kg.d)组。使用游标卡尺测量肿瘤的长度和宽度。使用V＝0.52×L×W 2公式计算肿瘤体积。每隔3天记录小鼠的体重，并在小鼠死亡当天记录肿瘤大小。28天后，处死裸鼠进行解剖，取出组织样品并进一步研究。结果见图6。其中A表示小鼠的体重变化。B表示肿瘤的体积及大小。C表示经P48处理后，磷酸化FLG、FRS2α、ERK1/2在体内的表达情况。D表示经P48处理后，Ki67在体内的表达情况。
综上所述：本发明人通过不懈努力，获得了短肽化合物P48，并发现其具有稳定的二级结构及相对较长的半衰期。此外，短肽化合物P48能有效靶向抑制FGFR1，在体内外表现出良好的抗肿瘤活性，有望成为癌症治疗的候选肽类抑制剂药物。
上述实施例对本发明的具体描述，只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限定，本领域的技术工程师根据上述发明的内容对 本发明作出一些非本质的改进和调整均落入本发明的保护范围之内。

Claims (17)

  1. 一种靶向FGFR的拮抗短肽，其特征在于：包括短肽化合物P48，所述短肽化合物P48可靶向结合FGFR的膜外免疫球蛋白域，短肽化合物P48的氨基酸序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH 2，其中FGFR为FGFR1、FGFR2和FGFR3中的至少一种。
  2. 根据权利要求1所述的靶向FGFR的拮抗短肽，其特征在于：所述短肽化合物P48可靶向结合FGFR1的膜外免疫球蛋白域。
  3. 如权利要求1所述的靶向FGFR的拮抗短肽作为活性成分在药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于：所述药物为通过拮抗体内FGFR1、FGFR2和FGFR3中的至少一种的信号通路治疗与FGFR异常失调相关的疾病。
  5. 根据权利要求4所述的应用，其特征在于：FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变。
  6. 根据权利要求4或5所述的应用，其特征在于：所述药物为治疗与FGFR1、FGFR2和FGFR3中的至少一种异常失调导致的肿瘤。
  7. 根据权利要求6所述的应用，其特征在于：FGFR拮抗短肽中的短肽化合物P48通过抑制FGFR1通路、FGFR2通路和FGFR3通路中的至少一种，抑制肿瘤细胞的生长和侵袭。
  8. 一种治疗与FGFR异常失调相关疾病的药物，其特征在于：包括如权利要求1所述的一种靶向FGFR的拮抗短肽或其药学上可接受的盐、酯。
  9. 根据权利要求8所述的药物，其特征在于：还包括药学上可接受的辅料。
  10. 根据权利要求9所述的药物，其特征在于：所述药物制剂为注射剂、片剂、胶囊剂、气雾剂、栓剂、膜剂、控释、缓释剂或纳米制剂。
  11. 一种核酸分子，其序列中包括编码如权利要求1所述的靶向FGFR的拮抗短肽其氨基修饰前的短肽的核酸序列，所述靶向FGFR的拮抗短肽其氨基修饰前的短肽序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser。
  12. 一种载体，其包含如权利要求11所述的核酸分子。
  13. 一种重组细胞，其包括如权利要求12所述的载体。
  14. 制备如权利要求1所述的靶向FGFR的拮抗短肽方法，所述方法包括培养如权利要求13所述的重组细胞和纯化得到靶向FGFR的拮抗短肽其氨基修饰前的短肽，所述靶向FGFR的拮抗短肽其氨基修饰前的短肽序列为Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser，然后将靶向FGFR的拮抗短肽其氨基修饰前的短肽端部通过氨基修饰得到如权利要求1所述的靶向FGFR的拮抗短肽。
  15. 如权利要求1所述的靶向FGFR的拮抗短肽作为诊断试剂的应用。
  16. 一种诊断与FGFR异常失调相关的疾病的方法，FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变，所述方法包括使用如权利要求1所述的靶向FGFR的拮抗短肽。
  17. 一种预防和/或治疗FGFR异常失调相关的疾病的方法，FGFR异常失调包括FGFR1、FGFR2和FGFR3中的至少一种的表达水平超过预先设定的阈值水平和FGFR1、FGFR2和FGFR3中的至少一种的突变，所述方法包括给予如权利要求8-10任一项所述的药物。
PCT/CN2020/134550 2019-12-10 2020-12-08 一种靶向fgfr的拮抗短肽 WO2021115258A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/768,296 US20240101600A1 (en) 2019-12-10 2020-12-08 Fibroblast growth factor receptor (fgfr)-targeting antagonistic short peptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911254125.6A CN110872341B (zh) 2019-12-10 2019-12-10 一种靶向fgfr1的拮抗短肽
CN201911254125.6 2019-12-10

Publications (1)

Publication Number Publication Date
WO2021115258A1 true WO2021115258A1 (zh) 2021-06-17

Family

ID=69717524

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/134550 WO2021115258A1 (zh) 2019-12-10 2020-12-08 一种靶向fgfr的拮抗短肽

Country Status (3)

Country Link
US (1) US20240101600A1 (zh)
CN (1) CN110872341B (zh)
WO (1) WO2021115258A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110872341B (zh) * 2019-12-10 2021-08-03 温州医科大学 一种靶向fgfr1的拮抗短肽

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003245A1 (en) * 1998-05-28 2000-01-20 Chugai Pharmaceutical Company Limited Peptide ligands for the human fibroblast growth factor (fgf) receptor
CN102399264A (zh) * 2011-08-12 2012-04-04 温州医学院 碱性成纤维细胞生长因子结合短肽衍生物及其应用
CN102453079A (zh) * 2010-11-01 2012-05-16 李校堃 特异性结合碱性成纤维细胞生长因子受体的肽
CN110872341A (zh) * 2019-12-10 2020-03-10 温州医科大学 一种靶向fgfr1的拮抗短肽

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224557B (zh) * 2012-08-08 2014-10-01 温州医学院 一种长效型成纤维细胞生长因子-23拮抗剂的开发
CN103432073A (zh) * 2013-09-11 2013-12-11 中国药科大学 一种肿瘤靶向纳米制剂及其制备方法
CN103992382B (zh) * 2014-05-14 2015-03-11 南方医科大学 一种靶向eps8与egfr结合的短肽及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003245A1 (en) * 1998-05-28 2000-01-20 Chugai Pharmaceutical Company Limited Peptide ligands for the human fibroblast growth factor (fgf) receptor
CN102453079A (zh) * 2010-11-01 2012-05-16 李校堃 特异性结合碱性成纤维细胞生长因子受体的肽
CN102399264A (zh) * 2011-08-12 2012-04-04 温州医学院 碱性成纤维细胞生长因子结合短肽衍生物及其应用
CN110872341A (zh) * 2019-12-10 2020-03-10 温州医科大学 一种靶向fgfr1的拮抗短肽

Also Published As

Publication number Publication date
US20240101600A1 (en) 2024-03-28
CN110872341A (zh) 2020-03-10
CN110872341B (zh) 2021-08-03

Similar Documents

Publication Publication Date Title
US20230002467A1 (en) Superagonists, partial agonists and antagonists of interleukin-2
US7785808B2 (en) Treatment of inflammation using α7 receptor-binding cholinergic agonists
CN112972492A (zh) Nk细胞中对细胞因子诱导的sh2蛋白的抑制
JP6921755B2 (ja) 脳腫瘍幹細胞の成長、遊走および浸潤性を低下させ脳腫瘍患者の生存率を改善する方法および組成物
EP0873998A2 (en) Receptor protein and its use
TW201632207A (zh) 細胞穿透抗體
WO2020171113A1 (ja) Ｆｇｆｒ１変異陽性脳腫瘍を治療するための医薬組成物及び治療方法
US8734775B2 (en) Chemokine derived peptides that bind with chemokine receptor CXCR3 and uses for chronic wound and angiogenesis inhibition treatments
WO2021115258A1 (zh) 一种靶向fgfr的拮抗短肽
WO2011003369A1 (zh) 一种新的肿瘤标志物
CN117730143A (zh) 通过缀合的n-末端甘氨酸修饰的细胞及其用途
Li et al. The role and therapeutic implication of endoplasmic reticulum stress in inflammatory cancer transformation
Deng et al. Cyclophilin A is the potential receptor of the Mycoplasma genitalium adhesion protein
CN110498850B (zh) 多肽、其衍生物及其在制备防治肿瘤的药物中的应用
WO2022174781A1 (zh) 多结构域融合蛋白及其应用
JP6050319B2 (ja) 活性ポリペプチドの結合親和力及び結合特異性を増強させる蛋白質骨格モジュール
US20200299374A1 (en) Anti-CCL8 Antibodies and Uses Thereof
KR100628917B1 (ko) 세포장해 단백질 및 그 이용
JP4694580B2 (ja) ヒトおよび哺乳動物の幹細胞由来神経生存因子
WO2019057120A1 (zh) LGR4和R-spondin结合抑制剂及其在肿瘤治疗中的用途
US20240166702A1 (en) Compositions and methods for treating breast cancer
TWI808063B (zh) 治療或預防癌症的方法
KR102658844B1 (ko) 펩티드 유도체 및 약학적 조성물
JP2009108013A (ja) Ｇタンパク質共役受容体に対する機能抗体およびその応用
Ao et al. Targeting GFPT2 to reinvigorate immunotherapy in EGFR-mutated NSCLC

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20900423

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 17768296

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20900423

Country of ref document: EP

Kind code of ref document: A1