A class of peptides targeting HER2 protein and their application in the preparation of peptide-conjugated drugs

Abstract

The present invention discloses a class of polypeptides targeting HER2 protein and their application in the preparation of polypeptide conjugate drugs. The sequence of the HER2-targeting polypeptide is: Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Xaa-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2. Among them, Xaa represents a sequence with a length of 1 to 7 amino acid residues, including at least one or more cysteine residues. The polypeptide provided by the present invention has HER2 targeting property. Using it as a carrier for small molecule anti-tumor drugs to prepare polypeptide conjugate drugs can deliver anti-tumor drugs to HER2-positive tumors directionally for specific targeted therapy.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a polypeptide specifically targeting HER2 protein and a polypeptide-coupled drug derived from the peptide and capable of binding to HER2. The present invention also discloses a preparation method thereof and the use of such compounds in the preparation of cancer targeted therapeutic drugs. Background Art

[0002] Malignant tumor is a major disease that seriously endangers human health. Its main characteristics include unlimited proliferation of tumor cells, escape from the body's growth inhibition, resistance to programmed cell death, induction of angiogenesis, promotion of invasion and metastasis, and immune escape.

[0003] Conventional treatments for malignant tumors mainly include surgical treatment, radiotherapy and chemotherapy. With the development of life science and technology, targeted tumor treatment methods have begun to emerge and become more mature. The premise of targeted cancer treatment is to find the right therapeutic target. Human epidermal growth factor receptor 2 (HER2), also known as erbB-2, is a tyrosine protein kinase receptor encoded by the ERBB2 gene on chromosome 17q12. HER2 is overexpressed in many human cancers, including breast cancer, gastric cancer, esophageal cancer, ovarian cancer and endometrial cancer. Especially in breast cancer, the incidence of this overexpression is almost 20%. HER2 overexpression is associated with higher tumor invasiveness, higher risk of recurrence, faster progression and poorer prognosis. Currently, a variety of clinical drugs targeting HER2 have been developed for cancer treatment.

[0004] Targeted peptides have small molecular weight, strong permeability blocking ability, and are easy to synthesize in large quantities. In addition, peptides can be repeatedly modified to change their affinity, charge, hydrophilicity, stability, and solubility. In tumor targeted therapy, targeted peptides have shown strong superiority and are an effective alternative to antibodies. In terms of application, targeting peptides can be used to construct peptide-coupled drugs, thereby delivering anti-tumor drugs to tumor cells expressing target proteins and exerting a cell-killing effect. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide that specifically targets HER2 protein, and a polypeptide-coupled drug derived from the peptide and capable of binding to HER2. The polypeptide has the characteristics of high selectivity, strong specificity, small molecular weight, easy availability, no immunogenicity, safety and reliability. The polypeptide-coupled drug can significantly increase the therapeutic effect of tumor drugs.

[0006] The first aspect of the present invention provides a polypeptide targeting HER2 protein, the general formula of its amino acid sequence is:

[0007] Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Xaa-Arg-I le-Lys-Pro-Arg-Lys-GLy-Tyr-Thr-Arg-NH2,

[0008] In the above peptide chain, X aa represents a sequence of 1 to 7 amino acid residues in length, including at least one or more cysteine ​​residues,

[0009] Preferably, X aa is a sequence of 3 or 5 amino acids in length,

[0010] More preferably, Xaa is Gly-Cys-Gly or Gly-Cys-Gly-Cys-Gly,

[0011] Some of the peptide sequences targeting HER2 protein are as follows:

[0012] Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Gly-Cys-Gly-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 (SEQ. ID NO: 1),

[0013] Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Gly-Cys-G ly-Cys-Gly-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 (SEQ. ID NO: 2),

[0014] The second aspect of the present invention provides a method for preparing a polypeptide targeting HER2 protein. The present invention adopts a microwave-promoted Fmoc / tBu orthogonal protection solid phase synthesis strategy to efficiently and quickly synthesize a peptide chain specifically targeting HER2.

[0015] The third aspect of the present invention provides the use of a polypeptide targeting HER2 protein in the preparation of a polypeptide-coupled drug, preferably the use of a polypeptide-coupled drug targeting HER2 protein

[0016] The fourth aspect of the present invention provides a class of polypeptide-coupled drugs, which include the polypeptide targeting HER2 protein, a cytotoxic drug and a linker, wherein the linker refers to a structure that connects the polypeptide targeting HER2 protein to the selected cytotoxic drug.

[0017] Preferably, the linker is linked to a cysteine ​​residue in the HER2 targeting polypeptide.

[0018] Further preferably, the linker structure is selected from any one of the following:

[0019]

[0020] Furthermore, the cytotoxic drug is a tumor chemotherapy drug, preferably gemcitabine, doxorubicin, paclitaxel, camptothecin, and more preferably camptothecin.

[0021] The polypeptide-coupled drug of the present invention utilizes the targeting effect of the polypeptide targeting HER2 protein on cancer cells overexpressing HER2 to deliver tumor chemotherapy drugs to tumors to exert targeted anti-cancer effects. Therefore, the tumor chemotherapy drug can be any clinically known or reported tumor chemotherapy drug.

[0022] Furthermore, the polypeptide-conjugated drug has a structure as shown in Formula A:

[0023]

[0024] Formula A

[0025]

[0026] The fifth aspect of the present invention provides the use of the polypeptide-coupled drug in the preparation of a cancer targeted therapeutic drug.

[0027] Furthermore, the cancer includes HER2-positive tumors, preferably HER2-positive breast cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, and bladder cancer.

[0028] Preferably, the cancer is HER2-positive breast cancer.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] The peptides of the present invention can specifically target HER2 positive cells with high selectivity, and the peptides of the present invention can be prepared by chemical synthesis method, with high purity, small molecular weight, strong specificity, no immunogenicity, and safety and reliability.

[0031] The polypeptide-coupled drug of the present invention introduces the cytotoxic drug camptothecin into a drug molecule, thereby increasing the therapeutic effect of the tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 SPR determination of affinity between peptide and HER2 protein

[0033] Figure 2Binding of polypeptide to HER2 positive or negative cells. (A) SEQ.ID NO.1; (B) SEQ.ID NO. 2

[0034] Figure 3 Distribution of peptides in SK-BR-3 tumor-bearing nude mice

[0035] Figure 4 Evaluation of the antitumor activity of peptide-drug conjugates in vivo DETAILED DESCRIPTION

[0036] The following abbreviations are used in this manual:

[0037] NMP: N-methylpyrrolidone; DIPEA: N,N'-diisopropylethylamine; DMF: dimethylformamide; DMSO: dimethyl sulfoxide; DCM: dichloromethane; Fmoc: N-9-fluorenylmethoxycarbonyl; DIC: N,N'-diisopropylcarbodiimide; DMAP: 4-dimethylaminopyridine; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBT: 1-hydroxy-benzotriazole; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrometry; Gly: glycine; Ser: serine; Ala: alanine; Thr: threonine; Val: valine; Ile: isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: histidine; Pro: proline; Asp: aspartic acid; Met: methionine; Glu: glutamic acid; Trp: tryptophan; Lys: lysine; Arg: arginine; Asn: asparagine; Gln: glutamine. The present invention is illustrated by the following examples, but these examples are not to be construed as limiting the present invention in any way.

[0038] Example 1

[0039] Microwave-enhanced solid phase synthesis of Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Gly-Cys-Gly-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 (SEQ. ID NO: 1)

[0040] (1) Swelling of resin

[0041] Weigh 50 mg of Fmoc-Rink amide-MBHA Resin (substitution amount 0.4 mmol / g), swell it with 7 mL of DCM for 30 min, filter out the DCM, swell it with 10 mL of NMP for 30 min, and finally rinse it with NMP, DCM, and 7 mL of NMP respectively.

[0042] (2) Microwave-assisted removal of Fmoc protecting groups

[0043] Put the swollen resin into the reactor, add 7mL of 25% piperidine / NMP (V / V) solution containing 0.1M HOBT, react in the microwave reactor for 1min, microwave power is 15W, reaction temperature is controlled within 50℃, use air compressor to cool, filter out the solution after the reaction is completed; add 7mL of 25% piperidine / NMP (V / V) solution containing 0.1M HOBT in the microwave reactor for another 4min, microwave power is 25W, reaction temperature is controlled at 50℃, use air compressor to cool. After the reaction is completed, filter out the solution and wash with NMP. Get the resin with the Fmoc protecting group removed from the initial connection.

[0044] (3) Microwave-assisted synthesis of Fmoc-Arg(Pbf)-Rink amide-MBHA Resin

[0045] Fmoc-Arg(Pbf)-OH (0.04 mmol), HBTU (0.04 mmol), HOBT (0.04 mmol) and DIPEA (0.08 mmol) were dissolved in 10 mL NMP, and the solution was added to the above resin. The reaction was carried out in a microwave reactor for 7 min, the microwave power was 25 W, the reaction temperature was controlled at 50 ° C, and the air was cooled by compressed air from an air compressor. After the reaction was completed, the reaction solution was filtered out, and the resin was washed 3 times with 7 mL of DCM and 7 mL of NMP.

[0046] (4) Detection of coupling efficiency

[0047] The coupling efficiency of the resin was qualitatively detected by the ninhydrin method or the bromophenol blue method. If the color reaction was negative, the next coupling cycle could be entered.

[0048] Ninhydrin method: Take a small amount of resin particles, wash them with ethanol, put them in a transparent vial, add 2 drops each of 5% ninhydrin ethanol, KCN pyridine solution (2mL 0.001M KCN ​​diluted in 98mL pyridine), and 80% phenol ethanol solution, heat at 100°C for 5 minutes. If the resin appears blue, it is positive.

[0049] Bromophenol blue method: Take a small amount of resin particles, wash them with dimethylacetamide, put them in a transparent vial, add 3 drops of 1% bromophenol blue dimethylacetamide solution, shake at room temperature for 3 minutes, if the resin appears blue, it is positive.

[0050] (5) Peptide chain extension

[0051] According to the sequence of the polypeptide, the above steps of deprotection and coupling are repeated to sequentially connect the corresponding amino acids, and the coupling microwave-promoted reaction time varies from 5 to 20 minutes to obtain a resin connected with the polypeptide sequence (SEQ.ID NO: 1).

[0052] (6) Cleavage of peptides on resin

[0053] The resin with the polypeptide sequence (SEQ.ID NO: 1) obtained above was placed in a reaction bottle, and 10 mL of the cleavage agent Reagent K (TFA / thioanisole / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added to each bottle. The mixture was shaken at 0°C for 30 minutes, and then reacted at room temperature for 3 hours. After the reaction was completed, the mixture was filtered, washed three times with a small amount of TFA and DCM, and the filtrate was combined. The filtrate was added to a large amount of ice ether to precipitate a white flocculent precipitate, and the crude product of the target polypeptide was obtained by refrigerated centrifugation.

[0054] The crude polypeptide was dissolved in 2 mL of water and directly purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse phase column (320 mm × 28 mm, 5 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20 min; flow rate 6 mL / min, detection wavelength 214 nm. The collected solution was lyophilized to obtain 25 mg of pure product. The theoretical relative molecular mass is 3325.8. ESI-MS m / z: found [M+3H] 3+ 1110.6, [M+4H] 4+ 833.1;calcu[M+3H] 3+ 1109.6, [M+4H] 4+ 832.5.

[0055] Example 2

[0056] Microwave-enhanced solid phase synthesis of Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Gly-Cys-Gly-Cys-Gly-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 (SEQ. ID NO: 2)

[0057] The polypeptide shown in SEQ.ID NO: 2 was prepared according to the method disclosed in Example 1. The theoretical relative molecular mass was 3484.7. ESI-MS m / z: found [M+3H] 3+ 1163.4, [M+4H] 4+ 872.4;calcu [M+3H]3+ 1162.6, [M+4H] 4+ 872.2.

[0058] Example 3

[0059] The surface plasmon resonance (SPR) method was used to detect the binding effects of SEQ.ID NO:1 and SEQ.ID NO:2 on HER2 protein respectively.

[0060] The human recombinant protein HER2 was coupled to the CM 5 chip, and the analyte was diluted into a concentration gradient (500, 250, 125, 62.5, 31.25 nM). Appropriate regeneration conditions (such as Glycine, pH 2.5) were used, the flow rate was set to 30 μL / min, the injection time of each cycle was set to 90 s, and the dissociation time was also 90 s. According to the Biacore instrument program flow, multi-cycle kinetic tests were performed, and finally the binding-dissociation curves were analyzed and fitted by Biacore T200 Evaluation Software to calculate affinity-related parameters.

[0061] The results are as follows Figure 1 As shown, the equilibrium dissociation constant K of SEQ.ID NO:1 peptide and HER2 D The equilibrium dissociation constant K between SEQ. ID NO:2 peptide and HER2 is 151 nM. D The peptide of the present invention has a strong binding effect on HER2 and can be used as a polypeptide targeting HER2.

[0062] Example 4

[0063] Immunofluorescence detection of the binding of SEQ.ID NO:1, SEQ.ID NO:2 to HER2-positive SK-BR-3 or HER2-negative MDA-MB-231 cells

[0064] HER2-positive SK-BR-3 cells or HER2-negative MDA-MB-231 cells were inoculated in confocal microplates at a density of 3000-5000 cells / plate. After 24 hours of culture, the culture medium in the plate was aspirated, and then 10 μM SEQ.ID NO.1 or SEQ.ID NO.2 peptide labeled with fluorescein isothiocyanate (FITC) was added, the cell nucleus was stained with hochest reagent, and the expression of HER2 was indicated by PE-labeled anti-HER2 antibody. Incubate in an ice bath in the dark for 30 minutes, wash with PBS, add 200 μL PBS, and observe the fluorescence signal using a laser confocal microscope.

[0065] from Figure 2It can be seen that both SEQ.ID NO: 1 and SEQ.ID NO: 2 peptides can bind to HER2-positive SK-BR-3 cells, but have almost no binding to HER2-negative MDA-MB-231 cells, indicating that the peptides of the present invention selectively and specifically bind to HER2-positive tumor cells and can be used as polypeptides that specifically target HER2.

[0066] Example 5

[0067] Evaluation of in vivo targeting ability of SEQ.ID NO:1 and SEQ.ID NO:2 peptides

[0068] Take SK-BR-3 cells and adjust the cell suspension concentration to 5×10 6 200 μL of cell suspension was inoculated in the armpit of each nude mouse. 3 Around 14:00 p.m., tumor-bearing nude mice were randomly divided into a control group and an experimental group. The control group was injected with FITC dye (10 μM, 200 μL) through the tail vein, and the experimental group was injected with FITC-labeled polypeptide (10 μM, 200 μL) through the tail vein. 90 min after the injection, all mice were killed and dissected, and the heart, liver, brain, kidney, spleen, lung and tumor were collected and observed and photographed using the IVIS Spectrum imaging system.

[0069] Depend on Figure 3 It can be seen that compared with the saline group, the strongest fluorescence signals were observed in the tumor tissues of the SEQ.ID NO:1 and SEQ.ID NO:2 groups, while the fluorescence signals of the FITC group were enriched in the kidneys and liver, indicating that the SEQ.ID NO:1 and SEQ.ID NO:2 peptides can be targeted and enriched in the tumor site.

[0070] Example 6

[0071] Synthesis of Conjugate 1

[0072] Weigh the peptide SEQ.ID NO:1 obtained in Example 1, dissolve it in a methanol / water mixed solvent, add the compound 4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl(2-(pyridin-2-yldisulfanyl)ethyl)carbonate, stir at room temperature overnight, and use LC-MS to detect the reaction progress. The reaction solution was filtered and directly purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse phase column (320mm×28mm, 5μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate was 6mL / min and the detection wavelength was 214nm. The peptide drug conjugate Conjugate 1 was purified, with a theoretical relative molecular mass of 3773.8. ESI-MS m / z: found [M+4H] 4+ 944.9, [M+5H] 5+ 755.8;calcu[M+4H] 4+ 944.5, [M+5H] 5+ The 755.8 structure is as follows:

[0073]

[0074] Example 7

[0075] Synthesis of Conjugate 2

[0076] Weigh the peptide SEQ.ID NO:1 obtained in Example 1, dissolve it in a methanol / water mixed solvent, add the compound 4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl 3-(pyridin-2-yldisulfanyl) propionate, stir overnight at room temperature, and use LC-MS to detect the reaction progress. The reaction solution is filtered and directly purified by preparative liquid chromatography. The chromatographic conditions are: C18 reverse phase column (320 mm×28 mm, 5μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate is 6mL / min and detection wavelength is 214nm. The peptide drug conjugate Conjugate 2 was purified and its theoretical relative molecular mass was 3760.3. ESI-MS m / z: found [M+4H] 4+ 941.3, [M+5H] 5+ 753.3;calcu[M+4H] 4+ 941.1, [M+5H]5+ The 753.1 structure is as follows:

[0077]

[0078] Example 8

[0079] Synthesis of Conjugate 3

[0080] Weigh the peptide SEQ.ID NO:1 obtained in Example 1, dissolve it in a methanol / water mixed solvent, add the compound 4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate, stir overnight at room temperature, and use LC-MS to detect the reaction progress. The reaction solution was filtered and directly purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse phase column (320mm×28mm, 5μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate was 6mL / min and the detection wavelength was 214nm. The peptide drug conjugate Conjugate 3 was purified and its theoretical relative molecular mass was 3867.4. ESI-MS m / z: found [M+4H] 4+ 968.0, [M+5H] 5+ 774.4;calcu[M+4H] 4+ 967.9, [M+5H] 5+ The 774.5 structure is as follows:

[0081]

[0082] Example 9

[0083] Synthesis of Conjugate 4

[0084] Weigh the peptide SEQ.ID NO:2 obtained in Example 2, dissolve it in a methanol / water mixed solvent, add the compound 4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl(2-(pyridin-2-yldisulfanyl)ethyl)carbonate, stir overnight at room temperature, and use LC-MS to detect the reaction progress. The reaction solution was filtered and directly purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse phase column (320mm×28mm, 5μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate was 6mL / min and the detection wavelength was 214nm. The peptide drug conjugate Conjugate 4 was purified, with a theoretical relative molecular mass of 4387.0. ESI-MS m / z: found [M+4H] 4+ 1098.3, [M+5H] 5+ 878.9;calcu[M+4H] 4+ 1097.8, [M+5H] 5+ The 878.4 structure is as follows:

[0085]

[0086] Example 10

[0087] Synthesis of Conjugate 5

[0088] Weigh the peptide SEQ.ID NO:2 obtained in Example 2, dissolve it in a methanol / water mixed solvent, add the compound 4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl 3-(pyridin-2-yldisulfanyl) propionate, stir overnight at room temperature, and use LC-MS to detect the reaction progress. The reaction solution was filtered and directly purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse phase column (320 mm×28 mm, 5 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20 min; flow rate was 6 mL / min and the detection wavelength was 214 nm. The peptide drug conjugate Conjugate 5 was purified and its theoretical relative molecular mass was 4355.0. ESI-MS m / z: found[M+4H] 4+ 1090.4, [M+5H] 5+ 872.4;calcu[M+4H] 4+1089.8, [M+5H] 5+ The 872.0 structure is as follows:

[0089]

[0090] Example 10

[0091] Synthesis of Conjugate 6

[0092] Weigh the peptide SEQ.ID NO:2 obtained in Example 2, dissolve it in a methanol / water mixed solvent, add the compound 4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate, stir overnight at room temperature, and use LC-MS to detect the reaction progress. The reaction solution is directly purified by preparative liquid chromatography after filtration, and the chromatographic conditions are: C18 reverse phase column (320mm×28mm, 5μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate is 6mL / min and detection wavelength is 214nm. The peptide drug conjugate Conjugate 6 was purified, with a theoretical relative molecular mass of 4569.1. ESI-MS m / z: found [M+4H] 4+ 1144.1, [M+5H] 5+ 915.5;calcu [M+4H] 4+ 1143.3, [M+5H] 5+ The 914.8 structure is as follows:

[0093]

[0094] Embodiment 11

[0095] In vitro antitumor activity studies

[0096] In order to study the in vitro antitumor activity of the conjugate, HER2-positive human breast adenocarcinoma cells SK-BR-3, human gastric cancer cells NCI-N87, human ovarian adenocarcinoma cells SK-OV-3, HER2-negative human breast cancer cells MDA-MB-231 and normal cells MCF-10A were selected to determine the cytotoxic effect of the conjugate.

[0097] Cells in the logarithmic growth phase were taken at 10 4The cells were inoculated at a density of 100 μL / well in a 96-well cell culture plate and cultured overnight at 37°C and 5% CO2. The test compound and the positive control were prepared into different concentrations and then added to the wells, with 3 replicates for each concentration. After incubation for 48 hours, 10 μL of CCK-8 solution was added to each well, and the incubation continued for 2-4 hours. The absorbance value of each well at 450 nm was detected by an ELISA instrument, and the survival rate of the cells at different concentrations was calculated. Then, the IC was calculated using Graphpad software. 50 .

[0098] The cytotoxicity of the conjugates is shown in Table 1

[0099] Table 1 Cytotoxicity of the conjugates a

[0100]

[0101] a All experiments were performed in triplicate

[0102] * P<0.05, ** P<0.01, *** P < 0.001 vs CPT; # P<0.05, ### P<0.001 vs Conjugate 4

[0103] Example 12

[0104] Determination of the antitumor efficacy of peptide-coupled drugs Conjugate 1 and Conjugate 4 in animal models SK-BR-3 cells were taken and the concentration of cell suspension was adjusted to 5×10 6 Each nude mouse was inoculated with 200 μL of cell suspension in the armpit. 3 The tumor-bearing nude mice were randomly divided into a blank control group, a positive control group and an experimental group, with 4 mice in each group. Vehicle, camptothecin (8 μmol / kg) and the preferred compound Conjugate 1 or Conjugate 4 (8 μmol / kg) were injected into the tail vein every three days. The tumor volume was measured every two days with a vernier caliper, and the calculation formula was: V = 0.5ab 2 (where a is the maximum length and b is the minimum length). Two weeks after administration, all nude mice were sacrificed, and the tumor tissues were isolated, measured in volume and weighed.

[0105] The results are as follows Figure 4As shown, the peptide conjugate drugs Conjugate 1 and Conjugate 4 have obvious tumor growth inhibitory effects on the SK-BR-3 tumor-bearing nude mouse model, indicating that the peptide conjugate drugs Conjugate 1 and Conjugate 4 have obvious anti-tumor activity.

Claims

1. A polypeptide targeting HER2 protein, characterized in that: The polypeptide sequence of the HER2 targeting polypeptide is selected from any one of the following sequences: Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Gly-Cys-Gly-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 (SEQ ID NO: 1), Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Gly-Cys-Gly-Cys-Gly-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 (SEQ ID NO: 2).

2. Use of the polypeptide according to claim 1 in the preparation of a polypeptide-coupled drug targeting HER2 protein, wherein the drug is used to treat HER2-positive breast cancer, ovarian cancer or gastric cancer.

3. A polypeptide-conjugated drug, characterized in that: It comprises the HER2-targeting polypeptide of claim 1, a cytotoxic drug and a linker, wherein the linker connects the HER2-targeting polypeptide of claim 1 to the cytotoxic drug.

4. The polypeptide-conjugated drug according to claim 3, characterized in that: The linker is connected to the cysteine ​​residue in the HER2-targeting polypeptide of claim 1.

5. The polypeptide-conjugated drug according to claim 4, characterized in that: The linker structure is selected from any of the following:

6. The polypeptide-conjugated drug according to claim 3, characterized in that: The cytotoxic drug is a tumor chemotherapy drug.

7. The polypeptide-conjugated drug according to claim 6, characterized in that: The cytotoxic drug is selected from gemcitabine, doxorubicin, paclitaxel or camptothecin.

8. The polypeptide-conjugated drug according to claim 7, characterized in that: The cytotoxic drug is selected from camptothecin.

9. The polypeptide-conjugated drug according to any one of claims 3 to 8, characterized in that: The polypeptide-coupled drug has a structure as shown in Formula A: Asn-Pro-Asn-Trp-Gly-Arg-Ser-Trp-Tyr-Asn-Gln-Arg-Phe-Lys-Fragment1-Arg-Ile-Lys-Pro-Arg-Lys-Gly-Tyr-Thr-Arg-NH2 Formula A 10. Use of the polypeptide-coupled drug according to any one of claims 3 to 8 in the preparation of a drug for treating HER2-positive breast cancer, ovarian cancer or gastric cancer.

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