Polypeptide targeting CLDN6 protein as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide targeting CLDN6 protein as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The invention relates to a polypeptide targeting CLDN6 protein. The structural general formula of the amino acid sequence of the polypeptide is X1IX2PYX3, wherein X1-X3 represents a sequence with the length of 1-7 amino acid residues, and the amino acid is any one of 21 natural amino acids and non-natural amino acids; the polypeptide can specifically target CLDN6 positive cells, is high in selectivity, and can be used as a drug delivery carrier or coupled with known components to form a drug combination, so that a better treatment effect is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a polypeptide targeting CLDN6 protein and a preparation method and application thereof. Background Art

[0002] Claudin-6 (CLDN6) is a member of the claudin membrane protein family, which consists of 27 different proteins characterized by four transmembrane domains and two extracellular loops. Various claudin (CLDN) family members are located at tight junctions between epithelial cells, where they are thought to play a key role in barrier function. Recently, CLDN6 has also been implicated in several key intracellular signaling pathways, including the YAP1-snail1 axis, the ASK1-p38 / JNK MAPK secretion signaling pathway, and CLDN6 signaling has been shown to activate ERα transcriptional activity in a ligand-independent manner, thereby promoting tumor progression in endometrial cancer. CLDN6 is also highly expressed in undifferentiated mouse and human stem cells and is thought to contribute to the tumorigenic potential of cultures containing human pluripotent stem cells. Although CLDN6 is widely expressed in cells and tissues during early stages of embryonic and fetal development, in adults, CLDN6 expression is primarily restricted to malignant tissues, where it has been implicated in the development, progression, and metastasis of certain cancers. It has been reported to be aberrantly expressed in ovarian, gastric, lung, endometrial, and cervical cancers.

[0003] Furthermore, in patients with ovarian and endometrial cancer, high levels of CLDN6 expression in malignant tissues were an independent prognostic marker for poor progression-free and overall survival. The combined data indicating that adult cancers have higher expression than normal tissues make CLDN6 an attractive target for the development of peptide-based therapies. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a polypeptide targeting the CLDN6 protein and a preparation method and application thereof, thereby solving the problems in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A polypeptide targeting CLDN6 protein, whose amino acid sequence structure is as follows: 1 IX 2 PX 3 ; where X 1 -X 3 It represents a sequence of 1-7 amino acid residues in length, wherein the amino acid is any one of the 21 natural amino acids and unnatural amino acids.

[0007] Furthermore, the amino acid sequences of the polypeptides are shown in SEQ.ID NO 1-SEQ.ID NO 10 respectively.

[0008] Use of the above-mentioned polypeptide targeting CLDN6 protein in the preparation of a drug or drug carrier for treating tumors with high expression of CLDN6 protein.

[0009] Furthermore, the tumors that highly express CLDN6 protein include: ovarian tumors, endometrial tumors and prostate tumors.

[0010] A drug for treating tumors with high expression of CLDN6 protein, comprising the above-mentioned polypeptide targeting CLDN6 protein.

[0011] A drug carrier for treating tumors with high expression of CLDN6 protein, comprising the above-mentioned polypeptide targeting CLDN6 protein.

[0012] Furthermore, the drug carrier is: a polypeptide coupled with any amino acid with a charge or different hydrophilicity to prepare a polypeptide hydrogel.

[0013] An immunoconjugate comprises the above-mentioned polypeptide.

[0014] Furthermore, it also includes chemotherapeutic agents, radioactive atoms, cytostatic agents, cytotoxic agents, immune checkpoint inhibitors, antibodies or antibody fragments.

[0015] The above-mentioned method for preparing a polypeptide targeting CLDN6 protein comprises the following steps:

[0016] S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter out DCM, swell it with NMP for 30 min, and finally rinse it with NMP, DCM, and NMP respectively;

[0017] S2, the swollen resin is placed in a reactor, and a piperidine / NMP solution containing HOBT is added to react, and after the reaction is completed, the solution is filtered off and washed with NMP;

[0018] S3, dissolving Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA in NMP, and then adding the solution to the depiperidinized resin for reaction. After the reaction is completed, the reaction solution is filtered out and washed with DCM and NMP;

[0019] S4, qualitatively detect the coupling efficiency of the resin using the ninhydrin method or the bromophenol blue method, and proceed to the next coupling cycle if the color reaction is negative;

[0020] S5, according to the sequence of the polypeptide, repeat S2 and S3 to sequentially connect the corresponding amino acids to obtain a resin connected with the polypeptide sequence;

[0021] S6, adding a cutting agent to cut the resin to obtain the polypeptide.

[0022] Beneficial effects of the present invention:

[0023] 1. The polypeptide of the present invention can specifically target CLDN6-positive cells with high selectivity, and the polypeptide of the present invention can be prepared by chemical synthesis, with high purity, small molecular weight, strong specificity, no immunogenicity, and safety and reliability.

[0024] 2. The polypeptide of the present invention can be used as a drug delivery carrier, or coupled with known components to form a drug combination, thereby obtaining a better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 is the mass spectrum of the amino acid sequence prepared in Example 1 of the present invention;

[0027] Figure 2 is the mass spectrum of the amino acid sequence prepared in Example 2 of the present invention;

[0028] Figure 3 is the mass spectrum of the amino acid sequence prepared in Example 3 of the present invention;

[0029] Figure 4 is the mass spectrum of the amino acid sequence prepared in Example 4 of the present invention;

[0030] Figure 5 is the mass spectrum of the amino acid sequence prepared in Example 5 of the present invention;

[0031] Figure 6 is the mass spectrum of the amino acid sequence prepared in Example 6 of the present invention;

[0032] Figure 7 is the mass spectrum of the amino acid sequence prepared in Example 7 of the present invention;

[0033] Figure 8 is the mass spectrum of the amino acid sequence prepared in Example 8 of the present invention;

[0034] Fig. 9 is the mass spectrum of the amino acid sequence prepared in Example 9 of the present invention;

[0035] Fig.10is the mass spectrum of the amino acid sequence prepared in Example 10 of the present invention;

[0036] Fig.11 It is a diagram of the CCK-8 assay results of the amino acid sequences prepared in Examples 1-10 of the present invention.

[0037] Fig.12 This is an immunofluorescence staining diagram of internalization after Example 1-10 of the present invention is connected with a FITC fluorescent group and co-incubated with cells. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] A polypeptide targeting CLDN6 protein, whose amino acid sequence structure is as follows: 1 IX 2 PX 3 ; where X 1 -X 3 It represents a sequence of 1-7 amino acid residues in length, wherein the amino acid is any one of the 21 natural amino acids and unnatural amino acids.

[0040] The amino acid sequences of the polypeptides are shown in SEQ.ID NO1 to SEQ.ID NO10, respectively; specifically: IGRIWPYFGG, IGKIWPYYGG, IGRIKPYRGG, IGRILPYYGG, RIWPYR, GRIWPYFG, IGRIRPYRGG, KIWPYY, GLINPYNG, GRIWPYRG.

[0041] The synthesis process of the above polypeptides (SEQ.ID NO1-SEQ.ID NO10) is introduced below through Examples 1-10;

[0042] Example 1

[0043] Solid phase synthesis of IGRIWPYFGG (SEQ.ID NO.1);

[0044] (1) Swelling of resin

[0045] Weigh 50 mg of Fmoc-Rink amide-MBHA Resin (substitution amount 0.4 mmol / g), swell it with 7 mL of dichloromethane (DCM) for 30 min, filter out DCM, swell it with 10 mL of N-methylpyrrolidone (NMP) for 30 min, and finally rinse it with 7 mL of NMP and DCM respectively.

[0046] (2) Removal of Fmoc protecting group

[0047] The swollen resin was placed in a reactor, and 7 mL of a 25% piperidine / NMP (V / V) solution containing 0.1 M 1-hydroxybenzotriazole (HOBT) was added and reacted for 25 min. After the reaction was completed, the solution was filtered off and washed with NMP to obtain a resin with the initially connected Fmoc protecting group removed.

[0048] (3) Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

[0049] Fmoc-Gly-OH (0.04 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (0.04 mmol), HOBT (0.04 mmol) and N,N-diisopropylethylamine (DIPEA) (0.08 mmol) were dissolved in 10 mL of NMP. The solution was added to the resin obtained in step (2) and reacted for 45 min. After the reaction was completed, the reaction solution was filtered off and the resin was washed three times with 7 mL of DCM and 7 mL of NMP respectively.

[0050] (4) Detection of coupling efficiency

[0051] 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.

[0052] Ninhydrin method: Take a small amount of resin particles, wash them with ethanol, put them in a transparent vial, add 5% ninhydrin ethanol, KCN pyridine solution (2ml 0.001M KCN ​​diluted in 98ml pyridine), 2 drops each of 80% phenol ethanol solution, heat at 100℃ for 5 minutes, if the resin appears blue, it is positive.

[0053] 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.

[0054] (5) Peptide chain extension

[0055] According to the sequence of the polypeptide, the above steps of deprotection and coupling were repeated to sequentially connect the corresponding amino acids to obtain a resin connected with the polypeptide sequence of IGRIWPYFGG (SEQ.ID NO.1).

[0056] (6) Cleavage of peptides on resin

[0057] The resin connected with the IGRIWPYFGG (SEQ.ID NO.1) polypeptide sequence 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 reaction bottle. The mixture was shaken at 0°C for 30 min, and then reacted at room temperature for 3 h. 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 icy ether to precipitate a white flocculent precipitate, and the crude product of the target polypeptide was obtained by refrigerated centrifugation.

[0058] 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 20% to 80%, 20 min; flow rate 7 mL / min, detection wavelength 214 nm; the collected solution was lyophilized to obtain 25 mg of pure product. Its theoretical relative molecular mass is 1165.366. ESI-MS m / z: found [M+H]+=1166.

[0059] The mass spectrum of IGRIWPYFGG (SEQ.ID NO.1) is as follows Figure 1 As shown, Figure 1 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.1), respectively. It can be seen from the figure that the purity of the peptide is 96.45%, and the molecular weight is the molecular weight of the target peptide.

[0060] Example 2

[0061] Solid phase synthesis of IGKIWPYYGG (SEQ.ID NO.2)

[0062] The only difference between the synthesis steps of this example and Example 1 is that the sequence of the synthetic polypeptide is different.

[0063] The solid phase synthesis theoretical relative molecular mass of IGKIWPYYGG (SEQ.ID NO.2) is 1153.356. ESI-MS m / z: found [M+H]+=1154.

[0064] The mass spectrum of IGKIWPYYGG (SEQ.ID NO.2) is as follows Figure 2 As shown, Figure 2 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.2), respectively. It can be seen from the figure that the purity of the peptide is 96.43%, and the molecular weight is the molecular weight of the target peptide.

[0065] Example 3

[0066] Solid phase synthesis of IGRIKPYRGG (SEQ.ID NO.3)

[0067] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0068] The solid phase synthesis theoretical relative molecular mass of IGRIKPYRGG (SEQ.ID NO.3) is 1116.336. ESI-MS m / z: found [M+H]+=1117.

[0069] The mass spectrum of IGRIKPYRGG (SEQ.ID NO.3) is as follows Figure 3 As shown, Figure 3 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.3), respectively. It can be seen from the figure that the purity of the peptide is 96.57%, and the molecular weight is the molecular weight of the target peptide.

[0070] Example 4

[0071] Solid phase synthesis of IGRILPYYGG (SEQ.ID NO.4)

[0072] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0073] The solid phase synthesis theoretical relative molecular mass of IGRILPYYGG (SEQ.ID NO.4) is 1108.306. ESI-MS m / z: found [M+H]+=1109.

[0074] The mass spectrum of IGRILPYYGG (SEQ.ID NO.4) is as follows Figure 4 As shown, Figure 4 (a) and (b) are HPLC and mass spectrometry of the peptide (SEQ.ID NO.4), respectively. It can be seen from the figure that the purity of the peptide is 98.21%, and the molecular weight is the molecular weight of the target peptide.

[0075] Example 5

[0076] Solid phase synthesis of RIWPYR (SEQ.ID NO.5)

[0077] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0078] The solid phase synthesis theoretical relative molecular mass of RIWPYR (SEQ.ID NO.5) is 890.05. ESI-MS m / z: found [M+H]+=891.

[0079] The mass spectrum of RIWPYR (SEQ.ID NO.5) is as follows Figure 5 As shown, Figure 5 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.5), respectively. It can be seen from the figure that the purity of the peptide is 97.37%, and the molecular weight is the molecular weight of the target peptide.

[0080] Example 6

[0081] Solid phase synthesis of GRIWPYFG (SEQ.ID NO.6)

[0082] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0083] The theoretical relative molecular mass of GRIWPYFG (SEQ.ID NO.6) synthesized by solid phase is 995.148. ESI-MS m / z: found [M+H]+=996.

[0084] The mass spectrum of GRIWPYFG (SEQ.ID NO.6) is as follows Figure 6 As shown, Figure 6 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.6), respectively. It can be seen from the figure that the purity of the peptide is 96.01%, and the molecular weight is the molecular weight of the target peptide.

[0085] Example 7

[0086] Solid phase synthesis of IGRIRPYRGG (SEQ.ID NO.7)

[0087] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0088] The solid phase synthesis theoretical relative molecular mass of IGRIRPYRGG (SEQ.ID NO.7) is 1144.346. ESI-MS m / z: found [M+H]+=1145.

[0089] The mass spectrum of IGRIRPYRGG (SEQ.ID NO.7) is as follows Figure 7 As shown, Figure 7(a) and (b) are HPLC and mass spectrometers of the peptide (SEQ.ID NO.7), respectively. It can be seen from the figure that the purity of the peptide is 96.01%, and the molecular weight is the molecular weight of the target peptide.

[0090] Example 8

[0091] Solid phase synthesis of KIWPYY (SEQ.ID NO.8)

[0092] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0093] The solid phase synthesis theoretical relative molecular mass of KIWPYY (SEQ.ID NO.8) is 869.03. ESI-MS m / z: found [M+H]+=870.

[0094] The mass spectrum of KIWPYY (SEQ.ID NO.8) is as follows Figure 8 As shown, Figure 8 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.8), respectively. It can be seen from the figure that the purity of the peptide is 96.49%, and the molecular weight is the molecular weight of the target peptide.

[0095] Example 9

[0096] Solid phase synthesis of GLINPYNG (SEQ.ID NO.5)

[0097] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0098] The theoretical relative molecular mass of GLINPYNG (SEQ.ID NO.9) synthesized by solid phase is 846.938. ESI-MS m / z: found [M+H]+=848.

[0099] The mass spectrum of GLINPYNG (SEQ.ID NO.9) is as follows Fig. 9 As shown, Fig. 9 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.9), respectively. It can be seen from the figure that the purity of the peptide is 97.34%, and the molecular weight is the molecular weight of the target peptide.

[0100] Example 10

[0101] Solid phase synthesis of GRIWPYRG (SEQ.ID NO.10)

[0102] The difference between the synthesis steps of this embodiment and embodiment 1 is that the sequence of the synthetic polypeptide is different.

[0103] The theoretical relative molecular mass of GRIWPYRG (SEQ.ID NO.10) synthesized by solid phase is 1004.158. ESI-MS m / z: found [M+H]+=1005.

[0104] The mass spectrum of GRIWPYRG (SEQ.ID NO.10) is as follows Fig.10 As shown, Fig.10 (a) and (b) are HPLC and mass spectra of the peptide (SEQ.ID NO.10), respectively. It can be seen from the figure that the purity of the peptide is 97.09%, and the molecular weight is the molecular weight of the target peptide.

[0105] Embodiment 11

[0106] Surface plasmon resonance (SPR): The affinity constants of the amino acid sequences prepared in Examples 1-10 (SEQ.ID NO.1-10) and CLDN6 protein were detected.

[0107] The human recombinant protein CLDN6 was coupled to the CM 5 chip, and the analyte was diluted into a concentration gradient (50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.0390625, 0.1953125, 0.09965625, 0nM), and appropriate regeneration conditions (such as Glycine 2.5) were used. The flow rate was set to 30 μL / min, and the injection time of each cycle was set to 90s, and the dissociation time was also 90s. According to the Biacore instrument program flow, a multi-cycle kinetic test was performed, and finally the Biacore T200Evaluation Software was used to analyze and fit the binding-dissociation curve, and the affinity-related parameters were calculated. The results are shown in Table 1 below;

[0108] Table 1: Summary of affinity constants (KD)

[0109]

[0110]

[0111] As can be seen from Table 1, the best affinity is <10 -8 , reaching 10nM level.

[0112] Example 12

[0113] CCK-8 cell killing experiment: The toxicity of the amino acid sequences (SEQ.ID NO.1-10) prepared in Examples 1-10 to the constructed ovarian cancer stable cell line with high expression of CLDN6 at different concentrations was detected.

[0114] Select cells in logarithmic growth phase with good growth status to make cell suspension, count them, and inoculate 5*103 cells per well in a 96-well plate. Inoculate the cell suspension (100μL / well): Set up 5 duplicate wells for each group. After the cells are iron-walled overnight, add different concentrations (1uM, 10uM, 100uM) of SEQ.ID NO.1-22 polypeptides and culture for 24 hours. Add CCK-8 reagent and use an ELISA reader for detection. The detection wavelength is 450-490nm and the reference wavelength is 600-650nm. Use Graphpad to calculate the toxicity of the polypeptide, and the results are as follows: Fig.11 As shown, Fig.11 The test results of SEQ.ID NO.1-10 are shown in the figure, indicating that each peptide segment has no obvious toxicity at different concentrations.

[0115] Example 13

[0116] Specifically, the ovarian cancer cell line OV90 was transfected with lentivirus to overexpress CLDN6 protein, the above 10 peptides were linked to FITC, the cells were incubated with anti-CLDN6 protein antibodies, FITC-containing peptides and DAPI, and the internalization of the drug and co-localization with the target protein were detected using a fluorescence microscope. The results are as follows: Fig.12 As shown in (a) and (b), it reflects the immunofluorescence staining images at different time points after the polypeptide sequences in Examples 1-10 are linked to the FITC fluorescent group and co-incubated with tumor cells. It can be seen from the figure that each peptide segment is well internalized and well co-localized with the target protein.

[0117] Specifically, the above-mentioned polypeptide is applied in the drug, and the original structure of the polypeptide is used without modification or after modification through a cleavable or non-cleavable linker (the modification includes but is not limited to the following types: cyclic peptides, peptidomimetics, small molecules, use of D-type amino acids, change of single or multiple amino acids in the polypeptide, modification of both ends of the polypeptide, modification of the polypeptide by biological macromolecules, physical encapsulation of the polypeptide, combined use of multiple modifications, etc.), and the subsequent modification includes but is not limited to the following types: polypeptide-conjugated drugs, radionuclide-conjugated drugs, small molecule-conjugated drugs, nucleic acid aptamer-drug conjugates, polypeptide oligonucleotide conjugates, immunostimulatory polypeptide-conjugated drugs, virus-like drug conjugates, polypeptide-siRNA conjugates, bicyclic peptide radioactive conjugates, antibody-biopolymer conjugates, degrader-antibody conjugates, polypeptide-siRNA drugs.

[0118] In summary, the polypeptide of the present invention can effectively target CLDN6-positive cells and is non-toxic itself, and can be used as an excellent targeting peptide, and thus has the potential to be used as a drug component or drug carrier for the treatment of ovarian cancer or other tumors that highly express CLDN6 (such as endometrial tumors, prostate tumors).

[0119] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0120] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A polypeptide targeting CLDN6 protein, characterized in that: The general formula of the amino acid sequence structure is: X1IX2PYX3; wherein X1-X3 represents a sequence of 1-7 amino acid residues in length, wherein the amino acid is any one of 21 natural amino acids and non-natural amino acids.

2. A polypeptide targeting CLDN6 protein according to claim 1, characterized in that: The amino acid sequences of the polypeptides are shown in SEQ.ID NO 1 to SEQ.ID NO 10 respectively.

3. Use of a polypeptide targeting CLDN6 protein according to claim 1 or 2 in the preparation of a drug or drug carrier for treating tumors with high expression of CLDN6 protein.

4. The use according to claim 3, characterized in that: The tumors that highly express CLDN6 protein include: ovarian tumors, endometrial tumors and prostate tumors.

5. A drug for treating tumors with high expression of CLDN6 protein, characterized in that: It comprises a polypeptide targeting CLDN6 protein as claimed in claim 1 or 2.

6. A drug carrier for treating tumors with high expression of CLDN6 protein, characterized in that: The invention comprises a polypeptide targeting CLDN6 protein as claimed in claim 1 or 2.

7. A drug carrier for treating tumors with high expression of CLDN6 protein according to claim 6, characterized in that: The drug carrier is: a polypeptide is coupled with any amino acid with a charge or different hydrophilicity to prepare a polypeptide hydrogel.

8. An immunoconjugate, characterized in that Comprising the polypeptide according to claim 1 or 2.

9. An immunoconjugate according to claim 8, characterized in that: Also included are chemotherapeutic agents, radioactive atoms, cytostatic agents, cytotoxic agents, immune checkpoint inhibitors, antibodies or antibody fragments.

10. The method for preparing a polypeptide targeting CLDN6 protein according to claim 1 or 2, characterized in that: The following steps are involved: S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter out DCM, swell it with NMP for 30 min, and finally rinse it with NMP, DCM, and NMP respectively; S2, the swollen resin is placed in a reactor, and a piperidine / NMP solution containing HOBT is added to react, and after the reaction is completed, the solution is filtered off and washed with NMP; S3, dissolving Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA in NMP, and then adding the solution to the depiperidinized resin for reaction. After the reaction is completed, the reaction solution is filtered out and washed with DCM and NMP; S4, qualitatively detect the coupling efficiency of the resin using the ninhydrin method or the bromophenol blue method, and proceed to the next coupling cycle if the color reaction is negative; S5, according to the sequence of the polypeptide, repeat S2 and S3 to sequentially connect the corresponding amino acids to obtain a resin connected with the polypeptide sequence; S6, adding a cutting agent to cut the resin to obtain the polypeptide.

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