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 amino acid sequence of the polypeptide is as shown in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7. 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

[0001] This application is a divisional application of application number 202510186223.X, entitled "A polypeptide targeting CLDN6 protein and its preparation method and application" (application date: 2025-02-20). Technical Field

[0002] This invention belongs to the field of medicinal chemistry technology, specifically relating to a polypeptide targeting CLDN6 protein, its preparation method, and its application. Background Technology

[0003] 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 and are considered to play a crucial role in barrier function. Recently, CLDN6 has also been associated with several key intracellular signaling pathways, including the YAP1-snail1 axis and the ASK1-p38 / JNK MAPK secretion signaling pathway. 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 believed to contribute to the tumorigenic potential of cultures containing human pluripotent stem cells. Although CLDN6 is widely expressed in cells and tissues during early embryonic and fetal development, in adults, CLDN6 expression is primarily confined to malignant tissues, and it is associated with the occurrence, progression, and metastasis of certain cancers. It has been reported to be abnormally expressed in ovarian cancer, gastric cancer, lung cancer, endometrial cancer, and cervical cancer.

[0004] Furthermore, in patients with ovarian and endometrial cancer, high levels of CLDN6 expression in malignant tissues are independent prognostic markers for poor progression-free survival and overall survival. The combined data indicating higher expression levels in adult cancers compared to normal tissues makes CLDN6 an attractive target for developing peptide-based therapies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a polypeptide targeting CLDN6 protein, its preparation method, and its application, thereby solving the problems in the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions: A polypeptide targeting the CLDN6 protein has the general formula of amino acid sequence structure: X1IX2PYX3; where X1-X3 represent sequences of 1-7 amino acid residues in length, and the amino acids are any one of 21 natural amino acids and non-natural amino acids.

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

[0008] The above-mentioned peptide targeting CLDN6 protein is used in the preparation of tumor drugs or drug carriers that treat 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 tumor treatment drug that highly expresses CLDN6 protein includes the aforementioned polypeptide that targets CLDN6 protein.

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

[0012] Furthermore, the drug carrier is a polypeptide coupled with any amino acid that is charged or has different hydrophilicity or hydrophobicity to form a polypeptide hydrogel.

[0013] Furthermore, the drug carrier comprises nanoparticles, and the polypeptide is coupled to the surface of the nanoparticles.

[0014] An immune conjugate comprising the aforementioned polypeptide.

[0015] Furthermore, this also includes chemotherapy agents.

[0016] Furthermore, the chemotherapeutic agent includes: radioactive atoms, cell growth inhibitors, cytotoxic agents, immune checkpoint inhibitors, antibodies or antibody fragments.

[0017] The above-mentioned method for preparing a polypeptide targeting CLDN6 protein includes the following steps: S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter off the DCM, swell it with NMP for 30 min, and finally wash it with NMP, DCM and NMP respectively. S2, the swollen resin is placed in the reactor, and a piperidine / NMP solution containing HOBT is added to react. After the reaction is completed, the solution is filtered off and washed with NMP. S3, Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA are dissolved in NMP, and this solution is added to the depiperidine-treated resin for reaction. After the reaction is completed, the reaction solution is filtered off and washed with DCM and NMP. S4. The coupling efficiency of the resin is qualitatively tested using the ninhydrin method or the bromophenol blue method. If the color reaction is negative, the next coupling cycle can begin. S5, following the sequence of the polypeptide, repeat S2 and S3 to sequentially attach the corresponding amino acids to obtain a resin with the polypeptide sequence attached. S6, a cutting agent is added to perform resin cutting to obtain the polypeptide.

[0018] The beneficial effects of this invention are: 1. The polypeptide of the present invention can specifically target CLDN6 positive cells with high selectivity. Furthermore, 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.

[0019] 2. The polypeptides of the present invention can be used as drug delivery carriers or coupled with known components to form drug combinations, thereby achieving better therapeutic effects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a mass spectrum of the amino acid sequence prepared in Example 1 of this invention; Figure 2 This is the mass spectrum of the amino acid sequence prepared in Example 2 of this invention; Figure 3 This is the mass spectrum of the amino acid sequence prepared in Example 3 of this invention; Figure 4 This is the mass spectrum of the amino acid sequence prepared in Example 4 of this invention; Figure 5 This is the mass spectrum of the amino acid sequence prepared in Example 5 of this invention; Figure 6 This is the mass spectrum of the amino acid sequence prepared in Example 6 of this invention; Figure 7 This is the mass spectrum of the amino acid sequence prepared in Example 7 of this invention; Figure 8 This is the mass spectrum of the amino acid sequence prepared in Example 8 of this invention; Figure 9 This is the mass spectrum of the amino acid sequence prepared in Example 9 of this invention; Figure 10 This is the mass spectrum of the amino acid sequence prepared in Example 10 of this invention; Figure 11 This is a diagram showing the CCK-8 assay results of the amino acid sequences prepared in Examples 1-10 of this invention; Figure 12These are immunofluorescence staining images of cells after being incubated with FITC fluorescent groups following examples 1-10 of this invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] A polypeptide targeting the CLDN6 protein has the general formula of amino acid sequence structure: X1IX2PYX3; where X1-X3 represent sequences of 1-7 amino acid residues in length, and the amino acids are any one of 21 natural amino acids and non-natural amino acids.

[0024] The amino acid sequences of the polypeptides are shown in SEQ.ID NO1-SEQ.ID NO10, respectively; specifically: IGRIPYFGG, IGKIWPYYGG, IGRIPYRGG, IGRIPYYGG, RIWPYR, GRIWPYFG, IGRIPYRGG, KIWPYY, GLINPYNG, GRIWPYRG.

[0025] The synthesis process of the above-mentioned polypeptides (SEQ.ID NO1-SEQ.ID NO10) will be described below through Examples 1-10; Example 1 Solid-phase synthesis of IGRIWPYFGG (SEQ.ID NO.1); (1) Swelling of resin Weigh 50 mg of Fmoc-Rink amide-MBHA Resin (substitution amount 0.4 mmol / g), swell in 7 mL of dichloromethane (DCM) for 30 min, filter to remove DCM, swell in 10 mL of N-methylpyrrolidone (NMP) for 30 min, and finally rinse with 7 mL of NMP and 7 mL of DCM respectively.

[0026] (2) Removal of Fmoc protecting groups 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. The reaction was carried out for 25 min. After the reaction was completed, the solution was filtered off and the resin was washed with NMP to obtain the resin with the initially attached Fmoc protecting group removed.

[0027] (3) Synthesis of Fmoc-Gly-Rink amide-MBHA Resin Fmoc-Gly-OH (0.04 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) (0.04 mmol), HOBT (0.04 mmol) and N,N-diisopropylethylamine (DIPEA) (0.08 mmol) were dissolved in 10 mL of NMP. This solution was then added to the resin obtained in step (2). The reaction was allowed to proceed 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.

[0028] (4) Detection of coupling efficiency The coupling efficiency of the resin can be qualitatively tested using the ninhydrin method or the bromophenol blue method. If the colorimetric reaction is negative, the next coupling cycle can begin.

[0029] 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 (2 ml of 0.001M KCN ​​diluted in 98 ml of pyridine), and 80% phenol ethanol solution, heat at 100℃ for 5 minutes. If the resin turns blue, it is positive.

[0030] Bromophenol blue method: Take a small amount of resin particles, wash them with dimethylacetamide, put them in a transparent bottle, add 3 drops of 1% bromophenol blue dimethylacetamide solution, shake for 3 minutes at room temperature, and if the resin turns blue, it is positive.

[0031] (5) Elongation of peptide chains Following the polypeptide sequence, the above deprotection and coupling steps were repeated to sequentially link the corresponding amino acids, resulting in a resin linked with the IGRIPYFGG (SEQ. ID NO. 1) polypeptide sequence.

[0032] (6) Cleavage of peptides on resin The resin containing the IGIWPYFGG (SEQ. ID NO. 1) polypeptide sequence obtained above was placed in a reaction flask, and 10 mL of Reagent K (TFA / anisole / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added to each flask. The mixture was first 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 filtrates were combined. The filtrate was added to a large amount of ice-cold diethyl ether to precipitate a white flocculent precipitate, which was then frozen and centrifuged to obtain the crude target polypeptide.

[0033] The crude polypeptide was dissolved in 2 mL of water and directly purified by preparative liquid chromatography (HPLC). The chromatographic conditions were: C18 reversed-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%–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.

[0034] The mass spectrum of IGRIWPYFGG (SEQ.ID NO.1) is as follows: Figure 1 As shown, Figure 1 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.1), respectively. As can be seen from the figure, the purity of the peptide is 96.45%, and the molecular weight is the molecular weight of the target peptide.

[0035] Example 2 Solid-phase synthesis of IGKIWPYYGG (SEQ. ID NO. 2) The only difference between this embodiment and Example 1 is the synthesis steps: the sequence of the synthesized polypeptide is different.

[0036] The theoretical relative molecular mass of IGKIWPYYGG (SEQ. ID NO. 2) for solid-phase synthesis is 1153.356. ESI-MS m / z: found [M+H]+= 1154.

[0037] The mass spectrum of IGKIWPYYGG (SEQ. ID NO. 2) is shown below. Figure 2 As shown, Figure 2 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.2), respectively. As can be seen from the figure, the purity of the peptide is 96.43%, and the molecular weight is the molecular weight of the target peptide.

[0038] Example 3 Solid-phase synthesis of IGRIKPYRGG (SEQ. ID NO. 3) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0039] The theoretical relative molecular mass of IGRIKPYRGG (SEQ. ID NO. 3) for solid-state synthesis is 1116.336. ESI-MS m / z: found [M+H]+= 1117.

[0040] The mass spectrum of IGRIKPYRGG (SEQ. ID NO. 3) is as follows: Figure 3 As shown, Figure 3 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.3), respectively. As can be seen from the figure, the purity of the peptide is 96.57%, and the molecular weight is the molecular weight of the target peptide.

[0041] Example 4 Solid-phase synthesis of IGRILPYYGG (SEQ. ID NO. 4) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0042] The theoretical relative molecular mass of IGRILPYYGG (SEQ. ID NO. 4) for solid-phase synthesis is 1108.306. ESI-MS m / z: found [M+H]+= 1109.

[0043] The mass spectrum of IGRILPYYGG (SEQ. ID NO. 4) is as follows: Figure 4 As shown, Figure 4 (a) and (b) are the HPLC and mass spectra of the peptide (SEQ.ID NO.4), respectively. As can be seen from the figures, the purity of the peptide is 98.21%, and the molecular weight is the molecular weight of the target peptide.

[0044] Example 5 Solid-phase synthesis of RIWPYR (SEQ. ID NO. 5) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0045] The theoretical relative molecular mass of RIWPYR (SEQ. ID NO. 5) for solid-phase synthesis is 890.05. ESI-MS m / z:found [M+H]+= 891.

[0046] The mass spectrum of RIWPYR (SEQ. ID NO. 5) is as follows: Figure 5 As shown, Figure 5 (a) and (b) are the HPLC and mass spectra of the peptide (SEQ.ID NO.5), respectively. As can be seen from the figures, the purity of the peptide is 97.37%, and the molecular weight is the molecular weight of the target peptide.

[0047] Example 6 Solid-phase synthesis of GRIWPYFG (SEQ. ID NO. 6) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0048] The theoretical relative molecular mass of GRIWPYFG (SEQ. ID NO. 6) obtained through solid-phase synthesis is 995.148. ESI-MS m / z: found [M+H]+ = 996.

[0049] The mass spectrum of GRIWPYFG (SEQ. ID NO. 6) is as follows: Figure 6 As shown, Figure 6 (a) and (b) are the HPLC and mass spectra of the peptide (SEQ.ID NO.6), respectively. As can be seen from the figures, the purity of the peptide is 96.01%, and the molecular weight is the molecular weight of the target peptide.

[0050] Example 7 Solid-phase synthesis of IGRIRPYRGG (SEQ. ID NO.7) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0051] The theoretical relative molecular mass of IGRIRPYRGG (SEQ. ID NO.7) for solid-phase synthesis is 1144.346. ESI-MS m / z: found [M+H]+= 1145.

[0052] The mass spectrum of IGRIRPYRGG (SEQ. ID NO.7) is as follows: Figure 7 As shown, Figure 7 (a) and (b) are the HPLC and mass spectra of the peptide (SEQ.ID NO.7), respectively. As can be seen from the figures, the purity of the peptide is 96.01%, and the molecular weight is the molecular weight of the target peptide.

[0053] Example 8 Solid-phase synthesis of KIWPYY (SEQ. ID NO. 8) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0054] The theoretical relative molecular mass of KIWPYY (SEQ. ID NO. 8) for solid-phase synthesis is 869.03. ESI-MS m / z: found [M+H]+ = 870.

[0055] The mass spectrum of KIWPYY (SEQ. ID NO. 8) is as follows: Figure 8 As shown, Figure 8 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.8), respectively. As can be seen from the figure, the purity of the peptide is 96.49%, and the molecular weight is the molecular weight of the target peptide.

[0056] Example 9 Solid-phase synthesis of GLINPYNG (SEQ. ID NO. 5) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0057] The theoretical relative molecular mass of GLINPYNG (SEQ. ID NO. 9) for solid-phase synthesis is 846.938. ESI-MS m / z: found [M+H]+= 848.

[0058] The mass spectrum of GLINPYNG (SEQ. ID NO. 9) is as follows: Figure 9 As shown, Figure 9 (a) and (b) are the HPLC and mass spectra of the peptide (SEQ.ID NO.9), respectively. As can be seen from the figures, the purity of the polypeptide is 97.34%, and the molecular weight is the molecular weight of the target polypeptide.

[0059] Example 10 Solid-phase synthesis of GRIWPYRG (SEQ. ID NO. 10) The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0060] The theoretical relative molecular mass of GRIWPYRG (SEQ. ID NO. 10) for solid-phase synthesis is 1004.158. ESI-MS m / z: found [M+H]+= 1005.

[0061] The mass spectrum of GRIWPYRG (SEQ. ID NO. 10) is as follows: Figure 10 As shown, Figure 10 (a) and (b) are the HPLC and mass spectra of the peptide (SEQ.ID NO.10), respectively. As can be seen from the figures, the purity of the polypeptide is 97.09%, and the molecular weight is the molecular weight of the target polypeptide.

[0062] Example 11 Surface plasmon resonance (SPR): The affinity constants of the amino acid sequences (SEQ. ID NO. 1-10) prepared in Examples 1-10 with the CLDN6 protein were detected.

[0063] Human recombinant protein CLDN6 was coupled to a CM 5 chip. The analyte was diluted to a concentration gradient (50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.0390625, 0.1953125, 0.09965625, 0 nM). Appropriate regeneration conditions (such as Glycine 2.5) were used, with a flow rate of 30 μL / min, an injection time of 90 s per cycle, and a dissociation time of 90 s. Following the Biacore instrument's workflow, multi-cycle kinetic tests were performed. Finally, the binding-dissociation curves were analyzed using Biacore T200 Evaluation Software, and affinity-related parameters were calculated. The results are shown in Table 1 below. As can be seen from Table 1, the optimal affinity is <10. -8 It reaches the 10nm level.

[0064] Example 12 CCK-8 cell killing assay: The toxicity of the amino acid sequences (SEQ.ID NO.1-10) prepared in Examples 1-10 to the constructed CLDN6-overexpressing ovarian cancer stable cell lines at different concentrations was detected.

[0065] Select healthy cells in the logarithmic growth phase to prepare a cell suspension, count the cells, and seed 5 cells per well. 103. Cell suspension was seeded in 96-well plates (100 μL / well): 5 replicates per group. After overnight incubation with iron walls, different concentrations (1 μM, 10 μM, 100 μM) of SEQ.ID NO.1—22 peptide were added, followed by 24 hours of incubation. CCK-8 reagent was then added, and the cells were detected using a microplate reader at a wavelength of 450-490 nm and a reference wavelength of 600-650 nm. The toxicity of the peptide was calculated using Graphpad, and the results are shown below. Figure 11 As shown, Figure 11 The results of the tests for SEQ.ID NO.1-10 are shown, indicating that there is no obvious toxicity at different concentrations of each peptide.

[0066] Example 13 Specifically, the ovarian cancer cell line OV90 was transfected with lentivirus to overexpress the CLDN6 protein. The aforementioned 10 peptides were linked to FITC. Cells were then incubated with an anti-CLDN6 protein antibody, FITC-conjugated peptides, and DAPI, respectively. Fluorescence microscopy was used to detect drug internalization and co-localization with the target protein. The results are as follows: Figure 12As shown in (a) and (b) in Examples 1-10, the immunofluorescence staining images at different time points after the polypeptide sequences in Examples 1-10 were linked to FITC fluorescent groups and co-incubated with tumor cells show that each peptide segment was well internalized and well co-localized with the target protein.

[0067] Specifically, the above-mentioned peptides are applied to drugs through linkers that can or cannot be cleaved, allowing for the application of the peptide's original structure without modification or after modification (modification includes, but is not limited to, the following types: cyclic peptides, peptide-like peptides, small molecules, use of D-type amino acids, alteration of one or more amino acids in the peptide, modification of both ends of the peptide, modification of the peptide by biomacromolecules, physical encapsulation of the peptide, combined use of multiple modifications, etc.). The modifications for subsequent application include, but are not limited to, the following types: peptide-conjugated drugs, radionuclide-conjugated drugs, small molecule-conjugated drugs, nucleic acid aptamer drug conjugates, peptide oligonucleotide conjugates, immunostimulatory peptide-conjugated drugs, virus-like drug conjugates, peptide-siRNA conjugates, bicyclic peptide radioconjugated drugs, antibody biopolymer conjugates, degradation agent-antibody conjugates, and peptide-siRNA drugs.

[0068] In summary, the polypeptide of the present invention can effectively target CLDN6-positive cells and is non-toxic, making it an excellent targeting peptide. Therefore, it 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 and prostate tumors).

[0069] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0070] The foregoing has shown and described 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. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A polypeptide targeting the CLDN6 protein, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.

7.

2. The use of the polypeptide targeting CLDN6 protein as described in claim 1 in the preparation of a drug carrier for treating ovarian tumors.

3. A drug, characterized in that, Includes a polypeptide targeting the CLDN6 protein as described in claim 1.

4. A drug carrier, characterized in that, Includes a polypeptide targeting the CLDN6 protein as described in claim 1.

5. A drug carrier according to claim 4, characterized in that, The drug carrier is a polypeptide hydrogel formed by coupling a polypeptide with any amino acid that is charged or has different hydrophilicity or hydrophobicity.

6. A drug carrier according to claim 4, characterized in that, The drug carrier comprises nanoparticles, and the polypeptide is coupled to the surface of the nanoparticles.

7. An immune conjugate, characterized in that, Includes a polypeptide targeting the CLDN6 protein as described in claim 1.

8. An immune conjugate according to claim 7, characterized in that, It also includes chemotherapy agents.

9. An immune conjugate according to claim 8, characterized in that, The chemotherapeutic agents include: radioactive atoms, cell growth inhibitors, cytotoxic agents, immune checkpoint inhibitors, antibodies or antibody fragments.

10. The method for preparing a polypeptide targeting CLDN6 protein according to claim 1, characterized in that, Includes the following steps: S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter off the DCM, swell it with NMP for 30 min, and finally wash it with NMP, DCM and NMP respectively. S2, the swollen resin is placed in the reactor, and a piperidine / NMP solution containing HOBT is added to react. After the reaction is completed, the solution is filtered off and washed with NMP. S3, Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA are dissolved in NMP, and this solution is added to the depiperidine-treated resin for reaction. After the reaction is completed, the reaction solution is filtered off and washed with DCM and NMP. S4. The coupling efficiency of the resin is qualitatively tested using the ninhydrin method or the bromophenol blue method. If the color reaction is negative, the next coupling cycle can begin. S5, following the sequence of the polypeptide, repeat S2 and S3 to sequentially attach the corresponding amino acids to obtain a resin with the polypeptide sequence attached. S6, a cutting agent is added to perform resin cutting to obtain the polypeptide.

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