Polypeptide targeting CLDN4 protein as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide targeting CLDN4 protein as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The amino acid sequence of the polypeptide of the targeted CLDN4 protein is as follows: QSGNWPYSIW, RSGNWPYSIQ or RSGNYPYSIW. The polypeptide can specifically target CLDN4 positive cells, is high in selectivity, and can be applied to preparation of drugs or drug carriers for treating ovarian cancer; the polypeptide can be prepared by a chemical synthesis method, and is high in purity, small in molecular weight, strong in specificity, free of immunogenicity, safe and reliable.

Description

[0001] This application is a divisional application of application number 202311439433.2, entitled "A polypeptide targeting CLDN4 protein and its preparation method and application" (application date: November 1, 2023). Technical Field

[0002] This invention belongs to the field of medicinal chemistry technology, specifically relating to a polypeptide targeting CLDN4 protein, its preparation method, and its application. Background Technology

[0003] Ovarian cancer encompasses various subtypes with varying incidence and mortality rates. The most common type is epithelial ovarian cancer (EOC), accounting for over 95% of ovarian cancer cases. Based on different pathogenesis, genetic basis, and histopathological features, ovarian cancer is further divided into five distinct subtypes: serous cystadenoma, serous cystadenocarcinoma, mucinous cystadenoma, mucinous cystadenocarcinoma, and borderline tumors. Ovarian cancer is a leading cause of death from gynecological malignancies and is often diagnosed at an advanced stage, with a lack of effective screening strategies. Therefore, the urgent need for early detection and innovative treatment strategies is emphasized.

[0004] CLDN4 is highly expressed not only in most ovarian cancers but also in many other types of tumors, such as pancreatic cancer and colorectal cancer. Currently, besides surgery, radiotherapy, chemotherapy, and combination therapy with monoclonal antibodies, there are no better treatment options for ovarian cancer. Therefore, a peptide targeting the CLDN4 protein is proposed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a polypeptide targeting CLDN4 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:

[0007] A polypeptide targeting the CLDN4 protein, comprising a first binding sequence L1 or a second binding sequence L2, wherein the first binding sequence L1 is: X1PAGNLYX2WX3; and the second binding sequence L2 is: X4SGNX5PYSIX6;

[0008] Where X1-X6 represent sequences of 1-7 amino acid residues in length, and the amino acids can be any of the 20 natural amino acids, with the following restrictions: X1 has at least one amino acid that is N, Q, R, K, W or I; X2 has at least one amino acid that is D, R, W or Y; X3 has at least one amino acid that is R, Y, W or F; X4 has at least one amino acid that is Y, W, R or Q; X5 has at least one amino acid that is Y, W or F; and X6 has at least one amino acid that is L, Y, W or Q.

[0009] Further, the amino acid sequence of the polypeptide is: NPAGNLYDWR, YSGNYPYSIL, QPAGNLYRWY, RPAGNLYRWY, QPAGNLYWWW, KPAGNLYWWF, QPAGNLYYWY, WPAGNLYYWW, QPAGNLYRWF, IPAGNLYWWY, WPAGNLYYWY, RPAGNLYFWY, WSGNWPYSIY, WSGNWPYSIW, RSGNWPYSIW, RSGNFPYSIW, QSGNWPYSIW, RSGNWPYSIQ, RSGNYPYSIW, YSGNWPYSIW, WSGNWPYSIQ, or WSGNWPYSIL.

[0010] The above-mentioned peptides are used in the preparation of drugs or drug carriers for treating ovarian cancer.

[0011] A drug comprising the aforementioned polypeptide.

[0012] Furthermore, the drug is prepared by coupling a polypeptide with doxorubicin or camptothecin via a linker that can or cannot be cleaved.

[0013] A drug carrier comprising the aforementioned polypeptide.

[0014] 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.

[0015] An immune conjugate comprising the aforementioned polypeptide.

[0016] Furthermore, immune conjugates also include chemotherapeutic agents, radioactive atoms, cell growth inhibitors, cytotoxic agents, immune checkpoint inhibitors, and antibodies or antibody fragments.

[0017] A method for preparing a peptide targeting the CLDN4 protein includes the following steps:

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] S6, a cutting agent is added to perform resin cutting to obtain the polypeptide.

[0024] The beneficial effects of this invention are:

[0025] 1. The polypeptide of the present invention can specifically target CLDN4 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.

[0026] 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

[0027] 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.

[0028] Figure 1 These are mass spectra of the amino acid sequences prepared in Examples 1-22 of this invention;

[0029] Figure 2 This is a diagram showing the CCK-8 sequence determination results of the amino acid sequences prepared in Examples 1-22 of this invention; Figure 3These are immunofluorescence staining images corresponding to the polypeptide sequences in Examples 1-22 of this invention. Detailed Implementation

[0030] 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.

[0031] A polypeptide targeting the CLDN4 protein, comprising a first binding sequence L1 or a second binding sequence L2, wherein:

[0032] The first binding sequence L1 is: X1PAGNLYX2WX3;

[0033] The second binding sequence L2 is: X4SGNX5PYSIX6;

[0034] Where X1-X6 represent sequences of 1-7 amino acid residues in length, and the amino acids can be any of the 20 natural amino acids, with the following restrictions: X1 has at least one amino acid that is N, Q, R, K, W or I; X2 has at least one amino acid that is D, R, W or Y; X3 has at least one amino acid that is R, Y, W or F; X4 has at least one amino acid that is Y, W, R or Q; X5 has at least one amino acid that is Y, W or F; and X6 has at least one amino acid that is L, Y, W or Q.

[0035] The following examples 1-22 illustrate the synthesis process of each binding sequence in a polypeptide;

[0036] Example 1

[0037] Solid-phase synthesis of NPAGNLYDWR (SEQ.ID NO.1);

[0038] (1) Swelling of resin

[0039] 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, then swell in 10 mL of N-methylpyrrolidone (NMP) for 30 min, and finally rinse thoroughly with 7 mL of NMP and 7 mL of DCM respectively.

[0040] (2) Removal of Fmoc protecting groups

[0041] 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.

[0042] (3) Synthesis of Fmoc-Asn(Pbf)-Rink amide-MBHA Resin

[0043] Fmoc-AsnPbf)-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 carried out 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.

[0044] (4) Detection of coupling efficiency

[0045] 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.

[0046] 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 0.001 M KCN ​​diluted in 98 ml pyridine), and 80% phenol ethanol solution, heat at 100℃ for 5 minutes. If the resin turns blue, it is positive.

[0047] 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.

[0048] (5) Elongation of peptide chains

[0049] 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 NPAGNLYDWR (SEQ.ID NO.1) polypeptide sequence.

[0050] (6) Cleavage of peptides on resin

[0051] The resin containing the NPAGNLYDWR (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℃ 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 ether to precipitate a white flocculent precipitate, which was then frozen and centrifuged to obtain the crude target polypeptide.

[0052] 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 1205.2950. ESI-MS m / z: found [M+H]+=1206, 1 / 2 [M+2H]2+=604.

[0053] Example 2

[0054] Solid-phase synthesis of YSGNYPYSIL (SEQ.ID NO.2)

[0055] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0056] The theoretical relative molecular mass of YSGNYPYSIL (SEQ.ID NO.2) for solid-phase synthesis is 1176.276. ESI-MS m / z:found [M+H]+=1177, 1 / 2[M+2H]2+=589.

[0057] Example 3

[0058] Solid-phase synthesis of QPAGNLYRWY (SEQ.ID NO.3)

[0059] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0060] The theoretical relative molecular mass of QPAGNLYRWY (SEQ.ID NO.3) for solid-phase synthesis is 1267.396. ESI-MS m / z:found [M+H]+=1268, 1 / 2[M+2H]2+=635.

[0061] Example 4

[0062] Solid-phase synthesis of RPAGNLYRWY (SEQ.ID NO.4)

[0063] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0064] The theoretical relative molecular mass of RPAGNLYRWY (SEQ.ID NO.4) for solid-phase synthesis is 1295.446. ESI-MS m / z:found [M+H]+=1296, 1 / 2[M+2H]2+=649.

[0065] Example 5

[0066] Solid-phase synthesis of QPAGNLYWWW (SEQ.ID NO.5)

[0067] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0068] The theoretical relative molecular mass of QPAGNLYWWW (SEQ.ID NO.5) for solid-phase synthesis is 1320.466. ESI-MS m / z:found [M+H]+=1321, 1 / 2[M+2H]2+=661.

[0069] Example 6

[0070] Solid-phase synthesis of KPAGNLYWWF (SEQ.ID NO.6)

[0071] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0072] The theoretical relative molecular mass of KPAGNLYWWF (SEQ.ID NO.6) for solid-phase synthesis is 1281.466. ESI-MS m / z:found [M+H]+=1282, 1 / 2[M+2H]2+=642.

[0073] Example 7

[0074] Solid-phase synthesis of QPAGNLYYWY (SEQ.ID NO.7)

[0075] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0076] The theoretical relative molecular mass of QPAGNLYYWY (SEQ.ID NO.7) for solid-phase synthesis is 1274.386. ESI-MS m / z:found [M+H]+=1275, 1 / 2[M+2H]2+=638.

[0077] Example 8

[0078] Solid-phase synthesis of WPAGNLYYWW (SEQ.ID NO.8)

[0079] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0080] The theoretical relative molecular mass of WPAGNLYYWW (SEQ.ID NO.8) for solid-phase synthesis is 1355.506. ESI-MS m / z:found [M+H]+=1357, 1 / 2[M+H]2+=679.

[0081] Example 9

[0082] Solid-phase synthesis of QPAGNLYRWF (SEQ.ID NO.5)

[0083] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0084] The theoretical relative molecular mass of QPAGNLYRWF (SEQ.ID NO.9) for solid-phase synthesis is 1251.396. ESI-MS m / z:found [M+H]+=1252, 1 / 2[M+2H]2+=627.

[0085] Example 10

[0086] Solid-phase synthesis of IPAGNLYWWY (SEQ.ID NO.10)

[0087] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0088] The theoretical relative molecular mass of IPAGNLYWWY (SEQ.ID NO.10) for solid-phase synthesis is 1282.456. ESI-MS m / z:found [M+H]+=1283, 1 / 2[M+2H]2+=642.

[0089] Example 11

[0090] Solid-phase synthesis of WPAGNLYYWY (SEQ.ID NO.11)

[0091] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0092] The theoretical relative molecular mass of WPAGNLYYWY (SEQ.ID NO.11) for solid-phase synthesis is 1332.466. ESI-MS m / z:found [M+H]+=1333, 1 / 2[M+2H]2+=667.

[0093] Example 12

[0094] Solid-phase synthesis of RPAGNLYFWY (SEQ.ID NO.12)

[0095] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0096] The theoretical relative molecular mass of RPAGNLYFWY (SEQ.ID NO.12) for solid-phase synthesis is 1286.436. ESI-MS m / z:found [M+H]+=1287, 1 / 2[M+H]2+=644.

[0097] Example 13

[0098] Solid-phase synthesis of WSGNWPYSIY (SEQ.ID NO.13)

[0099] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0100] The theoretical relative molecular mass of WSGNWPYSIY (SEQ.ID NO.13) for solid-phase synthesis is 1272.376. ESI-MS m / z:found [M+H]+=1273, 1 / 2[M+2H]2+=637.

[0101] Example 14

[0102] Solid-phase synthesis of WSGNWPYSIW (SEQ.ID NO.14)

[0103] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0104] The theoretical relative molecular mass of WSGNWPYSIW (SEQ.ID NO.14) for solid-state synthesis is 1295.416. ESI-MS m / z:found [M+H]+=1296, 1 / 2[M+2H]2+=649.

[0105] Example 15

[0106] Solid-phase synthesis of RSGNWPYSIW (SEQ.ID NO.15)

[0107] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0108] The theoretical relative molecular mass of RSGNWPYSIW (SEQ.ID NO.15) for solid-phase synthesis is 1265.386. ESI-MS m / z:found [M+H]+=1266, 1 / 2[M+2H]2+=634.

[0109] Example 16

[0110] Solid-phase synthesis of RSGNFPYSIW (SEQ.ID NO.16)

[0111] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0112] The theoretical relative molecular mass of RSGNFPYSIW (SEQ.ID NO.16) for solid-phase synthesis is 1226.346. ESI-MS m / z:found [M+H]+=1227, 1 / 2[M+H]2+=614.

[0113] Example 17

[0114] Solid-phase synthesis of QSGNWPYSIW (SEQ.ID NO.17)

[0115] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0116] The theoretical relative molecular mass of QSGNWPYSIW (SEQ.ID NO.17) for solid-phase synthesis is 1237.336. ESI-MS m / z:found [M+H]+=1238, 1 / 2[M+2H]2+=620.

[0117] Example 18

[0118] Solid-phase synthesis of RSGNWPYSIQ (SEQ.ID NO.18)

[0119] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0120] The theoretical relative molecular mass of RSGNWPYSIQ (SEQ.ID NO.18) for solid-phase synthesis is 1207.306. ESI-MS m / z:found [M+H]+=1208, 1 / 2[M+2H]2+=605.

[0121] Example 19

[0122] Solid-phase synthesis of RSGNYPYSIW (SEQ.ID NO.19)

[0123] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0124] The theoretical relative molecular mass of RSGNYPYSIW (SEQ.ID NO.19) for solid-phase synthesis is 1242.346. ESI-MS m / z:found [M+H]+=1243, 1 / 2[M+2H]2+=622.

[0125] Example 20

[0126] Solid-phase synthesis of YSGNWPYSIW (SEQ.ID NO.20)

[0127] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0128] The theoretical relative molecular mass of YSGNWPYSIW (SEQ.ID NO.20) for solid-phase synthesis is 1272.376. ESI-MS m / z:found [M+H]+=1273, 1 / 2[M+H]2+=637.

[0129] Example 21

[0130] Solid-phase synthesis of WSGNWPYSIQ (SEQ.ID NO.21)

[0131] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0132] The theoretical relative molecular mass of WSGNWPYSIQ (SEQ.ID NO.21) for solid-phase synthesis is 1237.336. ESI-MS m / z:found [M+H]+=1238, 1 / 2[M+2H]2+=620.

[0133] Example 22

[0134] Solid-state synthesis of WSGNWPYSIL (SEQ.ID NO.22)

[0135] The difference between the synthesis steps and those in Example 1 is that the sequence of the synthesized polypeptide is different.

[0136] The theoretical relative molecular mass of WSGNWPYSIL (SEQ.ID NO.22) for solid-phase synthesis is 1222.356. ESI-MS m / z:found [M+H]+=1223, 1 / 2[M+H]2+=612.

[0137] Among them, the mass spectra of Examples 1-22 are as follows Figure 1 As shown, Figure 1 (a)-(v) are the mass spectra corresponding to the amino acid sequences in Examples 1-22, respectively. As can be seen from the figures, each synthesized main product is the target peptide.

[0138] Example 23

[0139] Surface plasmon resonance (SPR): The affinity constants of the amino acid sequences (SEQ.ID NO.1-22) prepared in Examples 1-22 with the CLDN4 protein were detected.

[0140] Human recombinant protein CLDN4 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 (e.g., Glycine 2.5) were used, with a flow rate of 30 μL / min and an injection time and dissociation time of 90 s per cycle. Following the Biacore instrument's protocol, 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. The optimal affinity reached <10-1. -8 It reaches the 10nm level.

[0141] Table 1: Summary of Affinity Constants (Kd)

[0142]

[0143] Example 24

[0144] CCK-8 cell killing assay: The toxicity of the amino acid sequences (SEQ.ID NO.1-22) prepared in Examples 1-22 to the constructed CLDN4-overexpressing ovarian cancer stable cell lines was detected at different concentrations.

[0145] Cells in good logarithmic growth phase were selected and cultured to prepare a cell suspension. Cells were counted, and 5*10³ cells were seeded per well in a 96-well plate (100 μL / well). Five replicates were set up per group. After overnight incubation with iron walls, different concentrations (1 μM, 10 μM, 100 μM) of the SEQ.ID NO.1—22 peptide were added, and the cells were cultured for 24 hours. 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 2 As shown in (a) and (b), Figure 2 (a) and (b) in the figure show the test results of SEQ.ID NO.1-11 and SEQ.ID NO.12-22, respectively, showing that none of the peptides have obvious toxicity.

[0146] Example 25

[0147] Specifically, the ovarian cancer cell line A2780 was transfected with lentivirus to overexpress the CLDN4 protein. The aforementioned 22 peptides were then linked to FITC. Cells were incubated with an anti-CLDN4 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 3 As shown in (a)-(f), the immunofluorescence staining patterns corresponding to the polypeptide sequences in Examples 1-22 are reflected. It can be seen from the figures that each peptide is well internalized and well co-localized with the target protein.

[0148] Specifically, the above-mentioned polypeptides are applied to drugs, wherein the drug components are: by using a linker that can or cannot be cleaved, any one of the above-mentioned polypeptides is coupled with various small molecule chemotherapy drugs, including but not limited to: doxorubicin, camptothecin, etc., to produce PDC drugs.

[0149] The polypeptide is connected to a "bullet" that can form a covalent bond. The connection method can be to insert the "bullet" at the NH2 end or COOH end of the polypeptide, or to mutate any one or more amino acids in the polypeptide into a "bullet". The "bullet" includes, but is not limited to, at least one or more of the following: cysteine, methionine, lysine, histidine, serine, threonine, tyrosine, modified non-natural amino acids, etc.

[0150] The peptide is coupled with any amino acid with a special charge or different hydrophilicity or hydrophobicity, including natural and non-natural amino acids, to form an environmentally responsive peptide hydrogel, which encapsulates chemotherapy drugs to kill tumors.

[0151] An immune conjugate, comprising a polypeptide, binds the polypeptide to an immune conjugate component, the immune conjugate component generally including: a chemotherapeutic agent, a radioactive atom, a cell growth inhibitor and a cytotoxic agent, an immune checkpoint inhibitor, an antibody or antibody fragment. Radioactive agents include, but are not limited to: alpha emitters, beta emitters, gamma emitters; cytotoxins include, but are not limited to: doxorubicin, kazimycin, anthracycline, etc.; immune checkpoint inhibitors include, but are not limited to: inhibitors of immune checkpoints such as CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, G1TR, B7H4, KIR, A2aR, CD27, CD70, DR3, and ICOS, etc.; antibodies or antibody fragments include, but are not limited to: antibodies or fragments of related proteins such as CD3, CD28, LFA-1, LFA-2, CD40L, etc.

[0152] In summary, the polypeptide of the present invention can effectively target CLDN4-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 CLDN4.

[0153] 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.

[0154] 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 CLDN4 protein, characterized in that, The amino acid sequence of the polypeptide is: QSGNWPYSIW, RSGNWPYSIQ, or RSGNYPYSIW.

2. The use of the polypeptide of claim 1 in the preparation of a drug or drug carrier for treating ovarian cancer.

3. A drug, characterized in that, Includes the polypeptide described in claim 1.

4. The drug according to claim 3, characterized in that, The drug is prepared by coupling a polypeptide with doxorubicin or camptothecin via a linker that can or cannot be cleaved.

5. A drug carrier, characterized in that, Includes the polypeptide described in claim 1.

6. The drug carrier according to claim 5, 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.

7. An immune conjugate, characterized in that, Includes the polypeptide described in claim 1.

8. The immune conjugate according to claim 7, characterized in that, It also includes chemotherapeutic agents, radioactive atoms, cell growth inhibitors, cytotoxic agents, immune checkpoint inhibitors, and antibodies or antibody fragments.

9. The method for preparing a polypeptide targeting CLDN4 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.

Images