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: YSGNYPYSIL, QPALNLYRWY or RPALNLYRWY. 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 the application number 202311439433.2, the title of which is a polypeptide targeting CLDN4 protein and its preparation method and application (the application date is November 1, 2023). TECHNICAL FIELD

[0002] The present application belongs to the technical field of pharmaceutical chemistry, and specifically relates to a polypeptide targeting CLDN4 protein and its preparation method and application. BACKGROUND

[0003] Ovarian cancer includes various subtypes with different morbidity and mortality. The most common type is epithelial ovarian cancer (EOC), which accounts for more than 95% of ovarian cancer cases. According to different pathogenesis, genetic basis and histopathological characteristics, ovarian cancer is further divided into 5 different subtypes: serous cystadenoma, serous cystadenocarcinoma, mucinous cystadenoma, mucinous cystadenocarcinoma and borderline tumor. Ovarian cancer is one of the main causes of gynecological malignant tumor death, and is usually diagnosed in the late stage, and there is currently a lack of effective screening strategies. It is worrying that ovarian cancer takes away about 185,000 lives worldwide each year. Therefore, the urgent need for early detection and innovative treatment strategies is emphasized.

[0004] CLDN4 is not only highly expressed in most ovarian cancers, but also in other types of tumors such as pancreatic cancer, intestinal cancer, etc. Currently, in addition to surgery, radiotherapy and chemotherapy, and combined monoclonal antibody treatment, there is no better treatment for ovarian cancer. Therefore, a polypeptide targeting CLDN4 protein is proposed. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a polypeptide targeting CLDN4 protein and its preparation method and application, which solves the problems in the prior art.

[0006] The purpose of the present application can be achieved by the following technical solutions:

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

[0008] wherein X1-X6 represents a sequence of 1-7 amino acid residues in length, wherein the amino acids can be any of the 20 natural amino acids, with the proviso that: at least one of the amino acids in X1 is N, Q, R, K, W or I; at least one of the amino acids in X2 is D, R, W, or Y; at least one of the amino acids in X3 is R, Y, W or F; at least one of the amino acids in X4 is Y, W, R or Q; at least one of the amino acids in X5 is Y, W or F; and at least one of the amino acids in X6 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] Use of the above polypeptide in the preparation of a medicament or a drug carrier for treating ovarian cancer.

[0011] A medicament comprising the above polypeptide.

[0012] Further, the medicament is a PDC drug prepared by coupling the polypeptide with doxorubicin or camptothecin via a cleavable or non-cleavable linker.

[0013] A drug carrier comprising the above polypeptide.

[0014] Further, the drug carrier is a polypeptide hydrogel prepared by coupling the polypeptide with any amino acid with a charge or different hydrophilicity / hydrophobicity.

[0015] An immunoconjugate comprising the above polypeptide.

[0016] Further, the immunoconjugate further comprises a chemotherapeutic agent, a radioactive atom, a cell growth inhibitor, a cytotoxic agent, an immune checkpoint inhibitor, and an antibody or an antibody fragment.

[0017] A method for preparing a polypeptide targeting CLDN4 protein, comprising 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 3are immunofluorescence staining diagrams corresponding to the polypeptide sequences in embodiments 1-22 of the present application. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0031] A polypeptide targeting 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] wherein X1-X6 represents a sequence of 1-7 amino acid residues in length, and the amino acids therein can be any one of 20 natural amino acids, with the limitation that: at least one amino acid in X1 is N, Q, R, K, W or I; at least one amino acid in X2 is D, R, W or Y; at least one amino acid in X3 is R, Y, W or F; at least one amino acid in X4 is Y, W, R or Q; at least one amino acid in X5 is Y, W or F; and at least one amino acid in X6 is L, Y, W or Q.

[0035] The synthesis process of each binding sequence in the polypeptide will be introduced below through the following embodiments 1-22.

[0036] Embodiment 1

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

[0038] (1) Resin swelling

[0039] Take Fmoc-Rink amide-MBHA Resin 50 mg (substitution amount 0.4 mmol / g), swell in 7 mL dichloromethane (DCM) for 30 min, filter off the DCM, then swell in 10 mL N-methyl pyrrolidone (NMP) for 30 min, and finally rinse with NMP and DCM 7 mL respectively.

[0040] (2) Removal of Fmoc protecting group

[0041] The swelled resin was put into a reactor, 7 mL of 25% piperidine / NMP (V / V) solution containing 0.1 M 1-hydroxybenzotriazole (HOBT) was added, and 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 until clean, to obtain the resin from which the Fmoc protecting group of the initial connection was removed.

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

[0043] Fmoc-Asn(Pbf)-OH (0.04 mmol), O-benzotriazolyl-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, and the solution was added to the resin obtained in step (2), and the reaction was carried out for 45 min; after the reaction was completed, the reaction solution was filtered off, and the resin was washed with 7 mL of DCM and NMP each for 3 times.

[0044] (4) Detection of coupling efficiency

[0045] The coupling efficiency of the resin was qualitatively detected by the ninhydrin method or the bromophenol blue method, and if the color reaction was negative, the next coupling cycle could be entered.

[0046] Ninhydrin method: a small amount of resin particles was washed with ethanol, and was put into a transparent vial, 5% ninhydrin ethanol, 2 drops of 80% phenol ethanol solution, and 2 drops of KCN pyridine solution (2 mL of 0.001 M KCN diluted in 98 mL of pyridine) were added, and heating was carried out at 100°C for 5 min; if the resin showed blue color, it was positive.

[0047] Bromophenol blue method: a small amount of resin particles was washed with dimethylformamide, and was put into a transparent vial, 3 drops of 1% bromophenol blue dimethylformamide solution were added, and shaking was carried out at room temperature for 3 min; if the resin showed blue color, it was positive.

[0048] (5) Extension of the peptide chain

[0049] According to the sequence of the polypeptide, the above-mentioned deprotection and coupling steps were repeated to sequentially connect the corresponding amino acids, to obtain the resin connected with the NPAGNLYDWR (SEQ. ID NO. 1) polypeptide sequence.

[0050] (6) Cleavage of the polypeptide on the resin

[0051] The resin with NPAGNLYDWR (SEQ. ID NO. 1) polypeptide sequence was put into a reaction bottle, 10 mL of cleavage reagent Reagent K (TFA / benzyl methyl sulfide / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added, and it was shaken at 0°C for 30 min, and then reacted at room temperature for 3 h; after the reaction was completed, it was suction filtered, washed with a small amount of TFA and DCM for three times, and the filtrates were combined; the filtrate was added into a large amount of ice ethyl ether to precipitate white flocculent precipitate, and the crude product of the target polypeptide was obtained by freezing centrifugation.

[0052] The polypeptide crude product was dissolved in 2 mL of water, and directly subjected to preparative liquid chromatography purification, and the chromatography conditions were as follows: C18 reverse phase column (320 mm x 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 was 7 mL / min, detection wavelength was 214 nm; the collected solution was freeze-dried to obtain 25 mg of pure product. The theoretical relative molecular mass was 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 of Example 1 is that the sequence of the synthesized polypeptide is different.

[0056] The solid phase synthesis of YSGNYPYSIL (SEQ. ID NO. 2) has a theoretical relative molecular mass of 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 of Example 1 is that the sequence of the synthesized polypeptide is different.

[0060] The solid phase synthesis of QPAGNLYRWY (SEQ. ID NO. 3) has a theoretical relative molecular mass of 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 procedure of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0064] Solid phase synthesis of RPAGNLYRWY (SEQ. ID NO. 4) has a theoretical relative molecular mass of 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 procedure of example 1 and example 3 is that the sequence of the synthesized polypeptide is different.

[0068] Solid phase synthesis of QPAGNLYWWW (SEQ. ID NO. 5) has a theoretical relative molecular mass of 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 procedure of example 1 and example 4 is that the sequence of the synthesized polypeptide is different.

[0072] Solid phase synthesis of KPAGNLYWWF (SEQ. ID NO. 6) has a theoretical relative molecular mass of 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 procedure of example 1 and example 5 is that the sequence of the synthesized polypeptide is different.

[0076] Solid phase synthesis of QPAGNLYYWY (SEQ. ID NO. 7) has a theoretical relative molecular mass of 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 step of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0080] Solid phase synthesis of WPAGNLYYWW (SEQ. ID NO. 8) has a theoretical relative molecular mass of 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 step of example 1 and example 3 is that the sequence of the synthesized polypeptide is different.

[0084] Solid phase synthesis of QPAGNLYRWF (SEQ. ID NO. 9) has a theoretical relative molecular mass of 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 step of example 1 and example 4 is that the sequence of the synthesized polypeptide is different.

[0088] Solid phase synthesis of IPAGNLYWWY (SEQ. ID NO. 10) has a theoretical relative molecular mass of 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 step of example 1 and example 5 is that the sequence of the synthesized polypeptide is different.

[0092] Solid phase synthesis of WPAGNLYYWY (SEQ. ID NO. 11) has a theoretical relative molecular mass of 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 procedure of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0096] Solid phase synthesis of RPAGNLYFWY (SEQ. ID NO. 12) has a theoretical relative molecular mass of 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 procedure of example 1 and example 13 is that the sequence of the synthesized polypeptide is different.

[0100] Solid phase synthesis of WSGNWPYSIY (SEQ. ID NO. 13) has a theoretical relative molecular mass of 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 procedure of example 1 and example 14 is that the sequence of the synthesized polypeptide is different.

[0104] Solid phase synthesis of WSGNWPYSIW (SEQ. ID NO. 14) has a theoretical relative molecular mass of 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 procedure of example 1 and example 15 is that the sequence of the synthesized polypeptide is different.

[0108] Solid phase synthesis of RSGNWPYSIW (SEQ. ID NO. 15) has a theoretical relative molecular mass of 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 procedure of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0112] Solid phase synthesis of RSGNFPYSIW (SEQ. ID NO. 16) has a theoretical relative molecular mass of 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 procedure of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0116] Solid phase synthesis of QSGNWPYSIW (SEQ. ID NO. 17) has a theoretical relative molecular mass of 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 procedure of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0120] Solid phase synthesis of RSGNWPYSIQ (SEQ. ID NO. 18) has a theoretical relative molecular mass of 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 procedure of example 1 and example 2 is that the sequence of the synthesized polypeptide is different.

[0124] Solid phase synthesis of RSGNYPYSIW (SEQ. ID NO. 19) has a theoretical relative molecular mass of 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 step of Example 1 and Example 2 is that the sequence of the synthesized polypeptide is different.

[0128] The solid phase synthesis of YSGNWPYSIW (SEQ. ID NO. 20) has a theoretical relative molecular mass of 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 step of Example 1 and Example 2 is that the sequence of the synthesized polypeptide is different.

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

[0133] Example 22

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

[0135] The difference between the synthesis step of Example 1 and Example 2 is that the sequence of the synthesized polypeptide is different.

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

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

[0138] Example 23

[0139] Surface plasmon resonance (SPR): detecting the affinity constant of the prepared amino acid sequences (SEQ. ID NO. 1-22) of Examples 1-22 to CLDN4 protein, respectively.

[0140] ​Human recombinant protein CLDN4 was coupled to a CM 5 chip, and 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, 0nM), using appropriate regeneration conditions (such as Glycine 2.5), the flow rate was set to 30 μL / min, and the injection time for each cycle was set to 90 s, and the dissociation time was also 90 s. According to the Biacore instrument procedure, the multi-cycle kinetic test was carried out, and finally the binding and dissociation curves were analyzed by Biacore T200 Evaluation Software to calculate the affinity related parameters, and the results are shown in Table 1 below, and the best affinity reached <10 -8 , reaching the level of 10 nM.

[0141] Table 1: Affinity constant (Kd) summary

[0142]

[0143] Example 24

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

[0145] Cells in good growth state in the logarithmic growth phase were selected to prepare cell suspension and counted, and 5*103 cells were inoculated in each well of a 96-well plate (100 μL / well). Five replicate wells were set up in each group, and after the cells were incubated overnight, different concentrations (1 uM, 10 uM, 100 uM) of SEQ. ID NO. 1-22 polypeptides were added and incubated for 24 hours. CCK-8 reagent was added, and an enzyme marker was used for detection, with a detection wavelength of 450-490 nm and a reference wavelength of 600-650 nm. The toxicity of the polypeptide was calculated using Graphpad, and the results are shown in (a) and (b) of Figure 2 , (a) and (b) of Figure 2 respectively show the test results of SEQ. ID NO. 1-11 and SEQ. ID NO. 12-22, and it is shown that each peptide segment has no obvious toxicity.

[0146] Example 25

[0147] Specifically, the ovarian cancer cell line A2780 is lentivirus transfected to overexpress CLDN4 protein, the 22 peptide segments described above are connected with FITC, the cells are incubated with anti-CLDN4 protein antibody, FITC-labeled polypeptide and DAPI, respectively, and the drug internalization and co-localization with the target protein are detected by fluorescence microscopy. The results are shown in (a)-(f) of Figure 3 The results are shown in (a)-(f) of

[0148] Specifically, the above polypeptides are applied in drugs, wherein the drug component is: any one of the above polypeptides is coupled with various small molecule drugs of chemotherapy by a cleavable or non-cleavable linker, including but not limited to: doxorubicin, camptothecin, etc., to prepare PDC drugs.

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

[0150] The polypeptide is coupled with any amino acid with special charge or different hydrophilicity or hydrophobicity, including natural and unnatural amino acids, to prepare an environment corresponding polypeptide hydrogel to load chemotherapy drugs for tumor killing.

[0151] An immunoconjugate includes a polypeptide, the polypeptide is combined with an immunoconjugate component, and the immunoconjugate component generally includes: a chemotherapeutic agent, a radioactive atom, a cell growth inhibitor and a cytotoxic agent, an immune checkpoint inhibitor, an antibody or an antibody fragment. The radioactive agent includes but is not limited to: alpha emitter, beta emitter, gamma emitter; the cytotoxin includes but is not limited to: doxorubicin, calicheamicin, anthracycline, etc.; the immune checkpoint inhibitor includes but is not limited to: CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, G1TR, B7H4, KIR, A2aR, CD27, CD70, DR3 and ICOS, etc. immune checkpoint inhibitors; the antibody or antibody fragment includes but is not limited to: CD3, CD28, LFA-1, LFA-2, CD40L, etc. antibodies or fragments of related proteins.

[0152] In summary, the polypeptide of the present application can effectively target CLDN4 positive cells, and is not toxic per se, and can be used as an excellent targeting peptide, thus having the potential as a pharmaceutical ingredient or a pharmaceutical carrier for the treatment of ovarian cancer or other tumors with high expression of CLDN4.

[0153] In the description of the present specification, the description referring to the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0154] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only illustrative of the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1. A polypeptide targeting the CLDN4 protein, characterized in that, The amino acid sequence of the polypeptide is: YSGNYPYSIL, QPAGNLYRWY, or RPAGNLYRWY.

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.

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