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: QPALNLYWWW, KPALNLYWWF or QPALNLYYWY. 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, invention name: A polypeptide targeting CLDN4 protein, its preparation method and application (application date: November 1, 2023). Technical Field

[0002] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a polypeptide targeting the CLDN4 protein, and a preparation method and application thereof. Background Art

[0003] Ovarian cancer comprises various subtypes with varying morbidity and mortality rates. The most common type is epithelial ovarian cancer (EOC), which accounts for over 95% of ovarian cancer cases. Based on distinct pathogenesis, genetic basis, and histopathological features, ovarian cancer is further classified into five distinct subtypes: serous cystadenoma, serous cystadenocarcinoma, mucinous cystadenoma, mucinous cystadenocarcinoma, and borderline tumor. Ovarian cancer is one of the leading causes of death from gynecological malignancies and is often diagnosed at an advanced stage, with a lack of effective screening strategies. Alarmingly, ovarian cancer claims an estimated 185,000 lives worldwide annually, highlighting the urgent need for early detection and innovative treatment strategies.

[0004] CLDN4 is highly expressed not only in most ovarian cancers but also in many other tumor types, such as pancreatic and colorectal cancers. Currently, there are no effective treatment options for ovarian cancer beyond surgery, chemotherapy, and combined monoclonal antibody therapy. Therefore, a peptide targeting the CLDN4 protein was proposed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a polypeptide targeting the CLDN4 protein and a preparation method and application thereof, thereby solving the problems in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A polypeptide targeting CLDN4 protein, comprising a first binding sequence L1 or a second binding sequence L2, wherein 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 acid can be any one of the 20 natural amino acids, with the following restrictions: X1 wherein at least one amino acid is N, Q, R, K, W or I; X2 wherein at least one amino acid is D, R, W, or Y; X3 wherein at least one amino acid is R, Y, W or F; X4 wherein at least one amino acid is Y, W, R or Q; X5 wherein at least one amino acid is Y, W or F; X6 wherein at least one amino acid 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 use of the above polypeptide in the preparation of drugs or drug carriers for treating ovarian cancer.

[0011] A drug comprising the above polypeptide.

[0012] Furthermore, in the drug, the polypeptide is coupled with doxorubicin or camptothecin via a cleavable or non-cleavable linker to prepare a PDC drug.

[0013] A drug carrier comprises the above polypeptide.

[0014] Furthermore, the drug carrier is: a polypeptide coupled with any amino acid with a charge or different hydrophilicity to prepare a polypeptide hydrogel.

[0015] An immunoconjugate comprising the above polypeptide.

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

[0017] A method for preparing a polypeptide targeting the CLDN4 protein comprises the following steps:

[0018] S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter out the DCM, swell it with NMP for 30 min, and finally rinse it with NMP, DCM, and NMP respectively;

[0019] S2, the swollen resin is placed in a reactor, and a piperidine / NMP solution containing HOBT is added for reaction. After the reaction is completed, the solution is filtered off and washed with NMP;

[0020] S3, dissolving Fmoc-Arg(Pbf)-OH, HBTU, HOBT, and DIPEA in NMP, and then adding this solution to the depiperidinized resin for reaction. After the reaction is completed, the reaction solution is filtered and washed with DCM and NMP;

[0021] S4, qualitatively detect the coupling efficiency of the resin using the ninhydrin method or the bromophenol blue method. If the color reaction is negative, the next coupling cycle can be entered;

[0022] S5, repeating S2 and S3 in sequence to connect corresponding amino acids according to the sequence of the polypeptide to obtain a resin with a polypeptide sequence attached;

[0023] S6, adding a cleavage agent to cleave the resin to obtain the polypeptide.

[0024] Beneficial effects of the present invention:

[0025] 1. The polypeptide of the present invention can specifically target CLDN4-positive cells with high selectivity. The polypeptide of the present invention can be prepared by chemical synthesis, has high purity, small molecular weight, strong specificity, no immunogenicity, and is safe and reliable.

[0026] 2. The polypeptide of the present invention can be used as a drug delivery vehicle, or coupled with known components to form a drug combination, thereby achieving better therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0028] Figure 1 is a mass spectrum of the amino acid sequence prepared in Example 1-22 of the present invention;

[0029] Figure 2 This is a diagram showing the results of CCK-8 assay of the amino acid sequences prepared in Examples 1-22 of the present invention; Figure 3This is the immunofluorescence staining image corresponding to the polypeptide sequences in Examples 1-22 of the present invention. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making any creative efforts shall fall 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] Wherein X1-X6 represents a sequence of 1-7 amino acid residues in length, wherein the amino acid can be any one of the 20 natural amino acids, with the following restrictions: X1 wherein at least one amino acid is N, Q, R, K, W or I; X2 wherein at least one amino acid is D, R, W, or Y; X3 wherein at least one amino acid is R, Y, W or F; X4 wherein at least one amino acid is Y, W, R or Q; X5 wherein at least one amino acid is Y, W or F; X6 wherein at least one amino acid is L, Y, W or Q.

[0035] The following examples 1-22 are used to describe the synthesis process of each binding sequence in the 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 it with 7 mL of dichloromethane (DCM) for 30 min, filter out DCM, and swell it with 10 mL of N-methylpyrrolidone (NMP) for 30 min. Finally, rinse it with 7 mL of NMP and 7 mL of DCM respectively.

[0040] (2) Removal of Fmoc protecting group

[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 and allowed to react for 25 minutes. After the reaction, the solution was filtered off and washed with NMP to obtain a resin with the initially attached Fmoc protecting group removed.

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

[0043] Dissolve Fmoc-AsnPbf)-OH (0.04 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (0.04 mmol), HOBT (0.04 mmol) and N,N-diisopropylethylamine (DIPEA) (0.08 mmol) in 10 mL of NMP, and then add this solution to the resin obtained in step (2) and react for 45 minutes. After the reaction is completed, the reaction solution is filtered off and the resin is washed three times with 7 mL each of DCM and NMP.

[0044] (4) Detection of coupling efficiency

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

[0046] 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 (2 ml 0.001M KCN ​​diluted in 98 ml pyridine), and 2 drops each of 80% phenol ethanol solution, heat at 100°C for 5 minutes. If the resin turns blue, it is positive.

[0047] Bromophenol blue method: Take a small amount of resin particles and wash them with dimethylacetamide. Put them into a transparent vial and add 3 drops of 1% bromophenol blue dimethylacetamide solution. Shake at room temperature for 3 minutes. If the resin turns blue, it is positive.

[0048] (5) Peptide chain extension

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

[0050] (6) Cleavage of peptides on resin

[0051] The resin obtained above with the NPAGNLYDWR (SEQ.ID NO.1) polypeptide sequence was placed in a reaction flask, 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. The mixture was shaken at 0°C for 30 minutes and then reacted at room temperature for 3 hours. After the reaction was completed, the mixture was filtered and 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 target polypeptide was obtained by refrigerated centrifugation.

[0052] The crude peptide was dissolved in 2 mL of water and directly purified by preparative liquid chromatography using the following conditions: a 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: 20% to 80% mobile phase B over 20 min; flow rate: 7 mL / min; detection wavelength: 214 nm. The collected solution was lyophilized to yield 25 mg of the pure product. Its theoretical molecular mass was 1205.2950. ESI-MS m / z: [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 in Example 1 is that the sequence of the synthetic polypeptide is different.

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

[0060] The solid phase synthesis theoretical molecular mass of QPAGNLYRWY (SEQ. ID NO. 3) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0064] The solid phase synthesis theoretical relative molecular mass of RPAGNLYRWY (SEQ. ID NO. 4) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0068] The solid phase synthesis theoretical molecular mass of QPAGNLYWWW (SEQ.ID NO. 5) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0072] The solid phase synthesis theoretical molecular mass of KPAGNLYWWF (SEQ. ID NO. 6) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0076] The solid phase synthesis theoretical relative molecular mass of QPAGNLYYWY (SEQ. ID NO. 7) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0080] The solid phase synthesis theoretical relative molecular mass of WPAGNLYYWW (SEQ.ID NO. 8) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0084] The solid phase synthesis theoretical molecular mass of QPAGNLYRWF (SEQ. ID NO. 9) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0088] The solid phase synthesis theoretical relative molecular mass of IPAGNLYWWY (SEQ.ID NO.10) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0092] The solid phase synthesis theoretical relative molecular mass of WPAGNLYYWY (SEQ. ID NO. 11) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0096] The solid phase synthesis theoretical molecular mass of RPAGNLYFWY (SEQ. ID NO. 12) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0100] The solid phase synthesis theoretical molecular mass of WSGNWPYSIY (SEQ. ID NO. 13) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0104] The solid phase synthesis theoretical molecular mass of WSGNWPYSIW (SEQ. ID NO. 14) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0108] The solid phase synthesis theoretical molecular mass of RSGNWPYSIW (SEQ. ID NO. 15) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0112] The solid phase synthesis theoretical molecular mass of RSGNFPYSIW (SEQ. ID NO. 16) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0116] The solid phase synthesis theoretical molecular mass of QSGNWPYSIW (SEQ. ID NO. 17) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0120] The solid phase synthesis theoretical relative molecular mass of RSGNWPYSIQ (SEQ. ID NO. 18) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0124] The solid phase synthesis theoretical molecular mass of RSGNYPYSIW (SEQ. ID NO. 19) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0128] The solid phase synthesis theoretical molecular mass of YSGNWPYSIW (SEQ. ID NO. 20) 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 in Example 1 is that the sequence of the synthetic polypeptide is different.

[0132] The solid phase synthesis theoretical molecular mass of WSGNWPYSIQ (SEQ. ID NO. 21) is 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 steps in Example 1 is that the sequence of the synthetic polypeptide is different.

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

[0137] Among them, the mass spectra of Example 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. It can be seen from the figures that the main products of each synthesis are the target peptides.

[0138] Example 23

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

[0140] The human recombinant protein CLDN4 was coupled to a 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, 0 nM). Appropriate regeneration conditions (such as Glycine 2.5) were used, the flow rate was set to 30 μL / min, the injection time for each cycle was set to 90 s, and the dissociation time was also 90 s. According to the Biacore instrument program flow, multi-cycle kinetics testing was performed, and finally the binding-dissociation curve was analyzed and fitted by Biacore T200 Evaluation Software, and affinity-related parameters were calculated. The results are shown in Table 1 below. The optimal affinity reached <10 -8 , reaching 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 at different concentrations to the constructed ovarian cancer stably transfected cell lines with high expression of CLDN4 was detected.

[0145] Select cells in the logarithmic growth phase with good growth status to prepare cell suspension, count them, and inoculate 5*103 cells per well in a 96-well plate (100μL / well): set up 5 replicate wells for each group. After the cells were iron-walled overnight, different concentrations (1uM, 10uM, 100uM) of SEQ.ID NO.1-22 peptide were added and cultured for 24 hours. CCK-8 reagent was added and detected using a microplate reader with a detection wavelength of 450-490nm and a reference wavelength of 600-650nm. The toxicity of the peptide was calculated using Graphpad. The results are shown in the figure. 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, indicating that each peptide segment has no obvious toxicity.

[0146] Example 25

[0147] Specifically, the ovarian cancer cell line A2780 was transfected with lentivirus to overexpress CLDN4 protein. The 22 peptides mentioned above were linked to FITC. The cells were incubated with anti-CLDN4 protein antibodies, FITC-containing 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 images corresponding to the polypeptide sequences in Examples 1-22 are reflected. It can be seen from the figures that each peptide segment is well internalized and well co-localized with the target protein.

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

[0149] The polypeptide is linked to a "warhead" capable of forming a covalent bond. The linking method may be to insert the "warhead" at the NH2 end or COOH end of the polypeptide, or to mutate any one or several amino acids in the polypeptide into a "warhead". The "warhead" 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 polypeptide is coupled with any amino acid with a special charge or different hydrophilicity, including natural and non-natural amino acids, to prepare an environmentally responsive polypeptide hydrogel, which encapsulates chemotherapy drugs and kills tumors.

[0151] An immunoconjugate comprises a polypeptide, wherein the polypeptide is conjugated to an immunoconjugate component, wherein the immunoconjugate component generally includes: a chemotherapeutic agent, a radioactive atom, a cytostatic and cytotoxic agent, an immune checkpoint inhibitor, an antibody or antibody fragment. Radiopharmaceuticals include, but are not limited to, alpha emitters, beta emitters, and gamma emitters; cytotoxins include, but are not limited to, doxorubicin, calicinomycin, anthracyclines, etc.; immune checkpoint inhibitors include, but are not limited to, inhibitors of 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 thereof to proteins related to 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 itself, and can be used as 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] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these 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 any one or more embodiments or examples.

[0154] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A polypeptide targeting CLDN4 protein, characterized in that The amino acid sequence of the polypeptide is: QPAGNLYWWW, KPAGNLYWWF or QPAGNLYYWY.

2. Use of the polypeptide according to claim 1 in the preparation of a drug or drug carrier for treating ovarian cancer.

3. A drug, characterized in that Comprising the polypeptide of 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 cleavable or non-cleavable linker to form a PDC drug.

5. A drug carrier, characterized in that Comprising the polypeptide of claim 1.

6. The drug carrier according to claim 5, characterized in that The drug carrier is: a polypeptide coupled with any amino acid with charge or different hydrophilicity to prepare a polypeptide hydrogel.

7. An immunoconjugate, characterized in that Comprising the polypeptide of claim 1.

8. The immunoconjugate according to claim 7, characterized in that Also included are chemotherapeutic agents, radioactive atoms, cytostatic agents, 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: The following steps are involved: S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter out the 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 for reaction. 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 this solution to the depiperidinized resin for reaction. After the reaction is completed, the reaction solution is filtered and washed with DCM and NMP; S4, qualitatively detect the coupling efficiency of the resin using the ninhydrin method or the bromophenol blue method. If the color reaction is negative, the next coupling cycle can be entered; S5, repeating S2 and S3 in sequence to connect corresponding amino acids according to the sequence of the polypeptide to obtain a resin with a polypeptide sequence attached; S6, adding a cleavage agent to cleave the resin to obtain the polypeptide.

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