Peptide-drug conjugate targeting nectin-4

Abstract

The present invention relates to a polypeptide, bicyclic peptide and peptide-drug conjugate targeting Nectin-4, and the use of the peptide-drug conjugate. The polypeptide targeting Nectin-4 has an improved half-life and enhanced stability. The peptide-drug conjugate PDC has a strong tumor cell killing or inhibitory effect. PDC can further improve the selectivity and killing effect of the drug in tumor cells while reducing the side effects caused by traditional treatment methods, thereby enabling more effective treatment of various types of cancers.

Description

Peptide conjugates targeting Nectin-4 Technical Field

[0001] This invention relates to the field of biomedical technology. Specifically, this invention relates to peptides, bicyclic peptides, and peptide-conjugated drugs targeting Nectin-4, as well as the uses of peptide-conjugated drugs. Background Technology

[0002] Cancer has become one of the leading diseases threatening human life and health. With the increasing global population aging and environmental pollution, the incidence and mortality rates of cancer continue to rise. According to statistics from the International Agency for Research on Cancer (IARC) of the World Health Organization, 19.3 million new cancer cases were diagnosed globally in 2020, with 4.57 million new cases in China, ranking first in the world. Although surgical treatment is effective for the resection of localized tumors, its effectiveness is very limited for advanced or systemic cancers. While radiotherapy and chemotherapy can kill tumor cells, they are often accompanied by severe side effects such as hair loss, nausea, vomiting, and immunosuppression, seriously affecting patients' quality of life and treatment adherence.

[0003] Therefore, finding more effective and targeted treatments has become an important research direction in the field of cancer treatment. Nectin-4 (also known as PVRL4, Polyovirus receptor-related protein 4), a type I membrane protein, belongs to the Nectin family within the immunoglobulin superfamily and is significantly overexpressed in various cancers. Nectin-4 expression is prevalent in tumors such as lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and head and neck cancer, and is closely related to malignant characteristics such as tumor proliferation, metastasis, and recurrence. Its normal expression in placental and embryonic tissues contrasts sharply with its abnormal expression in cancer, making it an ideal target for targeted cancer therapy.

[0004] Peptide-drug conjugates (PDCs), as a novel targeted therapy strategy, link cytotoxic payloads with homing peptides that target specific receptors on the surface of tumor cells, thereby improving therapeutic precision while reducing toxicity to normal tissues. Compared with traditional antibody-drug conjugates (ADCs), PDCs have significant advantages such as smaller molecular weight, stronger ability to penetrate tumor tissue, lower immunogenicity, and lower production costs, making them a new hot topic in anti-tumor drug research. PDCs also exhibit superior pharmacokinetic properties and good stability, thus becoming the next generation of targeted anticancer drugs after small molecule targeted drugs, monoclonal antibodies, and ADCs.

[0005] Although some peptide-conjugated drugs, such as BT8009, have entered clinical trials and shown some efficacy, their efficacy remains relatively weak, especially when compared with existing drugs (such as Enfortumab Vedotin). Therefore, developing more stable and effective specific targeted PDCs remains a key technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0006] In view of the aforementioned deficiencies and shortcomings of the prior art, the inventors designed a Nectin-4-targeting peptide with improved half-life and enhanced stability, and constructed a novel PDC drug by combining this Nectin-4-targeting peptide with a cytotoxin. The peptide and PDC drug of this invention exhibit potent tumor cell killing or inhibitory effects, further improving the selectivity and killing effect of the drug in tumor cells while reducing the side effects of traditional treatment methods.

[0007] As is well known in the art, a polypeptide is a molecular structure in which multiple amino acids are linked together by amide bonds.

[0008] The structures of some of the amino acids mentioned in this article are shown below.

[0009] Therefore, in a first aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide having an amino acid sequence comprising or consisting of a general formula (I) or the reverse sequence (I') of general formula (I):

[0010] Ci-X1-X2-X3-Cii-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -Ciii (I)

[0011] Ciii-X12-X11-X10-X9-X8-X7-X6-X5-X4-Cii-X3-X2-X1-Ci (I')

[0012] In the formula, X1 is Pro; X2 is 1Nal or 2Nal; X3 is D-Asp or D-Lys; X4 is Met; X5 is hArg; X6 is Asp or His; X7 is Trp or 5fW; X8 is S; X9 is Thr; X 10 For Pro; X 11 For Hyp;X 12 For Trp; Ci, Cii, and Ciii are Cys or hCys. As shown in D-Asp, the prefix "D" indicates that the amino acid is a D-type amino acid.

[0013] In one embodiment, the polypeptide of the present invention comprises modified or unmodified natural or non-natural amino acids.

[0014] In one embodiment, the polypeptide of the present invention is PEGylated.

[0015] In one embodiment, the polypeptide of the present invention has one or more of the following modifications: fatty acid modification, glycosylation modification, phosphorylation modification, liposome modification, disulfide bond modification, and deuteration modification.

[0016] In one embodiment, the peptide of the present invention specifically targets the Nectin-4 protein.

[0017] In one embodiment, the amino acid sequence of the polypeptide of the present invention is selected from the group consisting of:

[0018] C i -P1-[1Nal]2-[dD]3-C ii -M4-[HArg]5-D6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 1);

[0019] C i -P1-[2Nal]2-[dD]3-C ii -M4-[HArg]5-D6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 2);

[0020] C i -P1-[1Nal]2-[dD]3-C ii -M4-[HArg]5-D6-[5fW]7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 3);

[0021] Ci-P1-[2Nal]2-[dD]3-C ii -M4-[HArg]5-D6-[5fW]7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 4);

[0022] C i -P1-[1Nal]2-[dK]3-C ii -M4-[HArg]5-H6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 5); and

[0023] C i -P1-[Nal]2-[dD]3-C ii -M4-[HArg]5-H6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 6).

[0024] In a preferred embodiment, the amino acid sequence of the polypeptide of the present invention is selected from the group consisting of:

[0025] Ci-P1-[2Nal]2-[dD]3-Cii-M4-[HArg]5-D6-W7-S8-T9-P10-[HyP]11-W12-Ciii (SEQ ID NO: 2);

[0026] Ci-P1-[1Nal]2-[dD]3-Cii-M4-[HArg]5-D6-[5fW]7-S8-T9-P10-[HyP]11-W12-Ciii (SEQ ID NO: 3);

[0027] Ci-P1-[2Nal]2-[dD]3-Cii-M4-[HArg]5-D6-[5fW]7-S8-T9-P10-[HyP]11-W12-Ciii (SEQ ID NO: 4);

[0028] and

[0029] Ci-P1-[Nal]2-[dD]3-Cii-M4-[HArg]5-H6-W7-S8-T9-P10-[HyP]11-W12-Ciii (SEQ ID NO: 6).

[0030] In a more preferred embodiment, the amino acid sequence of the polypeptide of the present invention is selected from the group consisting of:

[0031] and

[0032] Ci-P1-[Nal]2-[dD]3-Cii-M4-[HArg]5-H6-W7-S8-T9-P10-[HyP]11-W12-Ciii (SEQ ID NO: 6).

[0033] In a second aspect, the present invention provides a bicyclic peptide comprising the polypeptide or a pharmaceutically acceptable salt thereof according to the first aspect and a cyclic peptide molecular scaffold.

[0034] In one embodiment, the cyclic peptide scaffold is TATA, with the structural formula as follows: The olefin double bond of the cyclic peptide scaffold is linked to three cysteine ​​residues of the polypeptide or its pharmaceutically acceptable salt to form a bicyclic system.

[0035] In one embodiment, the structure of the bicyclic peptide is shown in formula (T1), (T2), (T3), or (T4):

[0036] In a preferred embodiment, the structure of the bicyclic peptide is shown in formula (T2) or (T3).

[0037] In a third aspect, the present invention provides a polypeptide-conjugated drug comprising a bicyclic peptide as described in the second aspect and a polytoxic group containing two or more toxins.

[0038] In a preferred embodiment, the bicyclic peptide and the polytoxin group are linked by a linker.

[0039] In one embodiment, the linker is a polysarcosine or PEG or a mixture thereof.

[0040] In one embodiment, the degree of polymerization of the connector is 0-20; more preferably, it is 0-10, and most preferably, it is 5-10; the degree of polymerization can be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0041] In one embodiment, the toxin is a microtubule inhibitor (such as MMAE), a camptothecin Top1 inhibitor (such as Camptothecin, SN-38, Irinotecan, icietecan), anthracyclines (such as Doxorubicin, Daunorubicin), paclitaxel, platinum-based chemotherapy drugs (such as cisplatin, carboplatin), diazacyclic chemotherapeutic agents (such as Pemetrexed), folded protease inhibitors (such as Bortezomib), rutin drugs (such as Vincristine, Vinblastine), radioisotope-labeled toxins, liposome-encapsulated chemotherapy drugs, etc.

[0042] In one embodiment, the bicyclic peptide and the linker are linked in the following manner: bicyclic peptide-Sar10-β-Ala-*1; where *1 is the linker site; the amino group on the Ci residue in the bicyclic peptide is linked to the carboxyl group of Sar10.

[0043] In one embodiment, the linker includes a spacer, which is -PABC-Cit-Val- or -PABC-cyclobutyl-Ala-Cit-, such as -PABC-Cit-Val-, where PABC represents p-aminobenzylcarbamate; -GGFG-.

[0044] In one embodiment, the spacer and the toxin are linked in the following ways: ①MMAE-PABC-Cit-Val-*2; or ②MMAE-PABC-cyclobutyl-Ala-Cit-*2; wherein the *2 position is connected to the carboxyl group of aspartic acid or glutamic acid, and the carboxyl group is connected to two ①, two ②, or ① and ② respectively, thereby forming a structure including:

[0045] In one embodiment, the spacer and the toxin are connected in the following ways: ① icietecan-GGFG-*2; or ② icietecan-PEGn-*2, n = 3-10; wherein *2 is connected to the carboxyl group of aspartic acid or glutamic acid, and the carboxyl group is connected to two ①, two ②, or ① and ② respectively, thereby forming a structure including:

[0046] In one embodiment, the polypeptide-conjugated drug of the present invention has the following structure:

[0047] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a polypeptide-conjugated drug and / or its deuterated form as described in the third aspect, and a pharmaceutically acceptable salt.

[0048] In one embodiment, the peptide-conjugated drug is used to treat related diseases, including, for example, bladder cancer, breast cancer, ovarian cancer, colorectal cancer (CRC), prostate cancer, and lung cancer.

[0049] In a fifth aspect, the present invention provides the use of the polypeptide-conjugated drug according to the third aspect in the preparation of a pharmaceutical composition for treating diseases of Nectin-4 overexpression, for example, cancers including bladder cancer, breast cancer, ovarian cancer, colorectal cancer (CRC), prostate cancer, and lung cancer.

[0050] This invention addresses the shortcomings of existing PDC drugs, resolving their insufficient efficacy in clinical applications, particularly in various types of cancer, enabling more precise targeted therapy and providing new treatment options for cancer patients. This not only offers a new direction for drug development in targeted cancer therapy but also lays the foundation for personalized and precise cancer treatment. The Nectin-4-targeting peptide conjugate proposed in this invention, through optimized linker and homing peptide design, improves the stability and pharmacokinetic properties of PDCs, demonstrating broad application prospects. Detailed Implementation

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Additionally, definitions and explanations of relevant terms are provided below for a better understanding of this disclosure.

[0052] In this document, the term “and / or” refers to and covers any and all possible combinations of more than one of the listed items.

[0053] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0054] It will be understood that the salt form of a polypeptide, bicyclic peptide, or polypeptide-conjugated drug is within the scope of this invention. When referring to a polypeptide, bicyclic peptide, or polypeptide-conjugated drug, this includes the salt form of said polypeptide, bicyclic peptide, or polypeptide-conjugated drug. Salts of polypeptides, bicyclic peptides, or polypeptide-conjugated drugs can be synthesized from a parent compound containing a base or acid moiety by conventional chemical methods, such as those described in *Pharmaceutical Salts: Properties, Selection, and Use*, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, *Hardcover*, 388 pages, August 2002. Typically, these salts can be prepared by reacting the free acidic or basic form of these compounds with a suitable base or acid in water, in an organic solvent, or in a mixture of both.

[0055] In this article, pharmaceutically acceptable salts generally refer to those salts that are harmless to the human body and can be used in pharmaceutical preparations. Their role is to improve the stability, solubility, and absorption of drugs. Common pharmaceutically acceptable salts include: sodium chloride (NaCl), sodium sulfate (Na2SO4), calcium chloride (CaCl2), sodium dihydrogen phosphate (NaH2PO4), ammonium chloride (NH4Cl), sodium acetate (NaAc), ammonium sulfate ((NH4)2SO4), sodium citrate (Na3C6H5O7), amino acid salts (such as monosodium glutamate), and sodium nitrite (NaNO2).

[0056] In one embodiment, the cysteine ​​amino residues of the bicyclic peptide of the present invention are linked to a linker, and the linker and the toxin are coupled to form the polypeptide-coupled drug (polypeptide-coupled toxin drug) of the present invention.

[0057] In one embodiment, the amino group of Asp or Glu in the bicyclic peptide of the present invention is linked to an acyl group such as glutaryl or succinylate via an amino acid condensation reaction; the β-Ala residue at the end of the linker-linked bicyclic peptide (such as BT1) is linked to another acyl group such as glutaryl or succinylate.

[0058] The present disclosure will be further described in detail below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the embodiments that do not specify specific conditions are performed under conventional conditions known in the art or under conditions recommended by the manufacturer.

[0059] Example

[0060] Example 1: Synthesis of Polypeptides

[0061] Synthesis of Sequence 1:

[0062] Ci-P1-[1Nal]2-[dD]3-Cii-M4-[HArg]5-D6-W7-S8-T9-P10-[HyP]11-W12-Ciii (SEQ ID NO: 1) is described in J.Med.Chem.2022,65,14337-14347, and was synthesized according to the method described in that document.

[0063] Synthesis of the intermediate (C2-M-HArg-HWSTP-HyP-W-C3-MBHA) of SEQ ID NO: 5 and SEQ ID NO: 6:

[0064] Take 8.3 g (0.72 g / mmol) of MBHA resin, add 6 mmol, swell for 30 min, filter and dry under vacuum, add 36 ml of 20% Pip. Dry under vacuum, wash 36 ml x 4, dry under vacuum, add 30 ml of DMF solution containing 2.5 eq of Fmoc-cys(trt)-OH, HOBt, and DIC (15 mmol each), shake at room temperature for 2 h. Filter, wash 2-3 times with DMF, then remove Fmoc with 36 ml of 20% Pip, shake for 30 min, filter, wash 3-5 times with 36 ml of DMF, then sequentially add Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Pro-OH, Fmoc-threonine, Fmoc-serine, Fmoc-L-histidine, Fmoc HArg-phf-, Fmoc-L-methionine, and Fmoc-cys(trt)-OH.

[0065] Divide this resin into two equal parts.

[0066] Synthesis of SEQ ID NO: 5:

[0067] Take half of the resin from the above experiment, calculate 3 mmol, and use the standard Fmoc amino acid synthesis method to sequentially extend the peptide chain and connect N-Boc-N'-Fmoc-L-lysine, Fmoc-3-(1-naphthyl)-L-alanine, Fmoc-proline and Fmoc-cys(trt)-OH respectively.

[0068] The peptide was cleaved from the resin using a mixture of 95% FTA and 5% DTT, and purified by preparative HPLC to obtain a white solid powder. This is SEQ ID NO: 5. Molecular weight: see Table 1.

[0069] Synthesis of SEQ ID NO: 6:

[0070] Take half of the resin from the above experiment, calculate based on 3 mmol, and use the standard Fmoc amino acid synthesis method to sequentially extend the peptide chain and connect FMOC-D-aspartic acid tert-butyl ester, Fmoc-3-(1-naphthyl)-L-alanine, Fmoc-proline and Fmoc-cys(trt)-OH respectively.

[0071] The peptide was cleaved from the resin using a mixture of 95% FTA and 5% DTT, and purified by preparative HPLC to obtain a white solid powder. This is SEQ ID NO: 6. Molecular weight: see Table 1.

[0072] Example 2: Synthesis of Bicyclic Peptides

[0073] SEQ ID NO: 5 (200 mg, 0.104 mmol), TATA (27.2 mg, 0.109 mmol), and (NH4)2CO3 (354 mg, 4.47 mmol) were dissolved in 20 mL of acetonitrile and reacted for 4 h. After the reaction was completed, the acetonitrile was removed by concentration, and the residue was dissolved in DMF and separated by reversed-phase HPLC to obtain 17.8 mg of bicyclic peptide T2.

[0074] Bicyclic peptides T1 and T3 were synthesized using the same method. The molecular weights of bicyclic peptides T1 to T3 are shown in Table 1.

[0075] Table 1: Theoretical molecular weight and mass spectra (M+2H) of linear peptides (polypeptides) and bicyclic peptides + Molecular weight:

[0076] Synthesis of the bicyclic peptide BT1 linked to the linker:

[0077] The following compounds were synthesized according to the method described in J. Med. Chem. 2022, 65, 14337-14347:

[0078] Take the MBHA-peptide of SEQ ID NO: 5, i.e. the intermediate before cleavage, and continue to link 10 Fmoc-sarcosine residues, then link Fmoc-β-alanine residues. Use TFA:DTT solution for cleavage, HPLC purification, and synthesize the linker-linked bicyclic peptide by referring to the synthesis method of bicyclic peptide BT1.

[0079] Synthesis of MMAE:

[0080] Val-Cit-PABC-MMAE, CAS: 644981-35-1, purchased from a manufacturer listed in Chemicalbook.

[0081] Example 3: Synthesis of the peptide-conjugated drug HZ-CP01

[0082] Step A: Synthesis of intermediate M3

[0083] Val-Cit-PABC-MMAE, M1 (2 eq, 0.89 mmol, 1 g), and Fmoc-glutamic acid (158 mg, 0.45 mmol) were dissolved in 10 mL of DMF. HATU (423 mg, 1.1 mmol) and DIPEA (172 mg, 1.3 mmol) were added, and the reaction was carried out for 2 h. 3 mL of piperidine was added to remove Fmoc. After the reaction was completed, the reaction solution was separated by preparative HPLC to obtain M3 (693 mg, 66%).

[0084] Step B: Synthesis of intermediate M5

[0085] M3 (100 mg, 0.042 mmol) and glutaric anhydride (5 mg, 0.044 mmol) were reacted and dissolved in 5 ml of DMF. 10 μL of DIPEA was added and the reaction was carried out for 2 h to obtain M4. HOSu (10 mg, 0.1 mmol) and EDCI (24 mg) were added to the reaction solution and the reaction was carried out for 2 h to obtain the active ester M5.

[0086] Step C: Synthesis of compound HZ-CP01

[0087] The active ester M5 and BT1 (124 mg, 0.042 mmol) were mixed, and 50 mg of DIPEA was added. The mixture was reacted for 2 h. The reaction solution was purified by preparative HPLC to obtain HZ-CP01 (15 mg, yield 5%). Specific mass spectrometry data are shown in Table 2.

[0088] Example 4: Synthesis of the peptide-conjugated drug HZ-CP02

[0089] HZ-CP02 was prepared according to the method in Example 3.

[0090] Step A: Preparation of the aspartic acid-linked compound, refer to Step A of Example 3.

[0091] Val-Cit-PABC-MMAE, M1, and Fmoc-aspartic acid were condensed to obtain M6. Fmoc was removed to obtain M7. M7 was reacted with glutaric anhydride to obtain M8. HOSu and EDCI were added to the reaction solution and reacted to obtain the active ester M9. The active ester M9 was reacted with BT1 to obtain the reaction solution of HZ-CP02. The solution was purified by Prep-HPLC to obtain the final product.

[0092] Example 5: Synthesis of the peptide-conjugated drug HZ-CP07

[0093] Step A, synthesis of cyclic peptide BT2:

[0094] The synthesis method of BT2 is the same as that of BT1 (as described in Example 2), except that the 5-fluorotryptophan at position 7 is replaced with tryptophan at position 7.

[0095] Step B: Synthesis of Fmoc-GGFG-CH2O-AcOH (F6)

[0096] F1 (1 g, 2.7 mmol) was dissolved in 50 mL of DCM, and then TsOH (172 mg, 1 mmol) and benzyl glycolate (4.49 g, 10 eq) were added. The reaction mixture was reacted overnight under reflux. The mixture was then extracted with EA, washed with water, dried over MgSO4, and filtered to obtain the crude product. The crude product was subjected to column chromatography with 10-100% EA and PE to give 1.2 g of product, yield 93.5%. LCMS: 475.6 (M+H)+.

[0097] Dissolve F2 in 50 ml DCM, add 1.7 ml diethylamine, and react overnight at room temperature. The reaction mixture was spotted by TLC; the starting material disappeared. The reaction solution was then concentrated to dryness and directly added to the next step.

[0098] F3 (1.261 g, 5 mmol) and F4 (2.756 g, 5.5 mmol) were dissolved in 20 mL of DCM, and HATU (2.2 g, 6 mmol) and DIPEA (1.29 g, 10 mmol) were added. The reaction was carried out at room temperature for 2 h until it was essentially complete. The resulting reaction solution was subjected to column chromatography to give a white solid F5 (2.5 g, 3.87 mmol), with a yield of 77%. LCMS: 736.4 (M+H)+.

[0099] The F5 obtained in the previous step (2 g, 2.7 mmol) was dissolved in 60 ml of methanol and 30 ml of EA, and 10% wet Pd / C (55% water content, 0.5 g) was added. The mixture was then purged with hydrogen three times and hydrogenated overnight at 2 atm. After filtration, the product F6 (1 g, 1.5 mmol) was obtained, with a yield of 55%.

[0100] Step C: Synthesis of GGFG-dxd(dxd-3)

[0101] Compound F6 (1.00 g, 1.55 mmol) was dissolved in DMF (5 mL), and HATU (647.40 mg, 1.70 mmol), mesylate of compound eczemab dxd-1 (1.00 g, 1.55 mmol), and DIPEA (400.34 mg, 3.10 mmol) were added sequentially. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solvent was removed under reduced pressure, and the concentrate was directly prepared by high performance liquid chromatography. The concentrate was then freeze-dried to give compound dxd-2 (1.05 g), with a yield of 60.6%.

[0102] The compound dxd-2 (1 g, 0.94 mmol) obtained in the previous step was dissolved in 10 mL of DCM, and diethylamine (1.86 mL, 17.9 mmol) was added. The mixture was reacted overnight at 25 °C. The resulting reaction solution was concentrated to dryness and subjected to Flash chromatography to give dxd-3 (470 mg, 0.559 mmol, 59.5% yield), an off-white solid. LCMS: 841.2 (M+H)+

[0103] Step D: Synthesis of HZ-CP07

[0104] HZ-CP07 was prepared by referring to the synthesis of M9 and HZ-CP02.

[0105] The solid dxd-3 (100 mg, 0.12 mmol) obtained in the previous step was mixed with glutaric anhydride (15 mg, 0.132 mmol) and dissolved in 10 mL of DMF. 30 μL of DIPEA was added, and the mixture was reacted for 2 h to obtain dxd-4. HOSu (30 mg, 0.3 mmol) and EDCI (72 mg) were added to the reaction solution, and the mixture was reacted for 2 h to obtain the active ester dxd-5. In the presence of DIEPA, a substitution reaction was carried out with the cyclic peptide BT2 to obtain the crude product. 35 mg of a white solid, HZ-CP07, was prepared, LCMS: 977.8 (M / 4+H). + .

[0106] Example 6: Synthesis of the peptide-conjugated drug HZ-CP08

[0107] Step A: Synthesis of the bitoxin intermediate dxd-7

[0108] The active ester is obtained by condensing dxd-3 with Fmoc-L-glutamic acid, removing Fmoc, reacting with glutaric anhydride, and then reacting with NHS.

[0109] Dissolve dxd-3 (2 eq, 0.6 mmol, 0.5 g) and Fmoc-L-glutamic acid (111 mg, 0.3 mmol) in 10 mL of DMF. Add HATU (423 mg, 1.1 mmol) and DIPEA (172 mg, 1.3 mmol) and react for 30 min. Add 3 mL of piperidine to remove Fmoc. After the reaction is complete, separate the reaction solution by preparative HPLC to obtain dxd-7 (350 mg, 0.195 mmol, yield 78%). LCMS: 897.8 (M / 2+H) + .

[0110] Step B: Synthesis of intermediate dxd-9

[0111] Dxd-7 (100 mg, 0.056 mmol) and glutaric anhydride (7 mg, 0.062 mmol) were reacted and dissolved in 5 ml of DMF. 10 μL of DIPEA was added, and the reaction was allowed to proceed for 2 h. No dxd-7 residue was observed. HOSu (10 mg, 0.1 mmol) and EDCI (24 mg) were added to the reaction solution, and the reaction was allowed to proceed for 2 h to obtain the active ester dxd-9.

[0112] Step C: Synthesis of HZ-CP08

[0113] The active ester dxd-9 and BT2 (150 mg, 0.05) were stirred and reacted for 2 h. The product was separated by HPLC to obtain 110 mg of the target product.

[0114] Comparative Example 1: Synthesis of the peptide-conjugated drug BT8009

[0115] BT8009 was synthesized using the method described in the references.

[0116] Next, the synthesized peptide-conjugated drugs HZ-CP01, HZ-CP02, HZ-CP07, HZ-CP08 and BT-8009 were characterized, as shown in Table 2 below.

[0117] Table 2: Characterization of HZ-CP01, HZ-CP02, HZ-CP07, HZ-CP08 and BT-8009

[0118] Example 5: Effect Evaluation

[0119] I. Plasma stability test

[0120] The stability of the 2 μM test compound was tested by incubating it in mouse or human plasma and then using an LCMS-MS instrument.

[0121] Table 3: Plasma stability of bicyclic peptides T1, T2, and T3

[0122] As can be seen from Table 3 above, bicyclic peptides T2 and T3 have better stability than T1.

[0123] II. Cell viability assays of peptide-conjugated drugs HZ-CP01, HZ-CP02, and BT-8009: IC50 (nM)

[0124] In vitro cell viability test:

[0125] The cell lines used in the following experiments are as follows: MBA-MD-468 (human breast cancer cells, highly expressing Nectin-4), T24 (bladder cancer cells, moderately or low expressing Nectin-4) and RT112 (human bladder cancer cells, highly expressing Nectin-4).

[0126] Experimental method: Celltiter Glo assay

[0127] 1. Prepare cells

[0128] 1.1 Cell Culture

[0129] All cells were adherent cells, and the experiments were conducted during the logarithmic growth phase.

[0130] 1.2 Preparation of cell suspension

[0131] Ensure cell viability is above 90%. Adjust to an appropriate concentration and seed 1000 cells into a 96-well plate, 180 μl of cell suspension per well.

[0132] 2. Preparation of test compounds

[0133] 2.1 Prepare DMSO stock solutions for the test compounds, with each test compound having a stock solution concentration of 5 mM.

[0134] 2.2 Prepare a 5mM working stock solution of the test compound. Add 4 μl of the compound stock solution to 196 μl of DMSO-free complete medium, and perform a 4-fold serial dilution to obtain 8 concentrations in a complete medium containing 2% DMSO. This is the working stock solution of the test compound (the compound concentration is 10 times the final concentration, with the highest concentration being 100 μM).

[0135] 2.3 Compound Treatment

[0136] Add 20 μl of the compound working stock solution (10-fold release of DMSO to a final concentration of 0.2%) to each well of a 96-well plate seeded with cells.

[0137] The final concentrations of the tested compounds were: 10000.00 nM, 2500 nM, 625 nM, 156.25 nM, 39.06 nM, 9.77 nM, 2.44 nM, and 0.61 nM.

[0138] 2.4 Setting of reference holes

[0139] Solvent control: 0.2% DMSO. Blank control: 96-well plate readings at 0 h after dosing.

[0140] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0141] 3. Melt the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Discard the drug-containing culture medium from the plate. Dilute the CTG reagent with PBS, adding 5 μl of CTG reagent to 10 μl of PBS. Add 150 μl of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake for 10 minutes (protect from light) to mix thoroughly. Equilibrate to room temperature for 5 minutes. Read the light signal value using a multi-mode microplate reader.

[0142] 4. Data Processing

[0143] 1) Inhibition rate (%) = (DMSO solvent control well reading - test sample well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0144] 2) Plot the graph to obtain the curve and calculate IC50, as shown in Table 4.

[0145] Table 4: IC50 (nM) of HZ-CP01, HZ-CP02 and BT-8009

[0146] As can be seen from Table 4 above, HZ-CP01 and HZ-CP02 significantly improved cell activity, showing a clear advantage over BT-8009.

[0147] III. In vivo efficacy testing of peptide-conjugated drugs HZ-CP01, HZ-CP02, HZ-CP08 and BT-8009

[0148] Test compound preparation: The test compound was prepared according to the method described in CN 112566651 A (Example 1, paragraph 0494).

[0149] Balb / c nude mice aged 6-8 weeks and weighing 18-22g were selected, and each mouse was inoculated with MBA-MD-468 cells (5×10⁻⁶). 6 When the tumor grows to 150-200mm... 3Mice were randomly divided into groups of five. The administration regimen was twice-weekly injections for three weeks. Tumor volume was measured every three days starting on day 7 after the first administration, and observation continued until day 21.

[0150] Experimental results showed that HZ-CP01, HZ-CP02 and HZ-CP08 exhibited good tumor inhibition effects in vivo.

[0151] All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated herein by reference in their entirety.

[0152] While this disclosure has been illustrated and described with reference to certain preferred embodiments, those skilled in the art should understand that the above description is a further detailed explanation of the disclosure in conjunction with specific embodiments, and should not be construed as limiting the specific implementation of this disclosure to these descriptions. Various changes in form and detail can be made by those skilled in the art, including some simple deductions or substitutions, without departing from the spirit and scope of this disclosure.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, wherein, The amino acid sequence of the polypeptide comprises or consists of the following general formula (I) or the reverse sequence of general formula (I): In-X1-X2-X3-In-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -What(I), In the formula, X1 is Pro; X2 is 1Nal or 2Nal; X3 is D-Asp or D-Lys; X4 is Met; X5 is hArg; X6 is Asp or His; X7 is Trp or 5fW; X8 is S; X9 is Thr; X 10 For Pro; X 11 For Hyp;X 12 For Trp; Ci, Cii, and Ciii are Cys or hCys.

2. The polypeptide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is selected from the group consisting of: Ci-P1-[1Nal]2-[dD]3-Cii-M4-[HArg]5-D6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -Ciii(SEQ ID NO:1): C i -P1-[2Nal]2-[dD]3-C ii -M4-[HArg]5-D6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 2); C i -P1-[1Nal]2-[dD]3-C ii -M4-[HArg]5-D6-[5fW]7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO:3); Ci-P1-[2Nal]2-[dD]3-Cii-M4-[HArg]5-D6-[5fW]7-S8-T9-P 10 -[HyP] 11 -W 12 -Ciii(SEQ ID NO:4)? C i -P1-[1Nal]2-[dK]3-C ii -M4-[HArg]5-H6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 5); and C i -P1-[Nal]2-[dD]3-C ii -M4-[HArg]5-H6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO:6)。 3. A bicyclic peptide comprising the polypeptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof and a cyclic peptide scaffold, preferably, the cyclic peptide scaffold being TATA with the structural formula [insert structural formula here]. The olefin double bond of the cyclic peptide scaffold is linked to the cysteine ​​residue of the polypeptide or its pharmaceutically acceptable salt to form a bicyclic system.

4. The bicyclic peptide according to claim 3, wherein, The bicyclic peptide targets nectin-4.

5. The bicyclic peptide according to claim 3 or 4, wherein, The structure of the bicyclic peptide is shown in formula (T1), (T2), (T3), or (T4):

6. A polypeptide-conjugated drug comprising a bicyclic peptide according to any one of claims 3 to 5 and a polytoxic group containing two or more toxins, preferably wherein the bicyclic peptide and the polytoxic group are linked by a linker.

7. The polypeptide conjugate according to claim 6, wherein, The linker is a polymeric sarcosine or PEG or a mixture thereof, preferably with a degree of polymerization of 0-20; more preferably with a degree of polymerization of 0-10, and most preferably with a degree of polymerization of 5-10; Preferably, the toxin is a microtubule inhibitor (such as melphalan MMAE), a camptothecin Top1 inhibitor (such as Camptothecin, SN-38, Irinotecan, icietecan), anthracyclines (such as Doxorubicin, Daunorubicin), paclitaxel, platinum-based chemotherapy drugs (such as cisplatin, carboplatin), diazacyclic chemotherapeutic agents (such as Pemetrexed), folded protease inhibitors (such as Bortezomib), rutin drugs (such as Vincristine, Vinblastine), radioisotope-labeled toxins, liposome-encapsulated chemotherapy drugs, etc.

8. The polypeptide conjugate according to claim 6 or 7, wherein, The bicyclic peptide and linker are linked in the following manner: bicyclic peptide-Sar10-β-Ala-*1; where *1 is the linker site; the amino group on the Ci residue in the bicyclic peptide is linked to the carboxyl group of Sar10.

9. The polypeptide-conjugated drug according to any one of claims 6 to 8, wherein, The linker includes a spacer, which is -PABC-Cit-Val- or -PABC-cyclobutyl-Ala-Cit-, such as -PABC-Cit-Val-, where PABC represents p-aminobenzylcarbamate; -GGFG-; Preferably, the spacer and the toxin have the following connection methods: ①MMAE-PABC-Cit-Val-*2; or ②MMAE-PABC-cyclobutyl-Ala-Cit-*2; wherein the *2 position is connected to the carboxyl group of aspartic acid or glutamic acid, and the two ends of the carboxyl group are connected to two ①, two ②, or ① and ② respectively, thereby forming a structure including:

10. The polypeptide-conjugated drug according to any one of claims 6 to 9, wherein, The spacer and toxin have the following connection modes: ① icietecan-GGFG-*2; or ② icietecan-PEGn-*2, n = 3-10; wherein *2 is connected to the carboxyl group of aspartic acid or glutamic acid, and the carboxyl group is connected to two ①, two ②, or ① and ② respectively, thereby forming a structure including:

11. The polypeptide-conjugated drug according to any one of claims 6 to 10, wherein, The polypeptide-conjugated drug has the following structure:

12. A pharmaceutical composition comprising any one of claims 6 to 11, a polypeptide-conjugated drug and / or its deuterated form, and a pharmaceutically acceptable salt.

13. The polypeptide-conjugated drug according to any one of claims 6 to 11, wherein, The peptide-conjugated drug is used to treat related diseases, including, for example, bladder cancer, breast cancer, ovarian cancer, colorectal cancer (CRC), prostate cancer, and lung cancer.

14. Use of the polypeptide-conjugated drug of any one of claims 6 to 11 in the preparation of a pharmaceutical composition for treating diseases of Nectin-4 overexpression, for example, said disease being cancer, including bladder cancer, breast cancer, ovarian cancer, colorectal cancer (CRC), prostate cancer, and lung cancer, etc.

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