Her2-targeting polypeptide-drug conjugate, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biomedicine, and particularly relates to an HER2-targeting polypeptide-drug conjugate, and a preparation method therefor and the use thereof. The polypeptide-drug conjugate provided in the present invention has good in-situ self-assembly performance in vivo, can bind to HER2 and increase the drug concentration in a tumor area by means of self-assembly at the target site, promotes drug endocytosis, and releases the drug in response to enzyme cleavage in lysosomes, thereby treating tumors having either high or low HER2 expression. In addition, the HER2-targeting polypeptide-drug conjugate provided in the present invention has the property of overcoming HER2 ADC drug tolerance.

Description

Polypeptide conjugate drugs targeting HER2 and preparation method and application thereof

[0001] Cross-reference

[0002] This application claims priority to Chinese Patent Application No. 2024108574949 entitled "Polypeptide Conjugate Drugs Targeting HER2 and Preparation Method and Application Thereof" filed on June 28, 2024, the entire disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, and in particular to a polypeptide conjugate drug targeting HER2 and a preparation method and application thereof. BACKGROUND

[0004] Cancer is the number one killer that threatens human health. Surgery can remove early-stage localized tumors, but it has little effect on advanced or systemic cancer. Radiotherapy and chemotherapy can cause side effects such as hair loss and vomiting. Targeted drugs are the star of the anti-tumor drug market. One of the main features of targeted drugs is that they act on specific targets. Each patient's condition is different, and the targeted drugs available for each patient are also different, to some extent achieving individualized treatment of tumors. From the demand trend of drugs, obvious efficacy and small side effects are the main demand direction of future product development. Under the driving of such market demand, the research and development and clinical application of targeted anti-tumor drugs will be one of the main development directions of the anti-tumor drug industry in the future.

[0005] The polymerization of human epidermal growth factor receptor 2 (HER2) leads to the phosphorylation of tyrosine residues and initiates multiple signaling pathways leading to cell proliferation and cancer. According to clinical statistics, the proportion of HER2-positive cancer mainly includes: breast cancer about 20-30%; gastric cancer about 10-30%; esophageal cancer about 17%; ovarian cancer about 20-30%; endometrial cancer about 21-47%; lung cancer about 20%; bladder cancer 12%, etc. In recent years, a variety of HER2-targeted drugs have been approved globally, including monoclonal antibodies, small molecule inhibitors and ADCs, which have improved the prognosis and quality of life of HER2-positive patients. Monoclonal antibodies include trastuzumab, pertuzumab, etc., whose molecular mechanism is to combine with HER2 receptor, prevent the formation of HER2 dimers, or carry out immune response, inhibit the decomposition of HER2, and finally destroy the downstream signaling pathway; small molecule tyrosine kinase inhibitors include lapatinib, afatinib and neratinib, which combine with HER2 extracellular tyrosine kinase to inhibit the phosphorylation of tyrosine kinase, thereby inhibiting the activity of the downstream signaling pathway; the mechanism of action of ADC drugs is to form a complex with the extracellular structure of HER2, induce endocytosis, and then make the cytotoxic drugs work. Currently, there are three HER2 ADC products approved for marketing globally, and Kadcyla is the first ADC drug approved for marketing for the treatment of solid tumors and the first anti-HER2 ADC product. Enhertu has become a new benchmark for anti-HER2 ADCs due to its outstanding performance in clinical trials. Videliximab (Aidixi, RC48) of Rongchang Biotech is the first domestic HER2 ADC. The wide indications and large patient population of HER2 ADCs mean that patients will inevitably develop resistance to HER2 ADCs as the medication time progresses, resulting in reduced or ineffective treatment. In order to cope with the resistance of HER2 ADC, further improvement of drug design is needed to inhibit drug resistance mutations and develop new targeted therapies.

[0006] Polypeptide conjugated drugs are to connect the targeting polypeptide and the anticancer agent with biological activity by using a specific linker, and to reduce the killing effect of the anticancer agent on normal cells by using the targeting of the polypeptide, and to enhance the antitumor effect. PDC has the characteristics of strong targeting, high specificity and low toxicity, and compared with ADC, the process is simple, the cost is low, and the economic burden of patients can be greatly reduced, so it is one of the hotspots of current biological and medical research. PDC is used to treat various diseases in clinical or preclinical research, for example, CN108135881B and CN106466485B disclose corresponding polypeptide drugs, but these are only linear polypeptides composed of chemotherapeutic drugs and natural amino acids, or linear polypeptides (or ligands) composed of two natural amino acids, which have poor stability and short half-life. Compared with antibody conjugated drugs, PDC has a smaller molecular weight and a shorter blood circulation time, resulting in poor effective enrichment effect. How to further improve the enrichment of PDC in tumor cells is a problem to be solved.

[0007] Therefore, the present application is provided. SUMMARY

[0008] To solve the above technical problems, the present application provides a HER2-targeting polypeptide conjugated drug, a preparation method and application thereof.

[0009] Specifically, the technical solutions of the present application are as follows:

[0010] In a first aspect, the present application provides a HER2-targeting polypeptide conjugated drug, and the structural formula is one of the following ①-④:

[0011] ①: P1-P2-L3-D;

[0012] ②: P1-P2-L2-L3-D;

[0013] ③: P1-L1-P2-L3-D;

[0014] ④: P1-L1-P2-L2-L3-D;

[0015] Wherein, P1 is a HER2 targeting unit; P2 is a self-assembly unit; D is an anti-tumor functional unit; L1, L2 and L3 are connecting units.

[0016] Wherein, L3 is selected from a peptide linker with lysosomal cleavage function.

[0017] Preferably, the self-assembly unit is a self-assembly polypeptide P2 selected from any one or a combination of at least two of polypeptide sequences NQFNLM, QILLWS, YYQNYQ, NFVNYS, GNNQQNY, ITSVV, FGFDP, YFTEF, ISDNL, ARVHVSE, KLYKVS, SVSLA, DCFILDH (described in the order of N-terminal to C-terminal amino acid sequence); wherein any amino acid can be selected from L-type or D-type.

[0018] More preferably, the self-assembly polypeptide P2 is selected from any one or a combination of at least two of polypeptide sequences GNNQQNY, ITSVV, FGFDP, YFTEF, ISDNL, ARVHVSE, KLYKVS, SVSLA, DCFILDH (described in the order of N-terminal to C-terminal amino acid sequence); wherein any amino acid can be selected from L-type or D-type.

[0019] The polypeptide conjugate provided by the present application has good in vivo in situ self-assembly performance, can bind to HER2 and increase the drug concentration in the tumor region by self-assembly at the target site, promote drug endocytosis, and release the drug by enzymatic cleavage in lysosomes to treat tumors with high or low expression of HER2.

[0020] Further preferably, L2 is selected from the following formula I or formula II:

[0021] In formula I or formula II, Aaa1 is 0-3 His, Aaa2 is 0-3 Ser, and p and q are respectively an integer in the range of 1-8;

[0022] More preferably, p and q are respectively an integer in the range of 2-6.

[0023] Further preferably, L3 is selected from any one or a combination of at least two of Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-PAB, Gly-Gly-Phe-Gly.

[0024] Further preferably, L1 is selected from the following formula III or formula IV:

[0025] In formula III or formula IV, m and n are respectively an integer in the range of 1-8;

[0026] More preferably, m and n are respectively an integer in the range of 2-6.

[0027] Further preferably, the HER2 targeting unit is a polypeptide targeting HER2 protein; the polypeptide is a linear peptide or a cyclic peptide consisting of 5-50 amino acids; wherein any amino acid can be selected from L type or D type.

[0028] More preferably, the polypeptide is a linear peptide or a cyclic peptide consisting of 7-20 amino acids.

[0029] More preferably, the polypeptide is selected from any one of polypeptide sequences CDGFYAC, YCDGFYACYMDV, FCDGFYACYMDV, YCDGFACYMDV, FCDGFACYMDV, CGPLPVDWYWC, CEWKFDPGLGQARC, CDYMTDGRAASKIC, KCCYSL, MARSGL, MCGVCLSAQRWT, SGLWWLGVDILG or a combination of at least two thereof; wherein any amino acid can be selected from L type or D type.

[0030] Further preferably, the anti-tumor functional unit is selected from a cytotoxic drug having a cancer treatment function; the cytotoxic drug is selected from at least one of a tubulin inhibitor, a DNA topoisomerase inhibitor, a DNA damaging agent, an anti-metabolite drug;

[0031] More preferably,

[0032] The tubulin inhibitor is selected from at least one of an auristatin derivative, a maytansine derivative, a taxol derivative;

[0033] And / or, the DNA topoisomerase inhibitor is selected from a camptothecin derivative;

[0034] And / or, the DNA damaging agent is selected from at least one of a calicheamicin derivative, an anthramycin derivative;

[0035] And / or, the anti-metabolite drug is selected from at least one of methotrexate, 5-fluorouracil.

[0036] More preferably, the cytotoxic drug is selected from an auristatin derivative and / or a camptothecin derivative.

[0037] The polypeptide conjugate drug obtained by the preferred scheme of the present application has stronger self-assembly ability, and after the formation of an assembly on the cell membrane by the HER2 receptor, it can be effectively internalized into the cell to play an anti-tumor role, has good tumor cell activity inhibition effect, and has high safety.

[0038] In a second aspect, the present application provides a preparation method of the polypeptide conjugate drug targeting HER2 as described in the first aspect above, which comprises the steps of resin activation, P1 amino acid condensation, L1 condensation, P2 amino acid condensation, L2 condensation, L3-D coupling and cyclization in sequence.

[0039] In a third aspect, the present application provides a derivative of the polypeptide conjugate targeting HER2 as described in the first aspect, wherein the derivative is a pharmaceutically acceptable salt or solvate of the polypeptide conjugate.

[0040] In a fourth aspect, the present application provides a pharmaceutical composition comprising the polypeptide conjugate targeting HER2 as described in the first aspect or the derivative as described in the third aspect.

[0041] In a fifth aspect, the present application provides a pharmaceutical preparation comprising the polypeptide conjugate targeting HER2 as described in the first aspect or the derivative as described in the third aspect, and a pharmaceutically acceptable carrier.

[0042] Preferably, the pharmaceutical preparation is in the form of a lyophilized powder, an injection, a tablet, a liposome or a nano-preparation.

[0043] In a sixth aspect, the present application provides use of the polypeptide conjugate targeting HER2 as described in the first aspect or the derivative as described in the third aspect in the preparation of an antitumor drug.

[0044] The tumor is a tumor with high or low expression of HER2.

[0045] Preferably, the tumor is at least one of breast cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, and skin cancer.

[0046] In a seventh aspect, the present application provides use of the polypeptide conjugate targeting HER2 as described in the first aspect or the derivative as described in the third aspect or the pharmaceutical composition as described in the fourth aspect or the pharmaceutical preparation as described in the fifth aspect in the prevention or treatment of a tumor disease.

[0047] The tumor disease is a tumor disease with high or low expression of HER2.

[0048] Preferably, the tumor disease is at least one of breast cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, and skin cancer.

[0049] Preferably, the use is in combination with one or more antitumor drugs. Beneficial effects:

[0050] The present application provides a polypeptide conjugate drug targeting HER2 and a preparation method and application thereof. The polypeptide conjugate drug provided by the present application has good in vivo in situ self-assembly performance, can bind to HER2 and improve the drug concentration in the tumor area through self-assembly at the target point, promote drug endocytosis, release the drug through enzyme cutting in lysosomes in response, and thus treat tumors with high or low expression of HER2. Meanwhile, the polypeptide conjugate drug targeting HER2 provided by the present application has the characteristic of overcoming HER2 ADC drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the present application or prior art, the drawings needed to be used in the embodiments or prior art description will be described below.

[0052] Fig. 1 is a scanning electron microscope imaging diagram of PDC-1 in different cell surfaces in the embodiment 5 of the present application, wherein the left side is MDA-MB-231 cells and the right side is NCI-N87 cells.

[0053] Fig. 2 is a confocal imaging diagram of PDC-FITC in NCI-N87 cells in the embodiment 5 of the present application.

[0054] Fig. 3 is the cell toxicity test result of PDC-1 on Enhertu drug resistant cell lines in the embodiment 6 of the present application. DETAILED DESCRIPTION

[0055] The present application provides a polypeptide conjugate drug targeting HER2 or a pharmaceutically acceptable salt or solvate thereof, which is designed by using in vivo in situ self-assembly technology, can bind to HER2 and improve the drug concentration in the tumor area through self-assembly at the target point, promote drug endocytosis, release the drug through enzyme cutting in lysosomes in response, and thus treat tumors with high or low expression of HER2. The polypeptide conjugate drug provided by the present application has four structural units of HER2 targeting peptide, self-assembly peptide, enzyme responsive peptide and toxin drug, enters the tumor site after blood circulation, activates the formation of oligomers on the HER2 receptor of tumor cell surface, improves the drug enrichment and retention and increases the receptor-mediated internalization at the same time; after the polypeptide conjugate drug is internalized into lysosomes, the cytotoxic drug molecules can be slowly released, further achieving the retention and slow release of cytotoxic drugs in tumor cells, and significantly improving the anti-tumor effect. Meanwhile, the polypeptide conjugate drug targeting HER2 provided by the present application has the characteristic of overcoming HER2 ADC drug resistance.

[0056] The present application first discloses the general formula of the polypeptide conjugate drug targeting HER2, which includes HER2 targeting peptide, self-assembly peptide, enzyme responsive peptide and toxin drug, and each component is connected by covalent bond or linking group, and the structural formula is P1-L1-P2-L2-L3-D.

[0057] In the present application, P1 is a HER2 targeting peptide (a polypeptide targeting HER2 protein).

[0058] In the present application, the linking group L1 is absent, or is selected from the following formula III or formula IV:

[0059] In formula III or formula IV, m and n are respectively an integer from 1 to 8.

[0060] Preferably, m is an integer from 2 to 6, and n is an integer from 2 to 6.

[0061] In the present application, P2 is a self-assembling polypeptide with intramolecular multiple hydrogen bonds.

[0062] In some embodiments, the self-assembling polypeptide P2 amino acid sequence used in the polypeptide conjugate drug of the present application can be selected from: NQFNLM, QILLWS, YYQNYQ, NFVNYS, GNNQQNY, ITSVV, FGFDP, YFTEF, ISDNL, ARVHVSE, KLYKVS, SVSLA, DCFILDH; wherein any amino acid can be selected from L type or D type.

[0063] The linking group L2 is selected from the following formula I or formula II:

[0064] In formula I or formula II: Aaa1 is 0-3 His, Aaa2 is 0-3 Ser, and p and q are respectively an integer in the range from 1 to 8.

[0065] L3 is a peptide linker with lysosomal cleavage function.

[0066] D is a cytotoxic drug (toxin drug) for cancer treatment.

[0067] In some embodiments, the polypeptide P1 amino acid sequence targeting HER2 protein used in the polypeptide conjugate drug of the present application is: YCDGFYACYMDV, CDGFYAC, YCDGFACYMDV or FCDGFACYMDV, wherein any amino acid can be selected from L type or D type.

[0068] In some embodiments, the linking group L1 used in the polypeptide conjugate drug of the present application can be selected from the following formula III or formula IV:

[0069] In formula III or formula IV, m is an integer from 2 to 6, and n is an integer from 2 to 6.

[0070] In some embodiments, the linker L2 used in the polypeptide conjugate drug of the present application can be selected from the following formula V, formula VI, formula VII, formula VIII, formula VIIII and formula X:

[0071] Any amino acid in formula V, formula VI, formula VII, formula VIII, formula VIIII and formula X can be selected from L-form or D-form, p is an integer from 2 to 6, q is an integer from 2 to 6.

[0072] In some embodiments, the peptide linker L3 having lysosomal cleavage function used in the polypeptide conjugate drug of the present application can be selected from valine-citrulline (Val-Cit-PAB), valine-alanine (Val-Ala-PAB), alanine-alanine (Ala-Ala-PAB), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0073] The present application also discloses a pharmaceutical composition comprising the polypeptide conjugate drug targeting HER2 provided by the present application or a pharmaceutically acceptable salt or solvate thereof.

[0074] In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly, parenterally or intraventricularly.

[0075] The present application also discloses a disease treatment method, which comprises administering to a patient in need a therapeutically effective amount of the conjugate compound provided by the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition provided by the present application.

[0076] In some embodiments, the disease is selected from a tumor with high or low expression of HER2.

[0077] In some embodiments, the tumor with high or low expression of HER2 is selected from breast cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer.

[0078] In some embodiments, the method provided by the present application further comprises administering one or more therapeutic agents in combination with the conjugate compound provided by the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition provided by the present application. Preferably, the therapeutic agent targets an anti-cancer therapeutic target, induces or enhances an immune response against cancer, or is a chemotherapeutic agent.

[0079] The present application also discloses a pharmaceutical preparation comprising the polypeptide conjugate drug of the present application or a pharmaceutically acceptable salt or solvate thereof. Preferably, the pharmaceutical preparation is in the form of a lyophilized powder, an injection solution, a tablet, a liposome, a nano-preparation.

[0080] The application also discloses a preparation method of the polypeptide conjugated drug targeting HER2, and the steps are as follows:

[0081] (1) Resin activation: the resin is put into a polypeptide solid-phase synthesis tube, and is activated after swelling.

[0082] (2) P1 amino acid condensation: 3-5 times equivalent of amino acids and condensing agents are added into the polypeptide solid-phase synthesis tube according to the amino acid sequence of the polypeptide in sequence, and a peptide resin after condensation of the first amino acid is obtained; the above "Fmoc deprotection-amino acid condensation" reaction steps are repeatedly performed until the last amino acid of the P1 part is reacted.

[0083] (3) L1 condensation: after the peptide resin is removed from the protection group, the Fmoc-protected L1 is continuously condensed.

[0084] (4) P2 amino acid condensation: the peptide resin repeatedly performs the "Fmoc deprotection-amino acid condensation" reaction steps until the last amino acid of the P2 part is reacted.

[0085] (5) L2 condensation: the peptide resin repeatedly performs the "Fmoc deprotection-amino acid condensation" reaction steps until the last amino acid of the L2 part is reacted.

[0086] (6) L3-D coupling: the peptide resin is directly coupled with the amino-protected L3-D part.

[0087] (7) Cyclization: the synthesized peptide resin is subjected to cleavage in a cleavage solution to obtain a crude peptide, and the polypeptide conjugated drug targeting HER2 is prepared after purification by preparative HPLC.

[0088] The polypeptide conjugated drug has a self-assembly capacity on the cell membrane surface, can be combined with the HER2 receptor on the cell membrane surface, and is assembled on the cell membrane surface to promote endocytosis of the drug. Compared with a relatively mature ADC therapy, the application provides a novel polypeptide conjugated drug. Based on the "in vivo in-situ self-assembly" technology, the HER2 targeting unit, the self-assembly unit and the enzyme response unit are integrated into the conjugated drug capable of carrying a cell toxin by using a modular design concept. The application integrates various advantages, improves the targeting effect, realizes enrichment of the drug at the target point, enhances the assembly stability, increases the uptake of the drug by cells and realizes drug slow release. Meanwhile, the polypeptide conjugated drug targeting HER2 provided by the application has the potential to overcome HER2 ADC drug resistance.

[0089] The term "HER2 targeting peptide" used in the application refers to a polypeptide having specific binding affinity to the selected HER2 target.

[0090] In some embodiments, the HER2 targeting peptide binds to the cell surface HER2 receptor with an affinity of 10 -6 ~10 -9 (Kd value).

[0091] In some embodiments, the HER2 targeting peptide is selected from a linear peptide or a cyclic peptide.

[0092] In some embodiments, the HER2 targeting peptide is preferably selected from a cyclic peptide.

[0093] In some embodiments, the HER2 targeting peptide has a length of 5 to 50 amino acids.

[0094] In some embodiments, the HER2 targeting peptide preferably has a length of 10 to 20 amino acids.

[0095] As used herein, the term "toxin drug" refers to a chemotherapeutic drug used to alleviate or treat cancer.

[0096] In some embodiments, the toxin drug (cytotoxic drug D used in the present application for cancer treatment) can be selected from a microtubulin inhibitor, a DNA topoisomerase inhibitor, a DNA damaging agent, and an antimetabolite.

[0097] In some embodiments, the microtubulin inhibitor can be selected from an Auristatin derivative, a Mertansine derivative, and a Taxol derivative.

[0098] In some embodiments, the DNA topoisomerase inhibitor can be selected from a Camptothecin derivative.

[0099] In some embodiments, the DNA damaging agent can be selected from a Calicheamicin derivative and an Anthramycin derivative PBD.

[0100] In some embodiments, the antimetabolite can be selected from methotrexate and 5-fluorouracil.

[0101] In some more preferred embodiments, the toxin drug is selected from an Auristatin derivative and a Camptothecin derivative.

[0102] As used herein, the term "self-assembly" refers to a technique in which basic molecular building blocks spontaneously form ordered structures. In the process of self-assembly, basic molecular building blocks spontaneously organize or aggregate into a stable, regularly geometrically appearing structure (nano, micro, etc.) based on non-covalent bond-based interactions.

[0103] The term "in vivo self-assembly in situ" as used herein refers to the introduction of exogenous molecules into a specific physiological and pathological environment, which form nanomaterials through intermolecular forces such as π-π stacking, electrostatic interaction, etc. Such materials have good biocompatibility and controllability, and can form nanoparticles, fibers and gels, etc. to play specific biological functions. The self-assembling molecules used in the present application are self-assembling polypeptides.

[0104] In some embodiments, the self-assembling polypeptides used in the present application have a length of 3 to 20 amino acids. Preferably, the self-assembling polypeptides of the present application comprise at least 1 hydrophobic amino acid.

[0105] The term "coupled" as used herein refers to the covalent linkage of two chemical groups, which can be a direct covalent bond between the two chemical groups, or an indirect linkage between the two chemical groups through a linker.

[0106] In some embodiments, the conjugate molecules provided by the present application comprise only a single toxin drug coupled to a plurality of cell interaction molecules.

[0107] In some embodiments, the conjugate molecules provided by the present application comprise a plurality of toxin drugs coupled to a plurality of cell interaction molecules.

[0108] The term "linking group" as used herein refers to a molecule or moiety that covalently links a toxin drug to a cell interaction targeting group.

[0109] In some embodiments, the linking group can contain two reactive functional groups, one for linking to the toxin drug and the other for linking to the assembly group or the targeting group.

[0110] In some embodiments, the reactive functional groups are different from each other.

[0111] In some embodiments, the functional groups comprise groups containing carboxyl reactive moieties and amine reactive moieties.

[0112] In some embodiments, the functional groups are the same as each other.

[0113] In the present application, the linking group should be stable enough to avoid accidental release of the toxin drug during blood circulation, to increase the effective amount of the toxin drug to the target cells or tissues and to avoid toxicity. At the same time, the linking group should be able to release the payload around or inside the target cells to effectively kill the target cells or block the function of the target cells.

[0114] In some embodiments, the linking group comprises at least one cleavable functional group. Preferably, the cleavable functional group is stable enough outside the target cells, but it cleaves to release the toxin drug after entering the target cells.

[0115] In some embodiments, the linker is a peptidic linker, which consists of straight or branched chain amino acids connected by peptide bonds.

[0116] In some embodiments, the peptidic linker can be cleaved by a protease that is highly or specifically expressed around or in the target cell, such as cathepsin B in lysosomes or endosomes.

[0117] The length of the peptidic linker used in the present application can be various. Preferably, the length of the peptidic linker of the present application is 1 to 10 amino acids.

[0118] In some embodiments, the length of the peptidic linker is preferably 2, 3, 4, or 5 amino acids.

[0119] In some embodiments, the peptidic linker with lysosomal cleavage function can be selected from: valine-citrulline (Val-Cit-PAB), valine-alanine (Val-Ala-PAB), alanine-alanine (Ala-Ala-PAB), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0120] In some embodiments, the polypeptide conjugate drug of the present application is a combination of 3 or 4 components selected from the above-mentioned HER2 targeting peptide, self-assembling peptide, enzyme-responsive peptide, and toxin drug module.

[0121] In some embodiments, the polypeptide conjugate drug of the present application is preferably compounds PDC-1 to PDC-16, the structural composition of which is shown in Table 1 and Table 2.

[0122] The amino acid sequences mainly involved in the present application are as follows:

[0123] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application. If not specifically indicated, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used can be obtained from commercial channels.

[0124] Example 1

[0125] This example provides a series of polypeptide conjugate drugs (PDC-1 to PDC-16, PDC-FITC), the molecular structure of which is P1-L1-P2-L2-L3-D, and the specific molecular composition is shown in Table 1.

[0126] Table 1: Molecular composition of polypeptide conjugate drugs Table 1: Molecular composition of polypeptide conjugate drugs

[0127] The present embodiment also provides a general synthetic method for the polypeptide conjugate drugs described above, which is used to synthesize the corresponding polypeptide conjugate drugs according to the standard polypeptide solid-phase synthesis method (SPPS). The general synthetic method is as follows:

[0128] 1. Preparation of experimental instruments and materials:

[0129] Dimethylformamide (DMF), piperidine, Rink amide resin, dichloromethane (DCM), ninhydrin reagent (ninhydrin and phenol), 1-hydroxybenzotriazole (HOBT), N,N'-diisopropylcarbodiimide (DIC), hexahydropyridine, triisopropylsilane (TIS), anhydrous ethanol, anhydrous ether, trifluoroacetic acid (TFA), methanol, Fmoc-alanine (Fmoc-Ala-OH), Fmoc-cysteine (Fmoc-Cys(Mob)-OH), Fmoc-aspartic acid (Fmoc-Asp(OtBu)-OH), Fmoc-glutamic acid (Fmoc-Glu(OtBu)-OH), Fmoc-phenylalanine (Fmoc-Phe-OH), Fmoc-glycine (Fmoc-Gly-OH), Fmoc-histidine (Fmoc-His(Trt)-OH), Fmoc-lysine (Fmoc-Lys(Boc)-OH), Fmoc-leucine (Fmoc-Leu-OH), Fmoc-asparagine (Fmoc-Asn(Trt)-OH), Fmoc-proline (Fmoc-Pro-OH), Fmoc-arginine (Fmoc-Arg(Pbf)-OH), Fmoc-serine (Fmoc-Ser(tBu)-OH), Fmoc-threonine (Fmoc-Thr(tBu)-OH), Fmoc-valine (Fmoc-Val-OH), Fmoc-tryptophan (Fmoc-Trp-OH), Fmoc-tyrosine (Fmoc-Tyr(tBu)-OH), NH2-OEG6-COOH, Fmoc-PEG4-NHS ester, glutaric anhydride, VC-PAB-MMAE, polypeptide solid-phase synthesis tube, etc.

[0130] 2. Preparation of experimental solutions:

[0131] Deprotection solution: mix hexahydropyridine and DMF in a volume ratio of 1:4;

[0132] Lysis solution: mix TFA, TIS and H2O, and the volume fractions of the mixed solutions are 92.5% TFA, 2.5% TIS and 2.5% H2O;

[0133] Ninhydrin test solution: one drop of ninhydrin and one drop of phenol.

[0134] 3. Specific operation steps:

[0135] (1) Fmoc (fluorenylmethyloxycarbonyl) deprotection: weigh 2.0 g Rink amide resin and put into the polypeptide solid-phase synthesis tube, add DMF to swell for 30 min. Remove DMF, and use deprotection solution for Fmoc deprotection reaction, and place on a shaker for 10 min. Remove the deprotection solution, and wash with DMF and DCM for 3 times. Take 10 mg Rink amide resin from the polypeptide solid-phase synthesis tube into a test tube, and wash with ethanol for 2 times. If it is dark blue after ninhydrin test, it is a positive result, and then the first amino acid is connected, and the amino acid condensation reaction is carried out.

[0136] (2) Amino acid condensation: according to the amino acid sequence, the first amino acid is connected to the resin. 3-5 equivalents of amino acid, DIEA, HOBT and DIC are respectively dissolved in 30 mL DMF, activated at 0°C for 5 min, and then put into the polypeptide solid-phase synthesis tube, and stirred to react. After 1 h, 10 mg Rink amide resin is taken from the polypeptide solid-phase synthesis tube into a test tube, and washed with ethanol for 2 times. If it is not colorless after ninhydrin test, it is a negative result, which proves that the condensation reaction is successful. Remove the liquid in the polypeptide solid-phase synthesis tube, and wash with DMF and DCM for 2 times to obtain the peptide resin after condensation of the first amino acid.

[0137] (3) Repeat the above "Fmoc deprotection-amino acid condensation" reaction steps for the obtained peptide resin until the last amino acid of the P1 module is reacted. Add the deprotection solution to remove the Fmoc protecting group of the amino group. Then add NH2-OEG6-COOH or Fmoc-PEG4-NHS ester to couple the hydrophilic module. Then continue the process of (2) to synthesize P2 and L2 modules.

[0138] (4) Repeat the above "Fmoc deprotection-amino acid condensation" reaction steps for the obtained peptide resin until the last amino acid of the L2 module is reacted. Add VC-PAB-MMAE or GGFG-DXD to couple the enzyme cutting and payload modules.

[0139] After the reaction is completed, wash the resin with DMF and DCM for 3 times, and with methanol for 2 times, and continue to dry for 20 min. Take the synthesized peptide resin from the polypeptide solid-phase synthesis tube, and lyse in the lysis solution for 2 h at room temperature. The lysis solution is first ice-bathed for 20 min. After the resin is filtered, it is evaporated to dryness on a rotary evaporator, and washed with anhydrous ether for 3 times under ice-bath condition. The crude peptide is purified by preparative reverse-phase HPLC, and the purity is detected by HPLC to be >94.7%. The obtained pure peptide is identified by mass spectrometry (MS, electrospray), and the measured molecular weight result is the same as the target molecular weight.

[0140] Example 2

[0141] This example takes PDC-1 as an example to provide the specific structural formula and synthesis method of PDC-1.

[0142] 1. The structural formula of PDC-1 is as follows:

[0143] 2. The synthesis method of PDC-1 is as follows:

[0144] Step I: Synthesis of fragment P1 protected peptide resin

[0145] Rink amide resin (5.0 g, degree of substitution: 1.2 mmol / g) was weighed into a solid-phase polypeptide synthesis tube, DMF was added to the shaker for 30 min, and then the DMF was removed. The Fmoc protecting group was removed with 20% piperidine in DMF (10 min*2), and the resin was washed with DMF 6 times. In a separate Erlenmeyer flask, Fmoc-Val-OH (8.0 g, 24.0 mmol), HOBt (3.2 g, 24.0 mmol) and DIEA (5.9 ml, 36.0 mmol) were dissolved in DMF and cooled to 0°C using an ice water bath, then DIC (3.7 ml, 24.0 mmol) was added and reacted for 5 min. The solution was added to the polypeptide synthesis tube and shaken for 5 hours. After the reaction was completed, the resin was washed with DMF 5 times.

[0146] Acetic anhydride (4 ml) and DIEA (4 ml) were dissolved in DMF (92 ml) to form a capping reagent. The capping reagent was added to the washed resin described above, and the capping was carried out at room temperature for 30 min on the shaker. The capping operation was repeated twice. After capping, the resin was washed with DMF 5 times, methanol was used for shrinkage for 20 min, and finally washed with methyl tert-butyl ether (MTBE) 3 times. The solvent was removed by suction, and Fmoc-Val-rink amide resin (6.3 g) was obtained. The degree of substitution was detected to be 0.8 mmol / g.

[0147] Weigh 6.3 g (5.0 mmol) of Fmoc-Val-rink amide resin (degree of substitution: 0.8 mmol / g) into the polypeptide synthesis tube, wash with DMF for 3 times, then swell for 30 min, and then remove the DMF. Remove the Fmoc protecting group with 20% piperidine in DMF (10 min*2), and wash with DMF for 6 times. Weigh Fmoc-Asp(OtBu)-OH (8.2 g, 20.0 mmol) and dissolve with DMF, and cool to 0°C using an ice water bath, then add DIEA (3.8 g, 30.0 mmol), HOBt (2.6 g, 20.0 mmol) and DIC (2.5 g, 20.0 mmol) and react for 5 min. Add the solution to the polypeptide synthesis tube and react for 3 hours. The reaction is completed when the ninhydrin test is negative. After removing the solvent, wash the resin with DMF for 6 times, and then remove the Fmoc protecting group with 20% piperidine in DMF. Repeat the above operation, and sequentially add each amino acid in the order from C-terminus to N-terminus in the peptide sequence. Coupling is performed in the order of Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Mob)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Mob)-OH, Fmoc-Tyr(tBu)-OH according to the peptide sequence. Finally, remove the Fmoc protecting group with 20% piperidine in DMF, wash the solution with DMF for 6 times, shrink with methanol twice (10 min), wash with methyl tert-butyl ether (MTBE) for 3 times, and dry the solvent to obtain the P1 protected peptide resin (15.0 g).

[0148] Step II: Synthesis of fragment P1-L1

[0149] Swell the obtained P1 protected peptide resin (15.0 g, 5.0 mmol) with DMF for 30 min, and remove the solvent. Weigh Fmoc-PEG4-NHS ester (5.8 g, 10.0 mmol) into another reaction bottle, and then add 100 mL of DMF and DIEA (0.8 g, 6.0 mmol), stir for 5 min, and then add the reaction solution to the polypeptide synthesis tube and shake for 2 h. The reaction is completed when the ninhydrin test is negative. Remove the solvent, wash the resin with DMF for 6 times, and then remove the Fmoc protecting group with 20% piperidine in DMF. Wash the solution with DMF for 6 times, shrink with methanol twice (10 min), wash with methyl tert-butyl ether (MTBE) for 3 times, and dry the solvent to obtain the P1-L1 protected peptide resin (16.0 g).

[0150] Step III: Synthesis of fragment P1-L1-P2

[0151] The resulting P1-L1 protected peptide resin (16.0 g, 5.0 mmol) was swelled in a peptide synthesizer with DMF for 30 min and the solvent was removed. In a separate flask, Fmoc-Tyr(tBu)-OH (9.1 g, 20.0 mmol) was weighed out and dissolved in DMF and cooled to 0 °C using an ice water bath. DIEA (3.8 g, 30.0 mmol), HOBt (2.6 g, 20.0 mmol) and DIC (2.5 g, 20.0 mmol) were added and allowed to react for 5 min. The solution was added to the peptide synthesizer and allowed to react for 3 h. The reaction was monitored by ninhydrin test and was negative. The solvent was removed and the peptide was washed 5 times with DMF. The Fmoc protecting group was removed using 20% piperidine in DMF. The above procedure was repeated and each amino acid was added in the order of C-terminal to N-terminal in the peptide sequence. Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH were coupled in that order. After the reaction was completed, the Fmoc protecting group was removed using 20% piperidine in DMF. The solution was washed 6 times with DMF, collapsed twice with methanol (10 min), washed 3 times with methyl tert-butyl ether (MTBE), and the solvent was removed to give P1-L1-P2 protected peptide resin (19.0 g).

[0152] Step IV: Synthesis of fragment P1-L1-P2-L2

[0153] The resulting P1-L1-P2 protected peptide resin (19.0 g, 5.0 mmol) was swelled in a peptide synthesizer with DMF for 30 min and the solvent was removed. In a separate flask, Fmoc-His(Trt)-OH (12.4 g, 20.0 mmol) was weighed out and dissolved in DMF and cooled to 0 °C using an ice water bath. DIEA (3.8 g, 30.0 mmol), HOBt (2.6 g, 20.0 mmol) and DIC (2.5 g, 20.0 mmol) were added and allowed to react for 5 min. The solution was added to the peptide synthesizer and allowed to react for 3 h. The reaction was monitored by ninhydrin test and was negative. The solvent was removed and the peptide was washed 5 times with DMF. The Fmoc protecting group was removed using 20% piperidine in DMF. The above procedure was repeated and Fmoc-His(Trt)-OH was coupled twice more. After the reaction was completed, the Fmoc protecting group was removed using 20% piperidine in DMF. The solution was washed 6 times with DMF.

[0154] Fumaric acid anhydride (0.7 g, 6.0 mmol) and DIEA (0.8 g, 6.0 mmol) were weighed out and dissolved in DMF, and cooled to 0°C using an ice water bath for 5 min. The solution was added to the polypeptide synthesis tube and shaken for 3 h. The reaction was terminated when the ninhydrin test was negative. The solvent was removed, the resin was washed with DMF 5 times, and methanol was used to collapse the resin twice (10 min), methy! tert-butyl ether (MTBE) was used to wash the resin 3 times, and the solvent was removed to obtain the P1-L1-P2-L2 protected peptide resin (21.0 g).

[0155] Step V: Synthesis of fragment L3-D

[0156] Fmoc-Val-OH (50.0 g, 145.0 mmol) and HOSu (17.0 g, 145.0 mmol) were dissolved in THF (500 ml), and DCC (18.0 g, 145.0 mmol) was dissolved in 200 ml of acetonitrile, and slowly added to the reaction solution while maintaining the internal temperature at about 0°C. The reaction solution was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with THF, and the filtrate was concentrated under reduced pressure to obtain a colorless oily crude product. The oily product was directly used in the next step without purification.

[0157] Citrulline (28.0 g, 160.0 mmol) was dissolved in THF (200 ml), and a sodium bicarbonate aqueous solution (100 ml, NaHCO310.0 g, 165.0 mmol) was added. The crude product Fmoc-Val-OSu (145.0 mmol) was dissolved in DME (300 ml), and then added to the reaction solution. The reaction solution was stirred at room temperature for 24 h. After the reaction was completed, 1M HCl aqueous solution (200 ml) was added to the system, and THF was removed by concentration under reduced pressure. Methyl tert-butyl ether (500 ml) was added to the residue to make a slurry, and filtered. The filter cake was repeatedly washed with methyl tert-butyl ether to make a slurry, and filtered. Finally, the filter cake was dried at 40°C under reduced pressure for 4 h to obtain the product (51.0 g) with a yield of 65%.

[0158] An appropriate amount of molecular sieves was added to the reaction flask, Fmoc-VC-OH (51.0 g, 104.0 mmol) and PABOH (26.0 g, 208.0 mmol) were weighed out, and DCM / MeOH=2 / 1 mixed solvent (60 ml) was added to dissolve and stir, and then EEDQ (5.2 g, 208.0 mmol) was added. The reaction system was stirred at room temperature for 24 h in the dark. After the reaction was completed, the molecular sieves were removed, and white solid was collected. Methyl tert-butyl ether (500 ml) was added to make a slurry, and filtered. The filter cake was washed with methyl tert-butyl ether, and the obtained white solid was dried at 40°C under reduced pressure to obtain 45.0 g with a yield of about 72%.

[0159] Fmoc-VC-PABOH (45.0 g, 75.0 mmol) was dissolved in 150 ml of anhydrous pyridine under nitrogen protection, and the reaction system was cooled to about 0°C. PNP (43.0 g, 150.0 mmol) was dissolved in 500 ml of DCM, and then slowly added to the reaction system. After the reaction system was kept at about 0°C for 10 min, the ice bath was removed, and the reaction was stirred at room temperature for 3 h. After the reaction was completed, 500 ml of EA and 200 ml of 1M aqueous HCl were added for extraction and separation, the organic layer was collected and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a light yellow oil. White solid was obtained by adding methyl tert-butyl ether, and the yield was 23.0 g, 40%.

[0160] Fmoc-VC-PAB-PNP (23.0 g, 30.0 mmol), pyridine (20.0 g, 250.0 mmol), and HOBT (8.1 g, 60.0 mmol) were weighed, added to DMF (200 ml) and stirred to dissolve, cooled to 0°C in an ice water bath, and then MMAE (14.3 g, 20.0 mmol) and DIEA (3.7 g, 36.0 mmol) were dissolved in 100 ml of DMF and stirred to dissolve. After dissolution, the above reaction system was added, and the temperature was raised to room temperature and stirred for 3 h. After the reaction was completed, Fmoc protection group was removed by adding 20% piperidine in DMF. After the reaction was completed, the reaction solution was concentrated, and then directly purified by a preparative column to obtain 11.3 g of white solid VC-PAB-MMAE, with a yield of 45%.

[0161] Step VI: Synthesis of fragment P1-L1-P2-L2-L3-D

[0162] P1-L1-P2-L2 protected peptide resin (21.0 g, 5.0 mmol) was weighed and placed in a solid-phase polypeptide synthesis tube, washed with DMF for 3 times, and shaken to swell for 30 min, and then the solvent was removed. VC-PAB-MMAE (3.4 g, 3.0 mmol), DIEA (2.6 g, 20.0 mmol), HOBT (1.3 g, 10.0 mmol), and DIC (1.2 g, 10.0 mmol) were dissolved in DMF, and then poured into the polypeptide synthesis tube, and shaken for 5 h at room temperature. After the reaction was completed, the solvent was removed, the resin was washed with DMF for 5 times, and then shrank with methanol for 2 times (10 min), washed with methyl tert-butyl ether (MTBE) for 3 times, and then the solvent was removed to obtain P1-L1-P2-L2-L3-D protected peptide resin (24.0 g).

[0163] Step VII: Cleavage of resin

[0164] TFA (285 ml), TIS (7.5 ml) and H2O (7.5 ml) were mixed and cooled to 0 °C. The P1-L1-P2-L2-L3-D protected peptide resin (24.0 g) was added to the cleavage solution and stirred at 0 °C for 3 h. The resin was removed by suction filtration and the filtrate was collected and concentrated. The crude product was washed with methyl tert-butyl ether for 3 times. The filter cake was collected and dried at 40 °C under vacuum to give a white solid crude product 11.2 g. The crude product was dissolved in DMF and directly purified by preparative HPLC. The white solid pure product 5.1 g was obtained by lyophilization.

[0165] Step VIII: disulfide bond cyclization

[0166] The P1-L1-P2-L2-L3-D (1 g, 0.24 mmol) was dissolved in methanol to give a 50 μM solution. 30% acetic acid aqueous solution was added to assist dissolution. Iodine (0.6 g, 2.4 mmol) was dissolved in methanol and added dropwise into the reaction solution in an ice water bath. The reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by HPLC. The reaction solution was concentrated and directly purified by preparative HPLC. The white solid 230 mg was obtained by lyophilization. The yield was 22%.

[0167] Example 3

[0168] This example provides the specific structural formula and synthesis method of PDC-FITC using PDC-FITC as an example.

[0169] The P1-L1-P2-L2 protected peptide resin (6 mmol) obtained after the coupling of glutaric anhydride in Example 1, Step IV was added into a solid phase peptide synthesis tube, followed by addition of DMF for swelling for 30 min. FITC (3.5 g, 9 mmol) and DIEA (2.3 g, 18 mmol) were dissolved in DMF and added into the peptide synthesis tube. The reaction was allowed to proceed for 2 h. The reaction was monitored by indole test. The reaction was completed. The resin was washed with DMF for 3 times and suctioned to dryness to give the P1-L1-P2-L2-FITC protected peptide resin.

[0170] Step 2: cleavage of the resin:

[0171] TFA (285 ml), TIS (7.5 ml) and H2O (7.5 ml) were mixed and cooled to 0 °C. The P1-L1-P2-L2-L3-D protected peptide resin (24.0 g) was added to the cleavage solution and stirred at 0 °C for 3 h. The resin was removed by suction filtration and the filtrate was collected and concentrated. The crude product was washed with methyl tert-butyl ether for 3 times. The filter cake was collected and dried at 40 °C under vacuum to give a white solid crude product 11.2 g. The crude product was dissolved in DMF and directly purified by preparative HPLC. The white solid pure product 5.1 g was obtained by lyophilization.

[0172] Step 3: disulfide cyclization

[0173] The cleaved pure P1-L1-P2-L2-FITC (1 g, 0.24 mmol) was weighed and dissolved in methanol to a concentration of 50 μM, and 30% acetic acid aqueous solution was added to assist dissolution. Iodine (0.6 g, 2.4 mmol) was weighed and dissolved in methanol, and was added dropwise into the reaction solution in an ice water bath, and was reacted at room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was concentrated and directly prepared and purified, and 230 mg of white solid was obtained after lyophilization, with a yield of 20%.

[0174] Example 4

[0175] This example takes PDC-5 as an example to provide the specific structural formula and synthesis method of PDC-5.

[0176] Step I: synthesis of fragment P1 protected peptide resin

[0177] Rink amide resin (5.0 g, degree of substitution: 1.2 mmol / g) was weighed and added to a solid-phase polypeptide synthesis tube, DMF was added to the shaker for 30 min, and then the DMF was removed. Fmoc protecting group was removed using 20% piperidine in DMF (10 min*2), and DMF was washed 6 times. In a separate Erlenmeyer flask, Fmoc-Val-OH (8.0 g, 24.0 mmol), HOBt (3.2 g, 24.0 mmol) and DIEA (5.9 ml, 36.0 mmol) were weighed and dissolved in DMF, and cooled to 0°C using an ice water bath, and then DIC (3.7 ml, 24.0 mmol) was added and reacted for 5 min. The solution was added to the polypeptide synthesis tube and shaken for 5 hours, and then washed with DMF 5 times.

[0178] Acetic anhydride (4 ml) and DIEA (4 ml) were dissolved in DMF (92 ml), and the mixture was mixed uniformly as a capping reagent. The capping reagent was added to the washed resin described above, and was capped and shaken at room temperature for 30 min, and the operation was repeated twice. After capping, DMF was used for washing 5 times, methanol was shrunk for 20 min, and finally methyl tert-butyl ether (MTBE) was washed 3 times, and the solvent was removed by suction to obtain Fmoc-Val-rink amide resin (6.3 g). The degree of substitution was detected to be 0.8 mmol / g.

[0179] Weigh 6.3 g (5.0 mmol) of Fmoc-Val-rink amide resin (degree of substitution: 0.8 mmol / g) into the polypeptide synthesis tube, wash with DMF for 3 times, then swell for 30 min, and then remove the DMF. Remove the Fmoc protecting group with 20% piperidine in DMF (10 min*2), and wash with DMF for 6 times. Weigh Fmoc-Asp(OtBu)-OH (8.2 g, 20.0 mmol) and dissolve with DMF, and cool to 0°C using an ice water bath, then add DIEA (3.8 g, 30.0 mmol), HOBt (2.6 g, 20.0 mmol) and DIC (2.5 g, 20.0 mmol) and react for 5 min. Add the solution to the polypeptide synthesis tube and react for 3 hours. The reaction is completed when the ninhydrin test is negative. After removing the solvent, wash the resin with DMF for 6 times, and then remove the Fmoc protecting group with 20% piperidine in DMF. Repeat the above operation, and sequentially add each amino acid in the order from C-terminus to N-terminus in the peptide sequence. Coupling is performed in the order of Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Mob)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Mob)-OH, Fmoc-Tyr(tBu)-OH according to the peptide sequence. Finally, remove the Fmoc protecting group with 20% piperidine in DMF, wash the solution with DMF for 6 times, shrink with methanol twice (10 min), wash with methyl tert-butyl ether (MTBE) for 3 times, and dry the solvent to obtain the P1 protected peptide resin (15.0 g).

[0180] Step II: Synthesis of fragment P1-L1

[0181] Swell the obtained P1 protected peptide resin (15.0 g, 5.0 mmol) with DMF for 30 min, and remove the solvent. Weigh Fmoc-PEG4-NHS ester (5.8 g, 10.0 mmol) into another reaction bottle, and then add 100 mL of DMF and DIEA (0.8 g, 6.0 mmol), stir for 5 min, and then add the reaction solution to the polypeptide synthesis tube and shake for 2 h. The reaction is completed when the ninhydrin test is negative. Remove the solvent, wash the resin with DMF for 6 times, and then remove the Fmoc protecting group with 20% piperidine in DMF. Wash the solution with DMF for 6 times, shrink with methanol twice (10 min), wash with methyl tert-butyl ether (MTBE) for 3 times, and dry the solvent to obtain the P1-L1 protected peptide resin (16.0 g).

[0182] Step III: Synthesis of fragment P1-L1-P2

[0183] The resulting P1-L1 protected peptide resin (16.0 g, 5.0 mmol) was swelled in a peptide synthesizer with DMF for 30 min and the solvent was removed. In a separate flask, Fmoc-Val-OH (6.8 g, 20.0 mmol), HOBt (2.7 g, 20.0 mmol) and DIEA (3.8 g, 30.0 mmol) were dissolved in DMF and cooled to 0 °C using an ice water bath, then DIC (2.5 g, 20.0 mmol) was added and reacted for 5 min. The solution was added to the peptide synthesizer and shaken for 3 h. The reaction was complete when the ninhydrin test was negative, the solvent was removed and the peptide was washed 5 times with DMF. The Fmoc protecting group was then removed using 20% piperidine in DMF. The above procedure was repeated and each amino acid was added in the order of C-terminal to N-terminal in the peptide sequence. Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH were coupled in that order according to the peptide sequence. After the reaction was complete, the Fmoc protecting group was removed using 20% piperidine in DMF, the solution was washed 6 times with DMF, shrunk twice with methanol (10 min), washed 3 times with methyl tert-butyl ether (MTBE), and the solvent was removed to give P1-L1-P2 protected peptide resin (18.0 g).

[0184] Step IV: Synthesis of fragment P1-L1-P2-L2

[0185] The resulting P1-L1-P2 protected peptide resin (18.0 g, 5.0 mmol) was swelled in a peptide synthesizer with DMF for 30 min and the solvent was removed. In a separate flask, Fmoc-His(Trt)-OH (12.4 g, 20.0 mmol), HOBt (2.7 g, 20.0 mmol) and DIEA (3.8 g, 30.0 mmol) were dissolved in DMF and cooled to 0 °C using an ice water bath, then DIC (2.5 g, 20.0 mmol) was added and reacted for 5 min. The solution was added to the peptide synthesizer and shaken for 3 h. The reaction was complete when the ninhydrin test was negative, the solvent was removed and the peptide was washed 5 times with DMF. The Fmoc protecting group was then removed using 20% piperidine in DMF. The above procedure was repeated and Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH were coupled. After the reaction was complete, the Fmoc protecting group was removed using 20% piperidine in DMF, the solution was washed 6 times with DMF.

[0186] Additionally, glutaric anhydride (0.7 g, 6.0 mmol) and DIEA (0.8 g, 6.0 mmol) were weighed out and dissolved in DMF, and cooled to 0°C using an ice water bath for 5 min. The solution was added to the polypeptide synthesis tube and shaken for 3 hours. The reaction was terminated by ninhydrin test negative, the solvent was removed, the resin was washed with DMF 5 times, and methanol was condensed twice (10 min), washed with methyl tert-butyl ether (MTBE) 3 times, and the solvent was removed to obtain the P1-L1-P2-L2 protected peptide resin (21.0 g).

[0187] Step V: Synthesis of fragment L3-D

[0188] Synthesis of dipeptide (GG): CTC resin (5 g, degree of substitution: 1.4 mmol / g) was weighed out and added to a solid-phase polypeptide synthesis tube, and DMF was added to shake and swell for 30 min, and then the DMF was removed. In a separate Erlenmeyer flask, Fmoc-Gly-OH (8.3 g, 28.0 mmol) and DIEA (5.4 g, 42.0 mmol) were dissolved in DMF and reacted for 5 min. The solution was added to the polypeptide synthesis tube and shaken for 3 hours. After the reaction was terminated, the resin was washed with DMF 5 times and DCM 3 times.

[0189] DIEA (5 ml) and methanol (15 ml) were dissolved in DCM (80 ml) to obtain a capping reagent. The capping reagent was added to the washed resin described above, and capping was performed at room temperature for 30 min on a shaker, and the operation was repeated twice. After capping was completed, the resin was washed with DMF 5 times, methanol was condensed for 20 min, and finally washed with methyl tert-butyl ether (MTBE) 3 times, and the solvent was removed to obtain Fmoc-Gly-CTC resin (7.5 g), and the degree of substitution was detected to be 0.8 mmol / g.

[0190] Weigh 7.5 g (6 mmol) Fmoc-Gly-CTC resin (substitution: 0.8 mmol / g) into the polypeptide synthesis tube, wash with DMF for 3 times, then swell for 30 min, and then remove DMF. Remove Fmoc protecting group with 20% piperidine in DMF (10 min*2), and wash with DMF for 6 times. Weigh Fmoc-Gly-OH (7.2 g, 24.0 mmol), DIEA (4.6 g, 36.0 mmol) and HOBt (3.2 g, 24.0 mmol) into DMF, and cool to 0°C with ice water bath, then add DIC (3.1 g, 24.0 mmol) and react for 5 min. Add the solution into the polypeptide synthesis tube, and react for 3 hours. The reaction is ended when the ninhydrin test is negative. After removing the solvent, wash the resin with DMF for 6 times. Shrink with methanol for 20 min, and finally wash with methyl tert-butyl ether (MTBE) for 3 times. Remove the solvent to obtain Fmoc-Gly-Gly-CTC resin (10.0 g).

[0191] Lysis of Fmoc-Gly-Gly-CTC resin: Mix TFA (380 ml), TIS (10 ml) and H2O (10 ml), and cool to 0°C after mixing. Take Fmoc-Gly-Gly-CTC resin (5.0 g) and add into the lysis solution, and stir at 0°C for 3 h. Remove the resin by suction filtration, concentrate the filtrate, and add methyl tert-butyl ether to pulp for 3 times. Remove the filtrate by suction filtration, collect the filter cake, and dry at 40°C under vacuum to obtain white solid crude product 3.0 g.

[0192] Synthesis of tripeptide (GGF): Weigh CTC resin (5 g, substitution: 1.4 mmol / g) into the solid-phase polypeptide synthesis tube, add DMF, and shake to swell for 30 min, and then remove DMF. In a separate Erlenmeyer flask, weigh Fmoc-Phe-OH (10.8 g, 28.0 mmol) and DIEA (5.4 g, 42.0 mmol) into DMF, and react for 5 min. Add the solution into the polypeptide synthesis tube, and shake to react for 3 hours. After the reaction is ended, wash with DMF for 5 times, and wash with DCM for 3 times.

[0193] Dissolve DIEA (5 ml) and methanol (15 ml) in DCM (80 ml), and mix uniformly to obtain a capping reagent. Add the capping reagent into the washed resin, and cap at room temperature for 30 min. Repeat the operation twice. After capping is ended, wash with DMF for 5 times, shrink with methanol for 20 min, and finally wash with methyl tert-butyl ether (MTBE) for 3 times. Remove the solvent to obtain Fmoc-Phe-CTC resin (6.0 g), and detect that the substitution is 0.8 mmol / g.

[0194] Weigh 6.0 g (4.8 mmol) of Fmoc-Phe-CTC resin (substitution degree: 0.8 mmol / g) into the polypeptide synthesis tube, wash with DMF for 3 times, then swell for 30 min, and then remove the DMF. Remove the Fmoc protecting group with 20% piperidine in DMF (10 min*2), and wash with DMF for 6 times. Weigh Fmoc-Gly-OH (5.7 g, 19.0 mmol), DIEA (3.7 g, 28.8 mmol) and HOBt (2.6 g, 19.0 mmol) and dissolve with DMF, and cool to 0°C with an ice water bath, then add DIC (2.4 g, 19.0 mmol) and react for 5 min. Add the solution to the polypeptide synthesis tube, and react for 3 hours. The reaction is ended when the ninhydrin test is negative. After removing the solvent, wash the resin with DMF for 6 times.

[0195] Wash the clean resin with 20% piperidine in DMF (10 min*2) to remove the Fmoc protecting group, and repeat the coupling of Fmoc-Gly-OH. After the coupling is ended, remove the solvent, and wash the resin with DMF for 6 times. Shrink with methanol for 20 min, and then wash with methyl tert-butyl ether (MTBE) for 3 times. After removing the solvent, obtain Fmoc-Gly-Gly-Phe-CTC resin (7.0 g).

[0196] Lysate Fmoc-Gly-Gly-Phe-CTC resin: mix TFA (380 ml), TIS (10 ml) and H2O (10 ml), and cool to 0°C after mixing. Take Fmoc-Gly-Gly-Phe-CTC resin (7.0 g) and add to the lysate, and stir at 0°C for 3 h. Remove the resin by suction filtration, concentrate the filtrate, add methyl tert-butyl ether and beat for 3 times, remove the filtrate by suction filtration, collect the filter cake, and dry at 40°C under vacuum to obtain white solid crude product 3.5 g (Fmoc-Gly-Gly-Phe-OH).

[0197] Intermediate A: Weigh Fmoc-Gly-Gly-OH (3.5 g, 7.0 mmol) and dissolve in a mixture of tetrahydrofuran (100 ml) and toluene (35 ml), add pyridine (0.7 g, 8.4 mmol) and lead tetraacetate (3.7 g, 8.4 mmol), and heat to reflux for 5 hours. After the reaction is ended, cool the reaction solution to room temperature, remove the insoluble matter by filtration through diatomite, and concentrate the filtrate under reduced pressure. Purify the obtained residue by silica gel column chromatography [petroleum ether: ethyl acetate = 9:1 (v / v) ~ ethyl acetate], and obtain white solid 2.7 g with a yield of about 75%.

[0198] Intermediate b: The above solid (2.7 g, 7.5 mmol) and benzyl glycolate (3.7 g, 22.5 mmol) were dissolved in tetrahydrofuran (30 ml) and cooled to 0 °C in an ice-water bath. Potassium tert-butoxide (1.7 g, 15 mmol) was added at 0 °C and the reaction mixture was stirred at room temperature for 30 min. The reaction solution was extracted with ethyl acetate and water at 0 °C and the organic phase was dried over sodium sulfate. The solvent was removed by distillation under reduced pressure and the resulting residue was dissolved in dioxane (30 ml) and water (10 ml). Sodium bicarbonate (0.8 g, 9 mmol) and 9-fluorenylmethyl chloroformate (1.9 g, 7.5 mmol) were added to the system and the reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction mixture was extracted with water and ethyl acetate, and the resulting organic layer was dried over sodium sulfate. The solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography [petroleum ether: ethyl acetate = 9: 1 (v / v) ~ ethyl acetate] to obtain 2.2 g of a colorless oil, with a yield of 60%.

[0199] Intermediate c: The above solid (2.2 g, 4.5 mmol) was dissolved in methanol (50 ml). Palladium-carbon catalyst (0.6 g, 10% w / w) was added and stirred at room temperature for 2 hours under hydrogen gas. The insoluble material was removed by filtration through diatomite, and the solvent was removed by distillation under reduced pressure to obtain a colorless solid, which was directly used in the next reaction.

[0200] Intermediate d: The above solid was dissolved in DMF (30 ml) and cooled to 0 °C in an ice-water bath. Methanesulfonic acid salt of exetecan (1.6 g, 3.0 mmol) and N-hydroxysuccinimide (0.8, 6.8 mmol) were added to the reaction mixture, followed by the addition of DIEA (1.2 g, 9 mmol) and DIC (0.9 g, 6.8 mmol), and the reaction mixture was stirred at room temperature for 3 days. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the resulting residue was purified by silica gel column chromatography [dichloromethane: methanol = 7:3 (v / v)] to obtain 2.0 g of a light brown solid, with a yield of about 82%.

[0201] Intermediate e: The above light brown solid (2.0 g, 2.5 mmol) was dissolved in DMF (30 ml), and piperidine (6 ml) was added and stirred at room temperature for 2 hours. The solvent was removed by concentration under reduced pressure to obtain a compound, which was used in the subsequent reaction.

[0202] Intermediate f: Fmoc-Gly-Gly-Phe-OH (2.5 g, 5.0 mmol), N-hydroxysuccinimide (1.2 g, 10.0 mmol), and intermediate e (2.0 g, 2.5 mmol) were dissolved in DMF (50 ml), and then DIC (1.3 g, 10.0 mmol) was added to the reaction system. The reaction was stirred at room temperature for 3 days, the solvent was removed under reduced pressure, and then the residue was purified by silica gel column chromatography [dichloromethane:methanol = 9:1 (v / v)] to obtain 2.4 g of a light yellow solid.

[0203] GGFG-DXD: The light yellow solid was dissolved in DMF (20 ml), piperidine (4 ml) was added, and stirring was performed at room temperature for 2 hours. The solvent was removed by concentration under reduced pressure to obtain compound GGFG-DXD (2.0 g), which was directly used in the subsequent reaction.

[0204] Step VI: Synthesis of fragment P1-L1-P2-L2-L3-D

[0205] P1-L1-P2-L2 protected peptide resin (21.0 g, 5.0 mmol) was weighed into a solid-phase polypeptide synthesis tube, DMF was added and washed 3 times, the shaker was swelled for 30 min, and the solvent was removed. Meanwhile, GGFG-DXD (2.0 g, 2.4 mmol), DIEA (1.3 g, 10.0 mmol), HOBT (0.8 g, 6.0 mmol), and DIC (0.7 g, 6.0 mmol) were dissolved in DMF, and then poured into the polypeptide synthesis tube, and the shaker was stirred at room temperature for 5 h. After the reaction was completed, the solvent was removed, the resin was washed 5 times with DMF, shrank twice with methanol (10 min), washed 3 times with methyl tert-butyl ether (MTBE), and the solvent was removed by suction to obtain P1-L1-P2-L2-L3-D protected peptide resin (23.0 g).

[0206] Step VII: Cleavage of resin

[0207] TFA (380 ml), TIS (10 ml), and H2O (10 ml) were mixed, and then cooled to 0°C. P1-L1-P2-L2-L3-D protected peptide resin (23.0 g) was added to the cleavage solution, and stirred at 0°C for 3 h. The resin was removed by suction filtration, the filtrate was concentrated, and methyl tert-butyl ether was added to make a slurry 3 times. The filtrate was removed by suction filtration, and the filter cake was collected and dried at 40°C under vacuum to obtain a white solid crude product 11.0 g. The crude product was dissolved in DMF, and directly prepared and purified. After lyophilization, a white solid pure product 3.8 g was obtained.

[0208] Step VIII: Disulfide bond cyclization

[0209] The pure P1-L1-P2-L2-L3-D (2.0 g, 0.53 mmol) was weighed and dissolved in methanol to a concentration of 50 μM, and 30% acetic acid aqueous solution was added to assist dissolution. Iodine (1.4 g, 5.3 mmol) was weighed and dissolved in methanol, and was added dropwise into the reaction solution in an ice water bath. The reaction was carried out at room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was concentrated and directly prepared and purified. After lyophilization, 440 mg of white solid was obtained, with a yield of 21%.

[0210] Example 5

[0211] In this example, the assembly properties of the polypeptide conjugate drugs (PDC-1 to PDC-16, PDC-FITC) obtained in the previous examples were determined.

[0212] The molecular structures and the determined molecular weights of the polypeptide conjugate drugs are shown in Table 2.

[0213] Table 2: Molecular structures of the compounds of the examples

[0214] The chemical structures of the polypeptide conjugate drugs (PDC-1 to PDC-16, PDC-FITC) are shown below:

[0215] 1. Determination of the critical assembly concentration of polypeptide conjugate drugs

[0216] In this experiment, the assembly properties of the polypeptide conjugate drugs in solution were investigated.

[0217] (1) Experimental method: The critical assembly concentration (CAC) of the polypeptide conjugate drugs in solution was determined by pyrene fluorescence method.

[0218] A 24 μg / mL pyrene PBS solution was prepared, and DMSO gradient dilution solutions of PDC-1 to PDC-16 were prepared. 10 μL of the pyrene solution and the polypeptide conjugate drug DMSO solution were added to 380 μL of the PBS solution, respectively, and incubated at room temperature for 1 h in the dark. The fluorescence intensities at 373 and 384 nm were measured by fluorescence spectrophotometry.

[0219] (2) Results and analysis

[0220] The critical assembly concentrations of the test compounds were analyzed by concentration-fluorescence intensity plot and are shown in Table 3.

[0221] Table 3: Critical assembly concentrations of the compounds of the examples

[0222] Critical assembly concentration assay results show that polypeptide conjugate drugs containing assembly polypeptide sequences have lower critical assembly concentrations, and PDC-4 without assembly polypeptide sequences has a critical assembly concentration higher than 500 μM. These results show that the compounds described in the present application have significantly enhanced self-assembly ability.

[0223] 2. Assembly performance of polypeptide conjugate drugs on cell surface

[0224] In this experiment, the polypeptide conjugate drug PDC-1 was used as a representative molecule to investigate its assembly performance on the surface of HER2-positive human gastric cancer cells (NCI-N87) and HER2-negative human breast cancer cells (MDA-MB-231).

[0225] (1) Experimental method

[0226] In each well of a 12-well plate, a silicon wafer treated with plasma on the surface was placed, and human gastric cancer NCI-N87 cell and human breast cancer MDA-MB-231 cell suspensions were added to each well of the 12-well plate at 1 mL per well, and cultured overnight. The culture medium was removed, and 1 mL of culture medium containing 20 μM of the test substance was added, and cultured for 4 h. Washed three times with PBS buffer (pH 7.4), and then fixed with a 4% polyformaldehyde solution for 2 h. Dehydrated with 10%, 30%, 50%, 70%, 90%, and 100% ethanol solutions diluted with PBS, respectively, for 10 min at each concentration. After the silicon wafer was dried, gold was sprayed, and scanning electron microscopy (SEM) imaging was performed.

[0227] (2) Results and analysis

[0228] According to the scanning electron microscopy imaging results shown in Figure 1, compared with HER2-negative MDA-MB-231 cells, PDC-1 formed uniform nano-assembly structures on the surface of HER2-positive NCI-N87 cell membranes, with a size of 100-1000 nm. These results show that the compounds described in the present application have HER2 receptor-induced self-assembly ability.

[0229] 3. Intracellular internalization properties of polypeptide conjugate drugs

[0230] In this experiment, the polypeptide conjugate PDC-FITC labeled with fluorescein was used as a tracer molecule to investigate the internalization properties of the above-mentioned polypeptide nano-conjugate drugs in NCI-N87 cells.

[0231] (1) Experimental method

[0232] NCI-N87 cell suspension 1 mL was added to a confocal dish and incubated overnight. The culture medium was removed and culture medium 1 mL containing 20 μM labeled FITC molecule 09130311 and 50 nM lysosome red fluorescent probe was added and incubated for 0.5 h and 2 h. Three times of washing with PBS buffer (pH 7.4) was performed and confocal imaging experiment was carried out. Green light band (520 nm-530 nm) and red light band (590 nm-690 nm) were collected using 488 nm and 561 nm laser channels.

[0233] (2) Results and analysis

[0234] According to confocal imaging Figure 2, PDC-FITC can be effectively internalized into cells after 2 h incubation and form fluorescent co-localization with lysosome. These results show that the compound of the present application can be effectively internalized into cells after the assembly is induced by HER2 receptor on cell membrane.

[0235] Example 6

[0236] The efficacy performance of the polypeptide conjugated drugs (PDC-1-PDC-16, PDC-FITC) listed in Table 2 of the preceding Example 5 was determined in this example.

[0237] 1. Cell toxicity test of polypeptide conjugated drug

[0238] This experiment investigated the cell toxicity of the above polypeptide conjugated drugs PDC-1-PDC-16 on HER2 positive human gastric cancer cells (NCI-N87) and HER2 negative human breast cancer cells (MDA-MB-231).

[0239] (1) Experimental method

[0240] NCI-N87 was cultured in RPMI-1640 complete culture medium containing 10% fetal bovine serum and MDA-MB-231 cells were cultured in DMEM complete culture medium containing 10% fetal bovine serum in a cell culture incubator at 37°C, 5% CO2, and the cells were subcultured every 2-3 days. NCI-N87 and MDA-MB-231 cells in logarithmic growth phase were seeded at 1 x 10 3Cells were seeded in 96-well plates and incubated at 37℃ in a 5% CO2 cell incubator for 8-12 hours. Prepare 4000 nM of polypeptide conjugated drugs PDC-1 to PDC-16 and positive control MMAE in culture medium (DMSO content 0.1%), and dilute with PBS to 2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM. Add 100 μL of different concentrations of test agent culture medium solution to each well, and incubate overnight at 37℃ in a 5% CO2 cell incubator. According to the manufacturer's protocol, add CCK-8 reagent, and incubate at 37℃ in a 5% CO2 cell incubator for 1 hour. Then use a microplate reader to measure the absorbance at 490 nm, and calculate the IC 50 values of the cytotoxicity of polypeptide conjugated drugs PDC-1 to PDC-16 and MMAE according to the compound concentration and luminescence signal value.

[0241] (2) Results and analysis

[0242] The IC 50 values of the cytotoxicity of polypeptide conjugated drugs PDC-1 to PDC-16 on human gastric cancer cells (NCI-N87) and human breast cancer cells (MDA-MB-231) are shown in Table 4.

[0243] Table 4: Cytotoxicity test results (IC 50 values) of the compounds of the examples, units (nM)

[0244] Taking polypeptide conjugated drug PDC-1 as an example, the test results show that PDC-1 has no obvious inhibitory activity on HER2-negative tumor cells MDA-MB-231, but has significant inhibitory activity on HER2-positive tumor cells NCI-N87, and cytotoxin MMAE has significant inhibitory activity on both HER2-positive and HER2-negative cells. These results show that polypeptide conjugated drug PDC-1 exerts tumor inhibitory activity through specific binding and internalization mechanism of HER2 receptor.

[0245] The compounds described in the present application all obtained similar results in this test, and in most cases, the compounds provided in the present application have significant inhibitory activity on HER2-positive tumor cells NCI-N87, with IC 50 values in the range of hundreds of nanomoles. The inhibitory activity of compound PDC-4 without assembling polypeptide sequence on tumor cells is weaker than that of other compounds described in the present application, indicating that the self-assembly ability of the polypeptide conjugated drugs described in the present application can enhance the inhibitory activity on tumor cells.

[0246] 2. Cytotoxicity test of polypeptide conjugated drugs on Enhertu-resistant cell lines

[0247] The experiment investigates the in vitro tumor cell inhibition activity of the above-mentioned polypeptide conjugate drug PDC-1 and the positive control drug Enhertu on Enhertu-induced drug-resistant human gastric cancer cells (NCI-N87-R) and human lung cancer cells (NCI-H2170-R).

[0248] (1) Experimental method

[0249] Enhertu-induced drug-resistant human gastric cancer cells (NCI-N87-R) and human lung cancer cells (NCI-H2170-R) were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum at 37°C in a 5% CO2 cell incubator, and the cells were passaged every 2-3 days. Cells in the logarithmic growth phase were seeded at 1x103 cells per well in a 96-well plate and incubated at 37°C in a 5% CO2 cell incubator overnight. Prepare a 3250nM PDC-1 medium solution (DMSO content 0.1%), dilute with PBS to 650nM, 130nM, 26nM, 5.2nM, 1nM, 0.2nM, 0.04nM, 0.008nM. Prepare a 50000ug / mL Enhertu medium solution, dilute to 10000ug / mL, 2000ug / mL, 400ug / mL, 80ug / mL, 16ug / mL, 3.2ug / mL, 0.64ug / mL, 0.128ug / mL. Add 100μL of different concentrations of PDC-1 or Enhertu medium solution per well with PBS, and incubate at 37°C in a 5% CO2 cell incubator for seven days (change the solution on the third day after administration). According to the manufacturer's protocol for Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573), the cells were treated, and then the luminescence signal was measured using a microplate reader. The IC 50 values of polypeptide conjugate drug PDC-1 and Enhertu cytotoxicity were calculated according to the compound concentration and luminescence signal value.

[0250] (2) Results and analysis

[0251] The results are shown in Figure 3 and Table 5. Human gastric cancer cells (NCI-N87) and human lung cancer cells (NCI-H2170) are sensitive to Enhertu, with IC 50 values of 0.049ug / mL and 0.092ug / mL, respectively, while drug-resistant human gastric cancer cells (NCI-N87-R) and human lung cancer cells (NCI-H2170-R) are resistant to Enhertu. The polypeptide conjugate drug PDC-1 can effectively inhibit the growth of Enhertu-resistant tumor cells, with an IC 50 value of 21nM.

[0252] Table 5: Cytotoxicity test results of the compounds in the examples (IC50) 50 value)

[0253] 3. In vivo antitumor efficacy studies of peptide-conjugated drugs

[0254] This experiment investigated the in vivo antitumor effects of the aforementioned peptide-conjugated drugs PDC-1 and PDC-6 in a human tumor cell line xenograft model (CDX).

[0255] (1) Experimental methods

[0256] First, tumor models were constructed by resuspending HER2-positive human gastric cancer cells (NCI-N87) and HER2-negative human breast cancer cells (MDA-MB-231) in PBS and Matrigel (1:1). Cell suspensions were then prepared at 5*10-1 ppm. 5 The inoculation dose was administered subcutaneously to the right axilla of the experimental animal (BALB / Cnud nude mouse). The model animals were housed in an IVC-grade animal facility at a temperature of 20–25°C and humidity of 40–70%, with 12 hours of light and 12 hours of darkness daily. Animals were housed in separate cages of four, with free access to food and water. Feed was irradiated for sterilization, and water was autoclaved. Quarantine and observation were conducted by a veterinarian at the animal center.

[0257] When the average tumor volume reaches 100-200 mm 3 In this study, uniformly shaped tumors were randomly assigned to groups based on tumor volume and body weight, and the selected animals were labeled. Each cell model was divided into 6 groups, with 8 animals in each group. Intravenous administration was used: the solvent group, the 1 mg / kg PDC-1 group, the 3 mg / kg PDC-1 group, the 5 mg / kg PDC-1 group, the 1 mg / kg PDC-6 group, the 3 mg / kg PDC-6 group, and the 5 mg / kg PDC-6 group were administered once every three days for three weeks; the 0.1 mg / kg MMAE group, the 5 mg / kg Enhertu group, and the 5 mg / kg RC48 group were administered once a week for three weeks. Observations were conducted for 28 consecutive days, with tumor size measured every two days using calipers. Changes in tumor volume and body weight were recorded for different mice. On the last day of the experiment, the animals were sacrificed, weighed, the tumors were removed, weighed, and photographed for recording.

[0258] The calculation of tumor-related parameters was based on the "Technical Guidelines for Non-Clinical Studies of Cytotoxic Antitumor Drugs" issued by the China National Medical Products Administration (NMPA). The antitumor activity evaluation index was the tumor inhibition rate (%), calculated as follows: Tumor inhibition rate (%) = (Average tumor weight in the negative control group - Average tumor weight in the treated group) / Average tumor weight in the negative control group × 100%.

[0259] Data analysis was performed using two-tailed analysis, and the statistical level was set at 5% or p≤0.05. The mean and standard deviation (Mean ± SEM) of each analysis index were calculated, including tumor volume, tumor weight, body weight, etc. Levene's test was used for homogeneity of variance; if there was no statistical significance (p>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA was statistically significant (p≤0.05), appropriate multiple comparison tests were used for comparative analysis.

[0260] (2) Results and analysis

[0261] The results are shown in Table 6. When treated with the compounds PDC-1 and PDC-6 of the present application at 1 mg / kg, the growth of HER2-positive human gastric cancer cells (NCI-N87) tumors was effectively delayed, and when the drug dosage was increased to 3 mg / kg, the anti-tumor activity was very obvious. The inhibition of tumors by the compounds PDC-1 and PDC-6 of the present application was dose-dependent, and at the same dosage of 5 mg / kg, the compounds PDC-1 and PDC-6 of the present application showed anti-tumor activity comparable to that of the same target ADC compared with the control drugs RC48 and Enhertu. Moreover, the compounds PDC-1 and PDC-6 provided by the present application had no obvious toxicity (based on animal body weight), and the animals were well tolerated.

[0262] In a similar manner, the anti-tumor activity of the compounds PDC-1 and PDC-6 in a HER2-negative human breast cancer cell (MDA-MB-231) tumor model was determined. The test results showed that the compounds PDC-1 and PDC-6 had almost no activity on the HER2-negative cell tumor model, and these results indicated that the polypeptide conjugate drugs of the present application act through a HER2-selective or HER2-specific mechanism.

[0263] Table 6: Tumor inhibition rate of the compounds of the examples on different transplanted tumor models, unit (%)

[0264] 4. Plasma stability test of polypeptide conjugate drugs

[0265] This experiment investigated the stability of the above polypeptide conjugate drugs PDC-1, PDC-5 and PDC-6 in the plasma of different animal species.

[0266] (1) Experimental method

[0267] The frozen plasma was thawed in cold tap water for 10-20 minutes. After the plasma was completely thawed, it was centrifuged at 3000 rpm for 5 minutes to remove the suspended and precipitated substances. The pH of the plasma was measured and adjusted to the range of pH = 7.40 ± 0.10 with 1% phosphoric acid solution or 1M sodium hydroxide solution. The working solution of PDC-1, PDC-5 and PDC-6 or the control compound procaine was added to the corresponding incubation plate, and three parallel holes were prepared for each sample. Then 196 μL of mouse, rat, monkey and human blank plasma was added to the incubation plate after the working solution was added. All samples were incubated in a 37°C water bath. The final incubation concentration of PDC-1, PDC-5 and PDC-6 and the control compound procaine was 1 μM. At the end of each incubation time point, the corresponding incubation plate was taken out, and 600 μL of acetonitrile solution containing 0.1% formic acid and 200 ng / mL tolbutamide was added to each corresponding sample hole to precipitate the protein. After all sample plates were sealed and shaken, they were centrifuged at 3000 rpm for 20 minutes. The supernatant was diluted with ultrapure water before detection, and all samples were mixed and analyzed by LC / MS / MS method.

[0268] (2) Results and analysis

[0269] The results are shown in Table 7. The half-lives of PDC-1, PDC-5 and PDC-6 in monkey and human plasma can be more than 10 hours, while the half-lives in rat and mouse plasma are shorter, which is related to the presence of carboxylesterase 1C (CES1c) in plasma, which can cleave the polypeptide linker.

[0270] Table 7: Plasma stability of example compounds in different species

[0271] In view of the above results, both in vitro and in vivo studies have shown that:

[0272] The polypeptide conjugate drug designed by the in situ self-assembly technology in the present application can form an assembly on the surface of tumor cells activated by HER2 receptor, increase drug enrichment and retention, and at the same time increase receptor-mediated internalization, and kill cells through the action of cytotoxin.

[0273] The cytotoxicity test on Enhertu-resistant cell lines shows that the polypeptide conjugate drug of the present application has significant inhibitory activity on Enhertu-resistant cell lines, and has the characteristics of overcoming HER2 ADC drug resistance.

[0274] In the in vivo anti-tumor efficacy in a human tumor cell line xenograft model (CDX), the polypeptide conjugate drug of the present application shows excellent efficacy in controlling or eliminating transplanted tumors in a dose- and receptor expression level-dependent manner, and does not cause weight loss or other obvious toxicity.

[0275] The above-described embodiments only express several implementation manners of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the protection scope of the present application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Industrial applicability

[0276] The present application provides a polypeptide conjugate drug targeting HER2 and a preparation method and application thereof. The polypeptide conjugate drug provided by the present application has good in vivo in situ self-assembly performance, can combine HER2 and improve the drug concentration in the tumor area through self-assembly at the target point, promote drug endocytosis, release the drug through enzymatic cutting in lysosomes, and thus treat tumors with high or low expression of HER2. Meanwhile, the polypeptide conjugate drug targeting HER2 provided by the present application has the characteristics of overcoming HER2 ADC drug resistance, has good economic value and application prospect.

Claims

1. A peptide-conjugated drug targeting HER2, characterized in that, Its structural formula is one of the following ① to ④: ①: P1-P2-L3-D; ②: P1-P2-L2-L3-D; ③: P1-L1-P2-L3-D; ④: P1-L1-P2-L2-L3-D; Wherein, P1 is the HER2 targeting unit; P2 is the self-assembly unit; D is the anti-tumor functional unit; L1, L2 and L3 are the connecting units; L3 is selected from peptide linkers with lysosomal cleavage function.

2. The HER2-targeting peptide conjugate according to claim 1, characterized in that, The self-assembly unit is a self-assembly polypeptide P2, which is selected from any one or a combination of at least two of the polypeptide sequences NQFNLM, QILLWS, YYQNYQ, NFVNYS, GNNQQNY, ITSVV, FGFDP, YFTEF, ISDNL, ARVHVSE, KLYKVS, SVSLA, and DCFILDH; wherein any amino acid can be selected from L-type or D-type. Preferably, the self-assembled polypeptide P2 is selected from any one or a combination of at least two of the polypeptide sequences GNNQQNY, ITSVV, FGFDP, YFTEF, ISDNL, ARVHVSE, KLYKVS, SVSLA, and DCFILDH; wherein any amino acid may be selected from L-type or D-type.

3. The HER2-targeting peptide conjugate according to claim 1, characterized in that, L2 is selected from either Equation I or Equation II: In Equation I or Equation II: Aaa1 is 0-3 His, Aaa2 is 0-3 Ser, and p and q are integers in the range of 1-8; Preferably, p and q are integers within the range of 2-6; And / or, L3 is selected from any one or at least a combination of two of Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-PAB, and Gly-Gly-Phe-Gly. And / or, L1 is selected from either Equation III or Equation IV: In Equation III or Equation IV, m and n are integers within the range of 1-8; Preferably, m and n are integers within the range of 2-6.

4. The HER2-targeting peptide conjugate according to any one of claims 1-3, characterized in that, The HER2 targeting unit is a polypeptide that targets the HER2 protein; the polypeptide is a linear peptide or cyclic peptide composed of 5-50 amino acids; wherein any amino acid can be selected from L-type or D-type. Preferably, the polypeptide is a linear peptide or a cyclic peptide composed of 7-20 amino acids.

5. The HER2-targeting peptide conjugate according to claim 4, characterized in that, The polypeptide is selected from any one or a combination of at least two of the polypeptide sequences CDGFYAC, YCDGFYACYMDV, FCDGFYACYMDV, YCDGFACYMDV, FCDGFACYMDV, CGPLPVDWYWC, CEWKFDPGLGQARC, CDYMTDGRAASKIC, KCCYSL, MARSGL, MCGVCLSAQRWT, and SGLWWLGVDILG; wherein any amino acid may be selected from L-type or D-type.

6. The HER2-targeting peptide-conjugated drug according to any one of claims 1-5, characterized in that, The anti-tumor functional unit is selected from cytotoxic drugs with cancer treatment functions; the cytotoxic drugs are selected from at least one of microtubule inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, and antimetabolites.

7. The HER2-targeting peptide conjugate according to claim 6, characterized in that, The microtubule inhibitor is selected from at least one of aureatin derivatives, maytansine derivatives, and paclitaxel derivatives; And / or, the DNA topoisomerase inhibitor is selected from camptothecin derivatives; And / or, the DNA damaging agent is selected from at least one of chachiomycin derivatives and aprotinin derivatives; And / or, the antimetabolite is selected from at least one of methotrexate and 5-fluorouracil.

8. The HER2-targeting peptide conjugate according to claim 6, characterized in that, The cytotoxic drugs are selected from auratestatin derivatives and / or camptothecin derivatives.

9. A method for preparing the HER2-targeting peptide-conjugated drug according to any one of claims 1-8, characterized in that, The process includes, in sequence, resin activation, P1 amino acid condensation, L1 condensation, P2 amino acid condensation, L2 condensation, L3-D coupling, and cyclization.

10. A derivative of the HER2-targeting peptide-conjugated drug according to any one of claims 1-8, characterized in that, The derivative is a pharmaceutically acceptable salt or solvate of the polypeptide-conjugated drug.

11. A pharmaceutical composition, characterized in that, Includes the HER2-targeting peptide conjugate of any one of claims 1-8 or the derivative of claim 10.

12. A pharmaceutical preparation, characterized in that, The drug includes the HER2-targeting peptide conjugate of any one of claims 1-8 or the derivative of claim 10, and further includes a pharmaceutically acceptable carrier. Preferably, the dosage form of the pharmaceutical preparation is a lyophilized powder for injection, an injection solution, a tablet, a liposome, or a nano-formulation.

13. The use of the HER2-targeting peptide conjugate of any one of claims 1-8 or the derivative of claim 10 in the preparation of an antitumor drug; The tumor is either HER2 highly expressed or lowly expressed; Preferably, the tumor is selected from at least one of breast cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, and skin cancer.

14. The use of the HER2-targeting peptide conjugate of any one of claims 1-8, the derivative of claim 10, the pharmaceutical composition of claim 11, or the pharmaceutical formulation of claim 12 in the prevention or treatment of tumor diseases; The tumor disease is one in which HER2 is highly or poorly expressed; Preferably, the tumor disease is selected from at least one of breast cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, and skin cancer.

15. The application according to claim 14, characterized in that, The drug is used in combination with one or more antitumor drugs and the HER2-targeting peptide conjugate of any one of claims 1-8, or the derivative of claim 10, or the pharmaceutical composition of claim 11, or the pharmaceutical formulation of claim 12.

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