A polypeptide-human serum albumin conjugate drug and a preparation method and application thereof

Abstract

The present application provides a kind of polypeptide-human serum albumin conjugated drug and its preparation method and application, the polypeptide-human serum albumin conjugated drug includes targeting tumor cell membrane protein polypeptide, human serum albumin and cytotoxin.The present application innovatively introduces HSA, and by the method of covalent coupling, targeting tumor cell membrane protein polypeptide and cytotoxin are connected on HSA, obtain the conjugated drug with good stability, cytotoxin is not easy to lose, the polypeptide-human serum albumin conjugated drug overcomes the technical defects of ADC large molecular weight, preparation is complicated and traditional PDC insufficient drug loading simultaneously, realizes the multiple technical effects of high drug loading, small molecular weight, easy preparation, with significant advantages and good application prospect.The preparation method provided by the present application is simple and easy to operate, does not need complex antibody expression and purification process, and the production cost is lower.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a polypeptide-human serum albumin conjugate drug, its preparation method, and its application. Background Technology

[0002] Epidermal growth factor receptor (EGFR) is an important transmembrane receptor on the cell membrane and has become one of the important targets for tumor treatment. Targeted therapies against EGFR have been widely used in clinical practice and have achieved significant efficacy. Tumor-targeted drugs against EGFR mainly include three categories: monoclonal antibodies, small molecule inhibitors, and antibody-drug conjugates (ADCs). Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been developed for three generations and are widely used in clinical practice, but they still face the serious challenge of tumor drug resistance. Monoclonal antibody drugs can block EGFR-related signaling pathways and can also exert indirect anti-tumor effects by mediating antibody-dependent cell-mediated cytotoxicity. However, monoclonal antibodies still have short pharmacokinetic characteristics and stability issues during manufacturing, transportation, and storage, which may lead to aggregation and protein denaturation.

[0003] Therefore, it is necessary to develop a novel EGFR-targeting drug. Summary of the Invention

[0004] EGFR-targeting adjuvants (ADCs) are novel targeted drugs formed by conjugating EGFR monoclonal antibodies with cytotoxins. After binding to target cells, they undergo endocytosis, releasing the conjugated cytotoxin and killing tumor cells. Peptide-conjugated drugs (PDCs) are another type of novel conjugate drug. Similar in composition to ADCs, they consist of a targeting ligand, a linker, and a payload. Compared to antibody targeting in ADCs, peptides, as the targeting group, offer many advantages, such as smaller molecular weight, simpler structure, ease of chemical modification, higher stability, solid-phase synthesis, simpler preparation methods, and lower cost. PDCs typically consist of 10 amino acids with a molecular weight of around 10 kDa, compared to the 160 kDa of ADCs. Their smaller molecular weight allows for rapid elimination through renal filtration, resulting in faster metabolism in vivo. However, PDCs generally consist of one targeting peptide conjugated to one cytotoxin drug. This invention introduces human serum albumin as a drug loading platform based on PDC. Its molecular weight is between 70-90 kDa, which is between that of PDC and ADC. One targeting peptide can be coupled with multiple cytotoxic drugs, which can both increase cytotoxic load and reduce preparation cost.

[0005] The technical solution of the present invention is as follows.

[0006] In a first aspect, the present invention provides a polypeptide-human serum albumin conjugate drug, wherein the polypeptide-human serum albumin conjugate drug comprises a polypeptide targeting tumor cell membrane protein, human serum albumin, and a cytotoxin.

[0007] The present invention relates to a polypeptide-human serum albumin conjugate drug in which a target tumor cell membrane protein polypeptide and a cytotoxin are covalently conjugated onto human serum albumin (HSA). Compared with existing PDC drugs, the innovative addition of HSA as a drug delivery "platform" allows one or more target tumor cell membrane protein polypeptides to carry multiple cytotoxins, achieving high-concentration drug accumulation at the tumor site, reducing the inconvenience to patients caused by frequent PDC administration, and also reducing the preparation cost of the conjugate drug.

[0008] Preferably, the tumor cell membrane protein polypeptide and the human serum albumin are covalently linked, the cytotoxin and the human serum albumin are covalently linked, and the molar ratio of the tumor cell membrane protein polypeptide, human serum albumin and cytotoxin is (1-10): 1: (1-59).

[0009] Preferably, the molar ratio of the tumor cell membrane protein polypeptide, human serum albumin and cytotoxin is 1:1:(6-19).

[0010] Preferably, the molar ratio of the tumor cell membrane protein polypeptide, human serum albumin and cytotoxin is 1:1:(6-8).

[0011] Preferably, the active site of the tumor cell membrane protein polypeptide forms a disulfide bond with the thiol group of the human serum albumin, or the active site of the tumor cell membrane protein polypeptide is covalently coupled to the amino group of the human serum albumin through a cross-linking agent.

[0012] Preferably, the tumor cell membrane protein polypeptide targets EGFR, and the tumor cell membrane protein polypeptide consists of 10-60 amino acid residues.

[0013] Preferably, the tumor cell membrane protein polypeptide is the targeting polypeptide NH2-CZ. EGFR:1907 -CONH2, the amino acid sequence of the target tumor cell membrane protein polypeptide is shown in SEQ ID No: 1.

[0014] Preferably, the targeting peptide NH2-CZ EGFR:1907 -CONH2 forms a disulfide bond with Cys residues in HSA via Cys residues or targets the polypeptide NH2-CZ. EGFR:1907 -CONH2 is covalently coupled to human serum albumin Lys via a cross-linking agent through the thiol group of Cys.

[0015] Preferably, the targeting peptide NH2-CZ EGFR:1907 The preparation method of -CONH2 includes: using L-Lys as the initial amino acid, linking the Fmoc-protected initial amino acid to a solid support, and removing the Fmoc protection; according to the targeting peptide NH2-CZ EGFR:1907 The amino acid sequence of -CONH2 is repeated as follows: coupling with Fmoc-protected amino acids, removing the Fmoc protecting group, to obtain the targeting peptide NH2-CZ linked on a solid support. EGFR:1907 -CONH2, cleavage, purification, to obtain the targeted peptide NH2-CZ EGFR:1907 -CONH2.

[0016] Preferably, the cytotoxic agent is selected from microtubule inhibitors, DNA damaging agents, and RNA polymerase II inhibitors.

[0017] Preferably, the microtubule inhibitor is selected from N... 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl)-matansine (DM1), N 2 '-Deacetyl-N 2 '-(4-mercapto-4-methyl-1-oxopentyl)-matansine (DM4), monomethylaurestatin E (MMAE), and monomethylaurestatin F (MMAF); the DNA damaging agent is selected from at least one of 7-ethyl-10-hydroxycamptothecin (SN38) and docalmicin; the transcription inhibitor is α-amaminine.

[0018] Secondly, the present invention provides a method for preparing the above-mentioned polypeptide-human serum albumin conjugate drug, comprising the following steps: Step 1): Add the target tumor cell membrane protein peptide to human serum albumin, stir and react, purify, and obtain peptide-human serum albumin (PHC). Step 2): Dissolve the polypeptide-human serum albumin (PHC) in a buffer solution, add a cross-linking agent and cytotoxin, stir the reaction, purify, and obtain the polypeptide-human serum albumin conjugate drug (PHDC).

[0019] Preferably, in step 1), the reaction is carried out in a solvent, wherein the solvent is DMSO.

[0020] Preferably, in step 1), 2,2'-dithiopyridine is also added during the reaction process.

[0021] Preferably, in step 1), the molar ratio of the tumor cell membrane protein polypeptide to the human serum albumin in the PHC is (1-10):1.

[0022] Preferably, in step 1), the molar ratio of the tumor cell membrane protein polypeptide to the human serum albumin in the PHC is 1:1.

[0023] Preferably, in step 1), the targeted tumor cell membrane protein polypeptide is the targeted polypeptide NH2-CZ. EGFR:1907 -CONH2, targeting peptide NH2-CZ EGFR:1907 -CONH2 forms a disulfide bond with Cys residues in HSA through Cys residues.

[0024] Preferably, in step 2), the crosslinking agent is selected from at least one of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), cyclohexane-1-carboxylic acid ester, and sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC). The NHS ester in SMCC or Sulfo-SMCC reacts with the lysine residue primary amine at pH 7-9 to form an amide bond. Furthermore, the maleimide group in SMCC or Sulfo-SMCC reacts with the thiol group at pH 6.5-7.5 to form a stable thioether bond. The crosslinking agent covalently couples different amounts of cytotoxins to the HSA, resulting in a stronger binding and better stability, which can reduce the potential harm caused by off-target effects of cytotoxins.

[0025] Preferably, in step 2), the molar ratio of human serum albumin to cytotoxicant in the polypeptide-human serum albumin conjugate is 1:(6-19).

[0026] More preferably, the molar ratio of human serum albumin to cytotoxicant is 1:(6-8).

[0027] Preferably, in step 2), the buffer solution is PBS buffer.

[0028] Thirdly, the present invention provides the use of the above-mentioned polypeptide-human serum albumin conjugate drug or the polypeptide-human serum albumin conjugate drug prepared by the above-mentioned preparation method in the preparation of a drug for treating tumors overexpressing EGFR.

[0029] Preferably, the tumor overexpressing EGFR is selected from at least one of lung cancer, head and neck squamous cell carcinoma, glioblastoma, colorectal cancer, and esophageal cancer.

[0030] More preferably, the tumor overexpressing EGFR is lung cancer.

[0031] The polypeptide-human serum albumin conjugate provided by this invention reaches the tumor site via blood transport, is recognized by the membrane protein EGFR on tumor cells, is internalized into the cells, and releases cytotoxins in lysosomes, leading to apoptosis and achieving a therapeutic effect.

[0032] The beneficial effects of this invention are as follows: 1. This invention provides a peptide-human serum albumin conjugate (HSA). This invention innovatively introduces an HSA and covalently conjugates a targeted peptide and a cytotoxin to the HSA, resulting in a conjugate with good stability and minimal cytotoxin loss. The molecular weight of this peptide-human serum albumin conjugate is 70-80 kDa, significantly lower than the 150-160 kDa molecular weight of traditional ADCs. Its smaller molecular size improves tissue permeability and solid tumor penetration, enhancing drug distribution in vivo. Furthermore, the drug loading ratio (DAR) of this peptide-human serum albumin conjugate is significantly better than that of traditional PDCs, achieving a drug loading level comparable to ADCs. While maintaining the advantage of low molecular weight, it significantly improves drug loading efficiency and enhances therapeutic potential. This invention also overcomes the technical shortcomings of large molecular weight and cumbersome preparation of ADCs, as well as the insufficient drug loading of traditional PDCs, achieving multiple technical advantages such as high drug loading, small molecular weight, and easy preparation, demonstrating significant advantages and promising application prospects.

[0033] 2. The present invention also provides a method for preparing the above-mentioned polypeptide-human serum albumin conjugate drug. The present invention uses human serum albumin and polypeptide as carrier backbone. The preparation method is simple and easy to implement, without the need for complex antibody expression and purification processes, resulting in lower production costs and easy large-scale production and quality control.

[0034] 3. The present invention also provides the application of the above-mentioned polypeptide-human serum albumin conjugate in the preparation of drugs against tumors overexpressing EGFR. Attached Figure Description

[0035] Figure 1 The targeted polypeptide NH2-CZ synthesized by solid-phase synthesis in Example 1 of this invention. EGFR:1907 -CONH2 mass spectrometry detection results; Figure 2 This is a mass spectrometry detection result of the polypeptide-human serum albumin conjugate drug in Example 2 of the present invention; Figure 3 The standard curves obtained by measuring the DM1 standard sample at a wavelength of 252 nm and the HSA standard sample at a wavelength of 280 nm in Example 2 of this invention are shown. Figure 4 This is a graph showing the results of an in vitro experiment on the inhibition of proliferation of human non-small cell lung cancer HCC-827 by the polypeptide-human serum albumin conjugate drug in Example 3 of the present invention. Figure 5 This is a comparative graph showing the tumor volume growth of human non-small cell lung cancer HCC-827 nude mice in the subcutaneous tumor treatment experiment of the PHDC group, DM1 group, PHC group and PBS group in Example 4 of the present invention; Figure 6 This image shows the tumor size results of non-small cell lung cancer HCC-827 nude mice in the PHDC group, DM1 group, PHC group and PBS group after subcutaneous tumor treatment experiment in Example 4 of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. However, these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0037] Balb / c nude mice and Balb / c mice (female, 5-6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; human non-small cell lung cancer HCC827 was purchased from the Chinese Academy of Sciences Cell Bank; ACN, DCM, DMF, DMSO, DIEA, TFA and human serum albumin (HSA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)matansine (DM1) was purchased from Shanghai Shengde Medical Technology Co., Ltd.; benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, triisopropylsilane and piperidine were purchased from Sinopharm Chemical Reagent Co., Ltd.; Rink amide MBHA resin and amino acids were purchased from Shanghai Jier Biochemical Technology Co., Ltd.; RPMI-1640 medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0038] Example 1 In this embodiment, the targeted polypeptide NH2-CZ was prepared. EGFR:1907 -CONH2, targeting peptide NH2-CZ EGFR:1907 The amino acid sequence of -CONH2 is SEQ ID No: 1. Targeting peptide NH2-CZ EGFR:1907 The preparation method of -CONH2 specifically includes the following steps: (1) On a peptide solid-phase synthesizer, 2 g of Rink amide MBHA resin with a degree of substitution of 0.375 mmol / g was weighed and placed into a reaction vessel. The corresponding program was set according to the target peptide sequence. DCM (dichloromethane) was added to swell the resin for 30 min. Then, the DCM was removed and the resin was washed three times with DMF (N,N-dimethylformamide). The resin was thoroughly dried after each wash. Then, a DMF solution of the amino acid Fmoc-L-Lys(Boc)-OH (3 mmol) protected by the first fluorene methoxycarbonyl (Fmoc) group at the C-terminus, the condensation reagent benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (4.5 mmol), and the organic base DIEA (9 mmol) was added. The reaction was carried out for 40 min. The solution was removed and the resin was washed with DMF, DCM, and DMF, respectively, to obtain Fmoc-L-Lys(Boc)-resin.

[0039] (2) Removal of Fmoc protecting groups from amino acids: A DMF solution containing 20% ​​(v / v) piperidine was added to the reaction flask to remove the Fmoc protecting groups at the amino terminus of the amino acids. After the reaction was completed, the resin was washed with DMF, DCM and DMF respectively to obtain NH2-Lys(Boc)- resin.

[0040] (3) Amide coupling: The second amino acid at the C-terminus of the target sequence was fixed onto the NH2-L-Lys(Boc)-resin obtained in step (2). Fmoc-protected amino acid Fmoc-L-Pro-OH (3 mmol), condensation reagent O-benzotriazole-tetramethylurea hexafluorophosphonate (HBTU) (4.5 mmol) in DMF solution, and base N,N-diisopropylethylamine (DIEA) (4.5 mmol) in DMF solution were added to the reaction flask. The reaction was carried out for 40 min. The solution was removed, and the mixture was washed with DMF, DCM, and DMF respectively to obtain Fmoc-Pro-Lys(Boc)-resin. Then, Fmoc was removed using the same method as in step (2) to obtain NH2-Pro-Lys(Boc)-resin. The process of coupling amino acids and removing Fmoc was repeated using the same method.Couple the resin sequentially with Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc- L-Val -OH, Fmoc-L-Leu-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L- Phe -OH, Fmoc-L-Ala-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Met-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Leu-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Pro-OH, Fmoc-L-Leu-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Ile-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Phe -OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc- L-Val -OH, Fmoc- L-Cys(Trt)-OH, followed by removing Fmoc, washing and drying,The resin-containing polypeptide NH2-CZ was obtained. EGFR:1907 -CONH2.

[0041] (4) Peptide cleavage: Weigh the peptide NH2-CZ containing resin. EGFR:1907 The mass of -CONH2, expressed as the volume of the lysis buffer and the amount of the resin-containing polypeptide NH2-CZ, is determined. EGFR:1907 A lysis buffer of appropriate volume was prepared using a 10:1 mass ratio of -CONH2. The lysis buffer consisted of 94% trifluoroacetic acid, 2% triisopropylsilane, 2% ethylenedithiol, and 2% water (by volume). The lysis buffer was then added to the resin-containing polypeptide NH2-CZ obtained in step (3). EGFR:1907 In CONH2, the mixture was stirred at room temperature for 3 hours. Subsequently, solid-liquid separation was performed, and the resulting filtrate was collected. Pre-cooled anhydrous diethyl ether was added to the filtrate, and the mixture was centrifuged to obtain a solid product. The solid was washed three times with pre-cooled anhydrous diethyl ether, and the precipitate was collected and vacuum dried to obtain the polypeptide NH2-CZ. EGFR:1907 -CONH2 crude product.

[0042] (5) Purification of the peptide: The peptide NH2-CZ obtained in step (4) was purified by high performance liquid chromatography (HPLC). EGFR:1907 The crude CONH2 product was purified using high-performance liquid chromatography (HPLC) with a reversed-phase C18 column (XBridge BEH C18 OBDPrep Column). Gradient elution was performed using acetonitrile and water as the mobile phases. Phase A consisted of acetonitrile (containing 0.1‰ TFA), and phase B consisted of water (containing 0.1‰ TFA). The gradient elution time was 15 min, the flow rate was 15 mL / min, and the elution conditions were: 30% A + 70% B for 2 min; 80% A + 20% B for 12 min; and 20% A + 80% B for 15 min. The detection wavelength was 210 nm. Mass spectrometry determined the molecular weight of the target component to be 6645.30, and the eluent was collected. Subsequently, acetonitrile was removed by concentration, and the product was lyophilized to obtain a white powder, which was the targeting peptide NH2-CZ. EGFR:1907 -CONH2.

[0043] The targeting peptide NH2-CZ synthesized in the solid phase EGFR:1907 -CONH2 was subjected to mass spectrometry analysis, and the mass spectrometry results are as follows: Figure 1 As shown, the results indicate that the targeting peptide NH2-CZ was successfully prepared. EGFR:1907 -CONH2.

[0044] Example 2 In this embodiment, cytotoxin N is covalently coupled to... 2 '-Deacetyl-N2 '-(3-mercapto-1-oxopropyl)matansin (DM1) and the targeting peptide NH2-CZ EGFR:1907 -CONH2 is conjugated to human serum albumin (HSA) to obtain a peptide-human serum albumin conjugate (PHDC). The specific steps include: (1) Take 0.1 g of the targeting polypeptide NH2-CZ prepared by the method in Example 1. EGFR:1907 -CONH2 and 0.005 g of 2,2'-dithiopyridine were added to 2 mL of DMSO and stirred at room temperature for 4 h. Then, the mixture was added to PBS buffer containing 0.7 g of HSA, the pH was adjusted to 8.0, and the reaction was continued at 4 °C with stirring to obtain crude peptide-human serum albumin (PHC). The crude peptide-human serum albumin (PHC) was placed in a dialysis membrane with a molecular weight cutoff (MWCO) of 30 kDa, and then placed in PBS buffer and ultrapure water in sequence. Dialysis was carried out overnight at 4 °C to obtain purified peptide-human serum albumin (PHC). In peptide-human serum albumin (PHC), the molar ratio of the tumor cell membrane protein peptide to the human serum albumin was 1:1.

[0045] (2) The purified polypeptide-human serum albumin (PHC) was dissolved in PBS buffer, and then 0.05 g of cross-linking agent SMCC was added to adjust the pH of the solution to 8.0. The reaction was continued at 4 °C with stirring for 4 h to obtain the product PHC-SMCC. Subsequently, the pH of the solution was lowered to 7.2, and 0.12 g of N2 was added. 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl)matansin (DM1) was stirred at 4 °C for 4 h to obtain crude peptide-human serum albumin conjugate (PHDC). The crude peptide-human serum albumin conjugate (PHDC) was placed in a dialysis membrane with a molecular weight cutoff (MWCO) of 30 kDa, and then successively placed in PBS buffer and ultrapure water. Dialysis was performed overnight at 4 °C, and then freeze-dried in a freeze dryer at -80 °C to obtain purified peptide-human serum albumin conjugate (PHDC).

[0046] Figure 2 The mass spectrometry results of the prepared peptide-human serum albumin conjugate (PHDC) are shown. The results show that the prepared peptide-human serum albumin conjugates have drug loading ratios (DAR) of 6, 8, 9, 12 and 19.

[0047] The mean drug loading ratio (DAR) of peptide-human serum albumin conjugate (PHDC) was obtained by measuring the concentrations of DM1 and HSA in the PHDC solution using Beer-Lambert's law, and then calculating the ratio of DM1 concentration to HSA concentration. Specifically, the steps included: measuring the absorbance of HSA standard at 280 nm and DM1 standard at 252 nm at different concentrations, and obtaining the linear relationship between concentration and absorbance value according to Beer-Lambert's law. The results are shown below. Figure 3 As shown, Figure 3 The standard curves were obtained by measuring the DM1 standard sample at a wavelength of 252 nm and the HSA standard sample at a wavelength of 280 nm. Figure 3 The left side shows the standard curve obtained by measuring the DM1 standard sample at a wavelength of 252 nm. Figure 3 The right side shows the standard curve obtained by measuring the HSA standard sample at a wavelength of 280 nm. Subsequently, a PHDC solution was prepared, with the concentrations of HSA and DM1 in the PHDC solution at... Figure 3 Within the linear range of the standard curve, the absorbance of the PHDC solution at 252 nm and 280 nm was measured, and the concentrations of HSA and DM1 in the PHDC solution were obtained according to the standard curve. The mean drug loading ratio (DAR) of the peptide-human serum albumin conjugate (PHDC) was calculated using the following formula: DAR= Among them, C DM1 The concentration of DM1, C HSA The concentration of HSA; The average drug loading ratio (DAR) of the peptide-human serum albumin conjugate (PHDC) was 6.8 after three measurements and the average value was taken.

[0048] Example 3 In this embodiment, the killing effect of the polypeptide-human serum albumin conjugate (PHDC) prepared in Example 2 on EGFR-overexpressing tumor cells was measured in vitro using the MTT assay. The human non-small cell lung cancer HCC827 cell line was used as an example of EGFR-overexpressing tumor cells in this embodiment. The experimental procedure included the following steps: (1) Weigh the PHDC, PHC and DM1 obtained in Example 2 and add them to sterilized PBS to prepare PHDC solution, PHC solution and DM1 solution of 100 μmol / L respectively.

[0049] (2) Add the 100 μmol / L PHDC solution, PHC solution and DM1 solution obtained in step (1) to the culture medium (90% RPMI-1640 basal medium + 10% high-quality fetal bovine serum (FBS)) to obtain culture media with a certain concentration gradient (0.005 nmol / L, 0.01 nmol / L, 0.05 nmol / L, 0.1 nmol / L, 0.5 nmol / L, 1 nmol / L, 5 nmol / L, 10 nmol / L and 50 nmol / L) for later use.

[0050] (3) After passage and centrifugation of normally growing human non-small cell lung cancer HCC827, the cell density was measured under an optical microscope, and the cells were added to 96-well plates (three wells per group, 5000 cells per well) and cultured overnight in a cell culture incubator.

[0051] (4) Remove the original culture medium from the 96-well plate and add culture medium prepared in step (2) with a certain concentration gradient (0.005 nmol / L, 0.01 nmol / L, 0.05 nmol / L, 0.1 nmol / L, 0.5 nmol / L, 1 nmol / L, 5 nmol / L, 10 nmol / L, 50 nmol / L) to the PHDC group, PHC group, and DM1 group respectively. A blank control group is set up (with drug-free culture medium (90% RPMI-1640 basal medium + 10% high-quality fetal bovine serum (FBS)) and placed in a cell culture incubator for 72 h.

[0052] (5) Remove the original culture medium from step (4), add the prepared MTT reagent, 10 μL per well, and incubate in a cell culture incubator for 4 h. Use a multi-functional microplate reader to read the absorbance value at 490 nm and process the data to obtain a normalized cytotoxicity result graph.

[0053] Figure 4 The graph shows the cell killing results of the PHDC, PHC, and DM1 groups. The vertical axis represents the relative value of cell viability, with the blank control group without any drugs as 100%. The results show that within the concentration range of 0.005 to 0.5 nmol / L, the cell killing effect of the PHDC group is significantly better than that of the DM1 and PHC groups.

[0054] Example 4 In this embodiment, the antitumor effect of the polypeptide-human serum albumin conjugate (PHDC) prepared in Example 2 was studied in mice. The selected tumor was a subcutaneous tumor of human non-small cell lung cancer HCC827 in nude mice. The specific steps included: (1) After digesting human non-small cell lung cancer HCC827 cells in the logarithmic growth phase, the cells were prepared into 2×10⁻⁶ cells using ice-cold PBS. 7 A cell suspension of 100 μL was injected into the right axilla of a female BALB / c Nude mouse using a sterile syringe.

[0055] (2) Wait until the mouse tumor volume reaches 150-200 mm 3 Mice were then randomly divided into four groups of three: PHDC, DM1, PHC, and PBS. The PHDC group received a dose of 4.5 mg / kg. The DM1 and PHC groups received equimolar amounts of DM1 and PHC, respectively. PHDC, DM1, and PHC were dissolved in PBS and brought to a final volume of 100 μL. The PBS group received 100 μL of PBS. Administered via tail vein injection, once a week, for a total of two administrations.

[0056] (3) Measure the volume of the mouse tumor with calipers every other day until day 24, then treat the mouse and remove the tumor.

[0057] Figure 5 A comparative graph showing the tumor volume growth of human non-small cell lung cancer HCC-827 nude mice in the subcutaneous tumor treatment experiment in the PHDC group, DM1 group, PHC group and PBS group; Figure 6 The figures show the tumor size results of HCC-827 non-small cell lung cancer nude mice in the PHDC, DM1, PHC, and PBS groups after subcutaneous tumor treatment experiments. As can be seen from the figures, the peptide-human serum albumin conjugate (PHDC) has a significant inhibitory effect on the growth of mouse tumors, and its anti-tumor effect is significantly better than that of DM1 and PHC. This indicates that the peptide-human serum albumin conjugate (PHDC) provided by this invention has great application value in the treatment of tumors overexpressing EGFR.

[0058] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A polypeptide-human serum albumin conjugate drug, characterized in that, The peptide-human serum albumin conjugate comprises a tumor cell membrane protein peptide, human serum albumin, and a cytotoxin; the molar ratio of the tumor cell membrane protein peptide, human serum albumin, and cytotoxin is 1:1:(6-19); the amino acid sequence of the tumor cell membrane protein peptide is shown in SEQ ID No: 1; the cytotoxin is N... 2 '-Deacetyl-N 2 '-(3-Mercapto-1-oxopropyl)-Maytansin; the targeted tumor cell membrane protein polypeptide forms a disulfide bond with the Cys residue in human serum albumin through the Cys residue; the cross-linking agent covalently couples the cytotoxin to the lysine residue of human serum albumin.

2. A method for preparing the polypeptide-human serum albumin conjugate drug according to claim 1, characterized in that, The steps include the following: Step 1): The targeted tumor cell membrane protein polypeptide is added to the human serum albumin, stirred and reacted, and purified to obtain polypeptide-human serum albumin; Step 2): Dissolve the polypeptide-human serum albumin in a buffer solution, add a cross-linking agent and cytotoxin, stir the reaction, purify, and obtain the polypeptide-human serum albumin conjugate drug.

3. The method for preparing the polypeptide-human serum albumin conjugate drug according to claim 2, characterized in that, In step 2), the crosslinking agent is selected from at least one of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester and sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester.

4. The use of the polypeptide-human serum albumin conjugate of claim 1 or the polypeptide-human serum albumin conjugate prepared by the preparation method of claim 2 or 3 in the preparation of a drug for treating tumors overexpressing EGFR, wherein the tumor overexpressing EGFR is lung cancer.

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