CN116270963A

CN116270963A - New application of polypeptide derivative

Info

Publication number: CN116270963A
Application number: CN202111564284.3A
Authority: CN
Inventors: 熊梦华; 袁月玲; 鲍燕; 李�杰
Original assignee: South China University of Technology SCUT; Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: South China University of Technology SCUT; Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2021-12-06
Filing date: 2021-12-20
Publication date: 2023-06-23

Abstract

The invention provides an application of a polypeptide derivative with a structure shown in a formula (I) in preparing an LMP inducer, wherein R is selected from C ₄ ‑C ₂₄ An alkyl group; r1 and R2 are respectively and independently selected from: H. c (C) ₁ ‑C ₆ Alkyl, or R1, R2, together with the carbon atom to which they are attached, form C ₃ ‑C ₈ A cycloalkenyl group; x+y is greater than 5. The polypeptide has electronegativity at physiological pH, low cytotoxicity to normal tissue, and can be converted into positive electricity under slightly acidic environment of tumor tissue to promoteIntake into cells; the polypeptide enters a lysosome through an endocytic way, has strong membrane rupture activity under the acidity of the lysosome, can cause permeabilization of the lysosome membrane and damage of the lysosome membrane, and causes the content in the lysosome to leak to cytoplasm, thereby inducing death of lysosome-dependent cells, having a selective high killing effect on tumor cells and being capable of inducing a strong anti-tumor immune effect.

Description

New application of polypeptide derivative

Technical Field

The invention relates to the technical field of high molecular polypeptide materials and medicines, in particular to a novel application of a polypeptide derivative in resisting tumors.

Background

Lysosomal membrane permeabilization (Lysosomal Membrane Permeabilization, LMP) is the process of lysosomal cathepsin and hydrolase leakage caused by lysosomal membrane damage, and is also a marker for lysosomal dependent cell death (lysoname-dependent Cell Death, LCD) and initiation of programmed cell death. Through regulating LMP, the effects of overcoming tumor cell apoptosis resistance and activating immunity can be realized, and the method has important significance for anti-tumor treatment. Studies have shown that due to the rapid division and expansion of tumor cells, their lysosomal volume increases and cathepsin activity increases, resulting in changes in the structure and function of the lysosomal membrane, making tumor cells more susceptible to increased sensitivity to various endogenous (p 53 activation, oxidative stress) and exogenous (cationic amphiphilic drugs) triggers of lysosomal membrane penetration. LMP triggered LCDs typically bypass the classical caspase-dependent apoptosis pathway, opening up new strategies for apoptosis resistance and drug-resistant tumor therapy. Furthermore, LM P can induce anti-tumor immune effect. In a colon cancer model with STAT3 gene mutation, STAT3 gene deletion causes lysosomal membrane disruption, and LMP promotes leakage of cathepsin into the cytoplasm; cathepsins enhance presentation of MHC-class I antigens in intestinal epithelial cells and transfer of peptide-MHC complexes to dendritic cells via cross-address, enhancing CD8 ⁺ Activation of T cells, induction of anti-tumor immunity; colorectal cancer mice are treated with an LMP inducer (chloroquine) to increase lymphocyte infiltration of cancerous tissues and survival rate.

Studies have shown that intervention or direct physical disruption of lysosomal membrane stability components (e.g., detergents and inhibitors of heat shock protein 70) or oxidation of lysosomal membranes can cause LMP. In addition, cytoskeleton directs lysosomal turnover through exocytosis and autophagy, and lysosomal swelling and instability can also be caused by disruption of the cytoskeleton, repositioning of actin filaments, and the like. Siramesine is a type of lysosome detergent containing amino groups, which can enter a lysosome through diffusion, amino side chains are protonated in the acidic environment of the lysosome to form positively charged cationic amino groups, the positively charged cationic amino groups are converted into the lysosome detergent, and LMP is induced by directly damaging a lysosome membrane, causing autophagosome accumulation or iron death, so that cell death is triggered. LMP is caused by targeting lysosomal sphingolipid metabolism, sphingosine, an unsaturated hydrocarbon-based chain-containing octadecyl amino alcohol, can be protonated in lysosomes and act as a detergent, causing dose-dependent lysosome rupture, inducing necrosis of cells. An increase in lysosomal pH or inhibition of cathepsin can be effective in reducing the lysosomal damaging effects of sphingosine. However, these reported lysosomal targeted drugs, like other anticancer drugs, are often limited in their application in clinical trials due to low selectivity for tumors, resulting in insufficient anticancer effects and concomitant serious side effects.

Disclosure of Invention

Based on the above, the invention discovers a polypeptide derivative, wherein the polypeptide is electronegative under physiological pH, has small cytotoxicity to normal tissues, is converted into electropositivity under the slightly acidic environment of tumor tissues, and promotes the cell uptake; the polypeptide enters a Lysosome through an endocytic way, has strong membrane rupture activity under the acidity of the Lysosome, can cause Lysosome Membrane Permeabilization (LMP), causes Lysosome membrane damage, and causes Lysosome content to leak to cytoplasm, thereby inducing Lysosome-dependent cell death (lysoname-dependent Cell Death, LCD), thereby having a selective high killing effect on tumor cells and being capable of inducing a strong anti-tumor immune effect.

The specific technical scheme is as follows:

use of a polypeptide derivative having a structure according to formula (I):

wherein R is selected from C ₄ -C ₂₄ An alkyl group;

r1 and R2 are respectively and independently selected from: H. c (C) ₁ -C ₆ Alkyl, or R1, R2, together with the carbon atom to which they are attached, form C ₃ -C ₈ A cycloalkenyl group;

x+y is greater than 5.

In some of these embodiments, R is selected from C ₈ -C ₂₀ An alkyl group.

In some of these embodiments, R is selected from C ₁₂ -C ₁₈ An alkyl group.

In some embodiments, R1, R2 are each independently selected from: H. methyl, ethyl, propyl, or R1, R2, together with the carbon atom to which they are attached, form a cyclopentenyl or cyclohexenyl group.

In some embodiments, one of R1 and R2 is H and the other is C ₁ -C ₃ An alkyl group.

In some of these embodiments, x+y is 5-20.

In some of these embodiments, x+y is 8-18.

In some of these embodiments, x+y is 10.

In some embodiments, x is 30-60% of x+y.

In some embodiments, x is 40-55% of x+y.

In some embodiments, x is 48-52% of x+y.

In some embodiments, x is 50% of x+y.

In some of these embodiments, the polypeptide derivative is selected from the following polymers:

the polypeptide derivative is applied to the preparation of medicines for preventing and/or treating tumors.

The application of the polypeptide derivative in preparing a vaccine for preventing and/or treating tumors.

The polypeptide derivative is applied to the preparation of an inducer for inducing anti-tumor immunity.

In some embodiments, the tumor is pancreatic cancer, colorectal cancer, breast cancer, colon cancer, lung cancer, liver cancer, melanoma, or glioma.

The invention also provides a medicine for preventing and/or treating tumors.

The specific technical scheme is as follows:

a medicine for preventing and/or treating tumor is prepared from active component and auxiliary materials and/or carrier, wherein the auxiliary materials and/or carrier are acceptable in medicine, and the active component comprises the polypeptide derivative.

In some embodiments, the active ingredient comprises a polypeptide derivative as described above and a combined immune checkpoint blocker.

In some of these embodiments, the combined immune checkpoint blocker is an Anti-PD-L1 antibody.

The invention also provides a vaccine for preventing and/or treating tumors.

The specific technical scheme is as follows:

a vaccine for preventing and/or treating tumors, which is a tumor cell treated with the above-mentioned polypeptide derivative.

In some embodiments, the tumor cell is a pancreatic cancer cell, colorectal cancer cell, breast cancer cell, colon cancer cell, lung cancer cell, liver cancer cell, melanoma cell, brain glioma cell.

The invention also discloses a preparation method of the vaccine for preventing and/or treating tumors.

The specific technical scheme is as follows:

a method for preparing a vaccine for preventing and/or treating tumors, comprising the steps of: and (3) treating tumor cells by using the polypeptide derivative, and collecting dying cells to obtain the vaccine.

In some embodiments, the method for preparing a vaccine for preventing and/or treating tumor comprises the following steps: plating the tumor cells at 0.8-1.2 million/dish at 80-120mm ³ Adding the polypeptide derivative diluted by serum-free DMEM into a culture dish, treating the tumor cells for 0.5-16 hours, discarding the supernatant, and collecting moribund cells to obtain the vaccine; the final concentration of the polypeptide derivative is 40 mug/mL-200 mug/mL.

The invention discovers a polypeptide derivative modified by anhydride, which is electronegative at physiological pH, does not damage normal tissue cells and has low cytotoxicity to the normal tissue; in the slightly acidic environment of tumor tissues, the polypeptide derivative is converted into electropositivity, so that the cell uptake can be promoted; and the polypeptide derivative can be converted into polypeptide with a spiral structure under the acidity of the Lysosome after entering the Lysosome through an endocytic way, has strong membrane rupture activity, can cause Lysosome Membrane Permeabilization (LMP), causes Lysosome membrane damage, and causes Lysosome content to leak to cytoplasm, thereby inducing Lysosome-dependent cell death (lysoome-dependent Cell Death, LCD), and further having high-selectivity and strong killing effect on tumor cells. In addition, the polypeptide derivative can induce a powerful anti-tumor immune effect.

Furthermore, the polypeptide derivative and the Anti-PD-L1 antibody of the combined immune checkpoint blocker are used in combination, so that the Anti-tumor effect and the effect of inducing a strong Anti-tumor immune effect can be further improved, and the treatment effect of curing the tumor can be achieved for partial tumor mice.

Drawings

FIG. 1 shows the synthesis of polypeptides of different alkyl chain lengths.

FIG. 2 shows GPC characterization results of PButLG10 with different carbon chain lengths.

FIG. 3 shows PButLG10 with different carbon chain lengths ¹ H-NMR characterization results.

FIG. 4 shows PButLG10-CA with different carbon chain lengths ¹ H-NMR characterization results.

FIG. 5 shows the results of circular dichroism characterization of membrane-broken polypeptide with different lengths of hydrophobic end group helical structures.

FIG. 6 shows the MTT assay for detecting the toxicity of membrane-broken polypeptides with different hydrophobic end-group helical structures to EMT6 cells.

FIG. 7 shows membrane rupture activity of membrane rupture polypeptide with different hydrophobic end group spiral structures in a hemolysis experiment.

FIG. 8 shows GPC characterization results of helical structure polypeptides with different degrees of polymerization.

FIG. 9 is a schematic diagram of a polypeptide having a helix structure with different degrees of polymerization ¹ H-NMR characterization results.

FIG. 10 shows a circular dichroscope for characterizing the secondary structure of helical polypeptides of different degrees of polymerization.

FIG. 11 shows membrane-rupture activity of helical structure polypeptides with different degrees of polymerization in a hemolysis experiment.

FIG. 12 is a schematic diagram showing the synthesis of a series of non-helical polypeptides.

FIG. 13 is a series of non-helical structure polypeptides ¹ H-NMR characterization results.

FIG. 14 shows membrane-disrupting activity of each of the non-helical polypeptides in a hemolysis assay.

FIG. 15 shows the determination of cytotoxicity of non-helical structure polypeptide against pancreatic cancer Panc02 cells by MTT method under physiological conditions (pH 7.4) or tumor slightly acidic environment (pH 6.8).

FIG. 16 shows the kinetics of helix recovery of non-helix polypeptides measured by round dichroism at different pH.

FIG. 17 is a graph showing the ability of liposome leakage experiments to determine the disruption of membrane structure by non-helical structure polypeptides at different pH conditions.

FIG. 18 shows particle size (A) and potential (B) of non-helical structure polypeptide measured by dynamic light scattering at pH 7.4 and pH 6.8.

FIG. 19 is a flow cytometry detection of intake of non-helical structural polypeptide by pancreatic cancer Panc02 cells at pH 7.4 and pH 6.8.

FIG. 20 is a graph showing the effect of MTT assay on the cytotoxicity of polypeptide P3/P4/P5 by low temperature inhibition of endocytosis.

FIG. 21 is a laser confocal microscope observation of FITC-labeled P3 polypeptide (P3-FITC) co-localize with lysosomes in steady-state LAMP1-mCherry pancreatic cancer Panc02 cells.

FIG. 22 shows the effect of endocytosis-inhibiting conditions on polypeptide P3 cellular uptake (A) and cytotoxicity (B-F).

FIG. 23 shows the detection of the damage of the integrity of the lysosomal membrane of tumor cells by the polypeptide P3 in Galctin3-GFP spot assay at pH 7.4 or pH 6.8.

FIG. 24 is a graph showing fluorescence changes of Galctin3-GFP and PI after treatment of cells with non-helical polypeptides at pH 6.8 (A-C); and a statistical plot of the time difference between the appearance of Galctin3-GFP spots and PI staining positivity (D).

FIG. 25 shows the lysosome morphology of the polypeptide P3 after treatment of Panc02 cells for 8h or 16h at pH 7.4 or pH 6.8 by transmission electron microscopy.

FIG. 26 is a laser confocal observation of the case of the leakage of the polypeptide P3 induced by lysosomes CTSB-mCherry at pH 7.4 or pH 6.8.

FIG. 27 is a chart showing the determination of the acidification inhibitors chloroquine and canavanine A by MTT method; effects of protease inhibitors E-64d and CA-074-ME on polypeptide P3 cytotoxicity.

Fig. 28 is the in vivo therapeutic effect of polypeptide P3 on a pancreatic cancer Panc02 subcutaneous model.

Fig. 29 shows in vivo therapeutic effects of polypeptide P3 on breast cancer EMT6 in situ model and colon cancer CT26 subcutaneous model.

FIG. 30 shows the in vivo therapeutic effect of a polypeptide P3 in combination with an anti-PD-L1 antibody on a pancreatic cancer Panc02 subcutaneous model.

FIG. 31 shows the in vivo therapeutic effect of polypeptide P3 in combination with anti-PD-L1 antibodies on a subcutaneous model of colon cancer MC 38.

FIG. 32 shows the in vivo therapeutic effect of a polypeptide P3 in combination with an anti-PD-L1 antibody on an in situ breast cancer E0771 model.

FIG. 33 shows the in vivo therapeutic effect of a polypeptide P3 in combination with an anti-PD-L1 antibody on an in situ model of breast cancer EMT 6.

FIG. 34 is the distal anti-tumor effect of the polypeptide P1/P3/P4/P5 in combination with anti-PD-L1 antibodies.

FIG. 35 shows the anti-tumor effect of the cellular vaccine after treatment with the polypeptide P1/P3/P4.

FIG. 36 shows the determination of the toxicity of different anhydride modified ratios of P3 polypeptide to tumor cells by MTT method.

Detailed Description

The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.

In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the compounds of the invention, when any variable (e.g., R ₁ 、R ₂ Etc.) occur more than once in any component, the definition of each occurrence is independent of the definition of each other occurrence. Also, combinations of substituents and variables are permissible provided that such combinations stabilize the compounds. The lines drawn from the substituents into the ring system indicate that the bond referred to may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any suitable carbon atom adjacent to the ring. It is to be understood that substituents and substitution patterns of the compounds of this invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that may be readily synthesized from readily available starting materials by techniques in the art and methods set forth below. If the substituent itself is substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, as long as the structure is stabilized.

The term "alkyl" as used herein is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, "C ₁ -C ₆ Alkyl "medium" C ₁ -C ₆ The definition of "includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, "C ₁ -C ₆ The alkyl group includes, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl.

The term "alkenyl" is an unsaturated monocyclic cyclic substituent containing a c=c double bond, the ring atoms of which are all carbon, for example: cyclopentenyl, cyclohexenyl, and the like.

The medicament for preventing and/or treating tumors of the present invention may be used in non-human mammals or humans.

The pharmaceutically acceptable auxiliary materials or carriers used in the medicament for preventing and/or treating tumor of the invention refer to: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity.

"compatibility" as used herein means that the components of the composition are capable of being admixed with the active ingredient of the present invention (the polypeptide derivative of formula I) and with each other without significantly reducing the efficacy of the active ingredient.

Pharmaceutically acceptable excipients used in the medicament for preventing and/or treating tumors of the present invention include, but are not limited to, one or more of the following materials: at least one of a solvent, excipient, filler, compatibilizer, binder, humectant, disintegrant, slow solvent, absorption accelerator, adsorbent, diluent, solubilizer, emulsifier, lubricant, wetting agent, suspending agent, flavoring agent, and perfume.

Examples of pharmaceutically acceptable auxiliary materials include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as

) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and the like.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

In these solid dosage forms, the active ingredient is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:

(a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

(b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia;

(c) Humectants, for example, glycerin;

(d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

(e) Slow solvents, such as paraffin;

(f) Absorption accelerators, for example quaternary amine compounds;

(g) Wetting agents, for example cetyl alcohol and glycerol monostearate;

(h) Adsorbents, such as kaolin; and

(i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

The solid dosage forms may also be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active ingredient in such a composition may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like. In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The compounds of formula I of the present invention may be used in combination with other drugs known to treat or ameliorate similar conditions. In combination, the original drug is administered in a constant manner & dose, while the compound of formula I is administered simultaneously or subsequently. When the compound of formula I is administered simultaneously with one or more other drugs, it is preferable to use a pharmaceutical composition containing one or more known drugs together with the compound of formula I. Drug combinations also include administration of a compound of formula I with one or more other known drugs over an overlapping period of time. When a compound of formula I is administered in combination with one or more other agents, the dosage of the compound of formula I or the known agent may be lower than when administered alone.

Drugs or active ingredients that may be used in combination with the compounds of formula i include, but are not limited to:

a combination immune checkpoint blocker, an estrogen receptor modulator, an androgen receptor modulator, a retinal-like receptor modulator, a cytotoxin/cytostatic agent, an antiproliferative agent, a protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protein kinase inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a cell proliferation and survival signaling inhibitor, a drug that interferes with the cell cycle checkpoint and an apoptosis inducer, a cytotoxic drug, a tyrosine protein inhibitor, an EGFR inhibitor, a VEGFR inhibitor, a serine/threonine protein inhibitor, a Bcr-Abl inhibitor, a c-Kit inhibitor, a Met inhibitor, a Raf inhibitor, a MEK inhibitor, an MMP inhibitor, a topoisomerase inhibitor, a histidine deacetylase inhibitor, a proteasome inhibitor, a CDK inhibitor, a Bcl-2 family protein inhibitor, an MDM2 family protein inhibitor, an IAP family protein inhibitor, a STAT family protein inhibitor, a PI3K inhibitor, an AKT inhibitor, an integrin blocker, an interferon- α, interleukin-12, a COX-2 inhibitor, p53, a p53 activator, a VEGF antibody, an EGF, and the like.

In some of these embodiments, the drugs or active ingredients that may be used in combination with the compounds of formula i include, but are not limited to: anti-PD-L1 antibody, aldesleukin, alendronic acid, interferon, al Qu Nuoying, allopurinol sodium, palonosetron hydrochloride, altretamine, aminoglutethimide, amifostine, amrubicin, an Ya pyridine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, minoxidil, 5-azacytidine, azathioprine, bacillus calmette-guerin or tice bacillus calmette-guerin, betadine, betamethasone acetate, betamethasone sodium phosphate formulation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, calcitonin, alezomib injection, capecitabine, carboplatin, kang Shide, cefesone, cil Mo Baijie, erythromycin, chlorambucil, cisplatin, cladribine, chloro Qu Linsuan, cyclophosphamide, cytarabine, carboxin, dacarbazine, dactinomycin D, dacarbazine dexamethasone, dexamethasone phosphate, estradiol valerate, dulinterleukin 2, dibaume, dulorelin, delazocine, diethylstilbestrol, dafukang, docetaxel, doxifluridine, doxorubicin, dronabinol, qin-166-chitosan complex, eligard, labyrinase, epirubicin hydrochloride, aprepitant, epirubicin, alfumagillin, erythropoietin, eplatin, levamisole, estradiol preparation, 17-beta-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphoric acid, fivelum, etoposide, fadrozole, tamoxifen preparation, feglastine, finasteride, feveridine, fluorouridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxymestane, flupirtine, fotemustine, 1-beta-D-arabinofuranosyl-cytothiadine-5' -stearoyl phosphate, fotemustine, fulvestrant, progastrin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine wafer capsule, goserelin, glatiramer hydrochloride, histrelin, and meflozin, hydrocortisone, erythro-hydroxynonyladenine, hydroxyurea, tetan iso Bei Moshan antibody, idarubicin, ifosfamide, interferon alpha 2A, interferon alpha 2B, interferon alpha nl, interferon alpha n3, interferon beta, interferon gamma la, interleukin 2, intron A, iressa, irinotecan, ketjel, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levamisole calcium levofolinate, sodium levothyroxine formulations, lomustine, lonidamine, dronabinol, nitrogen mustard, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogens, 6-borazine, mesna, methotrexate, methyl aminolevulinate, miltefosine, melomycin, mitomycin C, mitotane, mitoquinone, trospine, doxorubicin citrate liposomes, nedaplatin, pegylated febuxostat, olpreninterleukin, neunogen, nilutamide, tamoxifen, NSC-631570, recombinant human interleukin 1-beta, octreotide, ondansetron hydrochloride, dehydrohydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate formulations, pegine, roxyprogesterone, euphorbia, pernicid, and the like, pennisetum, streptozotocin, pilocarpine hydrochloride, bicubicin, plicamycin, porphin sodium, prednimustine, setprednisolone, prednisone, beclomethamine, procarbazine, recombinant human erythropoietin, raltitrexed, liratio, etidronate rhenium-186, mevalhua, dynamics stretch-A, romidepide, pilocarpine hydrochloride tablet, octreotide, sarustine, semustine, sirolimus, sibutramine, sibutrazol, sodium methylprednisolone, palustric acid, stem cell therapy, streptozocin, strontium chloride-89, levothyroxine sodium, tamoxifen, tamsulosin, testolazine, taxotere, temozolomide, teniposide, testosterone, thioguanine, thiotepa, somatostatin, temozolomide, toldronic acid, topotecan, tolnaftate, tolizumab, toxidan trastuzumab, trocounter, treoshu, tretinoin, methotrexate tablet, trimethamine, trimetraxazole, triptorelin acetate, trastuline pamoate, ulipraline, uridine, valrubicin, valdecolonil, vinblastine, vincristine, vinlamide, vinorelbine, vitamin Lu Liqin, dexpropimine, neat-Ding Sizhi, pivalonine, paclitaxel protein stabilized formulation, acolbifene, interferon r-lb, affinitak, aminopterin, alzoxifene, asorisnil, atomestane, atrasentan, BAY43-9006, avastin, CCI-779, CDC-501, celebantam, cetuximab, crizotrope, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dIM, dutasteride, edoxin, irinotecan, flunine, valirbestrol, bivalirudin, amiloride, daphne hydrochloride, daphne, holmium-166 DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, milbexifene, mi Nuoqu acid ester, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovalproate, norlabratex, olimarson, onco-TCS, oside, paclitaxel polyglutamate, sodium silk-miate, PN-401, QS-21, quaternary, R-154, raloxifene, ranpirnase, 13-cis-retinoic acid, satraplatin, orcalcitol, T-138067, tarceva, docosahexaenoic acid paclitaxel, thymol, prostaglandin furin, tipifarnib, tiramimine, TLK-286, toremio, 7R, valproan, prandin, ibvanadn, R-154, raloxifene, MID-100, and combinations thereof.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Example 1: synthesis and characterization of membrane-broken polypeptide with different hydrophobic end group spiral structures

(1) In this example, hexadecylamine, dodecylamine and N-octylamine are used as initiator to initiate ring-opening polymerization of glutamic acid-derived N-carboxylic anhydride (NCA) to obtain polypeptide C with terminal alkyl chains of different lengths _n PButLG10 (n=8, 12, 16). Polypeptide C by click reaction _n The side group of PButLG10 is modified by primary amine to obtain polypeptide C with different length alkyl chains at the end _n -PButLG10-CA(n＝8，12, 16) is shown in fig. 1 (for the synthetic method, refer to patent CN 111548388A).

(2) Polypeptide C for synthesizing alkyl chains with different lengths _n GPC characterization of PButLG10, from the results of FIG. 2, it can be seen that the different polypeptides C _n The retention time of PButLG10 is approximately the same and the PDI is small; indicating successful synthesis of narrowly dispersed polypeptide. As shown in fig. 3, by ¹ H-NMR characterization of polypeptide C _n The degree of polymerization of PButLG10, from the integrated area of the nuclear magnetism, can be calculated for each polypeptide to be about 10. As shown in FIG. 4, the polypeptide C obtained _n -PButLG ₁₀ -CA (n=8, 12, 16) for nuclear magnetic characterization, from which it can be seen that the peak of the pendant double bond is completely disappeared, indicating that the click reaction is complete.

(3) The synthesized series of helical structure polypeptide is characterized by a circular dichroscope, and the main operation is as follows: samples with a final concentration of 0.1mg/ml were placed in a round dichroism chromatograph quartz dish with a path length of 0.05cm for detection. Ellipticity [ theta ]]=mdeg x 1000/(d×c) where mdeg is the experimental measurement, d is the optical path (mM), c is the concentration (mM), the resulting molar ellipticity [ θ ]]The unit is deg.cm ² ·dmol ^-1 . As shown in FIG. 5, C ₁₆ -PButLG-CA、C ₁₂ -PButLG-CA and C ₈ The PButLG-CA has good absorption at 208nm and 220nm and similar absorption strength, which shows that the three materials have stronger alpha spiral structure, and the helicity is more than 90 percent. The hydrophobicity of the hydrophobic alkyl chain end groups has no obvious effect on the helicity of the polypeptide.

(4) And determining the killing effect of different hydrophobic end group helical structure membrane-breaking polypeptide on tumor cells by adopting an MTT method. The specific operation is as follows: EMT6 tumor cells are spread in 96-well plates according to 1 ten thousand cells per well, and are used after being cultured overnight; preparation of Membrane-rupture polypeptide (C) at a series of concentrations Using complete Medium ₈ -PButLG-CA、C ₁₂ -PButLG-CA and C ₁₆ -PButLG-CA) at a final drug concentration of 10-160 μg/mL, while taking the drug-free group as a positive control group; taking out the pore plate, removing the culture medium supernatant, adding corresponding culture medium containing the medicine according to 100 mu L/pore, and placing the cells in a 37 ℃ incubator for culture; at a specific time point, cells are removed and the contents are removed Adding culture medium containing 0.5mg/mLMTT into the supernatant, and culturing at 37deg.C for 2-4 hr; removing supernatant, adding 100 μl of dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) into each well, and shaking in a shaker at low speed in the absence of light for 10 min to dissolve the crystals completely; absorbance at 490nm was measured using an enzyme-labeled instrument, and absorbance for the experimental group incubated with drug was defined as I _{Experimental group} The absorbance of the drug-and cell-free medium group is defined as I _{Negative control} Cells without drug were used as positive control, absorbance was defined as I _{Positive control} The method comprises the steps of carrying out a first treatment on the surface of the And then according to the formula [ (I) _{Experimental group} -I _{Negative control} )/(I _{Positive control} -I _{Negative control} )]Cell viability was calculated by 100% and plotted. The results are shown in FIG. 6, C with hydrophobic end groups of different lengths ₈ -PButLG-CA、C ₁₂ -PButLG-CA and C ₁₆ The PButLG-CA has rapid killing activity on tumor cells, after the cells are treated by 80 mug/mL of polypeptide for 1 hour, the killing efficiency of three kinds of helical structure polypeptide on EMT6 tumor cells reaches more than 80%, and the killing efficiency of helical structure polypeptide with different hydrophobic end groups on tumor cells is not obviously different.

(5) And (3) testing the membrane rupture activity of the polypeptide with the spiral structure with different hydrophobic end groups by adopting a erythrocyte hemolysis experiment. The main operation is as follows: after the sheep whole blood is gently shaken up, 1mL of whole blood is taken into a 50mL centrifuge tube, diluted to 25mL (namely 4% sheep blood is prepared) by 1X PBS, and temporarily stored at 4 ℃ for standby; each polypeptide (C) was purified using DMSO ₈ -PButLG-CA、C ₁₂ -PButLG-CA and C ₁₆ -PButLG-CA) into a stock solution of 50 mg/ml; c with PBS ₈ -PButLG-CA、C ₁₂ -PButLG-CA and C ₁₆ -PButLG-CA stock solution was diluted to 512 μg/mL, followed by gradient dilution with PBS containing equivalent DMSO content to give a series of 2X drug stock solutions of each polypeptide at drug concentrations of 8-1024 μg/mL; taking the prepared 2X medicine solution in an EP tube, adding 4% sheep blood with equal volume, and finally, lightly blowing and mixing the solution by a liquid transfer device, wherein the concentration of the working solution of each polypeptide is 4-512 mug/mL; meanwhile, the final concentration of 0.1% Triton X is used as a positive control and PBS solution is used as a negative control; all samples were placed in a 37 ℃ incubator for 1 hour; taking outThe sample is placed in a centrifuge and centrifuged at 1000rpm for 5 minutes at room temperature; after centrifugation, 100 μl of each sample supernatant was gently pipetted into a 96-well plate; the absorbance at 576nm was measured using an enzyme-labeled instrument; the absorbance of the experimental group incubated with the drug was defined as I _{Experimental group} The absorbance of the control incubated with PBS and red blood cells was defined as I _{Negative control} The absorbance of the control incubated with red blood cells at a final concentration of 0.1% Triton-X100 was defined as I _{Positive control} The method comprises the steps of carrying out a first treatment on the surface of the And then according to the formula [ (I) _{Experimental group} -I _{Negative control} )/(I _{Positive control} -I _{Negative control} )]The haemolysis rate of the red blood cells was calculated by 100%. As can be seen from the results shown in FIG. 7, C ₁₆ The membrane rupture activity of PButLG-CA is the strongest, and the hemolysis rate is close to 100% when the concentration of polypeptide is 128 mu g/mL;

C ₁₂ -the membrane rupture activity of PButLG-CA is secondary, the rate of hemolysis at a concentration of polypeptide of 512 μg/mL is close to 100%; and C is ₈ The membrane rupture activity of PButLG-CA was the weakest among the three, and the hemolytic activity was about 20% at a concentration of 512. Mu.g/mL. From the above results, it is known that the membrane-breaking activity of the three helical structure polypeptides has a positive correlation with the length of their hydrophobic end groups; under the condition of keeping the spiral structure and the electropositivity of the polypeptide unchanged, the hydrophobic end group can effectively adjust the membrane rupture activity of the polypeptide with the spiral structure, and the longer the hydrophobic end group is, the stronger the membrane rupture activity is.

Example 2: synthesis and characterization of membrane-broken polypeptide with spiral structures with different polymerization degrees

By the same method as in example 1, a helical structure polypeptide C having polymerization degrees of 18, 10 and 5 was synthesized using hexadecylamine as an initiator, respectively ₁₆ -PButLG ₅ 、C ₁₆ -PButLG ₁₀ And C ₁₆ -PButLG ₁₈ The effect of the degree of polymerization on the helical structure polypeptide was compared. C (C) ₁₆ -PButLG ₅ 、C ₁₆ -PButLG ₁₀ And C ₁₆ -PButLG ₁₈ GPC characterization results of (2) ¹ The results of the H-NMR characterization are shown in FIG. 8 and FIG. 9, respectively. And modifying the side group by using a method of clicking the sulfhydryl ethylamine on the side group to respectively obtain polypeptide C ₁₆ -PButLG ₅ -CA、C ₁₆ -PButLG ₁₀ -CA and C ₁₆ -PButLG ₁₈ Characterization of the secondary structure of the helical structure polypeptide with different degrees of polymerization (method is the same as in example 1), as can be seen from the results in FIG. 10, absorption intensity at 208nm and 222nm is reduced at the degree of polymerization of 5, indicating that the secondary structure of the polypeptide is reduced after the degree of polymerization is reduced; when the polymerization degree is 10 or 18, the polypeptide has a stronger spiral structure; indicating that a certain number of degrees of polymerization are required to maintain the helical structure of the polypeptide. Meanwhile, a hemolysis experiment is adopted to test the membrane rupture activity of the series of polypeptides, and the main experimental operation is the same as that of example 1. As can be seen in figure 11 of the drawings,

C ₁₆ -PButLG ₅ -CA、C ₁₆ -PButLG ₁₀ -CA and C ₁₆ -PButLG ₁₈ CA shows concentration-dependent hemolytic activity below 64 mug/mL, and the hemolytic activity of the CA and the CA are similar; the change of the polymerization degree of 5-18 has little influence on the membrane rupture activity of the polypeptide with the spiral structure.

Example 3: synthesis and characterization of non-helical structure polypeptide

(1) This example selects C with the strongest rupture activity ₁₆ -PButLG ₁₀ The CA polypeptide is taken as a template peptide (named P1), the helical structure polypeptide P1 is modified by different anhydrides (maleic anhydride, citraconic anhydride, 3,4,5, 6-tetrahydrophthalic anhydride and 2, 3-dimethyl maleic anhydride), and the modification ratio of the anhydrides is 50 percent. P2, P3, P4 and P5 were obtained respectively, the reaction formula is shown in FIG. 12, (for the synthetic method, refer to patent CN 111548388A). P2, P3, P4 and P5 ¹ The H-NMR characterization result is shown in FIG. 13, wherein A is P2, B is P3, C is P4, and D is P5.

(2) The helicity of the polypeptide was measured by a circular dichroscope (the method is the same as that of example 1), the helicity of the obtained broken membrane polypeptide P1/P2/P3/P4/P5 is shown in Table 1, the helicity of the helix structure P1 is about 90%, the modified P2/P3/P4/P5 by anhydride (butenedioic anhydride/citraconic anhydride/cis-aconitic anhydride/2, 3-dimethylmaleic anhydride) shows a non-helical structure, the helicity is remarkably reduced to about 26.3%,16.9%,14.7% and 15.5%, the modification of the anhydride is shown to effectively shield the helical structure of the polypeptide, and thus a series of non-helical structure polypeptides are obtained.

TABLE 1 helicity of the series of polypeptide

(3) And a hemolysis experiment is adopted to test the membrane rupture capacity of the polypeptide P1 with the spiral structure and the polypeptide P2/P3/P4/P5 modified by the anhydride under the physiological condition. For specific procedures reference is made to example 1. As shown in the results of FIG. 14, the helicity of the polypeptide was correlated with its membrane-disrupting activity, and P1 with high helicity was concentration-dependent hemolytic activity, inducing hemolysis of nearly 100% of erythrocytes at a concentration of 100. Mu.g/mL; the modified polypeptide P2/P3/P4/P5 has obviously reduced helicity, and the hemolytic activity is obviously reduced, the polypeptide P2/P3/P4 has almost no hemolytic activity under the concentration of 100 mug/mL, the polypeptide P5 has a small amount of hemolytic activity under the high concentration, and the hemolytic rate is about 10% under the concentration of 100 mug/mL. The above results demonstrate that anhydride modification can effectively shield the spiral structure of the membrane-rupture polypeptide, thereby reducing the membrane-rupture activity under physiological conditions and reducing the damage to normal tissue cells under physiological conditions.

Example 4 toxicity of the polypeptide to tumor cells at physiological pH (pH 7.4) and tumor slightly acidic (pH 6.8)

The toxicity of the series of polypeptides to tumor cells at physiological pH (pH 7.4) and tumor slightly acidic (pH 6.8) was examined by MTT method, and the procedure is described in example 1. As the results in fig. 15 show, P2 below 320 μg/mL causes no significant toxicity when tumor cells were treated for 24 hours at physiological conditions pH 7.4 or tumor slightly acidic pH 6.8; cell killing by P3 showed significant differences between pH 7.4 and pH 6.8, 320. Mu.g/mL of P3 showed no significant cell killing by 24h incubation of cells at pH 7.4, whereas 160. Mu.g/mL of P3 killed about 80% of tumor cells at pH 6.8; in addition, 20-320 μg/mL of P4 and P5 showed significant cell killing after 24 hours of treatment of cells at pH 7.4 and pH 6.8. The results show that under the condition that the cells are treated by the polypeptide drug with the concentration of 20-320 mug/mL for 24 hours, P3 has better selectivity compared with P4 and P5, has no obvious toxicity at physiological pH, and has good killing activity in the tumor slightly acidic environment.

Example 5: investigation of the action pathway of P3-Selective killing of tumor cells

(1) The ability of the polypeptide P2/P3/P4/P5 to recover the helical structure at different pH values was determined by circular dichroism. Dissolving P2/P3/P4/P5 in methanol to form a stock solution; taking a proper amount of P2/P3/P4/P5 stock solution in a glass bottle, adding 1mM PBS solutions with different pH values (pH 7.4/pH 6.8/pH 6.0/pH 5.5/pH 5.0/pH 4.5) into the stock solution, and diluting the P1/P2/P3/P4/P5 to 0.1mg/mL; the sample was stirred at room temperature and sampled at a specific time point for circular dichroism detection. Spiral recovery (%) = [ (H-H) _7.4 )/(H _1.0 -H _7.4 )]X 100%. H represents the helicity of the polypeptide, H _7.4 Represents the helicity of the polypeptide at pH 7.4, H _1.0 Shows the helicity of the polypeptide at a pH of 1.0 when the helix is fully recovered. As shown in fig. 16, P2 has no apparent helical structure transition at pH 4.5-pH 7.4, which corresponds to its lack of cytotoxicity under physiological conditions and tumor slightly acidic environment; p3 exhibits a rapid helical conformational transition at pH 4.5, pH 5.0 and pH 5.5, a slower conformational transition at pH 6.0, and no apparent conformational transition at pH 7.4 and pH 6.8; p4 has a distinct helical conformational transition at pH 4.5, pH 5.0, pH 5.5 and pH 6.0, whereas there is no distinct helical conformational transition at pH 7.4 and pH 6.8; p5 turns very rapidly at pH 4.5, pH 5.0, pH 5.5, pH 6.0 and pH 6.8, returning to a fully helical structure almost within 1 minute at low pH, without significant helical conformational change in the short term at pH 7.4.

(2) The experiment adopts a liposome leakage experiment simulating tumor cell membranes to test the damage to the membrane structure after the polypeptide recovers the spiral structure at different pH values. Liposome preparation 1,1' - [1, 4-phenylenedi (methylene) was prepared using 1, 2-Dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (1, 2-dipalmitoyl-sn-glycero-3-phospho-L-serine, DPPS) and 1,2-di (cis-9-oleoyl) -sn-glycero-3-phosphocholine (DOPC) (molar ratio 3:1) to mimic tumor cell membranes ]Liposomes of bis (1-pyridinium) Dibromide (DPX) and 8-aminonaphthalene-1, 3, 6-trisulfonic acid disodium salt (ANTS); by co-incubation with a polypeptideThe polypeptide was incubated to simulate the destruction of tumor cell membranes. The cation quencher DPX and the polyanion ANTS fluorophore are encapsulated in the liposome, and the ANTS fluorescence is quenched; when liposomes are destroyed, ANTS and DPX dilute into the surrounding medium, the fluorescence of ANTS increases due to the reduced quenching effect of DPX; from this, the ratio of liposome destruction can be calculated. The main operation is as follows: chloroform was dissolved in DPPS and DOPC to 25mg/mL, DOPC (12.00 mg, 15. Mu. Mol) and DPPS (3.85 mg, 5. Mu. Mol) were taken in a 50mL round bottom flask, and 2mL chloroform was added and dissolved well to give a clear colorless solution. Chloroform was removed by rotary evaporation in a water bath at 37℃and maintained free from boiling during rotary evaporation. After 30 minutes of spin-steaming at 100mbar vacuum, the mixture was dried overnight under vacuum using an oil pump to form a uniform white lipid film at the bottom of the round-bottomed flask. Buffer 1 (10 mM Na was used ₂ HPO ₄ 、10mM K ₂ HPO ₄ ) Dissolving DPX to 25mM and ANTS to 90mM; 1mL of DPX solution and 1mL of LANTS solution are respectively taken and mixed with an EP tube for standby. 2mL of the DPX/ANTS mixture was added to the liposome membrane and hydrated by spin-steaming at 37℃under normal pressure for about 1 hour. The hydrated liposome solution was repeatedly freeze-thawed 10 times in liquid nitrogen and 37 ℃ water bath. Transferring the liposome solution into a liposome extruder, extruding through 400nm filter membrane, and repeatedly extruding for 10-20 times. The liposome solution after the membrane filtration was applied to a G-50 sephadex column using buffer 2 (10 mM Na ₂ HPO ₄ 90mM NaCl) as eluent, and purifying to remove non-entrapped fluorescent molecules; the obtained liposome solution coated with DPX/ANTS fluorescent dye is preserved at 4deg.C and used within 48 hr, and diluted 5-10 times before use. Selection buffer (10 mM Na) ₂ HPO ₄ 、10mM K ₂ HPO ₄ And 90mM NaCl) as a dilution of the polypeptide, the pH of the buffer was adjusted to 7.4, 6.8, 6.0, 5.5, 5.0 and 4.5 using 2M sodium hydroxide solution and 6M hydrochloric acid solution; respectively taking proper amounts of P2, P3, P4 and P5 in buffers with different pH values, and incubating at 37 ℃ overnight, wherein the final concentration of the medicine is 80 mug/mL; taking 50 mu L of the P2, P3, P4 and P5 solutions after the pre-incubation in a black ELISA plate, and adding 50 mu L of a liposome solution carrying DPX/ANTS fluorescent dye; the negative control was 50. Mu.L blank buffer and 50. Mu.L liposome solution, and the positive control was 50. Mu.LLTriton-X100 (0.2% w/v) and 50. Mu.L of liposome solution; standing at room temperature in a dark place for 5-10 minutes, and measuring the fluorescence intensity at 360/536nm by using an enzyme-labeled instrument; the fluorescence intensity of the experimental group incubated with the drug was defined as F _{Experimental group} The absorbance of the control incubated with blank buffer and liposomes was defined as F _{Negative control} The absorbance of the control incubated with liposomes at a final concentration of 0.1% Triton-X100 was defined as F _{Positive control} The method comprises the steps of carrying out a first treatment on the surface of the And then according to the formula [ (F) _{Experimental group} -F _{Negative control} )/(F _{Positive control} -F _{Negative control} )]The fluorescence leakage rate of the liposome was calculated by 100%. As the results of fig. 17 show, P2 did not cause leakage of the ANTS dye at all pH 4.5-pH 7.4; p3 induced about 71.9%, 46.0% and 32.1% leakage of liposomes at pH 4.5, pH 5.0 and pH 5.5, respectively, while no significant leakage of liposomes occurred at pH7.4, pH 6.8 and pH 6.0; p4 caused 84.0%, 70.7%, 77% and 41.2% liposome leakage at pH 4.5, pH 5.0, pH 5.5 and pH 6.0, respectively, without significant leakage at pH7 and pH 6.8; p5 caused significant liposome leakage at pH 4.5, pH 5.0, pH 5.5, pH 6.0 and pH 6.8, 83.2%, 80.9%, 76.6%, 63.1% and 39.2%, respectively, with no significant leakage at pH 7.4. The results show that, consistent with the results of the recovery of the spiral structure, the polypeptide with the spiral structure recovered under the condition of a certain pH can recover the membrane rupture activity.

(3) From the foregoing experiments, it can be found that P3 undergoes a significant helix transition and resumes membrane rupture activity at a pH below 5.5, whereas the pH of the slightly acidic environment in tumor tissue is typically around 6.8, and the acidity does not cause a transition of the P3 helix, i.e., P3 does not have a membrane rupture effect in the slightly acidic environment in tumor tissue. The inventor further researches that P3 plays a role in killing tumor through an endocytic pathway, and the specific steps are as follows: under the slightly acidic environment of tumor tissues, the polypeptide derivative P3 can be converted into electropositivity from electronegativity, so that cell uptake can be promoted, then the polypeptide can enter a Lysosome through an endocytosis way, polypeptide with a spiral structure can be converted under the acidity of the Lysosome, so that strong membrane breaking activity can be obtained, lysosome Membrane Permeabilization (LMP) can be caused, lysosome membrane damage can be caused, lysosome content can be leaked to cytoplasm, and Lysosome-dependent cell death (Lysosome-dependent Cell Death, LCD) can be induced, so that the polypeptide has high-selectivity and strong-effect killing effect on tumor cells.

(4) The synthesized polypeptide with the non-spiral structure has an amphipathic structure and can self-assemble in aqueous solution to form nano micelle particles. The specific operation is as follows: 50mg of polypeptide is dissolved in 1mL of DMSO and fully dissolved for standby; taking 20 mu L of polypeptide mother liquor in a centrifuge tube, and respectively adding 1mL of PBS (1 mM) with pH of 7.4 and pH of 6.8 to resuspend polypeptide; the polypeptide solution is loaded into a potential/particle size special test cell of a nano-particle analyzer, and the particle size and potential are tested. The test results are shown in FIG. 18A, in which the polypeptide self-assembles in aqueous solution to form nano-micelles with particle sizes of about 200-400nm, and P2/P3/P4 has no significant change in particle size at pH 7.4 and pH 6.8, while P5 has an increase in particle size at pH 6.8 over that at pH 7.4. As can be seen from FIG. 18B, the potentials of P2/P3/P4 at pH 7.4 were-17.4 mV, -13.8mV and-14.5 mV, respectively, exhibiting electronegativity; the potential of P5 is about 0mV, showing electroneutrality; at pH 6.8, the P2/P3/P4/P5 potential was changed from neutral or electronegative to neutral or electropositive, respectively-0.74 mV, 16.8mV, 31.4mV and 32.4mV.

(5) The difference in potential at pH 7.4 and pH 6.8 affects the uptake of the polypeptide drug by the cells, which is the basis for selective killing of the polypeptide; the experiment adopts flow cytometry to detect the difference of the intake of the polypeptide by the cells at pH 7.4 and pH 6.8, and the specific operation is as follows: pancreatic cancer Panc02 cells are spread in 24-well plates according to 10 ten thousand per well, and are used after being cultured overnight; the polypeptide (P2, P3, P4 and P5) was prepared in a series of concentrations using media at pH 7.4 and pH 6.8, respectively, the drug was added to the cells at a final concentration of 20. Mu.g/mL, and the culture was continued for about 2 hours in an incubator at 37 ℃; taking out cells, removing the medicine-containing supernatant, and cleaning the cells for 2-3 times by using normal-temperature PBS; cells were digested by adding pancreatin, centrifuged at 1000rpm at 4℃for 5 minutes to collect cells, and the cells were resuspended in PBS (1% PBA) containing 1% BSA; passing the cells through a 200-mesh screen, adding PI with the final concentration of 10 mug/mL, dyeing for 10 minutes, and then loading the cells into a machine for detection; the detection channels adopt 488/525nm and 561/610nm; analysis was performed with software flowjo V10. As a result, as shown in FIG. 19, the ratios of P2/P3/P4/P5 were 13.5, 9.4, 4.2 and 3.8, respectively, in the cell uptake difference ratio at pH 6.8 and pH 7.4; because P2 is not cytotoxic under normal physiological conditions and in the acidic environment of the cells, it does not exhibit selectivity even though its uptake is maximally different at pH 6.8 from pH 7.4; uptake of P3 differs twice at pH 6.8 and pH 7.4, and finally P4 and P5.

(6) This experiment further investigated whether the polypeptide exerts a killing effect via the endocytic pathway. Respectively placing Panc02 cells and a series of polypeptide solutions at pH 7.4 and 6.8 in 4 ℃ for precooling for 30 minutes; adding a pre-chilled polypeptide solution to the cells; treatment of P5, P4 and P3 at 2, 8 and 24 hour time points, respectively, and detection of cell viability of the polypeptide at the respective time points; meanwhile, cells and polypeptide liquid medicine are incubated at 37 ℃ to serve as a control. The results are shown in fig. 20, and the treatment at 4 ℃ can effectively inhibit the toxicity of P3 and P4 to cells at pH 6.8, indicating that P3 and P4 exert killing effect on tumor cells through endocytic pathway; however, treatment at 4 ℃ failed to inhibit the cytotoxicity of P5 at pH 7.4 and pH 6.8, indicating that P5 did not exert a killing effect on tumor cells via the endocytic pathway.

(7) Based on the above results, it is considered that P3, which has a good effect of selectively killing tumor cells, acts through an endocytic pathway, and the digestion site where the cells take up substances through the endocytic pathway is generally in lysosomes, and the experiment further studied the co-localization of P3 and lysosomes.

Synthesis of FITC-tagged polypeptide P3-FITC: p1 (10 mg, 0.0035 mmol), citraconic anhydride (1.97 mg, 0.0175 mmol), 5-fluorescein isothiocyanate (FITC, 1.36 mg, 0.0035 mmol) and triethylamine (3.5 mg, 0.035 mmol) were mixed in DMSO (1 ml), stirred at room temperature for 12 hours, and the resulting mixture was dialyzed (mwco=1 kDa) against distilled water for 24 hours, and lyophilized to give P3-FITC for use.

After digestion of the pancreatic cancer Panc02 cells of stable LAMP1-mCherry, the cells were isolated at 2X 10 ⁵ The cells of each/mL are added into a glass-bottom cell confocal culture dish and are used after being cultured overnight in a constant-temperature cell incubator; preparation of the medium containing 160. Mu.g/mL with pH 7.4 and 6.8, respectivelyIs added to cells and treated for about 1 hour; removing the medicine-containing culture medium, and cleaning the cells for 2 times by using a normal-temperature DMEM culture medium; adding trypan blue solution with the final concentration of 0.04% into the cells, incubating on ice for about 10 minutes, and washing the cells for 2 times by using a normal-temperature DMEM culture medium; DMEM medium containing 5. Mu.g/mLHoechst was added to the cells and incubated at room temperature for about 15 minutes in the absence of light; removing the culture medium supernatant, cleaning the cells for 2 times by using a normal temperature DMEM culture medium, adding a proper amount of culture medium to cover the cells, and then imaging; photographing is carried out by adopting Olympic Games turntable confocal, 345/455nm, 495/519nm and 578/603nm channels are adopted, and a 60x oil lens is adopted for a lens. The results are shown in FIG. 21, where cells uptake P3-FITC at pH 6.8 more than at pH 7.4; meanwhile, the P3-FITC and the lysosomal protein LAMP1-mCherry have good co-localization, which indicates that the P3-FITC enters the cell lysosome through an endocytic pathway.

(8) The result of the fluorescence imaging shows that P3 enters cells through an endocytic pathway and has good co-localization with lysosomes; the experiment uses endocytosis inhibitors with different action mechanisms, such as Wortmannin (giant cell potion inhibitor), chlorpromazine hydrochloride (chlorpromazine, inhibiting clathrin-mediated endocytosis), beta-cyclodextrin (mβCD, nidogen-dependent endocytosis inhibitor), 2-Deoxy-D-glucose (2-Deoxy-D-glucose, energy dependent inhibitor) and sodium azide (NaN ₃ Energy-dependent inhibitors), the effect of which endocytic pathway P3 enters the cell and the effect of endocytic inhibition on P3 cytotoxicity were studied further using MTT method, and specific experimental procedures are described in example 1. The results are shown in fig. 22, where cells were pre-treated with specific concentrations of endocytic inhibitors prior to co-incubation with P3; the endocytic inhibitor can effectively reduce the uptake and cytotoxicity of the cells to the P3-FITC; the results further demonstrate that P3 mediates killing of tumor cells by endocytic pathways, and that P3 enters cells via clathrin, megaloblastic, multiple energy-dependent endocytic pathways.

Example 6: research on action mechanism of P3 killer tumor cells

(1) The results of example 5 demonstrate that P3 enters the lysosome via the endocytic pathway, this exampleThe interaction of P3 with lysosomes was further investigated. First, the effect of P3 on lysosomal membrane integrity was tested using galectin 3 (Galctin 3) spot experiments. After digestion of pancreatic cancer Panc02 cells stably transformed with Galctin3-GFP, the cells were isolated at 2X 10 ⁵ The cells of each/mL are added into a glass-bottom cell confocal culture dish and are used after being cultured overnight in a constant-temperature cell incubator; preparing polypeptide P3 containing 80-160 mug/mL by using culture mediums with pH of 7.4 and 6.8 respectively, adding the polypeptide P3 into cells, incubating for 12-16 hours, imaging, and observing galectin 3-GFP spot formation in the cells; real-time photographing is carried out by adopting Olympic Games turntable confocal, 495/519nm channels are used, and a 60x oil mirror is adopted for a lens. The results are shown in FIG. 23, where P3 did not induce cells to form Galctin3-GFP spots at pH 7.4, and the Galctin3-GFP protein was uniformly distributed in the cytoplasm, indicating that the cell lysosome membrane remained intact under these conditions; however, a large number of Galctin3-GFP spots appeared at pH 6.8, indicating that the lysosomal membrane was broken, galctin3-GFP in the cytoplasm penetrated through the broken lysosomal membrane into the lysosomal cavity and bound with high affinity to galactose residues of glycoprotein on the lysosomal membrane, and finally Galctin3-GFP spots were formed.

(2) To demonstrate that P3 destroyed lysosomes before cell death was induced, rather than directly destroying cell membrane killer cells, galctin3-GFP spot/Propidium Iodide (PI) staining real-time imaging experiments were performed to indicate cell death with PI and nuclear staining. After digestion of pancreatic cancer Panc02 cells stably transformed with Galctin3-GFP, the cells were isolated at 2X 10 ⁵ The cells of each/mL are added into a glass-bottom cell confocal culture dish and are used after being cultured overnight in a constant-temperature cell incubator; preparation of a polypeptide (P3/P4/P5) containing 160. Mu.g/mL each using a medium with pH 6.8, addition to cells, and incubation in an incubator at 37 ℃; at a specific time point, cells were removed and PI was added to the cells at a final concentration of 1 μg/mL; imaging is carried out subsequently, the formation of galectin 3-GFP spots in the cells is observed, and the fluorescence changes of the galectin 3-GFP and PI of the cells are monitored; real-time shooting is carried out by adopting Olympic Games turntable confocal, 495/519nm and 578/603nm channels are adopted, and a 60x oil mirror is adopted for a lens. Taking the time point of occurrence of Galctin3-GFP spots as zero point, and recording the fluorescence changes of Galctin3-GFP and PI of cells before and after the zero pointThe method comprises the steps of carrying out a first treatment on the surface of the And the time difference between the appearance of Galctin3-GFP spots and PI staining positivity was statistically mapped. The results are shown in FIG. 24, panel A shows that P3 first induces the formation of the marker Galctin3-GFP spot for lysosomal membrane damage, and according to the statistical results, PI staining positivity occurs after about 150 minutes. There is a time difference of about 150 minutes between lysosomal damage and cell death, indicating that P3 is responsible for lysosomal damage and then cell death, i.e., P3 is responsible for cell death by lysosomal membrane permeation. As shown in panels B and C, after treatment of cells with P4 and P5, the dispersed galectin 3-GFP fluorescence in the cytoplasm of the cells first decreased, with concomitant increase in PI fluorescence, before the appearance of galectin 3-GFP spots; and according to the statistics of the D graph, the time difference between the occurrence of Galctin3-GFP spots and the positive PI staining is nearly 0 minutes, which indicates that lysosomal membrane damage and cell death occur simultaneously; i.e., P4 and P5 do not induce cell killing by lysosomal membrane permeation.

(3) Pancreatic cancer Panc02 cells at 1×10 ⁷ 100mm was added to each dish ³ In a culture dish, placing in a 37 ℃ incubator for overnight incubation for use; preparation of polypeptide P3-Fe containing 320. Mu.g/mL by using culture media of pH 7.4 and 6.8, respectively (preparation method comprises the steps of dissolving ferrocenylacetic acid (30.5 mg,0.125 mmol), EDCI (26.84 mg,0.14 mmol) and NHS (16.11 mg,2.3 mmol) in 500. Mu.L of methanol, stirring for 4 hours, adding 2mL of DMF, adding 356mg of P1. Stirring at room temperature for 24 hours, adding 70mg of citraconic anhydride, stirring at room temperature for 12 hours, dialyzing in ultra-pure water for 24 hours by using a dialysis bag with a molecular weight cut-off of 1kDa, freeze-drying and storing in a refrigerator at-20 ℃); adding the cells into an incubator at 37 ℃ for incubation; cells were collected at a specific time point using a cell scraper, and centrifuged (1000 rpm/5 min) at room temperature; 2.5% glutaraldehyde was added, and the cells were fixed at room temperature for 1 hour, and then transferred to 4℃for overnight storage; washing with PBS three times for 10-15 min each; cells were fixed for 1-2 hours with osmium tetroxide, and rinsed three times with PBS for 10-15 minutes each time; gradient dewatering the cells with gradient concentration (25%, 50%,75%,90% and 100%) of ethanol, sequentially adding ethanol of corresponding concentration into the cells, soaking for 10-15 min, and most Soaking in anhydrous acetone for 10-15 min; cells were immersed in acetone: embedding solution = 3:1 (v/v) for 0.5 hours; soaking in acetone: embedding liquid=1:1 (v/v) for 4 hours; then placing the mixture into a pure embedding solution, and preserving the mixture at 4 ℃ overnight; placing the sample into an embedding plate, baking at 37 ℃ for 24 hours, and then baking at 60 ℃ for 48 hours; the samples were cut into approximately 100nm thick slices using an ultramicrotome (EMUC 7, leica) and stained with uranium dioxane acetate for 20 minutes, lead citrate for 12 minutes, and finally observed using a transmission electron microscope. As a result, as shown in FIG. 25, after P3-Fe treatment of cells for 8 hours or 16 hours at pH 7.4, the lysosomal contents of the cells were uniformly distributed, and the lysosomal membrane structure was complete. Compared with the P3-Fe treated cells for 8 hours under the condition of pH 6.8, punctate particles can be seen in the lysosome cavity, the content is unevenly distributed, the lysosome membrane is in a sheet shape, the structure of the lysosome membrane is incomplete, and punctate particles are visible in a cytoplasmic area around the lysosome; when P3-Fe is used for treating cells for 16 hours, lysosomes are further expanded, breakage and weight are increased, cytoplasmic hollow bubble structures are increased, cell nuclei are found to be deformed, and the cells are in a dying state.

(4) As is clear from the above-mentioned transmission electron microscope results, the lysosome membrane was broken, and the substances in the lysosome cavity were leaked to the cytoplasm. The lysosome is rich in various hydrolases, and the experiment takes Cathepsin B (CTSB) as a research target to construct a Panc02 cell stably transformed into CTSB-mCherry, and P3 is detected by laser confocal detection to induce leakage of the lysosome content. After digestion of pancreatic cancer Panc02 cells stably transformed with CTSB-mCherry, the cells were isolated as 2X 10 ⁵ The cells of each/mL are added into a glass-bottom cell confocal culture dish and are used after being cultured overnight in a constant-temperature cell incubator; preparing polypeptide P3 containing 80-160 μg/mL with culture medium of pH 7.4 and pH 6.8, adding into cells, and incubating at 37deg.C in incubator for 12-16 hr; taking out the cells, and replacing the drug-containing culture medium with fresh culture medium; imaging is then carried out to observe the dispersion of the cells CTSB-mCherry from lysosomes into the cytoplasm; live cell photographing was performed using Olympic Games turnplate confocal, using 578/603nm channels and 60x oil mirrors. As a result, as shown in FIG. 26, CTSB-mCherry fluorescence in P3-treated cells was shown to be punctiform at pH 7.4, which is described asThe clear CTSB-mCherry is mainly located in the lysosome, and the lysosome membrane structure remains intact. In addition to dotted CTSB-mCherry fluorescence, diffuse CTSB-mCherry fluorescence was also present in the cytoplasm after treatment with P3 at pH 6.8, indicating that the structural integrity of the lysosome membrane was compromised and CTSB-mCherry had leaked from the lysosome into the cytoplasm of the cell.

(5) By combining the experimental results of example 5, the polypeptide P3 needs to be converted into a spiral structure below pH 5.5 to exert the effects of membrane rupture and killing, the acidity in the lysosome cavity in normal cells is generally between pH 4.5 and pH 5.5, and after P3 enters the lysosome through an endocytic pathway, the polypeptide P3 can be converted into the spiral structure under the physiological acidity of the lysosome to recover the spiral structure, and the lysosome membrane is destroyed, so that the effect of killing tumor cells is exerted. If the lysosomal acidity is inhibited, the conformational transition of P3 is inhibited, the killing effect is reduced, and the influence of an acidification inhibitor on P3 cytotoxicity is verified by adopting an MTT method, and the specific experimental method is referred to in example 1. As shown in a and B in fig. 27, cells were co-incubated with P3 at pH 6.8 using specific concentrations of the acidification inhibitors Chloroquine (CQ) and canavanine a (ConcanamycinA, CMA). The result shows that the acidification inhibitor can effectively reduce the killing of P3 to tumor cells; thus, P3 was countered by the induction of cell death by lysosomal injury. In addition, as shown in C and D in FIG. 27, the killing effect of P3 was also significantly reduced by the specific concentrations of the cathepsin inhibitor E-64D and CA-074-ME co-treated with P3 at pH 6.8, demonstrating that P3-induced leakage of lysosomal cathepsin is an important cause of cell death.

Example 7: in vivo tumor inhibition experiment of P3 polypeptide

This example tested the tumor suppression effect of P3 by in vivo experiments with a C57 mouse pancreatic cancer tumor subcutaneous model (Panc 02 cells), a Balb/C mouse colorectal cancer tumor subcutaneous model (CT 26 cells) and a Balb/C mouse breast cancer tumor in situ model (EMT 6 cells). The operation is as follows:

female mice (C57 BL/6,6-8 weeks) were subcutaneously injected 1X 10 in the back ⁶ individual/mL Panc02 cell suspension (100 μl) to establish a pancreatic cancer subcutaneous tumor model; in female mice (Balb/c, 6-8 weeks) backPartial subcutaneous injection 1×10 ⁶ individual/mL CT26 cell suspension (100 μl) to establish a subcutaneous tumor model of colorectal cancer; breast pad injection number two 3X 10 in female mice (Balb/c, 6-8 weeks) ⁵ individual/mL EMT6 cell suspension (50 μl) to establish breast cancer in situ tumor models. When the tumor grows to 50-100 mm ³ (tumor volume = length x width/2). After the grouping, the administration was by tail vein injection (15 mg/kg, or 5mg/kg, once a day); in addition, a negative control group was set, and only an equal volume of solvent was injected. Tumor size was measured using vernier calipers and mouse body weight was recorded.

The in vivo tumor inhibiting effect of the Panc02 model is shown in fig. 28, and it can be seen from the tumor growth curve that the tumor volume of the P3 treatment group (15 mg/kg by tail vein injection once daily for one week) is significantly inhibited compared with the control group. The mice experienced no difference in body weight between the experimental and control groups throughout the treatment period, and the treatment process did not result in weight loss in the mice. Meanwhile, the weight results of the isolated tumor show that the weight of the P3 treatment group (15 mg/kg by tail vein injection once daily for one week) is obviously reduced compared with that of the control group, and the weight of the isolated tumor is consistent with that of the tumor growth curve. In vivo tumor inhibition effects of EMT6 and CT26 tumor models are shown in FIG. 29, and it can be seen that P3 polypeptide (5 mg/kg by tail vein injection once daily for two consecutive weeks) also has better inhibition effects on EMT6 and CT26 tumor models (FIG. 29).

Example 8: p3 polypeptide combined with anti-PD-L1 antibody treatment induces anti-tumor immune response

The embodiment further researches the in-vivo anti-tumor curative effect of the P3 polypeptide combined immune checkpoint blocker anti-PD-L1 antibody on the basis of inhibiting the growth of an in-vivo tumor model by the P3 polypeptide. A C57 mouse pancreatic cancer tumor subcutaneous model (Panc 02 cells), a C57 mouse colon cancer tumor subcutaneous model (MC 38 cells), a C57 mouse breast cancer tumor in situ model (E0771 cells) and a Balb/C mouse breast cancer tumor in situ model (EMT 6 cells) were separately constructed for the combined efficacy test, and the main procedure was as in example 7. Results as shown in fig. 30, C57 mice loaded with the Panc02 subcutaneous tumor model were given 15mg/kg of P3 polypeptide for 5 consecutive days, which significantly inhibited the growth of Panc02 tumors relative to the untreated control group; intravenous injection of 1.0mg/kg of anti-PD-L1 antibody has a small amount of inhibition effect on the Panc02 subcutaneous tumor model; the inhibition effect of the P3 polypeptide and the anti-PD-L1 antibody combined treatment group on Panc02 subcutaneous tumor is more obvious than that of the two single-drug treatment groups, so that the growth speed of the tumor can be effectively slowed down and the growth of the tumor can be inhibited; both tumor volume monitoring and tumor weight were significantly reduced in the combination treatment compared to the single drug treatment. Meanwhile, the weight change of each group of mice after treatment is not different, which indicates that the treatment dosage of the P3 polypeptide and the anti-PD-L1 antibody under the treatment strategy is safe for the mice.

As shown in fig. 31 and 32, in the MC38 colon cancer tumor subcutaneous model and the E0771 breast cancer tumor in-situ model, the P3 polypeptide (15 mg/kg) single drug treatment group or the anti-PD-L1 antibody (0.75 mg/kg) single drug treatment group has a certain inhibition effect on different tumor models, but the P3 polypeptide (15 mg/kg) and the anti-PD-L1 antibody (0.75 mg/kg) combined treatment strategy shows more excellent treatment effect, which can not only effectively inhibit tumor growth, but also enable partial tumor mice to achieve the effect of tumor cure. As shown in FIG. 33, in the EMT6 breast cancer tumor in-situ model, the effect of the combination treatment of the P3 polypeptide (15 mg/kg) and the anti-PD-L1 antibody (0.6 mg/kg) on tumors is also obviously better than the effect of the single treatment of the P3 polypeptide (15 mg/kg) or the anti-PD-L1 antibody (0.6 mg/kg).

Example 9: polypeptide P3 can induce strong far-end anti-tumor immunity

P3 needs to be activated under the acidity of lysosomes to generate conformational transition, and P3 can destroy lysosome membranes after restoring a spiral structure, so that substances such as cathepsin in lysosomes are promoted to leak into cytoplasm, and then tumor cell death is caused. In order to compare the anti-tumor immune effects of the polypeptide P3 with those of other polypeptides, the present example used a remote tumor model to test the anti-tumor immune effects of each polypeptide. The results are shown in FIG. 34, where the No. 1 EMT6 tumor was implanted in the No. 1 breast pad on the left side of the lying Balb/C mouse; on the day prior to group treatment, no. 2 EMT6 tumors were implanted on the right breast pad of the lying Balb/C mice; group treatment was then performed according to tumor volume No. 1, group settings as in fig. 34; after grouping, the polypeptide drug was given by continuous intratumoral injection 125 μg/dose for 1 day/dose for 3 times; on day 2 after termination of the administration of the polypeptide, intravenous administration of the anti-PD-L1 antibody (0.75 mg/kg) was started for 2 days/time for 3 times in total; and the tumor sizes of the tumors No. 1 and No. 2 were monitored by using a vernier caliper, respectively, and the body weights of the mice were recorded. The results of fig. 34 (6 mice per group, CR in the figure referring to the proportion of "complete cure", e.g. "cr=3/6" referring to the disappearance/cure of tumors in 3 of the 6 mice in the group) show that intratumoral administration of P1, P4 and P5 or treatment with anti-PD-L1 antibodies in No. 1 had no significant inhibition of in situ tumors; and the P3 intratumoral injection or the P3+ anti-PD-L1 antibody combined treatment group has the best in-situ inhibition effect. For distal tumor No. 2, neither treatment with P1, P4, and P5, nor with anti-PD-L1 antibodies, inhibited growth of distal tumors; p3 has a certain far-end inhibition effect when singly treated, and the cure rate of the far-end tumor is about 50%; and the P3 combined anti-PD-L1 antibody combined treatment group shows more excellent far-end inhibition effect, and the cure rate of the far-end tumor is close to 100%. Meanwhile, the weight results of the tumors are consistent with the results, and together, the polypeptide P3 can induce strong anti-tumor immunity more than other polypeptides.

Example 10: the polypeptide P3 induced whole cell vaccine can provide strong anti-tumor protection

In this example, the vaccine experiment was used to test that polypeptide P3 further activated the anti-tumor immunity of the body after inducing tumor cell death, and compared with other polypeptides. EMT6 cells were plated at 1 million/dish at 100mm ³ A culture dish is treated by adding medicine after overnight; the polypeptide was diluted with serum-free DMEM, and 40. Mu.g/mL of P1, 160. Mu.g/mL of P3, and 160. Mu.g/mL of P4 were added to the cells (the drug concentration means the final concentration after the addition of the cells), and the EMT6 cells were treated for 0.5 hours, 4 hours, and 16 hours, respectively; at a specific time point, collecting cells by using a cell scraper after removing supernatant, and preparing a dying cell vaccine; meanwhile, an appropriate amount of EMT6 cells were collected and repeatedly frozen and thawed 10 times in a water bath at 37℃in liquid nitrogen as a whole cell vaccine control. Injecting the cell vaccine into the breast pad 1 on the left side of a lying Balb/C mouse according to the ratio of about 250 ten thousand per mouse, and performing tumor vaccine immunization; 11 days after vaccine injection, on the right side of lying Balb/C mice, no. 2Breast pad implantation number 2 EMT6 tumor; subsequently, the tumor size of tumor No. 2 was monitored and the body weight of the mice was recorded. The results as shown in fig. 35 (6 mice per group, CR in the figure means the "complete cure" ratio, e.g. "cr=6/6" means that the tumors of the 6 mice in the group all disappeared/healed) indicate that the freeze-thawed cells, the P1-treated cell vaccine and the P4-treated cell vaccine can prevent or inhibit the growth of tumors to some extent, with tumor-free ratios of 16.7%, 50% and 50% for the mice, respectively; whereas the P3 treated cell vaccine showed a more potent tumor preventing effect with a tumor-free ratio of 100%. The results indicate that polypeptide P3 can induce a more potent anti-tumor immune effect than other polypeptides.

Example 11: the polypeptide P3 with different anhydride modification ratios shows killing activity on tumor cells

The experimental result shows that the polypeptide P3 obtained by modifying the amino side chain of the polypeptide P1 with the helical structure by using citraconic anhydride has good selective killing to tumor cells, has no obvious toxicity to the cells under the physiological condition of pH 7.4, and can induce lysosome-dependent death of the tumor cells under the slightly acidic condition of pH 6.8. In order to study the killing of other anhydride modified proportion of P3 polypeptide to tumor cells, the embodiment adopts citraconic anhydride to modify the amino side chain of the polypeptide P1 with a spiral structure by 20%, 30% and 40%, and the toxicity of the series of materials after the cells are treated for 24 hours is verified by an MTT method. As shown in FIG. 36, when the citraconic anhydride modification ratio was 20%, the polypeptide P3 had significant cytotoxicity at pH 7.4 and pH 6.8, and killed 95% or more of the cells at 80. Mu.g/mL and 40. Mu.g/mL, respectively. 160 μg/m of polypeptide P3 killed more than 95% of cells at pH 7.4 or 80 μg/m of polypeptide P3 at pH 6.8 at a citraconic anhydride modification ratio of 30%. When the citraconic anhydride modification ratio is 40%, the polypeptide P3 has no obvious toxicity to cells at pH 7.4; however, at pH 6.8, 160. Mu.g/m of polypeptide P3 killed more than 95% of the cells. The above results indicate that the higher the modification ratio, the stronger the selectivity of the polypeptide P3 when the ratio of citraconic anhydride modified helical polypeptide is below 50%; the modification proportion is reduced, and the selectivity of polypeptide P3 is reduced; at modification ratios below 30%, the polypeptide P3 is also significantly cytotoxic to cells at pH 7.4.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the following embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. Use of a polypeptide derivative having a structure according to formula (I):

wherein R is selected from C ₄ -C ₂₄ An alkyl group;

x+y is greater than 5.

2. The polypeptide derivative according to claim 1, wherein R is selected from C ₈ -C ₂₀ An alkyl group.

3. The polypeptide derivative according to claim 2, wherein R is selected from C ₁₂ -C ₁₈ An alkyl group.

4. The polypeptide derivative according to claim 1, wherein R1, R2 are each independently selected from: H. methyl, ethyl, propyl, or R1, R2, together with the carbon atom to which they are attached, form a cyclopentenyl or cyclohexenyl group.

5. The polypeptide derivative according to claim 1, wherein one of R1 and R2 is H and the other is C ₁ -C ₃ An alkyl group.

6. Polypeptide derivative according to claim 1, characterized in that x+y is 5-20, preferably 8-18, more preferably 10.

7. Polypeptide derivative according to any one of claims 1 to 6, characterized in that x is 30 to 60%, preferably 40 to 55%, more preferably 48 to 52%, more preferably 50% of x+y.

8. The polypeptide derivative according to claim 1, characterized in that it is selected from the following polymers:

9. use of a polypeptide derivative according to any one of claims 1 to 8 for the preparation of a medicament for the prevention and/or treatment of tumors.

10. Use of a polypeptide derivative according to any one of claims 1 to 8 for the preparation of a vaccine for the prevention and/or treatment of tumors.

11. Use of a polypeptide derivative according to any one of claims 1 to 8 for the preparation of an inducer for inducing anti-tumor immunity.

12. The use according to any one of claims 9 to 11, wherein the tumour is pancreatic cancer, colorectal cancer, breast cancer, colon cancer, lung cancer cells, liver cancer cells, melanoma cells, brain glioma cells.

13. A medicament for preventing and/or treating tumors, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the polypeptide derivative as claimed in any one of claims 1 to 8.

14. A medicament for the prevention and/or treatment of tumors as claimed in claim 13, wherein said active ingredient comprises said polypeptide derivative and a combined immune checkpoint blocker.

15. The medicament for preventing and/or treating tumors according to claim 14, characterized in that said combined immune checkpoint blocker is an Anti-PD-L1 antibody.

16. Vaccine for the prevention and/or treatment of tumors, characterized in that it is a tumor cell treated with a polypeptide derivative according to any one of claims 1 to 8.

17. The vaccine for preventing and/or treating tumors according to claim 16, characterized in that said tumor cells are pancreatic cancer cells, colorectal cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, liver cancer cells, melanoma cells, brain glioma cells.

18. A method of preparing a vaccine for the prevention and/or treatment of tumors according to claim 16 or 17, characterized by the steps of: and (3) treating tumor cells by using the polypeptide derivative, and collecting dying cells to obtain the vaccine.

19. A method for preparing a vaccine for the prevention and/or treatment of tumors according to claim 18, characterized in that it comprises the following steps: plating the tumor cells at 0.8-1.2 million/dish at 80-120mm ³ Adding the polypeptide derivative diluted by serum-free DMEM into a culture dish, treating the tumor cells for 0.5-16 hours, discarding the supernatant, and collecting moribund cells to obtain the vaccine; the final concentration of the polypeptide derivative is 40 mug/mL-200 mug/mL.