CN111423497A - Antagonistic peptide, copolymer and nano assembly thereof, and preparation method and application thereof - Google Patents
Antagonistic peptide, copolymer and nano assembly thereof, and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention provides an antagonistic peptide, a copolymer and a nano assembly thereof, and a preparation method and application thereof, wherein the antagonistic peptide is PD-L1 antagonistic peptide which is named as M-APP and has the following structure:
the copolymer IR780-M-APP with a clear structure is prepared by taking M-APP as a raw material, and can be self-assembled to form a nano-assembly, so that the problem of poor immunogenicity in immunotherapy is solved, the utilization rate of immune antagonistic peptide is improved, the problem of poor IR780 water solubility can be solved by synergistic photodynamic therapy, the effect of phototherapy is improved, and the curative effect of immunotherapy is enhanced.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to antagonistic peptide, a copolymer and a nano assembly thereof, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The present invention relates to a method for treating melanoma, and more particularly, to a method for treating melanoma, which comprises administering to a patient in need thereof an effective amount of a synthetic polypeptide, wherein the effective amount of the synthetic polypeptide is a tumor candidate, wherein the therapeutic effect of the synthetic polypeptide is high, the stability of the synthetic polypeptide is high, the immunogenicity of the synthetic polypeptide is low, the organ or tumor is good, and the tumor is resistant to various kinds of malignant tumors, and the immune cell penetration of the synthetic polypeptide is high, and the immune cell is small enough to be used as a cancer antigen.
The IR780 iodide (IR780) is a typical near-infrared dye, and has good characteristics of photo-thermal and photodynamic, so that the IR780 iodide has good clinical application prospect, but the inventor finds that the IR780 has poor water solubility, is difficult to deliver in vivo and is a factor limiting the wide use of the IR780 iodide, and the IR780 serving as a photosensitizer generates singlet oxygen with short action time and limited action distance, so that the ideal treatment effect is difficult to achieve.
Disclosure of Invention
A complex system in the Tumor Microenvironment (TME) including various cell types, cell secretion factors, surrounding matrix, various enzymes and special physical factors such as low pH, hypoxia and interstitial fluid pressure plays an important role in Tumor therapy. The inventor finds in research that the combination of phototherapy and immune checkpoint antagonism can significantly improve the tumor treatment effect. The photodynamic therapy directly kills tumor cells by laser irradiation and simultaneously releases tumor-associated antigens, and the injury-associated molecular patterns generated by secretion are recognized by some receptors, so that the synthesis and release of immune effect factors are accelerated, and the immune effect is exerted to induce an organism to respond to the killing of tumors.
In order to solve the defects in the prior art, the invention provides an immune checkpoint antagonist peptide M-APP by adopting a method of combining phototherapy and immune checkpoint antagonism, a copolymer of the antagonist peptide M-APP and phototherapy drugs (or photosensitizers), a nano-assembly and a preparation method thereof, and a preparation method and application thereof. The copolymer of the antagonistic peptide M-APP and the phototherapy drug (or photosensitizer) and the nano-assembly thereof are tumor microenvironment-responsive, and can be applied to antitumor immunotherapy and photodynamic therapy.
The technical scheme of the invention not only overcomes the defects that phototherapy drugs such as IR780 with strong hydrophobicity are difficult to deliver in vivo, difficult to effectively accumulate at tumor parts, short action time of generated singlet oxygen, limited action distance and difficult realization of ideal treatment effect, but also enhances phototherapy effect, enables the immune checkpoint antagonist peptide to be exposed by breaking after reaching TME and combined with PD-L1 on the surface of tumor cells through special design of compounds, thereby inhibiting tumor immune escape process, improving utilization rate of in vivo immunotherapy of the antagonist peptide, enhancing the effect of the immunotherapy of the antagonist peptide and remarkably improving anti-tumor effect.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, the invention provides an immune checkpoint antagonist peptide, designated M-APP, having the structure shown below:
the M-APP has the structural characteristics that the M-APP consists of mercaptopropionic acid, four methionines, an MMP-2 response part P L G L VG (-Pro-L eu-Gly-L eu-Val-Gly-) and an anti-PD-L1 peptide DPPA-1 part (-NYSKPTDRQYHF, the molecular formula is-Asn-Tyr-Ser-L ys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe-NH2) The compositions, respectively, play a role in linking IR780, modulating assembly structure, performing MMP2 enzyme response fragmentation in TEM and playing an immunotherapeutic role.
In a first aspect of the present invention, the present invention provides a method for preparing the immune checkpoint antagonist peptide M-APP as described in the first aspect above, comprising linking mercaptopropionic acid, four methionines, MMP-2 response moiety P L G L VG (-Pro-L eu-Gly-L eu-Val-Gly-) and anti-PD-L1 peptide DPPA-1(-NYSKPTDRQYHF) by reaction to obtain M-APP of the present invention, which can be prepared by those skilled in the art according to the structural features of M-APP designed by the present invention.
The preparation method comprises the steps of firstly carrying out Resin swelling, soaking 10 g of Rink amide AM Resin (the substitution degree of the Resin is 0.35mmol/g) in 200ml of dichloromethane for 15 minutes, after the Resin expands, extracting dichloromethane, secondly removing amino protection, adding 200m L hexahydropyridine/DMF solution, stirring by using nitrogen, reacting for 2 times at room temperature for 5 minutes and 15 minutes, washing the Resin by using DMF after the reaction is finished, adding a small amount of Resin into an assay reagent, heating for 3 minutes at 100 ℃, determining that the color of the solution and the Resin is blue or red, judging that the amino Fmoc protection is removed, then carrying out condensation reaction, adding 2 times of molar number of Fmoc-D-Phe-OH and HBTU, dissolving by using 200m L, adding 2 times of molar number of DIEA, stirring by using nitrogen, reacting for 1 hour at room temperature, and washing the Resin by using DMF after the reaction is finished.
And then, repeating the steps 2-3, sequentially connecting each protected amino acid to complete the synthesis of the sequence, soaking the resin with dichloromethane and ether, pumping, separating the polypeptide from the resin, adding 150M L TFA lysate, reacting in a constant temperature shaking table for 1-3h, reacting at the temperature of about 15-40 ℃, rotating at the speed of 120 r/min, separating out a crude product, filtering to remove the resin, adding anhydrous ether into the filtrate, centrifuging at 2500rpm to obtain a solid, adding anhydrous ether to wash, centrifuging again, repeating for multiple times, drying to obtain a crude polypeptide, further purifying, dissolving the crude product, filtering by using a 0.45 mu M membrane, sampling, performing gradient elution by using a solvent acetonitrile solution and a solvent (0.1% trifluoroacetic acid and 99.9% pure water solution), collecting a main peak solution, combining the main peak solutions to obtain a fine peptide liquid, detecting by using HP L C, calculating the content, performing secondary purification, concentrating by using a rotary evaporator, removing residual organic solvent and partial water through reduced pressure concentration, freeze-drying, and performing freeze-drying on the concentrated product to obtain an APP product.
The molecular formula of the raw material DPPA-1 involved in the preparation process is H-Asn-Tyr-Ser-L ys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe-NH2DPPA-1 can be synthesized according to Chang, h. -n., L iu, b. -y., Qi, y. -k., Zhou, y., Chen, y. -p., Pan, k. -m., L i, w. -w., Zhou, x. -m., Ma, w. -w.,. Fu, c. -y., Qi, y. -m., 201582 iu, L. and Gao, y. -F. (bloking of the PD-1/PD-L1 Interaction by a D-Peptide Antagonist for Cancer immunology. expected. chem.int.ed.,54:11760-11764.doi: 10.1002/anie.06225, which is incorporated herein in its entirety.
In a third aspect of the invention, the invention provides a photosensitizer-PD-L1 antagonist peptide copolymer, designated IR780-M-APP, having the structure shown below:
in a fourth aspect, the present invention provides a process for preparing the copolymer IR780-M-APP described in the third aspect above, which comprises reacting IR780 with M-APP by chlorination.
In some embodiments of the invention, the method comprises mixing IR780 and M-APP, adding triethylamine, and reacting under an inert gas atmosphere.
In some embodiments of the invention, the IR780 and M-APP are separately dissolved in a solvent and mixed to react; the solvent of IR780 is selected from N, N-Dimethylformamide (DMF), dichloromethane (CH)2Cl2) One or more of ethanol (EtOH); the solvent of M-APP is selected from N, N-Dimethylformamide (DMF), water (H)2O) is used.
In some embodiments of the invention, when IR780 and M-APP are the same solvent, both N, N-dimethylformamide, the reactions described in the preparation methods of the invention proceed more fully.
In the embodiment of the invention, to obtain a better yield, the molar ratio of the IR780, the M-APP and the triethylamine is 1-10: 0.5-2: 15-25, especially when the ratio is 5:1:20, the reaction is better carried out, and the yield is higher.
The reaction of the invention can be carried out at room temperature, the reaction time is 10-48h, and the longer the reaction time in the range, the more sufficient the reaction is carried out.
In order to obtain more pure IR780-M-APP, in one or more embodiments of the present invention, purification and refinement processes may be performed, for example, the solvent is removed from the reacted material to obtain a crude product, the crude product is dissolved with methanol and then purified by rotary evaporation under reduced pressure, and the temperature of the purification process is not too high.
In a fifth aspect of the invention, the invention provides a nano-assembly formed by self-assembly of the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP described in the third aspect above, denoted as IR780-M-APP NPs.
In a sixth aspect of the invention, the invention provides a composition or pharmaceutical formulation comprising an immune checkpoint antagonist peptide M-APP as described in the first aspect above or a photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP as described in the third aspect above or nanoassembly IR780-M-APP NPs as described in the fifth aspect above;
alternatively, the composition or the pharmaceutical preparation may further comprise at least one pharmaceutically acceptable adjuvant.
The pharmaceutical preparation can be a liquid preparation or a solid preparation, such as an injection or a powder injection, and one skilled in the art can select a proper adjuvant or solvent according to needs.
In a seventh aspect of the invention, the invention provides a nano-formulation formed by self-assembly (self-assembly) of IR780-M-APP as described in the third aspect above, i.e. IR780-M-APP spontaneously forms ordered structures or nano-aggregates (or also called nano-assemblies); alternatively, the nanoformulation comprises the nano-assembly IR780-M-APP NPs described in the fifth aspect above.
In some embodiments of the invention, the nanoformulation is an intravenous injection.
In the eighth aspect of the present invention, the present invention provides a method for preparing a nano preparation, which comprises dissolving the IR780-M-APP compound described in the third aspect above in an organic solvent, dropping water into the organic solvent of the IR780-M-APP under ultrasound, self-assembling the IR780-M-APP compound to form a nano aggregate, and removing the organic solvent.
In some embodiments of the present invention, the organic solvent is selected from one or more of methanol, ethanol, and dimethyl sulfoxide, and methanol is preferred because methanol has a relatively low boiling point and facilitates removal by reduced pressure rotary evaporation.
In some embodiments of the invention, the mass to volume ratio of IR780-M-APP to organic solvent is 1:0.1 to 0.5, especially when the ratio is 1:0.25, the uniformity effect is better.
In a ninth aspect, the present invention provides the use of an immune checkpoint antagonist peptide M-APP as described in the first aspect above in the manufacture of a medicament for immunotherapy.
In some embodiments of the invention, the immunotherapeutic drug is a PD-L1 antagonist drug.
PD-L1 in various tumor cells such as melanoma cells, ovarian cancer cells, lung cancer cells and the like is highly up-regulated, metallomatriptase 2(MMP2) in TME is highly expressed, and the photosensitizer-PD-L1 antagonist peptide assemblies (IR780-M-APP NPs) of the invention can break sensitive bonds in the structure to release L AG-APP under the action of MMP2, and can be combined with PD-L1 on the surface of the tumor cells to play an immunotherapy role.
In a tenth aspect of the present invention, there is provided the use of the immune checkpoint antagonist peptide M-APP of the first aspect above or the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP of the third aspect above or the nano-assembly IR780-M-APP NPs of the fifth aspect above or the composition or pharmaceutical formulation of the sixth aspect above for the preparation of a photodynamic therapy medicament which is a photodynamic anti-tumour medicament.
The IR780-M-APP NPs have EPR effect and can be accumulated at a tumor site, MMP2 connected with DPPA-1 under the action of MMP2 enzyme breaks at a position responding to amino acid sequence P L G L VG to form L AG-APP after the IR780-M-APP reaches the tumor site, the exposed part of APP with the PD-L antagonist activity is combined with PD-L on the surface of tumor cells under the action of MMP2 enzyme, further the immune process of the tumor is inhibited, the utilization rate of the antagonist peptide in vivo immunotherapy is improved, the immunotherapy effect of the antagonist peptide is enhanced, meanwhile, the rest part after the breakage enters the tumor cells through endocytosis, the phototherapy effect is exerted under the action of laser, the defects that the single use of IR780 with poor water solubility, the in vivo delivery is difficult, the single use of singlet oxygen with short action time, the treatment effect with limited action distance are obviously improved, the phototherapy effect is obviously improved, the effects of the melanoma cells as a subject to be treated are obviously improved, the experiment of inhibiting the melanoma cells are carried out, the experiment by using MMP 780-M780-APP 780-NPs as a research subject, the experiment shows that the MMP 780-MMP-5-MMP-5-MMP-inhibitory effect is less than that the MMP-inhibitory effect of the MMP-inhibitory effect, the MMP-inhibitory effect of the MMP-780, the MMP-780, the defect that the defect of the defect that the malignant melanoma-NPs is less-780-MMP-780 toxicity-780, the.
In a tenth aspect of the present invention, the invention provides a use of the immune checkpoint antagonist peptide M-APP of the first aspect or the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP of the third aspect or the nano-assembly IR780-M-APP NPs of the fifth aspect or the composition or pharmaceutical preparation of the sixth aspect for the preparation of an immunotherapeutic drug or an antineoplastic drug.
In the embodiment of the invention, the in vitro melanoma cell inhibition experiment and the melanoma solid tumor inhibition experiment are carried out on the IR780-M-APP NPs, and the results show that the tumor volume growth of mice in the R780-M-APP NPs group is slower than that of the IR780 group, the tumor inhibition effect of the IR780-M-APP NPs preparation is obviously better than that of the IR780 group and the M-APP group which are independently administrated, and the tumor inhibition rate is close to 86%. The combination of the immunity and the phototherapy of the nano preparation is proved to enhance the effect of resisting the melanoma.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention synthesizes the novel nanometer preparation of the photodynamic molecule IR780 and PD-L1 antagonistic peptide self-assembly nanometer particle for the first time, the preparation not only solves the difficulties of strong hydrophobicity of IR780 and difficult drug delivery in vivo, increases accumulation at tumor parts, but also can further increase the treatment effect of immunotherapy and enhance the curative effect of phototherapy.
(2) The nano preparation prepared by the invention is simple and green, and accords with the resource saving in the aspect of green chemistry.
(3) The nano preparation prepared by the invention has uniform shape, the particle size is about 150nm, the nano preparation is suitable for intravenous injection, and can be accumulated in tumor parts through EPR effect.
(4) According to the invention, the nanoparticles are broken after reaching TME through the sensitive bond P L G L VG, so that the M-APP immune checkpoint antagonist peptide is combined with PD-L1 on the surface of a tumor cell, the process of tumor immune escape is inhibited, the utilization rate of in-vivo immunotherapy of the M-APP antagonist peptide is improved, and the effect of the M-APP antagonist peptide immunotherapy is enhanced.
(5) In vitro cell experiments show that the IR780-M-APP NPs preparation has stronger cytotoxicity to melanoma cells, and the preparation has obvious effect. The nano preparation has good biocompatibility and low dark toxicity; and the tumor inhibition rate can reach more than 86% in a drug effect experiment for inhibiting solid melanoma, and the drug has a strong tumor inhibition effect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a carbon spectrum (A) of M-APP prepared in example 1 of the present invention, and a mass spectrum (B) and a hydrogen spectrum (C) of IR780-M-APP prepared in example 2.
FIG. 2 is a TEM image of IR780-M-APP NPs prepared in example 3.
FIG. 3 shows the results of the experiment on the survival rate of tumor cells in the experiment for inhibiting B16-F10 melanoma cells in example 4, wherein A is the result without laser irradiation and B is the result after laser irradiation (L represents the abbreviation of laser L aser).
FIG. 4 is a graph showing the results of in vivo antitumor experiments using the IR780-M-APP NPs preparation of example 5, wherein A is a graph showing a tumor volume-time curve, and B is a graph showing the results of tumor weight and tumor inhibition rate.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
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. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1Synthesis of M-APP
(M-APP polypeptide structure: mercaptopropionic acid-L-Met-P L G L VG-DPPA-1)
The preparation process of M-APP is as follows:
firstly carrying out Resin swelling, soaking 10 g of Rink amide AM Resin (the substitution degree of the Resin is 0.35mmol/g) in 200m L dichloromethane for 15 minutes, after the Resin is swelled, drawing out dichloromethane, secondly, removing amino protection, adding 200m L piperidine/DMF solution, stirring with nitrogen, reacting for 2 times at room temperature for 5 minutes and 15 minutes, washing the Resin with DMF after the reaction is finished, adding a small amount of Resin into a color testing agent, heating for 3 minutes at 100 ℃, determining that the solution and the Resin are blue or red in color, determining that the amino oc FmFmprotection is removed, then, carrying out condensation reaction, adding 2 times of Fmoc-D-Phe-OH and HBTU, dissolving with 200m L DMF, adding 2 times of DIEA, stirring with nitrogen, reacting for 1 hour at room temperature, and washing the Resin with DMF after the reaction is finished.
And then, repeating the steps 2-3, sequentially connecting each protected amino acid to complete the synthesis of the sequence, soaking the resin with dichloromethane and ether, pumping, separating the polypeptide from the resin, adding 150Ml TFA lysate, reacting in a constant temperature shaking table for 1-3h, reacting at the temperature of 15-40 ℃ at the speed of 120 r/min, then, precipitating a crude product, filtering to remove the resin, adding anhydrous ether into the filtrate, centrifuging by a centrifuge (2500rpm) to obtain a solid, adding the anhydrous ether to wash, centrifuging again, repeating for multiple times, drying to obtain the crude polypeptide, further purifying, dissolving the crude product, filtering by a 0.45 mu M membrane, sampling, gradient eluting by using a solvent acetonitrile solution and a solvent (0.1% trifluoroacetic acid and 99.9% pure water solution), collecting a main peak solution, merging the main peak solutions to obtain a fine peptide liquid, detecting by HP L C, calculating the content, performing secondary purification, concentrating by a rotary evaporator, removing residual organic solvent and partial water through reduced pressure concentration, freeze-drying, and obtaining a freeze-dried product, wherein the freeze-dried product is shown in an APP-drying machine M-1-APP.
Wherein, the molecular formula of the raw material DPPA-1 involved in the preparation process is H-Asn-Tyr-Ser-L ys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe-NH2DPPA-1 may be synthesized according to Chang, h. -n., L iu, b. -y., Qi, y. -k., Zhou, y., Chen, y. -p., Pan, k. -m., L i, w. -w., Zhou, x. -m., Ma, w. -w., Fu, c. -y., Qi, y. -m., L iu, L. and Gao, y. -F. (2015), Blocking of the PD-1/PD-L1 Interactionby a D-Peptide Antagonist for Cancer immunology. angel. chem.int.ed.,54:11760-11764.doi: 10.1002/anie.06225, which is incorporated herein in its entirety.
Example 2IR780-M-APP molecular Synthesis
IR780 was weighed accurately on an analytical balance, dissolved in anhydrous N, N-Dimethylformamide (DMF) solvent and placed in a round bottom flask. M-APP (prepared as described in example 1) was weighed out, dissolved in anhydrous DMF and added to a stirring solution of IR780 in DMF and Triethylamine (TEA) was added and reacted under argon at room temperature for 48 hours, wherein the mass ratio of IR780, M-APP and TEA was 5:1: 20. After the reaction is finished, dimethyl sulfoxide is dialyzed to remove anhydrous DMF and unreacted IR780, dialyzed overnight, then is dialyzed by methanol to remove dimethyl sulfoxide, methanol in the solution is removed by reduced pressure evaporation, and is dried overnight in vacuum, so that the IR780-M-APP product is obtained. The structure identification related map of IR780-M-APP is shown in FIG. 1B and FIG. 1C:
1. flight mass spectrum (ESI-TOF-MS):
weighing about 1mg of IR780-M-APP, dissolving in methanol, measuring the molecular weight of the solution by using a flight mass spectrometer, and recording the relative molecular mass of the compound. As shown in FIG. 1B, the mass spectrum result can confirm that the molecular ion peak [ M +2H ]3181.62Da of the raw material molecule IR780-M-APP appears in the newly synthesized molecule, and the successful synthesis of IR780-M-APP is confirmed.
2. Nuclear magnetic spectrum:
weighing about 5mg of IR780-M-APP, dissolving deuterated dimethyl sulfoxide (DMSO-d6) and placing in a nuclear magnetic tube, measuring the nuclear magnetic resonance hydrogen spectrum of the tube by a 400MHz nuclear magnetic resonance hydrogen spectrometer, and recording the chemical shift value (ppm) of the compound. The results are shown in FIG. 1C, and the nuclear magnetic results confirmed the successful synthesis of IR 780-M-APP.
Example 3Preparation of IR780-M-APP NPs
Precisely weighing 2 mg of IR780-M-APP, dissolving in 500 mu L methanol, placing in a round bottom flask, performing ultrasonic treatment, dripping 2M L film-passing water into the methanol solution of IR780-M-APP, continuously performing ultrasonic treatment (ultrasonic condition: 100W, 30min) to form nano aggregates, performing reduced pressure evaporation to obtain a nano particle solution of IR780-M-APP, namely nano preparation IR780-M-APP NPs, wherein TEM results are shown in figure 2, and the morphology of the prepared IR780-M-APP NPs is nano particle morphology, so that the successful preparation is verified.
Example 4In vitro cytotoxicity test of IR780-M-APP NPs preparation
1. Culture of cells
Malignant invasive melanoma B16-F10 cells were selected as experimental subjects.
Frozen cells (B16-F10 cells) were harvested and cultured in RPMI-1640 medium at 37 ℃ under 5% CO2Culturing under the condition, carrying out passage when the cells are recovered and grow to high density, transferring the cells to a culture bottle according to a proportion, continuously culturing and counting the cells.
2. Cytotoxicity test
B16-F10 cells were collected in logarithmic growth phase and diluted to about 3.0 × 10 with medium4(iii)/M L. the test target compounds were divided into 3 groups, i.e., IR780 (CAS: 207399-07-3) group, IR780-M-APP NPs group and IR780-M-APP NPs + MMP2 group, wherein IR780-M-APP NPs were obtained from example 3, and IR780-M-APP NPs + MMP2 was prepared by adding MMP2 enzyme to IR780-M-APP NPs at about 2nM, and the solutions of the test target compounds IR780, IR780-M-APP Ps and IR780-M-APP NPs + MMP2 were diluted to 5. mu.M, 4. mu.M, 2. mu.M, 1. mu.M, 0.5. mu.M in culture medium, and the cells were diluted to 6 × 103Adding each well (200 μ L) into 96-well plate, allowing the plate to adhere overnight, discarding the culture medium, adding target compound solution 200 μ L with different concentrations, setting 3 multiple wells, setting the wells without drug as control group, culturing at 37 deg.C for 4 hr, discarding the upper layer of culture medium containing drug, and replacing with fresh culture medium, wherein the IR780, IR780-M-APP NPs preparation, IR780-M-APP NPs + MMP2 are irradiated with 808nm laser (power is 1W/cm each)2Irradiation time of 2min), in order to investigate the dark toxicity of IR780, IR780-M-APP NPs preparation and IR780-M-APP NPs + MMP2 solution, a dark toxicity hole is established at the same time, all treatments are consistent except laser irradiation, incubation is continued for 20h, MTT solution (5mg/M L) of 10 mu L is added into each hole, incubation is continued for 4h, then liquid in the hole is discarded, DMSO of 120 mu L is added into each hole for dissolution, the absorbance at 570nm is measured by an enzyme labeling detector, and the inhibition rate is calculated according to the following formula:
wherein A iscDenotes the absorbance of the wells of cells not treated with the drug, AsampleThe absorbance of the wells of the cells treated with each sample is indicated.
The experimental results of the survival rate of melanoma cells of B16-F10 under different concentrations of different target compound groups to be tested are shown in FIG. 3, wherein, A is the result without laser irradiation, and B is the result after laser irradiation (L represents the abbreviation of laser L aser).
As can be seen from FIG. 3B, compared with IR780, the cell survival rates of the group of IR780-M-APP NPs and the group of IR780-M-APP NPs + MMP2 are both reduced, and the group of IR780-M-APP NPs + MMP2 is significantly reduced, compared with IR780, the group of IR780-M-APP NPs can not only be treated by photodynamic of IR780, but also can expose PD-L1 antagonist peptide (APP) under the action of MMP2 enzyme, so as to play a role in immunotherapy, and the inhibition effect on malignant invasive melanoma cells is significantly better than that of IR 780.
As can be seen from FIG. 3A, the dark toxicity of the group IR780, group IR780-M-APP NPs and group IR780-M-APP NPs + MMP2 to the test cells is less than 20%, indicating that the drug toxicity is small, especially, when the drug concentration is less than 5.0. mu.M, the dark toxicity to the test cells is less than 10%, and when the drug concentration is not more than 2. mu.M, the dark toxicity to the test cells is lower.
Through the cell inhibition rate experiment, the conclusion can be drawn that: the IR780-M-APP NPs prepared in the embodiment of the invention can simultaneously enhance the curative effects of phototherapy and cancer immunity to maximize the curative effect of tumors.
Example 5In vivo anti-tumor experiment of IR780-M-APP NPs preparation
C57 mouse axillary tumor (100 ten thousand B16-F10 cells each) with tumor length of 100mm3On the left and right, the treatment was randomly divided into 6 groups of 7 individuals, each group was treated with intravenous injection of physiological saline (NS), M-APP (obtained in example 1), IR780, and IR780-M-APP NPs (obtained in example 3), and the groups were irradiated with excitation light of 808nm laser at 1W/cm for 6 hours after the administration of tail vein injection25 min. Changes in tumor volume in mice were recorded during treatment. After 7 treatments, the mice were sacrificed by cervical dislocation, tumors were removed, weighed and tumor inhibition rate was calculated.
Wherein, the calculation formula of the tumor volume is as follows:
l denotes the maximum diameter (mm) and W denotes the minimum diameter (mm).
Wherein, the calculation formula of the tumor inhibition rate is as follows:
wherein, wcIs the average tumor weight (g), w of the mice in the saline groupsampleIs the average tumor weight (g) of the other groups of mice.
The result is shown in figure 4, the IR780-M-APP NPs prepared in the embodiment of the invention have EPR effect, can be accumulated at a tumor site, are broken at the position of MMP2 response amino acid sequence P L G L VG of the connection of DPPA-1 under the action of MMP2 enzyme to form L AG-APP after reaching the tumor site, exposed PD-L1 antagonist peptide (APP) is combined with PD-L1 on the surface of a tumor cell, so that the tumor immune escape process is inhibited, the utilization rate of the antagonist peptide in vivo immunotherapy is improved, the immunotherapy effect of the antagonist peptide is enhanced, meanwhile, the rest part after breaking enters the tumor cell through endocytosis, the phototherapy effect is exerted under the action of laser, the defects that the single use of the IR780 is poor, the in vivo delivery is difficult, the singlet oxygen action time is short, the action distance is limited, the treatment effect is poor are obviously improved, as shown in figure 4, the volume of mice in the group of the IR780-M-APP 780 is slower than that the IR-M-APP combined melanoma inhibition effect is obviously enhanced, and the melanoma inhibition effect is obviously improved.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An immune checkpoint antagonist peptide designated M-APP, having the structure shown below:
2. a method of preparing the immune checkpoint antagonist peptide of claim 1 comprising reacting mercaptopropionic acid, four methionines, an MMP-2 response moiety P L G L VG, and-NYSKPTDRQYHF of the anti-PD-L1 peptide DPPA-1 to link.
3. A photosensitizer-PD-L1 antagonistic peptide copolymer, named IR780-M-APP, has the following structure:
4. a process for preparing the photosensitizer-PD-L1 antagonist peptide copolymer of claim 3, comprising chlorination reaction of IR780 with M-APP, wherein M-APP is as described in claim 1;
preferably, the method comprises the steps of mixing IR780 and M-APP, adding triethylamine, and reacting under an inert gas atmosphere to obtain the compound;
preferably, the IR780 and the M-APP are respectively dissolved in a solvent and then mixed for reaction;
preferably, the solvent of IR780 is selected from one or more of N, N-dimethylformamide, dichloromethane, ethanol;
preferably, the solvent of M-APP is N, N-dimethylformamide and/or water;
preferably, IR780 is the same solvent as M-APP, both N, N-dimethylformamide;
preferably, the molar ratio of the IR780, the M-APP and the triethylamine is 1-10: 0.5-2: 15-25, preferably 5:1: 20;
preferably, the reaction temperature is room temperature, and the reaction time is 10-48 h.
5. A nano-assembly formed by self-assembly of the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP as described in claim 3, denoted as IR780-M-APP NPs.
6. A composition or pharmaceutical preparation comprising the immune checkpoint antagonist peptide M-APP as described in claim 1 or the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP as described in claim 3 or the nano-assembly IR780-M-APP NPs as described in claim 5;
or, it further comprises at least one pharmaceutically acceptable adjuvant;
preferably, the pharmaceutical formulation is a nano-formulation, preferably comprising the nano-assembly IR780-M-APP NPs as described in claim 5;
preferably, the nano-formulation is an intravenous injection.
7. A method for preparing a nano-formulation, which comprises dissolving the IR780-M-APP of claim 3 in an organic solvent, dropping water into the solution under ultrasound, self-assembling the IR780-M-APP to form a nano-assembly, and removing the organic solvent to obtain the nano-formulation;
preferably, the organic solvent is selected from one or more of methanol, ethanol and dimethyl sulfoxide, and is preferably methanol;
preferably, the mass-to-volume ratio of IR780-M-APP to organic solvent is 1:0.1 to 0.5, preferably 1: 0.25.
8. Use of the immune checkpoint antagonist peptide of claim 1 in the preparation of an immunotherapeutic drug;
preferably, the immunotherapeutic drug is a PD-L1 antagonist drug.
9. Use of the immune checkpoint antagonist peptide M-APP of claim 1 or the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP of claim 3 or the nanoassembly IR780-M-APP NPs of claim 5 or the composition or pharmaceutical formulation of claim 6 for the preparation of an immunotherapeutic drug or for the preparation of an antineoplastic drug.
10. Use of the immune checkpoint antagonist peptide M-APP of claim 1 or the photosensitizer-PD-L1 antagonist peptide copolymer IR780-M-APP of claim 3 or the nanoassembly IR780-M-APP NPs comprising claim 5 or the composition or pharmaceutical formulation of claim 6 in the preparation of a photodynamic therapy medicament.
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