阅读说明：本技术 拮抗肽、其共聚物及纳米组装体、及其制备方法和应用 (Antagonistic peptide, copolymer and nano assembly thereof, and preparation method and application thereof ) 是由 栾玉霞 姜悦 王宁宁 于 2020-03-16 设计创作，主要内容包括：本发明提供一种拮抗肽、其共聚物及纳米组装体、及其制备方法和应用,该拮抗肽为PD-L1拮抗肽,其命名为M-APP,结构如下所示：<Image he="132" wi="700" file="DDA0002412840300000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明以M-APP为原料制备得到结构清晰的共聚物IR780-M-APP,其可自组装形成纳米组装体,不仅解决了免疫治疗中免疫原性差的问题,提高了免疫拮抗肽的利用率,还可以通过协同光动力治疗,解决IR780水溶性差的问题,提高了光疗治疗的效果,增强了免疫治疗的疗效。(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.)

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.

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.