CN111544588B

CN111544588B - Immunity active peptide-biliverdin conjugate, preparation method and application thereof

Info

Publication number: CN111544588B
Application number: CN202010399124.7A
Authority: CN
Inventors: 闫学海; 邢蕊蕊; 常蕊; 邹千里
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2020-05-12
Filing date: 2020-05-12
Publication date: 2021-09-28
Anticipated expiration: 2040-05-12
Also published as: CN111544588A

Abstract

The present disclosure relates to a kind of immune active peptide-biliverdin conjugate, its preparation method and application in cancer diagnosis, and/or tumor immunotherapy, and/or tumor "photothermal immunotherapy" (tumor photothermal therapy and combined immunotherapy)) The use of (1). The conjugate disclosed by the disclosure can stimulate an organism to generate a tumor immune effect, and can relieve and/or eliminate tumor inflammation, remold a tumor inflammatory microenvironment and realize photo-thermal immune cancer diagnosis and treatment. The conjugate has high biocompatibility, good stability and prolonged half-life, is prepared by chemically synthesizing immune active peptide and biliverdin, has a peptide end with an immune regulation function, and a pigment end with functions of tumor imaging diagnosis, tumor photothermal ablation, immunoinflammatory microenvironment regulation and the like, can obviously enhance an anti-tumor effect, and effectively inhibits tumor metastasis and recurrence.

Description

Immunity active peptide-biliverdin conjugate, preparation method and application thereof

Technical Field

The invention belongs to the field of 'photothermal immunity' anti-tumor medicines, and particularly relates to an immune active peptide-biliverdin conjugate, a preparation method thereof and application thereof in cancer diagnosis and/or tumor immunotherapy and/or tumor 'photothermal immunity' therapy (tumor photothermal therapy and combined immunotherapy).

Background

The traditional tumor treatment means such as surgery, chemotherapy, radiotherapy and the like all face side effects with different degrees, and the photothermal immunotherapy is a product combining photothermal tumor treatment and tumor immunotherapy, is a safe, accurate and broad-spectrum novel tumor treatment method, and has better treatment effect on late-stage tumors with metastatic and multiple focuses. The basic principle is that the photothermal immune drug is gathered at a tumor position in a targeted manner in an active or passive mode, and under the excitation of laser with specific wavelength, the drug gathered at the tumor position absorbs photothermal and converts the photothermal into heat energy, so that the tumor temperature is locally increased, tumor cells are effectively killed, and the tumor development process is inhibited or tumor tissues are eliminated; the tumor antigen produced by photothermal therapy and the immune activity part in the photothermal immune medicine activate or enhance the function of the immune system of the organism, enhance the activation of immune cells and the release of immune related factors, thereby further inhibiting the relapse and the metastasis of tumors.

Various photothermal preparations, immunopeptide preparations, methods of preparation, and uses thereof have been disclosed: for example, the application of a ferrite nano biomaterial in the preparation of a targeted tumor diagnosis and treatment medicament (publication No. 106310255A) is disclosed; a graphene-based photothermal formulation (publication No. CN 107080844A); a controllable preparation method of copper seleno compound nano-sheets for tumor photothermal therapy (publication No. 106902352B); an organic small molecule (3, 6-di (2-thienyl) -2, 5-dihydropyrrolo [3, 4-c ] pyrrole-1, 4-Diketone (DPP) derivative) nano tumor photothermal therapeutic agent and a preparation method thereof (publication No. 106008525B); a nanometer photothermal therapeutic agent formed by polymer formed by covalent bond of mPEG-PLGA and/or PEG-PLGA and porphyrin compounds, and its preparation method (publication No. 105327348A). These disclosed photothermal agents all exhibit high photothermal conversion efficiency and effectively inhibit tumor growth, but still have general problems: 1) long-term biosafety needs to be studied; 2) the metabolic mechanism has not been elucidated; 3) metastasis and recurrence of tumors following photothermal therapy. For another example, anti-tumor related peptides and related anti-cancer vaccine compositions have been disclosed for eliciting anti-tumor immune responses against colorectal cancer (publication No. 103360466 a); a tumor-associated peptide composition and related anti-cancer vaccine for the treatment of gliomas and other cancers has been disclosed (publication No. 102170901 a); a pharmaceutical composition (FOXP3 SIRNA-protamine-anti-CD 25 antibody complex) (publication No. 101455840A) and the like capable of enhancing an anti-tumor immune response have been disclosed. Meanwhile, related immunomodulatory peptide drugs such as thymopentin for injection, recombinant human interferon alpha-2 b injection, and corious versicolor glycopeptide capsule have been commercialized, but the published or commercialized immunomodulatory peptide drugs have the following problems: 1) the half-life period of the micromolecule is short and the micromolecule is easy to degrade; 2) the immune effect is weak; 3) the composition is complex, and the immune-related adverse events are serious. Therefore, there is a need for further development of novel tumor therapeutic agents and therapeutic methods based on the presently disclosed photothermal agents and immunological agents.

The combination of light treatment and immunotherapy is expected to further inhibit the metastasis and recurrence of tumor on the basis of tumor ablation, thereby bringing better survival benefit for tumor patients. Rakutene medical company develops an antibody-conjugated drug (ADC) consisting of cetuximab (cetuximab) and IRDye700DX for the combination of photodynamic therapy and immunotherapy, specifically, high tumor specificity is realized by the targeted delivery mediated by the cetuximab, and tumor ablation is realized by the photodynamic effect of IRDye700 DX. At present, in a clinical test aiming at local recurrent head and neck cancer, the technology achieves 50 percent of remission rate, 16.7 percent of complete remission rate and 86.7 percent of disease control rate. The treatment effect and the better biological safety, but the structure is complex, and the synthesis and the production are difficult to a certain degree. Meanwhile, the photodynamic therapy has strong dependence on oxygen and is not suitable for hypoxic tumors. The corresponding tumor 'photothermal immunotherapy' shows significant advantages, especially its broad spectrum and accuracy. The success of the therapy depends mainly on the high and low photothermal conversion efficiency of the medicine and the strong and weak immune effect. Meanwhile, the biological safety, immune-related adverse events (irAEs), metabolic mechanisms and the like of the photothermal immune preparation are also the key points for successfully implementing tumor photothermal immune therapy.

The endogenous pigment biliverdin is a secondary metabolite of hemoglobin in an animal body, has a definite metabolic mechanism, and has multiple biological activities (oxidation resistance, anti-inflammation, anti-tumor and the like). Biliverdin is a bioactive pigment with a linear tetrapyrrole structure, has remarkable near-infrared absorption, can realize effective conversion of near-infrared light to heat energy (patent publication No. 109224073A), and has wide application prospects in the fields of development of tumor treatment medicines and photo-thermal anti-tumor. Researches prove that the tumor inflammatory microenvironment can further promote the metastasis and recurrence of tumors, and the tumor inflammatory microenvironment is reversed by utilizing the anti-inflammatory activity of biliverdin molecules to realize tumor immunotherapy, so that no public report is provided. Meanwhile, small molecule immunoactive peptides are substances having specific amino acid sequences and multiple biological functions (immunoregulation, anti-tumor, etc.), and gradually exhibit unique advantages in the field of biological medicine.

A conjugate formed by a biliverdin molecule and an immunoactive peptide molecule through a chemical synthesis method, a preparation method thereof and application thereof in the aspect of photo-thermal immunity tumor treatment have not been reported. The immune active peptide-biliverdin conjugate disclosed by the invention has remarkable advantages in the fields of cancer diagnosis, and/or tumor immunotherapy, and/or tumor 'photo-thermal immunity' therapy: 1) the half-life period is prolonged and the stability is enhanced relative to the disclosed immune active peptide and the composition thereof; 2) has high biological safety, single component and definite metabolism mechanism in vivo; 3) the immune active peptide end can stimulate an organism to generate tumor immune response and enhance the immune function; the biliverdin end can realize cancer diagnosis and photothermal therapy, and simultaneously can relieve and eliminate tumor inflammation and remold a tumor inflammatory microenvironment. In conclusion, the molecular conjugate, the related preparation, the dosage form and the preparation method thereof have important significance for promoting clinical application of the molecular conjugate in tumor treatment, and have great application potential in eliminating primary tumors, inhibiting tumor metastasis, relapse and the like.

Disclosure of Invention

The invention discloses an immune active peptide-biliverdin conjugate, a preparation method thereof and application thereof in tumor imaging, tumor immunotherapy and tumor photo-thermal immunization treatment. The disclosed conjugates have the following advantages: 1) the half-life period is prolonged and the stability is enhanced relative to the disclosed immune active peptide and the composition thereof; 2) has high biological safety, single component and definite metabolism mechanism in vivo; 3) the immune active peptide end can stimulate an organism to generate tumor immune response and enhance the immune function; the biliverdin end can realize multi-modal tumor imaging and tumor photothermal therapy, realize tumor ablation, relieve and eliminate tumor inflammation, remold a tumor inflammatory microenvironment and prevent tumor metastasis and recurrence.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a class of immunologically active peptide-biliverdin conjugates, characterized in that said structure follows formula i or ii or iii, and salts, isomers, derivatives thereof which do not affect their pharmaceutical function:

wherein the content of the first and second substances,

m is selected from the following non-metal atoms or ions of the following non-metal elements: H. si, P, or an ion selected from the following metal atoms or metal elements: mg, Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Y, Ru, Rh, Pd, In, Sn, Pt, Au, Eu, Gd, Tb, Dy, Er, Yb, Lu, Tc, Tl, and radioactive isotopes thereof, and non-radioactive isotopes thereof; the number of M is 1-4, and the specific number of M is different due to different valence states of M;

R₁and R₂Each independently represents an active peptide having an immunoregulatory function and has the amino acid sequence X₁～X₂₂Any one of any group of:

X₁: ovalbumin peptide: SIINFEKL (8), EQLESIINFEKLTE (14), ISQAVHAAHAEINEAGR (17)

X₂: HPV16E7 peptide: PDRAHYNI (8), TLGIVCPI (8), RAHYNIVTF (9), YMLDLQPETT (10), GQAEPDRAHYNIVTF (15)

X₃：NYSKPTDRQYHF(12)、KHAHHTHNLRLP(12)

X₄：HVIHEGTVVI(10)、HVVHEGTVVI(10)

X₅：KVPRNQDWL(9)、FLWGPRALV(9)

X₆：YLEPGPVTA(9)、IMDQVPFSV(9)

X₇：MLLAVLYCL(9)、YMDGTMSQV(9)

X₈：TKPR(4)

X₉：GQPR(4)

X₁₀：CAPE(4)

X₁₁：RKEVY(5)

X₁₂：RKDVY(5)

X₁₃：LVVTPW(6)

X₁₄：FLGFPT(6)

X₁₅：PDRAHYNI(8)

X₁₆：FKFEFKFE(8)

X₁₇：ALCNTDSPL(9)

X₁₈：KIFGSLAFL(9)

X₁₉：KTKCKFLKKC(10)

X₂₀：QQKFQFQFEQQ(11)

X₂₁：PLYKKIIKKLLES(13)

X₂₂：HSLGKWLGHPDKF(13)

X₂₃：VHFFKNIVTPRTP(13)

X₂₄：EIIVTHFPFDEQNCSMK(17)

X₂₅：(SNTSESF)2KFRVTQ-LAPKQIKE-NH2(29)。

In a second aspect, the invention provides a conjugate according to the first aspect, wherein R1 and R2 are the same or different.

In a third aspect, the invention provides a conjugate according to the first to second aspects, wherein R is₁And R₂Is any sequence as described above, and can also be a peptide or protein containing any sequence as described above, or a derivative of any sequence as described above, or an amino acid, peptide or protein with similar functions;

preferably, the active site of the immune active peptide is at the non-N end, and the non-active end is formed by condensing with the C end of biliverdin through peptide bonds;

still preferably, said immunologically active peptide has the following amino acid sequence, or comprises the following sequence, or is a derivative of the following sequence, or is an amino acid, peptide or protein with similar function:

X₁：SIINFEKL(8)

X₃：NYSKPTDRQYHF(12)

X₅：FLWGPRALV(9)

X₆：YLEPGPVTA(9)、IMDQVPFSV(9)

X₇：YMDGTMSQV(9)

X₁₅：PDRAHYNI(8)

X₁₈：KIFGSLAFL(9)

X₂₃：VHFFKNIVTPRTP(13)

wherein, the derivative is a peptide molecule modified by phenyl, carbobenzoxy, tert-butyloxycarbonyl, beta-naphthamido, N- (3-indoleacetyl) or N-fluorenylmethoxycarbonyl group or a key molecule fragment thereof.

In a fourth aspect, the present invention provides the conjugates of the first to third aspects, which are characterized in that the molecular conjugates and preparations or dosage forms based on, derived from, such molecular conjugates:

the method comprises the following steps: a preparation or dosage form system formed by chemical bonding, physical adsorption, loading or wrapping; and assemblies, multimers or aggregates formed by weak intermolecular interactions;

wherein the preparation or dosage form comprises solution, lotion, suspension, tablet, gel or patch.

In a fifth aspect, the present invention provides a method for preparing the conjugate according to the first to fourth aspects, characterized by comprising the steps of:

(1) m is H:

a. adding biliverdin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS) and anhydrous Dimethylformamide (DMF) into a reactor in sequence, and uniformly mixing;

the concentration of the biliverdin is 0.1-500 mM, preferably 1-100 mM;

the concentration of EDC & HCl is 0.1-1000 mM, preferably 1-200 mM;

the concentration of the NHS is 0.1-1000 mM, preferably 1-200 mM;

the mass concentration ratio of the biliverdin to the EDC & HCl to the NHS is 1:1: 0.5-1: 20:20, preferably 1:1: 0.8-1: 5: 10;

b. stirring the mixture obtained in the step (a) at room temperature in a dark place, wherein the reaction time is 12-48 h, preferably 12-24 h;

c. adding water to the mixture obtained in the step (b) while stirring, and collecting precipitates;

d. adding anhydrous DMF into the precipitate obtained in the step (c), wherein the mass ratio of the precipitate to the DMF is 1:100, preferably 1: 5;

e. adding immunoactive peptide and anhydrous triethylamine into the anhydrous DMF solution of the precipitate obtained in the step (d), and stirring at room temperature in a dark place;

the concentration of the immunoactive peptide is 0.01-2000 mM, preferably, the concentration is 0.1-500 mM;

the concentration of the anhydrous triethylamine is 0.01-4000 mM, preferably 0.1-1000 mM;

the stirring time is 4-96 h, preferably 12-24 h;

f. adjusting the pH value of the mixed solution obtained in the step (e) to 3.5-7.5, preferably, the pH value is 4.0-6.0;

the pH value is adjusted by adding alkaline substances or acidic substances:

preferably, the alkaline substance is any one or a mixture of two or more of sodium hydroxide, potassium hydroxide and sodium carbonate;

preferably, the acidic substance is any one or a mixture of two or more of hydrochloric acid, sulfuric acid and nitric acid;

the pH value can also be adjusted by an aqueous solution dialysis method;

g. collecting the precipitate of step (f) and purifying by size exclusion chromatography;

h. recrystallizing the material from step (g) to obtain pure molecular conjugate.

(2) M is a metal atom or ion other than H:

i. dissolving biliverdin and excessive metal acetate in methanol, wherein the mass concentration ratio of biliverdin to metal salt is 1: 1-1: 100, preferably 1: 2-1: 20;

j. (ii) stirring the methanol solution of step (i) at a temperature in the range of 20 ℃ to 60 ℃, preferably 35 ℃ to 60 ℃ for 4 hours;

k. performing rotary evaporation on the solution obtained in the step (j) to remove the solvent to obtain a solid;

purifying the solid obtained in the step (k) by using a reversed phase chromatographic column to obtain the biliverdin-metal complex;

and m, according to the steps a-h, synthesizing the biliverdin-metal complex-immunoactive peptide conjugate.

(3) M is a non-metal atom or ion other than H:

n, dissolving biliverdin and non-metal halide or acyl chloride salt in an organic solvent pyridine or DMF, wherein the mass concentration ratio of the biliverdin to the non-metal halide or acyl chloride salt is 1: 1-1: 100, preferably 1: 2-1: 20;

o, stirring the mixed solution obtained in the step (14) at a certain temperature in a dark place for reaction, wherein the temperature is 20-100 ℃, and preferably 35-65 ℃; the reaction time is 2-8 h, preferably 4-6 h;

removing the solvent from the solution obtained in the step (15) by rotary evaporation;

purifying the solid obtained in the step (16) by using a reverse phase chromatographic column to obtain the biliverdin-nonmetal complex;

and r, according to the steps a-h, synthesizing the biliverdin-nonmetal complex-immunoactive peptide conjugate.

In a sixth aspect, the invention provides a conjugate as described in the first to fifth aspects and a preparation method thereof, wherein the conjugate has a "photothermo-immune" anti-tumor use.

In a seventh aspect, the present invention provides the use of the sixth aspect, wherein the conjugate molecule gathered at the tumor site is irradiated by laser with a specific wavelength to complete the conversion of light energy to heat energy, thereby achieving tumor ablation, and at the same time, the in-situ tumor specific antigen can be generated after the tumor site is irradiated by light; the conjugate can further activate the immune response of an organism, eliminate the tumor inflammatory microenvironment, enhance the specific immune response of the organism, realize the tumor immunotherapy and further prevent the tumor metastasis and recurrence. The tumor treatment system realizes the combination of tumor photothermal treatment and tumor immunotherapy, and is characterized in that the combination of tumor ablation, immunoregulation and tumor inflammatory microenvironment regulation is realized, and the tumor treatment effect is obviously improved;

wherein

The above-mentionedThe laser wavelength of the tumor ablation is 635nm, 660nm, 680nm, 730nm, 808nm, 980nm, 1064nm, preferably 730nm and 808 nm; the laser intensity is 0.05-2.5W/cm²Preferably, the laser intensity is 0.2-1.2W/cm²；

The tumor immunoregulation function is to enhance antigen recognition, uptake and presentation; enhance the activation, proliferation and differentiation of immune cells; synergy of one or two or more of the effects in increasing secretion of immunocytokines;

the regulation function of the tumor inflammatory microenvironment is the synergy of one or two or more than two of the functions of inhibiting inflammatory cells, inhibiting the secretion of inflammatory related factors and blocking intracellular signal pathways.

In an eighth aspect, the invention provides the use of the sixth to seventh aspects, wherein the conjugate is used for tumor diagnosis and monitoring before, during and after "photothermo-immune" treatment, including nuclear magnetic resonance imaging, radionuclide imaging, and photoacoustic imaging.

In a ninth aspect, the invention provides the use of the eighth aspect, wherein the conjugate is for magnetic resonance imaging of tumors, M is preferably selected from the following atoms or ions: mn, Fe, Cu, Eu, Gd, Dy.

In a tenth aspect, the invention provides the use of the eighth aspect, wherein the conjugate is for radionuclide imaging of tumors, M is preferably selected from the following atoms or ions:^64,67Cu、⁹⁹mTc、¹⁹⁵Pt、^67,68Gd、²⁰¹Tl、⁶⁰Co、¹¹¹In、⁵¹Cr。

in an eleventh aspect, the invention provides the use of the eighth aspect, wherein the conjugate is for photoacoustic imaging of tumors, M is preferably selected from the following atoms or ions: H. and Zn.

In a twelfth aspect, the present invention provides the use according to the sixth to eleventh aspects, wherein the tumor is a primary tumor or a metastatic tumor, and is selected from single-or multiple-tumor such as brain cancer, head and neck cancer, esophageal cancer, breast cancer, lung cancer, stomach cancer, liver cancer, colon cancer, pancreatic cancer, lymph cancer, melanoma, ovarian cancer, cervical cancer, prostate cancer, and bladder cancer, and preferably the tumor is a superficial tumor such as head and neck cancer, breast cancer, melanoma, cervical cancer, prostate cancer, and pancreatic cancer, or a tumor with a high surgical risk.

In a thirteenth aspect, the present invention provides the use according to the sixth to eleventh aspects, wherein the use can be combined with tumor treatment strategies such as surgery, chemotherapy, radiotherapy, immunotherapy, etc.

In a fourteenth aspect, the present invention provides the use of the thirteenth aspect, for the treatment of residual tumor lesions, and/or metastatic tumor lesions after surgery.

In a fifteenth aspect, the invention provides the use of the thirteenth aspect, for a combination of chemotherapy and "photothermal immune" treatment;

wherein the chemotherapy drug comprises one or more of cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, carmustine, lomustine, semustine, nimustine, methotrexate, pemetrexed, loratrexed, raltitrexed, fluorouracil, capecitabine, gemcitabine, ancitabine, cytarabine, tegafur, floxuridine, doxifluridine, idodine, vinblastine, vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, docetaxel, albumin-bound paclitaxel, camptothecin, irinotecan, topotecan, rubitecan, doxorubicin (adriamycin), epirubicin, pirarubicin, amide, ifosfamide, etoposide, or derivatives thereof, preferably cisplatin, paclitaxel, docetaxel, or derivatives thereof, Doxorubicin (adriamycin);

preferably, the amount of chemotherapeutic agent is 5% to 30%, more preferably 10% to 15% of the conventional amount.

In a sixteenth aspect, the invention provides the use of the thirteenth aspect, for a combination of radiotherapy and "photothermo-immune" treatment;

preferably, the radiotherapy dose is 5% to 40% of the conventional dose, and more preferably, 5% to 20%.

In a seventeenth aspect, the invention provides the use of the thirteenth aspect, for a combination of immunotherapy and "photothermo-immune" therapy;

preferably, the immunotherapy drug comprises antibodies, cytokines, molecular vaccines, cellular vaccines, biological response modifiers, immunosuppressants, and traditional Chinese medicine monomer components;

still more preferably, thymic factor, indoleamine 2, 3-dioxygenase inhibitor, interferon, interleukin;

still more preferably, the immunopharmaceutical dose is 10% to 50% of the conventional dose, still more preferably, 15% to 30%.

Eighteenth, the invention provides the conjugate of the first to seventeenth aspects, the preparation method thereof and the application in tumor treatment, which have the advantages of high biological safety, good stability, difficult drug resistance generation, clear metabolic mechanism, prolonged half-life period and the like, and can significantly enhance the tumor treatment effect.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) at present, no 'photothermal immune' molecule, preparation and dosage form based on biliverdin and relevant reports of tumor immunotherapy and/or 'photothermal immune' therapy are reported, and particularly, the conjugate disclosed by the invention can improve the photothermal effect of biliverdin;

(2) the molecular conjugate of the immune active peptide-biliverdin is obtained by taking endogenous biliverdin and the immune active peptide as initial raw materials through a chemical synthesis means, has high biological safety, good stability and definite metabolic mechanism, and can effectively solve the problems of serious immune related adverse events (irAEs), poor biocompatibility and the like;

(3) multifunctional synergy can be realized in the aspect of tumor treatment: the immune active peptide end can stimulate an organism to generate tumor immune response and enhance the immune function; the biliverdin end can realize tumor imaging and tumor photothermal treatment, realize tumor ablation, relieve and eliminate tumor inflammation, remold a tumor inflammatory microenvironment and reduce the tumor metastasis rate and the recurrence rate.

Drawings

Fig. 1 is a (a) molecular structure diagram of the conjugate prepared in example 1, and a cyclic (3) photothermal temperature rise curve of the biliverdin molecule (b) and the conjugate molecule (1c), indicating that the conjugate molecule has a better temperature rise effect and more excellent cyclic stability.

FIG. 2 is a result of a cell activity experiment of the conjugate prepared in example 2, showing that the prepared conjugate is highly biosafety and has no obvious cytotoxicity to human umbilical vein endothelial cell HUVEC;

FIG. 3 is a result of a cell activity experiment of the conjugate prepared in example 4, showing that the prepared conjugate is highly biosafety and has no significant cytotoxicity to mouse skin melanoma cells B16-F10;

FIG. 4 is the relative fluorescence intensity of positive BMDCs at different time points in example 4, indicating that BMDCs can successfully take up the conjugate, which lays the foundation for further tumor immunotherapy;

FIG. 5 shows the effect of the conjugate prepared in example 5 on the maturation of dendritic cells, which indicates that the molecules of the conjugate can promote the maturation of dendritic cells and lay the foundation for further implementation of tumor immunotherapy;

FIG. 6 shows the specific binding results of the conjugate prepared in example 6 (FITC-labeled) to DU-145 cell line and LNCaP cell line;

FIG. 7 shows the in vitro temperature increase of the conjugate obtained in example 7 under laser irradiation, and the results show that the conjugate molecules have better photothermal conversion effect, which lays the foundation for the realization of "photothermal immunity" tumor treatment;

FIG. 8 is the inhibitory behavior of the conjugate described in example 8 against tumors in the absence of light, indicating that the conjugate molecule has potential immunological antitumor activity;

FIG. 9 is a tumor suppression curve (a) and a recurrence curve (b) of the conjugate described in example 9, indicating that the conjugate has a better "photothermo-immune" tumor treatment effect and is effective in preventing recurrence of tumors;

FIG. 10 is a graph of the levels of the immune-related factors described in example 10, showing that the conjugates up-regulate the humoral immunity and down-regulate the immunosuppressive behavior in the absence and presence of light, indicating that the conjugates have "photothermal immune" tumor treatment;

FIG. 11 is a transmission electron microscope photograph of the conjugate molecular gelator of example 11, showing a structured fiber network structure;

FIG. 12 is a statistical plot of the results of the emulsion form of the conjugate described in example 12 for mouse bladder cancer tumor imaging, demonstrating the cancer diagnostic ability of the conjugate;

FIG. 13 is a graph of CD4 in mouse spleen and draining lymph nodes with the conjugate described in example 13⁺T and CD8⁺T cell change curve, showing that the conjugate has immune effect and 'photo-thermal immune' effect;

FIG. 14 is a radionuclide imaging of the conjugate described in example 14 (a), tumor hyperthermia in situ (b), and CD8 in mouse tumors⁺T cells (c) and CD8⁺The expression (d) condition of CD107 molecule on the surface of T cell proves that the conjugate can realize cancer diagnosis and 'photothermal immunity' tumor treatment;

FIG. 15 is a graph of the combined inhibitory effect of the conjugate of example 15 in combination with an immunological formulation against B16-F10 tumors and Lewis tumors, showing that the combination of "photothermal immunity" with immunotherapy significantly enhances the antitumor effect;

FIG. 16 is a graph of the combined inhibitory effect of the conjugate of example 16 in combination with low doses of chemotherapeutic agents on B16-F10 tumors and Lewis tumors, showing that the combination of "photothermal immunity" and chemotherapy significantly enhances the anti-tumor effect (a) and is effective in reducing the effect (body weight, B) on the survival status of mice;

FIG. 17 is a graph of the effect of the conjugate described in example 17 on vital organs (heart, liver, spleen, lung, kidney) showing that there is no severe damage to vital organs.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1:

the biliverdin-SIINFEKL conjugate is obtained by chemical synthesis according to the following steps: weighing a certain amount of biliverdin, EDC & HCl, NHS and DMF, sequentially adding into a reactor and uniformly mixing; stirring the obtained mixture at room temperature for 24 hours in a dark place; then adding water for stirring, and collecting precipitate; adding anhydrous DMF into the obtained precipitate, uniformly mixing, adding SIINFEKL peptide and anhydrous triethylamine, and stirring at room temperature in a dark place for reacting for 24 hours; collecting the precipitate from the above reaction and purifying by size exclusion chromatography; the resulting material was recrystallized to give pure molecular conjugates. Wherein the concentration of biliverdin is 100mM, the concentration of EDC & HCl is 100mM, the concentration of NHS is 50mM, and the concentration of peptide is 200 mM. The 1HNMR information of the prepared conjugates is as follows:

1HNMR(600MHz)δ＝11.95～13.00(3H),10.68(s,1H),8.85(s,1H),8.56(s,1H),8.40(s,1H),8.32(s,2H),8.21(s,2H),8.08(s,1H),7.50(s,1H),7.41(s,1H),7.30(m,3H),7.16(m,3H),7.03(q,2H),6.93(s,1H),6.49(t,2H),5.57(s,1H),5.20(m,4H),4.16(m,1H),3.95(m,1H),4.55(m,2H),4.92(m,1H),4.34(m,2H),4.94(m,1H),4.84(m,1H),4.61(1H),4.44(m,2H),3.44(m,1H),3.18(m,1H),2.81(m,1H),2.69(m,4H),2.49～2.42(7H),2.35(m,3H),2.23(m,2H),2.12(m,6H),2.06(m,2H),1.95(m,3H),1.49(m,1H),1.75(m,4H),1.55(m,6H),1.25(m,2H),1.11(m,6H),0.90～1.00(12H).

fig. 1(a) is a molecular structure diagram of the conjugate prepared in example 1, and fig. 1(b) is a cyclic (3) photothermal temperature rise curve of the biliverdin molecule and the conjugate molecule (fig. 1c), indicating that the conjugate molecule has better temperature rise effect and more excellent cyclic stability.

Example 2:

biliverdin molecules and SIINFEKL were chemically synthesized according to example 1 to obtain biliverdin-SIINFEKL conjugates. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. And incubating the prepared conjugates with different concentration gradients and human umbilical vein endothelial cells HUVEC in a dark place, and evaluating the biological safety of the conjugates by adopting an MTT colorimetric method. FIG. 2 is a result of a cell activity experiment of the conjugate prepared in example 2, showing that the prepared conjugate is highly biosafety and has no obvious cytotoxicity to human umbilical vein endothelial cell HUVEC.

Example 3:

firstly, biliverdin and excessive zinc acetate are chemically synthesized to obtain biliverdin-Zn metal complex, and the experimental method is as follows: dissolving biliverdin and excessive zinc acetate in methanol solution, stirring at 60 deg.C for 4 hr, removing solvent by rotary evaporation to obtain solid, and purifying with reversed phase chromatographic column to obtain biliverdin-Zn complex, wherein the mass concentration ratio of biliverdin to zinc acetate is 1: 5. The biliverdin-Zn metal complex and NYSKPTDRQYHF are prepared into biliverdin-Zn-NYSKPTDRQYHF conjugate according to the chemical synthesis method. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. And (3) incubating the prepared conjugates with different concentration gradients and mouse skin melanoma cells B16-F10 in a dark place, and evaluating the biological safety of the conjugates by adopting an MTT colorimetric method. FIG. 3 is a result of a cell activity experiment of the conjugate prepared in example 3, showing that the prepared conjugate is highly biosafety and has no significant cytotoxicity to mouse skin melanoma cells B16-F10.

Example 4:

firstly, chemically synthesizing biliverdin and excessive ferrous chloride to obtain biliverdin-Fe metal complex, and preparing the biliverdin-Fe-YMDGTMSQV conjugate from the biliverdin-Fe metal complex and YMDGTMSQV according to the chemical synthesis method. Weighing a certain mass of conjugate, pre-dissolving the conjugate by a trace amount of organic solvent, completely dissolving the conjugate in a PBS solution, filtering and sterilizing, and adjusting the pH value to be neutral. And (3) labeling the conjugate by using fluorescein, incubating the labeled conjugate with mouse bone marrow-derived dendritic cell BMDCs, and detecting the uptake behavior of the conjugate by the BMDCs by using a flow cytometer. FIG. 4 is the relative fluorescence intensity of BMDCs in example 4, shown to be positive, at different time points, indicating that BMDCs are able to successfully uptake the conjugate, which lays the foundation for further tumor immunotherapy.

Example 5:

biliverdin molecule and KIFGSLAFL were subjected to the above chemical synthesis to obtain biliverdin-KIFGSLAFL conjugate. Weighing a certain mass of conjugate, pre-dissolving the conjugate by a trace amount of organic solvent, completely dissolving the conjugate in a PBS solution, filtering and sterilizing, and adjusting the pH value to be neutral. The prepared conjugate molecule solution is co-cultured with dendritic cells taken from peripheral blood of a non-small cell lung cancer model mouse, after 24 hours, the dendritic cells are collected, washed and fluorescently labeled, and CD80, CD83 and CD86 on the cell surface are detected by using a flow cytometer to evaluate the promotion effect of the conjugate on the maturation of the dendritic cells. FIG. 5 shows the effect of the conjugate prepared in example 5 on the maturation of dendritic cells, and the results show that the conjugate molecules can promote the maturation of dendritic cells, thereby laying the foundation for further implementation of tumor immunotherapy.

Example 6:

biliverdin molecule and FLWGPRALV were subjected to the above chemical synthesis to obtain biliverdin-FLWGPRALV conjugate. Weighing a certain mass of conjugate, directly dissolving the conjugate in a PBS solution, stirring to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. The prepared conjugate is co-incubated with a human prostate cancer DU-145 cell line and an LNCaP cell line, flow cytometry analysis is carried out, and statistics is carried out by using SPSS 12.0. FIG. 6 shows the specific binding results of the conjugate prepared in example 6 (FITC-labeled) to DU-145 cell line and LNCaP cell line.

Example 7

Firstly, chemically synthesizing biliverdin and excessive manganese acetate tetrahydrate to obtain a biliverdin-Mn metal complex, and preparing the biliverdin-Mn-YLEPPGPVTA conjugate by using the biliverdin-Mn metal complex and YLEPPVTA according to the chemical synthesis method. A conjugate is weighed to a certain mass,after the trace organic solvent is pre-dissolved, the mixture is completely dissolved in PBS solution, and then is filtered and sterilized, and the pH value is adjusted to be neutral. 1mL (concentration of 0.2 mgmL) of the conjugate was added^-1) Placing in a laser (0.3W/cm) at 730nm²) The irradiation was carried out for 10min to investigate the temperature rise of the conjugate solution. FIG. 7 shows the in vitro temperature rise of the conjugate obtained in example 7, and the results show that the conjugate molecule has better photothermal conversion effect, and lays the foundation for realizing the tumor treatment of 'photothermal immunity'.

Example 8

Biliverdin molecule and IMDQVPFSV were subjected to the above chemical synthesis to obtain biliverdin-IMDQVPFSV conjugate. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. Constructing a C57BL/6 mouse model according to a standard tumor mouse modeling mode, inoculating a mouse breast cancer cell 4T1 subcutaneously, feeding in an SPF (specific pathogen free) environment, observing the growth condition of the tumor at any time, and waiting until the tumor volume grows to be 80-100 mm on average³After left and right, relevant experiments were performed. Mice were divided into 2 groups (10 mice each) and the experimental group was intraperitoneally injected with 100uL of the above conjugate (concentration of 0.2 mgmL) on

days

1, 2, 4, and 8^-1) The blank group was injected with the same quality of physiological saline. Mice were monitored for tumor volume growth within 28 days. Fig. 8 is the inhibitory behavior of the conjugate described in example 8 against tumors in the absence of light, indicating that the conjugate molecule has potential immunological anti-tumor activity.

Example 9

Biliverdin molecule and QQKFQFQFEQQ were subjected to the above chemical synthesis to obtain biliverdin-QQKFQFQFEQQ conjugate. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. Constructing a C57BL/6 mouse model according to a standard tumor mouse modeling mode, inoculating a mouse colon cancer cell ct-26 subcutaneously, feeding in an SPF (specific pathogen free) environment, observing the growth condition of the tumor at any time until the tumor volume grows to 80-100 mm on average³After left and right, relevant experiments were performed. Mice were divided into four groups: blank (saline), conjugate (no light)Control), conjugate (light control), 10 mice per group. Administered once on

days

1, 3, 8 and 12, with a dose concentration of 2mgkg^-1. Mice with conjugate (light group) were laser irradiated once 4h after the first day of administration with the following parameters: the laser intensity is 0.5W/cm²The laser wavelength is 808 nm. Mice were monitored for tumor suppression throughout the treatment cycle (cycle 45 days). Mice were completely cleared of tumor at day 29 after conjugate (light group) treatment and mice were monitored for recurrent behavior of tumor from day 30 to day 45. FIG. 9 is a tumor suppression curve (a) and a recurrence curve (b) of the conjugate described in example 9, indicating that the conjugate has a better "photothermo-immune" tumor treatment effect and is effective in preventing tumor recurrence.

Example 10

Firstly, chemically synthesizing biliverdin and excess gadolinium chloride hexahydrate to obtain biliverdin-Ga metal complex, and preparing the biliverdin-Ga-FKFEFKFE conjugate by using the biliverdin-Ga metal complex and FKFEFKFE according to the chemical synthesis method. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. Constructing a Pan02 in-situ model of C57BL/6 mouse pancreatic cancer according to a standard tumor mouse modeling mode, feeding the model in an SPF (specific pathogen free) environment, observing the growth condition of the tumor at any time until the tumor volume grows to be 80-100 mm on average³After left and right, relevant experiments were performed. Mice were divided into four groups: blank (saline), conjugate (no light), conjugate (light), 10 mice per group. Administered once on

days

1, 3, 8 and 12, with the administration concentration of 4mgkg^-1. Mice with conjugate (light group) were laser irradiated once 4h after the first day of administration with the following parameters: the laser intensity is 0.5W/cm²The laser wavelength is 730 nm. On day 15, mice were euthanized and tumor tissues from each group of mice were harvested and the content of immune-related factors including IFN-. gamma.with immune enhancing effect and IL-4 and IL-10 with immune suppressing effect in the supernatants of each group was measured by ELISA. FIG. 10 shows the levels of the immune-related factors described in example 10, indicating that the conjugates were able to function in the absence and presence of lightThe organism immunity is up-regulated, and the immunosuppressive behavior is down-regulated, which shows that the conjugate has better tumor treatment effect of photothermal immunity.

Example 11

And (3) preparing the biliverdin molecule and the LVVTPW according to the chemical synthesis method to obtain the biliverdin-LVVTPW conjugate. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, and adding water to form a fiber preparation of the conjugate. The concentration of the conjugate was 5mgmL^-1. FIG. 11 is a transmission electron microscope photograph of the conjugate molecular gelator of example 11, showing a structured fiber network structure.

Example 12

Firstly, chemically synthesizing biliverdin and excessive manganese chloride to obtain a biliverdin-Mn metal complex, and preparing the biliverdin-Mn-ALCNTDSPL conjugate from the biliverdin-Mn metal complex and ALCNTDSPL according to the chemical synthesis method. The conjugate was loaded into PLGA particles to prepare a conjugate emulsion. According to a standard tumor mouse modeling mode, a C57BL/6 mouse bladder cancer MB49 in-situ model and an MBT-2 in-situ model are constructed, the mice are raised in an SPF (specific pathogen free) environment, the growth condition of the tumor is observed along with time, and when the tumor volume grows to be 80-100 mm on average³After left and right, relevant experiments were performed. And (3) injecting the conjugate emulsion preparation intravenously, and after 6 hours, placing the mouse under a photoacoustic imager and a nuclear magnetic resonance imager to detect the photoacoustic signal and the nuclear magnetic resonance signal intensity of the tumor position. Fig. 12 is a statistical plot of the results of the emulsion form of the conjugate described in example 12 for mouse bladder cancer tumor imaging, demonstrating the cancer diagnostic ability of the conjugate.

Example 13

Biliverdin molecule and EQLESIINFEKLTE were subjected to the above chemical synthesis to obtain biliverdin-EQLESIINFEKLTE conjugate. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. Establishing BALB/C cervical carcinoma U14 mouse, and administering via abdominal cavity at a concentration of 5mgkg^-1. At

days

2, 4, and 7 after administration, detection was performed by immunofluorescence staining and flow cytometryExploration of CD4 in spleen and draining lymph nodes of mice⁺T and CD8⁺Content of T cells. FIG. 13 is a graph of CD4 in mouse spleen and draining lymph nodes with the conjugate described in example 13⁺T and CD8T cells, showing that the conjugate has immune effect and 'photothermal immune' effect.

Example 14

Mixing biliverdin with excessive amount⁹⁹mTc co-incubation to obtain radioactive labeled biliverdin, and adding biliverdin⁹⁹The biliverdin-one is prepared by mTc and ISQAVHAAHAEINEAGR according to the chemical synthesis method⁹⁹mTc-ISQAVHAAHAEINEAGR conjugates. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of DMSO solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. The conjugate was injected intravenously into tumor model mice (BALB/C, mouse mammary tumor cells C127, initial tumor volume of about 100 mm)³) And monitoring the enrichment condition of the tumor position by a single photon emission Computed Tomography (CT) instrument. As a result, at 4h after administration, the conjugate was found to be most clearly imaged at the tumor site, and the accumulation amount reached the highest value, providing a window for tumor treatment. Under the time window, the tumor position is irradiated by laser (the laser wavelength is 730nm, and the power is 0.2W/cm)²) The tumor site is monitored for temperature changes using a near infrared imaging device. Method for staining CD8 in mouse tumor by adopting fluorescence immunostaining⁺T cells and CD8⁺Expression of CD107 molecules on the surface of T cells was monitored to assess immune effects. FIG. 14 is a radionuclide imaging of the conjugate described in example 14 (a), tumor hyperthermia in situ (b), and CD8 in mouse tumors⁺T cells (c) and CD8⁺The expression (d) condition of the CD107 molecule on the surface of T cells proves that the conjugate can realize cancer diagnosis and 'photothermal immunity' tumor treatment.

Example 15

The biliverdin molecule and PDRAHYNI are prepared into biliverdin-PDRAHYNI conjugate according to the chemical synthesis method. Weighing a certain mass of conjugate, dissolving in a trace amount of organic solution in advanceDirectly dissolving in PBS solution, stirring for dissolving, filtering for sterilization, and adjusting pH value to neutral. Establishing a mouse skin melanoma B16-F10C 57BL/6 mouse model and a mouse lung cancer Lewis C57BL/6 mouse model, and carrying out combination of photothermal immunity treatment and immunotherapy. The administration concentration of the conjugate was 3mgkg^-1The dosage of the immune drug interferon is 20U/mouse. The dosing window was the log phase of tumor growth with an initial volume of 300mm³The inhibitory behaviour on the tumour is monitored. FIG. 15 is a graph of the combined inhibitory effect of the conjugate of example 15 in combination with an immunological formulation against B16-F10 tumors and Lewis tumors, showing that the combination of "photothermal immunity" with immunotherapy significantly enhances the antitumor effect.

Example 16

Biliverdin molecule and MLLAVLYCL were subjected to the above chemical synthesis to obtain biliverdin-MLLAVLYCL conjugate. Weighing a certain mass of conjugate, dissolving the conjugate in a trace amount of organic solution in advance, directly dissolving the conjugate in a PBS solution, stirring the solution to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. Establishing a mouse skin melanoma B16-F10C 57BL/6 mouse model and a mouse lung cancer Lewis C57BL/6 mouse model, and carrying out the combination of 'photoimmunization' treatment and chemotherapy. The administration concentration of the conjugate was 3mgkg^-1The administration concentration of the chemotherapy drug adriamycin is 1mgkg^-1. The dosing window was tumor growth into log phase with an initial volume of 400mm³The inhibitory behaviour on the tumour is monitored. FIG. 16 is a graph showing the combined inhibitory effect of the conjugate of example 16 in combination with low doses of chemotherapeutic agents on B16-F10 tumors and Lewis tumors, showing that the combination of "photothermal immunity" and chemotherapy significantly enhances the anti-tumor effect (a) and is effective in reducing the effect on mouse body weight.

Example 17

Firstly, the biliverdin and excess terbium trichloride hexahydrate are chemically synthesized to obtain the biliverdin-Tb metal complex, and the biliverdin-Tb metal complex and VHFFKNIVTPRTP are prepared to obtain the biliverdin-Tb-VHFFKNIVTPRTP conjugate according to the chemical synthesis method. Weighing a certain mass of conjugate, directly dissolving the conjugate in a PBS solution, stirring to dissolve the conjugate, filtering and sterilizing the solution, and adjusting the pH value to be neutral. Establishing mouse skinMelanoma B16-F10C 57BL/6 mouse model is administrated by intravenous injection at intervals of 5 times, and the administration dose is 2mgkg^-1. After 30 days, mouse tissues were taken, and the major organ index was measured to evaluate the biosafety of the conjugates. FIG. 17 is a graph of the effect of the conjugate of example 17 on vital organs, showing that there is no severe damage to vital organs.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

<110> institute of Process engineering of Chinese academy of sciences

<120> immune active peptide-biliverdin conjugate, preparation method and application thereof

<130> CP2020028

<141> 2020-05-12

<160> 36

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Unknown (Unknown)

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<211> 17

<212> PRT

<213> Unknown (Unknown)

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Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly

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Arg

<210> 4

<211> 8

<212> PRT

<213> Unknown (Unknown)

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Pro Asp Arg Ala His Tyr Asn Ile

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<211> 8

<212> PRT

<213> Unknown (Unknown)

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Thr Leu Gly Ile Val Cys Pro Ile

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<210> 6

<211> 9

<212> PRT

<213> Unknown (Unknown)

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Arg Ala His Tyr Asn Ile Val Thr Phe

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<211> 10

<212> PRT

<213> Unknown (Unknown)

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Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr

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<212> PRT

<213> Unknown (Unknown)

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<211> 12

<212> PRT

<213> Unknown (Unknown)

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<212> PRT

<213> Unknown (Unknown)

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<212> PRT

<213> Unknown (Unknown)

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His Val Ile His Glu Gly Thr Val Val Ile

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<211> 10

<212> PRT

<213> Unknown (Unknown)

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His Val Val His Glu Gly Thr Val Val Ile

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<213> Unknown (Unknown)

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Lys Val Pro Arg Asn Gln Asp Trp Leu

1 5

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<211> 9

<212> PRT

<213> Unknown (Unknown)

<400> 14

Phe Leu Trp Gly Pro Arg Ala Leu Val

1 5

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<211> 9

<212> PRT

<213> Unknown (Unknown)

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Tyr Leu Glu Pro Gly Pro Val Thr Ala

1 5

<210> 16

<211> 9

<212> PRT

<213> Unknown (Unknown)

<400> 16

Ile Met Asp Gln Val Pro Phe Ser Val

1 5

<210> 17

<211> 9

<212> PRT

<213> Unknown (Unknown)

<400> 17

Ile Met Asp Gln Val Pro Phe Ser Val

1 5

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<212> PRT

<213> Unknown (Unknown)

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Tyr Met Asp Gly Thr Met Ser Gln Val

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<213> Unknown (Unknown)

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Thr Lys Pro Arg

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Gly Gln Pro Arg

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<213> Unknown (Unknown)

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Cys Ala Pro Glu

1

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Arg Lys Glu Val Tyr

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Arg Lys Asp Val Tyr

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<213> Unknown (Unknown)

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Leu Val Val Thr Pro Trp

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Phe Leu Gly Phe Pro Thr

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<213> Unknown (Unknown)

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Pro Asp Arg Ala His Tyr Asn Ile

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<212> PRT

<213> Unknown (Unknown)

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Phe Lys Phe Glu Phe Lys Phe Glu

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<212> PRT

<213> Unknown (Unknown)

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Ala Leu Cys Asn Thr Asp Ser Pro Leu

1 5

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<212> PRT

<213> Unknown (Unknown)

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Lys Ile Phe Gly Ser Leu Ala Phe Leu

1 5

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<211> 10

<212> PRT

<213> Unknown (Unknown)

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Lys Thr Lys Cys Lys Phe Leu Lys Lys Cys

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<212> PRT

<213> Unknown (Unknown)

<400> 31

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<211> 13

<212> PRT

<213> Unknown (Unknown)

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<211> 13

<212> PRT

<213> Unknown (Unknown)

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His Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe

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<211> 13

<212> PRT

<213> Unknown (Unknown)

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<213> Unknown (Unknown)

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20

Claims

1. A class of immunologically active peptide-biliverdin conjugates, or salts thereof that do not affect their pharmaceutical function, characterized in that said conjugates follow the structure of formula i or formula ii or formula iii;

(iii)

wherein

M is selected from the following non-metal atoms or ions of the following non-metal elements: H. si, P, or an ion selected from the following metal atoms or metal elements: mg, Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Y, Ru, Rh, Pd, In, Sn, Pt, Au, Eu, Gd, Tb, Dy, Er, Yb, Lu, Tc, Tl, and radioactive isotopes thereof, and non-radioactive isotopes thereof; the number of M is 1-4;

X₁: ovalbumin peptide: SIINFEKL, EQLESIINFEKLTE, ISQACVHAAHAEINEAGR;

X₂: HPV16E7 peptide: PDRAHYNI, TLGIVCPI, RAHYNIVTF, YMLDLQPETT, GQADPALALITTF;

X₃：NYSKPTDRQYHF、KHAHHTHNLRLP；

X₄：HVIHEGTVVI、HVVHEGTVVI

X₅：KVPRNQDWL、FLWGPRALV

X₆：YLEPGPVTA、IMDQVPFSV

X₇：MLLAVLYCL、YMDGTMSQV

X₈：TKPR

X₉：GQPR

X₁₀：CAPE

X₁₁：RKEVY

X₁₂：RKDVY

X₁₃：LVVTPW

X₁₄：FLGFPT

X₁₅：PDRAHYNI

X₁₆：FKFEFKFE

X₁₇：ALCNTDSPL

X₁₈：KIFGSLAFL

X₁₉：KTKCKFLKKC

X₂₀：QQKFQFQFEQQ

X₂₁：PLYKKIIKKLLES

X₂₂：HSLGKWLGHPDKF

X₂₃：VHFFKNIVTPRTP

X₂₄：EIIVTHFPFDEQNCSMK

X₂₅：(SNTSESF)₂KFRVTQ-LAPKQIKE-NH₂。

2. the conjugate of claim 1, wherein R is₁And R₂The same or different.

3. The conjugate of claim 1, wherein R is₁And R₂Is any of the above sequences, and can also be a peptide or protein containing any of the above sequences, or a derivative of any of the above sequences;

4. The conjugate of any one of claims 1 to 3, which relates to a molecular conjugate and to formulations or dosage forms based on, derived from, or derived from such a molecular conjugate:

5. A process for the preparation of a conjugate according to any one of claims 1 to 4, comprising the steps of:

(1) m is H:

the concentration of the biliverdin is 0.1-500 mM;

the concentration of EDC & HCl is 0.1-1000 mM;

the concentration of the NHS is 0.1-1000 mM;

the mass concentration ratio of the biliverdin to the EDC & HCl to the NHS is 1:1: 0.5-1: 20: 20;

b. stirring the mixture obtained in the step (a) at room temperature in a dark place, wherein the reaction time is 12-48 h;

d. adding anhydrous DMF into the precipitate obtained in the step (c), wherein the mass ratio of the precipitate to the DMF is 1: 100;

the concentration of the immunoactive peptide is 0.01-2000 mM;

the concentration of the anhydrous triethylamine is 0.01-4000 mM;

the stirring time is 4-96 hours;

f. adjusting the pH value of the mixed solution obtained in the step (e) to 3.5-7.5;

the pH value is adjusted by adding an alkaline substance or an acidic substance or by an aqueous solution dialysis method;

h. recrystallizing the material from step (g) to obtain a pure molecular conjugate;

(2) m is a metal atom or ion other than H:

i. dissolving biliverdin and excessive metal acetate in methanol, wherein the mass concentration ratio of biliverdin to metal salt is 1: 1-1: 1000;

j. (ii) stirring the methanol solution of step (i) at a temperature in the range of 20 ℃ to 60 ℃ for 4 hours;

m, according to the steps a-h, synthesizing the biliverdin-metal complex-immunoactive peptide conjugate;

(3) m is a non-metal atom or ion other than H:

n, dissolving biliverdin and non-metal halide or acyl chloride salt in an organic solvent pyridine or DMF, wherein the mass concentration ratio of the biliverdin to the non-metal halide or acyl chloride salt is 1: 1-1: 100;

o, stirring the mixed solution obtained in the step (14) at a certain temperature in a dark place for reaction, wherein the temperature range is 20-100 ℃; the reaction time is 2-8 h;

6. Use of a conjugate according to any one of claims 1 to 4 for the preparation of a "photothermo-immune" anti-tumour preparation.

7. The use of claim 6, wherein said formulation simultaneously effects tumor ablation, immunomodulation and tumor inflammatory microenvironment modulation.

8. Use of a conjugate according to any one of claims 1 to 4 for the preparation of a nuclear magnetic resonance imaging agent for tumours, M being selected from the group consisting of the following atoms or ions: one or more of Mn, Fe, Cu, Eu, Gd and Dy.

9. Use of a conjugate according to any one of claims 1 to 4 for the preparation of a radionuclide imaging agent for tumors, M being selected from the group consisting of the following atoms or ions:^64,67Cu、⁹⁹mTc、¹⁹⁵Pt、^67,68Gd、²⁰¹Tl、⁶⁰Co、¹¹¹In、⁵¹one or more of Cr.

10. Use of a conjugate according to any one of claims 1 to 4 for the preparation of a photoacoustic imaging agent for tumors, M being selected from the group consisting of the following atoms or ions: H. one or more of Zn.