Tumor immunotherapy method capable of amplifying antigen on the basis of proximity labeling reaction

Abstract

Provided is a tumor immunotherapy method capable of amplifying an antigen on the basis of a proximity labeling reaction. The present disclosure can amplify a recognition signal of tumor cells, so as to facilitate the recognition of same by related active molecules or cells, thereby specifically killing the tumor cells. The present disclosure may be applied to in-vivo antigen labeling and achieve controlled covalent labeling and catalytic amplification of antigens.

Description

Tumor immunotherapy method for amplifying antigens based on proximity labeling reaction

[0001] Cross-reference to Related Applications

[0002] This application claims the benefit of and priority to Chinese Patent Application No. 202411315005.3, titled “Tumor immunotherapy method for amplifying antigens based on proximity labeling reaction,” filed September 19, 2024, including any sequence listing and drawings, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biotechnology, and specifically to a tumor immunotherapy method for amplifying antigens based on proximity labeling reaction. BACKGROUND

[0004] Current immunotherapies targeting tumor antigens have achieved remarkable success in treating hematological cancers, such as monoclonal antibody drugs and chimeric antigen T cell therapy (CAR-T). These therapies have shown significant efficacy in treating certain types of hematological cancers, such as leukemia and lymphoma. However, they still face many challenges in treating solid tumors.

[0005] One of the key obstacles to achieving effective anti-tumor immunotherapy lies in the heterogeneity of tumor antigen expression. The cell heterogeneity within a solid tumor is high, and even within the same tumor, different regions of cells can express different antigens. This heterogeneity makes it difficult for single antigen targeting therapy to comprehensively cover all tumor cells, resulting in poor treatment efficacy.

[0006] In addition, some tumor cells have insufficient number of surface antigens, and more critically, insufficient aggregation of antigens, making it difficult to elicit a meaningful immune response. It is difficult to find tumor antigens that are highly specific and not expressed or lowly expressed in normal cells. This lack of specific antigens increases the difficulty of treatment and can cause potential off-target effects (attacks on normal cells), and antigen deficiency easily triggers immune escape phenomena. These are challenges faced by both antibody-dependent cellular cytotoxicity and cytotoxic lymphocyte activation.

[0007] To address the issue of antigen heterogeneity, one potential approach is to design optimized CAR-T cells that can recognize tumor cells with low expression of antigens, but this requires complex protein engineering design for each CAR structure, and often has limited efficacy.

[0008] In addition, some techniques have recently been developed, such as increasing the availability of surface antigens by inhibiting endocytosis, intratumor injection of amphiphilic ligands that insert into the cell membrane, or transduction of tumor cells with oncolytic viruses to induce expression of exogenous antigens. However, these methods can only moderately increase the level of antigens at present, and cannot control their spatial distribution, so the therapeutic effect is not satisfactory.

[0009] In view of the above, there is an urgent need in the art to develop an effective treatment method for tumors with low antigen levels (e.g., below the threshold value). SUMMARY

[0010] The purpose of the present disclosure is to provide a method for antigen amplification and efficient immunotherapy for target cells with low antigen levels (e.g., below the threshold value).

[0011] In a first aspect of the present disclosure, an active ingredient combination is provided, which comprises:

[0012] (a) an artificial antigen carrying a reactive group;

[0013] (b) a catalyst; and

[0014] (c) an immunotherapeutic agent;

[0015] wherein, under the catalysis of the catalyst, the artificial antigen is covalently linked to a reactive group on the cell surface through the reactive group carried by the artificial antigen, thereby labeling the artificial antigen on the cell surface to obtain a cell with the cell surface labeled with the artificial antigen, and then the immunotherapeutic agent is used to specifically target the artificial antigen, thereby triggering an immune response.

[0016] In some embodiments, the artificial antigen carrying a reactive group is selected from the group consisting of an artificial antigen carrying a precursor group of an active intermediate, an artificial antigen carrying a reaction partner group of an active intermediate.

[0017] In some embodiments, the reactive group on the cell surface is selected from the group consisting of a reaction partner group of an active intermediate, a precursor group of an active intermediate.

[0018] In some embodiments, the reactive group on the cell surface is an amino acid in a cell surface protein.

[0019] In some embodiments, the amino acid includes tyrosine, histidine.

[0020] In some embodiments, the active ingredient combination comprises:

[0021] (a) an artificial antigen carrying a reactive group;

[0022] (b) a biological enzyme; and

[0023] (c) an immunotherapeutic agent;

[0024] In some embodiments, the active ingredient combination comprises:

[0025] (a) an artificial antigen carrying a reactive group;

[0026] (b) a light / sound sensitizer targeting a cell surface antigen; and

[0027] (c) an immunotherapeutic agent.

[0028] In some embodiments, the active ingredient combination comprises:

[0029] (a) an artificial antigen carrying a precursor group of an active intermediate;

[0030] (b) a light sensitizer targeting a cell surface antigen; and

[0031] (c) an immunotherapeutic agent;

[0032] wherein, under catalysis of the light sensitizer, the precursor group of the active intermediate is converted into the active intermediate, the artificial antigen is covalently linked to a reactive partner group of the active intermediate on the cell surface via the carried active intermediate, thereby labeling the artificial antigen on the cell surface to obtain a cell surface artificial antigen-labeled cell, and the immunotherapeutic agent is then used to specifically target the artificial antigen, thereby triggering an immune response.

[0033] In some embodiments, the active ingredient combination comprises:

[0034] (a) an artificial antigen carrying a reactive partner group of an active intermediate;

[0035] (b) a sound sensitizer targeting a cell surface antigen; and

[0036] (c) an immunotherapeutic agent;

[0037] wherein, under catalysis of the sound sensitizer, a precursor group of the active intermediate on the cell surface is converted into the active intermediate, the artificial antigen is covalently linked to the active intermediate via the carried reactive partner group of the active intermediate, thereby labeling the artificial antigen on the cell surface to obtain a cell surface artificial antigen-labeled cell, and the immunotherapeutic agent is then used to specifically target the artificial antigen, thereby triggering an immune response.

[0038] In some embodiments, the reactive group is selected from the group consisting of a phenolic compound, an amine compound, a hydrazide compound, a quinone compound, an aryl-ureido compound, an aryl-azide compound, a bis-aziridine compound, or a transpeptidation substrate.

[0039] In some embodiments, the artificial antigen is a hapten.

[0040] In some embodiments, the hapten is selected from the group consisting of fluorescein (FITC), biotin, dinitro (DNP), or Galα1-3Galβ1-4GlcNAc-R (αGal).

[0041] In some embodiments, the hapten carrying the phenolic compound is a phenolic-labeled probe substrate.

[0042] In some embodiments, the phenolic-labeled probe substrate is selected from the group consisting of fluorescein (FITC)-phenol, biotin-phenol, biotin-xx-phenol, alkyne phenol (AP), dinitro (DNP)-phenol, Galα1-3Galβ1-4GlcNAc-R (αGal)-phenol, or a bio-macromolecule containing a phenolic group.

[0043] In some embodiments, the hapten carrying the amine compound is an amine-labeled probe substrate.

[0044] In some embodiments, the amine-labeled probe substrate includes fluorescein (FITC)-amine, biotin-amine.

[0045] In some embodiments, the catalyst is selected from the group consisting of a photo / sonosensitizer, or a biological enzyme.

[0046] In some embodiments, the photosensitizer is selected from the group consisting of a porphyrin photosensitizer, a heavy metal iridium ruthenium tin osmium complex photosensitizer, an acridine orange photosensitizer, a fluorescein photosensitizer, an eosin photosensitizer, an oxazine photosensitizer, a methyl blue photosensitizer, a phycocyanin photosensitizer, a rhodamine photosensitizer.

[0047] In some embodiments, the porphyrin photo / sonosensitizer is a photo / sonosensitizer assembled from porphyrin and zirconium atom.

[0048] In some embodiments, the heavy metal iridium ruthenium tin osmium complex photosensitizer is a heavy metal iridium photosensitizer.

[0049] In some embodiments, the biological enzyme is selected from the group consisting of a peroxidase, a biotin ligase, a glycosyltransferase, a tyrosinase, a ubiquitin ligase, a transpeptidase, a protein photo-sonosensitizer.

[0050] In some embodiments, the sonosensitizer is selected from the group consisting of porphyrin-based sonosensitizer, phthalocyanine-based sonosensitizer, rhodamine-based sonosensitizer, inorganic nanomaterial-based sonosensitizer, and sonosensitizer that generates reactive oxygen species through sonoporation effect.

[0051] In some embodiments, the immunotherapeutic agent comprises one or more components selected from the group consisting of monospecific antibody, bispecific antibody, multispecific antibody, monoclonal antibody, or polyclonal antibody.

[0052] In some embodiments, the monospecific antibody is an antibody that specifically recognizes FITC.

[0053] In some embodiments, the bispecific antibody comprises:

[0054] (d1) a first antibody element that specifically recognizes a recognition sequence of an artificial antigen; and

[0055] (d2) a second antibody element that specifically recognizes a recognition sequence of an immune cell.

[0056] In some embodiments, the bispecific antibody is a BiTE.

[0057] In some embodiments, the BiTE comprises:

[0058] (d1) a first antibody element that specifically recognizes a single-chain antibody (ScFV) of FITC; and

[0059] (d2) a second antibody element that specifically recognizes a single-chain antibody of CD3 of T cell.

[0060] In some embodiments, the multispecific antibody comprises:

[0061] (e1) a first antibody element that specifically recognizes a single-chain antibody of FITC; and

[0062] (e2) two or more antibody elements that specifically recognize two or more other single-chain antibodies.

[0063] In some embodiments, the monoclonal antibody is a FITC monoclonal antibody.

[0064] In some embodiments, the polyclonal antibody comprises a FITC monoclonal antibody.

[0065] In some embodiments, the ratio of the number of the artificial antigens (N1) to the number of the cell surface antigens (N0) (N1 / N0) on the cell surface is ≥ 5, preferably ≥ 10, more preferably ≥ 50, most preferably ≥ 100 or more.

[0066] In some embodiments, the density of the artificial antigen in the cell surface marker-bearing cell is ≥ 60% per cell, preferably ≥ 70% per cell, more preferably ≥ 80% per cell, most preferably 60-90% per cell.

[0067] In some embodiments, the photo / sonosensitizer is a photo / sonosensitizer targeting a cell surface antigen.

[0068] In some embodiments, the photo / sonosensitizer targeting a cell surface antigen has a targeting element.

[0069] In some embodiments, the targeting element specifically binds to a cell surface associated antigen or marker.

[0070] In some embodiments, the cell surface associated antigen or marker is a tumor specific antigen (TSA), a tumor associated antigen (TAA), or a marker on the surface of a tumor cell.

[0071] In some embodiments, the tumor specific antigen, tumor associated antigen, or marker is selected from the group consisting of folate receptor, hyaluronan receptor, HER2, mesothelin, or a combination thereof.

[0072] In some embodiments, the hyaluronan receptor is CD44.

[0073] In some embodiments, the photo / sonosensitizer targeting a cell surface antigen has a structure shown in Formula I: (Z1-L)n-Z2(I)

[0074] In the formula,

[0075] Z1 is a targeting element;

[0076] L is nothing, a covalent bond, a non-covalent bond, physical adsorption;

[0077] Z2 is a photo / sonosensitizer;

[0078] n is an integer ≥ 1.

[0079] In some embodiments, Z1 is selected from the group consisting of folate, hyaluronan, HER2 antibody, mesothelin antibody, or a combination thereof.

[0080] In some embodiments, L is selected from the group consisting of a covalent bond, a non-covalent bond, physical adsorption, or a combination thereof.

[0081] In some embodiments, the photo / sonosensitizer is a photo / sonosensitizer targeting a cell surface antigen.

[0082] In some embodiments, the biological enzyme is a biological enzyme targeting a cell surface antigen.

[0083] In some embodiments, the cell surface targeting bio-enzyme has a targeting element.

[0084] In some embodiments, the targeting element is selected from the group consisting of a cholesterol, or a HER2 antibody.

[0085] In some embodiments, the targeting element specifically binds to a cell surface associated antigen or marker.

[0086] In some embodiments, the cell surface associated antigen or marker is selected from the group consisting of a phospholipid membrane, HER2, or a combination thereof.

[0087] In some embodiments, the covalent attachment is a proximity labeling reaction.

[0088] In a second aspect of the present disclosure, there is provided a kit comprising the active ingredient combination of the first aspect of the present disclosure.

[0089] In a third aspect of the present disclosure, there is provided use of the active ingredient combination of the first aspect of the present disclosure, for the manufacture of a medicament or a composition for:

[0090] (i) antigen amplification of a predetermined antigen on a target cell surface via a proximity labeling reaction; and

[0091] (ii) immunotherapy of a target cell.

[0092] In some embodiments, the target cell comprises a cell selected from the group consisting of a tumor cell, or a senescent cell.

[0093] In some embodiments, the tumor cell comprises a skin cancer cell, a melanoma cell, a breast cancer cell, a lung cancer cell, a colorectal cancer cell, an ovarian cancer cell, an ovarian adenocarcinoma cell, a gastric cancer cell, a lymphoma cell, a leukemia cell, a uterine cancer cell, an endometrial cancer cell, a liver cancer cell, a pancreatic cancer cell, a pancreatic ductal adenocarcinoma cell, a thyroid cancer cell, a thymus cancer cell, a kidney cancer cell, a prostate cancer cell, a prostate adenocarcinoma cell.

[0094] In some embodiments, the tumor cell is selected from the group consisting of a M109 melanoma skin tumor cell, a 4T1 breast tumor cell, a M109 lung cancer cell, a CT26 colon cancer cell, a SK-OV-3 human ovarian adenocarcinoma cell, a gastric cancer cell, a colon cancer cell, or a combination thereof.

[0095] In some embodiments, the predetermined antigen is a cell surface associated antigen or marker.

[0096] In some embodiments, the cell surface associated antigen or marker is a tumor cell surface associated antigen or marker.

[0097] In some embodiments, the tumor cell surface associated antigen or marker is selected from the group consisting of folate, hyaluronic acid, HER2, mesothelin, or a combination thereof.

[0098] In some embodiments, the immunotherapy comprises immunokilling.

[0099] In some embodiments, the immunokilling is achieved by significantly activating T cells.

[0100] In some embodiments, the "significantly activating T cells" means that the T cell activation marker is significantly increased and / or the cytokine secretion is significantly increased.

[0101] In some embodiments, the T cell activation marker is CD69.

[0102] In some embodiments, the "significantly increased" means that the ratio of the percentage of CD69 positive T cells Q1 to the reference value / standard value Q0 (Q1 / Q0) is ≥200%, preferably ≥300%.

[0103] In some embodiments, the cytokine comprises TNF-α, IFN-γ.

[0104] In some embodiments, the "significantly increased" means that the ratio of the level / concentration / amount of cytokine P1 to the reference value / standard value P0 (P1 / P0) is ≥200%, preferably ≥300%.

[0105] In some embodiments, the reference value / standard value is the level / concentration / amount measured when no or part of the preparation or composition is used.

[0106] In a fourth aspect, the present disclosure provides a method for artificially labeling a target cell surface in vivo or in vitro, comprising the steps of:

[0107] (a) providing a cell, and an artificial antigen carrying a reactive group and a catalyst in the active ingredient combination of the first aspect of the present disclosure; and

[0108] (b) artificially labeling the surface of the cell, wherein the artificial antigen is covalently linked to the reactive group on the surface of the cell by a covalent linkage reaction between the reactive group carried by the artificial antigen and the reactive group on the surface of the cell under the catalysis of the catalyst, thereby labeling the artificial antigen on the surface of the cell to obtain a cell with an artificial antigen labeled on the cell surface.

[0109] In some embodiments, the catalyst is selected from the group consisting of a light / sound sensitizer targeting a cell surface antigen, or a biological enzyme.

[0110] In some embodiments, before step (a), further comprising a method for preparing a catalyst, which is a light / sound sensitizer targeting a cell surface antigen, the method comprising the following steps:

[0111] (1) mixing a light / sound sensitizer and a transition metal element in an organic solvent environment to obtain a light / sound sensitizer dispersion;

[0112] (2) adding a targeting element to the light / sound sensitizer dispersion and performing a cross-linking reaction, thereby obtaining a light / sound sensitizer targeting a cell surface antigen.

[0113] In some embodiments, the targeting element specifically binds to a cell surface associated antigen or marker.

[0114] In some embodiments, the targeting element is selected from the group consisting of polyethylene glycol-folic acid, hyaluronic acid, or a HER2 antibody.

[0115] In some embodiments, in step (2), a modifying element and a targeting element are added to the light / sound sensitizer dispersion, and a cross-linking reaction is performed, thereby obtaining a light / sound sensitizer targeting a cell surface antigen;

[0116] wherein the modifying element modifies the light / sound sensitizer dispersion, and the targeting element is obtained by cross-linking reaction with the modifying element, thereby obtaining a light / sound sensitizer targeting a cell surface antigen.

[0117] In some embodiments, the modifying element is selected from the group consisting of phospho-polyethylene glycol-carboxyl (PO3-PEG-COOH), phospho-methoxy polyethylene glycol (PO3-mPEG), polydopamine (pDA), or a combination thereof.

[0118] In some embodiments, in step (2), the cross-linking reaction is performed in the presence of a zero-length cross-linking agent.

[0119] In some embodiments, the zero-length cross-linking agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide.

[0120] In some embodiments, before step (a), further comprising a method for preparing a catalyst, which is a biological enzyme, the method comprising the following steps:

[0121] (1) protein expression and purification to obtain a recombinant biological enzyme modified with a targeting element; or

[0122] (2) transfecting the cell with a recombinant plasmid expressing a bioenzyme to allow the cell to express the bioenzyme.

[0123] In some embodiments, the cell is a living cell.

[0124] In some embodiments, the living cell is a cell with a targetable site.

[0125] In some embodiments, the cell with a targetable site is a tumor cell.

[0126] In some embodiments, the tumor cell is selected from the group consisting of CD44 overexpressing mouse breast cancer 4T1 cells, folate receptor overexpressing mouse melanoma M109 cells, mouse lung cancer cell line M109 cells, HER2 overexpressing mouse colon cancer cells CT26 cells, or a combination thereof.

[0127] In some embodiments, the artificial antigen carrying a reactive group is a phenolic group-labeled probe substrate.

[0128] In some embodiments, the phenolic group-labeled probe substrate is selected from one or more components consisting of FITC-phenol, biotin-phenol, biotin-xx-phenol, alkynyl phenol, dinitrophenol, Galα1-3Galβ1-4GlcNAc-R (αGal)-phenol, or a bio-macromolecule containing a phenolic group.

[0129] In some embodiments, the hapten carrying the amine group compound is an amine group-labeled probe substrate.

[0130] In some embodiments, the amine group-labeled probe substrate includes fluorescein (FITC)-amine, biotin-amine.

[0131] In some embodiments, the catalyst is selected from the group consisting of a cell surface antigen-targeting photo / sonosensitizer, or a bioenzyme.

[0132] In some embodiments, the cell surface antigen-targeting photo / sonosensitizer is selected from the group consisting of a hyaluronic acid-modified photo / sonosensitizer, a folate-modified photo / sonosensitizer, or an antibody-targeting modified photo / sonosensitizer.

[0133] In some embodiments, the antibody-targeting modified photo / sonosensitizer is a HER2-targeting antibody.

[0134] In some embodiments, the photo / sonosensitizer is a far-red light-excited photo / sonosensitizer.

[0135] In some embodiments, the bioenzyme is selected from the group consisting of a cholesterol-modified horseradish peroxidase, a HER2 antibody-fused β-galactosidase, or a tyrosinase.

[0136] In some embodiments, the method is non-diagnostic and non-therapeutic.

[0137] In a fifth aspect of the present disclosure, there is provided a therapeutic system or device, comprising:

[0138] (z1) an antigen amplification module configured to administer to a subject the artificial antigen carrying the reactive group and the catalyst in the active ingredient combination of the first aspect of the present disclosure, so that in the subject's body, under the catalysis of the catalyst, the artificial antigen is covalently linked to the reactive group on the cell surface via the carried reactive group, thereby labeling the artificial antigen on the cell surface; and

[0139] (z2) optionally, an immunotherapy module configured to administer to the subject the immunotherapeutic agent in the active ingredient combination of the first aspect of the present disclosure, which is targeted to the artificial antigen.

[0140] In some embodiments, the therapeutic system or device further comprises:

[0141] (z3a) a light source module configured to irradiate a predetermined site of the subject to catalyze the covalent linkage of the reactive group carried by the artificial antigen to the reactive group on the cell surface, wherein the light source module provides a red light source.

[0142] In some embodiments, the therapeutic system or device further comprises:

[0143] (z3b) an acoustic source module configured to ultrasonically irradiate a predetermined site of the subject to catalyze the covalent linkage of the reactive group carried by the artificial antigen to the reactive group on the cell surface, wherein the acoustic source module provides an ultrasonic wave.

[0144] In some embodiments, the subject is a mammal.

[0145] In some embodiments, the subject is selected from the group consisting of a mouse, or a human.

[0146] In some embodiments, the reactive group on the cell surface is an amino acid in a cell surface protein.

[0147] In some embodiments, the amino acid comprises: tyrosine, histidine.

[0148] In some embodiments, the therapeutic system or device further comprises:

[0149] (z4) an artificial antigen detection module configured to detect the density or amplification level of the artificial antigen.

[0150] In some embodiments, the artificial antigen detection module detects the density of the artificial antigen by detecting a detectable signal emitted by the artificial antigen.

[0151] In some embodiments, the detectable signal comprises fluorescence, chemiluminescence, or a combination thereof.

[0152] In some embodiments, the red light has a wavelength of > 600 nm, preferably > 660 nm, more preferably > 680 nm, most preferably 682 nm.

[0153] In some embodiments, the light source module provides a deep red light source.

[0154] In some embodiments, the light source module is a red light emitter.

[0155] In some embodiments, the red light emitter is selected from the group consisting of a red LED light source, a 682 nm laser.

[0156] In some embodiments, the sound source module is an ultrasonic probe.

[0157] In some embodiments, the ultrasonic probe has a power of 0.25-6 W / cm 2 , preferably 0.5-3 W / cm 2 .

[0158] In some embodiments, the predetermined site is selected from the group consisting of a lesion site, a region or tissue enriched with a catalyst, a region or tissue enriched with an artificial antigen carrying a highly reactive group.

[0159] In some embodiments, the artificial antigen carrying a reactive group is selected from the group consisting of a phenolic group-labeled probe substrate, an amine group-labeled probe substrate.

[0160] In some embodiments, the catalyst is a light / sound sensitizer targeting a cell surface antigen.

[0161] In some embodiments, the phenolic group-labeled probe substrate is administered by intravenous injection or direct intratumoral injection.

[0162] In some embodiments, the phenolic group-labeled probe substrate is administered in a volume of 25-200 μL, preferably 50-100 μL.

[0163] In some embodiments, the phenolic group-labeled probe substrate is administered at a concentration of 10-200 μM, preferably 20-100 μM.

[0164] In some embodiments, the amine-based labeling probe substrate is administered in a volume of 25-200 μL, preferably 50-100 μL.

[0165] In some embodiments, the amine-based labeling probe substrate is administered at a concentration of 0.25-10 mM, preferably 0.5-5 mM.

[0166] In some embodiments, the photo / sonosensitizer targeting a cell surface antigen is administered by intravenous injection or direct intratumoral injection.

[0167] In some embodiments, the photo / sonosensitizer targeting a cell surface antigen is administered in a volume of 45-220 μL, preferably 90-110 μL.

[0168] In some embodiments, the photo / sonosensitizer targeting a cell surface antigen is administered at a concentration of 0.05-0.1 μg / μL, preferably 0.5-2 μg / μL.

[0169] In some embodiments, the bispecific antibody is a BiTE.

[0170] In some embodiments, the BiTE is administered by intravenous injection.

[0171] In some embodiments, the BiTE is administered at a concentration of 0.1-2 μg / μL, preferably 0.2-1 μg / μL.

[0172] In some embodiments, the intravenous injection is tail vein injection.

[0173] In some embodiments, the "irradiation with a light source module" is irradiation of the lesion site of the subject with a red light source, thereby achieving antigen amplification.

[0174] In some embodiments, the irradiation is for a duration of 20-120 min, preferably 30 min.

[0175] In some embodiments, the irradiation is at a height of 3-20 cm, preferably 5-10 cm.

[0176] In some embodiments, the ultrasound is for a duration of 5-30 min, preferably 10 min.

[0177] In a sixth aspect of the present disclosure, a treatment method is provided, comprising the steps of:

[0178] (a) administering to a subject a preparation or composition, the preparation or composition performing antigen amplification on a predetermined antigen on the surface of a target cell in the subject by proximity labeling reaction, thereby labeling the surface of the target cell with artificial antigens; and

[0179] (b) immunotherapy with an immunotherapeutic agent against the artificial antigens.

[0180] In some embodiments, the preparation or composition comprises: artificial antigens carrying reactive groups; and a catalyst.

[0181] In some embodiments, the catalyst is selected from the group consisting of a photo / sonosensitizer, or a biological enzyme.

[0182] In some embodiments, the target cell is selected from the group consisting of a tumor cell, or a senescent cell.

[0183] In some embodiments, the predetermined antigen is a tumor cell surface associated antigen or marker.

[0184] In some embodiments, the tumor cell surface associated antigen or marker is a tumor cell surface associated antigen or marker.

[0185] In some embodiments, the immunotherapeutic agent against the artificial antigens is used for immunotherapy against tumors.

[0186] In some embodiments, the immunotherapeutic agent against the artificial antigens is one or more components selected from the group consisting of a monospecific antibody, a bispecific antibody, a multispecific antibody, a monoclonal antibody, or a polyclonal antibody.

[0187] In some embodiments, the monospecific antibody is an antibody that specifically recognizes FITC.

[0188] In some embodiments, the bispecific antibody is a BiTE.

[0189] In some embodiments, the BiTE comprises a first antibody element of a single-chain antibody that specifically recognizes FITC, and a second antibody element of a single-chain antibody that specifically recognizes CD3 of a T cell.

[0190] In some embodiments, the multispecific antibody comprises a first antibody element of a single-chain antibody that specifically recognizes FITC, and two or more antibody elements that specifically recognize two or more other single-chain antibodies.

[0191] In some embodiments, the monoclonal antibody is a FITC monoclonal antibody.

[0192] In some embodiments, the polyclonal antibody comprises a FITC monoclonal antibody.

[0193] It should be understood that, within the scope of the present disclosure, each of the technical features of the present disclosure described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to constitute a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0194] FIG. 1 is a schematic diagram of a preparation method of a target-modified photo / acoustic sensitizer.

[0195] FIG. 2A is a schematic diagram of a PCN-mediated in vivo protein labeling strategy to amplify and enhance tumor immunogenicity by hapten labeling according to the present disclosure.

[0196] FIG. 2B is a transmission electron microscope (TEM) image of a PCN photo / acoustic sensitizer constructed according to the present disclosure.

[0197] FIG. 2C is an EDX-mapping elemental analysis of a PCN photo / acoustic sensitizer.

[0198] FIG. 2D is a Western blotting (WB) image of PCN-mediated BSA biotin labeling under light excitation, and a WB image of PCN-mediated BSA biotinylation reaction under different inhibitors.

[0199] FIG. 2E is a schematic diagram of PCN-mediated BSA labeling amino acid sites and mass spectrometry data under light excitation.

[0200] FIG. 2F is a Western blotting (WB) image of PCN-mediated BSA biotin labeling under ultrasound excitation, and a WB image of PCN-mediated BSA biotinylation reaction under different inhibitors.

[0201] FIG. 2G is a schematic diagram of PCN-mediated BSA labeling amino acid sites and mass spectrometry data under ultrasound excitation.

[0202] FIG. 3A is a schematic diagram of functional modification of PCN targeted labeling according to the present disclosure.

[0203] FIG. 3B is an immunofluorescence image of functional modification of PCN targeted corresponding receptor cell labeling.

[0204] FIG. 3C is a WB image of PCN-mediated cell biotinylated protein labeling under light / acoustic excitation.

[0205] FIGS. 3IE-3G are statistical diagrams of cell biotinylated protein enrichment effect.

[0206] FIG. 4A is a schematic diagram of PCN catalyzed FITC substrate targeted labeling of tumor cells.

[0207] Figure 4B is a confocal image of PCN catalyzing FITC substrate to target label tumor cells.

[0208] Figure 4C is a statistic of the enrichment fold of hapten FITC on the surface of target cell membrane.

[0209] Figure 4D is a fluorescence image of functionalized PCN compared with unmodified PCN for cell targeting labeling.

[0210] Figure 4E is a schematic diagram of ultrasound catalyzing PCN to target label tumor cells.

[0211] Figure 4F is a confocal image of biocatalysts peroxidase (HRP), tyrosinase (BmTyr), glycosyltransferase (pGal), miniSOG enzyme, sortase and tin metal catalyst (pMAP) catalyzing substrate to label hapten FITC to tumor cells.

[0212] Figure 5A is a schematic diagram of macrophages phagocytosing labeled tumor cells.

[0213] Figure 5B is a confocal image of macrophages phagocytosing labeled tumor cells.

[0214] Figure 5C is a statistic of the phagocytosis effect of macrophages on labeled tumor cells.

[0215] Figure 5D is a schematic diagram of T cells killing labeled tumor cells.

[0216] Figure 5E is a statistic of cytokines TNF, IFN secretion during T cells killing labeled tumor cells.

[0217] Figure 5F is a statistic of LDH of T cells killing labeled tumor cells in vitro.

[0218] Figure 5H is a schematic diagram of T cells killing labeled patient-derived tumor samples in vitro.

[0219] Figure 5I is a statistic of apoptosis of T cells killing patient-derived tumor organoids in vitro.

[0220] Figure 5J is a statistic of T cells activated by PATCH-treated patient-derived tumor pieces.

[0221] Figures 5K and 5L are immunofluorescence and cytokine secretion statistics of T cells activated by PATCH-treated patient-derived tumor pieces.

[0222] Figure 6A is a schematic diagram and timeline of PATCH treatment of tumors in mice.

[0223] Figure 6B is a confocal image of PATCH-labeled FITC in tumor tissue of mice.

[0224] Figure 6C is a confocal image of the PATCH labeled FITC in mouse tumor tissue by i.v. (intravenous injection) / i.t. (intratumoral injection).

[0225] Figure 6D is a graph of tumor size over time in mouse tumor PATCH treatment.

[0226] Figure 6E is a photograph of mouse tumor after PATCH treatment.

[0227] Figure 6F is a graph of tumor size over time in mouse tumor PATCH treatment.

[0228] Figure 6G is a graph of apoptosis in mouse tumor after PATCH treatment.

[0229] Figure 6H is a schematic diagram of mouse PATCH treatment of tumor cells on both sides and a time axis.

[0230] Figure 61 is a graph of tumor size over time in mouse treatment side.

[0231] Figure 6J is a graph of tumor size over time in mouse non-treatment side.

[0232] Figure 6K is a graph of survival over time in mice.

[0233] Figure 7A is a schematic diagram of mouse tumor treatment with bispecific antibody and a time axis.

[0234] Figure 7B is a photograph of mouse tumor after PATCH treatment.

[0235] Figure 7C is a graph of tumor size over time in mouse tumor PATCH treatment.

[0236] Figure 7D is a summary graph of tumor size over time in mouse tumor PATCH treatment.

[0237] Figure 7E is a schematic diagram of mouse treatment of tumor cells on both sides with bispecific antibody and a time axis.

[0238] Figure 7F is a graph of tumor size over time in mouse treatment side.

[0239] Figure 7G is a graph of tumor size over time in mouse non-treatment side.

[0240] Figure 7H is a graph of survival over time in mice.

[0241] Figures 8A and 8B are schematic diagrams of ultrasound-activated PATCH treatment of tumors in mice and a time axis.

[0242] Figure 8C is a confocal image of ultrasound-activated PATCH labeled FITC in mouse tumor tissue.

[0243] FIG. 8D is a graph of tumor growth curve and survival curve of ultrasound-activated PATCH treatment of tumors in mice.

[0244] FIG. 8E is a timeline of ultrasound-activated PATCH treatment of tumors in PBMC humanized mice.

[0245] FIG. 8F is a graph of tumor growth curve of ultrasound-activated PATCH treatment of tumors in PBMC humanized mice.

[0246] FIG. 9A is a timeline of PATCH combined with monoclonal antibody treatment of tumors in mice.

[0247] FIG. 9B is a graph of tumor size over time of tumor PATCH combined with monoclonal antibody treatment in mice.

[0248] FIG. 9C is a graph of tumor size over time of tumor control group in mice. DETAILED DESCRIPTION

[0249] The inventors, through extensive and in-depth research, after solid and sufficient experiments, first used proximity labeling technology for solid tumor antigen amplification to enhance the effect of immunotherapy, forming the PATCH treatment strategy. This strategy uses a functional catalyst targeting tumor cell membranes, such as a light / sound sensitizer, which, when activated, such as by light, catalyzes the formation of reactive groups from hapten probes, such as catalyzing the formation of fluorescein-phenol from fluorescein-phenoxy radicals, thereby labeling the hapten probe to the tumor cells, enhancing the immunogenicity of the tumor cells, so that the labeled tumor cells are recognized by the immune system, achieving specific killing of the tumor. On this basis, the present disclosure is completed.

[0250] TERMINOLOGY

[0251] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0252] Unless otherwise indicated, percentages and parts are by weight.

[0253] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 30" includes all values between 29 and 31 and (e.g., 29.1, 29.2, 29.3, 29.4, etc.).

[0254] As used herein, the terms "immunotherapeutic agent", "therapeutic agent for immunotherapy" can be used interchangeably, and all refer to drugs that mimic or alter components of the immune system, such as cytokines, fusion proteins, monoclonal antibodies, etc., to improve the body's ability to fight disease. In the present disclosure, the immunotherapeutic agents used include, but are not limited to, PATCH, and antibodies that can target cell surface markers, bispecific antibodies, such as anti-FITC, BiTE, CAR-T, etc.

[0255] As used herein, the term "lesion" or "lesion site" refers to the part of the body where the lesion occurs. In the present disclosure, preferred lesion sites include, but are not limited to, the site where tumor cells are located, including the tumor and the surrounding area.

[0256] Active ingredient combination and antigen amplification method of the present disclosure

[0257] The present disclosure provides an active ingredient combination for antigen amplification of a predetermined antigen on the surface of a target cell by proximity labeling technology, and immunotherapy by an immunotherapeutic agent targeting the antigen.

[0258] Preferably, the active ingredient combination of the present disclosure is as described in the first aspect of the present disclosure.

[0259] In the present disclosure, in view of the problems of insufficient antigen quality or quantity in tumor treatment under prior art conditions, and the lack of cell surface proximity labeling tools in vivo, the present disclosure first develops a proximity labeling system that can be used in vivo animals, and a method for immunotherapy using the labeling system.

[0260] Specifically, taking tumor cells as an example, the present disclosure provides a strategy for significantly enhancing tumor antigenicity by extensively amplifying designed antigens on the surface of tumor cells in vivo, called PATCH. In this preferred example, the catalyst in the active ingredient combination is a functionalized photo / acoustic sensitizer, and the artificial antigen carrying a reactive group is a hapten probe, such as fluorescein-phenol (FITC-phenol), FITC-amine, etc.

[0261] Preferably, the active ingredient combination includes a functionalized photo / acoustic sensitizer (PCN) targeting tumor cell membranes, etc., and a hapten probe fluorescein-phenol (FITC-phenol) for photo-activated proximity labeling or FITC-amine (FITC-amine) for ultrasound-activated proximity labeling.

[0262] Preferably, the present disclosure performs antigen amplification by light-driven method.

[0263] Preferably, the light / sound sensitizer is modified by a functional group of hyaluronic acid (HA) or folic acid (FA) to specifically recognize tumor cells, so as to obtain the ability of active targeting of CD44 overexpressing or folic acid receptor overexpressing tumor cells.

[0264] In one embodiment, the light / sound sensitizer is modified by a functional group of hyaluronic acid (HA) or folic acid (FA) to specifically recognize tumor cells, so as to obtain the ability of active targeting of CD44 overexpressing or folic acid receptor overexpressing tumor cells.

[0265] In the present disclosure, the light activation condition is not particularly limited, and the preferred light source includes but is not limited to red LED light source, 682nm laser and other light sources capable of irradiating sufficient light intensity.

[0266] In the present disclosure, the representative phenol-based labeling probe substrate includes but is not limited to FITC-phenol, biotin-phenol (BP), biotin-xx-phenol (BxxP), alkyne phenol (AP), dinitrophenol, Galα1-3Galβ1-4GlcNAc-R (αGal)-phenol and other molecules containing phenol groups.

[0267] In the present disclosure, the representative amine-based labeling probe substrate includes but is not limited to FITC-amine.

[0268] In the present disclosure, the target cell is not particularly limited, and the representative target cell includes but is not limited to tumor cells such as CD44 overexpressing mouse breast cancer cell line 4T1 cells, FA receptor overexpressing mouse melanoma cell line M109 cells, mouse lung cancer cell line M109 cells and HER2 overexpressing SK-OV-3 breast cancer cells.

[0269] The present disclosure is particularly suitable for objects with fewer specific antigens on the surface of target cells. Since the method of the present disclosure can efficiently amplify antigens based on proximity labeling reaction, it can be widely used in various antigen low-expression or high-expression tumor models by changing the targeting group or using antibodies for targeting.

[0270] It should be understood that in the present disclosure, the PATCH in vivo treatment strategy includes but is not limited to immunotherapy based on FITC vaccine stimulated anti-FITC antibody and immune cell therapy based on bispecific T cell antibody (BiTE) targeting, which can be widely used in Car-T therapy, monoclonal antibody therapy and other immunotherapies.

[0271] Proximity labeling

[0272] As used herein, the terms "proximity labeling", "proximity labeling reaction" can be used interchangeably, which is a new technology for proximity protein biotinylation using enzyme-catalyzed production of a broad reactive intermediate. This technology is currently only used to identify intermolecular interactions. In the present disclosure, the application of this broad reactivity to in vivo antigen labeling can achieve the unique advantage of catalytic amplification of covalent labeling of antigens at high local density with precise spatiotemporal control. However, proximity labeling on the cell surface is a long-term challenge: since the use of peroxidase (such as APEX and HRP) for catalysis requires the use of hydrogen peroxide with high toxicity, which causes biological toxicity; since biotin ligase (such as BioID and TurboID) requires ATP, it is inactivated outside the cell and cannot be used outside the cell; the recently developed photocatalysts, most of which are blue light activated, are limited in their use in vivo due to the poor penetration of blue light. In summary, it is necessary to expand the current proximity labeling composition to evaluate its therapeutic potential.

[0273] PATCH

[0274] As used herein, the term "PATCH" refers to Proximity Amplification and Tagging of Cytotoxic Haptens, which is a strategy for significantly enhancing the antigenicity of tumors by broad amplification of predetermined antigens on the surface of tumor cells in vivo.

[0275] Specifically, in the present disclosure, the strategy on the one hand uses functionalized photo / acoustic sensitizers (PCN) targeting tumor cell membranes and the like, so that the PCN of the present disclosure targets tumor cell membranes; on the other hand, by means of light activation and the like, the hapten probe fluorescein-phenol (FITC-phenol) is used to achieve proximity labeling of predetermined target antigens on the surface of tumor cells, significantly increasing the antigen density on the surface of tumor cells, or forming high-density antigen clusters, so as to achieve amplification of antigen signals for tumor cells with insufficient antigen expression or low degree of antigen aggregation, and then trigger receptor crosslinking on immune cells, activate downstream signaling, and achieve specific killing of tumors.

[0276] PATCH-based immunotherapy

[0277] The terms "PATCH therapy", "PATCH in vivo treatment strategy" can be used interchangeably, both of which refer to PATCH-based immunotherapy.

[0278] The present disclosure also provides a PATCH-based immunotherapy, comprising the steps of:

[0279] (a) administering to a subject a preparation or composition, causing the preparation or composition to antigenically amplify a predetermined antigen on the surface of a target cell in the subject by proximity labeling, thereby labeling the target cell surface with artificial antigens; and

[0280] (b) immunotherapy with an immunotherapeutic agent against the artificial antigens.

[0281] In some embodiments, the immunotherapeutic agent against the artificial antigens is used for immunotherapy against tumors.

[0282] With the immunotherapy method using PATCH of the present disclosure, diseases that cannot be treated or cannot be effectively treated by immunotherapy due to insufficient number of antigens can be treated.

[0283] Photosensitizers and sonosensitizers

[0284] As used herein, the term “photosensitizer / sonosensitizer”, “PCN”, “PCN photosensitizer / sonosensitizer” refers to a class of nanoenzymes, specifically refers to immobilized inorganic metal complexes, immobilized enzymes or inorganic nanoparticles and other biological materials with nanomaterials or nanostructures with enzymatic catalytic activity, which can catalyze biochemical substrates under mild environmental conditions. In this paper, the photosensitizer / sonosensitizer used is a photosensitizer / sonosensitizer self-assembled from porphyrin and zirconium atoms, and the surface thereof can be functionally modified.

[0285] As used herein, the term “photosensitizer / sonosensitizer targeting cell surface antigens” refers to a photosensitizer / sonosensitizer that can specifically recognize cell surface antigens or markers. In this paper, the photosensitizer / sonosensitizer targeting cell surface antigens can specifically target tumor cells, which are formed by modifying the surface of the porphyrin photosensitizer / sonosensitizer self-assembled from porphyrin and zirconium atoms with folic acid or hyaluronic acid. Among them, the folic acid is modified on the surface of the photosensitizer / sonosensitizer by polyethylene glycolation, and the phosphate group connected thereto is covalently connected to the zirconium atom, and the hyaluronic acid is modified on the surface of the photosensitizer / sonosensitizer by supramolecular force and physical adsorption. The folic acid modified photosensitizer / sonosensitizer or the hyaluronic acid modified photosensitizer / sonosensitizer can target tumor cells overexpressing folate receptors or CD44.

[0286] Among them, the sonosensitizer catalyzes the substrate by acoustic cavitation. As used herein, the terms “sonic cavitation effect” and “sonic cavitation” can be used interchangeably, which refers to a dynamic process of generating microbubbles (cavitation nuclei) under the action of ultrasonic waves (frequency usually 20 kHz-10 MHz), accompanied by the generation of active oxygen. The term “sonosensitizer producing active oxygen by sonic cavitation effect” refers to a sonosensitizer that can be excited to produce cavitation effect under ultrasound, generating high-energy conditions locally, and the energy released can be transferred to the surrounding oxygen to produce highly active intermediates.

[0287] As used herein, the term "bioenzyme" includes bioenzymes that can specifically recognize cell surface antigens or markers, i.e., cell surface-targeted bioenzymes, and bioenzymes that are enriched on cell surfaces. In the present disclosure, the cell surface-targeted bioenzymes are cholesterol-modified horseradish peroxidase, HER2 antibody chimeric protein with β-galactosidase. Among them, the cholesterol-modified horseradish peroxidase targets the horseradish peroxidase to the tumor cell by interacting with the phospholipid membrane on the tumor cell membrane through cholesterol; the HER2 antibody chimeric protein with β-galactosidase is ZHER-βGal, and ZHER is a HER2 antibody that can target tumor cells, thereby targeting β-galactosidase to HER2-overexpressing tumor cells. The bioenzymes enriched on the cell surface are non-targeted tyrosinase in the present disclosure, which is expressed around the cell by transfection into the expression vector of the tumor cell. Tyrosinase oxidizes the phenolic group to an ortho-phenolic intermediate, thereby triggering the proximity labeling reaction in the tumor environment.

[0288] artificial antigen

[0289] As used herein, the term "artificial antigen" refers to a class of synthetic antigens comprising small molecule compounds and carriers. Among them, the small molecule compounds have reactivity without immunogenicity, i.e., lack of T cell epitopes and cannot directly induce the body to produce corresponding specific antibodies. Therefore, the small molecule compound can only be called a hapten or an incomplete antigen, which needs to be coupled with a carrier according to immunological principles to form an artificial antigen, thereby obtaining immunogenicity. The artificial antigen indirectly induces B cells to proliferate and differentiate with the help of T cell epitopes, and then produces specific antibodies.

[0290] In the present disclosure, the artificial antigen is a small molecule hapten carrying a phenolic group, an amine group, an aryl azide, a quinone, and a transpeptidation substrate, wherein the small molecule hapten includes but is not limited to FITC, biotin. In an embodiment, the body is induced to produce anti-FITC antibodies by injecting FITC-KLH vaccine adjuvant, thereby triggering a corresponding immune response. Those skilled in the art can understand that any hapten or artificial antigen carrying a reactive group (such as a phenolic group or an amine group) that can achieve covalent reaction can be used as one of the active ingredients for antigen amplification by proximity labeling technology in the present disclosure.

[0291] reactive group

[0292] As used herein, the "reactive group" refers to a type of functional group that can participate in covalent labeling reactions in artificial antigens or their targets (such as protein surface groups) under the excitation of catalysts (such as photo / sonosensitizers), which specifically includes the following two types:

[0293] (1) Precursor groups of active intermediates: structural units that can be converted into active intermediates (such as free radicals, quinones, carbenes, etc.) under catalytic conditions (such as light or ultrasonic excitation), such as phenolic hydroxyl groups, aromatic amines, benzaldehydes, azides, etc.

[0294] (2) Reaction partner groups of active intermediates: structural units that can rapidly covalently react with the above-mentioned active intermediates, such as primary amines, aromatic amines, hydrazides, sulfhydryl groups, etc.

[0295] Active intermediates can rapidly covalently react with reactive groups in a local space, thereby achieving spatially directed labeling of artificial antigens. In a light labeling system, the phenolic hydroxyl groups carried by the artificial antigen generate phenoxy radicals under the action of a photosensitizer, and react with the tyrosine on the surface of the protein; in an ultrasonic labeling system, singlet oxygen is generated under the mediation of a sonosensitizer, which oxidizes the histidine residues on the surface of the protein to generate imidazole aldehyde intermediates, which then react with the primary amine or hydrazine group on the artificial antigen to form a stable covalent bond. Although the "reactive groups" in the two mechanisms are derived from different sources, they are both achieved by the locally generated active intermediates to achieve spatially selective labeling.

[0296] Specifically, the reaction intermediates can be derived from artificial antigens (such as phenol groups generating phenoxy radicals under light conditions), or from target cell surface proteins (such as histidine being oxidized to imidazole aldehyde intermediates under ultrasonic conditions), and ultimately all react with reactive groups through the formation of covalent bonds to achieve effective labeling of artificial antigens, thereby enhancing the immunogenicity of cells.

[0297] Common active intermediates include free radicals, carbonium ions, carbanions, carbenes, nitrenes, imidazole aldehydes, etc. Among them, free radicals are neutral active intermediates, which have an unpaired electron atom or molecule; carbonium ions and carbanions are charged active intermediates; carbenes are neutral active intermediates, which have two unpaired electrons; nitrenes are also neutral active intermediates, which have four unpaired electrons; and imidazole aldehyde is a neutral active intermediate with high electrophilicity. The phenoxy radical or imidazole aldehyde group used in the present disclosure usually has a half-life of less than 1 ms. In proximity labeling technology, the active intermediates used should also have the characteristic of small diffusion range, which means that the target substances or cells are more accurately labeled, thereby avoiding false positive labeling.

[0298] As used herein, the reactive groups used include phenol groups, benzoquinones, amine compounds, etc. Among them, phenol can form phenoxy radicals through oxidation. In biological systems, this reaction is usually catalyzed by peroxidase in the presence of hydrogen peroxide, or by using a light / sonosensitizer or an engineered light-oxygen-voltage domain under blue light irradiation.

[0299] Phenol group labeling probe substrate

[0300] In the present disclosure, a preferred class of hapten carrying reactive groups is phenolic group-labeled probe substrates.

[0301] As used herein, the term "phenolic group-labeled probe substrates" refers to a class of photosensitizer substrates that are connected to phenol and can be used as antigens or signal detection sites. In the present disclosure, the phenolic group-labeled probe substrates include, but are not limited to, FITC-phenol, biotin-phenol, biotin-xx-phenol (BxxP), alkyne phenol (AP), dinitrophenol, Galα1-3Galβ1-4GlcNAc-R (αGal)-phenol, and other phenolic group-containing molecules. In the phenol, the hydroxyl group can form a phenoxy radical after losing a hydrogen atom. The phenoxy radical is a common active intermediate, which can be obtained by thermodynamic or photochemical methods, of which the most commonly used is photochemical method. In photochemical reaction, the phenoxy radical can be excited by ultraviolet or visible light to form a reaction intermediate, which participates in chemical reaction. In the present disclosure, the phenolic group-labeled probe substrates form phenoxy radicals by exciting with deep red light in the presence of photosensitizer, thereby connecting the carried labeled probes to target cells.

[0302] As used herein, the term "FITC" is fluorescein isothiocyanate, which is a widely used green fluorescent derivative in biology. In addition to high absorption rate and excellent fluorescence quantum yield, the isothiocyanate group of FITC can bind to amino, sulfhydryl, imidazole, tyrosine, carbonyl and other groups on proteins, thereby realizing labeling of various biological macromolecules, bioactive substances and other proteins such as antibodies, lectins, etc. In the present disclosure, FITC is labeled on the surface of target cells by proximity labeling method, as a cell surface antigen, thereby being recognized by anti-FITC antibody to initiate immune killing of target cells.

[0303] Amine group-labeled probe substrates

[0304] In the present disclosure, a preferred class of hapten carrying reactive groups is amine group-labeled probe substrates.

[0305] As used herein, the term "amine group-labeled probe substrates" refers to a class of sonosensitizer substrates that are connected to amine groups and can be used as antigens or signal detection sites. In the present disclosure, the amine group-labeled probe substrates include, but are not limited to, fluorescein (FITC)-amine, biotin-amine. In the sonosensitizer, singlet oxygen is generated in the presence of ultrasound; the singlet oxygen forms an imidazole aldehyde intermediate by oxidizing the histidine on the cell surface; the intermediate covalently bonds with the amine group, thereby connecting the carried labeled probes to target cells

[0306] Antigen amplification

[0307] As used herein, the term "antigen amplification" refers to increasing the number of antigens or labels that can be recognized by a target substance. In the present disclosure, antigen amplification is performed on tumor cells using a proximity labeling technique, i.e., using a catalyst such as a light / sound sensitizer targeting folate receptors, CD44, or HER2 on the surface of tumor cells, a biological enzyme targeting phospholipid membranes or HER2, and a tyrosinase catalytically activated to activate artificial antigens carrying reactive groups such as phenol group labeled probe substrates (e.g., FITC-phenol and biotin-phenol), methylenebenzoquinone group labeled probe substrates, etc., to covalently bind antigens or labels to the surface of tumor cells. Among them, the label can be a hapten that is immunogenic only in a specific environment. A large number of antigens or labels can be bound to the cell surface using this method, thereby achieving the effect of antigen amplification. The corresponding antibody of the antigen or label can trigger an immune response.

[0308] Tumor-specific antigens and tumor-associated antigens

[0309] As used herein, the terms "tumor-specific antigen (TSA)" and "tumor-associated antigen (TAA)" refer to antigen molecules present on tumor cells. Among them, tumor-specific antigens are antigen molecules that are only expressed in tumor cells and not expressed in normal tissue cells; while tumor-associated antigens are antigen molecules that are highly expressed in tumor cells and only have a small amount of expression in specific tissues or normal cells. Tumor-specific antigens and tumor-associated antigens can be recognized by the immune system and attacked as foreign substances, and can be used as targets for cancer immunotherapy. In the present disclosure, tumor-specific antigens and tumor-associated antigens serve as targets for the above-mentioned modified light / sound sensitizers, for positioning the light / sound sensitizers near tumor cells, so that they can perform the catalytic function of light / sound sensitizers in the tumor environment. Common tumor-specific antigens include alpha-fetoprotein (AFP) and the like. Common tumor-associated antigens include folate receptors, hyaluronic acid receptors, HER2, mesothelin, etc. The tumor-associated antigens used in the present disclosure are folate receptors, hyaluronic acid receptors CD44, and HER2.

[0310] Bispecific antibodies

[0311] As used herein, the term "bispecific antibody" refers to an antibody that can specifically target two antigens or two different epitopes of one antigen at the same time. Currently, there are three main types of bispecific antibodies used for immunotherapy: (1) cytotoxic effector cell redirection factor, i.e., by specific binding to TAA / TSA and T cell receptor or CD3 complex, cytotoxic effector T cells are directed to tumor cells; (2) tumor-targeted immunomodulatory factor, i.e., designed to bind to TAA / TSA and immunomodulatory receptors (such as CD40). These immunomodulatory factors are usually in an inactive state before binding to tumor antigens, so as to super-localize immune stimulation to the tumor environment, effectively reducing the risk of immune-related side effects. (3) Dual immunomodulatory factor, i.e., binding to two different immunomodulatory targets, resulting in blockade of inhibitory targets, depletion of suppressor cells or activation of effector cells.

[0312] As used herein, the term "BiTE" is a bispecific T cell engager, which is a bispecific antibody that can bind to TAA and CD3 complex at the same time. Due to the limited number of T cells in the tumor environment, and the expression of TAA in normal tissues or cells, the direct use of BiTE for the immunotherapy of tumors is limited to a certain extent.

[0313] In order to avoid the limitations of BiTE, in the present disclosure, a bispecific antibody can be used to recruit immune cells to target tumor cells for immunokilling. The bispecific antibody used is a cytotoxic effector cell redirection factor, which has a first antibody element that can target an expanded antigen on tumor cells, and a second antibody element that can target immune cells. The first antibody element includes but is not limited to anti-FITC antibody, the second antibody element includes but is not limited to a molecule that can bind to CD3 complex, and the immune cells include but are not limited to T cells. In addition, engineered T cells, i.e., CAR-T cells, can also be used for immunotherapy. By incorporating the antibody to the expanded antigen into T cells, it can target the expanded antigen, and in turn target the tumor cells where the expanded antigen is located, so as to kill the tumor cells by immunotherapy. Accordingly, other engineered tumor-killing cells, such as CAR-NK cells, CAR-macrophages, etc., can also be used for immunotherapy.

[0314] Therapeutic system of the present disclosure

[0315] The present disclosure also provides a therapeutic system or device for use in conjunction with the methods of the present disclosure.

[0316] Generally, the therapeutic system of the present disclosure comprises:

[0317] (z1) an antigen amplification module configured to apply the artificial antigen carrying reactive groups and the catalyst of the first aspect of the present disclosure to a subject, so that in the body of the subject, under the catalysis of the catalyst, the artificial antigen carrying reactive groups and the reactive groups on the cell surface undergo a covalent linkage reaction, thereby labeling the artificial antigen on the cell surface; and

[0318] (z2) an optional immunotherapy module configured to apply the immunotherapeutic agent of the first aspect of the present disclosure to the subject, the immunotherapeutic agent being targeted to the artificial antigen.

[0319] In the therapeutic system of the present disclosure, preferably, a light source module is included and deep red light is used for irradiation treatment. Preferably, a sound source module is included and ultrasound is used for ultrasonic treatment.

[0320] As used herein, the term "light source module" refers to a type of component or device that can provide a light source. In the present disclosure, the PATCH contains a light-activated catalyst, such as a photosensitizer formed by porphyrin and zirconium atom, so that the light source module is used for irradiation to activate the photosensitizer, thereby completing the proximity labeling reaction. As used herein, the terms "light excitation" and "light activation" are used interchangeably to refer to a process of using light to act on a catalyst to make the catalyst catalytically active.

[0321] As used herein, the term "deep red light" refers to red light with a longer wavelength, usually with a wavelength exceeding 660 nm. Because the wavelength of deep red light is long, it has stronger penetration and lower energy than blue light, so it can better penetrate the surface of the body to act on the lesion site, and is safer.

[0322] As used herein, the term "sound source module" refers to a type of component or device that can provide a sound source. In the present disclosure, the PATCH contains a sound-activated catalyst, such as a porphyrin-based sonosensitizer, so that the sound source module is used for ultrasonic treatment to activate the sonosensitizer, thereby completing the proximity labeling reaction.

[0323] The main advantages of the present disclosure include:

[0324] (1) The technology presented in the present disclosure first specifically labels the hapten on the cell surface by proximity labeling technology, and uses the antibody or bispecific antibody of the hapten as an immunotherapeutic agent to achieve effective elimination of tumor cells. Compared with traditional antigen recognition methods, the technology presented in the present disclosure forms high-density antigen clusters through hapten proximity labeling, can amplify the antigen signal of tumor cells with insufficient antigen expression or low antigen aggregation degree, and then trigger the receptor crosslinking on immune cells, activate the initiation of endogenous tumor-specific T cells, produce the same treatment effect on untreated distal tumors, achieve specific killing of tumors, and have potential treatment potential for tumors with extensive metastases. The antigen labeling and targeted immunotherapy presented in the present disclosure provide a method for enhancing anti-tumor immunity by amplifying tumor antigens.

[0325] (2) In the present disclosure, a preferred method for amplifying a predetermined antigen on the surface of tumor cells based on proximity labeling technology utilizes PCN photo / acoustic sensitizers. Compared with traditional proximity labeling biological enzymes, PCN photo / acoustic sensitizers have more stable chemical properties, diverse functions, and are easy to modify, and different targeting groups can be used according to different target cells. PCN photo / acoustic sensitizers use photocatalytic labeling means, and do not require H2O2 or ATP and other essential components required by traditional labeling biological enzymes in reaction conditions, and can achieve precise and safe labeling reactions under laser induction in vivo. Compared with other blue light catalytic labeling methods, the red light catalysis used in the present disclosure has the advantages of stronger penetration brought by long wavelength, and lower energy biological safety. The above labeling system does not need to be expressed in vivo, and can be delivered into tumor tissues through intravenous blood circulation. It can achieve efficient and targeted labeling of tumor cells without introducing any exogenous DNA or RNA.

[0326] The present disclosure will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. The experimental methods in the following examples are not specified, and the methods are generally carried out under conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0327] Experimental methods

[0328] Preparation method of target modified photo / acoustic sensitizer

[0329] (S1) Meso-Tetra(4-carboxyphenyl)porphine (TCPP), zirconium chloride (ZrCl4), benzoic acid are mixed into solvent N,N-dimethylformamide (DMF) and stirred at 80-110°C for 4-6h. The light / sonic sensitizer PCN dispersion is obtained by centrifugation using DMF and washing with anhydrous ethanol. The mass ratio of TCPP / ZrCl4 to benzoic acid is TCPP:ZrCl4:benzoic acid = 1:3:30. The porphyrin is a typical photo sensitizer (PS) and sono sensitizer (SS), which can absorb energy and enter the excited state when exposed to appropriate laser and ultrasound irradiation, and ultimately transfer energy to adjacent molecules in the process of returning to the ground state, thereby generating a variety of highly active intermediates for further photo / sonic chemical reactions.

[0330] Alternatively, the constructed PCN light / sonic sensitizer can be constructed by replacing the central metal ion, such as Fe, Zn, and other transition metal elements that can coordinate, to realize the construction of the framework material.

[0331] (S2) Single phosphate-polyethylene glycol-folic acid (PO3-PEG-FA) or hyaluronic acid (HA) is added to the PCN dispersion. Stirring is carried out at room temperature to obtain FA-PCN and HA-PCN with targeting group modification. The combination of PO3-PEG-FA and PCN is covalent binding of phosphate groups and PCN surface zirconium atoms, and the combination of HA and PCN is supramolecular force and physical adsorption between HA and PCN nanoparticles. The mass ratio of PO3-PEG-FA to PCN is 6-10:1. The prepared targeted PCN stock solution has a concentration of 0.5-2 μg / μL (Figure 1).

[0332] Alternatively, the targeting modified light / sonic sensitizer can be prepared as HER2-PCN by replacing the targeting group, carboxylating PCN with PO3-PEG-COOH, activating with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and coupling with a targeting antibody through condensation reaction with amino groups. pDA-PCN can also be prepared by depositing polydopamine (pDA) as an amino conjugate on the surface of PCN, and covalently modifying PO3-mPEG to improve stability, and then coupling the targeting antibody with pDA to prepare HER2-PCN, such as HER2-PCN formed by HER2 antibody (Figure 1).

[0333] Proximity labeling method of PCN light / sonic sensitizer excited by light

[0334] (S3) For the labeling of proteins, the PCN suspension in S1 is mixed with proteins, and in the presence of a solution of phenol-based biotin, the labeling reaction is excited by red light irradiation; or in the presence of a solution of amine-based biotin, the labeling reaction is excited by ultrasound. For the targeted labeling of cells, the targeted modified FA-PCN, HA-PCN or HER-PCN suspension in S2 is added to the culture solution of the corresponding cells, and the PCN photo / sonosensitizer is targeted to the cell surface by co-culturing at 37°C. Then, a phenol-based probe substrate is added, and the labeling reaction is excited by red light irradiation; or an amine-based substrate probe is added, and the labeling reaction is excited by ultrasound. The working concentration of the PCN is 0.05-0.1 μg / μL, the co-culturing time of the PCN with the cells is 30 min-2 h, the phenol-based substrate probe is phenol-based biotin and phenol-based fluorescein (FITC-phenol), and the working concentration is 20-100 μM. The red light source is an LED red light panel or a 682 nm laser. The amine-based substrate probe is amine-based biotin and amine-based fluorescein (FITC-amine), and the working concentration is 0.5-5 mM. The light irradiation condition is that the protein, cell or clinical patient-derived tumor tissue is placed in a transparent container, such as a cell culture dish, and irradiated on the light panel, such as using a laser light source at a distance of 5-10 cm. The ultrasound instrument is a medical ultrasound probe, and the power is 0.5-3 W / cm 2 The protein, cell or clinical tumor tissue is placed in a liquid container, and the bottom of the container is uniformly coated with an ultrasonic coupling agent at the contact part with the ultrasound probe. For example, for the omics research under precise proximity labeling, preferably, the PCN is co-cultured with the cells, and the cells are washed several times to remove as much free PCN photo / sonosensitizer as possible. Then, the phenol-based substrate probe is added and irradiated.

[0335] Alternatively, the protein can be most proteins containing tyrosine residues, and the cell can be a cell having a targetable site.

[0336] Method for treating tumors in vivo by photo / sonic excitation of PCN photo / sonosensitizer

[0337] (S4) The targeted modified FA-PCN, HA-PCN or HER2-PCN suspension in S2 is delivered into the tumor-bearing mouse body by tail vein injection. After the PCN photo / sonosensitizer is enriched in the tumor tissue by in vivo circulation, active targeting and passive targeting effects of the nanoparticles, the immunogenic FITC-phenol / amine substrate is delivered into the tumor tissue by intratumoral injection. Then, a 682 nm laser is used for irradiation for 30 min, or a 2 W / cm 2Ultrasound probe for 10 min, which is a complete PATCH treatment process. The target modified FA-PCN, HA-PCN or HER2-PCN injection volume is 100 μL, the concentration is 0.5-2 μg / μL, the FA-PCN corresponds to the M109 cell line, the HA-PCN corresponds to the 4T1 cell line, the HER2-PCN corresponds to the CT26-HER2 or other HER2 overexpression cell line, and the mouse is a Balb / c female 6-8 week old mouse or a PBMC humanized mouse. The volume of the FITC-phenol substrate is 50-100 μL, and the concentration is 20-100 μM. The volume of the FITC-amine substrate is 50-100 μL, and the concentration is 0.5-5 mM. Subsequently, the anti-tumor effect is achieved by the anti-FITC antibody produced by the pre-inoculation of the FITC vaccine, or by the administration of the FITC specific BiTE. The FITC vaccine uses FITC conjugated keyhole limpet hemocyanin (KLH) mixed with Freund's adjuvant to stimulate the production of anti-FITC antibodies. The FITC specific BiTE is administered through the tail vein, and the administration concentration is 0.2-1 μg / μL.

[0338] Alternatively, the PATCH process can also be achieved by intratumoral injection of PCN, intratumoral injection of FITC-phenol substrate, or tail vein injection of PCN, tail vein injection of FITC-phenol substrate. Alternatively, the tumor-bearing mouse model used includes, but is not limited to, subcutaneous inoculation model, orthotopic tumor model, and other tumor models that can be treated by laser / ultrasound.

[0339] Example 1: Preparation of photoexcited proximity labeled photo / acoustic sensitizer PCN and detection of its properties

[0340] This embodiment relates to the preparation of a photoexcited proximity labeled photo / acoustic sensitizer PCN and the detection of its properties. The photo / acoustic sensitizer is self-assembled by high-temperature reaction of ZrOCl2 and benzoic acid with TCPP in DMF solvent. The specific preparation method and the steps of proximity labeling protein are described above in the preparation method of the target modified photo / acoustic sensitizer, and the proximity labeling method of the photoexcited PCN photo / acoustic sensitizer.

[0341] After functional modification, the photo / acoustic sensitizer can achieve the enhancement of tumor immunogenicity by extensive expansion of designed antigens on the surface of tumor cells in vivo (Figure 2A).

[0342] Figure 2B is a TEM image of PCN, which presents a uniform morphology of about 40 nm in size. Figure 2C is an elemental analysis image of PCN, Zr, N, and O are the unique elements of PCN.

[0343] PCN-mediated proximity labeling is based on the active intermediates generated by photochemical reaction. The present disclosure labels bovine serum albumin (BSA) with biotin in vitro under red light excitation. The phenoxy radical intermediates generated by photochemical reaction can be covalently coupled with the phenol residues of tyrosine on the surface of the protein. Neither the substrate probe BP nor PCN alone in the labeling system can label BSA. However, when PCN and BP coexist in the system, there is an obvious labeling signal under red light excitation. When there are active oxygen (ROS) inhibitors such as sodium ascorbate, water-soluble vitamin E (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Trolox) and sodium azide in the system, no labeling signal can be generated, proving that the proximity labeling is completed by the active intermediates mediated by ROS (Figure 2D).

[0344] As shown in Figure 2E, mass spectrometry analysis of the BSA cleavage peptide fragments after photoexcitation labeling detected that multiple tyrosine sites on the surface of BSA were biotinylated. Mass spectrometry analysis of BSA after biotin labeling showed that one of the two tyrosine sites in the same specific peptide fragment was significantly biotinylated. Biotinylation signals were detected in multiple cleavage peptides.

[0345] PCN-mediated proximity labeling is based on the active intermediates generated by photochemical reaction. The present disclosure labels bovine serum albumin (BSA) with biotin in vitro under red light excitation. The phenoxy radical intermediates generated by photochemical reaction can be covalently coupled with the phenol residues of tyrosine on the surface of the protein. Neither the substrate probe BP nor PCN alone in the labeling system can label BSA. However, when PCN and BP coexist in the system, there is an obvious labeling signal under red light excitation. When there are active oxygen (ROS) inhibitors such as sodium ascorbate, water-soluble vitamin E (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Trolox) and sodium azide in the system, no labeling signal can be generated, proving that the proximity labeling is completed by the active intermediates mediated by ROS (Figure 2D).

[0346] As shown in Figure 2G, mass spectrometry analysis of the BSA cleavage peptide fragments after photoexcitation labeling detected that multiple histidine sites on the surface of BSA were biotinylated.

[0347] Example 2: Preparation of proximity labeling enzyme with functional modification of targeting group

[0348] In this embodiment, based on the proximity labeling light / sound-sensitive agent in Example 1, further preparation of proximity labeling enzymes HA-PCN, FA-PCN, HER2-PCN with functional modification of targeting groups (Figure 3A).

[0349] The specific method is as follows: by mixing hyaluronic acid (HA) with PCN suspension, the HA is modified on the surface of PCN by physical adsorption under magnetic stirring, to prepare HA-PCN. By covalently connecting the monophosphate group to the Zr atom on the surface of PCN, PO3-PEG-FA is modified on the surface of PCN to prepare FA-PCN. After PCN is functionalized by polydopamine and PO3-mPEG, HER2 targeting antibody is coupled to prepare HER2-PCN (Figure 1).

[0350] HA-PCN realizes the targeted labeling of CD44 positive tumor cells by binding to the CD44 overexpressed on the surface of tumor cells. FA-PCN realizes the targeted labeling of FR positive tumor cells by binding to the folate receptor FR overexpressed on the surface of tumor cells. HER2-PCN realizes the targeting of HER2 positive tumor cells by binding to the HER2 receptor overexpressed on the surface of tumor cells. The above three labeling processes are shown in Figure 3D.

[0351] The tumor cell lines used in this embodiment are CD44 overexpressing mouse breast cancer 4T1 cells and FR overexpressing mouse melanoma M109 cells, and HEK293 cells are used as negative controls. For HER2 targeting, the engineered HER2 stable cell line CT26-HER2 of CT26 is used, and the wild type CT26 cells are used as negative controls.

[0352] This embodiment demonstrates two different targeted labeling, and by replacing the modified targeting group with other specific targeting groups or antibodies, targeted labeling of different cell types and different protein sites can also be achieved.

[0353] This embodiment demonstrates the proximity labeling of biotin phenol probe (BxxP) and biotin amine probe (BA) as the substrate, aiming to facilitate the later separation of labeled proteins for studying their omics, and by replacing the labeling substrate with other different active intermediates, targeted labeling of different types of modified target cells and target proteins can also be achieved.

[0354] As shown in FIG. 3B, compared with CT26 cells, HER2-PCN can target to the surface of CT26-HER2 cells overexpressing HER2, and the labeling signal is concentrated on the cell membrane surface, while there is no obvious signal on CT26 cells; compared with CT26 cells, HER2-PCN can target to the surface of CT26 cells overexpressing HER2, and the labeling signal is concentrated on the cell membrane surface of CT26-HER2 cells, while there is no obvious signal on CT26 cells. Compared with HEK293 cells, HA-PCN can target to the surface of 4T1 cells overexpressing CD44, and the labeling signal is concentrated on the cell membrane surface of 4T1 cells, while there is no obvious signal on HEK293 cells; compared with HEK293 cells, FA-PCN can target to the surface of M109 cells overexpressing folate receptor, and the labeling signal is concentrated on the cell membrane surface of M109 cells, while there is no obvious signal on HEK293 cells.

[0355] As shown in FIG. 3C, only in the presence of red light, laser or ultrasound, the intracellular protein can be labeled by the substrate, and in the absence of light or in the absence of HA-PCN, there is no labeling signal on the protein.

[0356] As shown in FIG. 3E, among the enriched proteins, the proteins with the most obvious enrichment effect are located on the cell surface and the cell membrane and cell interstitium and cell junction, and most of the proteins are involved in cell migration, cell adhesion and intercellular junction.

[0357] As shown in FIGS. 3F and 3G, among the enriched proteins, the proteins significantly enriched are mostly membrane proteins, including CD44 protein combined with HA, and the enriched proteins are all related to CD44.

[0358] Example 3: PATCH can efficiently activate immune activity

[0359] This example shows that the hapten FITC phenol probe (FITC phenol) is a substrate for proximity labeling, and different types of antigen amplification can be achieved by replacing the labeling substrate with other different types of antigen groups.

[0360] As shown in FIG. 4A, PCN can target to target cells through modified groups, catalyze phenol substrate to phenoxy radical, and then label target cells.

[0361] As shown in FIG. 4B, compared with single affinity connection such as HA-FITC, FA-FITC, HER2 antibody-FITC, HA-PCN, FA-PCN, HER2-PCN have stronger signal, and can target to 4T1 cells overexpressing CD44 compared with HEK293T cells. FA-PCN has stronger signal compared with single affinity connection FA-FITC, and can target to M109 cells overexpressing folate receptor compared with HEK293T cells. HER2-PCN has stronger signal compared with single affinity connection HER2 antibody-FITC, and can target to CT26-HER2 cells overexpressing folate receptor compared with CT26 cells. After statistics, the enrichment degree of cell membrane antigen after PCN photo / acoustic sensitizer labeling is 4-5 times higher than that of single affinity connection (FIG. 4C).

[0362] As shown in FIG. 4D, HER2-PCN mediated PATCH can target to the surface of CT26-HER2 cells overexpressing HER2 compared with non-target PCN without antibody modification, and the labeling signal is concentrated on the cell membrane surface, while there is no obvious signal on CT26 cells.

[0363] As shown in FIG. 4E, ultrasound triggered PATCH has obvious FITC targeting labeling effect on CT26-HER2 cells compared with no ultrasound treatment.

[0364] As shown in FIG. 4F, tumor cell labeling of hapten FITC probe can also be achieved by catalysis of biological enzymes horseradish peroxidase (HRP), tyrosinase (BmTyr) or galactosidase (βGal). First, cholesterol covalently modified HRP is used to localize HRP on the tumor cell membrane surface through the interaction of cholesterol and phospholipid membrane. Under the trigger of hydrogen peroxide, HRP catalyzes the high activity reaction intermediate of phenoxy radical from phenol group, and then realizes the labeling and enrichment of hapten FITC on the tumor cell membrane through protein binding labeling.

[0365] It can also be achieved by transfecting recombinant plasmid expressing BmTyr in tumor cells to make the cells express BmTyr to oxidize phenol group to highly electrophilic activity o-benzoquinone intermediate. The o-benzoquinone intermediate can be used to realize the labeling of FITC labeled tumor cells by reacting with the nucleophilic group on the surface of the protein.

[0366] It can also be achieved by catalytic labeling of βGal. First, the HER2 targeting nanobody ZHER-βGal is expressed and purified in prokaryotes, and βGal is targeted to the surface of CT26-HER2 cells overexpressing HER2 through ZHER, and βGal activates the probe containing methylene phenylquinone gFQM group, which can be used to realize the targeting labeling of tumor cells.

[0367] Also, the miniSOG can be expressed in the tumor cells by transfecting the tumor cells with a recombinant plasmid expressing the miniSOG. The miniSOG is membrane localized. Upon blue light irradiation, singlet oxygen is generated. The amino group containing substrate probe is catalytically labeled to the cell membrane of the tumor cells via an energy transfer pathway.

[0368] Also, the Sortase can be expressed in the tumor cells by transfecting the tumor cells with a recombinant plasmid expressing the Sortase. The Sortase is membrane localized. The N-terminal FITC labeled AALPETGG sequence polypeptide is labeled to the cell membrane via a transpeptidation reaction, achieving the FITC labeling of the tumor cells.

[0369] Also, the azido aryl probe containing can be labeled to the tumor cell membrane via an electron transfer pathway upon red light irradiation by a photo / sonosensitizer tin metal catalyst mediated μMAP photocatalytic labeling strategy in the system.

[0370] Example 4: PATCH can strongly activate immune activity

[0371] In this example, the HA-PCN, FA-PCN or HER2-PCN shown in Example 2 is used to target and label 4T1 cells and patient derived tumor fragments (PDTF) with hapten probe FITC-phenol / amine. The killing of the targeted cells by immune cells after antigen amplification is studied in vitro.

[0372] In this example, the strength of the Antibody-Dependent Cellular Phagocytosis (ADCP) effect mediated by anti-FITC antibody is studied using primary isolated mouse macrophages (Figure 5A), and the strength of the T cell receptor (TCR) effect mediated by engineered FITC targeted BiTE is studied using primary isolated mouse T cells (Figure 5D).

[0373] As shown in Figure 5A, the labeled tumor cells can be recognized by the antibody, and the macrophages can recognize the antibody and then phagocytose and eliminate the tumor cells.

[0374] As shown in Figure 5B, the PATCH group has a large number of macrophages gathered around the tumor cells, but the macrophages in the other groups do not have obvious aggregation.

[0375] As shown in Figure 5C, the results are the quantitative statistics of the macrophage phagocytosis.

[0376] As shown in Figure 5D, the labeled tumor cells can be recognized by the bispecific antibody, and the T cells are summoned to release cytokines near the tumor cells.

[0377] As shown in FIG. 5E, in the PATCH+BiTE group, the amount of toxic cytokines TNF-a and IFN-g released by T cells was significantly higher than that in other groups. In the PATCH group without BiTE, TNF-a was slightly higher than that in other control groups, while TNF-a in the PATCH+BiTE group was more than 2 times higher than that in other groups, and IFN-g was more than 3 times higher than that in other groups. As shown in FIG. 5F, the lactate dehydrogenase (LDH) in the PATCH+BiTE group, i.e., the cell killing ability, was higher, which was more than 2 times higher than that in other groups.

[0378] As shown in FIG. 5G, under the condition of equal amount of lymphocyte-specific protein tyrosine kinase (LCK), the phosphorylation level of the PATCH+BiTE group was the highest, and the LCK of the HA-FITC+BiTE group also had a certain phosphorylation, which proved the significant activation of the TCR pathway.

[0379] In this example, patient-derived tumor fragments were used to evaluate whether PATCH-mediated receptor activation could activate the response of immune cells to tumor tissue and overcome immune suppression (FIG. 5H). First, three tumor organoid models were established, which were constructed using HER2+ tumor cells from breast cancer, gastric cancer and colorectal cancer patients, respectively. After being labeled with PATCH and co-incubated with autologous PBMCs, BiTE treatment enabled 50% of HER2+ breast and gastric cancer cells and up to 90% of HER2 + Colorectal cancer cells were effectively eliminated, verifying the PATCH-induced T cell killing effect in this organoid model (FIG. 5I).

[0380] As shown in FIG. 5L, the analysis range was expanded to a total of 8 HER2 + Patient-derived tumor tissue fragments (breast cancer x 1, gastric cancer x 1, colorectal cancer x 6). These tissue fragments were freshly cut 1 mm 3 Tumor tissue, co-cultured with PBMCs from the same patient to preserve the structure and composition of the tumor microenvironment as much as possible. With CD69 expression on the cell surface as a marker of T cell activation, it was found that PATCH significantly promoted T cell activation in 7 / 8 tissue fragments (activation level increased by 1.6 to 20 times compared with the untreated control), and only 1 colorectal cancer sample did not respond (FIG. 5J).

[0381] As shown in FIG. 5K and L, TCR activation enhanced by antigen clustering can be synergistic with immune checkpoint inhibition. The results of further experiments showed that in tumor tissue fragments treated with PD-1 antibody and PATCH in combination, both fluorescence imaging of CD69 and detection of effector T cell cytokines were slightly enhanced compared with treatment with PATCH alone.

[0382] Overall, these results demonstrate that PATCH is potent and broadly applicable in inducing T cell activation in the tumor microenvironment.

[0383] Example 5: PATCH-mediated tumor treatment effect

[0384] After determining that PATCH treatment can effectively activate immune activity in Example 4, in this example, the PATCH-mediated tumor treatment effect after intravenous injection of HA-PCN photo / acoustic sensitizer and intratumoral injection of antigen probe FITC-phenol is further evaluated.

[0385] In this example, the use of PCN under PATCH treatment is tail vein injection, the administration of antigen probe FITC-phenol is direct intratumoral injection, and the mice are inoculated with FITC vaccine before PATCH treatment, and anti-FITC antibody is produced by mixing FITC-coupled hemocyanin (KLH) with Freund's adjuvant.

[0386] The process is shown in FIG. 6A. The mice were injected with an immunoadjuvant containing FITC-KLH 8 days and 1 day in advance, respectively, and were injected with HA-PCN 1 to 2 weeks after inoculation of 4T1 tumor, and were injected with substrate and laser treatment the next day after injection, and the treatment lasted for 3 to 4 times.

[0387] First, it is studied whether the red light activated PCN photo / acoustic sensitizer labeling system can effectively label and enrich antigen probe FITC-phenol on 4T1 tumor. And the targeting treatment effect of HA functional modification of PCN photo / acoustic sensitizer on tumor tissue is evaluated. The method is as follows:

[0388] Each Balb / c mouse with 4T1 tumor was injected with antigen probe FITC-phenol intratumorally, and the labeling reaction was performed with 682 nm laser, and then tumor tissues were extracted from mice injected with PBS, PCN and HA-PCN, and the positions of FITC signal were imaged by slicing.

[0389] The results show that by CLSM observation, no observable FITC signal is observed from the tissues of mice injected with PBS and PCN, while the mice treated with HA-PCN nanoparticles (PATCH group) show obvious FITC aggregation within the tumor boundary (FIG. 6B). At the same time, in addition to the above delivery mode of tail vein injection of PCN and intratumoral injection of FITC-phenol, the delivery mode of tail vein injection of PCN and intratumoral injection of FITC-phenol can also achieve FITC labeling in tumor tissue under light excitation (FIG. 6C).

[0390] These results show that the HA-functionalized HA-PCN photo / sonosensitizers can effectively target the tumor microenvironment for FITC-hapten labeling. The modification of HA-PCN can significantly improve the labeling efficiency of tumor tissues in vivo, and achieve the targeted labeling of antigen probes in vivo.

[0391] Next, the effect of PATCH therapy was examined by using 4T1 tumor models, which were subcutaneously implanted and evaluated for the effect of HA-functional modification of HA-PCN on the targeted treatment of tumor tissues. In this experiment, FITC-KLH vaccine-enhanced Balb / c mice with 4T1 tumors were randomly divided into different groups when the average tumor area reached about 30 mm 2

[0392] As shown in FIG. 6D, the 4T1 tumor volume was significantly reduced using HA-PCN and antigen probe FITC-phenol (PATCH group) under 682 nm laser. HA-modified HA-PCN can significantly improve the proliferation inhibition effect of 4T1 tumor, which is reflected in the PCN-treated control group. These results are consistent with the above-mentioned PATCH-mediated FITC labeling results in tumor tissue sections.

[0393] In addition, in order to evaluate the photodynamic therapy (PDT) effect caused by the ROS generated by PCN in PATCH anti-tumor therapy, the PATCH antigen probe FITC-phenol group was designed to eliminate the influence of antigen. At the same time, the HA-FITC group was used to evaluate the antigen modification under the binding of receptor-signal molecules without PATCH-mediated antigen extensive expansion, and the pure antigen probe FITC-phenol treatment was used as a control (PATCH without PCN group).

[0394] As shown in FIG. 6D, after PATCH treatment from day 7 to day 22, the 4T1 tumor volume under PATCH treatment was significantly reduced, while the PATCH without PCN, PATCH without FITC and HA-FITC groups showed slight volume reduction (FIG. 6E). The 4T1 tumor volume treated by PATCH showed the highest anti-tumor effect, with a significant degree of tumor volume reduction until almost disappearing, indicating the strong ability of the antigen extensive expansion strategy (FIG. 6F).

[0395] In addition, the apoptosis of each group was measured after the end of the whole treatment. As shown in FIG. 6G, the proportion of tumor cells in the PATCH treatment group was significantly increased.

[0396] ​As shown in FIG. 6H, mice were injected with FITC-KLH-containing immunoadjuvant 8 days in advance and 1 day in advance, respectively, and injected with HA-PCN two weeks after inoculating 4T1 tumors on both sides of the mice, and the effect of diffusion on the other side was studied by injecting the substrate and laser treatment on one side (1° tumor) the next day. The treatment lasted for three times.

[0397] As shown in FIG. 61, the tumor area of the treated side of the PATCH group decreased significantly after treatment, and the final tumor area was less than 50 square millimeters, while the tumor area of the control group increased significantly, and the final tumor area was more than 150 square millimeters and the mice died.

[0398] As shown in FIG. 6J, the tumor area of the treated side of the PATCH group was basically stable and no longer grew after treatment, and the final tumor area was less than 50 square millimeters, while the tumor area of the control group increased significantly, and the final tumor area was more than 150 square millimeters and the mice died.

[0399] As shown in FIG. 6K, the control group mice began to die around day 20, and around day 30, the control group mice basically all died, and the PATCH treatment group continued to 30 days without mouse death.

[0400] Example 6: Anti-tumor therapeutic effect of PATCH combined with BITE

[0401] This example aims to study the anti-tumor effect of PATCH treatment on antigen amplification after BiTE-mediated cell immune killing in vivo as determined in Example 4. In this example, PATCH combined with BiTE therapy in tumor models is attempted to be evaluated.

[0402] In this experiment, PATCH combined with BiTE therapy mainly relies on intravenous injection of HA-PCN, intratumoral injection of probe FITC-phenol, and irradiation with 682 nm laser, and intravenous injection of FITC-specific BiTE (FIG. 7A).

[0403] When the average area of 4T1 tumors on Balb / c mice reached about 40 mm 2 , they were randomly divided into different groups, then intravenously injected with HA-PCN photo / acoustic sensitizer, and then injected with FITC-specific BiTE via the tail vein. The BiTE sequence is as follows:

[0404] The amino acid sequence of the FITC-specific murine CD3 BiTE is as follows:

[0405] The amino acid sequence of the FITC-specific human CD3 BiTE is as follows:

[0406] The 4T1 tumor volume of the PATCH combined with BiTE treatment group was significantly reduced until almost disappeared from the 8th day to the 15th day after the injection of HA-PCN photo / acoustic sensitizer on the 7th day after the inoculation of tumor, which indicated that the PATCH combined with BiTE strategy had excellent anti-tumor effect.

[0407] As shown in FIG. 7B, the tumor size of the PATCH treatment group was significantly smaller than that of the control group.

[0408] As shown in FIG. 7C, the tumor size of the PATCH treatment group was significantly reduced after treatment, and the final tumor size was less than 20 square millimeters, while the other three groups had no significant downward trend after treatment, and the final tumor size was more than 20 square millimeters.

[0409] As shown in FIG. 7D, the tumor size of the PATCH treatment group was significantly reduced after treatment, and the final tumor size was less than 20 cubic millimeters, while the other control groups had no significant downward trend after treatment, and the final tumor size was more than 20 cubic millimeters.

[0410] As shown in FIG. 7E, the mice were inoculated with tumors on both sides, injected with PCN materials on the 7th day after the inoculation of tumor, and injected with substrates and laser treatment on one side (1° tumor) the next day to study the diffusion effect on the other side. BiTE antibody was injected 8 hours after the end of treatment, and the treatment was performed twice.

[0411] As shown in FIG. 7F, the area of the 1° tumor treated by the PATCH group was significantly reduced after treatment, and the final tumor area was less than 50 square millimeters, while the tumor area of the control group was significantly increased, and the final tumor area was more than 150 cubic millimeters.

[0412] As shown in FIG. 7G, the tumor area of the 2° tumor treated by the PATCH group was basically stable and no longer grew after treatment, and the final tumor area was less than 50 square millimeters, while the tumor area of the control group was significantly increased, and the final tumor area was more than 150 cubic millimeters.

[0413] As shown in FIG. 7H, the control group mice started to die around the 25th day, and the control group mice basically all died around the 29th day, and the PATCH treatment group continued to 30 days without mouse death.

[0414] Example 7: Ultrasound-activated PATCH against HER2 + Tumor treatment effect

[0415] This example aims to evaluate the anti-tumor effect of ultrasound-activated PATCH after determining the anti-tumor effect of light-activated PATCH on antigen amplification in Example 6.

[0416] In this experiment, the sonication PATCH combined with BiTE therapy mainly relied on intravenous injection of HER2-PCN, intratumoral injection of probe FITC-amine and use of 2W / cm 2 ultrasound treatment and intravenous injection of FITC-specific BiTE (Figure 8A).

[0417] When the average area of CT26-HER2 tumors in Balb / c mice reached about 40mm 2 , they were randomly divided into different groups, then intravenous injection of HER2-PCN, followed by tail vein injection of FITC-specific BiTE.

[0418] Mice were injected with HER2-PCN on the 19th, 23rd, and 27th days after tumor inoculation. From the 19th to the 35th day, the CT26-HER2 tumor volume of the ultrasound-activated PATCH combined with BiTE treatment group was significantly reduced until almost disappeared, which indicated that the ultrasound-activated PATCH combined with BiTE strategy had excellent anti-tumor effect.

[0419] As shown in Figure 8B, the timeline of the ultrasound-activated PATCH treatment group tumor.

[0420] As shown in Figure 8C, there was no observable FITC signal in the tumor tissue of the control mice without PCN injection and without ultrasound treatment, and there was obvious FITC signal in the tumor tissue of the mice injected with PCN and ultrasound-activated PATCH group. It was proved that the FITC label in the tumor tissue was activated by sound.

[0421] The tumor size of the PATCH treatment group was significantly reduced after treatment, and the final tumor size was less than 20 square millimeters, while the other three groups had no significant downward trend after treatment, and the final tumor size was more than 100 square millimeters (Figure 8D left). The survival curves of each group were further detected. The results showed that the survival curve of the PATCH treatment group was significantly greater than that of the control mice (Figure 8D right).

[0422] The timeline of the ultrasound-activated PATCH combined with BiTE therapy for the treatment of PBMC humanized NKG mouse tumors is shown in Figure 8E left. It mainly relied on intravenous injection of HER2-PCN, intratumoral injection of probe FITC-amine and use of 2W / cm 2 ultrasound treatment and intravenous injection of FITC-specific BiTE (Figure 8A). When the average area of SK-OV-3 tumors in PBMC humanized NKG mice reached about 20mm 2 , they were randomly divided into different groups, then intravenous injection of HER2-PCN, followed by tail vein injection of FITC-specific BiTE (Figure 8E).

[0423] As shown in FIG. 8F, the tumor size of the PATCH treatment group significantly decreased after treatment, and the final tumor size was less than 10 square millimeters, while the other three groups had no significant downward trend after treatment, and the final tumor size was greater than 40 square millimeters.

[0424] Example 8: Anti-tumor therapeutic effect of PATCH combined with monoclonal antibody

[0425] In this example, PATCH combined with monoclonal antibody (mAb) therapy in tumor models was attempted to be evaluated.

[0426] In this experiment, PATCH combined with mAb therapy mainly relied on intravenous injection of HER-PCN, intratumoral injection of probe FITC-phenol and irradiation with 682 nm laser, or intratumoral injection of FITC-amine and treatment with 2 W / cm 2 ultrasound, and intraperitoneal injection of FITC-specific monoclonal antibody (FIG. 9).

[0427] When the average area of the CT26-HER2 tumor carried by the Balb / c mice reached about 30 mm 2 , they were randomly divided into different groups, then intravenous injection of HER2-PCN photo / acoustic sensitizer, intratumoral injection of FITC hapten probe and photo / acoustic treatment, followed by intraperitoneal injection of anti-FITC monoclonal antibody. The control groups were set as injection of PBS, PATCH (photo / acoustic) treatment without injection of mAb, injection of HER2 mAb without PATCH treatment group, respectively.

[0428] The mice were injected with HER2-PCN photo / acoustic sensitizer on the 14th day after tumor inoculation, and the CT26-HER2 tumor volume of the PATCH combined with mAb treatment group significantly decreased until almost disappeared from the 16th day to the 30th day, which indicated that the PATCH combined with mAb strategy had excellent anti-tumor effect.

[0429] As shown in FIG. 9B, the tumor size of the PATCH combined with mAb treatment group significantly decreased, and the final tumor size was less than 10 square millimeters. As shown in FIG. 9C, the other four control groups had no significant downward trend, and the final tumor size was greater than 50 square millimeters.

[0430] Discussion

[0431] The application of immunotherapy in solid tumors is limited by the lack of effective targeted antigens, and few targeted antigens meet the following requirements: one is that there is enough antigen density on the surface of tumor cells to prevent escape and drug resistance; two is that it has high tumor specificity to reduce the targeting of healthy tissues. In this disclosure, we call the strategy PATCH, which solves these existing challenges by spatiotemporally amplifying exogenously introduced FITC hapten probes on the surface of tumor cells through light-excited proximity labeling. When used in combination with FITC-KLH vaccine or FITC-specific BiTE, PATCH triggers the activation of immune effector cells (such as macrophages and T cells) and anti-tumor immune response, resulting in tumor shrinkage and significantly prolonged survival in multiple syngeneic mouse solid tumor models. Importantly, PATCH-guided tumor killing triggers the activation of antigen-presenting cells, leading to endogenous immune attack on untreated distal tumors.

[0432] PATCH provides a new choice for antigen-engineered immunotherapy and has unique advantages. First, the use of catalytic labeling reactions ensures that cell surface-targeted PCN photo / sonosensitizers can repeatedly convert various FITC-phenol substrates, thus significantly increasing the amount of antigen (Figure 4). Second, the highly reactive phenoxyl radical intermediate covalently labels the proximal available proteins in the nanoscale range, and this labeling can locally produce high-density antigen clusters. Recent studies have confirmed that the enhancement of antigen intensity can effectively trigger immune receptor aggregation and immune effector cell activation. Previous studies have attempted to use FITC-coupled tumor-targeting ligands, such as FA-FITC, FITC-specific antibodies, or CAR-T cells, to achieve anti-tumor effects. However, the anti-tumor response triggered by these non-covalent, non-amplified, and non-clustered ligands is far less effective than PATCH (Figures 7 and 8), and can ultimately lead to antigen escape and drug resistance. At the same time, the specificity of PATCH in this disclosure can further control the labeling range through light irradiation to avoid off-target toxicity in vivo, which greatly expands the application range of the PATCH strategy. Most of the tumors shown in this disclosure overexpress antigens such as CD44 and FOLR (folate receptor), and the direct treatment targeting antigen selection often has problems in terms of selectivity, because antibody drug conjugates or CAR-T cells targeting tumor-associated antigens (such as HER2, CD44, and mesothelin) all or mostly exhibit potential off-target toxicity due to off-target cell expression. However, in this disclosure, they are suitable for the treatment strategy of PATCH regardless of their expression in healthy tissues outside the tumor or their success in current immunotherapy.

[0433] It is important to note that although the current protocol of the present disclosure involves intravenous injection of the photo / sonosensitizer and intratumoral injection of FITC-phenol, potent FITC labeling can also be achieved by intravenous co-administration of the catalyst and substrate (Figure 7B). Moreover, the cure of the tumor model constructed by the present disclosure only requires 2-3 doses of PATCH treatment (Figures 7 and 8), which greatly improves the feasibility of its clinical application.

[0434] In summary, the present disclosure demonstrates that photoexcitation proximity labeling provides a novel strategy for cell antigen engineering to achieve high density and high specificity of tumor surface antigens, which means that the PATCH strategy can improve the efficacy of cancer immunotherapy that requires a certain threshold level of antigens. With the rapid development of in vivo proximity labeling tools currently used to study molecular interactions, PATCH opens the door to another new application of these technologies. Combined with other advances in cancer immunotherapy, it is expected that the optimization and application of PATCH will improve the current antitumor efficacy of solid tumors.

[0435] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present disclosure upon reading and understanding the above lecture of the present disclosure, and it is intended that the present disclosure encompass all such alterations and modifications within the scope of the appended claims.

Claims

1. A combination of active ingredients, characterized in that, The active ingredient combination comprises: (a) an artificial antigen carrying a reactive group; (b) a catalyst; and (c) an immunotherapeutic agent, wherein, under the catalysis of the catalyst, the artificial antigen is covalently linked to a reactive group on the cell surface through the reactive group carried by the artificial antigen, thereby labeling the artificial antigen on the cell surface to obtain a cell with the cell surface labeled with the artificial antigen, and then the immunotherapeutic agent is used to specifically target the artificial antigen, thereby triggering an immune response.

2. The active ingredient combination as claimed in claim 1, characterized in that wherein, the reactive group is selected from the group consisting of a phenol compound, an amine compound, a hydrazine compound, a benzoquinone compound, an aryl-urea compound, an aryl azide, a bis-aziridine compound, or a transpeptidation substrate; the artificial antigen is a hapten, wherein the hapten is selected from one or more components of the group consisting of fluorescein (FITC), biotin, dinitro (DNP), or Galα1-3Galβ1-4GlcNAc-R (αGal); the catalyst is selected from the group consisting of a photo / sonosensitizer, or a biological enzyme, wherein the photo sensitizer is selected from the group consisting of a porphyrin photo sensitizer, a heavy metal iridium ruthenium tin osmium complex photo sensitizer, an acridine orange photo sensitizer, a fluorescein photo sensitizer, an eosin photo sensitizer, an oxazine photo sensitizer, a methyl blue photo sensitizer, a phycocyanin photo sensitizer, a rhodamine photo sensitizer; the sonosensitizer is selected from the group consisting of a porphyrin sonosensitizer, a phthalocyanine sonosensitizer, a rhodamine sonosensitizer, an inorganic nanomaterial sonosensitizer, a sonosensitizer producing reactive oxygen species through acoustic cavitation effect; the biological enzyme is selected from the group consisting of a peroxidase, a biotin ligase, a glycosyltransferase, a tyrosinase, a ubiquitin ligase, a transpeptidase, a protein photo sensitizer; the immunotherapeutic agent comprises one or more components selected from the group consisting of a monospecific antibody, a bispecific antibody, a multispecific antibody, a monoclonal antibody, or a polyclonal antibody, wherein the bispecific antibody is BiTE, and the monoclonal antibody is a FITC monoclonal antibody.

3. The active ingredient combination as claimed in claim 1, characterized in that wherein, the ratio of the number of the artificial antigen labeled on the cell surface N1 to the number of the cell surface antigen N0 (N1 / N0) is ≥5, preferably ≥10, more preferably ≥50, and most preferably ≥100 or more; in the cell with the cell surface labeled with the artificial antigen, the density of the artificial antigen is ≥60% per cell, preferably ≥70% per cell, more preferably ≥80% per cell, and most preferably 60%-90% per cell.

4. The active ingredient combination as claimed in claim 2, characterized in that wherein, the photo / sonosensitizer is a cell surface antigen targeting photo / sonosensitizer.

5. The active ingredient combination as claimed in claim 4, characterized in that The cell surface antigen targeting photo / sonosensitizer has a structure shown in Formula I: (Z1-L)n-Z2 (I) in the formula, Z1 is a targeting element; L is nothing, a covalent bond, a non-covalent bond, or physical adsorption; Z2 is a photo / sonosensitizer; n is an integer ≥1.

6. Active ingredient combination according to claim 5, characterized in that Z1 is selected from the group consisting of folic acid, hyaluronic acid, a HER2 antibody, a mesothelin antibody, or a combination thereof.

7. Active ingredient combination according to claim 6, characterized in that L is selected from the group consisting of a covalent bond, a non-covalent bond, physical adsorption, or a combination thereof.

8. A kit characterized in that, The kit contains the active ingredient combination of claim 1.

9. Use of the active ingredient combination as claimed in claim 1, characterized in that for preparing a preparation or composition for: (i) antigen amplification of a predetermined antigen on the surface of target cells through proximity labelling reaction; and (ii) immunotherapy of the target cells.

10. A method of artificially antigenic labelling a target cell surface in vivo or in vitro, characterised in that, The method comprises the steps of: (a) providing cells, and the artificial antigen and catalyst in the active ingredient combination of claim 1, which carry reactive groups; and (b) labelling the surface of the cells with the artificial antigen, wherein, under the catalysis of the catalyst, the artificial antigen is covalently linked to the reactive groups on the surface of the cells through the reactive groups carried by the artificial antigen, so as to label the artificial antigen on the surface of the cells, and obtain cells with artificial antigen labelled on the cell surface.

11. The method of claim 10, wherein, Before step (a), the method further comprises a method for preparing a catalyst, which is a light / acoustic sensitizer targeting cell surface antigens, and the method comprises the following steps: (1) mixing the light / acoustic sensitizer and the transition metal element in an organic solvent environment to obtain a light / acoustic sensitizer dispersion; (2) adding a targeting element to the light / acoustic sensitizer dispersion and performing cross-linking reaction, so as to obtain a light / acoustic sensitizer targeting cell surface antigens.

12. A treatment system or device, characterized by The treatment system or device comprises: (z1) an antigen amplification module, which is configured to apply the artificial antigen and catalyst in the active ingredient combination of claim 1, which carry reactive groups, to a subject, so that in the body of the subject, under the catalysis of the catalyst, the artificial antigen is covalently linked to the reactive groups on the surface of the cells through the reactive groups carried by the artificial antigen, so as to label the artificial antigen on the surface of the cells; and (z2) optionally, an immunotherapy module, which is configured to apply the immunotherapy agent in the active ingredient combination of claim 1 to the subject, and the immunotherapy agent is targeted to the artificial antigen.

13. The treatment system or device of claim 12, wherein, The treatment system or device further comprises: (z3a) a light source module, which is configured to irradiate a predetermined part of the subject to catalyze the covalent linkage of the reactive groups carried by the artificial antigen to the reactive groups on the surface of the cells, and the light source module provides a red light source.

14. The treatment system or device of claim 12, wherein, The treatment system or device further comprises: (z3b) a sound source module, which is configured to ultrasonically irradiate a predetermined part of the subject to catalyze the covalent linkage of the reactive groups carried by the artificial antigen to the reactive groups on the surface of the cells, and the sound source module provides ultrasonic waves.

15. The treatment system or device of claim 13 or 14, wherein, The treatment system or device further comprises: (z4) an artificial antigen detection module, which is configured to detect the density or amplification level of the artificial antigen.

16. A treatment method, characterized in that, The method comprises the steps of: (a) applying a preparation or composition to a subject, so that the preparation or composition performs antigen amplification of a predetermined antigen on the surface of target cells in the subject through proximity labelling reaction, so as to label the surface of the target cells with artificial antigens; and (b) immunotherapy with an immunotherapy agent against the artificial antigen.

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