CN116421738B - Oncolytic virus for capturing antigen and pharmaceutical composition, preparation and application thereof - Google Patents

Oncolytic virus for capturing antigen and pharmaceutical composition, preparation and application thereof Download PDF

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CN116421738B
CN116421738B CN202310171283.5A CN202310171283A CN116421738B CN 116421738 B CN116421738 B CN 116421738B CN 202310171283 A CN202310171283 A CN 202310171283A CN 116421738 B CN116421738 B CN 116421738B
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antigen
oncolytic virus
capturing
tumor
mal
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刘福囝
徐臣
陈丽婷
黄翰伟
段石杰
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First Hospital of China Medical University
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Abstract

An oncolytic virus for capturing antigen and a pharmaceutical composition, preparation and application thereof belong to the field of nano-drugs. The oncolytic virus for capturing the antigen is an oncolytic virus modified by an active group with an antigen capturing function. The tumor antigen which is cracked by the oncolytic virus can be captured and delivered to the lymph node to enhance the specific anti-tumor immunity and improve the adaptive immunity of the anti-tumor by carrying out the universal modification active group on the outer part of the oncolytic virus without affecting the killing capacity of the oncolytic virus. Finally, when the oncolytic virus capturing the antigen and indoleamine 2,3 are added with an oxidase inhibitor, the local negative feedback in the tumor immune microenvironment is overcome, and the combined scheme can induce lasting immune memory and inhibit tumor recurrence.

Description

Oncolytic virus for capturing antigen and pharmaceutical composition, preparation and application thereof
Technical Field
The invention relates to the field of nano-drugs, in particular to an oncolytic virus for capturing antigens, and a pharmaceutical composition, a preparation method and an application thereof.
Background
Cancer morbidity and mortality are still rising rapidly worldwide. In current clinical practice, early stage tumors are treated by surgery, while late stage tumors are treated by radiation, chemotherapy and immunotherapy. Whether it is intensity modulated radiotherapy, small molecule chemotherapy drugs or immune related checkpoint inhibitors, the drug has the problems of large toxic and side effects, low specificity, multiple drug resistance and the like. Oncolytic virus immunotherapy is a novel approach to the treatment of cancer that selectively destroys tumor tissue without unduly damaging normal non-cancerous cells. At present, a plurality of oncolytic virus medicaments are marketed in batches. In 2003, P53 gene modified adenovirus (genicine, china) was the first approved oncolytic virus product. Talimogene laherparepvec (T-VEC) is a transgenic herpes simplex virus type 1 (Herpes simplex virus type, HSV-1) that replicates and expresses the immune activating protein Granulocyte-macrophage colony stimulating factor (Granulocyte-macrophage colony-stimulating factor, GM-CSF) in tumor cells. In 2015, U.S. food and drug administration (Food and Drug Administration, FDA) and European drug administration (European Medicines Agency, EMA) approved T-VECs for the treatment of advanced melanoma. In addition, RIGVIR is also sold in Lativia, grragia and Amania. All of these oncolytic viral drugs are intratumoral injections, aimed at refractory malignant patients resistant to two-wire and even three-wire treatment regimens. In recent years, with the good safety of oncolytic viruses in clinical practice, a large number of oncolytic virus clinical trials have been conducted. From 2000 to 2020, there have been 97 clinical trials using OVs, treating more than 3000 cancer patients, but the results indicate that oncolytic viruses are ineffective against solid tumors with an objective remission rate of only 9%. Thus, increasing the efficacy of oncolytic viruses, particularly against metastatic and recurrent lesions, is an urgent clinical need.
The antitumor activity of oncolytic viruses is generally mediated by two mechanisms: first, oncolytic viruses are able to induce tumor cell lysis; secondly, after oncolytic virus lyses tumor cells, specific tumor-associated antigens are released, promoting T cells to recognize tumor antigens, thereby establishing tumor-specific T cell immunity. The selective replication of the virus in tumor cells was initially thought to play a major role, but recent research results suggest that the anti-tumor efficacy of oncolytic viruses is mediated by immune responses rather than direct oncolysis. The adaptive immunity has three main advantages, namely, good safety; secondly, the specificity is strong, and besides killing primary tumor, the medicine has strong curative effect on metastasis; the last point is that it can produce immunological memory function and resist re-invasion of tumor. These advantages are also consistent with clinical use of oncolytic viruses.
Oncolytic Viruses (OVs) entering and being tested clinically are mostly genetically engineered to locally release co-stimulatory molecules, chemokines, cytokines and immune checkpoint inhibitors to enhance the therapeutic effects of OVs. OVs have the potential to induce immunogenic cell death, leading to the release of tumor antigens, risk-associated molecular patterns and type I interferons. These immunogenic substances, in particular tumor antigens, can activate systemic tumor-specific immune responses, leading to tumor regression, whether distant or uninfected. However, the immune response to activation is often limited and insufficient to effectively attack the tumor, mainly due to poor immunogenicity and efficiency of use of soluble tumor antigens. Thus, increasing the immunogenicity and efficiency of utilization of tumor antigens released after oncolysis may be a potential strategy to increase OVs clinical response.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an oncolytic virus for capturing an antigen, and a pharmaceutical composition, preparation and application thereof. Finally, when the oncolytic virus capturing the antigen and Indoleamine 2,3-dioxygenase (IDO) inhibitor are used, the local negative feedback in the tumor immune microenvironment is overcome, and the combined scheme can induce lasting immune memory and inhibit tumor recurrence.
The oncolytic virus for capturing the antigen provided by the invention complements the oncolytic virus which is subjected to internal modification by genetic engineering, and provides a new strategy for overcoming the clinical bottleneck of poor curative effect of oncolytic virus solid tumors. The oncolytic virus for capturing the antigen has the characteristics of good biocompatibility, strong stability, high safety, high bioavailability and the like.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
the invention provides an oncolytic virus for capturing an antigen, which is an oncolytic virus modified by an active group with an antigen capturing function.
The oncolytic virus is preferably expressed Telomerase (TERT) promoter and AD 5 Enhancer genetically engineered oncolytic adenovirus AD 11 。
The active group with the antigen capturing function is one of NHS (N-Hydroxy succinimide) -PEG (Polyethylene glycol) -Mal (Maleimide) groups and PEI (poly (ethylene imine)) -PEG-PEI groups. Wherein Mal binds to the protein (tumor derived antigen) by forming stable thioether bonds; PEI binds to proteins (antigens of tumor origin) by ionic interactions.
The preparation method of the oncolytic virus for capturing antigen comprises the following steps:
and incubating OVs with excessive active groups with antigen capturing function at normal temperature, removing the active groups without reaction through a filter membrane and an ultrafiltration tube, and re-adding PBS (phosphate buffer solution) into the ultrafiltration tube for resuspension to obtain the oncolytic virus capturing the antigen.
Preferably, the oncolytic virus that captures the antigen is AD 11 -NHS-PEG-Mal(AD 11 -Mal)，AD 11 -PEI-PEG-PEI(AD 11 -PEI). The AD 11 Rich amino groups are present on the polymer and can be bonded with NHS esterCombining; AD (analog to digital) converter 11 Obviously negative electricity can be combined with positive electricity groups.
Preferably, the method for preparing oncolytic viruses that capture antigens comprises: AD is added with 11 Incubation with NHS-PEG-Mal, PEI-PEG-PEI at different concentrations at room temperature (20-25 deg.), removing unreacted active groups by a filter membrane and an ultrafiltration tube, and staining PEG with barium iodide to determine the maximum preparation amount of reaction (the PEG band is not obviously widened and deepened). Wherein, AD is calculated by the ratio of solute number to virus particle number 11 ：NHS-PEG-Mal(3.2×10 6 /vp)；AD 11 ：PEI-PEG-PEI(1.6×10 6 /vp)。
Preferably, in the active group with antigen capturing function, the molecular weight of PEG is 1-5Kda, and the molecular weight of PEG is more than 5Kda, so that the infection activity of oncolytic virus is weakened.
Preferably, in the super-separation tube, the low-speed centrifugation revolution is 5000-10000rpm, and the centrifugation is carried out for 5-10min;
preferably, the filter membrane used has a pore size of 0.45 μm and 0.22. Mu.m;
preferably, the ultrafiltration tube used has a molecular weight cut-off of 50-100kDa;
preferably, the oncolytic virus that captures the antigen is stored at-80 ℃ for later use.
The particle size of the oncolytic virus for capturing the antigen prepared by the invention is 116-120nm, the dispersion index PDI is 0.2-0.21, the particle size is more uniform (PDI is less than 0.3), the nano particles in the range are stable, the lymph node enrichment effect is realized, the antigen presenting cell uptake is facilitated, and the targeting and bioavailability of the antigen and the adjuvant can be improved.
Before the active group with antigen capturing function is incubated, hydration reaction is needed, and the specific process of the hydration reaction is as follows:
uniformly mixing one powder of active groups NHS-PEG-Mal and PEI-PEG-PEI with an antigen capturing function with PBS in a proportion of 1-10mg/ml, and carrying out hydration reaction to obtain a solution, wherein the hydration reaction is carried out at room temperature for at least 30mins to obtain an aqueous solution of the active groups with the antigen capturing function, and preserving the aqueous solution at 4 ℃;
preferably, the incubation is: uniformly mixing an aqueous solution of active groups with an antigen capturing function with oncolytic viruses, and then performing ultrasonic treatment at room temperature; wherein the ultrasonic time is 30-60min, and the room temperature is 20-25 ℃.
Further, the ultrasonic power is 3-5 w.
The present invention contemplates that peripheral mature T cells will produce a large number of Treg cells upon sustained antigen stimulation; therefore, the invention also provides a pharmaceutical composition for overcoming the Tregs-mediated immunosuppressive microenvironment by inhibiting IDO-mediated tryptophan to kynurenine conversion by combination.
A pharmaceutical combination comprising an oncolytic virus capturing an antigen and an IDO inhibitor.
Preferably, the IDO inhibitor is preferably 1-methyltryptophan (1-methyl-trphan, 1-MT) 1-MT.
The invention relates to an application of an oncolytic virus for capturing an antigen or a pharmaceutical composition thereof, which is an application of a medicine for treating malignant solid tumor diseases.
The application is as follows: 1-MT and AD 11 -Mal，AD 11 One of PEI is mixed together and is used for administration by intratumoral injection.
Compared with the prior art, the invention has the following beneficial effects:
oncolytic viruses are novel immunotherapies, but their mediated in situ killing and anti-tumor immunity generally do not have a durable effect on solid tumors. In preclinical studies and clinical trials, there are generally two approaches to enhance the anti-tumor efficacy of oncolytic viruses. First, deletion of non-essential viral genes to enhance tumor cell replication and reduce viral pathogenicity; OVs are then equipped with co-stimulatory molecules, chemokines, cytokines and immune checkpoint inhibitors to release the immunosuppressive microenvironment surrounding the tumor. These strategies rely on recombinant viral gene expression of one or more transgenes. The present invention is a strategy for killing tumors using oncolytic viruses while capturing antigens and delivering and activating antigen presenting cells to enhance anti-tumor immune efficacy. The active groups of Mal, PEI and the like which can capture antigen and AD 11 Ligation to capture self-solubilised released tumour antigen. This is quite different from previous strategies to enhance oncolytic viral efficacy and may be synergistic with these strategies. First, modification of the oncolytic viral surface does not affect the expression of its transgene. Second, expression of transgenic oncolytic viruses that promote viral replication will promote antigen release, while expression of transgenic oncolytic viruses that improve the immune microenvironment will attract more APCs to the vicinity of the tumor, in concert with strategies for releasing and capturing antigen.
Utilization of AD of the present invention 11 Nano-sized of (a) by AD 11 Capturing tumor antigen in situ and transferring it into draining lymph node, thereby improving the utilization efficiency of released tumor antigen, the invention utilizes AD 11 The surface contains abundant chemical reaction sites, and the surface is modified with active groups to capture antigens released in situ by microwave ablation, radiotherapy or photothermal therapy. Then, the oncolytic virus capturing the antigen is effectively drained to the lymph node due to its nano-sized effect, thereby improving the utilization efficiency of the released tumor antigen and inducing a strong anti-tumor immune response. In addition, AD 11 Is immunogenic and thus can assist tumor antigens in activating an immune response.
Drawings
FIG. 1A is AD in example 1 11 Electron microscope and particle size characterization of tumor vaccine, scale is 100nm;
FIG. 1B is an AD of example 1 11 -electron microscopy and particle size characterization of Mal tumor vaccine, scale 100nm;
FIG. 2 is AD in example 2 11 AD and AD 11 -the potential of PEI tumor vaccine;
FIG. 3A is AD in example 3 11 Mal and AD 11 Transfection efficiency for TC-1 cells at titers of 10pfu/cell and 20 pfu/cell;
FIG. 3B is AD in example 3 11 Mal and AD 11 Killing of TC-1 cells at titers of 10pfu/cell and 20 pfu/cell;
FIG. 4A shows AD in example 4 11 AD (analog to digital) converter 11 -particle size change of Mal after capture of cell lysate;
FIG. 4B shows AD in example 4 11 AD (analog to digital) converter 11 -relative mass of cell lysate that Mal can capture;
FIG. 5A shows AD in example 5 11 AD (analog to digital) converter 11 -the relative abundance of Mal captured DAMPs;
FIG. 5B shows AD in example 5 11 AD (analog to digital) converter 11 -the relative abundance of the Mal captured neoantigen;
FIG. 6A is a flow cytometry quantitative determination of Cy5.5 after uptake of Cy5.5-labeled cell lysate by BMDCs for 5h in example 6 + Percentage of BMDCs;
FIG. 6B is a graph showing the cell maturation of BMDCs, as quantified by flow cytometry, 5 hours after treatment in example 6.
FIG. 7A is an intratumoral injection of free SH-Cy5.5 and AD in example 7 11 -Mal-SH-Cy5.5, in vitro fluorescence image of draining lymph nodes after 12h, scale 50nm;
FIG. 7B is an intratumoral injection AD of example 7 11 Immunofluorescent staining of draining lymph nodes 12h after Mal-SH-Cy5.5. Fluorescent B220 and CD3 antibodies were used to label B cell and T cell regions, respectively;
FIG. 8A is a tumor growth curve of primary and distant tumors in example 8;
FIG. 8B is the tumor weight of primary and distant tumors after 14 days in example 8;
FIG. 9A shows the proportion of cytotoxic T lymphocytes from primary and distant tumors as quantified by flow cytometry in example 8;
FIG. 9B shows the proportion of regulatory T lymphocytes from primary and distant tumors as quantified by flow cytometry in example 8;
FIG. 10A is a flow cytometry analysis of CD8 in example 8 + Spleen cell CD3 after treatment of T cell failure mouse model + CD8 + Killer T lymphocytes;
FIG. 10B is a schematic diagram of CD8 in example 8 + Tumor growth curves for primary and distant tumors in the T cell failure mouse model;
FIG. 11A is a graph showing tumor growth curves of primary and distant tumors of mice pre-existing immune models after administration in example 9;
FIG. 11B is a photograph of a tumor of example 9 after the treatment of a pre-existing immune model mouse, on a scale of 1cm;
FIG. 12A is a graph showing tumor growth curves of primary and distant tumors of the combination model mice after drug administration in example 10;
FIG. 12B is a photograph of a tumor of example 10 after the end of treatment of the combined model mice, on a scale of 1cm;
FIG. 13A shows the proportion of cytotoxic T lymphocytes from primary and distant tumors after treatment in combination with model mice in example 10, as quantified by flow cytometry;
FIG. 13B is a graph showing the proportion of regulatory T lymphocytes from primary and distant tumors after treatment in combination with model mice in example 10, as quantified by flow cytometry;
FIG. 14 shows the control group and AD in example 11 11 -tumor growth curve of distant tumors in mal+1-MT group after tumor cell restimulation;
FIG. 15 shows the control group and AD in example 11 11 -mal+1-MT group distant tumors, picture of distant tumors after tumor cell restimulation, scale 1cm.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
In the following examples, telomerase (TERT) promoter and AD were expressed 5 Enhancer-engineered oncolytic virus AD 11 Is a gift of Beijing biological targeted therapy technology limited company and Zhengzhou university.
In the following examples, NHS-PEG 1000 Mal is representative of the active group capturing the antigen, purchased from Alasdine (Shanghai).
In the following examples, 1-MT was purchased from Shanghai Seiyaka Biotechnology Co., ltd.
Example 1
In this example, an oncolytic virus AD that captures an antigen was constructed by the following method 11 Mal, method of:
(1) mu.L of Mal-PEG 1000 NHS solution (1 mg/mL) with 100. Mu.L of AD 11 (1×10 9 pfu/mL) was mixed and incubated at room temperature for 1h under ultrasound. Centrifuging at 10000rpm at 4deg.C for 5-10min, and removing excessive Mal-PEG by ultracentrifugation 1000 -NHS；
(2) Ultrafiltration of the solution obtained in (1) using a 100kDa ultrafiltration tube to remove unreacted NHS-PEG 1000 Mal, dissolving residues in the filter membrane with PBS, namely AD 11 -Mal solution and kept for future use at-80 °;
(3) The AD obtained was measured by means of a transmission electron microscope (FEI, tecnai G2S-TWIN, 200 kV) and a laser particle sizer (Malvern, zetasizer Nano ZS, UK) 11 AD (analog to digital) converter 11 Morphological and particle size characterization of Mal tumor vaccine as shown in FIG. 1, wherein FIG. 1A is naked AD 11 A transmission electron microscope image of the tumor vaccine with the particle size of about 101.1nm and a dispersion index (PDI) of 0.004; FIG. 1B shows the prepared AD 11 Mal tumor vaccine, spherical with edge compared to AD 11 Blurring, a relatively uniform particle size, a particle size of about 117.9nm, and a dispersion index (PDI) of 0.209; AD with bare 11 In comparison, the particle size is increased by about 15nm, and the change of the shape of the electron microscope and the increase of the particle size indicate that we successfully prepare AD 11 -Mal。
Example 2
In this example, an oncolytic virus AD that captures an antigen was constructed by the following method 11 -PEI, the method being:
(1) 600. Mu.L of PEI-PEG 1000 PEI solution (1 mg/mL) with 100. Mu.L of AD 11 (1×10 9 pfu/mL) was mixed and incubated at room temperature for 1h under ultrasound. Centrifuging at 10000rpm at 4deg.C for 5-10min, and removing excessive PEI-PEG by ultracentrifugation 1000 -PEI；
(2) Ultrafiltration of the solution obtained in (1) using a 100kDa ultrafiltration tube to remove unreacted PEI-PEG 1000 -PEI, dissolving residues in the filter membrane with PBS, namely AD 11 -PEI solution and at-80 DEGStoring for standby;
(3) The AD obtained was subjected to a laser particle sizer (Malvern, zetasizer Nano ZS, UK) 11 AD (analog to digital) converter 11 Potential characterization of PEI tumor vaccine, as shown in FIG. 2, potential was determined by AD 11 From-10 mV to AD 11 PEI 15mV, indicating that we were successful in preparing AD 11 -PEI。
Mal is safer than PEI and is easier to produce. Thus, the following examples are presented as AD 11 Mal is an example.
Example 3
The present embodiment aims at verifying AD 11 Infection and killing function of Mal on tumors.
(1) Taking TC-1 cells in logarithmic phase in incubator, adding culture medium to adjust cell suspension concentration to 1×10 7 cell/ml was plated in 48-well cell culture plates with 100. Mu.l cell suspension per well, at which time the cell density in each well to be measured in each 48-well plate was 1X 10 6 And (3) setting 18 experimental holes. The cell suspension was diluted to a concentration of 1X 10 by adding the culture medium 5 cell/ml was plated in 96-well cell culture plates at 100. Mu.l cell suspension per well, at which time the cell density in each well to be measured in each 96-well plate was 1X 10 4 Holes, a total of 27 experimental holes were set.
(2) Cells were cultured conventionally for 14-16h, and the plates were removed when the cells were fully adherent but not proliferating.
(3) For cells in 24-well plates, 18 experimental wells were divided into groups A, B, 9 wells, 10ml PBS was added to the first 3 wells of group A, and 1X 10 was added to the middle 3 wells 9 Ad of pfu/cell 11 10ml, i.e. 10pfu/cell per well, and finally 1X 10 is added to 3 wells 9 Ad of pfu/cell 11 Mal 10ml, i.e.10 pfu/cell per well. 20ml PBS was added to the first 3 wells of group B, and 1X 10 was added to the middle 3 wells 9 Ad of pfu/cell 11 20ml, 20pfu/cell per well, and finally 1X 10 in 3 wells 9 Ad of pfu/cell 11 Mal 20ml, 20pfu/cell per well.
(4) After the cells were cultured for 24 hours conventionally, the cells were taken out and fluorescence of GFP in the cells was detected by a flow cytometer, as shown in FIG. 3A, 10pfu/cell and 20pfu/cell for AD 11 -MalUptake and transfection efficiency of TC-1 cells and naked AD 11 And consistent.
(5) For cells in 96-well plates, 27 wells were divided into groups A, B, C, 9 wells, 10ml PBS was added to the first 3 wells of group A, and 1X 10 was added to the middle 3 wells 9 AD of pfu/cell 11 10ml, i.e. 1000pfu/cell per well, and finally 1X 10 is added to 3 wells 9 AD of pfu/cell 11 Mal 10ml, 1000pfu/cell per well. The doses for groups B and C were reduced 10-fold in sequence, resulting in 100pfu/cell and 10pfu/cell per well.
(6) After conventional culturing of the cells for 3Day, the cells were removed, 100ul of CCK8 mixed solution (CCK8: 1640 medium: 1:10) was added to each well, incubated in a 37℃incubator for 0.5h, and the light absorption measured at 450nm by CCK8. Only cells cultured in medium served as positive control (maximum OD) and medium served as negative control (minimum OD). Cell viability was calculated as follows: cell viability (%) = (OD experiment-OD negative control)/(OD positive control-OD negative control) ×100. As shown in FIG. 3B, AD in 1000pfu/cell,100pfu/cell and 10pfu/cell 11 Efficiency of Mal to kill TC-1 cells and naked AD 11 And consistent.
The oncolytic virus of this example is AD with GFP fluorescence 11 . The cells are modified non-small cell lung cancer (TC-1) cancer cell lines that express the human CD46 receptor.
Example 4
The present embodiment aims at verifying AD 11 Capture ability of Mal for tumor antigen.
(1) Preparation of antigen in vitro:
collection of 3X 10 7 TC-1 cells of (A) in a 15ml centrifuge tube, placing the cells into an ultrasonic cell pulverizer for pulverization at a temperature of: 4 °, power: 35W, time: 5s for a total of 20min. Centrifuging TC-1 cell lysate, 12000 r.s, 5min, removing cell debris deposited on the bottom of the tube, and storing the cell lysate in a-80 deg. refrigerator for use.
(2) After antigen capture, AD 11 AD (analog to digital) converter 11 Variation of Mal particle size
PBS and AD 11 And AD (analog to digital) 11 -Mal(5×10 6 pfu) is dissolved in cell lysisIn the solution (2 mg/ml), the unbound cell lysate was removed by centrifugation at 12000rpm using a super centrifuge tube (MWCO 200 KD) after incubation at 4℃for 60 min. The residue in the filter was dissolved in PBS as a control group after antigen capture, as shown in FIG. 4A, AD 11 And AD (analog to digital) 11 Mal size was changed after incubation, but AD 11 Particle size change ratio AD of Mal 11 More remarkable.
(3) Capturing relative mass of cell lysate
By AD 11 Or AD (analog to digital) 11 -post-Mal protein concentration minus pre-Mal protein concentration, thereby profiling AD 11 Or AD (analog to digital) 11 Total protein mass captured by Mal. As shown in FIG. 4B, AD 11 Mal ratio AD 11 More cell lysate was captured.
Example 5
The present embodiment aims at verifying AD 11 Mal captures the components of the antigen lysate.
This is of no significance if the captured protein is not an antigen capable of mediating a tumor-specific immune response. Thus, the present invention identifies AD by mass spectrometry 11 And AD (analog to digital) 11 Diversity and composition of Mal captured proteins. Briefly, raw data were obtained by protein extraction, quantification, detection, cleavage and desalting, component separation and mass spectrometry detection. 67 raw data files were analyzed using the proteome finder version 2.2 (sequence HT, sammer femto science). The peak list is searched in the mus_museulus_uniprot_2022_1_27.fasta (86515 sequences) database. AD (analog to digital) converter 11 The relative abundance of Mal captured proteins was determined by dividing the sample abundance (normalized) value by the TC-1 tumor cell abundance (normalized) of each protein. To improve the quality of the analysis results, and reduce the false positive rate, the proteome finder software further filters the search results. A peptide profile match with a confidence level of more than 99% is authentic and a protein comprising at least one unique peptide fragment (unique peptide fragment) is authentic. The invention only retains the credible peptide and protein, and carries out error discovery rate verification to remove the large error ratePeptides and proteins at 1%.
The present invention compares mass spectral data with existing tumor mutations and neoantigen libraries to determine if the captured proteins contain DAMPs and neoantigens expressed by TC-1 cells. As shown in FIG. 5A, with AD 11 In comparison with AD 11 Mal captures a higher proportion of DAMPs, a class of pro-inflammatory molecules, which have been shown to elicit an innate or adaptive immune response. Notably, AD 11 Mal captured high mobility histone B1 (high mobility group protein B1, HMGB 1), a marker of immunogenic cell death, which has been demonstrated to enhance anti-tumor immunity. As shown in FIG. 5B, the present invention discovers AD 11 And AD (analog to digital) 11 Mal successfully captured multiple neoantigens, respectively. However, in AD 11 The abundance of the new antigen captured in Mal is significantly higher than AD 11 。
Example 6
The present embodiment aims at verifying AD 11 Whether or not antigen uptake and maturation of dendritic cells is promoted after Mal capture of antigen.
Labelling of cell lysates with Cy5.5 followed by AD 11 Or AD (analog to digital) 11 Mal capture followed by incubation with bone marrow dendritic cells (bone marrow-derived dendritic cells, BMDCs) at the same cell lysate concentration. The specific operation is as follows: first, cy5.5-NHS was reacted with cell lysate in a dark room for 12h (mass ratio 1:100). The free Cy5.5-NHS was then dialyzed (molecular weight cut-off: 10000) against phosphate buffer and removed for 24 hours. Cy5.5-labeled cell lysates were added to BMDCs (10 6 Individual cells). Finally, PBS and AD are added respectively 11 (10 pfu/cell) and AD 11 Mal (10 pfu/cell). After 5h incubation at 37C, fluorescence of cy5.5 in BMDCs was analyzed by flow cytometry to assess antigen uptake. The maturity of the cells was assessed using anti-mouse CD80 antibody (104707, clone number: 16-10A1, dilution ratio: 1:40) and anti-mouse CD86 antibody (105014, clone number: GL-1, dilution ratio: 1:20).
AD compared to cell lysate-Cy5.5 group as shown in FIG. 6A 11 Cell lysate-Cy5.5 group and AD 11 Mal-cell lysateMore Cy5.5 was detected in the group-Cy5.5 + BMDCs, suggesting AD 11 And AD (analog to digital) 11 Mal can mix captured cell lysates-Cy5.5 into BMDCs. Wherein AD is 11 Mal clearly can capture more cell lysates to BMDCs.
The antigen-transferring cells need to be stimulated to mature to cause subsequent tumor-adaptive immune killing, DAMPs contained in the cell lysate stimulate the antigen-transferring cells to mature, and AD 11 DC cell maturation may also be activated due to its own immunogenicity. As shown in FIG. 6B, in AD 11 The highest proportion of mature antigen-transmitting cells observed in the Mal-cell lysate group suggests that the manner in which the antigen-transmitting cells are stimulated to mature has a synergistic effect.
Example 7
Purpose of this example to verify AD 11 -Mal can drain tumor antigens to lymph nodes.
Drainage of tumor antigens to lymph nodes is critical for induction of anti-tumor immune responses. To evaluate AD 11 Whether Mal is able to drain tumor antigens to lymph nodes in vivo, the invention uses AD 11 Mal captures SH-Cy5.5 (AD 11-Mal-SH-Cy5.5), and free SH-Cy5.5 and AD 11 Mal-SH-Cy5.5 was subcutaneously injected into TC-1 tumor-bearing C57BL/6 mice. The specific scheme is as follows: in darkroom, AD 11 Mal was reacted with Cy5.5-PEG-SH for 12h (mass ratio 100:1), and free Cy5.5 was removed by dialysis. AD is added with 11 -Mal-SH-Cy5.5(10 6 pfu), cy5.5-PEG-SH and PBS were injected into TC-1 tumors in C57BL/6 mice. After 12h, draining lymph nodes were collected for in vitro fluoroscopy using a Maestro system (Caliper, us, IVIS spectroscopy). The inguinal lymph nodes were then rapidly frozen for sectioning. Sections were stained with DAPI, anti-cd 3 and anti-b 220 and examined with a laser scanning confocal microscope (3 DHISTECH, hungary, panoramic MIDI, panoramic 250FLASH, panoramic DESK). As shown in FIG. 7A, the Cy5.5 fluorescent signal is only at AD 11 Detection in draining lymph nodes isolated from Mal-SH-Cy5.5 group lymph nodes. As shown in FIG. 7B, the B cell and T cell regions in the lymph nodes were labeled with fluorescent B220 and CD3 antibodies, AD 11 Mal-SH-Cy5.5 is mainly concentrated in the T cell region, withFacilitating activation of antigen-specific T cells.
Example 8
Purpose of this example to verify AD 11 Mal anti-tumor efficacy and anti-tumor mechanism.
To evaluate AD 11 Anti-tumor effect of Mal, at day-7, 3X 10 6 The TC-1 cells were subcutaneously injected into the dorsal part (primary tumor) of female C57BL/6 mice (6-8 weeks old). Day-4, 3X 10 6 The TC-1 cells were injected on the other side of the back (distant tumor). On days 0, 2, 4 and 6, the primary tumor was injected intratumorally with different drugs (AD 11 And AD (analog to digital) 11 Mal, dose 5X 10 7 pfu). Mice were analyzed for primary tumor, distant tumor, spleen and serum on day 14. Tumor volume and body weight were measured every two days when the drug was injected. The formula for calculating tumor volume (V) is: v=1/2 ab 2 Wherein a and b are the major and minor axes of the tumor, respectively.
AD compared to saline control 11 And AD (analog to digital) 11 Mal significantly inhibited primary tumor growth, but AD alone 11 Mal still has strong inhibition of distal tumor growth (FIGS. 8A, 8B), suggesting AD 11 Mal elicits a distal immune response against tumors.
Tumor tissue was digested into single cell suspensions to assess infiltration of immune cells. As shown in fig. 9a,9 b: AD (analog to digital) converter 11 Cytotoxic T lymphocytes (CD 3) in primary and distant tumors of the Mal group + CD8 + Cells) were highest. However, in AD 11 In the Mal group, regulatory T lymphocytes (Treg, CD3 + CD4 + CD25 + Cells) ratio was also significantly increased.
To investigate CD8 + T cells in AD 11 Key role in Mal mediated anti-tumor action, CD8 was established by intraperitoneal injection of CD8 monoclonal antibody according to the invention + T cell depletion mouse model, i.e., 50 μg of anti-CD 8 monoclonal antibody (affinity immunity, U.S. A., ab-131-126 and 2 mg/mL) was intraperitoneally injected on days-8, -6, -2, 0, 2, 6 and 10 of the mouse. CD3 in all experimental groups + CD8 + The proportion of T cells is extremely lowIs shown in CD8 (FIG. 10A) + Successful establishment of the T cell depleting mouse model. As shown in FIG. 10B, AD 11 Or AD (analog to digital) 11 Anti-tumor effect of Mal on CD8 + The disappearance of the T cell depleting mouse model suggests that both direct and systemic anti-tumor activity is associated with immune responses.
Example 9
Purpose of this example to verify AD 11 Anti-tumor efficacy of Mal in mice with existing anti-viral antibodies.
In china, most people have low immunity in childhood, are infected with adenovirus, and produce neutralizing antibodies against adenovirus. When these humans are treated with adenovirus-based OVs, pre-existing immunity may eliminate OVs and affect the efficiency of the treatment. To explore this challenge, the present invention addresses low doses of AD by pre-injecting virus, i.e., on days-42, -35 and-14 11 (5×10 6 pfu) was subcutaneously injected into mice to construct a drug for AD 11 An immunized mouse model. Thus, it was found that AD 11 Mal still showed the most potent anti-tumor ability against primary and distant tumors (FIG. 11A, FIG. 11B).
Example 10
Purpose of this example to verify AD 11 -antitumor efficacy of Mal and 1-MT combination therapy and antitumor mechanism thereof.
Tumor immunotherapy can improve anti-tumor efficacy, but often is accompanied by negative feedback modulation. Sustained antigen delivery and pro-inflammatory stimulation significantly increases expression of immune checkpoints in tumors and lymph nodes. The present invention introduces 1-MT into the combination treatment regimen. 1-MT is a tryptophan analog with pleiotropic effects on the downstream guanine-aryl hydrocarbon receptor pathway. 1-MT promotes CD4 + T lymphocytes differentiate towards Th17 helper T lymphocytes, reducing regulatory T lymphocytes. AD (analog to digital) converter 11 Or AD (analog to digital) 11 The amount of Mal and the injection mode are the same, the 1-MT dose is 0.5mg/kg, and the injection mode is intratumoral injection. As shown in fig. 12A and 12B, AD 11 The inhibition of primary and distant tumor growth by Mal+1-MT is significantly stronger than AD 11 Mal, especially the treatment effect of distant tumors. Worth of itNote that AD 11 Mal+1-MT treatment resulted in complete regression of 20% of the tumors in mice.
For primary tumors, and AD 11 AD compared to Mal group 11 The killer T lymphocytes of the Mal+1-MT group were slightly elevated (FIG. 13A). Remarkably, the proportion of tregs was significantly reduced in both the primary and distant tumors, indicating that the tumor immunosuppressive microenvironment was relieved (fig. 13B).
Example 11
Purpose of this example to verify AD 11 Whether combined therapy of Mal and 1-MT induces long-term immunological memory
OVs are often used in patients with advanced tumors where the tumor frequently recurs and the prognosis is very poor. Thus, it is important to successfully induce immunological memory during treatment, and the present invention subcutaneously inoculates 3X 10 on the right side of C57BL/6 mice 6 TC-1 cells, forming a primary tumor. Then, physiological saline and AD were injected into the primary tumor on days 6, 8, 10 and 12, respectively 11 、AD 11 -Mal、1-MT、AD 11 +1-MT，AD 11 Drugs such as mal+1-MT 4 times (once every other day). Tumor volume growth of the primary tumor was measured every other day. Day 66, AD 11 Subcutaneous inoculation of Mal+1-MT group Living mice with left dorsal 3X 10 6 TC-1 cells, forming distant tumors. Healthy mice of the same week age served as controls. As shown in fig. 14 and 15, AD was compared to the rapid growth of distant tumors in the control group 11 Distal tumor growth was significantly reduced in Mal+1-MT treated mice, nearly 30% (2/7) of which showed complete resistance to tumor challenge.

Claims (8)

1. An oncolytic virus for capturing an antigen, characterized in that the oncolytic virus for capturing an antigen is an oncolytic virus modified by an active group with an antigen capturing function;
the active group with the antigen capturing function is an NHS-PEG-Mal group;
the oncolytic virus expresses telomerase promoter and AD 5 Enhancer genetically engineered oncolytic adenovirus AD 11 。
2. A method of producing an antigen-capturing oncolytic virus of claim 1, comprising:
and incubating the OVs and active groups with antigen capturing function at normal temperature, removing the active groups which do not react through a filter membrane and an ultrafiltration tube, and re-adding PBS (phosphate buffered saline) into the ultrafiltration tube for re-suspension to obtain the oncolytic virus for capturing the antigen.
3. The method for producing an antigen-capturing oncolytic virus according to claim 2, wherein the antigen-capturing oncolytic virus is prepared by subjecting AD to a method comprising the steps of 11 Incubating with NHS-PEG-Mal at normal temperature, removing non-reactive groups through a filter membrane and an ultrafiltration tube, and adopting barium iodide to dye PEG to determine the maximum preparation amount of reaction; wherein, AD is calculated by the ratio of solute number to virus particle number 11 : NHS-PEG-Mal 3.2X10 6 /vp。
4. The method of claim 2, wherein the incubation is: uniformly mixing an aqueous solution of active groups with an antigen capturing function with oncolytic viruses, and then performing ultrasonic treatment at room temperature; wherein the ultrasonic time is 30-60min, and the room temperature is 20-25 ℃;
and/or, in the ultrafiltration tube, adopting a low-speed centrifugation revolution of 5000-10000rpm, and centrifuging for 5-10min; and/or the filter membrane pore size used is 0.45 μm and 0.22 μm; and/or the ultrafiltration tube used has a molecular weight cut-off of 50-100kDa.
5. The method for producing an oncolytic virus for a captured antigen according to claim 2, wherein the particle size of the oncolytic virus for a captured antigen produced is 116-120nm, and the dispersion index PDI is 0.2-0.21.
6. A pharmaceutical combination comprising the oncolytic virus of the capture antigen of claim 1 and an IDO inhibitor.
7. The pharmaceutical composition of claim 6, wherein the IDO inhibitor is 1-methyltryptophan.
8. Use of an oncolytic virus of claim 1 capturing an antigen or a pharmaceutical combination of claim 6 for the manufacture of a medicament for the treatment of a malignant solid tumor.
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