CN117982638A - Application of immunoadjuvant combined PD-1 antibody medicament in treating pancreatic cancer - Google Patents
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Abstract
The invention discloses an application of an immunoadjuvant combined PD-1 antibody medicament in treating pancreatic cancer, and relates to the field of biological medicines. The immune adjuvant containing QS-21 and Poly (inosinic acid) Poly I: C as active ingredients is independently used as an in-situ tumor vaccine, so that pancreatic cancer can be effectively treated or improved, the immune adjuvant can be combined with PD-1 antibody medicines, the drug resistance of pancreatic cancer patients to PD-1 antibody medicines can be reduced or reversed, the tumor microenvironment of pancreatic cancer patients can be changed, specific anti-tumor immune memory and systemic anti-tumor immune effects can be induced, the anti-tumor effects of the immune adjuvant and the PD-1 antibody medicines are obviously improved, and a new approach is provided for effective treatment of pancreatic cancer.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an application of an immunoadjuvant combined with a PD-1 antibody medicament in treating pancreatic cancer.
Background
Tumor in situ vaccine (in situ vaccination, ISV) is an immunotherapeutic approach, and after a small amount of immunomodulatory agent (adjuvant) is directly injected into tumor by intratumoral injection with tumor as an own vaccine, a high concentration of immunostimulating product can be obtained in situ to obtain therapeutic effect. The advantage of tumor in situ vaccines is that local delivery allows for multiple combination therapies while preventing significant systemic exposure and off-target toxicity. Although the primary epitope for a particular cancer is not determined, one can therefore trigger an immune response against a tumor neoantigen or tumor-associated antigen without the need to characterize it. Such immunostimulation can induce strong tumor immune initiation locally, while producing systemic (distant) tumor responses, due to circulation of appropriately activated anti-tumor immune cells. While addressing many of the limitations of current cancer immunotherapy development, intratumoral immunotherapy also offers a unique opportunity to better understand the dynamics of cancer immunity by allowing continuous and multifocal biopsies at each tumor injection.
Tumor in situ vaccines aim to stimulate the first three steps of the anticancer immune cycle: cancer antigen release and presentation, immune cell activation and immune cell activation. Once immune cells are activated, they still need to complete the remaining few steps: peripheral mobilization, penetration into the cancer site, recognition of cancer cells, and triggering cytotoxicity to cancer cells. Thus, the drug resistance mechanisms of anti-cancer immunity, particularly in TMEs, still reduce the efficacy of cancer vaccines, and methods of enhancing cancer vaccines are currently being explored.
Immunotherapy such as Immune Checkpoint Inhibitors (ICI) and Adoptive Cell Therapy (ACT) has radically altered cancer therapies, particularly metastatic cancers, with long-term remission and survival for some patients that were previously thought incurable. However, while immunotherapy can produce a sustained response, ICI monotherapy typically has only about 20% response to solid tumors, and many patients eventually develop secondary resistance. Once immune cells enter the tumor immune microenvironment (TME), a number of mechanisms are created to induce resistance against tumor immunity, including cancer cell intrinsic factors, immunosuppressive cells, and immunosuppressive environments.
The unique tumor microenvironment of pancreatic cancer is one of the important reasons that the malignancy of pancreatic cancer is far higher than that of other tumors, and is also one of the main reasons that patients with pancreatic cancer are insensitive to radiotherapy and chemotherapy and have poor prognosis. The tumor microenvironment of the pancreatic cancer contains a large amount of compact stroma components, so that on one hand, favorable conditions are provided for the growth of pancreatic cancer cells, on the other hand, compact fiber tissues press blood vessels in the pancreatic cancer tissues, so that anti-tumor drugs cannot enter the cancer tissues to play a role, and meanwhile, the compact fiber tissues prevent the infiltration of immune effector cells such as CD8+ T cells and NK cells in the tumor tissues, so that the tumor tissues evade the monitoring of an immune system.
About 30% of pancreatic cancer patients have been found to have positive expression of PD-L1. However, clinical data indicate that single drug treatment with PD-1/PD-L1 inhibitors has poor efficacy in pancreatic cancer patients.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an application of an immunoadjuvant combined with PD-1 antibody medicament in treating pancreatic cancer.
The invention is realized in the following way:
In a first aspect, embodiments of the present invention provide the use of a composition comprising, as active ingredients: immunoadjuvants and PD-1 antibody drugs; the active ingredients of the immunoadjuvant comprise QS-21 and Poly inosinic acid PolyI: C.
In a second aspect, embodiments of the present invention provide a pharmaceutical composition comprising: the composition of the preceding examples.
In a third aspect, embodiments of the present invention provide a kit comprising: the composition described in the previous examples.
In a fourth aspect, embodiments of the present invention provide the use of an immunoadjuvant as described in the previous embodiments for the manufacture of a product for the treatment or co-treatment of pancreatic cancer.
The invention has the following beneficial effects:
The invention discovers that the immune adjuvant containing QS-21 and Poly (inosinic acid) Poly I: C as active ingredients is independently used as an in-situ tumor vaccine, can effectively treat or improve pancreatic cancer, is combined with PD-1 antibody medicaments, can reduce or reverse the drug resistance of pancreatic cancer patients to PD-1 antibody medicaments, can also change the tumor microenvironment of pancreatic cancer patients, induces specific anti-tumor immune memory and systemic anti-tumor immune effects, ensures that the anti-tumor effects of the immune adjuvant and the PD-1 antibody medicaments are obviously improved, and provides a new approach for the effective treatment of pancreatic cancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the comparison of the antitumor effects of three different adjuvants;
FIG. 2 is a schematic diagram of a AYK103,103 combined PD-1 administration route for treating pancreatic cancer in PD-1 resistant mice;
FIG. 3 is a graph showing the tumor growth curve of AYK103,103 in combination with PD-1 for the treatment of pancreatic cancer in PD-1 resistant mice;
FIG. 4 is a tumor tissue anatomy of AYK103 in combination with PD-1 for treatment of PD-1 resistant mouse pancreatic cancer;
FIG. 5 is a graph showing the number of CD8+ T cells in a tumor immune microenvironment for AYK a combination PD-1 treatment of PD-1 resistant mouse pancreatic cancer;
FIG. 6 is a graph showing the number of CD4+ T cells in a tumor immune microenvironment for AYK103 in combination with PD-1 for treatment of pancreatic cancer in PD-1 resistant mice;
FIG. 7 is a graph showing the number of CD8+CD69+ T cells in a tumor immune microenvironment of AYK and PD-1 in combination with PD-1 for treatment of pancreatic cancer in PD-1 resistant mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Therapeutic tumor vaccines are classified into 4 major classes, namely, cell vaccines, protein/synthetic peptide vaccines, nucleic acid vaccines and viral vector vaccines, depending on the source of tumor antigen expression. However, these 4 tumor vaccines have limitations, respectively, and cell vaccines have weak immunogenicity and secrete various inhibitory factors, which can lead to poor vaccine effect and kill healthy cells in vivo; nucleic acid vaccine delivery efficiency is weak and stability is poor; viral vector vaccines cannot be vaccinated repeatedly, otherwise the immune response generated by the body will attack the viral vector vaccine that re-enters our body.
In order to remedy the defects of the vaccine, the embodiment of the invention provides an in-situ tumor vaccine. The vaccine has no antigen participation, and the tumor tissue autoantigen is utilized to form an in-situ vaccine in situ on the tumor by injecting a novel immunological adjuvant system AYK103,103 into the tumor. Compared with nucleic acid and virus vector vaccine, the method avoids the problem of low tumor delivery efficiency, and has higher stability and safety. The adjuvant system has the characteristics of low cost, period, low drug resistance and the like, and can improve immunogenicity through multiple injections. Compared with the traditional subunit vaccine and gene vaccine, the tumor in-situ vaccine (immunoadjuvant) provided by the invention does not contain antigen and hapten, and has higher safety. Compared with cell therapies such as DC vaccine, the immunoadjuvant provided by the invention has low price, short period, low toxicity and good broad spectrum, is easier to be used in combination with other therapies, and provides more choices for developing clinical new tumor clinical treatment strategies.
The tumor in-situ vaccine can activate antigen presenting cells in an immune microenvironment in tumor tissues, so that the antigen presenting cells present tumor neoantigens and tumor specific antigens in the tumor tissues into drainage lymph nodes to generate specific T cell immunity. Through research and exploration of pancreatic cancer, the inventor discovers and verifies that the immunological adjuvant of specific active ingredients (QS-21 and Poly I: C) adopted as an in-situ tumor vaccine is combined with PD-1 antibody medicines, so that the microenvironment of pancreatic cancer tumor can be effectively changed, the proportion of T cells in the microenvironment of pancreatic cancer tumor can be increased, the poor effect of PD-1 antibody medicine treatment is improved, and meanwhile, the immunological adjuvant has the effects of inducing specific anti-tumor immunological memory and systemic anti-tumor immunological effect.
The immunoadjuvant and the combined treatment of the immunoadjuvant and the PD-1 antibody medicament can be applied to effectively treating pancreatic cancer patients (I phase, II phase, III phase and IV phase), pancreatic cancer patients with drug resistance to the PD-1 antibody medicament and medium and late stage (III phase and IV phase) pancreatic cancer patients with first-line treatment failure.
Specific technical scheme
In one aspect, embodiments of the present invention provide the use of a composition comprising, as active ingredients, the active ingredients of which: immunoadjuvants and PD-1 antibody drugs; the active ingredients of the immunoadjuvant comprise QS-21 and Poly inosinic acid PolyI: C.
Polyinosinic acid PolyI: C, polyinosinic-polycytidylic acid is a toll-like receptor 3 (TLR 3) agonist, is a synthetic double-stranded RNA (dsRNA), CAS number: 42524-50-0.
QS-21, CAS number 141256-04-4, molecular formula: c 92H148O46, molecular weight: 1990.14.
In some embodiments, the in situ tumor vaccine is administered by intratumoral injection of pancreatic cancer, and the in situ tumor vaccine is formed in situ in the tumor by utilizing the inherent antigen-binding immune adjuvant (AYK 103,103) in the tumor, so as to activate the anti-tumor immune response in the tumor tissue and improve the immune memory.
In some embodiments, the PD-1 antibody drug is administered intraperitoneally.
In some embodiments, the final concentration of QS-21 in the immunoadjuvant is 50 to 300 μg/mL. The final concentration may specifically be in the range of between any one or any two of 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 μg/mL.
In some embodiments, the final concentration of polyinosinic acid PolyI: C in the immunoadjuvant is 400-3000 μg/mL. The final concentration may specifically be in the range of any one or between any two of 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000 μg/mL.
In some embodiments, the immunoadjuvant further comprises: a liposome carrier;
In some embodiments, in the immunoadjuvant, the mass ratio of QS-21, poly I: C, and DOPC is 1:6 to 10:15 to 25, and specifically may be any one or any two of 1:6:16、1:6:18、1:6:20、1:6:22、1:6:24、1:8:16、1:8:18、1:8:20、1:8:22、1:8:24、1:10:16、1:10:18、1:10:20、1:10:22、1:10:24.
In some embodiments, the liposome carrier comprises: any one or more of cationic liposomes and neutral liposomes.
In some embodiments, the cationic liposome comprises: DOTAP, DOTMA, DC-Chol.
In some embodiments, the neutral liposome comprises: DOPC, DSPC, DPPC, any one or more of the following.
In some embodiments, the mass ratio of cationic liposome to neutral liposome is 1: 8-12. Specifically, the range between any one or any two of 1:8, 1:9, 1:10, 1:11 and 1:12 can be adopted.
In some embodiments, the cationic liposome has a particle size of 100 to 300nm. The particle size may specifically be in the range between any one or any two of 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300nm.
In some embodiments, the method of preparing the cationic liposome comprises:
Mixing 1, 2-dioleoyl-3-trimethyl ammonium chloride propane, dioleoyl phosphatidylcholine and cholesterol according to a mass ratio of 1:8-12:3-4 to obtain a first mixture;
The cationic liposome is prepared by taking the first mixture as a raw material and adopting any one of an ethanol injection method, a film dispersion method, an ultrasonic dispersion method and a reverse evaporation method.
In some embodiments, the mixing mass ratio of 1, 2-dioleoyl-3-trimethylammonium propane chloride (DOTAP), dioleoyl phosphatidylcholine (DOPC), and cholesterol may specifically be in a range between any one or any two of 1:8:3, 1:8:4, 1:9:3, 1:9:4, 1:10:3, 1:10:4, 1:11:3, 1:11:4, 1:12:3, 1:12:4.
In some embodiments, the neutral liposomes have a particle size of 100 to 300nm. The particle size may specifically be in the range between any one or any two of 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300nm.
In some embodiments, the method of preparing neutral liposomes comprises:
mixing dioleoyl phosphatidylcholine (DOPC) and cholesterol according to a mass ratio of 3-5:1 to obtain a second mixture;
and taking the second mixture as a raw material, and adopting any one of an ethanol injection method, a film dispersion method, an ultrasonic dispersion method and a reverse evaporation method to prepare the neutral liposome.
In some embodiments, the mixing mass ratio of dioleoyl phosphatidylcholine to cholesterol may be in a range between any one or any two of 3:1, 3.5:1, 4:1, 4.5:1, 5:1.
In some embodiments, the product is any one of a drug, a reagent, and a kit.
The technical scheme of the invention has no special limitation on PD-1 antibody medicines, and the PD-1 antibody medicines disclosed in the prior art can be selected. In some embodiments, the PD-1 antibody drug comprises: any one or a combination of a plurality of palbociclib monoclonal antibody, nal Wu Liyou monoclonal antibody, atilizumab, divali You Shan antibody, singal di Li Shan antibody, terlipressin Li Shan antibody, tirelib monoclonal antibody, karellib monoclonal antibody, s Lu Lishan antibody, prairin monoclonal antibody, p An Puli monoclonal antibody, en Wo Lishan antibody, sapalib monoclonal antibody and kadulcitol.
In some embodiments, the treating or adjunctively treating pancreatic cancer comprises treating or adjunctively treating a patient with stage I, stage II, stage III, stage IV pancreatic cancer.
In some embodiments, the treating or adjunctively treating pancreatic cancer comprises treating or adjunctively treating a patient with pancreatic cancer that is resistant or intolerant to PD-1 antibody drugs.
In some embodiments, the treating or adjunctively treating pancreatic cancer comprises treating or adjunctively treating a patient with intermediate-and-late pancreatic cancer who failed first-line therapy.
In another aspect, embodiments of the present invention provide a pharmaceutical composition comprising: a composition as described in any of the preceding examples.
Equivalent substitutions can be made for "drug" and "pharmaceutical composition". The pharmaceutical composition comprises the active ingredients in the invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulates, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorption carriers.
In another aspect, embodiments of the present invention provide a kit comprising: a composition as described in any of the preceding examples.
In addition, the embodiment of the invention also provides the use of the immunoadjuvant as described in any of the previous embodiments for the preparation of a product for the treatment or adjuvant treatment of pancreatic cancer.
"Treating" in the present invention includes preventing or alleviating a condition, reducing the rate at which a condition is raised or developed, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof.
For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination of the foregoing. For tumors, "treatment" includes clearing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or slowing tumor progression, or some combination thereof.
The formulation of therapeutic drugs or pharmaceutical compositions and their subsequent administration (administration) is within the ability of those skilled in the art. Administration depends on the severity and responsiveness of the disease state to be treated, the period of treatment lasting from days to months, or until a cure is achieved or a reduction in the disease state is achieved. The optimal dosing schedule may be calculated from measurements of drug accumulation in the patient. The optimal dosage, method of administration and repetition rate can be readily determined by one of ordinary skill in the art. The optimal dosage may vary depending on the relative potency of the active ingredient, and can generally be estimated based on the EC50 that is effective in an effective in vitro and in vivo animal model.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Experimental animal use license number: SYXK (su) 2018-0019.
Example 1: AYK103 an immunological adjuvant (tumor in situ vaccine) and its preparation.
An immunoadjuvant AYK103 comprising the following three components: QS-21, poly I: C and a liposome carrier.
(1) QS-21 at a final concentration of 100 μg/ml in the immunoadjuvant;
(2) Poly I: C, final concentration in immunoadjuvant was 800. Mu.g/ml;
(3) Liposome carrier: the liposome carrier is a cationic liposome preparation.
The preparation method of the immunoadjuvant AYK is as follows:
(1) Preparation of cationic liposome:
The mass ratio of 1, 2-dioleoyl-3-trimethyl ammonium chloride propane (DOTAP), dioleoyl phosphatidylcholine (DOPC) and cholesterol is 1:10:3, preparing; after the three lipid components are uniformly dispersed, preparing and obtaining the cationic liposome by using an ethanol injection method (in other embodiments, a film dispersion method, an ultrasonic dispersion method and a reverse evaporation method can be adopted); the preparation steps of the ethanol injection method are as follows: sequentially using extrusion films with the pore diameters of 200nm and 100nm for extrusion molding after ultrafiltration, and finally filtering and sterilizing by a sterile filter.
(2) QS-21 and Poly I C, DOPC are mixed according to the mass ratio of 1:8:20 are mixed and stirred uniformly to prepare the immunoadjuvant AYK103,103.
Example 2: the effects of different immunoadjuvants are compared.
Setting 3 experimental groups immune adjuvants AYK, AYK102 and AYK, AYK were prepared as AYK, AYK101, AYK102 described in example 1, respectively, and the differences were as follows, except for AYK103 in example 1:
AYK 101A 101 replaces Poly I: C with MPLA, the final concentration of MPLA is 200 μg/mL;
AYK 102A 102 replaced QS-21 with MPLA with a final concentration of MPLA of 200 μg/mL.
Experimental method
Construction of B16-OVA cell line melanoma tumor model: mouse melanoma B16-OVA cell line. Culture conditions: RPMI 1640 medium+10% (volume fraction) fbs+1% (volume fraction) diabody. The mouse melanoma B16-OVA cell line was centrifuged at 1000r/min for 5 minutes. Cell viability (viability > 90%) was measured by 0.4% (volume fraction) trypan blue exclusion, cells were washed 3 times with PBS and centrifuged at 1000r/min for 5 min. PBS was added to adjust the cell concentration to 5X 10 6/ml, 100. Mu.l of the cell suspension was pipetted into the axilla of 20 female mice, and the subcutaneous tumor volumes of female C57BL/6J mice were determined on day 5 after tumor inoculation.
The subcutaneous tumor volumes of female C57BL/6J mice were measured on day 5 after tumor inoculation, and the mice were divided into 4 experimental groups according to tumor volumes by a random grouping method, specifically as follows: liposome control (empty liposome negative control without drug coated, 100 μl/dose), AYK (100 μl/dose), AYK (100 μl/dose), AYK (100 μl/dose), 103 (100 μl/dose). Each group had 10 females. The first administration was performed on the day after grouping (day 9 after tumor inoculation), the second intratumoral administration was performed on day 12 after tumor inoculation, and the last intratumoral administration was performed on day 15 after tumor inoculation. The body weight and diet of the animals are observed daily, the body weight change of the mice is monitored, and the tumor size change is detected every three days.
Experimental results
As a result, referring to FIG. 1, it was found that AYK103 (Poly (I: C) +QS21) had a favorable effect of suppressing tumor growth in three adjuvants AYK, 101, AYK, 102, AYK, and the tumor suppression rate was about 96%.
Example 3: construction of FC1242 cell line pancreatic cancer model.
The cell line is a mouse pancreatic cancer FC1242 cell line. Culture conditions: RPMI 1640 medium+10% (volume fraction) fbs+1% (volume fraction) diabody. Animal species and strain: c57BL/6J. Grade: SPF stage. Age of animals: 6-8 weeks of age. The ingestion method comprises the following steps: can be taken freely. Adaptation period label: cage cards and cut-toe methods were used as animal identification markers during environmental adaptation. According to the experimental animal numbering method of the mechanism, the experimental animal is marked, and the animal marking information is marked on the cage card. The white cage cards are uniformly used during the environment adaptation period, and the experiment numbers, animal species, cage numbers, animal numbers, room time, remark information and the like are noted. Numbering after grouping: cage cards and toes are used as animal identification markers.
The mouse pancreatic cancer FC1242 cell line was centrifuged at 1000r/min for 5 minutes. Cell viability (viability > 90%) was measured by 0.4% (volume fraction) trypan blue exclusion, cells were washed 3 times with PBS and centrifuged at 1000r/min for 5 min. Cell concentration was adjusted by adding PBS, 100. Mu.l of the cell suspension was pipetted under the skin of 20 female mice, each mouse was inoculated with 5X 10 5 cells, and the subcutaneous tumor volume of female C57BL/6J mice was measured on day 4 after tumor inoculation.
Example 4: AYK103 treatment of PD-1 resistant mouse pancreatic cancer in combination with PD-1.
Experimental method
Grouping and dosing regimen: female C57BL/6J mice (FC 1242 cell line pancreatic cancer model prepared in example 3) were each assayed for subcutaneous tumor volume on day 4 post-tumor inoculation and were divided into 4 experimental groups according to tumor volume by a random grouping method, as follows:
Group 1 (vehicle+igg): blank liposome (Vehicle, 100 μl/dose) +immunoglobulin IgG (BioXCell IgG,100 μg/dose);
Group 2 (vehicle+anti-PD-1 mAb): vehicle (100. Mu.L/min) +anti-PD-1mAb (BioXcell company PD-1 antibody, 100. Mu.g/min);
group 3 (AYK 103): AYK103 group 103 (100 μl/dose);
Group 4 (anti-PD-1 mabs+ayk 103): anti-PD-1mAb (100. Mu.g/min) + AYK103 groups 103 (100. Mu.L/min).
Each group had 10 females. The first intratumoral administration AYK103,103 was performed on the day after grouping (day 5 after tumor inoculation), and the intraperitoneal administration of PD-1 mab was performed on the second day (day 6 after tumor inoculation). A second intratumoral administration AYK103,103 (as in example 1) was performed on day 7 after the tumor inoculation, and a second intraperitoneal administration of PD-1 mab was performed the following day. A third intratumoral administration AYK103,103 was performed on day 10 after tumor inoculation, and a third intraperitoneal administration of PD-1 mab was performed on the following day. The body weight and diet of the animals are observed daily, the body weight change of the mice is monitored, and the tumor size change is detected every two days. After the last dose, observation was continued for 7 days. The volume, route and frequency of administration are shown in detail in figure 2.
Tumor volume measurement: dosing was started on day 4 after tumor inoculation, animal body weight was monitored daily, tumor volume was measured every three days using the method of measuring tumor diameter, and the antitumor activity of the test drug was dynamically observed. The length and the short diameter of the tumor were measured by a vernier caliper, and the volume of the tumor was calculated, and the tumor volume=1/2×the long diameter×the short diameter 2.
Experimental results
The results are shown in fig. 3 and 4: reflecting the growth of subcutaneous tumors of pancreatic cancer in mice injected with Vehicle+IgG, vehicle+PD-1 mab, AYK103+IgG and AYK103+PD-1 mab in combination. Compared with the injection of Vehicle+IgG, the injection AYK +IgG group tumor growth is obviously inhibited, which shows that AYK103 has the effect of treating the pancreatic cancer of mice. Whereas injection of vehicle+pd-1 mab had little effect on pancreatic cancer growth in mice. For the group of the combined injection AYK & lt103+ & gt PD-1 monoclonal antibodies, the growth of the pancreatic cancer of mice is obviously inhibited, and the effect is better than that of the group of the injection AYK & lt103+ & gt IgG, which shows that the combined use of AYK & lt103+ & gt PD-1 monoclonal antibodies can generate the combined synergistic effect.
Example 5: AYK 103A 103 in combination with PD-1 alters the tumor immune microenvironment in mouse pancreatic cancer.
Experimental method
The tissue treatment method comprises the following steps:
1. Tumor tissue sampling: removing neck from the mice constructed in example 4, carefully poking the hair, skin and tissue of the mice with surgical scissors and elbow forceps, gently freeing the complete pancreatic cancer subcutaneous tumor, weighing, recording and photographing, and reserving 0.1-0.5g of each tumor tissue, and placing in precooled 1 XPBS;
2. Tumor tissue single cell fluid preparation: preparing a clean 5mL EP tube according to the group number, cutting the tumor tissue into tissue blocks with the size of 5mm in the tube, re-suspending the tumor tissue by using prepared tissue digestive juice [ RPMI-1640 culture medium, 0.5mg/mL Collagenase D and 0.1mg/mL Dnase I ] according to the volume of 4mL digestive juice/sample, transferring the tumor tissue into a GENTLE MACS C tube, running a program 37C_Multi_F, and removing the C tube from a GENTLE MACS tissue processor after the program is finished; washing the filter with RPMI-1640, filtering the grinding fluid with 70 μm filter membrane, collecting cell suspension with 10mL EP tube, centrifuging (400 g,5min, room temperature), discarding supernatant, preparing 1 Xerythrocyte lysate with pure water, re-suspending cell precipitate with 2mL of 1 Xerythrocyte lysate/volume of each sample, standing at room temperature for 5min, adding 5mL of 1 XPBS to terminate lysis, centrifuging (400 g,5min, room temperature), discarding supernatant;
Flow dyeing method:
Live/read staining: the cell pellet was resuspended in 100. Mu.L Live/Dead diluent/sample volume with formulated Live/Dead diluent (Near IR at a 1:500 ratio with 1 XPBS), incubated at 4℃in the dark for 30min,1mL of 1 XPBS was terminated, a small amount of cell suspension was aspirated from each set of samples for Live/Dead Shan Yangguan use, and the supernatant was discarded by centrifugation (400 g,5min, room temperature).
Flow antibody staining: the cell pellet was resuspended in a volume of 100. Mu.L of mixed dye solution/sample (PE/Cy 7-CD3, FITC-CD4, BV510-CD8, PE-CD25, BV421-CD69, APC-PD-1 at a ratio of 1:800, diluted with 1 XPBS), incubated at 4℃for 30min in the absence of light, the staining stopped with 1mL of 1 XPBS, and the supernatant centrifuged (400 g,5min, room temperature) was discarded. Each antibody was stained for use with Beads 0.2. Mu.L/tube and the staining method was the same.
Sample loading: the flow cytometry detection is carried out, blank control tube and single positive tube are firstly subjected to voltage definition, after the voltage is fixed, the cell count of each group of samples is recorded, and the cell/gtumor unit conversion is carried out by using the mass of the sampled tumor tissue.
Experimental results
The results are shown in FIGS. 5, 6 and 7.
As can be seen from fig. 5, cd8+ T cells in tumor tissues injected with AYK (Poly (I: C)/QS 21) +pd-1 mab were significantly higher than that in blank liposome control (Vehicle) +igg group, blank liposome control (Vehicle) +pd-1 mab, AYK (Poly (I: C)/QS 21) +igg group, demonstrating that AYK in combination with PD-1 mab can help infiltration of cd8+ T cells into tumor.
As can be seen from fig. 6, cd4+ T cells in tumor tissues injected with AYK (Poly (I: C)/QS 21) +pd-1 mab were significantly higher than that in blank liposome control (Vehicle) +igg group, blank liposome control (Vehicle) +pd-1 mab, AYK (Poly (I: C)/QS 21) +igg group, demonstrating that AYK in combination with PD-1 mab can help infiltration of cd4+ T cells into tumors, helping cd8+ T cells to function.
Expression of CD69 is up-regulated after CD 8T cells are activated, and thus cd8+cd69+ T cells are activated CD 8T cells. As shown in fig. 7, cd8+cd69+ T cells in tumor tissues of AYK (Poly (I: C)/QS 21) +pd-1 mab group were significantly higher than those of blank liposome control (Vehicle) +igg group, blank liposome control (Vehicle) +pd-1 mab, AYK (Poly (I: C)/QS 21) +igg group, which indicates that AYK in combination with PD-1 mab can increase the number of activated CD 8T cells in tumor tissues, thereby better exerting the effect of killing tumor.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Use of a composition for the preparation of a product for the treatment or co-treatment of pancreatic cancer, characterized in that the active ingredients of the composition comprise: immunoadjuvants and PD-1 antibody drugs; the active ingredients of the immunoadjuvant comprise QS-21 and poly inosinic acid PolyI:C.
2. The use according to claim 1, wherein in the immunoadjuvant the final concentration of QS-21 is 50 to 300 μg/mL.
3. The use according to claim 1, characterized in that in the immunoadjuvant the final concentration of Poly inosinic acid Poly I: C is 400-3000 μg/mL.
4. The use according to claim 1, wherein the immunoadjuvant further comprises: a liposome carrier;
Optionally, the liposome carrier comprises: any one or more of cationic liposomes and neutral liposomes.
5. The use according to claim 4, wherein the cationic liposome has a particle size of 100 to 300nm.
6. The use according to claim 5, wherein the method for preparing cationic liposomes comprises:
Mixing 1, 2-dioleoyl-3-trimethyl ammonium chloride propane, dioleoyl phosphatidylcholine and cholesterol according to a mass ratio of 1:8-12:3-4 to obtain a first mixture;
The cationic liposome is prepared by taking the first mixture as a raw material and adopting any one of an ethanol injection method, a film dispersion method, an ultrasonic dispersion method and a reverse evaporation method.
7. The use according to claim 1, wherein the product is any one of a medicament, a reagent and a kit.
8. A pharmaceutical composition, comprising: a composition as claimed in any one of claims 1 to 7.
9. A kit, comprising: a composition as claimed in any one of claims 1 to 7.
10. Use of an immunoadjuvant according to any one of claims 1 to 7, for the preparation of a product for the treatment or co-treatment of pancreatic cancer.
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