CN115463161A - Application of oncolytic virus in preparation of pharmaceutical composition for treating osteosarcoma - Google Patents
Application of oncolytic virus in preparation of pharmaceutical composition for treating osteosarcoma Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to an application of oncolytic virus in preparation of a pharmaceutical composition for treating osteosarcoma, wherein the pharmaceutical composition provided by the invention comprises oncolytic virus echolucent and an anti-PD-1 antibody, the drug has specific adding time and dosage in the using process, the therapeutic effect is achieved by increasing the number of immune cells in osteosarcoma, and a K7M2.Wt cell test and a nude mouse transplanted tumor model prove that the pharmaceutical composition provided by the invention can play roles of synergy and attenuation and tumor growth inhibition, so that the effect of improving weight reduction caused by tumors is achieved. The invention realizes the combination of the oncolytic virus echol and the anti-PD-1 antibody for preparing the medicine for treating osteosarcoma, enlarges the application range of the echol and provides a strategy for the research of novel osteosarcoma related medicines.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of oncolytic virus in preparation of a pharmaceutical composition for treating osteosarcoma.
Background
An echol (recombinant human adenovirus type 5 injection) mainly comprises recombinant human adenovirus type 5 particles with the names of deletion of E1B-55kD and E3 region gene fragments (78.3-85.8 mu); patients with advanced nasopharyngeal carcinoma who are refractory to conventional radiotherapy or radiotherapy plus chemotherapy and are palliatively treated with a 5-FU, cisplatin chemotherapy regimen are tried in combination with the aforementioned chemotherapy regimen. The injection amount is generally 1.5 × 1012 vp/day (3 pieces), and the distribution amount of each lesion should be proportionally injected according to the size of the tumor lesion. Before use, the product is taken out from the storage environment at the temperature of-20 ℃, completely melted at room temperature and gently mixed. The product is generally diluted to 30% of the total tumor volume with physiological saline, and can be adjusted properly according to specific tumor conditions. The needle is inserted from the tumor edge under the skin, and the liquid medicine is injected into the tumor edge and the tumor uniformly. If the tumor volume is less than or equal to 10cm < 3 >, the injection is uniformly injected in a radial shape in the whole tumor body; if the tumor volume is >10cm3, the tumor volume is divided into five quadrants, and the injections are given to one quadrant every day. The adverse reactions mainly comprise nausea, vomiting, diarrhea, abdominal pain, bronchitis, gastroenteritis, hepatitis, cystitis and conjunctivitis, are generally self-limiting, and can recover automatically after stopping taking the medicine.
PD-1 is a programmed death receptor 1 and is an important immunosuppressive molecule. Is a membrane protein of 268 amino acid residues, belonging to the immunoglobulin superfamily. It was originally cloned from apoptotic mouse T-cell hybridoma 2b4.11. The immunoregulation taking PD-1 as a target point has important significance for resisting tumor, infection, autoimmune disease, organ transplantation survival and the like. The ligand PD-L1 can also be used as a target spot, and the corresponding antibody can also play the same role. The binding of PD-1 and PD-L1 initiates the programmed death of T cells, allowing tumor cells to gain immune escape. For the treatment of locally advanced or metastatic melanoma after failure of previous standard therapy.
In 2018, 12 months and 17 days, the national drug administration has a conditional approval for the first domestic anti-PD-1 monoclonal antibody, terepril monoclonal antibody injection (trade name: tuoyi), to be marketed. The approved Terepril monoclonal antibody is a fully human monoclonal antibody of an anti-PD-1 antibody receptor developed and developed by Suzhou Zhonghe biological medicine corporation, and can relieve the immune suppression of tumor cells on the immune cells by blocking the PD-1 of T lymphocytes and blocking the combination of the T lymphocytes and the PD-L1 on the surface of the tumor cells, so that the immune cells can play the immune role of anti-tumor cells again to kill the tumor cells. However, the remission rate of the melanoma is only 17.3%, so that the melanoma has certain limitation in the process of preparing a medicament for treating the tumor.
On the basis, chinese patent CN1780632B discloses a method for treating primary cancer and metastatic cancer, which is implemented by removing cancer cells in tumor, i.e. adding a lytic agent into tumor cells to form primary tumor cells, and further applying in vivo stimulation without contacting with a distal tumor, so as to remove specific tumor cells, but this method requires adding a specific lytic agent, such as oncolytic bacteria, isolated nucleic acid expression constructs, etc., and also requires applying external stimulation such as high-frequency electromagnetic pulses, etc., so that it is very easy to damage normal cells, and has no specificity in treatment.
Chinese patent CN110461346A discloses the use of an oncolytic virus alone or in combination with a checkpoint inhibitor for the treatment of cancer, wherein the oncolytic virus can be used for the treatment of B-cell lymphoma, colorectal cancer, melanoma, etc., and the checkpoint inhibitor is CTLA-4, an anti-PD-1 antibody or a PD-L1 blocker. Moreover, although the medicine can be used for treating cancer, some side effects still exist in the specific taking process, the medicine is harmful to human bodies, and the medicine is only suitable for metastatic cancer, is limited in application range and has certain toxicity.
In conclusion, the prior art generally has the defects of poor treatment effect, low specificity, high toxicity, certain side effect and the like in the process of preparing the tumor treatment medicine.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a kit for specifically inhibiting the proliferation of K7M2.Wt of osteosarcoma cells and application thereof. The pharmaceutical composition provided by the invention contains the echogenic viruses echogenic and the anti-PD-1 antibody, so that whether the echogenic viruses echogenic can enhance the anti-PD-1 antibody anti-osteosarcoma treatment effect can be determined, and the synergistic attenuation effect on the osteosarcoma treatment can be achieved.
In order to achieve the purpose, the invention adopts the technical effects that:
use of an oncolytic virus for the manufacture of a pharmaceutical composition for the treatment of osteosarcoma, said pharmaceutical composition comprising an oncolytic virus and an immunosuppressive molecule antibody.
Preferably, the oncolytic virus is echol.
Preferably, the immunosuppressive molecule antibody is an anti-PD-1 antibody.
The invention also provides application of the oncolytic virus in preparing a pharmaceutical composition for treating osteosarcoma, wherein the pharmaceutical composition comprises echolucin and an anti-PD-1 antibody.
Preferably, the anti-PD-1 antibody is from BioXcell, lot No. 800121A1ZB.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
Preferably, the dosage form of the pharmaceutical composition is one of injection, freeze-dried powder and nano-liposome.
The invention also provides application of the oncolytic virus in preparing a composition for increasing the number of immune cells in osteosarcoma, wherein the composition comprises echolucin and an anti-PD-1 antibody.
Preferably, the immune cells are T lymphocytes and DC cells.
For test animals, the pharmaceutical composition provided by the invention has specific administration dosage and administration timing when being taken. Wherein the dosage of the hecogenin in the medicine is 1 × 10 9 vp/mm 3 Tumor volume/time; the administration time is once every 3 days from 7 days after the experimental animal is inoculated with the tumor cells, and the administration is carried out after the anti-PD-1 antibody is administered; the dosage of the anti-PD-1 antibody in the medicament is 2.5mg/kg, the administration timing is once every 3 days from 7 days after the experimental animal is inoculated with the tumor cells, and the administration is carried out before echolucent.
In the transplantation tumor model of K7M2 inoculated mice, oncolytic virus echori was given intratumoral injection (1 x 10) 9 vp/mm 3 Tumor volume/time, qd, D7-D9, D11-D13, D15-D17, D19-D21, D23-D26, D28-D31, D33-D35, D37-D39), tail vein injection of anti-PD-1 antibody (2.5 mg/kg, i.p., qw, D10, D17, D24, D31)), and an effect test for treating osteosarcoma.
The anti-PD-1 antibody treatment can obviously inhibit tumor growth, improve weight loss caused by tumors and increase immune cell infiltration in tumor tissues; the echolocation oncolytic virus ancymi is used for inhibiting tumor growth, improving weight loss caused by tumors and promoting immune cell infiltration including cell infiltration of a lymphatic system and a medullary system in cooperation with anti-PD-1 antibody treatment. Deep sequencing analysis shows that the combined treatment of the oncolytic virus ancorum and the anti-PD-1 antibody shows different transcription characteristics, but the ancorum and the anti-PD-1 antibody have synergistic effect, particularly on the regulation and control of immunity and metabolism. These results suggest that echolucin, an oncolytic virus, can cooperate with anti-PD-1 antibody treatment to exert synergistic attenuation effects, and that the mechanism of improving tumor killing effect by combined administration may be realized by increasing antigen presentation, regulating T lymphocyte phenotypic characteristics and enhancing anti-PD-1 antibody killing effect.
The invention analyzes and evaluates the value of ancorui for treating osteosarcoma by evaluating whether the ancorui and the anti-PD-1 antibody can achieve the effect of synergy and attenuation in treating osteosarcoma by combined use, and provides a new example for synergy and attenuation with the anti-PD-1 antibody.
Compared with the prior art, the invention has the following advantages: the invention combines the anti-PD-1 antibody and the oncolytic virus echol, can treat osteosarcoma, has obvious treatment effect and synergy and attenuation effects, and can provide reference and basis for applying the oncolytic virus echol to other tumor treatments.
Drawings
FIG. 1 is a diagram of a specific dosing regimen;
FIG. 2 is an analysis of survival of animals after administration;
FIG. 3 is a graph of tumor volume change following administration;
FIG. 4 is a graph of weight change in animal carcinostatic animals;
FIG. 5 is a graph showing the results of HE staining and immunofluorescence staining;
FIG. 6 is a graph showing the results of tissue analysis of the ratio of immune cells (CD 45+ cells) and CD3+ T lymphocytes to the total cells in tumor tissues;
FIG. 7 is a graph of changes in myeloid and lymphoid cells in a tissue analyzed tumor tissue immune cell population;
FIG. 8 is a graph showing the specific changes of T cells (CD 3+ cells) and B cells in lymphocytes of tumor tissue in tissue analysis;
FIG. 9 is a graph showing the specific changes of CD4+ cells and CD8+ cells in CD3+ T lymphocytes of tumor tissues in tissue analysis;
FIG. 10 is a graph showing the specific changes of macrophages and Dendritic Cells (DCs) in myeloid lineage cells of a tissue analyzed tumor tissue;
FIG. 11 is a graph showing the specific changes in two subsets of CD11b + DCs and CD103+ DCs in dendritic cells from tissue analyzed tumor tissues;
FIG. 12 is a graph of the alteration of TAM1 and TAM2 subtypes in macrophages of tissue analyzed tumor tissues;
FIG. 13 is a graph of serum analysis of the changes in the liver function biomarkers ALT and AST of each experimental group;
FIG. 14 is a graph of histopathological analysis of liver at 2-fold magnification for each experimental group.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The specific techniques or conditions not mentioned in this example were followed by procedures and apparatus instructions in accordance with conventional techniques in the art; the reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.
The oncolytic virus ancorui can be purchased from Shanghai three-dimensional biotechnology, inc.; the anti-PD-1 antibody is commercially available from zhengzhou bosch biotechnology limited (BioXcell) under lot number 800121A1ZB; the osteosarcoma cell K7M2.Wt is derived from the cell bank of Chinese academy of sciences.
Example 1 animal dosing test
1. Test subjects: 40 BALB/c mice are averagely divided into 8 cages, each cage is 5, each group is 2 cages, and the labels are marked on the cages in sequence;
2. the test process comprises the following steps: the test was divided into 4 groups, namely NTC (simple negative control group), anti-PD-1 antibody treatment group, anti-PD-1 antibody + oncolytic virus echol combination treatment group, and oncolytic virus echol treatment group. When administered, the dosage of the anti-PD-1 antibody treatment was 2.5mg/kg, i.e., 0.35mL of the antibody stock solution (7.18 mg/mL) was taken, and 3.65mL of PBS (10% glycerol) was added to obtain a working solution, which was injected subcutaneously into each animal at 0.1mL. The dose of echolucin for oncolytic virus is 2 multiplied by 10 10 vp/mm 3 (ii) a NTC group added PBS; in the combination treatment group, the dosage of the anti-PD-1 antibody is 2.5mg/kg, and the dosage of the oncolytic virus echol is 1x10 9 vp/mm 3 Tumor volume/time;
after the animal grows the tumor and the tumor volume is measured, the administration volume is calculated according to the tumor volume on the previous day on the next day for administration. Animals were received (D-14), after 14 days of acclimation, K7M2.Wt tumour cells (D0) were inoculated, raised for 7 days and weighed (D7), and anti-PD-1 antibody and oncolytic virus ancorum treatment were given as course of treatment, wherein the anti-PD-1 antibody was administered once a week by tail vein injection. The echolucin treatment of the oncolytic virus adopts an intratumoral injection method, the administration lasts for 3 days and 2 days, and the administration is circulated for 5 cycles, and the specific administration scheme is shown in figure 1.
3. And (3) test results: specific test results are shown in fig. 2, fig. 3 and fig. 4, and as can be seen from fig. 2, the survival time of animals is remarkably prolonged in the anti-PD-1 antibody and oncolytic virus treatment group compared with the anti-PD-1 antibody group alone; as can be seen from fig. 3, the anti-PD-1 antibody-oncolytic virus treatment group showed significantly lower tumor growth rate and significantly smaller tumor volume than the anti-PD-1 antibody treatment group alone, compared to the anti-PD-1 antibody treatment group alone, suggesting that the combination treatment group had a good anti-tumor effect; as can be seen from FIG. 4, the growth curves of the animals are also different between 4 groups, the body weights of the animals in the anti-PD-1 antibody and oncolytic virus treatment group are relatively kept stable, and the weight average of the tumor-removed bodies of the other 3 groups of animals is obviously reduced gradually. Therefore, the survival rate of the group treated by the anti-PD-1 antibody and the oncolytic virus is generally higher than that of other groups, the change of the tumor volume is rapid, the volume is smaller than that of other groups, and the change of the body weight after tumor removal is minimum.
Example 2 histopathological analysis
The experiment as described in example 1 above was repeated simultaneously with 5 animals per group, and tumor tissue was taken on day 30 after tumor inoculation and histopathological analysis was performed, and the results are shown in fig. 5 of the specification. As can be seen in FIG. 5, HE staining revealed an increase in the necrotic area of tissues in the oncolytic virus-anti-PD-1 antibody-treated group. Immunofluorescence results show that 3 intervention treatment groups all have increased infiltration of immune cells in tumor tissues compared with a control group, and a combined treatment group is obviously better than a single treatment group.
The inventors further used the tissue analysis for the proportion of immune cells (CD 45+ cells) and CD3+ T lymphocytes in tumor tissues. The relevant results are described in the specification and shown in figure 6, and as can be seen from figure 6, compared with the NTC control group, the proportion of immune cells and lymphocytes is increased in the tumor tissues in the treatment of 3 groups, wherein the effect of the combination treatment group is most obvious, and the effect of the oncolytic virus combined with the anti-PD-1 antibody is stronger than the increase value of the single treatment group. Indicating that the combination treatment group has increased tumor tissue immune cell infiltration, and the increase is mainly increased by CD3+ T lymphocytes.
The change of the myeloid line cell and lymphoid line cell sub-population is shown in the figure 7 of the specification; as can be seen from fig. 7, the proportion of immune cells and lymphocytes increased in the tumor tissue was observed in all of the 3 groups of treatments compared to the NTC control group, wherein the effect of the combination treatment group was most significant. In the lymphocyte population, T cells and B cells are changed as shown in the figure 8 of the specification, and as can be seen from the figure 8, the infiltration of the T cells in the tumor tissue is obviously increased and the B cells are obviously reduced in the combination treatment group compared with the NTC group. It is believed that T cells are a major contributor to the anti-tumor effect of immunotherapy, while B cells exert a complex effect that reduces the anti-tumor effect. Therefore, the immune cell infiltration formed by combining the oncolytic virus and the anti-PD-1 antibody in a tumor microenvironment, particularly the anti-tumor effect of the anti-tumor virus can be obviously enhanced by increasing the infiltration of T lymphocytes. The change of the composition ratio of CD4+ T cells and CD8+ T cells is shown in figure 9 of the specification; the CD8+ T lymphocyte infiltration rate of the combination treatment group was increased, while the CD4+ T lymphocyte infiltration rate was decreased. The CD8+ T lymphocyte is a lymphocyte population mainly killing tumors in the T lymphocyte, and the CD4+ T cell contains a plurality of subgroups and has complex action; we analyzed the results of the change in the ratio of T lymphocytes by the combination treatment group and found that the ratio of CD8+ T lymphocytes is significantly increased, which suggests that the ratio of CD8+ T lymphocytes, which are the main tumor-killing groups, can be significantly increased by the combination treatment group.
In the aspect of myeloid cells, we mainly analyze the composition ratio of Tumor-associated macrophages (TAMs) and Dendritic Cells (DCs), mainly the change of DC cells can be seen in fig. 10 of the specification, but the ratio of TAMs is not changed significantly. The DC cell can recognize antigen in tumor immunity and presents to lymphocyte to kill. The oncolytic virus reduced the proportion of DC cells in this study, but the combination treatment group resulted in an increase in the proportion of DC cells, suggesting that the combination treatment group could restore the effect of DC and promote the enhancement of anti-tumor effect. To further analyze DC cell subsets, we analyzed changes in both CD11b + DC and CD103+ DC subsets in tumor tissues based on CD11b and CD103 expression, as shown in fig. 11; from the results, it can be seen that the anti-PD-1 antibody caused an increase in the CD103+ DC subpopulation ratio, whereas oncolytic virus treatment caused a decrease in the CD103+ DC cell population, and the combination treatment group balanced the DC cell ratio, improving CD103+ DC cell infiltration of the tumor microenvironment. CD103+ DC cells may recruit lymphocytes to participate in tumor immunity, suggesting that combination therapy may reprogram the tumor microenvironment, promoting enhanced anti-tumor therapeutic efficacy. FIG. 12 shows the changes of TAM1 and TAM2 subtypes in two subgroups of TAM, and it can be seen from FIGS. 10 and 12 that TAM1 and TAM2 are significantly changed although total macrophages are not significantly changed; TAM1 in a tumor microenvironment can secrete Th1 cytokines to play a role in proinflammatory and antitumor, while TAM2 promotes angiogenesis and tumor invasion by secreting Th2 cytokines, and our results show that oncolytic viruses can remarkably promote infiltration of TAM1 in the tumor microenvironment and reduce accumulation of TAM 2. The combined treatment group shows that the combined use of the anti-PD-1 antibody and the oncolytic virus can promote the infiltration of the TAM1 and reduce the accumulation of the TAM2, which indicates that the anti-PD-1 antibody combined with the oncolytic virus can also be regulated and controlled by the subgroup of the TAM to enhance the anti-tumor effect of the oncolytic virus.
To characterize the anti-PD-1 antibody in combination with the oncolytic virus echol, we performed toxicity studies, in particular functional analysis of liver, an important organ for virus accumulation. As shown in figure 13 of the specification. Serum analysis of the change of ALT and AST as liver function biomarkers in different experimental groups shows that the anti-PD-1 antibody, the oncolytic virus echol and the combined treatment of the two do not cause significant change of liver function. FIG. 14 shows histopathological analysis of liver tissues in each experimental group; the results showed no obvious abnormalities in the liver in each group. As can be seen from FIGS. 13-14, the combined use of the oncolytic virus echol and the anti-PD-1 antibody for treating osteosarcoma has no hepatotoxicity and good safety.
Finally, it should be noted that the above-mentioned embodiments are only illustrative for the principle, performance and efficacy of the present invention, and are not meant to limit the present invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (9)
1. Use of an oncolytic virus for the manufacture of a pharmaceutical composition for the treatment of osteosarcoma, said pharmaceutical composition comprising an oncolytic virus and an immunosuppressive molecular antibody.
2. The use according to claim 1, wherein the oncolytic virus is echol.
3. The use of claim 1, wherein the immunosuppressive molecule is an anti-PD-1 antibody.
4. Use of an oncolytic virus for the manufacture of a pharmaceutical composition for the treatment of osteosarcoma, said pharmaceutical composition comprising echol and an anti-PD-1 antibody.
5. The use according to any one of claims 3 to 4, wherein the anti-PD-1 antibody is from BioXcell under the batch number 800121A1ZB.
6. The use of claim 1 or 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
7. The use of claim 1 or 4, wherein the pharmaceutical composition is in the form of one of injection, lyophilized powder, and nanoliposome.
8. Use of an oncolytic virus for the preparation of a composition for increasing the number of immune cells in osteosarcoma, said composition comprising echol and an anti-PD-1 antibody.
9. The use of claim 8, wherein the immune cells are T lymphocytes and/or DC cells.
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| CN116271008A (en) * | 2022-12-30 | 2023-06-23 | 广东天普生化医药股份有限公司 | Pharmaceutical composition containing An Kerui and Carilizumab and application thereof |
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