CN115120744A - Application of recombinant human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in preparation of anti-tumor drugs - Google Patents
Application of recombinant human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in preparation of anti-tumor drugs Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to an application of a recombinant human endostatin adenovirus and an anti-PD-1 antibody or an anti-PD-L1 antibody in preparation of an anti-tumor medicine. The invention aims to solve the technical problem of providing a medicament with better anti-tumor effect. The technical scheme for solving the technical problem is to provide an anti-tumor medicament which contains adenovirus for coding human endostatin and an anti-PD-1 antibody or an anti-PD-L1 antibody as main active ingredients. The combination of the adenovirus containing the coding human endostatin and the immunotherapy of the anti-PD-1 antibody or the anti-PD-L1 antibody is an effective method for treating tumors and has good application prospect.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an application of recombinant human endostatin adenovirus and an anti-PD-1 antibody or an anti-PD-L1 antibody in preparation of an anti-tumor medicine.
Background
Angiogenesis refers to the generation of new blood vessels from existing blood vessels, and tumor angiogenesis can provide oxygen and nutrients for tumors, and is an important process for the growth and progression of tumor cells. Tumor growth results in a decrease in oxygen content in the tumor microenvironment, which promotes the expression of pro-angiogenic factors (e.g., vascular endothelial growth factor, epidermal growth factor, etc.) and thus angiogenesis. The tumor blood vessels have tortuous shapes and more branches, and the structural and functional abnormalities of the blood vessels can cause serious influence on blood perfusion, thereby causing tumor tissue hypoxia and hindering drug delivery and diffusion. Therefore, the development of anti-angiogenic drugs to block blood supply is one of the effective ways to treat malignant and non-malignant neovascular diseases. Folkman proposed the theory of tumor angiogenesis as early as 1971, and proved by experiments that anti-angiogenic drugs can effectively inhibit tumor growth. Through many years of research, a plurality of anti-angiogenesis drugs are developed at present, mainly targeting angiogenesis promoting factors and receptors thereof, such as antibody drugs bevacizumab and small molecule inhibitor sunitinib, and are approved by FDA in the united states for treating a plurality of tumors such as lung cancer, metastatic colorectal cancer, ovarian cancer, and the like. Endostatin is an endogenous anti-angiogenic molecule with a molecular weight of 20 kDa. In 2005, the chinese FDA approved endostatin (endostar) with proprietary intellectual property rights for the treatment of non-small cell lung cancer. Ad-Endostatin (Ad-E) is an adenovirus encoding human Endostatin (see Chinese patent ZL200510021720.7 "a recombinant human Endostatin adenovirus and its preparation method and use"), and early studies show that: Ad-E has inhibitory effect on the growth of lung cancer, SKOV3 ovarian cancer and CT26 colon cancer, and the recombinant adenovirus has already completed phase I clinical research at present.
With the continuous and intensive research, it is found that the anti-angiogenesis therapy has certain limitations, and some tumors such as prostate cancer, pancreatic cancer and the like are not sensitive to anti-angiogenesis drugs. In glioblastoma, bevacizumab responds significantly initially but for a limited duration, is susceptible to drug resistance leading to poor prognosis and limited patient survival. Bevacizumab also failed to achieve the expected therapeutic effect in clinical studies on metastatic breast cancer. The regulation of tumor angiogenesis is influenced by a plurality of factors and receptors, and the inhibition of one signaling pathway may not influence other signaling pathways, so that the search for new therapeutic approaches is crucial for tumor treatment. At present, endostatin is mainly combined with radiotherapy, chemotherapy and the like clinically to improve the cure rate of cancer patients and prolong the survival time of the patients.
Activated CD4 + T cell, CD8 + Various cells such as T cells, natural killer T cells and the like can express PD-1 molecules, and PD-L1 is a ligand of PD-1 and is often expressed on the surface of tumor cells. Studies have shown that in tumor tissues of tumor patients, the binding and interaction of PD-1 and PD-L1 renders T cells unable to recognize tumor cells, leading to tumor immune escape. At present, tumor immune escape caused by PD-1/PD-L1 becomes a hotspot field for researching tumor treatment, and inhibitors of PD-1 and PD-L1 can block the combination of PD-1/PD-L1, so that T cell failure is relieved, effector T lymphocytes are activated, tumor immunosuppression is reduced, and T cell activity is recovered, and then tumor cells are identified and killed. At present, antibodies aiming at PD-1 and PD-L1 are approved to be used for treating various tumors, FDA has approved an anti-PD-L1 antibody to treat bladder cancer and melanoma, the anti-PD-1 antibody is widely used in more than 10 tumors such as melanoma, non-small cell lung cancer, liver cancer, renal cell carcinoma, head and neck cancer, DNA mismatch deletion type colorectal cancer and the like, the tumor growth is relieved after treatment, and the survival period is prolonged. However, as intensive and continuous research progresses, the blocking treatment of the PD-1/PD-L1 inhibitor has a plurality of limitations: the single medicine treatment response rate is not high, and is generally between 20 and 40 percent; the effect difference is large aiming at different tumors, and a plurality of insensitive tumors exist; even in sensitive tumors, there are significant individual differences, and only a portion of the population can benefit; the remission rate is large and unstable, and even patients are in the processA case where the disease progresses rapidly without receding after the use of an inhibitor; secondary resistance phenomena exist, and studies have reported that up to 60-70% of patients are estimated to develop resistance to treatment with immune checkpoint inhibitors. Current studies indicate that there are many reasons for primary resistance of tumors to PD-1/PD-L1 inhibitors, mainly: infiltration T lymphocytes in a tumor microenvironment are reduced, tumor mutation antigen load is reduced, expression of Th1 type chemokines is silenced due to epigenetic abnormality, expression of important immune signal molecules is reduced, and immune inhibitory molecules are highly expressed to further unbalance immune regulation and the like.
Disclosure of Invention
The invention aims to provide a medicament with better anti-tumor effect. The technical scheme for solving the technical problem is to provide an anti-tumor medicament which contains adenovirus for coding human endostatin and an anti-PD-1 antibody or an anti-PD-L1 antibody as main active ingredients.
Wherein, the amino acid sequence coded and expressed by the adenovirus coding the human endostatin in the medicine is the human endostatin shown in SEQ ID No. 1.
SEQ ID No.1：
HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK。
Furthermore, the adenovirus in the anti-tumor medicament is replication-defective adenovirus. Further, the adenovirus is a type 5 replication-defective adenovirus.
Wherein, the N end of the human endostatin in the anti-tumor drug is also connected with a signal peptide.
Wherein the signal peptide in the antitumor drug is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor type hEGF signal peptide, human immunoglobulin type kappa chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-2 signal peptide.
Furthermore, when the signal peptide in the antitumor drug is the human IL-2 signal peptide, the amino acid sequence of the human endostatin connected with the human IL-2 signal peptide is shown as SEQ ID No. 2.
SEQ ID No.2：
MYRMQLLSCIALSLALVTNSAHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK。
Wherein, the coding nucleotide sequence of the human endostatin in the antitumor drug is shown in SEQ ID No. 3.
SEQ ID No.3：
CACAGCCACCGCGACTTCCAGCCGGTGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGCGGGGCATCCGCGGGGCCGACTTCCAGTGCTTCCAGCAGGCGCGGGCCGTGGGGCTGGCGGGCACCTTCCGCGCCTTCCTGTCCTCGCGCCTGCAGGACCTGTACAGCATCGTGCGCCGTGCCGACCGCGCAGCCGTGCCCATCGTCAACCTCAAGGACGAGCTGCTGTTTCCCAGCTGGGAGGCTCTGTTCTCAGGCTCTGAGGGTCCGCTGAAGCCCGGGGCACGCATCTTCTCCTTTGACGGCAAGGACGTCCTGAGGCACCCCACCTGGCCCCAGAAGAGCGTGTGGCATGGCTCGGACCCCAACGGGCGCAGGCTGACCGAGAGCTACTGTGAGACGTGGCGGACGGAGGCTCCCTCGGCCACGGGCCAGGCCTCCTCGCTGCTGGGGGGCAGGCTCCTGGGGCAGAGTGCCGCGAGCTGCCATCACGCCTACATCGTGCTCTGCATTGAGAACAGCTTCATGACTGCCTCCAAGTAG。
Furthermore, the coding nucleotide sequence of the human endostatin connected with the IL-2 signal peptide is shown in SEQ ID No. 4.
SEQ ID No.4：
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGGCCCACAGCCACCGCGACTTCCAGCCGGTGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGCGGGGCATCCGCGGGGCCGACTTCCAGTGCTTCCAGCAGGCGCGGGCCGTGGGGCTGGCGGGCACCTTCCGCGCCTTCCTGTCCTCGCGCCTGCAGGACCTGTACAGCATCGTGCGCCGTGCCGACCGCGCAGCCGTGCCCATCGTCAACCTCAAGGACGAGCTGCTGTTTCCCAGCTGGGAGGCTCTGTTCTCAGGCTCTGAGGGTCCGCTGAAGCCCGGGGCACGCATCTTCTCCTTTGACGGCAAGGACGTCCTGAGGCACCCCACCTGGCCCCAGAAGAGCGTGTGGCATGGCTCGGACCCCAACGGGCGCAGGCTGACCGAGAGCTACTGTGAGACGTGGCGGACGGAGGCTCCCTCGGCCACGGGCCAGGCCTCCTCGCTGCTGGGGGGCAGGCTCCTGGGGCAGAGTGCCGCGAGCTGCCATCACGCCTACA TCGTGCTCTGCATTGAGAACAGCTTCATGACTGCCTCCAAGTAG。
The medicine is prepared by mixing adenovirus for coding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody into a composition, and independently packaging in the same packaging container in a separated mode or respectively and independently packaging in packaging containers.
Meanwhile, the invention also provides the application of the adenovirus for coding the humanized endostatin and the anti-PD-1 antibody or the anti-PD-L1 antibody in preparing anti-tumor drugs. Wherein, the adenovirus for coding the human endostatin in the application is combined with an anti-PD-1 antibody or an anti-PD-L1 antibody.
Wherein, the amino acid sequence coded and expressed by the adenovirus for coding the human endothelial chalone in the application is the human endothelial chalone shown in SEQ ID No. 1.
Furthermore, the adenovirus in the anti-tumor medicament is replication-defective adenovirus. Further, the adenovirus is a type 5 replication-defective adenovirus.
Wherein, the N end of the human endostatin in the anti-tumor drug is also connected with a signal peptide.
Wherein the signal peptide used in the above application is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor hEGF signal peptide, human immunoglobulin kappa chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide and human IL-2 signal peptide.
Furthermore, when the signal peptide in the anti-tumor drug is a human IL-2 signal peptide, the amino acid sequence of the human endostatin connected with the human IL-2 signal peptide is shown in SEQ ID No. 2.
Wherein, the coding nucleotide sequence of the human endostatin in the antitumor drug is shown in SEQ ID No. 3.
Furthermore, the coding nucleotide sequence of the human endostatin connected with the IL-2 signal peptide is shown in SEQ ID No. 4.
Wherein, the adenovirus for coding the human endostatin in the anti-tumor drugs in the application and the anti-PD-1 antibody or the anti-PD-L1 antibody are mixed into a composition and are independently packaged in the same packaging container in a separated way or are respectively and independently packaged in the packaging containers.
The present invention also provides a method of treating a tumor comprising the step of administering to a patient having a tumor an effective amount of an adenovirus encoding human endostatin and an effective amount of an anti-PD-1 antibody, or administering an effective amount of an adenovirus encoding human endostatin and an effective amount of an anti-PD-L1 antibody.
Further, the adenovirus of human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody in the above-described method may be administered simultaneously or separately during the course of treatment.
Among them, the adenovirus in the above-mentioned medicament is preferably a replication-defective adenovirus.
Wherein, the tumor in each technical scheme can be selected from colorectal cancer, breast cancer, non-small cell lung cancer, liver cancer, colon cancer, kidney cancer, ovarian cancer, head and neck cancer, endometrial cancer, melanoma and bladder cancer.
The invention has the beneficial effects that: the invention can inhibit angiogenesis, improve tumor inhibition microenvironment, enhance T cell tumor infiltration, reduce immunosuppression, inhibit M2 type macrophage polarization in tumors and the like by drug combination treatment of adenovirus (Ad-E) encoding human endostatin and anti-PD-1 or anti-PD-L1 antibody. Experiments show that the two combined treatments can effectively inhibit the growth of the tumor, eliminate the tumor and have no relapse, and no obvious toxic or side effect is seen. The invention has the advantages that the combination of the Ad-E anti-angiogenesis therapy and the anti-PD-1 antibody immunotherapy or the anti-PD-L1 antibody immunotherapy is an effective method for treating tumors, provides a theoretical basis for further clinical relevant experiments, and has good application prospect.
Drawings
FIG. 1. anti-tumor activity of α PD-1 (anti-PD-1 antibody)/α PD-L1 (anti-PD-L1 antibody) in mouse tumor models. A. Alpha PD-1 treats B16F10 tumors. B. Alpha PD-1 treats MC38 tumors. C. Alpha PD-L1 treats MC38 tumors. ns: not significant (nonognificant).
FIG. 2 Ad-E treatment effectively inhibited tumor growth. A and B: B16F10(A) and MC38(B) tumor volumes were significantly reduced following Ad-E treatment; Ad-E post-treatment B16F10, flow analysis of tumor groupsKnitted CD4 + And CD8 + An increase in the proportion of T cells; d: WB detection of PD-L1 protein expression following B16F10 after Ad-E treatment; e: PD-L1mRNA expression was detected by RT-PCR after B16F10 following Ad-E treatment. ns is not significant (notnsigniicant)<0.05，**p<0.01，***p<0.001
FIG. 3 Ad-E in combination with alpha PD-1 treatment enhances antitumor activity. A: the combined treatment of Ad-E and alpha PD-1 effectively inhibits the growth of MC38 tumor; b: tumor specimens were immunostained with CD31 (red) and DAPI nuclear stain (blue) to detect angiogenesis.
Figure 4.Ad-E in combination with α PD-L1 treatment enhanced antitumor activity. Ad-E and alpha PD-L1 in combination effectively inhibit MC38 tumor growth;
FIG. 5 shows the change of tumor microenvironment after in vitro detection and combination. A and B: flow cytometry analysis of CD8 in tumor samples + And PD-1 + Proportion of T cells (T cells). C and D: flow cytometry analysis of CD11b in tumor samples + F4/80 + Macrophages and CD206 + Proportion of macrophages (TAMs). P<0.05，**p<0.01。
FIG. 6 tumor tissue transcriptomics analysis after Ad-E in combination with α PD-1 treatment. A: the number of differentially expressed genes was found in each group of samples, with Up (Up) on the left and Down (Down) on the right in each group of data; b: and (3) carrying out enrichment analysis on the differential gene GO after the combination treatment of Ad-E and alpha PD-1.
FIG. 7 preliminary evaluation of the safety of Ad-E in combination with anti- α PD-1 treatment. H & E staining was used to detect pathological changes in the organs.
Detailed Description
The present invention will be described in more detail below with reference to specific embodiments.
A great deal of creative work in the early stage of the invention discovers that the adenovirus (recombinant human endostatin adenovirus, Ad-endostatin, abbreviated as Ad-E or ADE) for coding the human endostatin can inhibit the growth of tumors and promote the infiltration of tumor T cells and the expression of PD-L1, thereby providing a basis for the combination of the anti-PD-1 antibody or the anti-PD-L1 antibody.
In the early work, the invention discovers that the application of the anti-PD-1 antibody can not show obvious anti-tumor effect in a B16F10 tumor model and an MC38 tumor model, and the anti-PD-L1 antibody treatment can not inhibit the growth of the MC38 tumor. Therefore, the Ad-E is combined with the anti-PD-1 antibody or the PD-L1 antibody to synergistically improve the anti-tumor effect. The invention discovers that the combination of Ad-E and an anti-PD-1 antibody or an anti-PD-L1 antibody can play a synergistic role, remarkably enhances the effect of inhibiting the tumor growth, and even shows the condition of tumor regression and no relapse. It is likely that tumor angiogenesis will lead to immunosuppressed tumor microenvironments: the method has the advantages of generating a mechanical barrier for T cell infiltration to reduce the T cell infiltration, inhibiting the antigen presentation of DC cells, improving the proportion of Tregs, bone marrow suppressor cells and M2 tumor-related macrophages, up-regulating the expression of PD-1 on the surface of the T cells and weakening the anti-tumor function of the T cells. Ad-E targeted angiogenesis can normalize blood vessels, increase immune cell infiltration and PD-L1 expression in tumor tissues, reduce tumor-induced immunosuppression, and promote the curative effect of tumor immunotherapy. The combination of the two can inhibit angiogenesis, improve tumor inhibition microenvironment, reduce immunosuppression and reduce chemotaxis of M2 type macrophages, thereby mutually enhancing the anti-tumor effect.
On the basis, the invention obtains various technical schemes to be protected.
One of them is to provide an antitumor drug containing adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody as main active ingredients. Namely, the anti-tumor medicament containing the adenovirus for coding the human endostatin and the anti-PD-1 antibody as main active components or the anti-tumor medicament containing the adenovirus for coding the human endostatin and the anti-PD-L1 antibody as main active components is provided.
Simultaneously provides the application of the adenovirus for coding the human endostatin and the anti-PD-1 antibody or the anti-PD-L1 antibody as main active ingredients in preparing anti-tumor drugs.
It will be appreciated by those skilled in the art that there are various combinations of the adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody as the main active ingredients in the above-mentioned drugs. For example, an adenovirus encoding human endostatin may be formulated with an anti-PD-1 or anti-PD-L1 antibody for administration as a combination.
The adenovirus for coding the human endostatin and the anti-PD-1 antibody or the anti-PD-L1 antibody can be respectively prepared into separate medicaments, and the medicaments are prepared by placing the medicaments in the same package. Such a medicament can be administered by simultaneously administering the two active ingredients or separately administering and controlling the respective amounts thereof, as desired. Of course, such a formulation can also facilitate long-term storage and improved stability of the two major active ingredients.
One skilled in the art can also formulate the adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody separately into separate dosage forms, but in separate packages for combined administration.
The formulation of these agents, the selection of the corresponding pharmaceutically acceptable auxiliary ingredients, and the selection of the corresponding packaging materials for the agents are easily accomplished by those skilled in the art.
In addition, the present invention provides a method for treating a tumor, the method comprising the steps of administering to a patient having a tumor an effective amount of an adenovirus encoding human endostatin and administering an effective amount of an anti-PD-1 antibody or an anti-PD-L1 antibody. Further, the adenovirus and the anti-PD-1 antibody or the anti-PD-L1 antibody of human endostatin in the above-described method may be administered simultaneously or separately during the course of treatment. Of course, the specific amounts of the above-described adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody administered can be suitably adjusted to achieve a better synergistic anti-tumor effect for different tumor types and for different specific patients.
The adenovirus encoding human endostatin used in the present invention may also be referred to as recombinant human endostatin adenovirus. The amino acid sequence of the human endostatin is shown in SEQ ID No. 1. In order to play a better role, a signal peptide can be connected to the N end of the human endostatin. The signal peptide is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor hEGF signal peptide, human immunoglobulin kappa chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-2 signal peptide. However, replication-defective adenoviruses are generally used for adenoviruses of the invention which encode human endostatin. Preferably, a type 5 replication-defective adenovirus is used.
The preparation of the gene encoding human endostatin can be performed using conventional methods for preparing recombinant adenoviruses in the art. In one embodiment of the present invention, an adenovirus encoding human endostatin prepared by the method described in the embodiment of chinese patent ZL200510021720.7 "a recombinant human endostatin adenovirus and its preparation method and use" is used.
The anti-PD-1 antibody or the anti-PD-L1 antibody in each technical scheme provided by the invention can be various common anti-PD-1 antibodies or anti-PD-L1 antibodies.
The present invention will be described in further detail by way of examples.
The experimental materials mainly used in the examples are as follows:
c57BL/6 mice were purchased from Wintonlifys, Beijing.
antibodies such as anti-CD4, anti-CD3, anti-CD8, anti-CD45, anti-CD11b, anti-F4/80, and anti-CD206 were purchased from BD Biosciences. PD-L1 antibody was purchased from Bio X cell
PD-1 antibody was purchased from Bio X cell (cat # BP 0146).
PD-L1 antibody was purchased from Bio X cell (cat BP 0101).
Adenovirus (recombinant human endostatin adenovirus, Ad-endostatin, abbreviated as Ad-E or ADE) encoding humanized endostatin, and unloaded adenovirus (Ad-Null) were provided by pharmaceutical corporation of Bailing, Guizhou, with an Ad-E concentration of 1 × 10 12 VP/mL; Ad-Null concentration 1.24X 10 12 VP/mL, cryo-preserved at-80 ℃ and diluted to the desired concentration just before use. Reference may also be made to chinese patent ZL200510021720.7, "a recombinant human endostatin adenovirus, and its preparation method and use" which describes the adenovirus encoding human endostatin used in the preparation examples.
Example 1 study of in vivo antitumor Activity of alpha PD-1 (anti-PD-1 antibody)
6-8 week-old female C57BL/6 mice were injected subcutaneously5×10 5 B16F10 melanoma cells or 2X 10 6 MC38 Colon cancer cells when the tumor volume reaches about 100mm 3 And then, random grouping is carried out. The method is divided into two groups: group alpha PD-1 or alpha PD-L1, treated with alpha PD-1 or alpha PD-L1, each intraperitoneal injection of 1200. mu.g of alpha PD-1 or alpha PD-L; the control group is isotype antibody (isotype antibody of alpha PD-1 is IgG2a, isotype antibody of alpha PD-L1 is IgG2b), and 200 mug of isotype antibody is injected into the abdominal cavity every time; the administration is 2 times for 1 week, 4 times in total.
The experimental results show that: in the B16F10 tumor model (fig. 1A), alpha PD-1 treatment did not inhibit tumor growth compared to the IgG2a control group; in the MC38 tumor model (fig. 1B), alpha PD-1 treatment had weak anti-tumor activity compared to the IgG2a control group, but there was no significant difference between the two groups. In alpha PD-L1 treatment of MC38 tumors (fig. 1C), compared to IgG2a control.
Example study of in vivo antitumor Activity of recombinant human endostatin adenovirus (Ad-E)
Ad-E therapy for inhibiting tumor growth
6-8 week-old female C57BL/6 mice were injected subcutaneously with 5X 10 5 B16F10 melanoma cells or 2X 10 6 MC38 colon cancer cells when the tumor volume reaches about 100mm 3 At this time, random grouping is performed.
(1) B16F10 melanoma model grouping and treatment:
control group: equal volume of sterile PBS;
Ad-Null group: 1X 10 9 pfuAd-Null/mouse, intratumorally injected once every 3 days for 4 times;
group Ad-E: 1X 10 9 pfuAd-E/mouse, intratumorally injected once every 3 days for 4 times;
(2) MC38 colon cancer model grouping and treatment:
control group: equal volume of sterile PBS;
Ad-Null group: 3X 10 8 pfuAd-Null/mouse, intratumorally injected once every 1 week for 2 times;
Ad-E group: 3X 10 8 pfuAd-E/mouse, intratumorally injected once every 1 week for 2 times;
tumor size was measured in mice every 3 days.
The experimental results show (fig. 2): in the B16F10 (FIG. 2A) and MC38 tumor models (FIG. 2B), Ad-E treatment significantly inhibited tumor growth (p <0.05) compared to the Control and Ad-Null groups.
Second, Ad-E detection of influence on tumor microenvironment
In B16F10 melanoma, model establishment, grouping and treatment were as in example two, and 3 days after the last administration, mice were sacrificed, tumor tissues were taken, and the T cell ratio in tumor tissues and the expression of PD-L1 in tumor tissues were detected by flow cytometry, WB and RT-PCR, respectively.
The results show that: Ad-Null and Ad-E significantly increased CD3 in tumor specimens compared to Control (FIG. 2C) + CD8 in cells + T cells and CD4 + Proportion of T cells (p)<0.05). CD3 in tumor specimens following Ad-E treatment compared to Ad-Null + CD8 in cells + T cells and CD4 + The proportion of T cells is also increased. Ad-E treatment was shown to increase T cell infiltration in tumor tissues.
The tumor tissues of the Control group hardly expressed PD-L1, whereas Ad-Null and Ad-E increased the expression of PD-L1 after treatment (FIGS. 2D and 2E). T cell infiltration and expression of PD-L1 thus provide a therapeutic basis for immune checkpoint inhibitors.
EXAMPLE three Ad-E in combination with alpha PD-1 for the study of the antitumor Effect
Combination of Ad-E and alpha PD-1 for inhibiting tumor growth
6-8 week-old female C57BL/6 mice were injected subcutaneously with 2X 10 6 MC38 colon cancer cells when the tumor volume reaches about 100mm 3 And then, random grouping is carried out.
IgG2a group: intratumoral injection of sterile 100. mu.l PBS + intraperitoneal administration of 200. mu.g IgG2 a;
group α PD-1: intratumoral injection of sterile 100 μ l PBS + intraperitoneal administration of 200 μ g α PD-1;
Ad-Null group: intratumoral injection of 3X 10 8 pfuAd-Null/mouse (100. mu.l) + equal volume of intraperitoneal sterile PBS;
Ad-E group: intratumoral injection of 3X 10 8 pfuAd-E/peritoneal (100. mu.l) + Isovoluminal Abdominal CavitySterile PBS;
Ad-Null + α PD-1 group: intratumoral injection of 3X 10 8 pfuAd-Null/pig (100. mu.l) + intraperitoneal administration of 200. mu.g of alpha PD-1;
Ad-E + α PD-1 group: intratumoral injection of 3X 10 8 pfuAd-E/mouse (100. mu.l) + intraperitoneal administration of 200. mu.g of alpha PD-1; Ad-Null and Ad-E were injected intratumorally once every 1 week for 2 times; IgG2a and α PD-1 were administered 2 times for 1 week for 4 times. Tumor size was measured in mice every 3 days.
The results show (fig. 3A): the tumors of the IgG2a group and the PD-1 antibody group mice grew very rapidly, indicating that the PD-1 antibody alone had poor therapeutic effect. Ad-Null and Ad-E have certain inhibition effect on tumor growth. The Ad-Null combination alpha PD-1 group has partial remission effect on tumor growth, while the Ad-E combination alpha PD-1 group has the best treatment effect, and 3 mice completely regress tumors, and the result shows that the Ad-E combination alpha PD-1 group can effectively inhibit tumor growth.
II, Ad-E combined with alpha PD-1 for inhibiting tumor angiogenesis
In MC38 colon cancer, model establishment, grouping and treatment modes are shown in example III, after administration, tumor tissues are taken, and blood vessel growth conditions in the tumor tissues are detected by immunohistochemical frozen sections and staining with CD31 antibody.
The results show (FIG. 3B) that the intensity of vascular staining was highest in the IgG2a group, while the vascular density was lowest in the Ad-E group and the Ad-E combined α PD-1 group, indicating that Ad-E combined α PD-1 was effective in improving angiogenesis in the tumor microenvironment.
EXAMPLE four Ad-E in combination with alpha PD-L1 antitumor Effect study
Ad-E combined with alpha PD-L1 for inhibiting tumor growth
6-8 week-old female C57BL/6 mice were injected subcutaneously with 2X 10 6 MC38 colon cancer cells when the tumor volume reaches about 100mm 3 And then, random grouping is carried out.
IgG2b group: intratumoral injection of sterile 100. mu.l PBS + intraperitoneal administration of 200. mu.g IgG2 b;
group α PD-L1: intratumoral injection of sterile 100 μ L PBS + intraperitoneal administration of 200 μ g α PD-L1;
Ad-Null group: intratumoral injectionRay 3X 10 8 pfuAd-Null/mouse (100. mu.l) + equal volume of intraperitoneal sterile PBS;
Ad-E group: intratumoral injection of 3X 10 8 pfuAd-E/single (100. mu.l) + equal volume of intraperitoneal sterile PBS;
Ad-Null + α PD-L1 group: intratumoral injection of 3X 10 8 pfuAd-Null/mouse (100 μ L) + intraperitoneal administration of 200 μ g α PD-L1;
Ad-E + α PD-L1 group: intratumoral injection of 3X 10 8 pfuAd-E/mouse (100. mu.l) + Abdominal administration of 200. mu.g α PD-L1;
Ad-Null and Ad-E were injected intratumorally once every 1 week for 2 times; IgG2b and α PD-L1 were administered 2 times for 1 week for 4 times. Tumor size was measured in mice every 3 days.
The results show (fig. 4): the mice of the IgG2b group and the PD-L1 antibody group have faster tumor growth, which indicates that the PD-L1 antibody has poor single treatment effect. The Ad-E group has the effect of inhibiting the growth of tumors, and the average tumor volume of the mice on the 25 th day is 1587mm 3 . The Ad-Null combined alpha PD-L1 group has partial relieving effect on tumor growth, and the average tumor volume of a mouse at the 25 th day of treatment is 918mm 3 . The Ad-E combined alpha PD-L1 group has the best treatment effect, and the average tumor volume of the mice at the 25 th day of treatment is 224mm 3 Compared with IgG2b, alpha PD-L1, Ad-Null and Ad-E groups, the gene has significant difference (p is less than 0.05). Indicating that Ad-E in combination with alpha PD-L1 is also capable of inhibiting tumor growth.
Example analysis of the Effect of five Ad-E in combination with alpha PD-1 treatment on in vitro tumor microenvironment
In MC38 colon cancer, model establishment, grouping and treatment modes are shown in example III, after administration, tumor tissues are taken, and flow cytometry is adopted to analyze CD8 in tumor microenvironment + And PD-1 + Proportion of T cells, and CD11b + F4/80 + Macrophages and CD206 + Proportion of macrophages.
The experimental results show that: the results show CD45 in tumor tissue after alpha PD-1 treatment + CD8 + The proportion of T cells did not improve. CD8 in Ad-Null, Ad-E and Ad-Null combination with alpha PD-1 treatment + The proportion of T cells increased, but there was no significant difference between the three groups. However, CD8 in Ad-E in combination with alpha PD-1 treatment + The infiltration ratio of T cells is obviously improved, and the T cells are obviously different from IgG2a and alpha PD-1 groups (p)<0.01) (fig. 5A). Simultaneous targeting of CD8 in tumor tissue + The detection of PD-1 on the surface of T cells shows that compared with other treatment methods, the CD8 is obtained after the treatment of Ad-E combined with alpha PD-1 + The proportion of the positive phenotype of PD-1 expressed by T cells is the lowest, and the positive phenotype is obviously different from that of the Ad-Null combined alpha PD-1 group (p)<0.05) (fig. 5B). Shows that the Ad-E combined with the alpha PD-1 treatment can increase the immune infiltration and activate CD8 + CTLs, relieve T cell failure.
Macrophages in tumor tissues are also detected, and compared with an IgG2a group, the alpha PD-1, Ad-Null and Ad-Null are combined with CD11b in the alpha PD-1 group + F4/80 + No significant difference was observed in the proportion of macrophages, whereas Ad-E combined with CD11b in the alpha PD-1 group + F4/80 + The proportion of macrophages is obviously reduced, and the proportion is obviously different from that of IgG2a, alpha PD-1 and Ad-Null combined alpha PD-1 (p)<0.05) (fig. 5C). And the Ad-E combined alpha PD-1 treatment can obviously reduce CD206 Hi Ratio of TAMs (p)<0.05) (fig. 5D). Shows that the combination of alpha PD-1 and Ad-Null with alpha PD-1 group Ad-E combined with alpha PD-1 treatment can relieve immunosuppression and inhibit M2 type macrophage polarization in tumor.
Example six Ad-E combination anti-alpha PD-1 treatment activates immune-related signaling pathways
Female C57BL/6 mice 6-8 weeks old were injected subcutaneously with 2X 10 6 MC38 colon cancer cells when the tumor volume reaches about 100mm 3 And then, random grouping is carried out.
IgG2a group: intratumoral injection of sterile 100 μ l PBS + intraperitoneal administration of 200 μ g IgG2 a;
group α PD-1: intratumoral injection of sterile 100 μ l PBS + intraperitoneal administration of 200 μ g α PD-1;
Ad-E group: intratumoral injection of 3X 10 8 pfuAd-E/single (100. mu.l) + equal volume of intraperitoneal sterile PBS;
Ad-E + α PD-1 group: intratumoral injection of 3X 10 8 pfuAd-E/mouse (100. mu.l) + intraperitoneal administration of 200. mu.g of alpha PD-1;
Ad-E was injected intratumorally once every 1 week for 2 times; IgG2a and α PD-1 were administered 2 times for 1 week for 4 times. After the administration is finished, tumor tissues are taken, transcriptome sequencing is carried out, and the change of signal paths in the tumor tissues is analyzed by bioinformatics.
The experimental results show that: in the 51912 gene probe set, the Ad-E combined with α PD-1 treatment group identified 377 differentially up-regulated genes and 120 differentially down-regulated genes compared to the IgG2a group. The α PD-1 antibody induced up-regulation of 72 genes and down-regulation of 10 genes. Ad-E treatment induced up-regulation of 22 gene expression and down-regulation of 6 genes (FIG. 6A). GSEA enrichment analysis is further carried out on the differential genes in the Ad-E combined alpha PD-1 treatment group, and the results show that the differential genes in the differential expression, such as immune system process, immune response and the like, are enriched more. Further GO bioprocess analysis is performed on genes enriched in the immune system process. The GO bioprocess enrichment suggests that differentially expressed genes are significantly enriched in T cell activity, leukocyte activity, adaptive immune response, interferon production, antigen processing and presentation, etc. (figure 6B). The Ad-E combined with the alpha PD-1 treatment can activate the microenvironment immune response of the body tumor.
EXAMPLE seven Ad-E Combined anti-alpha PD-1 treatment safety preliminary evaluation
6-8 week old female C57BL/6 mice were injected subcutaneously with 2X 10 6 MC38 colon cancer cells, when tumor volume reaches approximately 100IgG2a group: intratumoral injection of sterile 100. mu.l PBS + intraperitoneal administration of 200. mu.g IgG2 a;
control group: without treatment
Group α PD-1: intratumoral injection of sterile 100 μ l PBS + intraperitoneal administration of 200 μ g α PD-1;
Ad-Null group: intratumoral injection of 3X 10 8 pfuAd-Null/mouse (100. mu.l) + equal volume of intraperitoneal sterile PBS;
Ad-E group: intratumoral injection of 3X 10 8 pfuAd-E/single (100. mu.l) + equal volume of intraperitoneal sterile PBS;
Ad-Null + α PD-1 group: intratumoral injection of 3X 10 8 pfuAd-Null/mouse (100. mu.l) + Abdominal administration of 200. mu.g IgG2 a;
Ad-E + α PD-1 group: intratumoral injection of 3X 10 8 pfuAd-E/mouse (100. mu.l) + intraperitoneal administration of 200. mu.g of alpha PD-1;
Ad-Null and Ad-E were injected intratumorally once every 1 week for 2 times; IgG2a and α PD-1 were administered 2 times for 1 week for 4 times. The body weight of the mice was measured every 3 days after tumor inoculation, and after treatment, the mouse organs were stained with H & E.
The results show that no significant abnormality in the body weight of the mice occurred during the treatment, and the combination treatment mode was not significantly toxic to the mice. And the H & E staining mode is adopted again to perform staining photographing observation on important organs such as the heart, the liver, the spleen, the lung and the kidney of the mice, and the like, and the result shows that the important organs have no obvious toxic or side effect (figure 7).
Sequence listing
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Claims (15)
1. An antineoplastic medicine is characterized by containing adenovirus for coding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody as main active components.
2. The antitumor agent as claimed in claim 1, wherein: the amino acid sequence coded and expressed by the adenovirus for coding the human endostatin is the human endostatin shown in SEQ ID No. 1; or; preferably, the human endostatin is endostatin coded by a nucleotide sequence shown in SEQ ID No. 3.
3. The antitumor agent as claimed in claim 3, wherein: the N end of the human endostatin is also connected with a signal peptide; alternatively, the adenovirus is a replication-defective adenovirus; preferably, the adenovirus is a type 5 replication-defective adenovirus.
4. The antitumor agent as claimed in claim 3, wherein: the signal peptide is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor type hEGF signal peptide, human immunoglobulin type kappa chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-2 signal peptide.
5. The antitumor agent as claimed in claim 2 or 3, wherein: the signal peptide is human IL-2 signal peptide, and the amino acid sequence of the human endostatin connected with the human IL-2 signal peptide is shown in SEQ ID No. 2.
6. The antitumor agent according to claim 5, characterized in that: the method comprises the following steps of (1); or the coding nucleotide sequence of the human endostatin connected with the IL-2 signal peptide is shown as SEQ ID No. 4.
7. The antitumor agent as claimed in any one of claims 1 to 6, wherein: in the anti-tumor medicament, the adenovirus for coding the humanized endostatin and the anti-PD-1 antibody or the anti-PD-L1 antibody are mixed into a composition, and are independently and separately packaged in the same packaging container or are respectively and independently packaged in the packaging containers.
8. The application of adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in preparing antitumor medicine.
9. Use according to claim 8, characterized in that: the adenovirus coding the human endothelial chalone codes and expresses the human endothelial chalone with the amino acid sequence shown in SEQ ID No. 1; or; preferably, the human endostatin is endostatin coded by a nucleotide sequence shown in SEQ ID No. 3.
10. Use according to claim 8, characterized in that: the N end of the human endostatin is also connected with a signal peptide; alternatively, the adenovirus is a replication-defective adenovirus; preferably, the adenovirus is a type 5 replication-defective adenovirus.
11. Use according to claim 10, characterized in that: the signal peptide is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor type hEGF signal peptide, human immunoglobulin type kappa chain signal peptide, human IL-2 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-12 signal peptide.
12. Use according to claim 11, characterized in that: when the signal peptide is a human IL-2 signal peptide, the amino acid sequence of the human endostatin connected with the human IL-2 signal peptide is shown in SEQ ID No. 2.
13. Use according to claim 13, characterized in that: the nucleotide sequence of the coding gene of the human endostatin is shown in SEQ ID No. 4.
14. Use according to any one of claims 8 to 13, characterized in that: in the anti-tumor medicament, the adenovirus for coding the humanized endostatin and the anti-PD-1 antibody or the anti-PD-L1 antibody are mixed into a composition, and are independently and separately packaged in the same packaging container or are respectively and independently packaged in the packaging containers.
15. The antitumor agent as claimed in any one of claims 1 to 7 or the use as claimed in any one of claims 8 to 14, wherein: the tumor is as follows: at least one of colorectal cancer, breast cancer, non-small cell lung cancer, liver cancer, colon cancer, kidney cancer, ovarian cancer, head and neck cancer, endometrial cancer, melanoma, and bladder cancer.
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