Use of recombinant human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in the preparation of anti-tumor drugs

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the use of recombinant human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in the preparation of anti-tumor drugs. The technical problem to be solved by the present invention is to provide a drug with good anti-tumor effect. The technical solution to the technical problem of the present invention is to provide an anti-tumor drug containing an adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody as main active ingredients. The co-treatment of adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody can effectively inhibit tumor growth, cause tumor regression without recurrence, and no obvious toxic side effects are observed, indicating that the combination of anti-angiogenic therapy containing adenovirus encoding human endostatin and immunotherapy with anti-PD-1 antibody or anti-PD-L1 antibody is an effective method for treating tumors and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a recombinant human endostatin adenovirus and an anti-PD-1 antibody or an anti-PD-L1 antibody in the preparation of an anti-tumor drug. Background Art

[0002] Angiogenesis refers to the generation of new blood vessels from existing blood vessels. Tumor angiogenesis can provide oxygen and nutrients to tumors and is an important process for the growth and progression of tumor cells. Tumor growth will lead to a decrease in the oxygen content of the tumor microenvironment, which will promote the expression of pro-angiogenic factors (such as vascular endothelial growth factor, epidermal growth factor, etc.) and thus promote angiogenesis. Tumor blood vessels are tortuous and have many branches. The structure and function of the vascular system are abnormal, which will have a serious impact on blood perfusion, leading to hypoxia in tumor tissue 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. As early as 1971, Folkman proposed the theory of tumor angiogenesis, and proved through experiments that anti-angiogenic drugs can effectively inhibit tumor growth. After years of research, a variety of anti-angiogenic drugs have been developed, mainly targeting pro-angiogenic factors and their receptors, such as antibody drugs bevacizumab and small molecule inhibitors sunitinib, which have been approved by the US FDA for the treatment of lung cancer, metastatic colorectal cancer, ovarian cancer and other tumors. Endostatin is an endogenous anti-angiogenic molecule with a molecular weight of 20kDa. In 2005, the Chinese FDA approved Endostatin (Endostar), which has independent 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"). Previous studies have shown that Ad-E has an inhibitory effect on the growth of lung cancer, SKOV3 ovarian cancer and CT26 colon cancer. The recombinant adenovirus has now completed Phase I clinical research.

[0003] As research continues to deepen, it has been found that anti-angiogenic therapy has certain limitations, and some tumors such as prostate cancer and pancreatic cancer are insensitive to anti-vascular drugs. In glioblastoma, the initial response to bevacizumab treatment is significant but the response time is limited. It is easy to develop drug resistance, resulting in poor prognosis and limited patient survival. Bevacizumab has also failed to achieve the expected therapeutic effect in clinical studies of metastatic breast cancer. The regulation of tumor angiogenesis is affected by multiple factors and receptors. The inhibition of one signaling pathway may not affect other signaling pathways. Therefore, finding new treatment methods is crucial for tumor treatment. At present, endostatin is mainly used in combination with radiotherapy, chemotherapy, etc. in clinical practice to improve the cure rate of cancer patients and prolong their survival time.

[0004] Activated CD4 + T cells, CD8 + T cells, natural killer T cells and other cells can express PD-1 molecules. PD-L1 is the 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 combination and interaction of PD-1 and PD-L1 will prevent T cells from recognizing tumor cells, leading to tumor immune escape. At present, tumor immune escape caused by PD-1 / PD-L1 has become a hot area for studying tumor treatment. Inhibitors of PD-1 and PD-L1 can block the combination of PD-1 / PD-L1 to relieve T cell exhaustion, activate effector T lymphocytes, reduce tumor immunosuppression, restore T cell activity, and then recognize and kill tumor cells. At present, antibodies against PD-1 and PD-L1 have been approved for the treatment of various tumors. The FDA has approved anti-PD-L1 antibodies for the treatment of bladder cancer and melanoma. Anti-PD-1 antibodies are currently widely used in more than 10 tumors, including melanoma, non-small cell lung cancer, liver cancer, renal cell carcinoma, head and neck cancer, and DNA mismatch-deficient colorectal cancer. After treatment, tumor growth is alleviated and survival is improved. However, with the continuous in-depth research, the blocking therapy of PD-1 / PD-L1 inhibitors also has many limitations: the response rate of monotherapy is not high, generally between 20% and 40%; the effect varies greatly for different tumors, and there are many insensitive tumors; even in sensitive tumors, there are significant individual differences, and only some people can benefit; the remission rate fluctuates greatly and is unstable, and there are even cases where the disease does not regress but progresses rapidly after the patient uses the inhibitor; there is secondary drug resistance, and some studies have reported that it is estimated that up to 60-70% of patients have developed resistance to immune checkpoint inhibitor treatment. Current studies have shown that there are many reasons for the primary resistance of tumors to PD-1 / PD-L1 inhibitors, mainly: a decrease in infiltrating T lymphocytes in the tumor microenvironment, a decrease in the tumor mutation antigen load, epigenetic abnormalities leading to silencing of Th1 chemokine expression, reduced expression of important immune signaling molecules, high expression of immunosuppressive molecules and thus imbalance of immune regulation. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a drug with good anti-tumor effect. The technical solution to the technical problem of the present invention is to provide an anti-tumor drug containing an adenovirus encoding human endostatin and an anti-PD-1 antibody or an anti-PD-L1 antibody as main active ingredients.

[0006] Wherein, the amino acid sequence encoded and expressed by the adenovirus encoding human endostatin described in the above-mentioned drug is the human endostatin shown in SEQ ID No.1.

[0007] SEQ ID No.1:

[0008] HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK.

[0009] Furthermore, the adenovirus in the anti-tumor drug is a replication-deficient adenovirus. Furthermore, the adenovirus is a type 5 replication-deficient adenovirus.

[0010] Wherein, the N-terminus of the human endostatin in the above-mentioned anti-tumor drug is also connected to a signal peptide.

[0011] Among them, the signal peptide described in the above-mentioned anti-tumor drug is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor hEGF signal peptide, human immunoglobulin κ chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-2 signal peptide.

[0012] Furthermore, when the signal peptide in the above anti-tumor drug is a human IL-2 signal peptide, the amino acid sequence of human endostatin connected to the human IL-2 signal peptide is shown in SEQ ID No.2.

[0013] SEQ ID No.2:

[0014] MYRMQLLSCIALSLALVTNSAHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK.

[0015] Wherein, the coding nucleotide sequence of human endostatin in the above-mentioned anti-tumor drug is shown as SEQ ID No.3.

[0016] SEQ ID No.3:

[0017] .

[0018] Furthermore, the nucleotide sequence encoding human endostatin connected to the IL-2 signal peptide is shown in SEQ ID No.4.

[0019] SEQ ID No.4:

[0020] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGGCCCACAGCCACCGCGACTTCCAGCCGGTGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGCGGGGCATCC GCGGGGCCGACTTCCAGTGCTTCCAGCAGGCGCGGGCCGTGGGGCTGGCGGGCACCTTCCGCGCCTTCCTGTCCTCGCGCCTGCAGGACCTGTACAGCATCGTGCGCCGTGCCGACCGCGCAGCCGTGCCCATCGTCAACCTC AAGGACGAGCTGCTGTTTCCCAGCTGGGAGGCTCTGTTCTCAGGCTCTGAGGGTCCGCTGAAGCCCGGGGCACGCATCTTCTCCTTTGACGGCAAGGACGTCCTGAGGCACCCCACCTGGCCCCAGAAGAGCGTGTGGCATGG CTCGGACCCCAACGGGCGCAGGCTGACCGAGAGCTACTGTGAGACGTGGCGGACGGAGGCTCCCTCGGCCACGGGCCAGGCCTCCTCGCTGCTGGGGGGCAGGCTCCTGGGGCAGAGTGCCGCGAGCTGCCATCACGCCTACA TCGTGCTCTGCATTGAGAACAGCTTCATGACTGCCTCCAAGTAG.

[0021] Among them, the adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody described in the above-mentioned medicine are mixed into a composition, independently packaged in the same packaging container, or separately packaged in packaging containers.

[0022] At the same time, the present invention also provides the use of adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody in the preparation of anti-tumor drugs. Wherein, the adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody in the above use are used in combination.

[0023] The amino acid sequence encoded and expressed by the adenovirus encoding human endostatin in the above-mentioned use is the human endostatin shown in SEQ ID No.1.

[0024] Furthermore, the adenovirus in the anti-tumor drug is a replication-deficient adenovirus. Furthermore, the adenovirus is a type 5 replication-deficient adenovirus.

[0025] Wherein, the N-terminus of the human endostatin in the above-mentioned anti-tumor drug is also connected to a signal peptide.

[0026] Among them, the signal peptide described in the above use is at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor hEGF signal peptide, human immunoglobulin κ chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-2 signal peptide.

[0027] Furthermore, when the signal peptide in the above anti-tumor drug is a human IL-2 signal peptide, the amino acid sequence of human endostatin connected to the human IL-2 signal peptide is shown in SEQ ID No.2.

[0028] Wherein, the coding nucleotide sequence of human endostatin in the above-mentioned anti-tumor drug is shown as SEQ ID No.3.

[0029] Furthermore, the nucleotide sequence encoding human endostatin connected to the IL-2 signal peptide is shown in SEQ ID No.4.

[0030] Among them, the adenovirus encoding human endostatin in the anti-tumor drug in the above-mentioned use is mixed with the anti-PD-1 antibody or the anti-PD-L1 antibody as a composition, and is independently packaged in the same packaging container or separately packaged in packaging containers.

[0031] The present invention also provides a method for treating tumors, comprising the steps of administering an effective amount of an adenovirus encoding human endostatin and an effective amount of an anti-PD-1 antibody to a patient suffering from a tumor, or administering an effective amount of an adenovirus encoding human endostatin and an effective amount of an anti-PD-L1 antibody.

[0032] Furthermore, the human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in the above method can be administered simultaneously, or separately during the course of treatment.

[0033] Wherein, the adenovirus described in the above-mentioned medicine is preferably a replication-deficient adenovirus.

[0034] Among them, the tumors in the above-mentioned technical solutions 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.

[0035] The beneficial effects of the present invention are: the combined treatment of drugs containing adenovirus (Ad-E) encoding human endostatin and anti-PD-1 or anti-PD-L1 antibodies can inhibit angiogenesis, improve tumor suppression microenvironment, enhance T cell tumor infiltration, reduce immunosuppression, inhibit intratumoral M2 macrophage polarization, etc. Experiments have shown that the combined treatment of the two can effectively inhibit tumor growth, cause tumor regression without recurrence, and no obvious toxic side effects are observed. The beneficial effects of the present invention illustrate that the combination of Ad-E anti-angiogenesis therapy and anti-PD-1 antibody immunotherapy or anti-PD-L1 antibody immunotherapy is an effective method for treating tumors, and also provides a theoretical basis for further clinical related experiments, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 . Antitumor activity of αPD-1 (anti-PD-1 antibody) / αPD-L1 (anti-PD-L1 antibody) in mouse tumor models. A. αPD-1 treatment of B16F10 tumors. B. αPD-1 treatment of MC38 tumors. C. αPD-L1 treatment of MC38 tumors. ns: nonsignificant.

[0037] Figure 2 Ad-E treatment effectively inhibited tumor growth. A and B: The tumor volume of B16F10 (A) and MC38 (B) was significantly reduced after Ad-E treatment; C: Flow cytometry analysis of CD4 + and CD8 + The proportion of T cells increased; D: WB detection of PD-L1 protein expression after Ad-E treatment B16F10; E: RT-PCR detection of PD-L1 mRNA expression after Ad-E treatment B16F10. ns: nonsignificant. *p<0.05, **p<0.01, ***p<0.001

[0038] Figure 3 Ad-E combined with αPD-1 treatment enhances anti-tumor activity. A: Ad-E and αPD-1 combined treatment effectively inhibits MC38 tumor growth; B: Tumor samples were immunostained for CD31 (red) and DAPI nuclear staining (blue) to detect angiogenesis.

[0039] Figure 4 .Ad-E combined with αPD-L1 treatment enhances anti-tumor activity. Ad-E and αPD-L1 combined treatment effectively inhibits MC38 tumor growth;

[0040] Figure 5 . In vitro detection of changes in the tumor microenvironment after combined application. A and B: Flow cytometry analysis of CD8 +and PD-1 + C and D: Flow cytometry analysis of CD11b in tumor samples + F4 / 80 + Macrophages and CD206 + The proportion of macrophages (TAMs). *p<0.05, **p<0.01.

[0041] Figure 6 .Transcriptomic analysis of tumor tissues after Ad-E combined with αPD-1 treatment. A: The number of differentially expressed genes found in each group of samples, with up-regulated (Up) on the left and down-regulated (Down) on the right in each group of data; B: GO enrichment analysis of differentially expressed genes after combined treatment of Ad-E and αPD-1.

[0042] Figure 7 .Preliminary evaluation of the safety of Ad-E combined with anti-αPD-1 therapy. H&E staining to detect organ lesions. DETAILED DESCRIPTION

[0043] The present invention is described in detail below through the introduction of specific implementation methods.

[0044] A lot of creative work in the early stage of the present invention found that the use of adenovirus encoding human endostatin (recombinant human endostatin adenovirus, Ad-endostatin, abbreviated as Ad-E or ADE) can inhibit tumor growth and promote tumor T cell infiltration and PD-L1 expression, providing a basis for combination with anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0045] In the previous work of the present invention, it was found that the administration of anti-PD-1 antibodies did not show obvious anti-tumor effects in both the B16F10 tumor model and the MC38 tumor model, and anti-PD-L1 antibody treatment also failed to inhibit the growth of MC38 tumors. Therefore, the present invention combines Ad-E with anti-PD-1 antibodies or PD-L1 antibodies to synergistically improve the anti-tumor effect. The present invention found that the combination of Ad-E and anti-PD-1 antibodies or anti-PD-L1 antibodies can play a synergistic role, significantly enhance the effect of inhibiting tumor growth, and even cause tumor regression without recurrence. It may be that tumor angiogenesis leads to an immunosuppressive tumor microenvironment: a mechanical barrier to T cell infiltration leads to a decrease in T cell infiltration, inhibits antigen presentation of DC cells, increases the proportion of Treg, bone marrow suppressor cells and M2 tumor-associated macrophages, upregulates PD-1 expression on the surface of T cells, and weakens the anti-tumor function of T cells. Ad-E targeting angiogenesis can normalize blood vessels, increase immune cell infiltration and PD-L1 expression in tumor tissues, reduce tumor-induced immunosuppression, and promote the efficacy of tumor immunotherapy. The combination of the two can inhibit angiogenesis, improve the tumor suppression microenvironment, reduce immunosuppression, and reduce the chemotaxis of M2 macrophages, thereby enhancing the anti-tumor effect.

[0046] On this basis, the present invention obtains the various technical solutions to be protected above.

[0047] One of them is to provide an anti-tumor drug containing adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody as main active ingredients. That is, to provide an anti-tumor drug containing adenovirus encoding human endostatin and anti-PD-1 antibody as main active ingredients, or an anti-tumor drug containing adenovirus encoding human endostatin and anti-PD-L1 antibody as main active ingredients.

[0048] Also provided is the use of an adenovirus encoding human endostatin and an anti-PD-1 antibody or an anti-PD-L1 antibody as main active ingredients in the preparation of an anti-tumor drug.

[0049] It can be understood by those skilled in the art that there can be multiple combinations of adenovirus encoding human endostatin and anti-PD-1 antibody or anti-PD-L1 antibody as the main active ingredient in the above-mentioned drug. For example, adenovirus encoding human endostatin and anti-PD-1 or anti-PD-L1 antibody can be formulated into a combined agent and administered together.

[0050] The adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody can also be prepared into separate medicaments, but placed in the same package to make medicines. Such medicines can be used to achieve simultaneous administration of the two active ingredients or separate administration and control of their respective dosages according to needs. Of course, such a preparation form can also facilitate the long-term storage and stability improvement of the two main active ingredients.

[0051] Those skilled in the art may also separately prepare the adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody into separate agents, but place them in separate different packages for the purpose of combined medication.

[0052] The preparation of the above-mentioned medicaments, the selection of corresponding pharmaceutically acceptable auxiliary ingredients, and the selection of corresponding medicament packaging materials are all easily accomplished by those skilled in the art.

[0053] In addition, the present invention also provides a method for treating tumors, which comprises the steps of administering an effective amount of adenovirus encoding human endostatin and an effective amount of anti-PD-1 antibody or anti-PD-L1 antibody to a patient suffering from a tumor. Furthermore, the adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody in the above method can be administered simultaneously, or separately during the treatment process. Of course, for different tumor types and different specific conditions of patients, the specific administration amounts of the adenovirus encoding human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody can be appropriately adjusted to obtain a better synergistic anti-tumor effect.

[0054] The adenovirus encoding human endostatin used in the present invention can also be referred to as a recombinant human endostatin adenovirus. The amino acid sequence of the human endostatin is shown in SEQ ID No. 1. In order to better exert its effect, a signal peptide may be further connected to the N-terminus of human endostatin. Commonly used signal peptides are at least one of human CD5 signal peptide, human CD14 signal peptide, human epidermal growth factor hEGF signal peptide, human immunoglobulin κ chain signal peptide, human IL-12 signal peptide, human IL-3 signal peptide, human IL-18 signal peptide or human IL-2 signal peptide. The adenovirus encoding human endostatin in the present invention generally uses a replication-deficient adenovirus. Preferably, a type 5 replication-deficient adenovirus is used.

[0055] The preparation of human endostatin encoding can be carried out using conventional methods for preparing recombinant adenovirus in the art. In one example provided by the present invention, adenovirus encoding human endostatin is prepared by the method described in the example of Chinese Patent ZL200510021720.7 "A recombinant human endostatin adenovirus and its preparation method and use".

[0056] The anti-PD-1 antibody or anti-PD-L1 antibody described in each technical solution provided by the present invention may be various common anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0057] The present invention is described in further detail below through examples.

[0058] The main experimental materials used in the examples are as follows:

[0059] C57BL / 6 mice were purchased from Beijing Weitonglihua Company.

[0060] 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

[0061] PD-1 antibody was purchased from Bio X cell (Cat. No. BP0146).

[0062] PD-L1 antibody was purchased from Bio X cell (Cat. No. BP0101).

[0063] Adenovirus encoding human endostatin (recombinant human endostatin adenovirus, Ad-endostatin, abbreviated as Ad-E or ADE) and empty adenovirus (Ad-Null) were provided by Guizhou Bailing Enterprise Group Pharmaceutical Co., Ltd. The concentration of Ad-E was 1×10 12 VP / mL; Ad-Null concentration 1.24×10 12 VP / mL, stored at -80℃, diluted to the required concentration before use. The adenovirus encoding human endostatin used in the examples can also be prepared with reference to the description in Chinese Patent ZL200510021720.7 "A recombinant human endostatin adenovirus and its preparation method and use".

[0064] Example 1 Study on the anti-tumor activity of αPD-1 (anti-PD-1 antibody) in vivo

[0065] Female C57BL / 6 mice aged 6-8 weeks were injected subcutaneously with 5×10 5 B16F10 melanoma cells or 2 × 10 6 MC38 colon cancer cells, when the tumor volume reaches about 100mm 3The patients were randomly divided into two groups: the αPD-1 or αPD-L1 group, which was treated with αPD-1 or αPD-L1, with 200 μg of αPD-1 or αPD-L1 injected intraperitoneally each time; the control group was treated with isotype antibodies (IgG2a for αPD-1 and IgG2b for αPD-L1), with 200 μg of isotype antibodies injected intraperitoneally each time; the administration was twice a week for a total of 4 times.

[0066] The experimental results showed that in the B16F10 tumor model ( Figure 1 A), αPD-1 treatment did not inhibit tumor growth compared with the IgG2a control group; in the MC38 tumor model ( Figure 1 B), αPD-1 treatment had a weak antitumor activity compared with the IgG2a control group, but there was no significant difference between the two groups. Figure 1 C), compared with the IgG2a control group.

[0067] Example 2 Study on the anti-tumor activity of recombinant human endostatin adenovirus (Ad-E) in vivo

[0068] 1. Ad-E treatment inhibits tumor growth

[0069] Female C57BL / 6 mice aged 6-8 weeks were subcutaneously injected with 5×10 5 B16F10 melanoma cells or 2 × 10 6 MC38 colon cancer cells, when the tumor volume reaches about 100mm 3 When, random grouping was performed.

[0070] (1) B16F10 melanoma model grouping and treatment:

[0071] Control group: equal volume of sterile PBS;

[0072] Ad-Null group: 1×10 9 pfuAd-Null / mouse, injected intratumorally once every 3 days, for a total of 4 times;

[0073] Ad-E group: 1×10 9 pfuAd-E / mouse, injected intratumorally once every 3 days, for a total of 4 times;

[0074] (2) MC38 colon cancer model grouping and treatment:

[0075] Control group: equal volume of sterile PBS;

[0076] Ad-Null group: 3×10 8 pfuAd-Null / mouse, injected intratumorally once every week, twice in total;

[0077] Ad-E group: 3×10 8 pfuAd-E / mouse, injected intratumorally once every week, for a total of 2 times;

[0078] The tumor size of mice was measured every 3 days.

[0079] The experimental results show that ( Figure 2 ): In B16F10( Figure 2 A) and MC38 tumor model ( Figure 2 In B), Ad-E treatment significantly inhibited tumor growth compared with the Control group and Ad-Null group (p<0.05).

[0080] 2. Detection of the effect of Ad-E on the tumor microenvironment

[0081] In B16F10 melanoma, the model establishment, grouping and treatment methods are shown in Example 2. Three days after the last administration, the mice were killed and the tumor tissues were taken. The proportion of T cells in the tumor tissues was detected by flow cytometry, and the expression of PD-L1 in the tumor tissues was detected by WB and RT-PCR.

[0082] The results showed that compared with the control ( Figure 2 C), Ad-Null and Ad-E significantly increased the expression of CD3 + CD8 + T cells and CD4 + Compared with Ad-Null, the proportion of CD3 T cells in tumor samples after Ad-E treatment was significantly higher than that in Ad-Null. + CD8 + T cells and CD4 + The proportion of T cells also increased, indicating that Ad-E treatment can increase T cell infiltration in tumor tissues.

[0083] The tumor tissues in the control group hardly expressed PD-L1, while the expression of PD-L1 was increased after treatment with Ad-Null and Ad-E ( Figure 2 D and 2E). Thus, T cell infiltration and PD-L1 expression provide a therapeutic basis for immune checkpoint inhibitors.

[0084] Example 3 Study on the anti-tumor effect of Ad-E combined with αPD-1

[0085] 1. Ad-E combined with αPD-1 inhibits tumor growth

[0086] Female C57BL / 6 mice aged 6-8 weeks were subcutaneously injected with 2×10 6MC38 colon cancer cells, when the tumor volume reaches about 100mm 3 When, random grouping was performed.

[0087] IgG2a group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg IgG2a;

[0088] αPD-1 group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg αPD-1;

[0089] Ad-Null group: 3×10 8 pfuAd-Null / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0090] Ad-E group: intratumoral injection of 3×10 8 pfuAd-E / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0091] Ad-Null+αPD-1 group: 3×10 8 pfuAd-Null / mouse (100 μl) + intraperitoneal administration of 200 μg αPD-1;

[0092] Ad-E+αPD-1 group: 3×10 8 pfuAd-E / mouse (100 μl) + intraperitoneal administration of 200 μg αPD-1; Ad-Null and Ad-E were injected intratumorally once a week, for a total of 2 times; IgG2a and αPD-1 were administered twice a week, for a total of 4 times. The tumor size of the mice was measured every 3 days.

[0093] The results show that Figure 3 A): The tumor growth of mice in the IgG2a group and the PD-1 antibody group was very fast, indicating that the PD-1 antibody alone was not effective. Ad-Null and Ad-E treatments had a certain inhibitory effect on tumor growth. The Ad-Null combined with αPD-1 group had a partial relief effect on tumor growth, while the Ad-E combined with αPD-1 group had the best treatment effect, and 3 mice had complete tumor regression. The results showed that Ad-E combined with αPD-1 could effectively inhibit tumor growth.

[0094] 2. Ad-E combined with αPD-1 inhibits tumor angiogenesis

[0095] In MC38 colon cancer, the model establishment, grouping and treatment methods are shown in Example 3. After the administration, the tumor tissue was taken and the blood vessel growth in the tumor tissue was detected by immunohistochemical frozen section staining with CD31 antibody.

[0096] The results show that Figure 3B): The vascular staining intensity was the highest in the IgG2a group, while the vascular density was the lowest in the Ad-E group and the Ad-E combined with αPD-1 group. The results showed that Ad-E combined with αPD-1 can effectively improve angiogenesis in the tumor microenvironment.

[0097] Example 4 Study on the anti-tumor effect of Ad-E combined with αPD-L1

[0098] 1. Ad-E combined with αPD-L1 inhibits tumor growth

[0099] Female C57BL / 6 mice aged 6-8 weeks were subcutaneously injected with 2×10 6 MC38 colon cancer cells, when the tumor volume reaches about 100mm 3 When, random grouping was performed.

[0100] IgG2b group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg IgG2b;

[0101] αPD-L1 group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg αPD-L1;

[0102] Ad-Null group: 3×10 8 pfuAd-Null / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0103] Ad-E group: intratumoral injection of 3×10 8 pfuAd-E / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0104] Ad-Null+αPD-L1 group: 3×10 8 pfuAd-Null / mouse (100 μl) + intraperitoneal administration of 200 μg αPD-L1;

[0105] Ad-E+αPD-L1 group: 3×10 8 pfuAd-E / mouse (100 μl) + intraperitoneal administration of 200 μg αPD-L1;

[0106] Ad-Null and Ad-E were injected intratumorally once a week for 2 times, IgG2b and αPD-L1 were administered twice a week for 4 times. The tumor size of mice was measured every 3 days.

[0107] The results show that Figure 4 ): The tumors in the IgG2b group and the PD-L1 antibody group grew faster, indicating that the PD-L1 antibody alone was not effective. The Ad-E group had an inhibitory effect on tumor growth. On the 25th day of treatment, the average tumor volume of the mice was 1587mm3 The Ad-Null combined with αPD-L1 group had a partial relief effect on tumor growth, and the average tumor volume of mice on the 25th day of treatment was 918mm 3 The Ad-E combined with αPD-L1 group had the best therapeutic effect. On the 25th day of treatment, the average tumor volume of mice was 224 mm 3 , which was significantly different from the IgG2b, αPD-L1, Ad-Null, and Ad-E groups (p < 0.05), indicating that Ad-E combined with αPD-L1 can also inhibit tumor growth.

[0108] Example 5 Analysis of the effect of Ad-E combined with αPD-1 treatment on the in vitro tumor microenvironment

[0109] In MC38 colon cancer, the model establishment, grouping and treatment methods are shown in Example 3. After the administration, the tumor tissue was taken and the number of CD8 in the tumor microenvironment was analyzed by flow cytometry. + and PD-1 + The proportion of T cells and CD11b + F4 / 80 + Macrophages and CD206 + The proportion of macrophages.

[0110] The experimental results showed that the CD45 in tumor tissues after αPD-1 treatment + CD8 + The proportion of T cells did not improve. + The proportion of T cells increased, but there was no significant difference among the three groups. However, in the Ad-E combined with αPD-1 treatment, CD8 + The proportion of T cell infiltration was significantly increased, and there was a significant difference compared with the IgG2a and αPD-1 groups (p<0.01)( Figure 5 A) At the same time, CD8 + The results showed that compared with other treatments, Ad-E combined with αPD-1 treatment had a significant effect on CD8 + The proportion of T cells expressing PD-1 positive phenotype was the lowest, and there was a significant difference compared with the Ad-Null combined with αPD-1 group (p<0.05) ( Figure 5 B) It shows that Ad-E combined with αPD-1 treatment can increase immune infiltration and activate CD8 + CTLs, relieve T cell exhaustion.

[0111] At the same time, macrophages in tumor tissues were also detected. Compared with the IgG2a group, CD11b+ F4 / 80 + There was no significant difference in the proportion of macrophages, but in the Ad-E combined with αPD-1 group, CD11b + F4 / 80 + The proportion of macrophages was significantly reduced, which was significantly different from the IgG2a, αPD-1 and Ad-Null combined with αPD-1 groups (p<0.05) ( Figure 5 C). Ad-E combined with αPD-1 treatment can significantly reduce CD206 Hi The proportion of TAMs (p<0.05) ( Figure 5 D). This indicates that Ad-E combined with αPD-1 treatment can alleviate immunosuppression and inhibit the polarization of intratumoral M2 macrophages compared with αPD-1 and Ad-Null combined with αPD-1.

[0112] Example 6 Ad-E combined with anti-αPD-1 therapy activates immune-related signaling pathways

[0113] Female C57BL / 6 mice aged 6-8 weeks were injected subcutaneously with 2×10 6 MC38 colon cancer cells, when the tumor volume reaches about 100mm 3 When, random grouping was performed.

[0114] IgG2a group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg IgG2a;

[0115] αPD-1 group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg αPD-1;

[0116] Ad-E group: intratumoral injection of 3×10 8 pfuAd-E / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0117] Ad-E+αPD-1 group: 3×10 8 pfuAd-E / mouse (100 μl) + intraperitoneal administration of 200 μg αPD-1;

[0118] Ad-E was injected intratumorally once a week for 2 times in total; IgG2a and αPD-1 were administered twice a week for 4 times in total. After the administration, tumor tissues were collected for transcriptome sequencing and bioinformatics analysis of changes in signal pathways in tumor tissues.

[0119] The experimental results showed that among the 51,912 gene probe groups, compared with the IgG2a group, the Ad-E combined with αPD-1 treatment group identified a total of 377 differentially upregulated genes and 120 differentially downregulated genes. αPD-1 antibody induced 72 genes to be upregulated and 10 genes to be downregulated. Ad-E treatment induced 22 genes to be upregulated and 6 genes to be downregulated ( Figure 6 A). GSEA enrichment analysis was further performed on the differentially expressed genes in the Ad-E combined with αPD-1 treatment group. The results showed that among the many differentially expressed genes, differentially expressed genes in the immune system process and immune response were more enriched. GO biological process analysis was further performed on the genes enriched in the immune system process. GO biological process enrichment suggested that differentially expressed genes were significantly enriched in T cell activity, leukocyte activity, adaptive immune response, interferon production, antigen processing and presentation, etc. ( Figure 6 B). This indicates that Ad-E combined with αPD-1 therapy can activate the body's tumor microenvironment immune response.

[0120] Example 7 Preliminary evaluation of the safety of Ad-E combined with anti-αPD-1 therapy

[0121] Female C57BL / 6 mice aged 6-8 weeks were injected subcutaneously with 2×10 6 MC38 colon cancer cells, when the tumor volume reached about 100IgG2a group: intratumoral injection of sterile 100μl PBS + intraperitoneal administration of 200μg IgG2a;

[0122] Control group: no treatment

[0123] αPD-1 group: intratumoral injection of sterile 100 μl PBS + intraperitoneal administration of 200 μg αPD-1;

[0124] Ad-Null group: 3×10 8 pfuAd-Null / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0125] Ad-E group: intratumoral injection of 3×10 8 pfuAd-E / mouse (100 μl) + equal volume of sterile intraperitoneal PBS;

[0126] Ad-Null+αPD-1 group: 3×10 8 pfuAd-Null / mouse (100 μl) + intraperitoneal administration of 200 μg IgG2a;

[0127] Ad-E+αPD-1 group: 3×10 8 pfuAd-E / mouse (100 μl) + intraperitoneal administration of 200 μg αPD-1;

[0128] Ad-Null and Ad-E were injected intratumorally once a week for 2 times in total; IgG2a and αPD-1 were administered twice a week for 4 times in total. The weight of mice was measured every 3 days after tumor inoculation, and the organs of mice were taken for H&E staining after the treatment.

[0129] The results showed that during the treatment, the mice's weight did not show any significant abnormalities, which preliminarily indicated that the combined treatment method had no significant toxicity to the mice. H&E staining was used again to stain and photograph important organs such as the heart, liver, spleen, lungs, and kidneys of the mice, and the results showed that no significant toxic side effects were observed on important organs ( Figure 7 ). Sequence Listing <110> Sichuan University <120> Use of recombinant human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in the preparation of anti-tumor drugs <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 183 <212> PRT <213> Artificial Sequence <400> 1 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala 35 40 45 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala 50 55 60 Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe 65 70 75 80 Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro 85 90 95 Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro 100 105 110 Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg 115 120 125 Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser 130 135 140 Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln 145 150 155 160 Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn 165 170 175 Ser Phe Met Thr Ala Ser Lys 180 <210> 2 <211> 204 <212> PRT <213> Artificial Sequence <400> 2 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala His Ser His Arg Asp Phe Gln Pro Val Leu His 20 25 30 Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 35 40 45 Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala 50 55 60 Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser 65 70 75 80 Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys 85 90 95 Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu 100 105 110 Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp 115 120 125 Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser 130 135 140 Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg 145 150 155 160 Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly 165 170 175 Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His Ala Tyr Ile Val 180 185 190 Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys 195 200 <210> 3 <211> 552 <212> DNA <213> Artificial Sequence <400> 3 cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60 ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120 gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct gtacagcatc 180 gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc tcaaggacga gctgctgttt 240 cccagctggg aggctctgtt ctcaggctct gagggtccgc tgaagcccgg ggcacgcatc 300 ttctcctttg acggcaagga cgtcctgagg caccccacct ggccccagaa gagcgtgtgg 360 catggctcgg accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg 420 gaggctccct cggccacggg ccaggcctcc tcgctgctgg ggggcaggct cctggggcag 480 agtgccgcga gctgccatca cgcctacatc gtgctctgca ttgagaacag cttcatgact 540 gcctccaagt ag 552 <210> 4 <211> 615 <212> DNA <213> Artificial Sequence <400> 4 atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60 gcccacagcc accgcgactt ccagccggtg ctccacctgg ttgcgctcaa cagccccctg 120 tcaggcggca tgcggggcat ccgcggggcc gacttccagt gcttccagca ggcgcgggcc 180 gtggggctgg cgggcacctt ccgcgccttc ctgtcctcgc gcctgcagga cctgtacagc 240 atcgtgcgcc gtgccgaccg cgcagccgtg cccatcgtca acctcaagga cgagctgctg 300 tttcccagct gggaggctct gttctcaggc tctgagggtc cgctgaagcc cggggcacgc 360 atcttctcct ttgacggcaa ggacgtcctg aggcacccca cctggcccca gaagagcgtg 420 tggcatggct cggaccccaa cgggcgcagg ctgaccgaga gctactgtga gacgtggcgg 480 acggaggctc cctcggccac gggccaggcc tcctcgctgc tggggggcag gctcctgggg 540 cagagtgccg cgagctgcca tcacgcctac atcgtgctct gcattgagaa cagcttcatg 600 actgcctcca agtag 615

Claims

1. An anti-tumor drug, characterized in that: The invention contains an adenovirus encoding and expressing human endostatin and an anti-PD-1 antibody or an anti-PD-L1 antibody as main active ingredients: the adenovirus is a type 5 replication-deficient adenovirus; the adenovirus encoding and expressing human endostatin encodes and expresses human endostatin with a human IL-2 signal peptide connected to the N-terminus, and the amino acid sequence of human endostatin connected to the human IL-2 signal peptide at the N-terminus is shown in SEQ ID No.

2.

2. The anti-tumor drug according to claim 1, characterized in that: The human endostatin is endostatin encoded by the nucleotide sequence shown in SEQ ID No.

3.

3. The anti-tumor drug according to claim 2, characterized in that: The coding nucleotide sequence of human endostatin with IL-2 signal peptide connected to the N-terminus is shown in SEQ ID No.

4.

4. The anti-tumor drug according to claim 1, characterized in that: The adenovirus encoding and expressing human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody in the anti-tumor drug are mixed into a composition, and are separately packaged in the same packaging container or separately packaged in packaging containers.

5. The anti-tumor drug according to any one of claims 1 to 4, characterized in that: The tumor is 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 or bladder cancer.

6. Use of an adenovirus encoding and expressing human endostatin and an anti-PD-1 antibody or an anti-PD-L1 antibody in the preparation of anti-tumor drugs; the adenovirus is a type 5 replication-deficient adenovirus; the adenovirus encoding and expressing human endostatin encodes and expresses human endostatin with a human IL-2 signal peptide connected to the N-terminus, and the amino acid sequence of human endostatin with a human IL-2 signal peptide connected to the N-terminus is shown in SEQ ID No.

2.

7. The use according to claim 6, characterized in that: The encoding nucleotide sequence of human endostatin is shown in SEQ ID No.

3.

8. The use according to claim 6, characterized in that: The coding nucleotide sequence of human endostatin with IL-2 signal peptide connected to the N-terminus is shown in SEQ ID No.

4.

9. The use according to claim 6, characterized in that: The adenovirus encoding and expressing human endostatin and the anti-PD-1 antibody or anti-PD-L1 antibody in the anti-tumor drug are mixed into a composition, and are separately packaged in the same packaging container or separately packaged in packaging containers.

10. The use according to any one of claims 6 to 9, characterized in that: The tumor is 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 or bladder cancer.

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